| Objectives Liver cancer is a high incidence malignant tumor in clinical diagnosis and treatment.LukS-PV is the S component of Panton-Valetine leukocidin(PVL),a toxin secreted by Staphylococcus aureus.Previous studies showed that LukS-PV could inhibit the proliferation of hepatocellular carcinoma cells and induce apoptosis and cycle arrest of hepatocellular carcinoma cells,but the specific mechanism was still unclear.In this study,high-throughput omics sequencing technology was used to explore the specific molecular mechanism of LukS-PV inhibiting proliferation,inducing apoptosis and cycle arrest of hepatocellular carcinoma cells,laying a theoretical foundation for its clinical application as a targeted inhibitor of hepatocellular carcinoma.Methods(1)After stimulating liver cancer HepG2 cells with PBS and 0.5 ?mol/L LukS-PV for 24 h,the proteins were extracted.Western blot was used to detect the changes of acetylation,succinylation,crozoylation and 2-hydroxyisobutyrylation after LukS-PV treatment.(2)RNA and protein were extracted from hepatocellular carcinoma HepG2 cells with PBS and 0.5 ?mol/L LukS-PV for 24 h,respectively,and then sent to transcriptometric sequencing and quantitative proteomics sequencing.HepG2,Bel-7402 and Hep3B cells were treated with different concentrations of LukS-PV(0,0.25,0.5,0.75 and 1 ?mol/L)for 24 h,and the changes in HDAC2 expression were detected by qRT-PCR and Western blot,respectively.(3)The expression levels of HDAC2 in different liver cancer cells and normal liver cells were detected,and the liver cancer cells were transfected with HDAC2-specific Si RNA or HDAC2 plasmid.(1)Changes in proliferation ability of liver cancer cells were detected by CCK-8 and Ed U methods,respectively.(2)FCM was used to detect the changes in apoptosis rate and cell cycle of hepatocellular carcinoma cells.(3)qRT-PCR and Western blot were used to detect the changes in the expression levels of apoptosis related proteins and cell cycle regulatory proteins.(4)HepG2,Hep3 B,Bel-7402 cells of liver cancer were treated with different concentrations of LukS-PV(0,0.25,0.5,0.75,1 ?mol /L)for 24 h.Then changes in m RNA expression levels of PTEN were detected by qRT-PCR and changes in expression levels of PTEN,AKT,and p-AKT were detected by Western blot.The expression levels of PTEN,AKT and p-AKT were detected by Western blot after transfection with HDAC2-specific Si RNA or HDAC2 plasmid.qRT-PCR was used to detect the changes in the expression level of mi R-3691-5p in HCC cells treated with LukS-PV.Changes in the expression of mi R-3691-5p after transfection with HDAC2-specific Si RNA or HDAC2 plasmid in HCC cells were detected.(5)Subcutaneous injection of hepatocellular carcinoma cells was performed to construct a nude mouse hepatocellular carcinoma model,which was divided into 2 groups and treated with PBS and LukS-PV respectively to monitor the tumor volume of the two groups.The mice were sacrificed 24 days later and the tumors were removed to compare the size and weight of the tumors between the two groups.Samples of the tumor were stained with HE,and the expressions of HDAC2 and Ki-67 were detected by immunohistochemistry.Results(1)After LukS-PV was applied to HepG2 cells,the overall acetylation level increased significantly.(2)Quantitative proteomics sequencing and transcriptometric sequencing showed that the expression of HDAC2 was down-regulated after LukS-PV treatment,while qRT-PCR and Western blot indicated that LukS-PV could significantly reduce the expression level of HDAC2 in liver cancer cells.(3)qRT-PCR and Western blot results showed that the expression level of HDAC2 in liver cancer cell lines was higher than that in normal liver cells,and the expression level of HDAC2 in HepG2 cells was higher,while the expression level of HDAC2 in bel-7402 cells was lower.After knocking down HDAC2 in HepG2 cells,the cell proliferation ability decreased and the apoptosis rate increased,cell cycle was blocked in G1 phase,the expression levels of Bax and p21 increased,and the expression levels of Bcl-2 and Cyclin D1 decreased.After the overexpression of HDAC2 in Bel-7402 cells,the cell proliferation capacity increased,the apoptosis rate decreased,the cell cycle conversion accelerated,the expression levels of Bax and p21 decreased,and the expression levels of Bcl-2 and Cyclin D1 increased.Overexpression of HDAC2 can resist the anticancer effect of LukS-PV.(4)After LukS-PV stimulation of liver cancer cell lines,qRT-PCR and Western blot results showed that the expression of PTEN increased,while Western blot results showed that the expression of p-AKTdecreased without any significant change of AKT.Knocking down HDAC2 can increase the expression of PTEN and decrease the expression of p-AKT.Overexpression of HDAC2 decreased the expression of PTEN,increased the expression of p-AKT,and resisted the effect of LukS-PV.qRT-PCR results showed that the expression of mi R-3691-5p was down-regulated after LukS-PV stimulation of HCC cell lines.After knocking down HDAC2 in HepG2 cells,the expression of mi R-3691-5p was down-regulated.After the overexpression of HDAC2 in Bel-7402 cells,the expression of mi R-3691-5p was up-regulated and the inhibition of LukS-PV on mi R-3691-5p was resisted.(5)After LukS-PV treatment,the volume and weight of subcutaneous tumors in nude mice were significantly lower than those in PBS group.HE staining showed that tumor tissues of nude mice treated with LukS-PV showed relatively large necrotic areas.Immunohistochemical results showed that HDAC2 and Ki-67 expression were down-regulated in tumor tissues of nude mice treated with LukS-PV group.Conclusions(1)The expression level of HDAC2 was down-regulated after LukS-PV treatment in liver cancer cells.(2)LukS-PV inhibited the proliferation of HCC cells and induced their apoptosis and cycle arrest by down-regulating HDAC2.(3)LukS-PV can up-regulate the expression of PTEN and down-regulate the expression of p-AKT by down-regulating HDAC2.(4)LukS-PV can inhibit the growth and proliferation of liver cancer in vivo. |
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