| Objectives Liver cancer is a common malignant tumor.C5 a receptor tissue(C5a R)high expressed in liver cancer,and is associated with poor prognosis.Luk S-PV is the S component of PV-staphylococcal(Panton-Valentine leukocidin,PVL)secreted by Staphylococcus aureus.Our previous study found that Luk S-PV can inhibit the proliferation and induce apoptosis of leukemia cells through C5aR.So,we speculate that Luk S-PV also has a similar biological role in liver cancer cells that highly express C5aR.This study was to investigate the effects of Luk S-PV on proliferation,apoptosis and invasion and metastasis of hepatoma cells,and to explore its mechanism to lay the foundation for its targeted drugs.Methods(1)Hepatoma cells Hep G2,Hep3 B,Huh7,SMMC-7721,Bel7402 were cultured in vitro,and were stimulated with different concentrations of Luk S-PV(0,0.25,0.5,0.75,1μM)for 24 H.(1)The effect of Luk S-PV on the proliferation of hepatoma cells was detected by CCK-8 method.(2)The effect of Luk S-PV on the cell cycle of liver cancer was detected by flow cytometry,and the expression of cyclin protein was detected by q RT.(3)The effect of Luk S-PV on apoptosis of hepatoma cells was detected by Annexin V/PI double staining flow cytometry.(4)The expression changes of Bax,Bcl-2,Caspase3 and Caspase8 were detected by Western blot.(2)Transwell assay was used to detect the effect of Luk S-PV on the migration and metastasis of hepatoma cells and the expression of EMT-related protein were detected by Western blot.(3)Liver cancer cells were pre-treated with ERK agonist,and stimulation with Luk S-PV,Western blot was used to detect the expression of EMT-related proteins.(4)Bel7402 cells and Hep G2 cells were transfected with lentiviral C5aR overexpression vector and lentiviral C5aR inhibitory expression vector,respectively,and screened with puromycin for subsequent experiments.The transfection efficiency was verified by real-time PCR and Westrn blot.The experiment was divided into three groups for experiments:(1)NC group(2)NC + Luk S-PV stimulation group(3)C5a R knockdown/overexpression+Luk S-PV stimulation group.The expression changes of E-cadherin and N-cadherin were detected by Western blot.Results(1)The inhibitory effect of Luk S-PV on liver cancer cells proliferation was concentration dependent.(2)Luk S-PV can induce liver cancer cell arrest in G0/G1 phase,up-regulate p21 gene expression,and down-regulate Cyclin A2 and Cyclin D1 expression.(3)Luk S-PV can induce apoptosis of hepatoma cells in a concentration-dependent manner.Westrn blot analysis revealed that Luk S-PV up-regulated the expression of Bax,Caspase3 and Caspase8 and down-regulated the expression of Bcl-2.(4)Luk S-PV can inhibit the migration and invasion of liver cancer cells.Compared with the control group,the expression of E-cadherin was significantly up-regulated after Luks-PV treatment,and the proteins of N-cadherin,snail,twist and vimentin were significantly down-regulated.(5)Compared with the control group,Luk S-PV can down-regulate the expression of ERK protein.After pretreatment with ERK agonist,the effect of Luk S-PV on inhibiting EMT is significantly attenuated.(6)After overexpression of C5aR,E-cadherin expression was significantly increased and N-cadherin expression was significantly down-regulated in the C5aR overexpressing + Luk S-PV-stimulated group compared with the NC group.After knocking down C5aR,E-cadherin expression was significantly down-regulated and N-cadherin expression was significantly increased in the C5aR knockdown + Luk S-PV-stimulated group compared with the NC group.Conclusions(1)Luk S-PV has the effect of inhibiting the proliferation of hepatoma cells and inducing apoptosis of hepatoma cells.(2)Luk S-PV can inhibit the expression of ERK protein and inhibit EMT of liver cancer cells.(3)Luk S-PV exerts anti-tumor effect by binding to C5aR. |
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