Studies On The Mechanisms Of HBV Intra-uterine Transmission By Proteomic And MiRNA Analysis

Hepatitis B virus (HBV), widely spread in China, which causes acute or chronic HBV infection and leads to hepatoma cirrhosis and eventually hepatocellular carcinoma has been a main threaten to public health. HBV could spread by blood, mother-infant transmission or sexual contact. Mother-infant transmission of HBV occurred in the perinatal period, so the blockade of the mother-infant transmission of HBV is the key to control HBV spread. The exploration of the mechanisms of HBV intra-uterine transplacental transmission is the emphasis and difficulty in the prevention of mother-infant transmission of HBV.HBV intra-uterine transmission could be caused by placenta leakage, placenta infection or peripheral blood mononuclear cell infection. However, the process of virus infects host cells is complicated, many molecules may play a role in this process. Till now, the underlying mechanisms of HBV intra-uterine transplacental transmission remain unclear and there are no effective measures to block the transmission. Therefore, it is requisite to explore the mechanisms of HBV intra-uterine transplacental transmission, which will conduce to the blockade of the HBV intra-uterine transmission.Proteome methods, which may be used to detecet the protein expression patterns in the diseased samples compared to the normal samples, are effectively means to clarify the mechanisms of disease and find novel therapeutic targets or biomarkers to serve as diagnostic indicators of disease. Besides that, the infection of virus may lead to the changes of miRNA(microRNA) expression in the host cells, so the interaction of HBV with host cells regulated by miRNA may play a role in HBV infection. This study is to detect protein expression patterns in the placentas derived from HBV-positive pregnant women by proteome methods compared to that of normal pregnant women, and then to explore the changes of miRNA expressions in placental trophoblasts infected with HBV on the basis of the establishment of cell model infected by HBV. Therefore, the mechanisms of HBV intra-uterine transmission may be clarified from new points of view on both proteome and miRNA. Methods1.The placentas derived from HBV-positive pregnant women were used as experimental objects and the placentas derived from HBV-negative pregnant women were used as control. By MALDI-TOF and ESI–MS/MS, the 2-DE map of normal placentas was established. The comparative proteome methods were used to detect protein expression patterns in the HBV-positive and negative placentas, and then the functions and roles of the proteins different in expression were analyzed.2.Placental trophoblast cells JEG-3 were treated with HBV positive or negative serum, HBsAg in the cell supernatant or in cells was determined by ELISA or immunohistochemistry SP method respectively, and HBV DNA in JEG-3 was detected by PCR. Besides that, apoptosis and necrosis of JEG-3 was determined by AnnexinⅤ/PI staining and then FACS detection. Transmission Electro Microscope was used to observe cell ultra structure and find virus granule.3.JEG-3 were treated with HBV positive or normal serum, miRNA chip was used to detect the differences in miRNA expression between the HBV positive serum treated cells and normal serum treated cells. Then the target genes of the miRNA different in expression were predicted and then their biological functions were analyzed.Results1.The 2-DE map of normal mature placenta was established. 405 protein spots were detected in the pH4-10 compositive map, 104 proteins correspond to 157 protein spots were identified, 24 proteins of which were found to correspond to multi-protein spots in the map, including structural proteins and scaffolding protein, et al. The functions of some identified proteins were unknown. 56 proteins displayed significant differences in expression in the placenta derived from HBV-positive placenta compared to the control placenta, and then 24 proteins were identified, 14 of these proteins were up-regulated while 10 proteins displayed reduced expression. These proteins different in expression include enzymes, regulated proteins, transport proteins, structural proteins, scaffolding protein, protective proteins and some proteins with unknown functions.2.HBsAg in cell supernatant of placental trophoblast cells JEG-3 treated with HBV-positive serum was positive. HBsAg and HBV DNA in JEG-3 cells were also positive. Besides that, the necrosis rate of JEG-3 cells treated with HBV-positive serum significantly increased compared to control cells, but the apoptosis rate was not changed. The changes of ultra structure of JEG-3 cells treated with HBV-positive serum was as follows: many vacuoles appeared in cytoplasma and lysosome increased significantly, besides that, virus-like granules were found in cytoplasma.3.Compared to cells treated with normal serum, some miRNA in JEG-3 cells treated with HBV-positive serum were up-regulated, including has-miR-197, has-miR-32, has-miR-125a-3p, has-miR-20a, has-miR-210 and has-miR-574-3p, there was no significantly down-regulated miRNA was detected.Conclusions1.The 2-DE map of normal mature placenta was established and 104 proteins were identified. Some proteins may have post-translational modifications, suggesting the important role of these proteins in the development of placenta. 24 proteins different in expression in placenta derived from HBV–positive pregnant women compare to control placenta were identified. HSPs, actin and Annecxin may play a role in the HBV intra-uterine transmission2.Placental trophoblast cells treated with HBV-positive serum could be infected by HBV, so the HBV infection cell model was established with the method which was most consistent to the conditions of HBV infection in vivo. HBV-positive serum may cause significant increase of lysosome and cell necrosis.3.miRNA expressed in JEG-3 cells treated with HBV-positive serum changed compared to control cells. Some up-regulated miRNA were detected, including has-miR-197, has-miR-32, has-miR-125a-3p, has-miR-20a, has-miR-210 and has-miR-574-3p, suggesting miRNA may play a role in the regulation of HBV infection.