Application of SERPINE1 inhibitor in preventing recurrence and metastasis after hepatocellular carcinoma microwave ablation

文档序号：1880464 发布日期：2021-11-26 浏览：22次中文

阅读说明：本技术 Serpine1抑制剂在肝细胞癌微波消融术后抗复发转移中的用途 (Application of SERPINE1 inhibitor in preventing recurrence and metastasis after hepatocellular carcinoma microwave ablation ) 是由刘嵘束敏峰韩红高洋范卓阳张巍杨国威王建华颜志平于 2021-08-23 设计创作，主要内容包括：本发明公开了SERPINE1抑制剂在肝细胞癌微波消融术后抗复发转移中的用途,属于生物医药技术领域。本发明通过建立亚致死热损伤HCCLM3皮下异种移植模型考察SERPINE1抑制剂PAI-039在亚致死热治疗后的体内作用,与对照组相比,PAI-039组显示出更小的异种移植体积,能够显著抑制肿瘤生长,免疫组化结果显示,PAI-039组的SERPINE1和VEGF表达水平显著降低,表明PAI-039可以抑制亚致死热处理后的体内异种移植。本发明提供的SERPINE1抑制剂为微波消融术后抗肝癌复发转移提供一种新的药物治疗方案,为临床上实现进一步提高肝癌患者预后提供了一种新的途径。(The invention discloses an application of an SERPINE1 inhibitor in recurrence-resistant metastasis after hepatocellular carcinoma microwave ablation, belonging to the technical field of biological medicines. The invention researches the in vivo effect of an SERPINE1 inhibitor PAI-039 after sublethal heat treatment by establishing a sublethal heat injury HCCLM3 subcutaneous xenograft model, compared with a control group, the PAI-039 group shows smaller xenograft volume and can obviously inhibit tumor growth, and immunohistochemical results show that the SERPINE1 and VEGF expression level of the PAI-039 group are obviously reduced, which indicates that the PAI-039 can inhibit in vivo xenograft after sublethal heat treatment. The SERPINE1 inhibitor provided by the invention provides a new drug treatment scheme for resisting liver cancer recurrence and metastasis after microwave ablation, and provides a new approach for further improving prognosis of liver cancer patients clinically.)

Application of an SERPINE1 inhibitor in preparation of a medicine for resisting recurrence and metastasis of liver cancer after microwave ablation.

2. The use of claim 1, wherein the medicament comprises a pharmaceutically acceptable carrier and an effective amount of an active ingredient which is an inhibitor of SERPINE 1.

3. The use as claimed in claim 2 wherein the SERPINE1 inhibitor comprises PAI-039.

Technical Field

The invention relates to an application of an SERPINE1 inhibitor in preventing recurrence and metastasis after hepatocellular carcinoma microwave ablation, belonging to the technical field of biological medicines.

Background

Hepatocellular carcinoma (HCC, hereinafter referred to as liver cancer) is the third place in malignant tumors in China, and the overall survival rate of five years is only 12.1%. Liver cancer treatment methods are diverse, such as surgical resection, liver transplantation, interventional therapy, microwave/radio frequency ablation, targeted drugs, systemic chemotherapy, and the like. In the specifications of primary liver cancer diagnosis and treatment (2019), microwave ablation is an important means for treating liver cancer. However, the recurrence and metastasis of residual cancer after microwave ablation are the key causes of death of patients, and further, the discovery of a new treatment means has great significance for improving the overall prognosis of liver cancer.

Angiogenesis is considered to be one of the most important pathological processes for tumor metastasis. In hepatocellular carcinoma (HCC), the physician can easily distinguish between malignant and benign lesions, since the tumor blood supply is generally abundant. Therefore, anti-angiogenesis therapy is an effective means for treating tumors in liver cancer. Vascular endothelial growth factor A (VEGF-A) has been a key angiogenic cytokine in malignant angiogenesis for many years. Many studies have reported "adverse effects" of VEGF in HCC. Although sorafenib is a VEGFR-targeted TKI inhibitor and has been FDA approved for the treatment of advanced or unresectable HCC for over a decade, the overall survival of patients with sorafenib is extended by only about 3 months.

Plasminogen activator inhibitor type I (also known as SERPINE1, PAI-1) is a member of the serine protease inhibitor superfamily and was first reported to decrease activation of Matrix Metalloproteinases (MMPs), thereby disrupting the balance of the cellular microenvironment. Recent studies have demonstrated the relationship between SERPINE1 and cancer. James et al disclosed that miR-30c inhibits carcinogenesis by blocking SERPINE1 in breast and lung cancer; a study from Teng et al demonstrated that the NKX2-1-AS1/miR-145-5 p-axis positively upregulates SERPINE1 translation in gastric cancer. The role played by SERPINE1 in HCC is unclear.

RNA methylation is an important modification at the post-transcriptional level in eukaryotic cells and has been shown to play a different role. More than half of the RNA methylation was N6-methyladenosine (m6A modification). To date, m6A modifications have been shown to be widely involved in the process of tumor metastasis. Chen et al demonstrated that the METTL3/m6A/GLS2 axis promotes the progression of esophageal squamous cell carcinoma; hou et al disclose that YTHDC1/m6A inhibits pancreatic ductal adenocarcinoma by up-regulating miR-30 d; chen et al show that m6A status determines the occurrence of lung metastasis of breast cancer. However, little is known about whether and how m6A modification plays a role in angiogenesis after sub-lethal heat treatment of HCC.

Recently, organoid technology has become an important model for the study of cancer biology, clinical transformation, and precise medicine. Patient-derived organoids (PDOs) can more accurately summarize tumor histology and genetics than traditional two-dimensional cell lines. Patient-derived graft models (PDX) are more expensive, time-consuming, and likely under mouse-specific tumor evolution. PDO is an in vitro three-dimensional (3-D) tumor model with the ability to self-renew and self-organize, and to retain the key features of the originating tumor. The HCC PDO model was used in previous studies to evaluate phenotypic changes following sublethal heat treatment and was found to exhibit high metastatic profiles, which may be associated with CD-47 mediated EMT effects. Thus, sublethal heat treatment can cause HCC invasion, suggesting that microwave ablation may lead to tumor metastasis. On the other hand, angiogenesis is generally closely associated with tumor metastasis.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: the recurrence and metastasis of residual cancer and other problems exist after the microwave ablation of hepatocellular carcinoma.

In order to solve the technical problems, the invention provides application of an SERPINE1 inhibitor in preparing a medicine for resisting liver cancer recurrence and metastasis after microwave ablation.

Preferably, the medicament comprises a pharmaceutically acceptable carrier and an effective amount of an active ingredient which is an inhibitor of SERPINE 1.

Preferably, the SERPINE1 inhibitor comprises PAI-039.

PAI-039 of the present invention, chemical name 1-benzyl-5- (4- (trifluoromethoxy) phenyl) -1H-indol-3-yl) oxyacetic acid; alpha-oxo-1- (phenylmethyl) -5- [4- (trifluoromethoxy) phenyl]-1H-indole-3-acetic acid, also known as Tiplasinin, Tiplaxtinin, WAY-168039, is a potent, orally active selective inhibitor of plasminogen activator inhibitor-1 (PAI-1) having the structure of formula (I), molecular weight 439.38, and formula C₂₄H₁₆F₃NO₄。

Compared with the prior art, the invention has the beneficial effects that:

the SERPINE1 inhibitor provided by the invention provides a new drug treatment scheme for resisting liver cancer recurrence and metastasis after microwave ablation, and provides a new approach for further improving prognosis of liver cancer patients clinically.

Drawings

FIG. 1A: heat maps showing the "first 25 increased genes" and "first 25 decreased genes" in the sublethal thermal injury cell model RNA-seq results;

FIG. 1B: venn plots based on three GO terms "angiogenesis", "forward regulated angiogenesis" and "extracellular secreted protein" and a specific "cross-gene" list of venn plots;

FIG. 1C: representing changes in SERPINE1 at the mRNA and protein levels.

FIG. 1D: indicates the overall survival of SERPINE 1-high and low expressing patients in the TCGA database;

FIG. 1E: representing the expression level of SERPINE1 at different stages of HCC;

FIG. 1F: representing typical protein levels of SERPINE1 in HPA database

FIG. 1G: representing the expression of SERPINE1 in HCC and para-cancerous tissues of patients receiving resection;

FIG. 1H: representing a schematic diagram for establishing a VX2 rabbit ablation model;

FIG. 1I: indicating successful establishment of VX2 liver cancer in situ before the ablation operation; representative tumors are 7X 7mm²(ii) a a: a tumor; b: normal liver tissue;

FIG. 1J: a schematic diagram representing microwave ablation of a rabbit model;

FIG. 1K: showing the general situation of ultrasonic image display 1 day, 3 days, 7 days and 14 days after microwave ablation; the method comprises the following steps: normal liver tissue; secondly, the step of: residual tumors; ③: ablating tissue;

FIG. 1L: residual tumor and normal liver around the ablated section before and after h.e. staining (× 10); a: a tumor; b: ablating tissue; c: a peripheral inflammatory site; d: normal liver tissue; )

FIG. 1M: relative mRNA levels and relative protein levels representing the residual rabbit liver cancer SERPINE1 at different time points;

FIG. 2A: shows that the HCCLM3 and Huh7 cell lines have reduced mRNA expression level and protein expression level after the SERPINE1 is knocked down under the culture condition of 37 ℃, and the mRNA expression level and the protein expression level of the SERPINE1 can be partially recovered under the culture condition of 46 ℃;

FIG. 2B: shows that the HCCLM3 and Huh7 cell lines have decreased mRNA transcription level and protein expression level of SERPINE1 by SERPINE1 inhibitor PAI-039 under the culture condition of 37 ℃, and the mRNA level and the protein expression level of SERPINE1 can be partially restored under the culture condition of 46 ℃;

FIG. 2C: shows that ELISA shows that the level of SERPINE1 in the extracellular supernatant is reduced after knocking down SERPINE1 under the culture condition of HCCLM3 at 37 ℃, the culture condition at 46 ℃ can partially recover the level of SERPINE1, while ELISA shows that the level of SERPINE1 in the extracellular supernatant is reduced after knocking down SERPINE1 under the culture condition of Huh7 at 37 ℃, and the culture condition at 46 ℃ cannot significantly recover the level of SERPINE 1;

FIG. 2D: tube formation assay for HUVECs, harvesting supernatants from HCCLM3 or Huh7 obtained under different treatment conditions to culture HUVECs for 48 hours, significantly reduced tube formation numbers after culture in extracellular supernatant after knockdown of SERPINE1 compared to supernatants directly using 37 ℃ culture conditions, and partially restored tube formation numbers using supernatants obtained using 46 ℃ culture conditions;

FIG. 2E: it was shown that in the tube formation test of HUVEC, collecting the supernatants from HCCLM3 or Huh7 obtained under different treatment conditions to culture HUVEC for 48 hours, the tube formation amount was significantly decreased using the culture supernatant after PAI-039 addition, compared to the supernatant directly using the 37 ℃ culture condition, while the tube formation amount was significantly increased using the supernatant of the 46 ℃ culture condition;

FIG. 2F: STRING analysis was shown to show that there are more proteins potentially associated with SERPINE1, and VEGF, SMAD2, SMAD3 and SMAD7 were significantly associated with SERPINE 1;

FIG. 2G: shows that the protein level expression of VEGF is obviously reduced after SERPINE1 is knocked down in HCCLM3 and Huh7 hepatoma cells, while the expression level of VEGF can be increased under the culture condition of 46 ℃;

FIG. 2H: shows that in HCCLM3 and Huh7 liver cancer cells, after SERPINE1 is knocked down, the relative expression level of VEGF in supernatant is obviously reduced, and the extracellular expression level of VEGF is increased after VEGF is added;

FIG. 2I: the HCCLM3 and Huh7 cells are cultured at 46 ℃, the VEGF expression is reduced after the SERPINE1 is knocked out, and the SERPINE expression has no obvious change after the VEGF is added;

FIG. 2J: tube formation assay displaying HUVECs, supernatants from HCCLM3 or Huh7 were collected and used to culture HUVECs for 48 hours; tube formation decreased after treatment with 46 ℃ + SERPINE1 knockdown supernatant and increased when VEGF treated cell supernatant was added;

FIG. 3A: shows that when METTL3 is knocked down in HCCLM3 or Huh7, the mRNA expression level of SERPINE1 is increased and the mRNA expression level of METTL3 is not obviously changed after PAI-1 is added;

FIG. 3B: represents the relative METTL3 and SERPINE1 protein levels when METTL3 is knocked down with/without SERPINE1 in HCCLM3 or Huh 7;

FIG. 3C: shows that in HCCLM3 or Huh7, the SERPINE1 is knocked down, and the SERPINE1mRNA expression level is not obviously changed;

FIG. 3D: shows relative METTL3 and SERPINE1 protein levels when SERPINE1 is knocked down in HCCLM3 or Huh 7;

FIG. 3E: a schematic diagram showing the m6A modification site predicted by SERPINE1 from RMBase;

FIG. 3F: shows that wild type or m6A consensus mutant SERPINE1 cDNA is fused with firefly luciferase reporter gene, and mutation of m6A consensus sequence of SERPINE1 relieves post-transcriptional repression of SERPINE1 and METTL3 in HCCLM3 and Huh 7;

FIG. 3G: representing the tubule formation experiment of HUVEC, collecting the supernatant from HCCLM3 or Huh7 and culturing HUVEC for 48 hours under the condition that METTL3 is knocked out at 46 ℃, and the proliferation and amplification capacity is improved after PAI-1 is added;

FIG. 3H: demonstrates an RNA stability assay under conditions of met tl3 with/without SERPINE1 knockdown at 46 ℃;

FIG. 3I: shows that in HCCLM3 or Huh7 cell lines, the transcriptional and translational levels of IGF2BP1 and SERPINE1 were significantly reduced when IGF2BP1 was knocked down;

FIG. 3J: it was shown in HCCLM3 or Huh7 that IGF2BP1 and SERPINE1 decreased significantly at the transcriptome and translation levels when SERPINE1 was knocked down;

FIG. 3K: displaying fusion of wild type or m6A consensus mutant SERPINE1 cDNA with firefly luciferase reporter gene, mutation of m6A consensus of SERPINE1 relieved post-transcriptional repression of SERPINE1 and IGF2BP1 in HCCLM3 and Huh 7;

FIG. 4A: the liver cancer organoid is represented by a three-dimensional cell mass formed by liver cancer cells, and the EMT characteristics are represented after heating;

FIG. 4B: showing that HE staining shows that the liver cancer organoid has the histological characteristics of liver cancer;

FIG. 4C: immunofluorescent staining indicated that PDO-1 exhibited typical characteristics of HCC: positive expression of AFP and negative expression of EpCAM, and the expression level of SERPINE1 was significantly higher in PDO heat treated at 46 ℃, and this effect could be attenuated by the addition of PAI-039;

FIG. 4D: immunofluorescent staining indicated that PDO-2 exhibited typical characteristics of HCC: positive expression of AFP and negative expression of EpCAM, and the expression level of SERPINE1 was significantly higher in PDO heat treated at 46 ℃, and this effect could be attenuated by the addition of PAI-039;

FIG. 4E: immunofluorescent staining indicated that PDO-3 exhibited typical characteristics of HCC: positive expression of AFP and negative expression of EpCAM, and the expression level of SERPINE1 was significantly higher in PDO heat treated at 46 ℃, and this effect could be attenuated by the addition of PAI-039;

FIG. 4F: showing the change of the expression level of SERPINE1 under the conditions of 37 ℃, 46 ℃ and 46 ℃ plus PAI-039, the expression level of SERPINE1 is obviously increased after heat treatment, and the expression level of SERPINE1 is obviously reduced after PAI-039 is added;

FIG. 5A: indicating that 10 female BALB/c nude mice received sub-lethal heat-treated HCCLM3 injections, of which 5 received saline, another 5 received PAI-039 every other day, and on day 25, the mice were sacrificed;

FIG. 5B: indicates that the PAI-039 injection obviously inhibits the growth of the tumor;

FIG. 5C: represents HE staining results of a control group and a PAI-039 group;

FIG. 5D: shows immunohistochemistry results, and compared with a control group, the expression level of SERPINE1 of the PAI-039 group is obviously reduced;

FIG. 5E: shows immunohistochemistry results, compared with a control group, the expression level of VEGF in the PAI-039 group is obviously reduced;

in each of the above figures, "NS": no significant difference was indicated; *: p < 0.05; **: represents p < 0.01; ***: p < 0.001; ****: representing p < 0.0001.

Detailed Description

In order to make the invention more comprehensible, preferred embodiments are described in detail below with reference to the accompanying drawings.

In the following examples, the specific experimental procedures used are as follows:

1. cell culture and processing

In vitro experiments were performed using HCC cell lines HCCLM3 and Huh7 cell lines; and rabbit orthotopic transplantation is carried out by using a rabbit squamous cell line VX 2. These three cells were from the liver cancer institute of Zhongshan hospital, university of double denier (Shanghai, China). VX2 cells have been widely used as a classical liver tumor model. Cells were cultured in Dulbecco's modified Eagle Medium (DMEM, Gibco, Gibco, Grand Island, NY, USA) containing 10% Fetal Bovine Serum (FBS) and antibiotics (penicillin (100U/ml)/streptomycin (0.1 mg/ml)). The culture conditions were 37 ℃ and 5% CO₂And humidifying the environment, and changing the liquid for 3 times every week.

2. Transfected and stable cell lines

Plasmid transfection was performed using Lipofectamine 2000 reagent (Life Technology, Thermo Fisher Scientific, DE, USA), pcDNA3.1-SERPINE1, pcDNA3.1-VEGF, and SERPINE1 virus (Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin) and 2 siRNAs (siMETTL3:5'-GCUACCUGGACGUCAGUAUTT-3' (SEQ ID NO: 1), siIGF2BP1:5'-TCTGCAACTCGTTCACCGT-3' (SEQ ID NO: 2)) were purchased from Genechem (Shanghai, China). The empty vector served as a negative control. The transfection procedure followed strictly the manufacturer's instructions for Lipofectamine 2000 reagent (Invitrogen). For one group, a total of 5X 10⁵Individual cells were seeded into wells of 6-well plates. After transfection, RT-qPCR analysis or western blot analysis was used to verify transfection efficiency.

3. RNA extraction and real-time PCR

Total RNA was extracted by RNA purification kit (EZbioscience, USA) according to the manufacturer's instructions. Then, RT-PCR was performed using 4 × Reverse Transcription Master Mix (EZbioscience), using a tip without DNAse and RNAase (YueYIBioTech, Shanghai, China). SYBR Green PCR kit (Yeasen, China) was used for qPCR. The expression level of each gene was normalized to that of ACTIN according to the 2-. DELTA.Ct method, and used as an internal control.

Primers for human and rabbit genes (synthesized by Sunya, china) were as follows:

human-actin:

ACTIN-F:ACCTTCTACAATGAGCTGCG(SEQ ID NO：3)；

ACTIN-R:CCTGGATAGCAACGTACATGG(SEQ ID NO：4)。

human-SERPINE 1:

SERPINE1-F：ACCGCAACGTGGTTTTCTCA(SEQ ID NO：5)；

SERPINE1-R：TTGAATCCCATAGCTGCTTGAAT(SEQ ID NO：6)。

human-METTL 3:

METTL3-F:TTGTCTCCAACCTTCCGTAGT(SEQ ID NO：7)；

METTL3-R:CCAGATCAGAGAGGTGGTGTAG(SEQ ID NO：8)。

human-IGF 2BP1:

IGF2BP1-F:GCGGCCAGTTCTTGGTCAA(SEQ ID NO：9)；

IGF2BP1-R:TTGGGCACCGAATGTTCAATC(SEQ ID NO：10)。

rabbit actin:

actin-F: TGGCTCTAACAGTCCGCCTAG (SEQ ID NO: 11);

actin-R: AGTGCGACGTGGACATCCG (SEQ ID NO: 12).

Rabbit-serpin 1:

Serpine1-F：GCCTCTAAGGACCGCAATGT(SEQ ID NO：13)；

Serpine1-R：GCCGTGCTCTGCTCATCTAT(SEQ ID NO：14)。

4. RNA-seq analysis

For RNA-Seq analysis, after constructing the library, sequencing data was manipulated using the github package SOAPnuke (v1.5.2) (https:// github. com) to delete the following elements: reading using a sequencing adapter; low mass readings. The clean readings are then converted to FASTQ format. HISAT2(v2.0.4) (http:// www.ccb.jhu.edu/software/HISAT) was used to draw clean readings. The spliced gene was then fused by Ericscript (v0.5.5) (http:// rnaseq-mat. sourceform. net /) or rMATS (V3.2.5) (https:// bowtiebo. sourceform. io /). Thereafter, the expression level of the gene (https:// githu. com/deweylab/RSEM) was calculated using RSEM (v 1.2.12). Finally, an analysis was made using R (https:// www.r-project. org /) for a Q value of 0.05 or less. Enrichment analysis of GO (http:// www.geneontology.org /) and KEGG (https:// www.kegg.jp /) for annotation of different expressed genes helped us understand phenotypic changes.

5. Western blot

Cells were harvested at the indicated times. In each pole, 20. mu.g of total protein was used for electrophoresis. The membrane was blocked with 5% skim milk for 1 hour at room temperature and then incubated with primary antibody overnight at 4 ℃. Subsequently, the corresponding secondary antibody was applied to the membrane and incubated at room temperature for 2 hours. Protein bands were visualized using a chemiluminescent ECL kit (Tanon, shanghai, china). The antibodies used were as follows: monoclonal anti- β -actin antibody # a5441 (Sigma); anti-Serpine 1(AB2) antibody # AV47470 (Sigma); β -actin (13E5) rabbit monoclonal antibody #4970 (CST); PAI-1(D9C4) rabbit monoclonal antibody #11907 (CST); anti-VEGFA antibody [ VG-1] # ab1316 (Abcam); METTL3(E3F2A) rabbitmab #86132 (CST); IGF2BP1# ab82968 (abcam).

6. GEPIA and HPA and STRING

By GEPIA2(http:// gepia2. cander-pku. cn) and TCGA database (http:// www.oncolnc. tissue /). The statistical method used was the Kaplan-Meier method using a log-rank test. The expression scores of HCC and normal liver tissues in the HPA database describe the estimated SERPINE1 levels. The STRING database (https:// STRING-db.org/cgi/input.pl) has been widely used to predict protein-protein interactions (PPIs), from which to look for proteins that might interact with SERPINE 1.

7. Ablation model

New Zealand white rabbits are adopted to establish the liver cancer in-situ model. Rabbits were first seeded subcutaneously with VX2 cells. Xenografts grow to about 1cm³Then, the mixture is cut into pieces (about 2 to 3mm)³) And is ready for transplantation. Rabbits were fasted 12 hours prior to xenograft orthotopic transplantation. At the beginning of the transplantation, rabbits were anesthetized with 2% sodium pentol and the skin was washed. After transplantation, the blood was stopped with gelatin sponge and penicillin was used in 40 ten thousand units for 3 consecutive days. The rabbit model VX2 was established for 2 weeks before ultrasonography (MyLab Tthread by Esaote, Italy) and the graft was 9.50. + -. 1.56mm (8mm-13 mm). The Laser ablation system was Echo Laser X4 (emission Laser wavelength was 1064nm, fiber diameter was 300 um). Power selection at ablation 4W, output energy of about 100J. 1/3-2/3 of tumor is destroyed, a residual tumor (partial ablation) model is established, and conditions for sub-lethal heat treatment are created. The lesions were then examined using color doppler ultrasound and shear wave viscoelasticity 3 days, 7 days and 14 days post ablation, respectively.

8. Luciferase reporter gene detection

Luciferase reporter gene assays were performed according to the manufacturer's instructions (11402ES60, Yeasen). HCCLM3 and Huh7 cells were seeded in 6-well plates and transfected with a broadtype SERPINE 1-responsive luciferase reporter construct (SERPINE1-WT), a mutant SERPINE 1-responsive luciferase reporter construct (SERPINE1-MUT), a broadtype METTL3 plasmid, or, correspondingly, IGF2BP 1. At 24h post-transfection, cell lysates were incubated with 10. mu.g/ml firefly and TK, respectively, for 10 min, using a dual-luciferase reporter detection system (Promega, Madison, Wis., USA) and a microplate luminometer (Promoka). Firefly luciferase activity was corrected for the corresponding renilla luciferase activity. Results are representative of three independent experiments.

9. Enzyme linked immunosorbent assay

After cell transfection, cell culture supernatants were collected. SERPINE1 and VEGF concentrations were analyzed using the human SERPINE1 kit (ab269373, abcam) and the VEGF-A human ELISA kit (BMS277-2, Thermofish, Invitrogen) according to the manufacturer's instructions.

10. RNA decay analysis

RNA attenuation assays were performed according to the study of Qing et al. Briefly, HCCLM3 and Huh7 cells were seeded at 50% confluence in 6 cm plates. After 24 hours, each 6 cm plate was re-inoculated into three 6 cm plates. After 48 hours, actinomycin D was added to 3mg/ml 6 hours, 3 hours and 0 hour before trypsinization and collection. Total RNA was purified by an on-column DNase-I digestion step. The amount of RNA was determined by RT-qPCR. The mRNA decay rate was calculated by the equation described by Qing et al as follows:

dC: the concentration of mRNA;

dt: a specific time;

-k_decay：mRNAa rate constant of decay;

c: the concentration of mRNA;

t: time;

first, the formula can be found:

dC/dt＝-k_decay·C；

the degradation constant can then be estimated as:

In(C/C₀)＝-k_decay·t；

C₀is the initial concentration of degradation, so the concentration of half of the degradation is calculated as:

In(1/2)＝-k_decay·t_1/2；

the time required for half of the RNA degradation was therefore:

t_1/2＝In2/k_decay。

11. subcutaneous xenograft of nude mice

To simulate a sub-lethal heat treatment subcutaneous implant model, HCCLM3 was heated at 46 ℃ for 10 minutes and seeded into 6 cm petri dishes to simulate a sub-lethal state, and then the petri dishes were replaced twice a day as described previously. After 2 days, 5X 10⁵Stably sublethal heat treated HCCLM3 cells were injected subcutaneously into BALB/C nude mice. 7 days after implantation, PBS was administered at 0.1ml every other day in the control group, while Tiplaxtin (PAI-039) was administered orally at 1mg/kg every other day in the PAI-039 group. After 25 days, the mice were sacrificed.

12. Organoids

HCC PDO organoids were established and experiments were performed using 3 PDOs. For the heating group, PDO was collected after centrifugation at 300g for 10 minutes, and then incubated at 46 ℃ for 10 minutes. PAI-1 inhibitor PAI-039 (final concentration: 10. mu. mol/L) was added to the medium of PDO culture for 4-5 days. The heated PDO was then passaged further and organoid photographs taken every 2-3 days before formalin fixation. PDO was previously embedded in 1% agarose (Biowest agarose, 111860), fixed with 4% phosphate buffered formalin, embedded in paraffin, and cut to 4 μm for hematoxylin-eosin and immunofluorescence analysis, using standard procedures.

In performing the immunofluorescent staining, the following primary antibodies were used: anti-EpCAM (1: 100; 21050-1-AP; Proteintech), anti-AFP (1: 200; 14550-1-AP; Proteintech), anti-PAI 1(1: 100; ab 66705); abcam). The second is an Alexa Fluor 488-conjugated goat anti-rabbit antibody (1: 200; GB 25303; ServiceBio, China) or a Cy 3-conjugated goat anti-rabbit antibody (1: 200; GB 21303; ServiceBio, China). Nuclei were counterstained with 4, 6-diamidino-2-phenylindole (Servicebio, China). Images were obtained with a confocal microscope (olympus).

13. Human liver cancer sample

During 2021, 5 HCC patients received biopsies in interventional radiology and were diagnosed as HCC. All patients provided written informed consent.

14. Tube formation test

Tubes were formed on Matrigel by HUVEC cells and supernatants from HCCLM3 or Huh7 from different transfection conditions were placed into HUVEC cells. After 9 hours, the tube formation was photographed.

15. Immunohistochemistry

Samples were dehydrated with different concentrations of ethanol and finally embedded in paraffin. Paraffin was sectioned into pieces and dried at 60 ℃ overnight. Serial paraffin sections were rehydrated in reduced concentration ethanol after deparaffinization. The SABC kit was used according to the manufacturer's instructions (BOSTER, Wuhan, China). The primary and secondary antibodies used were human SERPINE1, PAI-1 antibody (ab66705, abcam), respectively. Finally, the sections were imaged by phase contrast microscopy (canon, japan).

Example 1

PAI-1 increases following sublethal ablation:

RNA-seq sequencing was performed on the sublethal heat treatment model and heatmaps of the first 25 up-regulated mrnas and the first 25 down-regulated mrnas were obtained, as shown in fig. 1A. Since tumor vessels are essential for tumor survival, venn plots are drawn between the "forward regulation of angiogenesis", "extracellular exosomes" terms based on GO. The results showed 8 "cross-genes", i.e., "SERPINE 1, rapgof 3, ECM1, PRKD2, PRKCA, ERAP1, CAST, GAB 1", where "SERPINE 1" had the most significant change, with at least a 16-fold increase in the sublethal heat-treated group over the control group, as shown in fig. 1B. This suggests that SERPINE1 may be a key protein for angiogenesis after sub-lethal heat treatment of HCC.

In addition, changes in mRNA and protein levels of SERPINE1 were verified. The mRNA and protein expression levels of SERPINE1 increased after sublethal heat treatment, as shown in figure 1C. The function of SERPINE1 in HCC was then examined by analyzing the TCGA database for overall survival in SERPINE1 high and low expressing patients, and the LIHC data set showed shorter survival for the SERPINE1 high expressing group, as shown in figure 1D. On the other hand, by examining the levels of SERPINE1 at different stages of HCC, it was found that the patients had higher SERPINE1 expression as the tumor stage progressed, as shown in fig. 1E. Analysis of the typical protein levels of SERPINE1 by the HPA database revealed that SERPINE1 expression was higher in HCC patients than in the corresponding patients, as shown in figure 1F, which is consistent with our case, and SERPINE1 was expressed higher in HCC in patients receiving resection than in para-cancerous tissues, as shown in figure 1G. To simulate HCC by sublethal heat treatment, an ablated rabbit HCC model was established using VX2, as shown in FIG. 1H, and FIG. 1I shows successful establishment of a VX2 liver cancer in situ model, with a representative tumor of 7X 7cm². The xenografts were precisely positioned and the ablation zone controlled using ultrasound and residual HCC was grown as shown in figure 1J with microwave ablation power of 4W and output energy of about 100J. General conditions of 1 day, 3 days, 7 days and 14 days after microwave ablation are observed through ultrasonic images, ultrasonic contrast is carried out on 7 days after ablation, and the result is shown in figure 1K. Residual HCC tissue survival was observed by microscopic observation of residual tumor and normal liver around the ablated section before and after h.e. staining, as shown in fig. 1L. By examining the relative mRNA expression level and protein expression level increase of SERPINE1, the results showed that the mRNA expression level and protein expression level of SERPINE1 were increased at 3 days and 7 days after ablation, and thereafter, the relative expression level of SERPINE1 was gradually decreased after 7 days of ablation, as shown in fig. 1M, compared to the non-ablated group.

Example 2

SERPINE1 promoted angiogenesis in part by VEGF after sublethal heat treatment:

in order to further study the specific mechanism of SERPINE1, SERPINE1 knockdown cells were established in experiments and treated with SERPINE1 inhibitor PAI-039, and the results showed that HCCLM3 and Huh7 cell lines reduced mRNA expression levels and protein expression levels after knockdown of SERPINE1 under 37 ℃ culture conditions, and that SERPINE1 partially restored mRNA expression levels and protein expression levels under 46 ℃ culture conditions, as shown in FIG. 2A. In addition, the HCCLM3 and Huh7 cell lines inhibited PAI-039 at 37 ℃ in the SERPINE1 inhibitor reduced mRNA transcript levels and protein expression levels in SERPINE1, and at 46 ℃ in the cell lines partially restored mRNA and protein expression levels in SERPINE1, as shown in FIG. 2B. Secretion of SERPINE1 in the supernatant was significantly reduced when SERPINE1 gene knockdown or PAI-039 was inhibited. Then enzyme-linked immunosorbent assay is carried out, ELISA shows that the level of SERPINE1 in the extracellular supernatant is reduced after knocking down SERPINE1 under the culture condition of HCCLM3 at 37 ℃, the level of SERPINE1 can be partially recovered under the culture condition at 46 ℃, while ELISA shows that the level of SERPINE1 in the extracellular supernatant is reduced after knocking down SERPINE1 under the culture condition of Huh7 at 37 ℃, and the level of SERPINE1 cannot be significantly recovered under the culture condition at 46 ℃, as shown in FIG. 2C. Thus, sublethal heat treatment rescued the partial effects of SERPINE1 knockdown or PAI-039 application in HCCLM3 cells, whereas the addition of PAI-039 inhibitors to Huh7 cells was not observed.

Supernatants from HCCLM3 or Huh7 obtained under different treatment conditions were collected and cultured for HUVEC for 48 hours. The number of tube formation was significantly reduced after culturing in the extracellular supernatant after using knocked-down SERPINE1 compared to the supernatant directly using 37 ℃ culture conditions, and the tube formation was partially restored by the supernatant obtained using 46 ℃ culture conditions, as shown in fig. 2D; supernatants from HCCLM3 or Huh7 obtained under different treatment conditions were collected and cultured for HUVEC for 48 hours. The number of tube formation was significantly reduced using the culture supernatant after PAI-039 addition compared to the supernatant directly using the 37 ℃ culture conditions, while the number of tube formation was significantly increased using the supernatant using the 46 ℃ culture conditions, as shown in FIG. 2E. Tube formation assays showed that HUVEC reduced the ability to form tubes after incubation with supernatant under conditions in which virus or PAI-039 inhibited SERPINE 1. Then, protein-protein interactions were analyzed by STRING (https:// STRING-db. org), and the results showed that there were more proteins potentially associated with SERPINE1, and some members of the SMAD family and VEGF were close to SERPINE 1; furthermore, in the TCGA LIHC dataset, SMAD2, SMAD3, SMAD7 and VEGFA were significantly correlated with SERPINE1, wherein the correlation coefficient R values of VEGF with SERPINE1 were as high as 0.37, strongly indicating that there is a correlation between SERPINE1 and VEGF, as shown in fig. 2F. In HCCLM3 and Huh7 hepatoma cells, the protein level expression of VEGF was significantly reduced after SERPINE1 knockdown, whereas the expression level of VEGF could be increased under 46 ℃ culture conditions, as shown in fig. 2G. In HCCLM3 and Huh7 hepatoma cells, the relative expression level of VEGF in the supernatant was significantly reduced after SERPINE1 knockdown, and the extracellular VEGF expression level was increased after VEGF addition, as shown in fig. 2H. As expected, VEGF decreased with inhibition of SERPINE1 in HCCLM3 and Huh7, and at the same time, secreted VEGF decreased. When HCCLM3 and Huh7 cells were cultured at 46 ℃, SERPINE1 was knocked out and VEGF expression decreased, and there was no significant change in SERPINE expression after VEGF addition, as shown in fig. 2I. Next, a tube formation assay of HUVECs was performed, and supernatants from HCCLM3 or Huh7 were collected and used to culture HUVECs for 48 hours. Tube formation decreased after treatment with 46 ℃ + SERPINE1 knockdown supernatant and increased when VEGF treated cell supernatant was added, as shown in figure 2J. Thus, the results indicate that additional VEGF can promote tube formation by inhibiting SERPINE 1.

Example 3

METTL3/IGF2BP1/m6A axial forward regulation SERPINE 1:

previous studies have shown that METTL3 promotes HCC invasion after sub-lethal heat treatment. Whereas angiogenesis is positively correlated with HCC invasion. Thus, it was next experimentally verified whether METTL3 could modulate SERPINE1 in angiogenesis. When METTL3 was knocked down in HCCLM3 or Huh7, the mRNA expression level of SERPINE1 increased and that of METTL3 did not change significantly after PAI-1 addition, as shown in FIG. 3A. Changes in relative METTL3 and SERPINE1 protein levels when met tl3 was knocked down with/without SERPINE1 in HCCLM3 or Huh7 are shown in figure 3B. The above results show that inhibition of METTL3 reduces SERPINE1 expression at both mRNA and protein levels, and that additional SERPINE1 may partially rescue SERPINE1 levels, but has no effect on METTL 3. In HCCLM3 or Huh7, after SERPINE1 was knocked down, there was no significant change in METTL3 mRNA expression levels, inhibition of SERPINE1 did not inhibit METTL3 expression, as shown in fig. 3C. The above data indicate that METTL3 may be located upstream of SERPINE 1. Since METTL3 is a methyltransferase, to verify whether SERPINE1 is regulated by METTL3 in the m6A mode, the m6A modification site of SERPINE1 was subsequently analyzed on RMbase 2.0(http:// rna.sys. edu.cn/RMBase). As a result, there were more than 40 predicted m6A sites, as shown in fig. 3E.

According to the prediction, the SERPINE1-mutant (SERPINE1-MUT) plasmid was constructed by selecting the m6A modification site (purple) with the highest motif score and the m6A site adjacent thereto. Luciferase assays using wild-type or m6A consensus mutant SERPINE1 cDNA fused to firefly luciferase reporter showed that SERPINE1 strongly increased firefly/renilla activity with METTL3, but SERPINE1-MUT failed, as shown in fig. 3F, mutations in m6A consensus of SERPINE1 relieved the post-transcriptional repression results of SERPINE1 and METTL3 in HCCLM3 and Huh 7. Subsequently, a tubule formation experiment of HUVEC was performed, and supernatants from HCCLM3 or Huh7 were collected and used to culture HUVEC for 48 hours. Under the condition that METTL3 was knocked out at 46 ℃, the proliferation amplification capacity was improved after PAI-1 addition, as shown in FIG. 3G. Thus, silencing of METTL3 may inhibit tubule forming ability, but overexpression of SERPINE1 may rescue this impact assay. RNA stability under METTL3 knockdown with/without SERPINE1 at 46 ℃ is shown in FIG. 3H. In HCCLM3 or Huh7 cell lines, IGF2BP1 and SERPINE1 were significantly decreased at the transcriptional and translational levels when IGF2BP1 was knocked down, as shown in fig. 3I. In HCCLM3 or Huh7, IGF2BP1 and SERPINE1 decreased significantly in transcriptome and translation levels when SERPINE1 was knocked down, as shown in fig. 3J. The wild-type or m6A consensus mutant SERPINE1 cDNA was fused to a firefly luciferase reporter gene, and the luciferase assay results are shown in FIG. 3K. The results show that mutations in the m6A consensus sequence of SERPINE1 abolished the post-transcriptional repression of SERPINE1 and IGF2BP1 in HCCLM3 and Huh 7.

Example 4

SERPINE1 was upregulated in organoids under sublethal heat treatment:

the HCC PDO model was further used to verify the function of SERPINE 1. HCC PDO exhibited epithelial phenotypes such as greater invasive pseudopodogenesis and irregular fibroblast-like shapes after 10 minutes of heat treatment at 46 ℃. With the addition of the SERPINE1 inhibitor PAI-039(10 μ M), the phenotype was reversed to some extent, as shown in figure 4A. HE staining indicated that PDO exhibited a pseudo-glandular histological structure, as shown in figure 4B. Immunofluorescent staining indicated that three PDOs exhibited characteristics typical of HCC, such as positive expression of AFP and negative for EpCAM, as shown in fig. 4C-4E. It was found by examining the expression level of SERPINE1 that the expression level of SERPINE1 was significantly higher in heat-treated PDO, and that this effect could be attenuated by the addition of PAI-039, as shown in FIGS. 4C-4F. The above results indicate that heat treatment also stimulated SERPINE1 expression in the three-dimensional PDO model, an effect that could be reversed by SERPINE1 inhibitor PAI-039.

Example 5

PAI-039 was effective in inhibiting in vivo SERPINE1 expression following sublethal heat treatment:

10 female BALB/c nude mice received sub-lethal heat-treated HCCLM3 injections, as shown in FIG. 5A. Of these, 5 received physiological saline, and the other 5 received PAI-039 every other day. At the end of the experiment on day 25, mice were sacrificed and the in vivo effect of the SERPINE1 inhibitor PAI-039 after sublethal heat treatment was examined by establishing a sublethal heat injury HCCLM3 subcutaneous xenograft model, the PAI-039 group showed a smaller xenograft volume compared to the control group, as shown in fig. 5B, and injection of PAI-039 significantly inhibited tumor growth. Both groups showed malignant growth as shown in figure 5C. Immunohistochemistry results showed a significant reduction in SERPINE1 expression levels in the PAI-039 group compared to the control group, as shown in fig. 5D, and as expected, the PAI-039 group showed less SERPINE1 expression compared to the control group. Notably, immunohistochemistry results showed a significant decrease in VEGF expression levels in PAI-039 group compared to the control group, as shown in fig. 5E. These data indicate that PAI-039 can inhibit in vivo xenografts following sub-lethal heat treatment.

The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way and substantially, it should be noted that those skilled in the art may make several modifications and additions without departing from the scope of the present invention, which should also be construed as a protection scope of the present invention.

Sequence listing

<110> Zhongshan Hospital affiliated to Fudan university

Application of <120> SERPINE1 inhibitor in preventing recurrence and metastasis after hepatocellular carcinoma microwave ablation

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 21

<212> DNA/RNA