| Background and objective:P8,also named as com1,was a tumor associated stress molecule. Previous studies reported expression changes of p8 in a wide array of tumor tissues including pancreatic,breast, thyroid and prostate cancer.P8 might be involved in the initiation and development of tumor via influencing cell growth, apoptosis and metastasis and so on. However,there were no any reports about the function of p8 in hepatocellular carcinoma(HCC).This study is aims to investigate the roles of p8 in HCC.Content:The research was performed in HCC cell lines. P8 expression was knocked down by RNA interference (RNAi) method. Cell proliferation,migration and colony formation were accessed in p8-knockdown cells to explore the roles of p8 in hepatocarcinogenesis..Method:1. Human p8 mRNA sequence was retrieved from GenBank. Two sites were selected as the target sequences of RNAi through online tool. Corresponding dsDNAs were generated from synthesized oligos, and then inserted into pSuperior.puro vector digested with two restriction enzymes. Plasmids were extracted and verified by sequencing.2. P8 expression in four kinds of HCC cell lines (BEL-7402,QSG-7703,SMMC-7721 and HepG2) was detected by Real-time PCR. HepG2 was selected for following experiments due to the highest level expression of p8. HepG2 cells were stably transfected with above RNAi plasmids and control vector respectively by lipofectamine reagent. After 3-week-selection with puromycin(0.5ng/ul), drug-resistant colonies were picked up from each group for extant culture Real-time PCR was employed to screen the efficiency of RNAi..3. Cell proliferation,migration and colony formation were accessed in p8-knockdown cells. Expression of CDK4,CyclinD1,p27,p21,p16,Bim,β-catenin and c-myc were detected by Western blot.Result:1. Two sites were selected as the target sequences of RNAi through online tool as following: p8i-1: 5′-GACCAAGCTGCAGAATTCA-3′and p8i-2: 5′-GACTCCAGC CTGGATGAAT-3′; Corresponding dsDNAs were successfully cloned into the pSuperior.puro and confirmed by DNA sequencing. These plasmids were named as pSuperior.puro/p8i-1 and pSuperior.puro/p8i-2 respectively.2. The expression of p8 was the highest in HepG2 among four cell lines. After transfected with control and RNAi constructs with lipofectamine reagent, HepG2 cells were grew in media with puromycin. Drug-resistant colonies were picked up from each group for amplified culture after 3 weeks of selection and were named as: HepG2/p8i-1,HepG2/p8i-2 and HepG2/v respectively. Real-time PCR result shown that p8 mRNA level was significantly down-regulated in HepG2/p8i-1 and HepG2/p8i-2 cells as compared with the control HepG2/v cells,.The knock-down rate of p8 mRNA was 76.6% and 94.9% respectively.3. MTT assay indicated that proliferation abilities of HepG2/p8i-1 and HepG2/p8i-2 cells were significantly lower than that of control HepG2/v cells. Plate colony formation experiment demonstrated that the single cell clone formation capacities of HepG2/p8i-1and HepG2/p8i-2 cells were obviously lower than that of control HepG2/v cells. (p<0.05 and p<0.01 respectively). Flow cytometry demonstrated increased G1 phase arrests in HepG2/p8i-1 and HepG2/p8i-2 cells. Transwell experiment shown decreased infilted cells in HepG2/p8i-1and HepG2/p8i-2 cells as compared with HepG2/v cells. ( p<0.01 and p<0.05 respectively). Western blot indicated that the expression of c-myc in HepG2/p8i-1and HepG2/p8i-2 cells was lower than that in HepG2/v cells. No significant difference was found in protein levels of CDK4, CyclinD1, p27, p21, p16, Bim andβ-catenin among HepG2/p8i-1, HepG2/p8i-2 and HepG2/v cells.Conclusion:Both of shRNAs we selected could efficiently knock down the expression of p8 in HepG2 cells.A serial of studies indicated that cell growth, colony formation and cell migration were significantly inhibited upon p8 knockdown, increased G1 phase arrest and decreased expression of c-myc expression were found in p8 knockdown cells. P8 might serve as a tumor promoting gene in HCC.
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