Validation Of The Inhibition Effect Of PreS1 Molecular Conjugate Carring SiHBX On The Expression Of HBX Gene In HepG2-HBX Cell Lines

Hepatitis B Virus(HBV) infection is a global pubulic healthy problem, chronic infection has a variable course after several decades, resulting in hepatic cirrhosis,liver cancer even to hepatic failure. HBV infection ratio in HCC patient is up to 50%-80%. Hepatitis B virus X protein has been suggested to play an important role in hepaocarinogenesis. Now licensed therapies that have been employed to eliminate HBV are some interferons and nucleic acid analogues, but there are some problems such as side effects, virus mutation escape. Therefore, the development of novel approaches to inhibit HBV replication is requiered.As a new therapeutic strategy, RNA interference may be commonly used in many fields to treat human tumors,virus infections or metabolic diseases . However, the key hurdles of RNA interference are the low stability of siRNA and the uneffective delivery system. At present, the siRNA delivery systems mainly inculde nanocarrier and molecular conjugate, and the molecuar conjugate used the CPPs and CTLs strategy.The surface proteins of HBV include large proteins(L),middle proteins(M) and small proteins(S). They all have the domains of S. Compared to S protein, M protein has another pres2 domain which consists of 55 amimo acids. Compared to M protein, the most outside of L protein has another pres1 domain which consists of 108 or 118 amimo acids. At present, it is not completely clear how HBV invades into liver cells, and there is no definite conclusion about the host specific receptors.Recent studies indicate that preS1 might be the ligand which binds to specific membrane receptors, thereby leading to HBV entering into liver cell. The principal binding domain of preS1 is N teminal, and the 21th to the 47th amino acids are suggested to play a critical role.Objective: This study aims to construct a HepG2-HBX cell line that stably expressing the hepatitis B virus protein X (HBX) and to explore the specific silencing effects of siRNA which is carried by preS1 molecular conjugate on the HBX gene, and finally to establish an experimental basis for subsequent therapeutic and preventive research in HBV infection. There is no related study reports about these described above.Methods: In this study, the role of HBV X gene was investigated by RNAi. The pCDNA3.1(+)/HBX-Flag plasmid containing the HBX sequence was transfected into HepG2 cell lines with lipofectamine 2000, and the HepG2-HBX cell line stably expressing the HBX gene was screened out with G418; RT-PCR and immunohistochemistry were employed to validate the expression of the HBX gene; the siHBX fragment targeting the HBX gene was chemically synthesized and transfected in to the HepG2-HBX cell line, and RT-PCR, real time quantitative RT-PCR and Western blotting were respectively performed to evaluate the the expression of HBX at both mRNA and protein levels in order to validate the specific silencing effects of siHBX on the HBX gene. Moreover, the pres1 molecule conjugate was chemically synthesized, and we tested the ability of the preS1 to bind to HepG2-HBX and the siRNA. The siRNA was then transfected into the HepG2-HBX cell line by preS1. Quantitative RT-PCR and Western blotting were respectively employed to validate the inhibition effects of siHBX on the expression of the HBX gene.Results: G418 tests showed that the pCDNA3.1(+)/HBX-Flag plasmid containing the HBX sequence was transfected into the HepG2, RT-PCR and immunohistochemistry validated that HBX19 highly expressed HBX gene. Compared to the empty control, the inhibition rate of the nagative group was 2.11%±0.04% and 1.02%±0.05% after 36h and 72h, the lipofectamine were 85.37±2.6% and 58.05%±4.00%, respectively. Moreover, HBX protein and the cell cycle progression were repressed analyzed by Western Blot and Flow cytomety, respectively.Further EMSA study showed that preS1 could effectively bind to siRNA (10mol:1mol), and siRNA was then transfected into the HepG2-HBX cell line by preS1 to inhibit the expression of the HBX gene. Compared to empty control, the inhibition rate of the nagative group was 11.97±6.3%, while the preS1 molecule conjugate and the lipofectamine were 85.24±2.6% and 77.80±6.1%, respectively.Conclusions: These results indicated that HepG2-HBX cell line stably expressing the HBX gene was successfully constructed and suggested the effctiveness and specificty of the siHBX to the HBX gene. Moreover, preS1 molecule conjugate could be used as a novel siRNA carrier to inhibit the expression of HBX gene in liver cancer cells, laying an experimental basis for the subsequent therapeutic research in HBV infection-related liver diseases.