阅读说明：本技术 一种Shank1通过与MDM2相互作用调节Klotho泛素化的机制研究 (Mechanism research of Shank1 for regulating Klotho ubiquitination through interaction with MDM2 ) 是由 吴剑卿 陈波 黄淑红 赵卫红 于 2021-08-26 设计创作，主要内容包括：本发明公开了一种Shank1通过与MDM2相互作用调节Klotho泛素化的机制研究,包括Shank1在非小细胞肺癌(NSCLC)中的作用以及对Klotho的影响研究和Shank1/mdm2/Klotho三者之间相互作用研究。本发明研究表明,Shank1是一个癌基因,通过与KL和MDM2结合,通过泛素降解下调KL,促进肺癌细胞的迁移、侵袭和增殖能力,参与非小细胞肺癌的细胞增殖、凋亡、转化、侵袭和肿瘤进展。(The invention discloses a mechanism research of Shank1 for regulating Klotho ubiquitination through interaction with MDM2, which comprises the research on the effect of Shank1 in non-small cell lung cancer (NSCLC) and the influence on Klotho and the interaction research between Shank1/MDM 2/Klotho. The research of the invention shows that the Shank1 is an oncogene, and through combination with KL and MDM2, KL is reduced through ubiquitin degradation, so that the migration, invasion and proliferation capacity of lung cancer cells are promoted, and the Shank1 participates in cell proliferation, apoptosis, transformation, invasion and tumor progression of non-small cell lung cancer.)

1. A mechanistic study by Shank1 in modulating Klotho ubiquitination through interaction with MDM2, characterized by: including the effects of Shank1 in non-small cell lung cancer (NSCLC) and the influence research on Klotho (KL) and the interaction research among Shank1/mdm2/Klotho, the following contents are specifically included:

role of A Shank1 in non-small cell lung cancer (NSCLC) and study of influence on Klotho

Shank1 upregulation in A-1NSCLC

The protein level of Shank1 in NSCLC tissues and adjacent normal lung tissues is detected by an immunohistochemical analysis method, and the result shows that 50 of 100 NSCLC tumor tissues show abnormal expression of Shank1, which is obviously higher than that of the corresponding adjacent normal lung tissues;

protein expression in 10 pairs of NSCLC tissues and corresponding adjacent normal lung tissues is detected by a Western blot analysis (Western blot) method, and the result shows that the expression of Shank1 in tumor tissues is increased compared with a corresponding normal lung tissue sample;

the high-risk group and the low-risk group are differentiated according to mRNA expression by using Kaplan-Meier analysis, and the result shows that the total survival rate of the NSCLC patient with high Shank1 expression is lower than that of the NSCLC patient with low Shank1 expression;

knockout of A-2Shank1 inhibits migration, invasion and induction of apoptosis in NSCLC cells

Transwell assays were performed on A549 and H1299 cells transfected with Shank1-shRNA (sh-SHANK1) or Shank1-shRNA (NC), and showed that down-regulation of Shank1 significantly reduced the number of transplanted and invasive A549 and H1299 cells, indicating that Shank1 is a positive regulator of NSCLC motility;

the apoptosis is detected by using flow cytometry to determine the inhibition effect of shRNA on cell death, and the result shows that the apoptosis cell proportion of a549 cell transfected by control Shank1-shRNA (NC) or sh-Shank1 is 4.17 percent and 9.42 percent respectively, which indicates that the reduction of the number of the proliferation cells of sh-SHANK1 is really caused by cell death, and the result of transfecting H1299 cells by using the same method is the same;

wherein, the Shank1-shRNA (sh-SHANK1) is hairpin RNA which can specifically reduce the expression of the SHANK, and the Shank1-shRNA (NC) is a negative control which can not reduce the expression of any gene;

a-3Shank1 overexpression decreases protein expression of Klotho (KL) in a dose-dependent manner

The Western blot method is adopted to carry out correlation research on the expression of the Shank1 and KL proteins of 6 NSCLC patients, and the result shows that the expression of Shank1 and KL in 4 tumor tissues is high, the expression of KL is low, the expression of Shank1 in 2 tumor tissues is low, and the expression of KL is high, which indicates that the expression of Shank1 in lung cancer tissues is remarkably and negatively correlated with the expression of KL;

b Shank1/mdm2/Klotho interaction study

B-1Shank1 interacts with KL to increase the ubiquitination of KL

Through transfection and co-immunoprecipitation experiments, the Shank1 and KL in A549 cells are found to have clear interaction, and the localization of endogenous Shank1 and KL is detected by using the Shank1 and KL antibodies, so that the results show that the Shank1 and the KL are partially co-localized, and the Shank1 obviously increases the ubiquitination of KL;

b-2MDM2 interacts with KL to increase ubiquitination of KL

Transfection of A549 cells with MDM2-HA and Flag-KL plasmids, binding of KL to MDM2 was observed by immunoblot analysis using MDM2-HA antibody after immunoprecipitation of KL with FLAG antibody, complex between Flag-KL and MDM2-HA was also detected after immunoprecipitation of MDM2-HA and immunoblotting with anti-Flag antibody, and localization of endogenous MDM2 and KL was detected with MDM2 and KL antibodies, indicating partial co-localization of MDM2 and KL;

then endogenous KL/MDM2 co-immunoprecipitation determination is carried out on A549 cells, lysates are immunoprecipitated by using an anti-MDM 2 antibody, KL is found to be co-immunoprecipitated with MDM2 at an endogenous level, and the MDM2/KL is proved to be capable of forming a compound in vitro and in vivo, MDM2 can reduce the expression of KL, and the action can be blocked by MG132 (proteasome inhibitor);

the constructs expressing MDM2 gene wt (HA mark) and DN mutant (C462A) are respectively transfected into A549 cells, and MDM2-wt can obviously increase the ubiquitination of KL, while the DN mutant (C462A) of MDM2 can not play the same role, and the results show that MDM2 can also be used as E3 ubiquitin ligase of KL, and Shank1 can regulate the interaction between the two;

b-3Shank1/KL/MDM2 can form a complex

After immunoprecipitation of MDM2 with specific antibodies, western blotting and immunoprecipitation test results of KL show that the correlation between KL and MDM2 can be significantly reduced by Shank1 gene knockout, KL expression is not affected by Shank1, interaction between KL and MDM2 can be increased by exogenous overexpression of Shank1, the interaction can be reduced by Shank1 silencing, and the correlation between KL and Shank1 cannot be affected by the expression level of MDM 2;

after carrying out an endogenous MDM2/KL co-immunoprecipitation test, the Shank1 co-immunoprecipitates with KL under an endogenous level, and the silent Shank1 can weaken the interaction between MDM2 and KL in A549 cells;

through co-immunoprecipitation analysis, it was found that Shank1 did not affect the association between p53 and MDM2, and furthermore, Shank1 was also associated with Mdm2/KL complex and affected complex formation, which indicates that Shank1/KL/MDM2 can form a complex and that Shank1 affected complex formation.

2. The mechanism of Shank1 for modulating Klotho ubiquitination by interacting with MDM2 as claimed in claim 1, wherein: the immunohistochemical analysis method included placing the dewaxed lung cancer paraffin tissue section in EDTA citric acid buffer (ph8.0), microwave repairing the antigen, then incubating the slide with primary antibody (anti-SHANK 1) overnight at 4 ℃, with normal rabbit IgG as negative control to ensure specificity, treating the slide with HRP-polymer coupled secondary antibody for 30min, developing with diaminobenzidine solution (DAB), counterstaining the nucleus with hematoxylin, image acquisition using Nikon (Nikon) inverted fluorescence microscope (Eclipse TE2000-U) HQ2 cold CCD camera and self-contained software, the anti-SHANK 1 model being ab154224, purchased from Abcam, the non-small cell lung cancer tissue chip (TMA) used in the immunohistochemical analysis method being purchased from outpot tech (china shanghai).

3. The mechanism of Shank1 for modulating Klotho ubiquitination by interacting with MDM2 as claimed in claim 1, wherein: the Kaplan-Meier is a survival rate calculation tool, is a website tool based on a cancer genome map database, a European genome phenotype archive (EGA) and a gene expression comprehensive database (GEO) (limited to affymetrix microarrays), calculates a log rank P value and a Hazard Ratio (HR) of a 95% Confidence Interval (CI) and performs mapping, and selects a GEPIA website for the Kaplan-Meier analysis, wherein the GEPIA website is applied to drawing survival maps based on thousands of TCGA samples.

4. The mechanism of Shank1 for modulating Klotho ubiquitination by interacting with MDM2 as claimed in claim 1, wherein: primary antibodies in the Western blot method comprise anti-SHANK 1(ab154224), anti-SHANK 3(ab93607), Bcl-2(ab32124, 1:1000), caspase3(ab13847, 1:1000), Bax (ab32503, 1:1000), AKT (ab8805, 1:1000), P-AKT (ab38449, 1:1000), cyclin D1(ab134175, 1:1000), P70(ab109393, 1:1, 000) and GAPDH (ab181602,1:5000), all of which are purchased from Abcam, and the relative expression of the target protein is calculated by protein/internal reference by using Quantity One software.

5. The mechanism of Shank1 for modulating Klotho ubiquitination by interacting with MDM2 as claimed in claim 1, wherein: the HEK-293, A549, H1299, H650 cells were cultured in Dulbecco Modified Eagle (DMEM) medium supplemented with 10% FBS and 1% penicillin streptomycin in humidified incubators at 37 ℃ and all experiments were completed 48 hours after transfection using Lipofectamin 2000(DNA: lipid 1:2.5) in Opti MEM according to the manufacturer's instructions (ThermoFisher, Waltham, MA), where HEK-293, A549, H1299, H650 cells and Dulbecco Modified Eagle (DMEM) medium were purchased from Kyoto Kay Biotech.

6. The mechanism of Shank1 for modulating Klotho ubiquitination by interacting with MDM2 as claimed in claim 1, wherein: the flow cytometry is specifically to stain NSCLC cells with annexin V-Fluorescein Isothiocyanate (FITC) and Propidium Iodide (PI), detect apoptosis by a flow cytometer, collect the cells by centrifugation after staining treatment, suspend cell particles obtained by centrifugation for 15min at 2500g/min in 500 mul of binding buffer solution, wherein the binding buffer solution is 5 mul L V-FITC and 5 mul of PI solution, incubate for 15min at room temperature, measure annexin V and PI staining by the flow cytometer (FACSCalibur), and analyze data by FlowJo software.

7. The mechanism of Shank1 for modulating Klotho ubiquitination by interacting with MDM2 as claimed in claim 1, wherein: the cell Transwell and invasion assay is specifically characterized in that before starting the assay, a coating buffer is prepared, the coating buffer comprises 0.01M, pH of 8.0 of Tris and 0.7% of NaCl, and is filtered in a sterile environment, and the invasion assay is specifically operated in that Matrigel matrix (purchased from ThermoFisher, Waltham, MA) is subpackaged, unfrozen in an environment at 4 ℃, then diluted with serum-free 1640 medium in a ratio of 1:6, 100 mul is added to each permeable support pore of a 24-well plate, incubated for 4 hours at 37 ℃, the permeable support membrane is removed without affecting the matrix gel layer, 100ul and 600ul of serum-free 1640 medium are respectively added to the inside and outside of the matrix gel layer, and incubated for 30 minutes at 37 ℃, the procedure of the Transwell migration assay is similar to the invasion assay, but without the Matrigel treatment, and the number of cells per empty is 5000.

8. The mechanism of Shank1 for modulating Klotho ubiquitination by interacting with MDM2 as claimed in claim 1, wherein: the cells used in the co-immunoprecipitation assay were HEK293 or a549, the Shank1 siRNA oligonucleotide previously tested in the Shank1 gene knock-out was synthesized from Invitrogen corporation with a target sequence of 5'-aagcttggcacgccaaaaaa-3', and the negative control oligonucleotide encoding siRNA was purchased from Invitrogen, not homologous to any known mammalian sequence.

9. The mechanism of Shank1 for modulating Klotho ubiquitination by interacting with MDM2 as claimed in claim 1, wherein: the plasmids encoding HA-and myc-tagged human Klotho were from b.chen et al (2012), b.chen et al (2019) reports, the plasmids encoding Shank1, MDM2 and related mutants were from Youbio, and other mutations were introduced by PCR and confirmed by dideoxy sequencing.

10. The mechanism of Shank1 for modulating Klotho ubiquitination by interacting with MDM2 as claimed in claim 1, wherein: the antibodies and reagents used in the assay were rabbit and mouse monoclonal anti-KL antibodies, rabbit anti-Shank 1 and rabbit anti-MDM 2 from Abcam, uk; detecting ubiquitination with a mouse anti-ubiquitin (P4D1) antibody; rabbit and mouse anti-FLAG antibodies, and rat monoclonal high affinity anti-HA antibodies were from Sigma-Aldrich (st louis, missouri); MG132 is available from MCE corporation, Annelberg, Mich; tissue culture media and reagents were from Invitrogen, purchased from ThermoFisher, Waltham, MA; restriction enzymes were purchased from new england biological laboratory (NEB) (Danvers, MA) and all other chemicals were purchased from Sigma Aldrich.

Technical Field

The invention relates to a research on expression and function of Shank1 in non-small cell lung cancer, in particular to a research on a mechanism that Shank1 regulates Klotho ubiquitination through interaction with MDM2, and belongs to the field of biomedical application.

Background

Lung cancer is one of the most common malignant tumors in the world, and has a high mortality rate. Non-small cell lung cancer (NSCLC) is the most common histological type of lung cancer, accounting for 85-90% of all lung cancer cases. A large number of non-small cell lung cancer patients are diagnosed as advanced. Despite great progress in various therapeutic approaches including surgery, 5-year survival rates remain < 15%. The molecular mechanisms of proliferation, invasion and metastasis of non-small cell lung cancer can undoubtedly provide beneficial guidance for the diagnosis and treatment of lung cancer.

H3 and multiple ankyrin repeat domain (SHANK)1-3 belong to a scaffold protein family SHANK protein, and are mainly present in the postsynaptic compact (PSD) region of neuronal excitatory synapses. The function of the SHANK family is critical to the normal function of the nervous system. Non-small cell lung cancer (Non-small cell lung cancer! NSCLC) is the leading cause of cancer-related death worldwide, with poor prognosis and a need for new therapeutic approaches in the clinic. SHANK1 is a scaffold protein that plays an important role in the normal function of neurons.

Shank1 plays a key role in cognition, and its dysregulation leads to severe impairment of learning and cognitive assessment. However, the expression and function of Shank1 in non-small cell lung cancer is not clear. In recent years, Shank1 has been reported to be abnormally highly expressed in colon cancer tissues and to be associated with patient prognosis. The SHANK1 gene knockout inhibits the survival of colon cancer cell lines and induces the apoptosis of the colon cancer cell lines through AKT/mTOR signaling pathway. The changed methylation of the SHANK1 CpG island can be used as a biomarker for the risk and diagnosis of chronic lymphocytic leukemia. These data indicate that SHANK1 may be a novel oncogene. However, it is not clear how SHANK1 plays a role in the tumor process.

The anti-aging gene Klotho (KL) is a potent tumor suppressor in many malignancies, including colorectal, glioma, melanoma, and ovarian cancers. Multiple signaling mechanisms, including IGF-1, FGF, and Wnt/β -catenin pathways, have been reported to be associated with Klotho tumor inhibitory activity; the specific mode of action of Klotho in cancer remains controversial, however, we previously reported a role for Klotho in NSCLC pathogenesis, suggesting that Klotho inhibits NSCLC proliferation and motility and triggers apoptosis by modulating IGF-1/insulin signaling and Wnt signaling pathways. Although numerous studies have demonstrated the important tumor-inhibiting effects of Klotho, information on the underlying molecular mechanisms of Klotho regulation is still limited at present. Recently, we found that rab8 is involved in membrane localization of KL and KL-induced abnormal regulation of Wnt signaling pathway. However, the function of other KL-interacting proteins still needs to be further investigated. Through immunoprecipitation and mass spectrometry, we found Shank1 to be a potential KL-binding protein.

In this application, the inventors disclose that Shank1 is abnormally upregulated in non-small cell lung cancer and participates in invasion and metastasis of non-small cell lung cancer cells by modulating MDM 2-dependent KL degradation.

Disclosure of Invention

The invention aims to solve the problems and provide a mechanism research of Shank1 for regulating Klotho ubiquitination through interaction with MDM 2.

The invention achieves the aim through the following technical scheme, and the mechanism research of Shank1 for regulating Klotho ubiquitination through interaction with MDM2 comprises the function of Shank1 in non-small cell lung cancer (NSCLC) and the influence research on Klotho (KL) and the interaction research among Shank1/MDM2/Klotho, and specifically comprises the following contents:

role of A Shank1 in non-small cell lung cancer (NSCLC) and study of influence on Klotho

Shank1 upregulation in A-1NSCLC

The protein level of Shank1 in NSCLC tissues and adjacent normal lung tissues is detected by an immunohistochemical analysis method, and the result shows that 50 of 100 NSCLC tumor tissues show abnormal expression of Shank1, which is obviously higher than that of the corresponding adjacent normal lung tissues;

protein expression in 10 pairs of NSCLC tissues and corresponding adjacent normal lung tissues is detected by a Western blot analysis (Western blot) method, and the result shows that the expression of Shank1 in tumor tissues is increased compared with a corresponding normal lung tissue sample;

the high-risk group and the low-risk group are differentiated according to mRNA expression by using Kaplan-Meier analysis, and the result shows that the total survival rate of the NSCLC patient with high Shank1 expression is lower than that of the NSCLC patient with low Shank1 expression;

knockout of A-2Shank1 inhibits migration, invasion and induction of apoptosis in NSCLC cells

Transwell assays were performed on A549 and H1299 cells transfected with Shank1-shRNA (sh-SHANK1) or Shank1-shRNA (NC), and showed that down-regulation of Shank1 significantly reduced the number of transplanted and invasive A549 and H1299 cells, indicating that Shank1 is a positive regulator of NSCLC motility;

the apoptosis is detected by using flow cytometry to determine the inhibition effect of shRNA on cell death, and the result shows that the apoptosis cell proportion of a549 cell transfected by control Shank1-shRNA (NC) or sh-Shank1 is 4.17 percent and 9.42 percent respectively, which indicates that the reduction of the number of the proliferation cells of sh-SHANK1 is really caused by cell death, and the result of transfecting H1299 cells by using the same method is the same;

wherein, the Shank1-shRNA (sh-SHANK1) is hairpin RNA which can specifically reduce the expression of the SHANK, and the Shank1-shRNA (NC) is a negative control which can not reduce the expression of any gene;

a-3Shank1 overexpression decreases protein expression of Klotho (KL) in a dose-dependent manner

The Western blot method is adopted to carry out correlation research on the expression of the Shank1 and KL proteins of 6 NSCLC patients, and the result shows that the expression of Shank1 and KL in 4 tumor tissues is high, the expression of KL is low, the expression of Shank1 in 2 tumor tissues is low, and the expression of KL is high, which indicates that the expression of Shank1 in lung cancer tissues is remarkably and negatively correlated with the expression of KL;

b Shank1/mdm2/Klotho interaction study

B-1Shank1 interacts with KL to increase the ubiquitination of KL

Through transfection and co-immunoprecipitation experiments, the Shank1 and KL in A549 cells are found to have clear interaction, and the localization of endogenous Shank1 and KL is detected by using the Shank1 and KL antibodies, so that the results show that the Shank1 and the KL are partially co-localized, and the Shank1 obviously increases the ubiquitination of KL;

b-2MDM2 interacts with KL to increase ubiquitination of KL

Transfection of A549 cells with MDM2-HA and Flag-KL plasmids, binding of KL to MDM2 was observed by immunoblot analysis using MDM2-HA antibody after immunoprecipitation of KL with FLAG antibody, complex between Flag-KL and MDM2-HA was also detected after immunoprecipitation of MDM2-HA and immunoblotting with anti-Flag antibody, and localization of endogenous MDM2 and KL was detected with MDM2 and KL antibodies, indicating partial co-localization of MDM2 and KL;

then endogenous KL/MDM2 co-immunoprecipitation determination is carried out on A549 cells, lysates are immunoprecipitated by using an anti-MDM 2 antibody, KL is found to be co-immunoprecipitated with MDM2 at an endogenous level, and the MDM2/KL is proved to be capable of forming a compound in vitro and in vivo, MDM2 can reduce the expression of KL, and the action can be blocked by MG132 (proteasome inhibitor);

the constructs expressing MDM2 gene wt (HA mark) and DN mutant (C462A) are respectively transfected into A549 cells, and MDM2-wt can obviously increase the ubiquitination of KL, while the DN mutant (C462A) of MDM2 can not play the same role, and the results show that MDM2 can also be used as E3 ubiquitin ligase of KL, and Shank1 can regulate the interaction between the two;

b-3Shank1/KL/MDM2 can form a complex

After immunoprecipitation of MDM2 with specific antibodies, western blotting and immunoprecipitation test results of KL show that the correlation between KL and MDM2 can be significantly reduced by Shank1 gene knockout, KL expression is not affected by Shank1, interaction between KL and MDM2 can be increased by exogenous overexpression of Shank1, the interaction can be reduced by Shank1 silencing, and the correlation between KL and Shank1 cannot be affected by the expression level of MDM 2;

after carrying out an endogenous MDM2/KL co-immunoprecipitation test, the Shank1 co-immunoprecipitates with KL under an endogenous level, and the silent Shank1 can weaken the interaction between MDM2 and KL in A549 cells;

through co-immunoprecipitation analysis, it was found that Shank1 did not affect the association between p53 and MDM2, and furthermore, Shank1 was also associated with Mdm2/KL complex and affected complex formation, which indicates that Shank1/KL/MDM2 can form a complex and that Shank1 affected complex formation.

Preferably, the immunohistochemical analysis method comprises placing the dewaxed paraffin tissue section of lung cancer in EDTA citrate buffer (ph8.0), microwaving the antigen, then incubating the slide with a primary antibody (anti-SHANK 1) overnight at 4 ℃, with normal rabbit IgG as a negative control to ensure specificity, treating the slide with HRP-polymer coupled secondary antibody for 30min, developing with diaminobenzidine solution (DAB), counterstaining the nuclei with hematoxylin, image acquisition using a Nikon (Nikon) inverted fluorescence microscope (Eclipse TE2000-U) HQ2 cold CCD camera and self-contained software, the anti-SHANK 1 model ab154224, purchased from Abcam, and the non-small cell lung cancer tissue chip (TMA) used in the immunohistochemical analysis method, purchased from Outdo Biotech (china shanghai).

Preferably, the Kaplan-Meier is a survival rate calculation tool, and is a website tool based on a cancer genomic profiling database, a european genome phenotype archive (EGA) and a gene expression integrated database (GEO) (restricted to affymetrix microarrays), and the Kaplan-Meier analysis selects a GEPIA website, which is applied to plot survival based on thousands of TCGA samples by calculating a Hazard Ratio (HR) of a log rank P value and a 95% Confidence Interval (CI) and plotting.

Preferably, the primary antibodies in the Western blot method include anti-SHANK 1(ab154224), anti-SHANK 3(ab93607), Bcl-2(ab32124, 1:1000), caspase3(ab13847, 1:1000), Bax (ab32503, 1:1000), AKT (ab8805, 1:1000), P-AKT (ab38449, 1:1000), cyclin D1(ab134175, 1:1000), P70(ab109393, 1:1, 000) and GAPDH (ab181602,1:5000), all of which are purchased from Abcam, and the relative expression of the target protein is calculated by protein/internal reference using Quantity One software.

Preferably, the HEK-293, A549, H1299, H650 cells were cultured in Dulbecco's Modified Eagle (DMEM) medium supplemented with 10% FBS and 1% penicillin streptomycin in humidified incubator at 37 ℃ and transfected with Lipofectamin 2000(DNA: lipid 1:2.5) in Opti MEM according to the manufacturer's instructions (ThermoFisher, Waltham, MA), all at 48 hours post-transfection, with HEK-293, A549, H1299, H650 cells and Dulbecco's Modified Eagle (DMEM) medium purchased from Kyoto Kayji Biotech.

Preferably, the flow cytometry is specifically to stain NSCLC cells with annexin V-Fluorescein Isothiocyanate (FITC) and Propidium Iodide (PI), detect apoptosis by a flow cytometer, collect the cells by centrifugation after staining, suspend cell particles obtained by centrifugation for 15min at 2500g/min in 500 μ L of binding buffer solution, wherein the binding buffer solution is 5 μ L V-FITC and 5 μ L PI solution, incubate for 15min at room temperature, measure annexin V and PI staining by a flow cytometer (FACSCalibur), and perform data analysis by FlowJo software.

Preferably, the cell Transwell and invasion assay is embodied by preparing a coating buffer comprising 0.01M, pH of 8.0 Tris and 0.7% NaCl before starting the assay and filtering in a sterile environment, the invasion assay being embodied by dispensing Matrigel matrix (purchased from ThermoFisher, Waltham, MA), thawing at 4 ℃ and then diluting with serum-free 1640 medium in a ratio of 1:6, adding 100 μ l to each permeable support well of a 24-well plate, incubating for 4 hours at 37 ℃, removing the permeable support membrane without affecting the matrix gel layer, and adding 100ul and 600ul of serum-free 1640 medium to the inside and outside of the matrix gel layer, respectively, incubating for 30 minutes at 37 ℃, the procedure of the Transwell migration assay being similar to the invasion assay but without Matrigel treatment, the number of cells per void being 5000.

Preferably, the cells used in the co-immunoprecipitation assay are HEK293 or a549, the Shank1 siRNA oligonucleotide previously tested in the Shank1 gene knock-out was synthesized from Invitrogen corporation with a target sequence of 5'-aagcttggcacgccaaaaaa-3', and the negative control oligonucleotide encoding siRNA was purchased from Invitrogen without homology to any known mammalian sequence.

Preferably, the plasmids encoding HA-and myc-tagged human Klotho are from the reports of b.chen et al (2012), b.chen et al (2019), the plasmids encoding Shank1, MDM2 and related mutants are from Youbio, and other mutations are introduced by PCR and confirmed by dideoxy sequencing.

Preferably, the antibodies and reagents used during the assay are rabbit and mouse monoclonal anti-KL antibodies, rabbit anti-Shank 1 and rabbit anti-MDM 2 antibodies from Abcam, uk; detecting ubiquitination with a mouse anti-ubiquitin (P4D1) antibody; rabbit and mouse anti-FLAG antibodies, and rat monoclonal high affinity anti-HA antibodies were from Sigma-Aldrich (st louis, missouri); MG132 is available from MCE corporation, Annelberg, Mich; tissue culture media and reagents were from Invitrogen, purchased from ThermoFisher, Waltham, MA; restriction enzymes were purchased from new england biological laboratory (NEB) (Danvers, MA) and all other chemicals were purchased from Sigma Aldrich.

The invention has the beneficial effects that: the invention discloses a novel method for degrading KL through an ubiquitin system by researching a mechanism of Shank1 for regulating Klotho ubiquitination through interaction with MDM2, and discloses a report that Shank1 is an oncogene, is combined with KL and MDM2, is used for reducing KL through ubiquitin degradation, promoting migration, invasion and proliferation capacity of lung cancer cells, and participates in cell proliferation, apoptosis, transformation, invasion and tumor progression of non-small cell lung cancer. The data disclosed by the invention provide a new mechanism for the effects of Shank1, KL and MDM2 in the progression of NSCLC.

Drawings

FIG. 1 is a graph of the expression of Shank1 detected by immunohistochemical staining of NSCLC tissue samples according to the present invention.

FIG. 2 is a graph showing the relative expression of the proteins of Shank1 and Shank1 in NSCLC samples assayed by the WesternBlot of the present invention, and tubulin, wherein T represents tumor cells and N represents normal cells.

FIG. 3 is a graphical representation of the prognostic survival of Shank1 for all NSCLC, adenocarcinoma, and squamous cell carcinoma patients of the present invention, requiring AffymetrixID 220563_ at (Shank 1).

Figure 4 is a graph of survival curves for non-smokers, and male patients in accordance with the present invention.

FIG. 5 is the expression diagram of KL protein of different lung cancer cell lines according to the present invention.

FIG. 6 is a graph showing the expression level of Shank1mRNA in A549 cells after transfection of Shank1-shRNAs and Shank1-shRNA (NC) respectively in the present invention.

FIG. 7 is a graph showing the comparison of the expression levels of Shank1mRNA in H1299 cells after transfection of Shank1-shRNAs and Shank1-shRNA (NC) in accordance with the present invention.

FIG. 8 is a graph showing the OD values of A549 cells at 48 and 72 hours after transfection of Shank1-shRNA (sh-SHANK1) and Shank1-shRNA (NC) according to the present invention, respectively.

FIG. 9 is a graph showing the OD values of H1299 cells at 48 and 72 hours after transfection of Shank1-shRNA (sh-SHANK1) and Shank1-shRNA (NC) according to the present invention, respectively.

FIG. 10 is a comparison of A549, H1299, and SK-MES-1 cell colonies after transfection of the present invention with Shank1-shRNA (sh-SHANK1) and Shank1-shRNA (NC), respectively.

FIG. 11 is a graph comparing the number of clones of A549, H1299 and SK-MES-1 cells after transfection of Shank1-shRNA (sh-SHANK1) and Shank1-shRNA (NC) respectively in accordance with the present invention.

FIG. 12A is a graph showing the detection of migration and invasion of A549 cells by the Transwell method according to the present invention.

Fig. 12B is a histogram of the number of cells migrated by a549 cells of the invention (. P < 0.05).

Fig. 12C is a histogram of the number of cells invaded by a549 cells of the invention (. P < 0.05).

FIG. 13A is a graph showing the migration and invasion of H1299 cells measured by the transwell method of the present invention.

Fig. 13B is a histogram of the number of cells migrated by H1200 cells of the invention (. P < 0.05).

Fig. 13C is a histogram of the number of cells invaded by H1200 cells of the invention (. P < 0.05).

FIG. 14 is a diagram of the detection of apoptosis of a549 cells by flow cytometry.

FIG. 15 is a graph of the detection of H1299 apoptosis by flow cytometry in accordance with the present invention.

FIG. 16 is a bar graph of the proportion of apoptosis of the WesternBlot assay A549 cells and H1299 cells of the invention (. about.P < 0.05).

FIG. 17A is a graph of the relative protein levels of Bax, C-Caspase-3, Bcl2 and GAPDH in cells of WesternBlot of the invention, A549 and H1299.

FIG. 17B is a bar graph of the fold difference between Bax, C-Caspase-3 and Bcl2 in A549 cells of the invention (. about.P < 0.05).

FIG. 17C is a bar graph of the fold difference between Bax, C-Caspase-3, Bcl2 in H1299 cells of the invention (. about.P < 0.05).

FIG. 18A is a graph of the WesternBlot assay of AKT, p-AKT, mTOR, p-mTOR, p70S6K, and GAPDH-related protein levels in A549 and H1299 cells of the invention.

FIG. 18B is a bar graph of fold difference between P-AKT, P-mTOR and P70S6K in A549 cells of the invention (. about.P < 0.05).

Figure 18C is a bar graph of fold difference between P-AKT, P-mTOR and P70S6K in H1299 cells of the invention (. about.pp < 0.05).

FIG. 19 is a bar graph of Shank1 and KL protein expression levels of 6 patients with NSCLC of the present invention.

FIG. 20A is a graph showing that protein expression of KL and relative expression of tubulin in the WesternBlot assay A549 cells of the present invention were preincubated with CHX and CHX + MG132 for 6 hours.

Fig. 20B is a bar graph of relative KL protein expression (. sp <0.05) of a549 cells of the invention pre-incubated for 6 hours with CHX and CHX + MG 132.

FIG. 21A is a diagram showing the results of immunoprecipitation with an anti-Flag antibody after lysis of A549 cells transfected with Shank1-myc and KL-Flag vectors by WesternBlot blot analysis of the present invention.

FIG. 21B is a graph showing the detection of the endogenous relationship of KL and Shank1 in adult rat lung tissue by immunoprecipitation in accordance with the present invention.

FIG. 22 is a graph showing the ubiquitination results of KL detected by the IP method and the Ub antibody WesternBlot blotting method of the present invention.

FIG. 23 is a horizontal view of the invention showing the relative Ub in FIG. 22.

FIG. 24 is a diagram showing that the lysate of A549 cells transfected with MDM2-HA and Flag-KL vector of the present invention is immunoprecipitated with anti-Flag antibody, and the immunoblotting analysis detects MDM2 and KL immunoprecipitated proteins.

FIG. 25 is a graph showing that the lysate of A549 cells transfected with MDM2-HA and Flag-KL constructs of the present invention was immunoprecipitated with anti-HA antibody, and the immunoblotting method was used to detect MDM2 and KL immunoprecipitated proteins.

FIG. 26 is a diagram of the subcellular co-localization of endogenous KL and MDM2 in A549 cells observed with a confocal microscope according to the present invention.

FIG. 27 is a graph showing the results of the co-immunoprecipitation assay of endogenous KL and MDM2 of the invention.

FIG. 28A is a graph showing the detection of KL protein levels in A549 cells transfected with vector or MDM2 by immunoblotting of the invention.

Figure 28B shows KL relative protein levels (× P <0.01) in figure 28A for the present invention.

FIG. 29A is a diagram showing the detection of KL immunoprecipitated protein by the IP method and Ub antibody WesternBlot blotting method of the present invention.

Fig. 29B shows relative levels of Ub in fig. 29A (× P <0.01) for the invention.

FIG. 30 is a graph showing the results of the present invention in detecting the lysates of A549 cells transfected with MDM2-HA, KL-Flag, and sh-Shank1 by immunoprecipitation and immunoblotting.

FIG. 31 is a diagram showing the results of the immunoblotting method of the present invention for detecting KL protein in A549 cell lysate of transfected Shank1-myc or sh-Shank1 vector.

FIG. 32A is a graph of immunoprecipitated proteins from MDM2 and Shank1 detected by immunoblotting of the invention.

FIG. 32B is a diagram of immunoprecipitated proteins from the immunoblotting assay for KL and Shank1 of the invention.

FIG. 33A is a graph of the immunoblot detection of immunoprecipitated proteins from A549 cell lysates co-transfected with Myc-Shank 1/or siShank1, MDM2-HA and Flag-KL constructs using anti-HA antibody immunoprecipitation in accordance with the present invention.

FIG. 33B is a diagram showing the detection of immunoprecipitated proteins from A549 cell lysates co-transfected with Myc-Shank1r, MDM 2-HA/or si-MDM2 and Flag-KL constructs by immunoprecipitation with anti-Myc antibody in accordance with the immunoblotting method of the invention.

FIG. 34 is a diagram of the immunoprecipitated protein from A549 cell lysate of transfected siShank1 detected by the immunoblotting method of the invention.

FIG. 35 is a graph showing the immunoprecipitation of A549 cell lysate from immunoprecipitated proteins of vectors co-transfected with Myc-Shank1 and/or sink 1, MDM2-HA and Flag-p53 by immunoprecipitation using anti-HA antibody in accordance with the present invention.

FIG. 36 is a graph of immunoprecipitated proteins from A549 cells transfected with the vector or the Shank1-myc construct of the invention by immunoblot analysis with anti-p 53 and p21 antibodies.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The antibodies and reagents used in the assay were rabbit and mouse monoclonal anti-KL antibodies, rabbit anti-Shank 1 and rabbit anti-MDM 2 from Abcam, uk; detecting ubiquitination with a mouse anti-ubiquitin (P4D1) antibody; rabbit and mouse anti-FLAG antibodies, and rat monoclonal high affinity anti-HA antibodies were from Sigma-Aldrich (st louis, missouri); MG132 is available from MCE corporation, Annelberg, Mich; tissue culture media and reagents were from Invitrogen, purchased from ThermoFisher, Waltham, MA; restriction enzymes were purchased from new england biological laboratory (NEB) (Danvers, MA) and all other chemicals were purchased from Sigma Aldrich.

Experimental data statistical analysis of quantitative data was performed using the sps 19 software. Data were analyzed using one-way analysis of variance (ANOVA), followed by Bonferroni/Dunn post hoc mean comparisons and multiple comparison corrections. p <0.05 was considered significantly different.

A mechanism study of Shank1 in regulating Klotho ubiquitination through interaction with MDM2 includes the role of Shank1 in non-small cell lung cancer (NSCLC) and the study of the influence on Klotho and the interaction study between Shank1/MDM 2/Klotho.

EXAMPLE I Effect of Shank1 in non-Small cell Lung cancer (NSCLC) and study of the Effect on Klotho

1.1 Shank1 upregulation in NSCLC

In order to identify the expression of Shank1 in NSCLC tissues, the protein level of Shank1 in NSCLC tissues (tissue chips) and adjacent normal lung tissues is detected by an immunohistochemical method, and the results are shown in figure 1, 50 of 100 NSCLC tumor tissues show that the expression of Shank1 is abnormal and is obviously higher than that of the corresponding adjacent normal lung tissues;

furthermore, the expression of proteins in 10 pairs of NSCLC tissues and corresponding adjacent normal lung tissues is detected by Western blot analysis, and the result is shown in figure 2, and the expression of Shank1 in tumor tissues is increased compared with the corresponding normal lung tissue samples;

using the Kaplan-Meier analysis of the Kaplan-Meie website (http:// kmplot. com), we differentiated the high risk group from the low risk group based on mRNA expression (log-rank test, p <0.01), and as a result, as shown in FIG. 3, the overall survival rate of NSCLC patients with high Shank1 expression is lower than that of low Shank1 expression, mainly occurring in adenocarcinoma rather than squamous carcinoma, so we are more concerned about the role of Shank1 in LUAD cells. Furthermore, we found that this correlation was only found in the non-smoking group or the male group (as shown in fig. 4). Furthermore, we examined the expression of Shank1 protein in various non-small cell lung cancer cell lines compared to BEAS-2 cells and found that its expression was elevated in most NSCLC cell lines (as shown in fig. 5); "

To investigate whether Shank1 plays an important role in non-small cell lung cancer, we used specific Shank1-shRNAs (shRNA1#, shRNA2#, shRNA3#) to prevent Shank1 expression, transfected a549 and H1299 cells with Shank1-shRNAs, and then examined the knock-out efficiency of siRNAs with qRT PCR, as shown in fig. 6 and 7, Shank1-shRNAs transfected a549 cells, Shank1-shRNA2 and Shank1-shRNA3 groups Shank1mRNA expression significantly decreased (p <0.01#, Shank1-shRNAs transfected H1299 cells, Shank1-shRNA 68642 and Shank1-shRNA2 groups Shank1mRNA expression significantly decreased (p <0.01#, as shown in fig. 6 and 7, as shown in the results, Shank 2a transfected cells and H1299 cells transfected with Shank 58549, Shank 4624-shRNA 549 p < 5730.5730;

as measured by CCK8, we observed that Shank1-shRNA (sh-Shank1) caused down-regulation of Shank1mRNA expression significantly decreased OD values (p <0.05) in a549 and H1299 cells at 48 and 72 hours, as shown in fig. 8 and 9, i.e., CCK8 experimental results showed that Shank1 gene knockout inhibited proliferation of a549 and H1299 cells;

we further studied the proliferation of A549, H1299 and SK-MES-1 cells by using clonogenic experiments, and we constructed stable knockout cell lines with sh-SHANK1 sequence in order to obtain longer knockout effect, as shown in FIG. 10 and FIG. 11, the clone number of the Shank1-shRNA (sh-SHANK1) group was reduced to about 60% of that of the Shank1-shRNA (NC) group in 3 cell lines, and the clonogenic experiments showed that SHANK1 gene knockout inhibited the growth of A549, H1299 and SK-MES-1 cells.

The immunohistochemistry method in the above example specifically comprises placing θ sections in EDTA citric acid buffer (ph8.0), microwave repairing the antigen, then incubating the slide with primary antibodies (anti-SHANK 1, ab154224, Abcam) overnight at 4 ℃, with normal rabbit IgG as a negative control to ensure specificity, treating the slide with HRP-polymer conjugated secondary antibody for 30min, developing with diaminobenzidine solution (DAB), counterstaining the nuclei with hematoxylin, and image acquisition using a nikon camera and software.

The non-small cell lung cancer tissue chip (TMA) used in the immunohistochemical method described above was purchased from Outdo Biotech (Shanghai core Biotech Co., Ltd., China).

Western blot analysis in the above examples, the primary antibody, which includes anti-SHANK 1(ab154224, Abcam), anti-SHANK 3(ab93607, Abcam), Bcl-2(ab32124, 1:1000), caspase3(ab13847, 1:1000), Bax (ab32503, 1:1000), AKT (ab8805, 1:1000), P-AKT (ab38449, 1:1000), cyclin D1(ab134175, 1:1000), P70(ab109393, 1:1000) and GAPDH (ab181602,1:5000), were purchased from Abcam. The relative expression of the target protein was calculated by protein/internal reference using Quantity One software.

In the above embodiment, the Kaplan-Meier analysis specifically includes performing data analysis using a TCGA database, calculating a log rank P value and a Hazard Ratio (HR) of a 95% Confidence Interval (CI), and plotting the calculated values. The GEPIA website is applied to plot survival maps based on thousands of samples of TCGA.

The Kaplan-Meier analysis is a web-site tool based on a cancer genomic profiling database, european genome phenotype archive (EGA) and gene expression integration database (GEO), limited to affymetrix microarrays.

In the above examples, the colony formation assay and cell proliferation assay were carried out by seeding A549, H1299 and SK-MES-1 cells at a density of 1000 cells per well in 6-well plates and culturing for 14 days until colonies were evident. Colonies were fixed in 10% formaldehyde at room temperature for 15min and stained with 1% crystal violet. CCK8 was used for cell proliferation assays. Performing trypsinization and counting on normally cultured NSCLC cells (namely A549 cells, H1299 cells and SK-MES-1 cells) to form a suspension, respectively inoculating 1000A 549 cells, H1299 cells and SK-MES-1 cells into each well of a 96-well plate, using 0.1% DMSO-treated cells as a control, adding 1 part of CCK8 reagent every 24 hours to detect the cell viability, after incubating for 90min at 37 ℃, using a microplate reader to measure the OD value of exciting light at the wavelength of 450nm, and drawing a proliferation curve according to the OD value, wherein the culture medium is DMEM culture medium and 10% fetal bovine serum.

In the above examples BEAS-2 cells were normal human bronchial epithelial cells isolated from necropsies of non-cancerous individuals.

In the above examples A549, H1299 and SK-MES-1 were obtained from Kakey biotech Inc. of Nanjing.

The CCK8 detection method in the above examples is disclosed in the invention patent "A method for analyzing the effect of Rab8 in the regulation of Klotho expression in non-small cell lung cancer" (No. CN109030835B), and is not described herein.

1.2 knockout of Shank1 inhibits migration, invasion and induction of apoptosis in NSCLC cells

To investigate the expression of Shank1 in cell migration and invasion, transwell assays were performed on A549 and H1299 cells transfected with Shank1-shRNA (sh-SHANK1)/Shank1-shRNA (NC). The results showed that down-regulation of Shank1 significantly reduced the number of transplanted and invasive a549 and H1299 cells (P <0.01), as shown in figures 12 and 13, indicating that Shank1 is a positive regulator of NSCLC motility.

To determine the inhibitory effect of shRNA on cell death, we examined apoptosis with a flow cytometer and the results are shown in figures 14-16, with control Shank1-shRNA (nc) and sh-Shank1 transfecting a549 cells at 4.17% and 9.42% apoptotic cell ratios, respectively, indicating that the reduction in the number of sh-Shank1 proliferating cells was indeed due to cell death (P <0.01), and the results for H1299 cells were the same.

To further investigate the molecular mechanism of Shank1 in regulating apoptosis, we used the western blot assay to detect the expression of apoptosis-related proteins. As shown in FIG. 17, Shank1 silencing significantly increased the expression of Bax and active Caspase-3, and decreased the expression of the anti-apoptotic protein Bcl-2. The result shows that the down-regulation of the Shank1 can promote the apoptosis by regulating apoptosis-related protein. To investigate the mechanism of inhibition of Shank1 on NSCLC cells by down-regulation, we investigated the detection of key proteins in the PI3K-AKT/mTOR signaling pathway using western blot experiments. The PI3K-AKT/mTOR signaling pathway plays an important role in tumor cell proliferation, cell cycle, apoptosis, movement and the like, and as a result, as shown in FIG. 18, the phosphorylation levels of AKT and mTOR of A549 and H1299 cells are significantly down-regulated when Shank1 is silenced (P <0.05), which indicates that the inactive AKT/mTOR signaling pathway of Shank1 is down-regulated in NSCLC cells.

In the above examples, the cell transwell and invasion assay was specifically carried out by preparing a coating buffer comprising 0.01M, pH consisting of 8.0 Tris and 0.7% NaCl, filtering the coating buffer under sterile conditions, dispensing Matrigel matrix (purchased from ThermoFisher, Waltham, MA), thawing the matrix in a 4 ℃ environment, diluting the matrix with serum-free 1640 medium at a ratio of 1:6, adding 100. mu.l to each permeable support well of a 24-well plate, incubating the matrix at 37 ℃ for 4 hours, carefully removing the permeable support membrane without affecting the matrix gel layer, adding 100ul and 600ul serum-free 1640 medium to the inside and outside of the matrix gel layer, respectively, and incubating the matrix gel layer at 37 ℃ for 30 minutes, before starting the assay. The procedure of the Transwell migration experiment was similar to the invasion experiment, but without Matrigel treatment, the number of cells per void was 5000.

The flow cytometry in the above examples specifically comprises staining NSCLC cells with annexin V-Fluorescein Isothiocyanate (FITC) and Propidium Iodide (PI), detecting apoptosis by flow cytometry, collecting cells by centrifugation after staining, suspending cell particles obtained by centrifugation at 2500g/min for 15min in 500 μ g/ml binding buffer of 5 μ L V-FITC and 5 μ L PI solution, incubating at room temperature for 15min, measuring annexin V and PI staining by flow cytometry (FACSCalibur), and analyzing data by FlowJo software.

1.3 Shank1 overexpression of proteins that decrease KL in a dose-dependent manner

To further study the relationship between the expression of the Shank1 and KL proteins, a Western blot method was used to perform a correlation study on the expression of the Shank1 and KL proteins in 6 NSCLC patients. The results are shown in FIG. 19, wherein Shank1 is highly expressed and KL is low in 4 tumor tissues; 2 tumor tissues Shank1 were low in expression, KL was high in expression. The results show that the expression of Shank1 in lung cancer tissues is obviously inversely related to the expression of KL.

The decrease in KL protein levels may be due to decreased KL synthesis or increased degradation. To determine the course of the decrease in Shank 1-regulated KL protein levels, a549 cells were preincubated with protein biosynthesis inhibitor (cycloheximide, CHX)/proteasome inhibitor (MG132) for 2 hours prior to the time that KL expression was detected, and the results are shown in figure 20, where the decrease in KL protein levels was blocked when proteasome degradation was inhibited, indicating that Shank1 may regulate KL protein levels via the degradation pathway rather than the biosynthesis pathway.

Example two study on the interaction between Shank1/mdm2/Klotho

2.1 Shank1 interacts with KL to increase the ubiquitination of KL

Chen et al (2019) report that Shank1 is a potential KL-interacting protein. To further study the interaction of Shank1 with KL, we transfected a549 cells with Shank1-myc and KL-Flag vectors, lysed with TNE buffer, then immunoprecipitated with anti-Flag antibody, and analyzed by Western blot, and then examined the endogenous association of KL with Shank1 in adult rat lung tissue by co-immunoprecipitation, as shown in fig. 21, and we found that Shank1 had a clear interaction with KL in a549 cells by transfection and co-immunoprecipitation experiments.

We used the Shank1 and KL antibodies to detect localization of endogenous Shank1 and KL, i.e., a549 cells transfected with vector or Shank1-myc, lysed with TNE buffer, and detected KL ubiquitination using anti-KL antibody immunoprecipitation, IP method, and Ub antibody western blot, indicating that Shank1 co-localizes with KL moieties (as shown in figure 22). Furthermore, as shown in figure 23, Shank1 significantly increased ubiquitination of KL, suggesting that Shank1 plays a role in modulating ubiquitination and degradation of KL.

In the above examples, transfection of cells included cell culture and transfection, specifically HEK-293, A549, H1299, H650 cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% FBS and 1% penicillin streptomycin in humidified incubator at 37 deg.C, and the cells were transfected using Lipofectamin 2000(ThermoFisher) (DNA: lipid 1:2.5) in Opti MEM according to the manufacturer's instructions. All experiments were completed 48 hours after transfection.

In the above examples, co-immunoprecipitation experiments were performed according to the report by b.chen et al (2019). Specifically, the cells (HEK293 or A549) are scraped from a cell culture dish, centrifuged for 15min at 2500 rpm in Phosphate Buffered Saline (PBS) and then collected, then the cells are suspended in immunoprecipitation buffer (IPB) containing 50%, the cells are lysed at 4 ℃, slowly rotated for 1h and centrifuged for 15min at 2500 rpm to remove debris, the supernatant is incubated overnight at 4 ℃ with 25-30 ul protein G agarose gel beads, the next day, washed 3 times with IPB and boiled and eluted for 5min in SDS buffer.

The immunoprecipitation buffer (IPB) contained 50mM Tris-Cl,2mM EDTA,250mM NaCl, 10% (v/v) glycerol, 0.5% NP-40,20mM NaF,1mM sodium orthovanadate, $10mM N-ethylmalemide%

2.2 MDM2 interacts with KL to increase ubiquitination of KL

According to reports of D.Wu et al (2018) and M.Wade et al (2013), MDM2 is one of the most effective negative regulators of p 53. As an E3 ubiquitin ligase, it is able to down-regulate p53 activity by modulating polyubiquitination of p53 and proteasome-mediated degradation targeting p 53; outputting p53 from the nucleus; or directly with p53 to block transactivation of key targets. According to the report of h.hou et al (2019), MDM2 is overexpressed in many malignant tumors, including lung cancer, breast cancer, liver cancer, esophageal cancer, stomach cancer, colorectal cancer, etc. MDM2 also promotes the degradation of p 21. In addition, MDM2 is associated with a variety of protein molecules, such as E2F, p19(Arf) and Ras mitogen-activated protein kinase (MAPK).

To investigate whether MDM2 could promote the degradation of KL by modulating its ubiquitination. First, we tested whether MDM2 could form a complex with KL. We transfected A549 cells with MDM2-HA and Flag-KL plasmids. After immunoprecipitation of KL with the FLAG antibody, binding of KL to MDM2 was observed by immunoblot analysis using the HA antibody (as shown in figure 24). After immunoprecipitation of HA and immunoblotting of anti-Flag antibodies, a complex between Flag-KL and MDM2-HA was also detected (as shown in FIG. 25). We stained with rabbit anti-KL (green) and mouse anti-MDM 2 (red) antibodies and examined the localization of endogenous MDM2 and KL with MDM2 and KL antibodies, indicating that MDM2 is partially co-localized with KL (as shown in figure 26). To test whether this interaction could occur under physiological conditions, we performed an endogenous KL/MDM2 co-immunoprecipitation assay on a549 cells, immunoprecipitation of rat brain lysates with mouse anti-MDM 2 or rabbit anti-KL antibodies, immunoprecipitation of lysates with anti-MDM 2 antibody, and we found that KL co-immunoprecipitated with MDM2 at the endogenous level (as shown in figure 27). The results show that MDM2/KL can form a complex in vitro and in vivo.

We also performed western blot to detect KL expression, transfection of a549 cells with vector or MDM2, treatment with MG132, solubilization with TNE buffer, immunoprecipitation with anti-KL antibody, detection of KL protein levels using immunoblotting, and the results are shown in fig. 28, which further demonstrate that MDM2 can reduce KL expression and that this effect can be blocked by MG132 (proteasome inhibitor).

Furthermore, we also tested whether MDM2 overexpression could enhance ubiquitination of KL. Constructs expressing the MDM2 gene MDM2-wt (HA-tag) and DN mutant (MDM2-C462A) were transfected separately into A549 cells, lysed with TNE buffer, and then immunoprecipitated with an anti-KL antibody. We found that MDM2-wt can significantly increase ubiquitination of KL, whereas the DN mutant of MDM2 (MDM2-C462A) does not perform the same function (as shown in figure 29). The above results indicate that MDM2 can also act as a KL's E3 ubiquitin ligase, and Shank1 can modulate the interaction between them.

In the above examples, the plasmids encoding HA-and myc-tagged human Klotho were from b.chen et al (2012), b.chen et al (2019) reports, the plasmids encoding Shank1, MDM2 and related mutants were from Youbio, and other mutations were introduced by PCR and confirmed by dideoxy sequencing.

2.3Shank1/KL/MDM2 Complex formation

The influence of Shank1 on the interaction between MDM2 and KL is analyzed through a gene knockout test, immunoprecipitation and immunoblotting are used for detecting the expression of KL protein in the lysate of A549 cells transfected with MDM2-HA, KL-Flag and sh-Shank1, and the immunoblotting is used for detecting the expression of KL protein in the lysate of A549 cells transfected with Shank1-myc or sh-Shank1 vectors, and the result shows that the Shank1 gene knockout can obviously reduce the correlation between KL and MDM2 (shown in figure 30), and the expression of KL is not influenced by Shank1 (shown in figure 31).

We immunoprecipitated A549 cell lysate with mouse anti-dm 2 or Rb anti-KL antibody, eluted the protein complex, detected immunoprecipitated proteins of MDM2, KL and Shank1 by immunoblotting, and the results are shown in FIG. 32,

we immunoprecipitated a549 cell lysates co-transfected with Myc-Shank 1/or siShank1, MDM2-HA and Flag-KL constructs using anti-HA antibody immunoprecipitation, and the results are shown in fig. 33A for immunoprecipitation proteins by immunoblotting; immunoprecipitated proteins were detected by immunoblotting using a549 cell lysates co-transfected with Myc-Shank1r, MDM2-HA and/or si-MDM2 and Flag-KL constructs by immunoprecipitation with anti-Myc antibodies, and as a result, association of KL with Shank1 was also observed by immunoblotting after immunoprecipitation of MDM2 with specific antibodies, as shown in fig. 33B. This association can also be found in immunoprecipitation of KL. Exogenous overexpression of Shank1 increased the interaction between KL and MDM2, silencing of Shank1 decreased this interaction (as shown in figure 33A), while the expression level of MDM2 did not affect the association between KL and Shank1 (as shown in figure 33B).

We transfected a549 cell lysate from siShank1, immunoblotted with KL antibody to detect immunoprecipitated proteins, and after performing the endogenous MDM2/KL co-immunoprecipitation assay, we found that Shank1 co-immunoprecipitated with KL at the endogenous level, and silent Shank1 attenuated the interaction between MDM2 and KL in a549 cells (as shown in figure 34).

Considering that MDM2 is an important E3 ubiquitin ligase for p53, we wanted to test whether Shank1 could also modulate the interaction between p53 and MDM2, and we performed co-immunoprecipitation analysis using anti-HA antibodies to co-transfect a549 cell lysates of Myc-Shank 1/or Shank1, MDM2-HA and Flag-p53 vectors, and as a result Shank1 did not affect the association between p53 and MDM2 as shown in fig. 35. Shank1 also is associated with the Mdm2/KL complex and affects complex formation.

We transfected a549 cells with vector or Shank1-myc construct, solubilized with TNE buffer, and immunoblotted with anti-p 53 and p21 antibodies as shown in figure 36, which shows that Shank1/KL/MDM2 can form a complex and that Shank1 affects complex formation.

In the above examples, the Shank1 gene knockout specifically, a previously tested Shank1 siRNA oligonucleotide was synthesized from Invitrogen (by ThermoFisher). The target sequence is 5'-aagcttggcacgccaaaaaa-3'. Negative control oligonucleotides encoding siRNA were not homologous to any known mammalian sequence and were purchased from Invitrogen.

In conclusion, Shank1 is an oncogene, and through combination with KL and MDM2, KL is down-regulated through ubiquitin degradation, so that the migration, invasion and proliferation capacity of lung cancer cells are promoted, and the oncogene participates in cell proliferation, apoptosis, transformation, invasion and tumor progression of non-small cell lung cancer.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned. "

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.