Effect Of Mir-145-3p On Biological Behavior Of Cervical Cancer By Targeting ZEB1

Part One Expression of mir-145-3p and ZEB1 in cervical cancer and their relationship with clinical featuresObjective: To analyze the relative expression levels of mir-145-3p and ZEB1 m RNA in cervical cancer,and to explore the relationship between them and the clinical characteristics of cervical cancer patients,so as to provide a new basis for the study of the molecular mechanism and progress of cervical cancer.Methods: From January 2020 to September 2021,32 cervical cancer tissue samples from patients with cervical squamous cell carcinoma and FIGO stage above IB2 confirmed by colposcopy biopsy or cervical conization pathology in the second hospital of Hebei Medical University were selected as the study group.32 normal cervical tissue samples from patients with benign gynecological diseases and negative cervical cancer screening were collected as the control group.The relative m RNA expressions of mir-145-3p and ZEB1 in cervical cancer and normal cervical tissues were detected by real-time fluorescent quantitative PCR(q RT-PCR)and compared.At the same time,the relationship between the relative expression of mir-145-3p and ZEB1 m RNA and the clinicopathological features of patients was analyzed.Results: 1.The relative expression of mir-145-3p in cervical cancer tissues was significantly lower than that in normal control tissues(P =0.00).2.The relative expression of ZEB1 m RNA in cervical cancer tissues was significantly higher than that in normal control tissues(P =0.00).3.In cervical cancer,the relative expression of mir-145-3p was low,while ZEB1 m RNA was high.There was a negative correlation between the two(r=-0.676).4.The relative expression of mir-145-3p and ZEB1 m RNA in cervical cancer was not related to age,menopausal status,HPV infection and TCT(P >0.05),but related to FIGO stage and tumor size(P <0.05).The metastasis of lymph nodes was associated with the high expression of ZEB1 m RNA,but not with the expression of mir-145-3p.Conclusions: 1.Compared with normal cervix,the relative expression of mir-145-3p in cervical cancer decreased,and the relative expression of ZEB1 m RNA increased significantly.2.The expression of mir-145-3p was low and ZEB1 m RNA was high in cervical cancer.There was a negative correlation between the two groups.3.The relative expression levels of mir-145-3p and ZEB1 m RNA in cervical cancer were not related to the patient’s age,menopausal status,HPV infection and TCT,but related to the patient’s FIGO stage and tumor size.Lymph node metastasis was associated with high expression of ZEB1 m RNA.Part Two Role of zinc finger E-box binding homeobox 1(ZEB1)in epithelial mesenchymal transformation of cervical cancerObjective: To study the expression of zinc finger E-box binding homeobox 1(ZEB1),E-cadherin and vimentin in cervical cancer and normal cervical tissues,so as to evaluate the role of ZEB1 in the transformation of cervical cancer epithelium to stroma.Methods: From January 2020 to September 2021,20 paraffin tissue samples of patients with cervical squamous cell carcinoma and FIGO stage above IB2 confirmed by colposcopy biopsy or cervical conization pathology in the second hospital of Hebei Medical University were selected as the study group,and 20 normal paraffin tissue samples of patients with benign gynecological diseases and negative cervical cancer screening were collected as the control group.(both the study group and the control group were selected from the cases in the first part of the study).The expression of ZEB1,E-cadherin and vimentin in tissues was determined and compared by immunohistochemistry.Results: 1.The positive rate of ZEB1 and vimentin expression in cervical cancer was significantly higher than that in normal cervical group,while the positive rate of E-cadherin expression was lower than that in normal cervical group.The difference was statistically significant(P <0.05).2.Pearson correlation analysis showed that the expression of ZEB1 was negatively correlated with the expression of E-cadherin,but positively correlated with the expression of Vimentin.Conclusions: 1.The positive rate of ZEB1 and Vimentin expression in cervical cancer was significantly higher than that in normal cervical group,while the positive rate of E-cadherin expression was lower than that in normal cervical group.2.The expression of ZEB1 was negatively correlated with that of E-cadherin,but positively correlated with that of Vimentin.3.ZEB1 down regulates the expression of E-cadherin,up regulates the expression of Vimentin and participates in the transformation of cervical squamous epithelial stroma.Part Three mi R-145-3p participates in the proliferation,migration,apoptosis and invasion of cervical cancer cells in vitro by targeting ZEB1Objective: Taking cervical cancer cell line C33 A as the research object,to construct the corresponding lentivirus plasmid to overexpress and silence mir-145-3p and ZEB1,so as to explore the mechanism of microrna-145-3p targeted regulation of ZEB1 to promote the transformation of cervical cancer epithelium to stroma and promote tumor progression,so as to provide reference for other clinical related studies.Methods: Human cervical cancer cell line C33 A was cultured and subcultured.According to the instructions of the kit,mir-145-3p-mimc,mir-145-3p inhibitor and Mir NC were transfected into C33 A cells with Lipofectamine 2000 transfection reagent at a dose of 20 nnmol / L,and the transfection efficiency was measured 48 hours after transfection;Cell cloning test to determine cell proliferation;Annexin V-FITC / PI double staining was used to detect apoptosis;Cell migration was measured by scratch test;Cell invasion was determined by Transwell analysis;Targetscan biological software was used to predict the related targets of mir-145-3p.It was found that there were complementary binding sites between the 3’UTR end of ZEB1 and mir-145-3p.The targeting relationship between them was determined by luciferase reporting system,and the luciferase activity was determined by luciferase activity kit;The amount of RNA was determined by real-time fluorescent quantitative PCR,and the concentration and purity of total RNA were determined by UV spectrophotometry;Western blot was used to detect the expression of E-cadherin and Vimentin in cells.Results: 1.Transfection efficiency of mi R-145-3p: 48 h after transfection into C33 A cells,the expression level of mir-145-3p was determined by RT-PCR.The results showed that compared with NC group,the expression level of mir-145-3p in mimics group was significantly increased and that in inhibitor group was significantly decreased.The difference was statistically significant(P < 0.05).2.Effects of overexpression and knockdown of mir-145-3p on proliferation and apoptosis of cervical cancer cells: Compared with NC group,the clonogenic ability of mimics group was significantly reduced and the apoptosis rate was significantly increased.The clonogenic ability of inhibitor group was significantly improved and the apoptosis rate was significantly reduced(P < 0.05).3.Effects of overexpression and knockdown of mir-145-3p on invasion and metastasis of cervical cancer cells: Compared with NC group,the migration and invasion ability of mimics group cells decreased significantly,and the migration and invasion ability of inhibitor group cells increased significantly,with statistical difference.4.Effects of overexpression and knockdown of mir-145-3p on the expression of EMT protein in cervical cancer cells: Compared with NC group,the expression of E-cadherin increased and Vimentin decreased in mimics group,while the expression of E-cadherin decreased and Vimentin increased in inhibitor group(P < 0.05).5.The analysis of double luciferase expression gene showed that mir-145-3p and ZEB1 were in the targeted regulation relationship.Compared with Mir NC,there was no significant difference between mir-145-3p mut and Mir NC(P > 0.05),and the luciferase activity of mir-145-3p wt decreased significantly.The difference was statistically significant(P < 0.05).6.Effect of ZEB1 overexpression on proliferation and apoptosis of cervical cancer cells: compared with the vector group,the clone forming ability of ZEB1 group was significantly enhanced and the apoptosis rate was significantly reduced(P < 0.05).7.Invasion and metastasis of cervical cancer cells induced by ZEB1 overexpression: compared with the vector group,the number of cell migration and invasion in ZEB1 group was significantly up-regulated(P < 0.05).8.Effect of ZEB1 overexpression on the expression of EMT protein in cervical cancer cells: compared with the vector group,the expression of E-cadherin in ZEB1 group decreased and the expression of Vimentin increased.The above differences were statistically significant.Conclusions: 1.48 h after transfection of C33 A cells,compared with NC group,the expression level of mir-145-3p in simulation group increased significantly,while the expression level of mir-145-3p in inhibitor group decreased significantly,the difference was statistically significant.2.Compared with the normal control group,the clone forming ability,migration and invasion ability and Vimentin expression of cells in the simulation group were significantly reduced,while the apoptosis rate and E-cadherin expression were significantly increased.In the inhibitor group,cell cloning ability,cell migration and invasion ability and Vimentin expression were significantly increased,while the apoptosis rate and E-cadherin expression were significantly decreased,which was statistically significant.3.The analysis of double luciferase expression gene showed that mir-145-3p and ZEB1 had targeted regulation relationship.Compared with Mir NC,there was no significant difference between mir-145-3p mut and Mir NC,and the difference of mir-145-3p wt was statistically significant;Compared with the vector group,the cell cloning ability,the number of cell migration and invasion and the expression of Vimentin in ZEB1 group were significantly increased,while the apoptosis rate and the expression of E-cadherin were significantly decreased.