| Cervical cancer is one of the most common gynecological malignancies in women.According to the World Cancer Report published by the World Health Organization(WHO)in 2018,the number of new cases of cervical cancer in women in the world is 569,847 in 2018,and the number of deaths is 311.365.The incidence was 13.1%and the mortality was 6.9%,which ranking fourth in female tumors.The morbidity and mortality in developing countries were significantly higher than those in developed countries.According to the China Population Cancer Statistics Report published in 2015,the morbidity and mortality of cervical cancer in the Chinese female population has been increasing year by year from 2000 to 2014.High-risk human papillomavirus HPV persistent infection is currently recognized as a major risk factor for the development of cervical cancer.But this process is slow,often lasting for decades.And only a very small number of HPV-infected patients progress to cervical cancer,indicating that in addition to persistent HPV infection,several other factors are involved in the carcinogenic process.Therefore,in-depth study of the carcinogenic mechanism of cervical cancer is of great significance for the accurate screening and treatment of cervical cancer.MicroRNAs(miRNAs)are a class of non-coding RNAs discovered in recent years,derived from endogenous chromosomes with an average length of about 22 nucleotides.The miRNA can complement the sequence of the 3' untranslated region of the gene mRNA,causing degradation of the target RNA or inhibiting its translation,thereby exerting a role in post-transcriptional regulation of gene expression.Depending on the function of the target gene regulated by it,miRNA can play a role as a proto-oncogene or a tumor suppressor gene,and plays an important role in the development,metastasis and drug resistance of malignant tumors.However,during the process of tumor formation,angiogenesis lost its normal regulation and abnormal angiogenesis increased.The process of tumor blood vessel growth includes steps such as vascular endothelial matrix degradation,endothelial cell migration,cell proliferation,and formation of a new basement membrane by endothelial cells.Neovascularization provides a large amount of nutrients for tumor cells,promotes rapid tumor growth and metastasis,and is an important mechanism for invasion,metastasis and drug resistance of malignant tumors.miR-9 is up-regulated in glioma,head and neck squamous cell carcinoma,and breast cancer,but down-regulated in colorectal cancer,colon cancer,and gastric cancer.In the early stage of patients with malignant breast cancer,elevated levels of miR-9 promote epithelial-mesenchymal transition(EMT)of tumor cells.And miR-9 can enhance the growth of blood vessels.thereby increasing the blood supply to the tumor and promoting tumor growth and invasion.Therefore,this subject is mainly devoted to the study of the mechanism of miR-9 in cervical carcinogenesis,angiogenesis and radiotherapy tolerance.The research content is mainly divided into three parts:Part I:Expression of miR-9-5p in cervical squamous cell carcinoma and its clinical significancePart II:Effect of miR-9-5p on proliferation and metastasis of cervical cancer cels and its mechanismPart III:The effect of miR-9-5p on the sensitivity of cervical cancer radiotherapy and its mechanismPart I:Expression of miR-9-5p in cervical squamous cell carcinoma and its clinical significancePurposes:miRNAs have been found to play an important and even decisive role as oncogenes or tumor suppressor genes in different types of cancer.Lymph node metastasis is the main metastasis pathway for cervical cancer,and its mechanism needs further study.The primary objective of this study was to screen for specific miRNAs associated with lymph node metastasis of cervical cancer and to clarify its relationship to clinical pathology and prognosis.Materials and Methods:The Cancer Genome Atlas(TCGA)database was used to analyze the differential expression of miRNAs in cervical lymph node metastasis positive and lymph node metastasis negative tissues.To further verify the expression of miR-9-5p in 22 cases of cervical lymph node metastasis and 22 cases of cervical lymph node metastasis-negative cancer,and to analyze the relationship between miR-9-5p expression and clinicopathological features of cervical cancer patients.Results:TCGA data analysis showed that miR-9-5p was significantly higher in patients with positive lymph node metastasis than in patients with negative lymph node metastasis.qRT-PCR results showed that mRNA expression of miR-9-5p was significantly higher in patients with positive lymph node metastasis than in non-metastatic patients.Patient survival analysis in the TCGA database suggested that patients with elevated miR-9-5p expression and patients with relatively low expression had no significant change in overall survival.The expression of miR-9-5p in 22 patients with positive lymph node metastasis of cervical cancer was significantly higher than that in patients without lymph node metastasis.The clinical and pathological data of 44 patients with cervical cancer were analyzed.The difference in expression of miR-9-5p at mRNA level was only Lymph node metastasis was associated with no significant difference in age,stage,differentiation,tumor size,and vascular invasion in patients with cervical cancer.Conclusion:MiR-9-5p is higher in cancer tissues with positive lymph node metastasis than in patients without lymph node metastasis.Part ?:Effect of miR-9-5p on proliferation and metastasis of cervical cancer cells and its mechanismPurposes:Previous studies have shown that miR-9 expression level is elevated in gliomas,head and neck squamous cell carcinoma,and breast cancer,playing an oncogene role;In the early stage of patients with malignant breast cancer,elevated miR-9 levels promote epithelial-mesenchymal transition(EMT)of tumor cells and promote tumor growth and invasion.Based on the results of the first part,we speculate that miR-9-5p may also be an oncogene in cervical cancer.The purpose of this part of the study was to investigate the effect of miR-9-5p on the proliferation,invasion and migration of cervical cancer cells,the effect on the angiogenic ability of HUVEC cells,and to identify the downstream target genes of miR-9-5p and explore its possible mechanism.Materials and Methods:The cervical cancer cell line SiHa was selected to up-regulate or down-regulate the expression of miR-9-5p in cells by transfecting miRs or inhibitors of miR-9-5p.The growth curve was measured by CCK-8 method in vitro,and the cell proliferation ability was measured.The number of cells passed through the Transwell experiment was used to detect the change of cell invasion and migration ability.The effect of miR-9-5p on the angiogenesis ability of HUVEC cells was examined by angiogenesis assay.Western blot was used to detect the effect of miR-9-5p on cervical cancer proliferation-related proteins,EMT-related molecules in EMT and angiogenesis-related proteins.The in vivo experiment used a subcutaneous tumor formation method in nude mice to compare the difference in tumorigenic ability.The miR-9-5p downstream target gene SOCS5 was screened by the prediction software Targetscan and miRanda.Immunohistochemistry was used to detect the difference of SOCS5 expression in cervical cancer tissues and normal cervical tissues.The dual luciferase reporter assay demonstrated whether SOCS5 could serve as a direct downstream target for miR-9-5p.The Rescue assay can detect whether overexpression of SOCS5 can reverse the promotion of miR-9-5p on cervical cancer cells.Western blot was used to detect changes in epithelial-mesenchymal transition-related molecules and angiogenesis-related molecules to confirm the effect of miR-9-5p interaction with SOCS5 on downstream pathways and cellular malignant behavior.Results:In vitro,compared with the control group,the proliferation of SiHa cells transfected with miR-9-5p mimics increased,and their invasion and migration ability was also enhanced;SiHa cells transfected with miR-9-5p inhibitor,The proliferative capacity is weakened,and the ability to invade and migrate is weakened.The cell supernatant transfected with miR-9-5p mimics was co-cultured with HUVEC cells.The number of blood vessels formed by HUVEC cells was increased compared with the control group,and the vascular structure was more mature.Western blot analysis showed that the expression of E-cadherin was significantly decreased in cells transfected with miR-9-5p mimics,and the expressions of N-cadherin,Snail,Vimentin,p-mTOR,p-VEGFR2,VEGFA were increased.In the transfected miR-9-5p inhibitor group,the trends of EMT-related molecules(such as E-cadherin and N-cadherin)and angiogenesis-related proteins were opposite to those of the mimics group.In vivo experiments,the tumorigenic ability of the nude mice in the miR-9-5p overexpression group increased.The predicted results of TargetScan and miRanda showed that miR-9-5p has a binding site to SOCS5 mRNA 3-UTR.Compared with normal cervical tissues,the expression of SOCS5 was low in cervical cancer tissues and negatively correlatedwith the expression of miR-9-5p.The results of the dual luciferase reporter gene assay showed that miR-9-5p mimics significantly reduced the fluorescence intensity of wild-type group(wt)cells without changing the fluorescence intensity of the mutant(mut)cells.The results of Rescue showed that SOCS5 overexpression increased the expression of SOCS5 in SiHa cells.The analysis of angiogenesis showed that SOCS5 overexpression could inhibit the effect of miR-9-5p on angiogenesis.Western blot showed that the effect of miR-9-5p was reversed after transfection of SOCS5 in miR-9-5p stably transfected cells.The expression of E-cadherin was increased.The expression of p-VEGFR2 and VEGFA was significantly decreased,and the expression of p-ERK was also decreased.Conclusion:MiR-9-5p promotes the proliferation,invasion and migration of cervical cancer cells by targeting inhibition of SOCS5 expression,and promotes the angiogenesis ability of HUVECs.Part III:The effect of miR-9-5p on the sensitivity of cervical cancer radiotherapy and its mechanismPurposes:Radiotherapy is one of the most effective means of treating cervical cancer,and miRNA plays an important role in radiotherapy tolerance and sensitivity.The purpose of this study was to investigate the effect of miR-9-5p on the sensitivity of cervical cancer radiotherapy,and further explore its related molecular mechanisms to provide new ideas and targets for improving the radiotherapy effect of cervical cancer.Materials and Methods:SiHa cells transfected with miR-9-5p mimics were irradiated with OGy and 4Gy gamma rays.SiHa cells transfected with miR-9-5p mimics were irradiated with y-rays of OGy and 4Gy,and the supernatant of the medium was co-cultured with HUVECs cells to analyze the changes of angiogenic ability of HUVECs cells.SiHa cells transfected with miR-9-5p mimics were transfected with GFP-LC3 lentivirus,and the number of autophagosomes was observed under gamma irradiation of 0 Gy and 4 Gy.Western blot was used to detect the expression of angiogenesis-related proteins and autophagy-related proteins in each group after radiotherapy.Results:miR-9-5p mimics significantly inhibited the killing effect of radiation on cervical cancer cells under 4Gyy-rays.After radiotherapy,the number of angiogenesis in HUVEC cells co-cultured with SiHa cells transfected with mimics and NC group was significantly reduced.The number of angiogenesis in transfected miR-9-5p mimics group was decreased less than that in NC group.The number of autophagosomes in SiHa cells transfected with miR-9-5p mimics group was less than that in NC group.After radiotherapy,the number of autophagosomes in both groups increased,but mimics group was increased less than NC group.Western blot analysis showed that the expression levels of autophagy-related proteins LC3 increased,p62 and p-ULKdecreased after radiotherapy,and the expression of angiogenesis-related proteins p-VEGFR2,p-ERK and VEGFA decreased,but the expression in mimics group was decreased less than that in NC group.Conclusion:MiR-9-5p causes radiotherapy resistance in cervical cancer cells by increasing angiogenesis and reducing autophagy.Innovation:1.For the first time,by analyzing the TCGA database,it was elucidated thatmiR-9-5p is highly expressed in lymph node metastasis-positive cervical cancer.2.Complete in vitro and in vivo experiments confirmed the important role of miR-9-5p in the development and angiogenesis of cervical cancer.3.It was first discovered that SOCS5 is a direct downstream target of miR-9-5p.4.It is shown for the first time that miR-9-5p can mediate the radiosensitivity of cervical cancer through autophagy. |
PDF Full Text Request