Knockdown Of AFP Represses The Proliferation Of Hepatoma Cell (HepG2) Via Regulating The Cell Cycle

Objective:Alpha-fetoprotein(AFP) is one of the major plasma proteins in mammalian fetuses,and high concentration of AFP in serum is found in connection with primary liver cancer and teratocarcinoma.Until now,little is known about its function/effect in pathogenesis of hepatoma.The objective of this paper is to analyze the ability of AFP,which modulates the proliferation of hepatoma cells,and the potential mechanisms involved.Methods:To knock down the expression of AFP in HEK293 cells and HepG2 cells,we prepared pENTR/U6 vector(pENTR/U6/AFPsiRNA) and recombinant adenovirus (Adv-AFPsiRNA) expressing the short interferencing RNA targeting AFP.The expression level of AFP in HEK293 cells transfected by pCDNA3.1/AFP+ pENTR/U6/AFPsiRNA and HepG2 cells infected by Adv-AFPsiRNA was determined by western blotting.The AFPmRNA level in HepG2 cells that AFP was knocked down was assayed by semi-quantity RT-PCR.The repression of cell proliferation in vitro and growth of hepatoma in vivo were examined by colony formation assay,growth curve and tumor xenograft in SCID mice,respectively. TUNEL assay was used to detect apoptosis;and cell cycle was assayed by flow cytometry.The gene expression analysis was determined by microarrays and the result was confirmed by semi-quantity RT-PCR.Results:1.Through western blotting,the potent and specific inhibition of AFP was conspicuous from the AFP-derived siRNA.Co-transfection of HEK293 cells with pCDNA3.1/AFP(reporter plasmid) and pENTR^TM/U6/AFPsiRNA led to a statistically significant decrease of AFP at 48 hours after the transfection compared with transfection of pCDNA3.1/AFP only.The knockdown level was 90%.Then,we tested the effect of Adv-AFPsiRNA on the endogenous AFP expression in HepG2 cells.Infection of HepG2 with Adv-AFPsiRNA at 20 MOI after 48 hours showed a statistically significant reduction of AFP expression. The knockdown level was 75%.Through semi-quantity RT-PCR,we confirmed that the AFPmRNA level was knockdown about 92%in HepG2 cells that AFP was knocked down by AFPsiRNA.2.Through colony formation assay and growth curve assay,we confirmed that the growth rate of HepG2 cells that AFP knock down was obviously slower than that of the control HepG2 cells.Colony formation capacity of cells infected with Adv-AFPsiRNA and Adv-LacZsiRNA were estimated over 20 days.Infection of Adv-AFPsiRNA resulted in a 85%decrease in the number of colonies. And growth curve result showed HepG2-B5 cells(the HepG2 stable cell line with low expression level of AFP which were from HepG2 cells after transfection with pSilencer2.1-U6neo/AFPsiRNA) were associated with a marked decrease in growth rate in standard proliferation assay compared with HepG2(-) cells.And in vivo experiment, Adv-AFPsiRNA caused a significant tumor-growth delay in contrast to the control groups.3.The in situ apoptosis detection and FACS assay showed that AFP knockdown did not induce cell apoptosis,but an obvious delay in the G₁/S transition of cell cycle.We assessed the proportion of apoptosis cells in Adv-AFPsiRNA or Adv-LacZsiRNA infected HepG2 cells on the 7th day using In Situ Cell Death Detection Kit(Roche).The result showed that there was no obvious cell apoptosis appeared in Adv-AFPsiRNA infected HepG2 cells,no difference between Adv-LacZsiRNA infected HepG2 cells and HepG2 cells untreated.In contrast,most treated HepG2 cells with 0.09mg/ml teniposide(VM-26) that is an apoptosis inducer showed markedly apoptosis.In FACS assay, HepG2-B5 and HepG2(-) cells were synchronized by 48 hours' culture in serum-free medium,and cell cycle entry was stimulated by adding 10%FBS.Twenty-four hours after adding FBS,there was a 7%and a 64%decrease in the proportion of cells with a G0/G1 DNA content in HepG2-B5(p=0.385) and HepG2(-) cells(p=0.004),respectively.The result indicated a reduced exit from quiescence in HepG2-B5 cells.4.We used a microarray platform to identify differentially expressed genes between HepG2 cells knocking down AFP(HepG2-B5) and the control HepG2 cells[HepG2(-)].The important genes involved in cell cycle and cell proliferation that changed≥2 fold included Cell division cycle 34,S-phase kinase-associated protein 2,C-src tyrosine kinase, Estrogen receptor binding site associated,antigen,9,Cyclin D1,etc. These genes' expression changes were confirmed by semi-quantity RT-PCR and the changes were accordance with these genes' function relating to cell cycle and cell proliferation that leaded to delay in the G₁/S transition of cell cycle.Conclusions:The results indicated that the potent and specific inhibition of AFP is conspicuous from the AFP-derived siRNA we designed.And AFP is highly related with the proliferation of HepG2 cells.The growth inhibitory effect of AFP does not result from cell apoptosis,but the delay in the G₁/S transition of cell cycle.The effects may show a new gene therapy pathway to hepatoma.