阅读说明：本技术 miR-140-3p在制备肺腺癌治疗药物中的应用 (Application of miR-140-3p in preparation of lung adenocarcinoma treatment medicine ) 是由 吴朔明 于 2020-07-10 设计创作，主要内容包括：本发明公开了miR-140-3p在制备肺腺癌治疗药物中的应用,属于生物医药技术领域。本发明证实了miR-140-3p可以通过抑制LUAD细胞中的Wnt/β-catenin信号传导,从而增强顺铂敏感性并减弱干细胞样特性。由此可知,miR-140-3p可用于制备肺腺癌治疗药物,为治疗肺腺癌(LUAD)的化学耐药性提供潜在的策略。(The invention discloses application of miR-140-3p in preparation of a lung adenocarcinoma treatment drug, and belongs to the technical field of biological medicines. The invention proves that miR-140-3p can enhance cis-platinum sensitivity and weaken stem cell-like characteristics by inhibiting Wnt/beta-catenin signaling in LUAD cells. Therefore, miR-140-3p can be used for preparing lung adenocarcinoma treatment medicines and provides a potential strategy for treating chemical resistance of lung adenocarcinoma (LUAD).)

The application of miR-140-3p in preparing a medicament for treating lung adenocarcinoma is disclosed, wherein the nucleotide sequence of miR-140-3p is shown as SEQID NO. 1.

2. Use according to claim 1, characterized in that: the miR-140-3p can enhance the sensitivity of lung adenocarcinoma cells to cisplatin.

3. Use according to claim 1, characterized in that: miR-140-3p can attenuate the stem cell-like properties of lung adenocarcinoma cells.

4. A medicament for treating lung adenocarcinoma, which is characterized in that: comprises cisplatin and miR-140-3p combined with a carrier, wherein the nucleotide sequence of the miR-140-3p is shown in SEQ ID NO. 1.

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of miR-140-3p in preparation of a lung adenocarcinoma treatment medicine.

Background

Currently, lung cancer remains one of the leading causes of death associated with cancer worldwide, while non-small cell lung cancer (NSCLC) is the predominant form of lung cancer, accounting for approximately 80% of the total number of lung cancers. In recent years, lung adenocarcinoma (LUAD) is the most rapidly developing subtype of non-small cell lung cancer. Cisplatin, which can alter DNA structure and function, is widely used as a first-line NSCLC therapeutic and to prolong patient survival, however, its efficacy is often compromised by the development of chemical resistance. Resistance of tumor cells to chemotherapy invariably exhibits more malignant behavior, such as higher proliferative capacity and greater resistance to apoptosis.

Tumor stem cells (CSCs) have recently received much attention because of their functions such as self-renewal, differentiation, stress, and drug resistance. CSCs have been reported in a variety of human cancers, including LUAD. Although CSCs are only a small fraction of tumor cells, they play a crucial role in tumor development, progression, and resistance to radiation and chemotherapy. Studies have demonstrated that CSCs are regulated by a number of signaling pathways, such as Wnt/β -catenin, Notch and Hedgehog. Thus, inhibition of CSCs by targeting the signaling pathway that regulates these cells may increase the sensitivity of lung cancer therapy.

miRNAs are short, non-coding RNA families that negatively regulate gene expression through a3 'untranslated region (3' UTR) that directly binds to its mRNA target, and are involved in the regulation of a variety of biological processes, such as cancer cell proliferation, apoptosis, sensitive chemotherapy, and CSCs desiccation. Previous studies have shown that many mirnas are up-or down-regulated in lung cancer, thereby enhancing resistance to chemotherapy and stem cell-like properties by modulating CSCs-related signaling pathways. However, the expression and function of miR-140-3p in LUAD is still unknown.

Disclosure of Invention

The invention aims to provide application of miR-140-3p in preparation of a lung adenocarcinoma treatment drug, and provides a potential strategy for treating chemical resistance of lung adenocarcinoma (LUAD).

In order to achieve the above object, the present invention adopts the following technical means:

application of miR-140-3p in preparation of lung adenocarcinoma treatment medicines.

The nucleotide sequence of the miR-140-3p is as follows:

TTTTGAATAAAGCAGCATTCAGTTGAAATAACCCTTTCTGTGGTAATTCCAAGTGAATATTTTTCTTCTAGGTGATGAGTTTCTACTTCCTCTGGTTTTTACAACAGGAAATGAAATGGTATCTAAAATAAACAAGCTGTTTATTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(SEQ ID NO.1)。

further, miR-140-3p can enhance the sensitivity of lung adenocarcinoma cells to cis-platinum.

Further, miR-140-3p can attenuate the stem cell-like properties of lung adenocarcinoma cells.

A medicament for treating lung adenocarcinoma comprises cisplatin and miR-140-3p combined with a carrier.

The invention proves that miR-140-3p can enhance cis-platinum sensitivity and weaken stem cell-like characteristics by inhibiting Wnt/beta-catenin signaling in LUAD cells. Therefore, miR-140-3p can play a role in an effective treatment strategy, and targeting miR-140-3p can be a strategy for improving cis-platinum sensitivity in LUAD.

Drawings

FIG. 1 is the result of miR-140-3p downregulation in LUAD in example 1.

FIG. 2 is a graph showing the effect of miR-140-3p on cis-platin sensitivity in example 2.

FIG. 3 is a graph of the effect of miR-140-3p on the stem cell-like properties of LUAD cells in example 3.

FIG. 4 shows that miR-140-3p attenuates the Wnt/beta-catenin signaling pathway in LUAD cells in example 4.

FIG. 5 is the effect of the Wnt/β -catenin signaling pathway in example 4 on the stem cell-like properties of LUAD cells mediated by miR-140-3 p.

FIG. 6 is a graph of the effect of the Wnt/β -catenin signaling pathway in example 4 on the cisplatin sensitivity of miR-140-3p on LUAD cells.

Detailed Description

According to published data from the GEO and TCGA databases, the inventors found that miR-140-3p expression in LUAD samples was lower than normal lung tissue and positively correlated with patient survival, suggesting that miR-140-3p may be a good prognostic factor for LUAD.

Cisplatin is widely used as a first-line chemotherapeutic agent in the treatment of many cancers, including LUAD. However, chemotherapy resistance remains a major challenge for treatment failure. Therefore, increasing cisplatin sensitivity of LUAD cells is an important strategy for effective cancer treatment. Through colony formation determination, MTT determination and Caspase-3 determination, the invention proves that the up-regulation of miR-140-3p enhances the sensitivity of LUAD cells to cis-platinum. Meanwhile, the expression of miR-140-3p is up-regulated, so that the number of CD133+ cells in LUC cells is reduced, the formation of tumor balls is weakened, and the expression of a pluripotency factor is reduced. That is, miR-140-3p may play a key role in modulating sensitivity to cisplatin and the stem cell-like properties of LUAD cells, and be a therapeutic target for LUAD.

In addition, the invention further researches the influence of miR-140-3p on the activation of the Wnt/beta-catenin signaling pathway, and finds that the up-regulation of miR-140-3p inhibits the activation of the Wnt/beta-catenin signaling pathway, and the reactivation of the Wnt/beta-catenin signaling pathway partially restores the cisplatin resistance and the cancer stem cell-like characteristics of the LUAD cells. At the same time, it is considered that the tumor suppressor P53 is central to many tumor-related events, such as sternness and treatment resistance. The present invention further investigated the P53 regulation in the cell lines selected in the present invention and found no difference at the protein level between miR-140-3P mock cells and control mock cells (data not shown), indicating that these miR-140-3P mediated phenotypes were independent of P53. In conclusion, the results of the invention show that miR-140-3 p/Wnt/beta-catenin signaling has important influence on the characteristics of cancer stem cells, and can play a new role in regulating the resistance of cis-platin in LUAD.

The technical scheme of the invention is further explained by combining specific examples. It is to be understood that the following examples are illustrative of the invention and are not to be construed as limiting the invention in any way.

The experimental method adopted in the embodiment of the invention is as follows:

1. cell culture and cell transfection

The human bronchial epithelial cell line BEAS-2B and the lung adenocarcinoma cell lines A549, H1299, H292 and Calu3 were from the American type culture Collection (ATCC, Rockville, Md., USA). BEAS-2B was cultured in BEBM Medium (Lonza Group Ltd. of Basel, Switzerland) and all lung adenocarcinoma cell lines were cultured in Roswell Park Memori Institute (RPMI)1640 medium (Gibco; Invitrogen; Thermo Fisher scientific, Inc.) of 100. mu.g/mL penicillin/streptomycin 10% fetal bovine serum (FBS; Gibco; Thermo Fisher scientific, Inc., Waltham, Mass., USA) under 5% CO at 37 ℃ under 5%₂。

Transfections, cells were cultured in six-well plates and obtained by Lipofectamine2000 (Invitrogen; Thermo Fisher Scientific, Inc.) with plasmids (pcDNA3.1-ctnnb1 or pcDNA3.1-Vector, GenePharma Company, Shanghai, China) or mimetics (miR-140-3p mimetic 5'-UACCACAGGGGGUAGAACCACGG-3' or control mimetic 5'-GCAAGAGACAAGCGCUUAGCC-3', GenePharmaCompany, Shanghai, China). In detail, 2. mu.g of plasmid or 50nM mimic were added to 200. mu.l of Opti-MEM (Gibco; Semmer Feishell science Co.) in one vial, and then 4. mu.L of Lipofectamine2000 was mixed with 200. mu.l of Opti-MEM medium in another vial. After 5 minutes of incubation at room temperature, the two vials were combined and incubated for an additional 20 minutes at room temperature. The mixture is added to the cell culture. After incubation for 6h at 37 ℃, the cell culture medium was replaced with fresh medium. Transfected cells were harvested at 48 hours.

RNA extraction and real-time qPCR assay

Total RNA from cultured cells was extracted using Trizol reagent (Invitrogen; Thermo Fisher scientific, Inc.) according to the manufacturer's protocol. For detection of mRNA or miRNA, cDNA was synthesized using Moloney murine leukemia virus RT kit (M-MLV, Promega Corporation) with dNTPs (Promega Corporation) and random primers (Promega Corporation). Specific primers for miR-140-3p and internal reference U6 were designed and synthesized by RiboBio. The temperature protocol for the reverse transcription reaction included synthesis of cDNA at 37 ℃ for 60 minutes and cDNA synthesis terminated at 80 ℃ for 2 minutes. Real-time qPCR was performed using SYBR Green real-time PCR Master Mix (Fermentas) and Bio Rad CFX96 system (Bio-Rad Laboratories, inc., Hercules, CA, usa) according to the manufacturer's instructions. The reaction conditions were as follows: 40 cycles were carried out at 94 ℃ for 2 minutes, 94 ℃ for 20 seconds and 60 ℃ for 30 seconds. By 2^-△△CtMethods relative mRNA or miRNA levels were calculated and normalized to GAPDH or U6, respectively (26). All measurements were repeated three times. Table 1 lists specific primers used for real-time qPCR analysis.

3. Cell viability assay

Cells were seeded into 96-well plates (4 × 10)³Cell expansion) and incubated overnight 48 hours after transfection. Freshly prepared cisplatin was added at the indicated concentrations. Treating for 24 hoursThereafter, 20. mu.L of 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) reagent (Sigma, St. Louis, Mo) was added to the cell culture and incubated at 37 ℃ for 4 hours. The culture supernatant was removed and dimethyl sulfoxide (DMSO) (100 μ LL wells) was added to each well to dissolve the methyl-thee. Absorbance was measured at 490nm using a microplate reader (Bio-Rad Laboratories, Inc.). All assays were performed in triplicate.

4. Colony formation assay

Cells were seeded into 24-well plates (10 × 10)⁴Cells/well) and incubated overnight 48 hours after transfection. RPMI1640 medium containing cisplatin (5. mu.g/. mu.L) was used to culture the cells for 24 hours. Colonies were then fixed and stained with crystal violet. The assay has been performed three times.

Caspase-3 Activity

Caspase-3 activity was assessed using the Caspase-3 assay kit (Sigma, St. Louis, MO). Cells were lysed after 24 hours of treatment with cisplatin (5. mu.g/mL). Assays were performed on 96-well microtiter plates by incubating 10. mu.L of cell lysate protein/sample in 10. mu.L of reaction buffer containing 10. mu.L of caspase-c (1% NP-40, 20mmol/L Tris-HCl pH 7.5, 137mmol/L NaCl and 10% glycerol). 3 substrates (2mmol/L Ac-DEVD-pNA). The lysates were incubated at 37 ℃ for 4 h. Caspase-3 activity in the samples was quantified by a microplate reader (Bio-Rad Laboratories, Inc.) at an absorbance of 405 nm. Caspase-3 activity was then assessed by calculating the ratio of OD 405nm of cisplatin-treated cells to untreated cells. The detailed analysis process is performed according to the manufacturer's protocol. The assay has been performed three times.

6. Flow cytometry analysis

Harvest 1 × 10⁶The cells were washed twice with PBS supplemented with 0.5% bovine serum albumin (BSA, fraction V; Gibco GrandIsland, N.Y., USA) and 2mM radioimmunoprecipitation assay (RIPA), and incubated with the diluted CD 133-PE antibody solution (#372803, BioLegend) for 15 minutes. Prior to flow cytometry analysis, cells were washed twice and then evaluated using a FACSCalibur flow cytometer (BD, heidelberg, germany) (28). The assay has been performed three times.

7. Tumor sphere formation assay

Transfected cells were seeded in six-well ultra-low attachment plates (Corning Inc., Corning, NY, USA) (3 × 10)³Cells/well) and cultured in dmemf 12 serum-free medium supplemented with 20nggml EGF, 20nggml bFGF, 5 μ ggml insulin, 0.4% BSA, 2% B-27 (Invitrogen, usa), for 6 days of treatment. The spheres were then photographed and counted.

8. Immunoblotting

After 48 hours of transfection, incubation on ice for 30 minutes, LUAD cells were lysed in RIPA lysis buffer (Beyotime biotech institute), protein concentration was determined according to the manufacturer's instructions using the bicinchoninic acid (BCA) assay kit (Pierce; ThermoFisher Scientific, Inc.), equal amounts of protein lysates (40. mu.g each) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to polyvinylidene fluoride membranes (PVDF, Millipore, MA, USA). the membranes were blocked with Tween 20 (ST) Tris buffer solution containing 5% skimmed milk powder for 1h, and then incubated overnight at 4 ℃ with PBS anti-catenin (Cat No. 32572, 1): 4000, Abcam), anti-Myc-Myc (Cat No. 10828-1-AP, 1: 1000, Proteitech catalog D35 β -caten (Cat No. 32572, 1), 1, 4000, Abcam), anti-Myc-Imc (Cat No. 10828-1-AP, 1, 1000, Cat No. 5000, 5000-B # 5000-5000, and 15 min after exposure to the substrate of Lab # Ab # 20, and using the enhanced luminescence, Abcamb # 20, Ab # 20, and Ab # 12, Ab # 20, and Ab # 11, and Ab # 12^TMSoftware2.0(Bio-rad laboratories, Inc.). ). GAPDH was used as an internal control. The expression at the protein level was quantitatively analyzed using ImageJ 1.41 software (NIH, Bethesda, MA, USA).

9. Luciferase assay

TCF reporter gene assay (TOPPFOP) was performed using the Dual-Glo luciferase assay kit (Promega Corporation, Madison, Wis., USA) to assess the activity of the Wntt β -catenin signaling pathway. For this assay, cells were seeded in 24-well plates and incubated overnight. Cells were then co-transfected with the ToppFop Flash carrier (100. mu.ng), the internal control pRL-TK Renilla luciferase carrier (10ng) and the control mimic or miR-140-3p mimic (50nM), respectively, using Lipofectamine 2000. Firefly and algal luciferase activities were assayed in triplicate 48 hours after transfection according to the manufacturer's instructions. Luciferase activity was normalized to renilla luciferase activity.

Statistical analysis

The results are shown as mean ± SD and analyzed using SPSS 19.0 software (SPSS inc., IBM, NY, USA). All experiments were repeated for at least three separate times. Differences between the two groups were assessed using student t-test, one-way ANOVA, followed by Dunnett's post-test to measure differences between the three groups and intra-group differences were determined using mixed design ANOVA. P values <0.05 were considered statistically significant.