Study On The Effect Of Chloride Ion On Biological Behavior Of Hepatocellular Carcinoma Cells And Its Mechanisms

Objective: Hepatocellular carcinoma(HCC)is one of the most common cancer worldwide with insufficiency in early diagnosis and rapid progression.To date,although there are many conventional treatments,for instance,chemotherapy,surgical resection,drug targeted therapy,and immunotherapy,its high rate of metastasis and recurrence lead to an extensive poor prognosis.Therefore,it is particularly important to explore the pathogenesis and emerging treatments.Chloride ion is the most abundant anion in the human body and participates in several physiological processes of the body,such as cell proliferation,apoptosis,and regulation of cell cycle.However,there are devoid of report investigating the relationship between hepatoma cells and chloride ion.Therefore,the present study aimed to elucidate the effect of chloride ion on the proliferation and apoptosis of HCC cells,and the possible mechanism to further clarify the relationship between chloride ion and HCC,and provide theoretical basis for the development and treatment of HCC.Methods:(1)The effect of chloride ion on the proliferation of SK-Hep1 and Huh7 cells were detected by the growth curve.(2)The effect of chloride ion on the migration and invasion of SK-Hep1 and Huh7 cells were detected by the Transwell assay.(3)CCK8(Cell Counting Kit-8)proliferation assay was used to detect the proliferation of SK-Hep1 and Huh7 cells.(4)The effect of chloride ion on the DNA synthesis of SK-Hep1 and Huh7 cells was detected by Edu assay.(5)Using flow cytometry(Annexin V-FITC / PI)to detect the effect of chloride ion on the apoptosis of SK-Hep1 and Huh7 cells.(6)The effect of chloride ion on the cell cycle of SK-Hep1 and Huh7 cells were detected by flow cytometry cell cycle assay.(7)After SK-Hep1 cells were treated with different concentrations of chloride ion for 48 h,differentially expressed genes were detected by gene chip and further tested by q PCR.(8)The effect of chloride ion on the protein expression levels of SK-Hep1 and Huh7 cells treated with different concentrations of chloride ion for 48 h was detected by Western Blot.Results:(1)The results of the growth curve experiment showed that after different concentrations of chloride ion(55.4m M,75.4m M,95.4m M,115.4m M,135.4m ML,150.4m M)treated SK-Hep1 and Huh7 cells for 7 days,compared with the control group(115.4m M),low chloride ion(55.4m M,75.4m M,95.4m M)and high chloride ion(135.4m M,150.4m M)inhibited the proliferation of SK-Hep1 and Huh7 cells(P<0.05).(2)Transwell assay showed that low chloride ion(55.4m M)and high chloride ion(150.4m M)inhibited the migration and invasion of SK-Hep1 and Huh7 cells compared with the control group(115.4m M).(3)Gene chip detection has found that high chloride ion(150.4m M)significantly modulate DNA replication-related genes in SK-HEP1 cells.(4)CCK-8 assay showed that after SK-Hep1 and Huh7 cells treated with high chloride ion(115.4m M,135.4m M,150.4m M)for 24 h,48h,and 72 h,compared with the control group(115.4m M),high chloride ion(150.4m M)inhibited the proliferation of SK-HEP1 and Huh7 cells(P<0.05).(5)Annexin V-FITC/PI double staining(flow cytometry)showed that SK-Hep1 and Huh7 cells treated with different concentrations of chloride ions(115.4 m M,135.4 m M,150.4 m M)for 24 h did not significantly induce apoptosis;while treated for 48 h at the same conditions,high chloride ions(150.4 m M)could significantly induce late apoptosis of SK-Hep1 and Huh7 cells compared with the control group(115.4 m M)(p<0.05).(6)cell cycle(flow cytometry)analysis revealed that SK-Hep1 and Huh7 cells were not significantly arrested in the cell cycle by different concentrations of chloride ion(115.4 m M,135.4 m M,150.4 m M)after treatment for 24 h.However,after treatment for48 h under the same condition,high chloride ion(150.4 m M)arrested SK-Hep1 and Huh7 cells in the G0/G1 phase compared with the control group(115.4 m M)(p < 0.05).(7)The cell proliferation assay(Edu)showed that after being treated with different concentrations of chloride ion(115.4 m M,135.4 m M,150.4 m M)for 24 h,SK-Hep1 and Huh7 cells were not inhibited the DNA synthesis in the S phase;after 48 h,high chloride ions(150.4 m M)could inhibit the DNA synthesis of SK-Hep1 and Huh7 cells in S phase compared with the control group(115.4 m M)(p < 0.05).(8)q PCR assay showed that SK-Hep1 and Huh7 cells were treated with different concentrations of chloride ion(115.4 m M,135.4 m M,150.4 m M)for 48 h,the DNA replication-related genes MCM2,MCM4,MCM7,MCM10,GINS2,GINS3,GINS4 and CDC45 were down-regulated at the m RNA level in the high chloride ion(150.4 m M)group compared with the control group(115.4 m M)(p < 0.05).However,in SK-Hep1 cells,but not in Huh7 cells,MCM3,MCM6 and GINS1 genes were significantly down-regulated(p < 0.05).(9)Western blot revealed that SK-Hep1 and Huh7 were treated with different concentrations of chloride ion(115.4 m M,135.4 m M,150.4m M)for 48 h,c-myc protein expression was down-regulated in the high chloride ion(150.4 m M)group compared with the control group(115.4 m M),and downstream DNA replication-related genes CDC45,MCM2,MCM4,and MCM7 were down-regulated at the protein expression level.Conclusion: Extracellular high chloride ion significantly inhibit the proliferation of hepatocellular carcinoma cells.Extracellular high chloride ion and low chloride ion inhibit the migration and invasion of hepatocellular carcinoma cells.The inhibition of HCC cell proliferation by extracellular high chloride ion may be related to the down-regulation of the expression of c-myc and CDC45,MCM2,MCM4 and MCM7,DNA replication-related genes,which leads to the cells being arrested in the S phase.