| Objective:To construct a lentiviral vector carrying siRNA to silence Id-1, and observe its inhibition of human hepatocellular carcinoma HepG2 of subcutaneous tumor in nude mice and its effect on ERK1/ 2 signaling pathway.Investigate a possible mechanism of their participation in the proliferation of HCC, and provide experimental evidence for a new therapeutic targets in primary hepatocellular carcinoma.Methods:Using Id-1 gene as target sequence, 5 lentiviral vector mediated shRNA were constructed, and collected lentiviral supernatants(3×106TU/ml),which then were transfected into HepG2 cell line.After screening the cell lines stable silencing the expression of Id-1,the cell lines with best silencing effect were iderntified by RT-QPCR.18 BALA/c nu/nu Male nude mice were randomly divided into 3 groups with equal number:the transfected experimental group(treated with Id-1-RNAi-Lentiv -irus),the negative control group(treated with GFP-lentivirus),and the control group(treated with PBS). subcutaneous injection in nude mice's right axillary with 0.2ml the best interferential efficiency cell lines,vector virus treated cell lines and nomal HepG2 cell lines, The concentration of all these cell lines were 5×106/ml. Continuous observation for 28 days, during which time the tumor volume and the weight of nude mice were measured every week, and the tumor growth curve was mapped.After 28 days,mice were killed and make the tumor specimens and perform routine pathological examination, moreover, the expression of Id-1,ERK1/2,p-ERK1/2 in mRNA and protein level were examined by Semi-quantitative RT-PCR and Western bolt,respectively.Resulte:Cell morphology changes not evident before and after transfection observed by inverted fluorescence microscope. RT-QPCR identified that sh31 liver cancer HepG2 stable cell lines have the best silence effect, reducing the expression of Id-1 more than 80% compared to stable empty virus vector cell lines while about 60% compared to normal HepG2 cell lines. Three to five days after tumor formation in nude mice, tumor nodules whose diameter is about 3mm-4mm subcutaneously can be touched and the percentage of tumorigenesis is 100%.Tumor grow much more sharply in negative control group and control group than in transfected experimental group and gradually increase in negative control group and control group with time. Pathology histology display that by naked eye observation, the size of tumor in transfected experimental group is obviously smaller than that in control group. however,in microscope, tumor cells of control group arrange densely and grow vigorously with various size and shape.In addition, these cells has little karyopyknosis and small flake necrosis areas meanwhile large darkly stained nuclear.Conversely,in transfected experimental group ,tumor cells are large necrosis and vaculation with homogeneous and red dye performance.Besides,most of the nucleus is completely dissolved and cell structure disappeared,while most cells are apoptosis and appear cytoplasmic concentration, condensation nuclei stained.RT-PCR results display that the mRNA expression levels of Id-1, ERK1/2, p-ERK1/2 in transfected experimental group are obviously decreased while the difference to those in control group are statistical significance(P<0.05).Western bolt results show that the protein expression levels of Id-1, ERK1/2 in transfected experimental group are also obviously decreased,the difference to those in control group are also statistical significance ( P <0.05).However,the protein expression level of ERK1/2 protein expression does not change significantly(P>0.05).Conclusion:Constructed a lentiviral vector carrying specific siRNA for Id-1 , then transfected it into the hepatoma cell line HepG2 to silence the expression of Id-1 gene stably and the way to establish a liver tumor model by transplanting subcutaneously in nude mice with hepatocellular carcinoma HepG2 cell line is effective and feasible. the growth of nude mice and the proliferation of tumor cell have been inhibited when Id-1 have been down-regulated, in adddtion,p-ERK1/2,the activated format of ERK1/2 are also decreased,which indicated that Id-1 may influence the development of liver cancer by regulate ERK1/2MAPK signaling pathway.The fact that Id-1 is highly expressed in hepatocellular carcinoma and down-regulation of the Id-1 could inhibit the proliferation of hepatocellular carcinoma suggest that inhibiting the expression of Id-1 may become a new treatment for liver cancer's gene therapy.
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