The Relationship Between Augmenter Of Liver Regeneration, Cytochrome P450 And HEPG2 In Drug Resistance In Vitro

Background and Objective:Augmenter of liver regeneration (ALR), a heat-stable hepatotrophic growth factor,can non-specificly stimulate regeneration of liver cells[1] and plays an important role in the occurrence and development of hepatocellular carcinoma[2-8]. Cytochrome P450 is one of the most important enzymes in drug metabolism, and CYP3A4 is the most important isozyme[9]. CYP3A4 can be expressed abundantly in human liver and small intestine, and has a relationship with the metabolism of a large number of drugs and side effects[10].CYP3A4 participates in the metabolism and transformation process of more than 50% drugs used presently[11], and plays a central role in the metabolism of chemotherapeutics, so people pay more attention to the relationship between CYP3A4 and the drugs resistance of chemotherapeutics. Research shows that ALR can reduce basal and induced CYP3A4 protein and mRNA expression and decrease its activity in primary human hepatocytes[12]. Presently,however,it is still unclear that the relationship between the effect of ALR on the expression of CYP3A4 and the drug resistance of HepG2. The expression and activity of CYP3A4 in HepG2/CDDP and drug sensitivity to CDDP of HepG2/CDDP cells in each group were investigated in this study with small interfering RNA (siRNA) targeting ALR by blocking the expression of hALR, to clarify whether ALR participated in the occurrence and development of the drug resistance of HepG2/CDDP cells by inhibiting the expression of CYP3A4.Methods1. HepG2/CDDP was established in vitro by stepwise increasing dose of CDDP and successive administration, and the resistant index was detected by MTT assay [13].2. The expressing siRNA plasmid psiALR-A targeting hALR and the unrelated control plasmid psiALR-B were constructed successfully and transfected into HepG2/CDDP with lipofectamine 2000 methods ,respectively. The expression of green fluorescent protein was observed under a fluorescent microscope to calculate transfection efficiency.â’ŠAccording to different plasmid transfection, HepG2/CDDP were divided into three groups: transfection group (transfected psiALR-A), control group (transfected psiALR-B) and blank control group (non-transfected with the recombinant plasmid). There are six duplicated wells in each group. The expression of hALR and CYP3A4 in each group cells were measured by immunocytochemistry,Western Blot and Real-time FQ-PCR. The activities of CYP3A4 in each group cells were detected by Chemiluminescence; Drug sensitivity to cDDP in each group cells was detected by MTT assay. Each experiment was repeated for three times.Resluts1. HepG2/CDDP was successfully established after 4 months with stable resistance to CDDP and the resistant index was 6.26.2. 24 hours after transfection, there are a large number of bright green fluorescent protein (GFP) expression in HepG2/CDDP cells . There are more than 70% cells with green fluorescent protein after transfected by the two plasmids. The transfection efficiency is high. The two groups of cells have basically the same transfection efficiency.3. 48 hours after transfection, the expression of hALR on protein and nucleotide level in each group cells: Compared with control group and blank control group, the level of hALR expression decreased significantly in transfection group, P<0.05. There is no obvious difference between control group and blank control group in expression of hALR,P>0.05. On protein and nucleotide level, it suggests that siRNA targeting hALR could significantly inhibit the expression of hALR.4. 48 hours after transfection, the change of CYP3A4 in the expression level and its activity in each group cells. The expression and activity of CYP3A4 were detected by Western Blot,Real-time FQ-PCR and Chemiluminescence in each group,and discovered that the expression and the activity of CYP3A4 increased significantly in the transfected group compared with the control group and the blank control group, P <0.05,but there was no obvious difference between control group and blank control group, P>0.05.5. 48 hours after transfection, drug sensitivity to CDDP in each group cells was detected by MTT assay. The result showed that the sensitivity to CDDP was decreased in transfection group; However, there was no obvious change in the control group and blank control group.Conclusion1. HepG2/CDDP cell line was induced successfully, possessing the basic characteristics of resistant cells.2. The expressing siRNA plasmid psiALR-A targeting hALR and the unrelated control plasmid psiALR-B were transfected into HepG2/CDDP cells with lipofectamine 2000 methods,respectively.The transfection efficiency is high.3. The psiALR-A can inhibited the expression of hALR in HepG2/CDDP cells significantly and specifically compared with psiALR-B.4. The expression and activity of CYP3A4 in transfection group cells increased significantly after the blocking of the expression of hALR.5. The sensitivity of cells in transfection group to CDDP was decreased after blocking the expression of ALR.