Influence Of HBV Up-regulation Of MiR-181a/362/382/19a On PTEN/HSPA5/HSPA2 Expression And Biological Behaviors Of Hepatoma Cells

【Background】 Epidemiological studies have shown that chronic HBV infection is an important cause of hepatocellular carcinoma.Hepatitis is endemic in China.Exploring the mechanisms how HBV infection influences the occurrence and development of liver cancer is of great significance for the prevention and treatment of liver cancer related to HBV infection.Recently it has been reported that abnormal expression of miRNAs is closely related to tumors.Previous research of our group found that HBV could cause miR-181a/362/382/19 a to be up-regulated in hepatoma cells and tissues.HSPA5 and HSPA2 may be their potential target genes.HSPA5 is an important molecular chaperone located in the endoplasmic reticulum,and its expression is increased under stress to maintain the stability of the endoplasmic reticulum and protect the cells from apoptosis.HSPA2 also belongs to the HSP70 family,with 84% homology with HSP70 amino acids,but currently its related study is less.The expression of HSPA5/HSPA2 in hepatocellular carcinoma tissues was significantly higher than that in the adjacent tissues,but it was lower in the Hep G2.215 cell line that stably expressed HBV in vitro than in the Hep G2 cell line.Therefore,the mechanisms of HBV up-regulation of miR-181a/362/382/19 a affecting the occurrence and development of hepatocellular carcinoma and the reasons and significance of the differential expression of HSPA5/HSPA2 between in vivo liver cancer tissues and in vitro cell models need to be further explored.We speculate that HBV infection may influence the expression of multiple miRNAs that regulate the expression of multiple target genes so as to influence multiple signaling pathways and thereby affecting the occurrence and development of liver cancer together.The common target gene PTEN ofmiR-181a/362/382/19 a was further screened for study.Based on previous studies,this study examined the effect of HBV infection on the expression of HSPA5/HSPA2/PTEN in hepatocellular carcinoma tissues and paracancerous tissues,determined whether miR-181a/362/382/19 a binds to HSPA5/HSPA2/PTEN m RNA 3’UTR,explored the differential role of miR-181a/362/382/19 atargeting between HSPA5/HSPA2 and PTEN and whether miR-181a/362/382/19 a influences the biological behaviors of hepatoma cells and the activation of PI3K/AKT signaling pathway through the regulation of PTEN.This study provides new experimental data for the mechanisms how HBV infection influences the occurrence and development of hepatocellular carcinoma,and also provides new ideas for the clinical treatment,diagnosis and prognosis of liver cancer associated with HBV infection.【Methods】 1.Detection of HSPA5,HSPA2 and PTEN expression in hepatocellular carcinoma tissues and paracancerous tissues with and without HBV infection Immunohistochemistry was used to detect the expression of HSPA5,HSPA2 and PTEN in hepatocellular carcinoma tissues and noncancerous tissues with HBV infection and HBV infection.2.Luciferase reporter gene analysis of miRNA targeting its target genes The 3’UTR sequence of the target gene that binds to the miRNA is predicted by the Target Scan website for preparing the region fragment and the corresponding mutation fragment.The wild region fragment and the corresponding mutation fragment were respectively inserted into the luciferase reporter gene vector,and the sequence was verified by sequencing to obtain wild type(WT)and mutant(MUT)luciferase reporter gene plasmids.These plasmids were co-transfected with mimics of miRNA respectively into Hep G2 cells.After 24 hours of culturing,the intensities of firefly luciferase and Renilla luciferase were measured,relative fluorescence intensities were calculated,and the differences were analyzed.3.Differential role of miR-181a/362 targeting between HSPA5 and PTEN Mi R-181a/362 mimics were respectively transfected into Hep G2 cells andmiR-181a/362 inhibitors were respectively transfected into Hep G2.215 cells.After 48 hours of culturing,the m RNA expressions of HSPA5 and PTEN in cells were detected simultaneously by real-time PCR and the protein expressions of HSPA5 and PTEN in cells were detected simultaneously by western blot.4.Differential role of miR-382/19 a targeting between HSPA2 and PTEN Mi R-382/19 a mimics were respectively transfected into Hep G2 cells and miR-382/19 a inhibitors were respectively transfected into Hep G2.215 cells.After 48 hours of culturing,the m RNA expressions of HSPA2 and PTEN in cells were detected simultaneously by real-time PCR and the protein expressions of HSPA2 and PTEN in cells were detected simultaneously by western blot.5.The effect of miR-181a/362/382/19 a on the proliferation of hepatocellular carcinoma cells via regulating PTEN Mi R-181a/362/382/19 a mimic or NC-mimic and PTEN overexpression plasmid or its empty vector were co-transfected into Hep G2 cells,while miR-181a/362/382/19 a inhibitor or NC-inhibitor and PTEN RNA interference plasmid or its empty vector were co-transfected into Hep G2.215 cells.The proliferation of each group was detected by CCK-8 kit after 48 hours of culturing.6.The effect of miR-181a/362/382/19 a on the migration of hepatocellular carcinoma cells via regulating PTEN Mi R-181a/362/382/19 a mimic or NC-mimic and PTEN overexpression plasmid or its empty vector were co-transfected into Hep G2 cells,while miR-181a/362/382/19 a inhibitor or NC-inhibitor and PTEN RNA interference plasmid or its empty vector were co-transfected into Hep G2.215 cells.Then the cells were subjected to the scratch test(the degree of scratch healing of each group was observed after 24 hours)and a transwell migration experiment(the number of migration cells of each group was counted after 24 hours).7.The effect of miR-181a/362/382/19 a on the invasion of hepatocellular carcinoma cells via regulating PTEN Mi R-181a/362/382/19 a mimic or NC-mimic and PTEN overexpression plasmid orits empty vector were co-transfected into Hep G2 cells,while miR-181a/362/382/19 a inhibitor or NC-inhibitor and PTEN RNA interference plasmid or its empty vector were co-transfected into Hep G2.215 cells.Then the cells were subjected to the transwell-Matrigel experiment,and the number of invasion cells of each group was counted after 48 hours.8.The effect of miR-181a/362/382/19 a on the PI3K/AKT signaling pathway of hepatocellular carcinoma cells via regulating PTEN Mi R-181a/362/382/19 a mimic or NC-mimic and PTEN overexpression plasmid or its empty vector were co-transfected into Hep G2 cells,while miR-181a/362/382/19 a inhibitor or NC-inhibitor and PTEN RNA interference plasmid or its empty vector were co-transfected into Hep G2.215 cells.Western blot was used to examine the protein expression of PTEN,phosphorylated AKT(p-AKT)and total AKT in the cells.【Results】 1.Expression of HSPA5 and HSPA2 in the hepatocellular carcinoma tissues Immunohistochemistry results showed the expression of HSPA5 and HSPA2 in HBV-infected paracarcinoma tissues was lower than that in HBV-uninfected paracarcinoma tissues,consistent with the previous result that the expression of HSPA5 and HSPA2 was lower in the Hep G2.215 cell line that stably expressed HBV than in the Hep G2 cell line.2.HSPA5 is the target gene of miR-181a/362 Luciferase reporter gene analysis showed that the relative fluorescence intensities of miR-181a/362 mimic and HSPA5 luciferase reporter wild-type(WT)plasmid co-transfecting into Hep G2 cells were significantly lower than its co-transfection with luciferase reporter gene mutant(MUT)plasmid,indicating that miR-181a-5p/362-3p can directly target the 3’ UTR of HSPA5 and that HSPA5 is the target gene of miR-181a/362.3.HSPA2 is the target gene of miR-382/19 a Luciferase reporter gene analysis showed that the relative fluorescence intensitiesof miR-382/19 a mimic and HSPA2 luciferase reporter wild-type(WT)plasmid co-transfecting into Hep G2 cells were significantly lower than its co-transfection with luciferase reporter gene mutant(MUT)plasmid,indicating that miR-382-5p/19a-3p can directly target the 3’ UTR of HSPA2 and that HSPA2 is the target gene of miR-382/19 a.4.PTEN is a common target gene of miR-181a/362/382/19 a Luciferase reporter gene analysis showed that the relative fluorescence intensities of miR-181a/362/382/19 a mimic and PTEN luciferase reporter wild-type(WT)plasmid co-transfecting into Hep G2 cells were significantly lower than its co-transfection with luciferase reporter gene mutant(MUT)plasmid,indicating that miR-181a/362-3p/382-5p/19 a can directly target the 3’UTR of PTEN m RNA and that PTEN is the target gene of miR-for 181a/362/382/19 a.5.Expression of PTEN in hepatocellular carcinoma tissues Immunohistochemistry results showed that the expression of PTEN in HBV-infected paracarcinoma tissues was lower than that in HBV-uninfected paracarcinoma tissues,suggesting that low expression of PTEN may play an important role in hepatocellular carcinogenesis.6.Differential role of miR-181a/362 targeting between HSPA5 and PTEN The results of real-time PCR and western blot showed that the m RNA and protein levels of HSPA5 and PTEN were decreased after the expression of miR-181a/362 was up-regulated respectively in Hep G2 cells,and the degree of PTEN decrease was greater than that of HSPA5,the m RNA and protein levels of HSPA5 and/or PTEN had an increase trend after the expression of miR-181a/362 was down-regulated respectively in Hep G2.215 cells,and there was no significant difference between the increase degree of PTEN and HSPA5.The results indicated that miR-181a/362 could inhibit the expression of HSPA5 and PTEN respectively,and the degree of inhibition of PTEN by miR-181a/362 up-regulation was greater than that of HSPA5.7.Differential role of miR-382/19 a targeting between HSPA2 and PTENThe results of real-time PCR and western blot showed that the m RNA and protein levels of HSPA2 and PTEN were decreased after the expression of miR-382/19 a was up-regulated respectively in Hep G2 cells,and the degree of PTEN decrease was greater than that of HSPA2,the m RNA and protein levels of HSPA2 and PTEN had an increase trend after the expression of miR-382/19 a was down-regulated respectively in Hep G2.215 cells,and there was no significant difference between the increase degree of PTEN and HSPA2.The results indicated that miR-382/19 a could inhibit the expression of HSPA2 and PTEN respectively,and the degree of inhibition of PTEN by miR-382/19 a up-regulation was greater than that of HSPA2.8.Mi R-181a/362/382/19 a regulates the proliferation of hepatocellular carcinoma cells via targeting PTEN The results of CCK-8 kit showed that up-regulation of miR-181a/362/382/19 a or interference with PTEN expression in Hep G2 cells could promote the proliferation of hepatoma cells,and overexpression of PTEN could antagonize the effect of miR-181a/362/382/19 a up-regulation on cell proliferation,down-regulation of miR-181a/362/382/19 a or overexpression of PTEN in Hep G2.215 cells could inhibit the proliferation of hepatoma cells,and interference with PTEN expression could antagonize the effect of miR-181a/362/382/19 a down-regulation on cell proliferation.The results suggest that miR-181a/362/382/19 a can regulate the proliferation of hepatoma cells via regulating the expression of PTEN.9.Mi R-181a/362/382/19 a regulates the migration of hepatocellular carcinoma cells via targeting PTEN The results of scratch test showed that up-regulation of miR-181a/362/382/19 a or interference with PTEN expression in Hep G2 cells could promote the scratch healing of hepatoma cells,and overexpression of PTEN could antagonize the effect of miR-181a/362/382/19 a up-regulation on cell scratch healing,down-regulation of miR-181a/362/382/19 a or overexpression of PTEN in Hep G2.215 cells could inhibit the scratch healing of hepatoma cells,and interference with PTEN expression could antagonize the effect of miR-181a/362/382/19 a down-regulation on cell scratchhealing.The results of transwell migration experiment showed that up-regulation of miR-181a/362/382/19 a or interference with PTEN expression in Hep G2 cells could increase the number of migration cells,and overexpression of PTEN could antagonize the effect of miR-181a/362/382/19 a up-regulation on cell migration,down-regulation of miR-181a/362/382/19 a or overexpression of PTEN in Hep G2.215 cells could decrease the number of migration cells,and interference with PTEN expression could antagonize the effect of miR-181a/362/382/19 a down-regulation on cell migration.Both the scratch test and transwell migration experiment suggest that miR-181a/362/382/19 a can influence the migration of hepatoma cells via regulating the expression of PTEN.10.Mi R-181a/362/382/19 a regulates the invasion of hepatocellular carcinoma via targeting PTEN The results of transwell-Matrigel experiment showed that up-regulation of miR-181a/362/382/19 a or interference with PTEN expression in Hep G2 cells could increase the number of invasion cells,and overexpression of PTEN could antagonize the effect of miR-181a/362/382/19 a up-regulation on cell invasion,down-regulation of miR-181a/362/382/19 a or overexpression of PTEN in Hep G2.215 cells could decrease the number of invasion cells,and interference with PTEN expression could antagonize the effect of miR-181a/362/382/19 a down-regulation on cell invasion.The results suggest that miR-181a/362/382/19 a can influence the invasion of hepatoma cells via regulating the expression of PTEN.11.Mi R-181a/362/382/19 a regulates PI3K/AKT signaling pathway by targeting PTEN The results of western blot showed the up-regulation of miR-181a/362/382/19 a or interference with PTEN in Hep G2 cells significantly could increase the phosphorylation of AKT,and overexpression of PTEN significantly could antagonize the effect of miR-181a/362/382/19 a up-regulation on the AKT phosphorylation,down-regulation of miR-181a/362/382/19 a or overexpression of PTEN in Hep G2.215 cells significantly could inhibit the phosphorylation of AKT,and interference withPTEN expression significantly could antagonize the effect of miR-181a/362/382/19 a down-regulation on the phosphorylation of AKT.The results suggest that miR-181a/362/382/19 a can promote the activation of PI3K/AKT signaling pathway in hepatoma cells via regulating PTEN.【Conclusions】 The results suggest that:(1)Relative to the hepatoma tissues,HSPA5 expression after HBV infection was lower in the paracancerous tissues where the blood supply was relatively normal,making it more sensitive to chemotherapeutic drugs and more prone to apoptosis and necrosis causing liver disturbance;(2)HBV up-regulated miR-181a/362 to inhibit the expression of their target gene HSPA5,thus providing an favoring environment for HBV stable replication.HBV up-regulated miR-382/19 a to inhibit the expression of their target gene HSPA2,making the expression of HSPA2 in the Hep G2.215 cell line lower.The significance of the HSPA2 decrease in Hep G2.215 cell line needs to be further studied;(3)miR-181a/362/382/19 a binds to PTEN 3’UTR and PTEN is a common target gene for miR-181a/362/382/19 a.HBV infection can up-regulate miR-181a/362/382/19 a expression and exert the comprehensive effects to significantly inhibit the expression of PTEN;(4)The role of miR-181a/362 targeting between HSPA5 and PTEN was differential,so did the role of miR-382/19 a targeting between HSPA2 and PTEN,and miR-181a/362/382/19 a had more significant inhibitory effect on PTEN;(5)Up-regulation of miR-181a/362/382/19 a or interference with PTEN could significantly increase the proliferation,migration and invasion of hepatoma cells,and overexpression of PTEN could antagonize the effect of miR-181a/362/382/19 a up-regulation on the proliferation,migration and invasion of hepatoma cells,suggesting that miR-181a/362/382/19 a influence the proliferation,migration and invasion of hepatoma cells via regulating the expression of PTEN;(6)miR-181a/362/382/19 a could promote the PI3K/AKT signaling pathway in hepatoma cells via regulating the expression of PTEN.