Preliminary Study On The Role Of Prostaglandin E2 Synthetase-1 And Its MiRNAs Signaling Pathway In Malignant Progress Of Hepatocellular Carcinoma

ObjectiveThe mPGES-1-RNAi lentivirus was constructed and infected with hepatocellular carcinoma cells.The proliferation invasion and migration of SMMC7721 and HepG₂after mPGES-1 gene interference were observed.The miRNAs chip method was used to detect the differential expression of miRNAs after HepG₂ mPGES-1 gene interference,and verify and analyze the correlation in order to find the role and possible molecular mechanism of mPGES-1 mediated miRNA signaling pathway in the malignant progress of hepatocellular carcinoma.Methods?1?Construct mPGES-1-RNAi lentivirus and infect hepatocellular carcinoma cells SMMC7721 and HepG₂ respectively.At the same time set up empty vector group?NC group?and uninfected group?Control group?.Western blot and PCR were used to detect the changes of mPGES-1 mRNA and protein expression in hepatocellular carcinoma cells SMMC7721 and HepG₂ before and after mPGES-1 gene interference.CCK-8,the wound health test and transwell invasion test were used to detect the changes of proliferation,migration and invasion ability of SMMC7721 and HepG₂ before and after mPGES-1 gene interference.?2?Enzyme linked immunosorbent assay?ELISA?was used to detect the content of prostaglandin E2?PGE₂?before and after mPGES-1 gene interference in hepatocellular carcinoma cell lines SMMC7721 and HepG₂.?3?The most significant miRNAs was screened by the miRNAs chip in hepatocellular carcinoma cells HepG₂ after mPGES-1 gene interference and miRNAs was verify by qPCR.Predict target genes of the most significant miR-146a-5p by TargetScan,PITA,and microRNAorg database.GO and KEGG Pathway analysis were performed on the target genes of miR-146a-5p.?4?The immunohistochemical showed that the expression of mPGES-1,cyclinD1and EGFR in liver and paracancerous liver tissues was detected and the correlation between them was analyzed.Results?1?The mPGES-1-RNAi lentivirus was successfully constructed and infected with the hepatocellular carcinoma cells SMMC7721 and HepG₂,and the infection rate of SMMC7721 is about 80%,and the infection rate of HepG₂ is about 90%.?2?Compared with the uninfected group?Control group?and the unloaded group?NC group?,the expression of mRNA and protein in the mPGES-1 gene interference group?LV-2 group?in the hepatocellular carcinoma cells SMMC7721 and HepG₂ was decreased significantly,and the difference was statistically significant?P<0.05?.?3?Compared with the uninfected group?Control group?and the unloaded group?NC group?,the PGE₂ content of the mPGES-1 gene interference group?LV-2 group?in the hepatocellular carcinoma cells SMMC7721 and HepG₂ was significantly decreased,and the difference was statistically significant?P<0.05?.?4?CCK8 and transwell invasion test results showed in the hepatocellular carcinoma cells SMMC7721 and HepG₂,the cell proliferation and invasion ability of mPGES-1 gene interference group?LV-2 group?were significantly decreased than the uninfected group?Control group?and the the unloaded group?NC group?,and the difference was statistically significant?P<0.05?.?5?In the wound health test showed that in the hepatocellular carcinoma cell SMMC7721,the cell migration capability of the mPGES-1 gene interference group?LV-2 group?were significantly decreased than the uninfected group?Control group?and the the unloaded group?NC group?,and the difference was statistically significant?P<0.05?;In the hepatocellular carcinoma cell HepG₂,the cell migration capability of the mPGES-1 gene silencing group?LV-2 group?were decreased partly,but the difference was not statistically significant?P>0.05?.?6?The miRNAs chip screening significant differential expression miRNAs and qPCR verification results:miR-146a-5p,miR-6889-5p,miR-3150b-3p,miR-4470 is up regulated,miR-20a-3p,miR-23a-5p,miR-629-5p,miR-7-1-3p,miR-6820-3p is down regulated,the up-regulation of miRNAs was the most obvious change of mR-146a-5p,the up regulation ratio was 4.12,the down-regulation of miRNAs was the most obvious change of miR-7-1-3p,and the down-regulation ratio was 3.70.However,the expression of miR-5191,miR-2276-3p and miR-892b was up-regulated,and the expression of miR-521 was down-regulated.The expression of these 4 miRNAs was not consistent with that of the microarray.?7?The target gene prediction of the most significant differential expression of miR-146a-5p and the results of GO analysis and KEGG analysis of target genes showed that there were 120 candidate target genes of miR-146a-5p,including cyclinD1 and EGFR,and the target gene of miR-146a-5p mainly regulated gene transcription,cell metastasis,small GTPase mediated signal transduction,dephosphorylation,T cell excitation and the target gene of miR-146a-5p mainly enriched such as pathways in cancer,Ras signaling pathways,Wnt signaling pathways,Hippo signaling pathways,ErbB signaling pathways,and so on.?8?The immunohistochemical staining showed that the high expression rate of mPGES-1 in liver cancer tissues was higher than that in the paracancerous liver tissues?77.78%vs.53.70%?,and the difference was statistically significant?P<0.05?;The high expression rate of cyclinD1 in the liver cancer tissues was higher than that in the paracancerous liver tissues?12.96%vs.1.85%?,and the difference was statistically significant?P<0.05?;EGFR in the liver tissues was statistically significant?P<0.05?.The high expression rate of EGFR in the paracancerous liver tissues was higher than that in liver cancer tissues?38.89%vs.72.22%?,and the difference was statistically significant?P<0.05?.The correlation of mPGES-1,cyclinD1 and EGFR were analyzed showed that mPGES-1 had a positive correlation with cyclinD1 and negative correlation with EGRF,while cyclinD1 was negatively correlated with EGFR,and the difference was statistically significant?P<0.05?.Conclusions?1?Successful construction of mPGES-1-RNAi lentivirus vector can effectively infect the hepatocellular carcinoma cells SMMC7721 and HepG₂,which can stably and significantly down regulate the expression of mPGES-1 gene.?2?Interfering mPGES-1 gene in the hepatocellular carcinoma cells can decrease the ability of proliferation,migration and invasion.?3?The expression profiles of miRNAs were changedin the HepG₂after interfering mPGES-1 gene,and mPGES-1 may affect the malignant progress of hepatocellular carcinoma by targeting cyclin D1 and EGFR by miR-146a-5p target.