The Effect Of MiR-146a On Chronic HBV Infection In HepG2.2.15 Cells

ObjectiveTo explore the effect of miR-146a on immune response of chronic HBV infection and the hepatocellular carcinoma.Methods1.The pre-miR-146a was amplified by PCR.Then the production was digested by the restriction enzyme of BamH ? and Hind ?,and it was cloned into the pmR-mCherry plasmid.the recombinant vector(pmR-146a)was verified by colony PCR?double enzyme digestion and sequencing.2.The recombinant vector was transfected into HepG2.2.15 cells by lip2000 as experimental group(lip2000+pmR-146a),while the empty plasmid group(lip2000+pmR-mCherry plasmid),the blank group(lip2000)and the knock down group(short for KD group,lip2000 +microFFTM has-miR-146a inhibitor)were set as controls.The fluorescent protein expression was observed under the fluorescence microscopy at 24?48h after post-transfection.The expression of miR-146a was evaluated by qPCR at 24?48h after post-transfection.3.The ultra purified plasmids or microFFTM has-miR-146a inhibitor were transfected into HepG2.2.15 cells as above.The expression of HBeAg and HBsAg were investigated by chemiluminescent assay at 24h?48h after post-transfection.The expression of NF-?B protein were detected by Western Blotting and the changes of TGF-? was evaluated by ELISA at 48h after post-transfection.4.The expression of c-Myc mRNA were evaluated by qPCR at 24h?48h after post-transfection and the expression levels of c-Myc protein were detected by Western blot at 48h after post-transfection.The cell proliferation was respectively detected at 24?48?72h after post-transfection by CCK-8,Results1.The colony PCR?double enzyme digestion and sequencing verified that the gene was inserted into the pmR-mCherry vector.2.At 24?48h after post-transfection,intracellular strong fluorescence were seen,the transfection efficiency was at 50%-60%contrasting without fluorescence.The expression of miR-146a in the experimental group was increased and the KD group was declined compared with blank group(P<0.01).The difference between the blank group with the empty plasmid group was no statistically significant(P>0.05).3.The chemiluminescent assay showed that the expression of HBsAg and HBeAg in the experimental group were increased and the KD group were declined compared with blank group(P<0.05),but the difference between the blank group with the empty plasmid group was no statistically significant(P>0.05).The expression of NF-?B protein,by western blot,in the experimental group were declined and the KD group were increased compared with blank group(P<0.05),then the difference between the blank group with the empty plasmid group was no statistically significant(P>0.05).the level of the TGF-? in the experimental group were increased and the KD group were declined compared with blank group(P<0.01),but the difference between the blank group with the empty plasmid group was no statistically significant(P>0.05).4.The qPCR showed that the level of c-Myc mRNA in the experimental group were declined and the KD group were increased compared with blank group(P<0.05),then the difference between the blank group with the empty plasmid group was no statistically significant(P>0.05).At the same time,the expression of c-Myc protein In the experimental group were declined and the KD group were increased compared with blank group(P<0.05),then the difference between the blank group with the empty plasmid group was no statistically significant(P>0.05).The cells profication show that the experimental group grew slower and the KD group grew faster compared with blank group(P<0.01),then the difference between the blank group with the empty plasmid group was no statistically significant(P>0.05).ConclusionThe pmR-146a eukaryotic overexpression vector is successfully constructed,this recombinant vector can express miR-146a stably.miR-146a can promote the replication of HBV through down-regulating the expression of NF-?B and up-regulating the expression of TGF-?.miR-146a can down-regulate the expression of c-Myc expression and suppress the cells proliferation,which can be one of potential targets for treating hepatocellular carcinoma.