Study On The Correlation Of MicroRNA Let-7f Involvement In The Invasion And Migration Of Pancreatic Cancer

ObjectiveTo investigate the influence of microRNA Let-7f on the growth,apoptosis,invasion and migration of pancreatic cancer cells,and try to disclose the role of Let-7f in the occurrence and development of pancreatic cancer and its possible mechanism.To provide a new target and experimental data for explore the early clinical diagnosis of pancreatic cancer and the development of anticancer drugs.Methods1、Real-time quantitative reverse transcription polymerase chain reaction(RT-PCR)was used to detect the expression difference of Let-7 mi RNAs in normal pancreatic cell line HPDE6C7 and pancreatic cancer cell line PANC-1 and Bx-PC3.2、Let-7f mimic and inhibitor were transfected into pancreatic cancer PANC-1 and Bx-PC3 cells respectively by functional acquisition and functional deletion test.The expression of Let-7f in cells after transfection was detected by RT-qPCR.CCK-8 assay was used to explore the effect of changing Let-7f expression in cells on cell proliferation.The influence of Let-7f expression on apoptosis was observed by Annexin V-FITC/PI double staining flow cytometry.Transwell was performed to analyze the ability of cell invasion and migration by regulating Let-7f expression.The expression of invasion related genes matrix metalloproteinases 11(MMP-11),ribonucleotide reductase M2(RRM2)and enhancer of Zeste homolog 2(EZH2)were detected by RT-qPCR.Western Blot was used to detect the expression of MMP-11 at protein level.3、 Construction of LV-Let-7f-PANC-1,which is a PANC-1 cell that stable overexpression of Let-7f.Then,LV-Let-7f-PANC-1 and control LV-NC-PANC-1 cells were injected into BALB/c nude mice,respectively.The size,quality and expression of MMP-11 in the tumor tissues of the two groups were compared.4、The plasma of 60 patients with pancreatic cancer was collected,and 60 healthy subjects were collected as control.The difference of Let-7f expression in plasma between the two groups was compared by RT-qPCR.Enzyme linked immunosorbent assay(ELISA)was used to detect the expression level of MMP-11 in two groups.The relationship between the expression level of Let-7f and clinicopathological factors in patients with pancreatic cancer was statistically analyzed.Meanwhile,the correlation between Let-7f and MMP-11 was statistically analyzed.Kaplan-Meier was used to analyze the relationship between the expression level of Let-7f and the prognosis of patients with pancreatic cancer.Results1、The results of RT-qPCR showed that only the expression of Let-7f in PANC-1 and Bx-PC3 cells in Let-7 mi RNAs was statistically lower than that of HPDE6C7cells(P<0.05).2、After transfection of Let-7f mimic,the expression level of Let-7f in PANC-1 and Bx-PC3 cells increased significantly(P<0.05).Oppositely,the expression level of Let-7f was significantly down regulated after transfection of Let-7f inhibitor(P<0.05).CCK-8assay showed that there was no significant difference in cell proliferation between Let-7f mimetic transfection group compared with control group and Let-7f inhibitor transfection group compared with control group(P>0.05).The results of flow cytometry showed that although the proportion of apoptotic cells in Let-7f mimic group was slightly larger than that in the control group,the number of apoptotic cells in Let-7f inhibitor group was slightly lower than that in the control group,but the difference was not statistically significant(P>0.05).Transwell assay showed that the number of cells passing through Matrigel matrix glue in the Let-7f mimetic group was significantly less than that in the control group(P<0.05),while there was no significant difference between the Let-7f inhibitor group and the control group(P>0.05).The migration test results were similar.Up regulation of Let-7f expression in pancreatic cancer cells could inhibit cell migration,but down regulate Let-7f expression and cell migration ability did not change significantly.The expression of MMP-11 in Let-7f mimetic group was significantly lower than that in the control group by RT-qPCR and Western Blot(P<0.05),while in the Let-7f inhibitor group was significantly higher than that in the control group(P<0.05).There was no significant change in expression of RRM2 and EZH2(P>0.05).3、In vitro,the mass of tumor in group LV-Let-7f-PANC-1 was significantly lowerthan that in group LV-NC-PANC-1(P<0.05),and the expression level of MMP-11 in tumor tissue was significantly lower than that in group LV-NC-PANC-1(P<0.05).4、The expression of Let-7f in the plasma of the patients with pancreatic cancer was significantly lower than that in the healthy control group(P<0.05).Let-7f low expression in the plasma was significantly associated with TNM stage(P=0.002),lymph node metastasis(P=0.013),and distant metastasis(P=0.007).On other hand,no significant difference was seen regarding patients’ gender(P=0.755),age(P=0.066),tumor location(P=0.435),tumor size(P=0.348)and differentiation degree(P=0.267).Pearson correlation analysis showed that the expression of Let-7f in plasma was negatively correlated with the expression of MMP-11(r=-0.678,P<0.001).The Kaplan-Meier results indicated that the median survival time of the patients with low expression of Let-7f was shorter than that of the high expression group of Let-7f,and prognosis of patients with low expression of Let-7f was worse(c2=18.618,P=0.000).Conclusions1、 Let-7f may be involved in the development of pancreatic cancer by regulating MMP-11 to inhibit the invasion and migration of pancreatic cancer cells.2、Let-7f is low expression in the plasma of the patients with pancreatic cancer,which can be used as an indicator of the clinical diagnosis and prognosis of pancreatic cancer.