The Effect And Mechanism Of MiR-30e-5p Targeting SNAI1 To Regulate EMT On Pancreatic Cancer

PART ? Expression of miR-30e-5p in pancreatic cancer and its relationship with clinicopathological characteristics of pancreatic cancerObjective: To investigate the expression of miR-30e-5p in pancreatic cancer and its relationship with clinicopathological characteristics of pancreatic cancer.Methods: From July 2016 to August 2019,48 paired pancreatic cancers and their adjacent pancreatic tissues were collected in the First Affiliated Hospital of Guangxi Medical University.All patients were diagnosed with pancreatic cancer by pathology.RNA was extracted using the miRNeasy FFPE kit,and q RT-PCR was used to detect the expression level of miR-30e-5p in the pancreatic cancer group and paracancerous group.Pearson correlation analysis was used to analyze the correlation between the expression level of miR-30e-5p and the age,gender and TNM stage of pancreatic cancer patients.Results:(1)The gene expression level of miR-30e-5p in the tissues of patients with pancreatic cancer was lower than that of paracancerous groups(P<0.01),the difference was statistically significant.(2)The expression levels of miR-30e-5p did not reach statistical significance in patients with different genders,ages and TMN stages.Conclusion: miR-30e-5p is significantly differentially expressed in pancreatic cancer and its adjacent tissues,suggesting that miR-30e-5p may play an important role in the development of pancreatic cancer.PART ? Effect of miR-30e-5p on the biological function of pancreatic cancercells in vitroObjective: To investigate the effect of miR-30e-5p on the biological function of human pancreatic cancer cells by transfecting miR-30e-5p in human pancreatic cancer cell lines in vitro.Methods: The liposome method was used to transfect negative control(NC),miR-30e-5p mimics and miR-30e-5p inhibitor in human pancreatic cancer cell lines Bx PC-3 and PANC-1.The cells were divided into NC group,miR-30e-5p mimics group and miR-30e-5p inhibitor group.The q RT-PCR method was used to detect the expression of miR-30e-5p in each group of Bx PC-3 and PANC-1;MTT method was used to detect the cell viability of each group of Bx PC-3 and PANC-1;the colony formation ability of each group of Bx PC-3 and PANC-1cells was detected by the cell colony formation test;The cell invasion test(Transwell method)was used to detect the invasion ability of each group of Bx PC-3 and PANC-1;the migration ability of each group in Bx PC-3 and PANC-1 cells was detected by cell migration test;flow cytometry was used to detect the apoptosis rate of each group of Bx PC-3 and PANC-1 cells;flow cytometry was used to detect the cell cycle of each group of Bx PC-3 and PANC-1 cells.Results:(1)The q RT-PCR experiment found that in Bx PC-3 and PANC-1cells,the gene expression level of miR-30e-5p in miR-30e-5p mimics group was significantly higher than that in NC group,and the differences were statistically significant(P<0.001).(2)Compared with the NC group,MTT experiment found that miR-30e-5p mimics group could inhibit the cell viability of Bx PC-3 and PANC-1 cells,and the differences were significant(P<0.05);while miR-30e-5p inhibitor group could increase the cell viability of Bx PC-3 and PANC-1,and the differences were significant(P<0.05).(3)Compared with NC group,cell clone formation experiment found that miR-30e-5p mimics group could inhibit the colony formation ability of Bx PC-3 and PANC-1 cells,the differences were statistically significant(P<0.001);miR-30e-5p inhibitor group could promote the colony forming ability,and the difference were statistically significant(P<0.001).(4)Cell invasion experiments showed that miR-30e-5p mimics group could inhibit the invasion ability of Bx PC-3 and PANC-1 cells compared with NC group,the differences were statistically significant(P<0.01);while miR-30e-5p inhibitor group could promote the invasive ability of Bx PC-3 and PANC-1,and the differences were statistically significant(P<0.05).(5)Compared with NC group,cell migration experiments showed that miR-30e-5p mimics group could inhibit the migration ability of Bx PC-3 and PANC-1 cells,the differences were statistically significant(P<0.01);while miR-30e-5p inhibitor group could promote the migration ability of Bx PC-3 and PANC-1cells,and the differences were statistically significant(P<0.05).(6)Apoptosis experiments showed that miR-30e-5p mimics group could promote the apoptosis of Bx PC-3 and PANC-1 cells compared with NC group,and the differences were statistically significant(P<0.01);miR-30e-5p inhibitor group could inhibit the apoptosis of Bx PC-3 and PANC-1 cells,and the differences were statistically significant(P<0.05).(7)Cell cycle experiments found that compared with the NC group,the miR-30e-5p mimics group and the miR-30e-5p inhibitor group failed to intervene in the cell cycle stages of Bx PC-3 and PANC-1.Conclusion: The high expression of miR-30e-5p in pancreatic cancer can significantly inhibit the proliferation,invasion,migration and promote apoptosis of pancreatic cancer cells,suggesting that miR-30e-5p may play a tumor suppressor role in the development of pancreatic cancer.PART ? The relationship between miR-30e-5p and its potential target gene SNAI1Objective: To predict miR-30e-5p potential target gene SNAI1 and binding site through miRNA-target prediction database,and verify whether it can bind to SNAI1 site by dual luciferase and the q RT-PCR.Methods: The Target Scan website was used to predict the miR-30e-5p target gene SNAI1 and its binding site,and 393 T cells were transfected by constructing WT and MUT vectors of SNAI1,and the cells were divided into WT+NC group,WT+miR-30e-5p mimics group,MUT+NC group and MUT+miR-30e-5p mimics group,the PCR was used to verify whether it could bind to the SNAI1 site.The q RT-PCR method was used to detect the expression level of SNAI1 in Bx PC-3 and PANC-1 cells after transfection with NC,miR-30e-5p mimics and miR-30e-5p inhibitor.Results:(1)Through miRNA target gene prediction website,which was found that miR-30e-5p had a binding site with SNAI1.(2)The dual luciferase report found that the relative luciferase reading of the WT+miR-30e-5p mimics group was significantly lower than that of the WT+NC group(P<0.01);there were no difference between the MUT+NC group and the MUT+miR-30e-5p mimics group.(3)Compared with the NC group,the q RT-PCR found that the expression level of SNAI1 gene in Bx PC-3 and PANC-1 cells decreased in the miR-30e-5p mimics group,with significant differences(P<0.001);conversely,the expression level of SNAI1 gene increased in the miR-30e-5p inhibitor group(P<0.05).Conclusion: miR-30e-5p may participate in the pathogenesis of pancreatic cancer by affecting the translation of target gene SNAI1 and thereby reducing protein expression.PART ? Effect of miR-30e-5p expression on the transcriptome of pancreatic cancer cellsObjective: To explore the genes that miR-30e-5p may regulate in pancreatic cancer cells,and to elucidate its possible mechanism in the development and progression of pancreatic cancer.Methods: PNAC-1 cells were transfected with NC,miR-30e-5p mimics and miR-30e-5p inhibitor by liposome transfection method,which were divided into NC,mimics and inhibitor three groups.The gene expression profiles of three groups of cells were detected by transcriptome sequencing technology,and the differentially expressed genes(DEGs)between cells in mimics group VS NC group and inhibitor group VS NC group were compared by bioinformatics method.Gene enrichment analysis of DEGs in mimics group and inhibitor group by gene ontology(GO)and pathway enrichment analysis of Kyoto Encyclopedia of Genes and Genomes(KEGG);the prognosis of DEGs in the mimics group and inhibitor group was analyzed;the mimics group and inhibitor group were enriched with the NC group as the control and the results were analyzed by gene set enrichment analysis(GSEA).Results:(1)After transfection of miR-30e-5p,253 DEGs were selected in the mimics group,and 63 DEGs were selected in the inhibitor group.The DEGs in the mimics group were mainly enriched in the expression of bidirectional regulatory genes and epigenetic,cellular proteins metabolic processes,etc.;the inhibitor group was mainly enriched in the biological processes of nucleic acid regulation,transcription regulation using DNA as a template,and DNA template transcription.(2)Prognostic analysis showed that DEGs in the mimics group and inhibitor group were closely related to the prognosis of pancreatic cancer patients.(3)GSEA analysis showed that GO in the mimics group was mainly enriched in regulating inflammatory response,regulating immune response and bidirectionally controlling the production of vascular endothelial growth factor.In the mimics group,KEGG could be significantly enriched in ?-catenin,MMP-14,Interleukin-7,NCAM1,and COMP signaling pathways.Conclusion: miR-30e-5p is very likely to affect the occurrence and development of pancreatic cancer through EMT.PART ? The molecular mechanism of miR-30e-5p targeting SNAI1 to regulate the tumor suppressive effect of EMT on pancreatic cancerObjective: To explore the molecular mechanism of miR-30e-5p targeting SNAI1 to regulate the tumor suppressive effect of EMT on pancreatic cancer.Methods: Lentivirus infection method was used to overexpression SNAI1(OE-SNAI1)lentivirus in human pancreatic cancer cell lines Bx PC-3 and PANC-1,and then transfected with NC,miR-30e-5p mimics and miR-30e-5p inhibitor,and cells were divided into OE-SNAI1+NC group,OE-SNAI1+miR-30e-5p mimics group and OE-SNAI1+ miR-30e-5p inhibitor group.The cell viability of each group of Bx PC-3 and PANC-1 cells was detected by MTT method;the colony formation ability of each group of Bx PC-3and PANC-1 cells was detected by cell colony formation;the cell invasion test(Transwell method)was used to detect the invasion ability of each group of Bx PC-3 and PANC-1 cells;the cell migration experiment was used to detect cell migration ability of each group in Bx PC-3 and PANC-1;flow cytometry was used to detect the apoptosis rate of each group of Bx PC-3 and PANC-1 cells;the cell cycle of each group of Bx PC-3 and PANC-1 cells was detected by flow cytometry;western blot was used to detect the expression of SNAI1 and E-cadherin,N-cadherin,MMP-9 of EMT-related proteins in each group of Bx PC-3 and PANC-1 cells.Results:(1)Compared with the OE-SNAI1+NC group,MTT experiment found that the OE-SNAI1+miR-30e-5p mimics group could inhibit the viability of Bx PC-3 and PANC-1 cells,and the differences were significant(P<0.05);while OE-SNAI1+miR-30e-5p inhibitor group increased cell viability,the differences were significant(P<0.05).(2)Cell clone formation experiments found that the OE-SNAI1+miR-30e-5p mimics group could inhibit the colony forming ability of Bx PC-3 and PANC-1 cells compared with the OE-SNAI1+NC group,and the differences were statistically significant(P<0.05),while the OE-SNAI1+miR-30e-5p inhibitor group could promote the colony forming ability,and the differences were statistically significant(P<0.05).(3)Compared with the OE-SNAI1+NC group,the cell invasion experiment showed that the OE-SNAI1+miR-30e-5p mimics group could inhibit the invasion ability of Bx PC-3 and PANC-1,and the differences were statistically significant(P<0.05);the OE-SNAI1+miR-30e-5p inhibitor group could promote the invasion ability,and the difference were statistically significant(P<0.05).(4)The cell migration experiment found that the OE-SNAI1+miR-30e-5p mimics group could inhibit the migration ability of Bx PC-3 and PANC-1 cells compared with the OE-SNAI1+NC group,and the differences were statistically significant(P<0.05);while the OE-SNAI1+miR-30e-5p inhibitor group could promote the migration ability,and the difference were statistically significant(P<0.05).(5)Compared with the OE-SNAI1+NC group,the apoptosis experiment showed that the OE-SNAI1+miR-30e-5p mimics group could promote the apoptosis of Bx PC-3and PANC-1 cells,the differences were statistically significant(P<0.05),the OE-SNAI1+miR-30e-5p inhibitor group had no effect on apoptosis.(6)Compared with the OE-SNAI1+NC group,cell cycle experiments found that the OE-SNAI1+miR-30e-5p mimics group had a lower proportion of PNAC-1 cells in the G1 phase(P<0.05),and the S phase ratio elevated(P<0.05),the remaining groups had no significant effect on the G1,S and G2 phases of the cell cycle.(7)Compared with NC group,western blot experiments found that the expression of E-cadherin protein was up-regulated in miR-30e-5p mimics group in Bx PC-3and PANC-1 cells(P<0.001);which was down-regulated in the miR-30e-5p inhibitor group in Bx PC-3 and PANC-1(P<0.05),and the differences were statistically significant.The expression of N-cadherin protein was down-regulated in the miR-30e-5p mimics group in Bx PC-3 cell(P<0.001);while was up-regulated in miR-30e-5p inhibitor group in Bx PC-3 and PANC-1cells(P<0.01),and the differences were statistically significant.The expression of SNAI1 protein was decreased in the miR-30e-5p mimics group in Bx PC-3and PANC-1 cells(P<0.05);while was up-regulated in miR-30e-5p inhibitor group of Bx PC-3 and PANC-1(P<0.01),and the differences were statistically significant.MMP-9 protein was down-regulated in miR-30e-5p mimics group of Bx PC-3 and PANC-1 cells(P<0.05);while was up-regulated in miR-30e-5p inhibitor group of PANC-1 cell(P <0.01),and the differences were statistically significant.Conclusion: miR-30e-5p may regulate tumor biological behaviors such as proliferation,migration,invasion and apoptosis of pancreatic cancer cells through the miR-30e-5p-SNAI1-EMT control axis.