| Objective: Hepatocellular carcinoma is a highly malignant tumor.It cannot be discovered in time,has a rapid progression and a poor prognosis.Despite active treat-ment,the majority of patients still die from tumor metastasis and recurrence.In order to be able to diagnose the disease earlier and find an effective treatment,it isextremely important to clarify the pathogenesis of liver cancer.Ion channel proteinsplay a key role in the development of tumors,and more and more researches havebeen done on the relationship between ion channels and liver cancer.SLC26A6 is a anion channel protein,it was found in the TCGA database that the expression in the liver of normal people was lower than that of liver cancer,and the survival curves of the two were statistically different.Based on the low expression of SLC26A6 in human normal liver,it is speculated that the upregulation of SLC26A6 gene may lead to the occurrence of liver cancer.The relationship between SLC26A6 and tumor has not been reported in domestic and foreign studies.This study aims to study the effect of SLC26A6 in primary liver cancer and its molecular mechanism,and provide a theoretical basis for pathogenesis and prevention of primary liver cancer.Methods:1.Immunohistochemistry was used to detect the expression of SLC26A6 in normal liver and liver cancer tissues;2.q RT-PCR was used to compare SLC26A6 m RNA expression in human normal liver cell line QSG-7701 and human hepatoma cell line SK-Hep1,Hep G2,LM3,MHCC-97 H and Huh7;3.Overexpression of SLC26A6 in QSG-7701 and SK-Hep1 cell line and knockdown of SLC26A6 in QSG-7701 and Huh7 cell line were achieved by lentiviral transfection,and q RT-PCR was used to confirm it;4.Proliferation of QSG-7701,SK-HEP1 and Huh7 cells were detected by CCK-8 experiment,growth curve assay and Ed U in three groups-normal control group,transfection control group,and SLC26A6 overexpression/knockdown group;5.The effects of SLC26A6 gene overexpression/knockdown on colony forming ability of QSG-7701 and Huh7 cells in three groups-normal control group,transfection control group,and SLC26A6 overexpression/knockdown group by plate colony forming assay;6.The tumor formation of human normal hepatocytes QSG-7701 overexpressing SLC26A6 gene was detected by subcutaneous tumor formation in nude mouse;7.The effects of SLC26A6 gene overexpression/knockdown on the cell cycle of QSG-7701,SK-HEP1 and Huh7 cells in three groups-normal control group,transfection control group,and SLC26A6 overexpression/knockdown group by flow cytometry cell cycle assay;8.Flow cytometry(Annexin V-FITC/PI)was used to detect the effect of knockdown SLC26A6 gene in Huh7 on the early apoptosis and late apoptosis in three groups-normal control group,transfection control group,and SLC26A6 knockdown group.Results:1.Immunohistochemistry revealed that there was a obviously lower expression of SLC26A6 in human normal liver than in primary liver cancer tissues(P < 0.001),in liver cancer tissues with different degrees of differentiation(low-differentiation,moderately differentiated and well-differentiated),the expression of SLC26A6 was significantly higher than that of paracancer tissues of liver cancer(P < 0.001),the worse the differentiation of liver cancer,the higher the expression of SLC26A6,irrespective of the patient's age,gender,tumor size,serum AFP,vascular invasion,and hepatitis B virus infection;2.The results of q RT-PCR showed that SLC26A6 expressed in all of the cell lines detected,and SLC26A6 was highest in human hepatoma cell line Hep G2,followed by Huh7 cells,the lowest expression in liver cancer cell line SK-Hep1;3.As demonstrated by q RT-PCR,SLC26A6 was overexpressed respectively in QSG-7701 and SK-Hep1 cell line(P < 0.05,P < 0.01);And SLC26A6 was knockdowned respectively in QSG-7701 and Huh7 cell line(P < 0.01);4.The results of CCK-8 showed that the proliferation of the SLC26A6 overexpression group of QSG-7701 and SK-Hep1 was significantly higher than that of the idling group(P< 0.05,P < 0.001);compared with the idling group,the growth of the knockdown group of QSG-7701 and Huh7 was significantly slower(P < 0.05,P < 0.01);5.The growth curve experiment showed that the overexpression groups of QSG-7701 and SK-Hep1 were significantly increased compared with the idling group(P < 0.01,P <0.001);compared with the idling group,the growth of the knockdown group of QSG-7701 and Huh7 was significantly slower(P < 0.05,P < 0.01).6.The results of Edu showed that the over-expression group of QSG-7701 and SK-Hep1 showed significantly more DNA synthesis in the S-phase than that in the idling group(P <0.05,P < 0.01);compared with the idling group,the knockdown group of QSG-7701 and Huh7 showed a significant decrease in DNA synthesis in the S phase(P < 0.001).7.The results of plate colony formation showed that the number and area of colonies in the overexpressing group were significantly increased in the QSG-7701 overexpression group compared with the idling group(P < 0.05,P < 0.01);compared with the idling group,the number and area of colonies in the knockdown group of QSG-7701 and Huh7 were significantly lower(P < 0.01,P < 0.001).8.The results of subcutaneous tumor formation in nude mice showed that there was a significant difference in the weight and volume of the tumor-forming tissue before and after the injection of the side of QSG-7701 overexpressing SLC26A6(P < 0.05),and HE staining was performed on the tumor-forming tissues,and a dense structure was found;9.Flow cytometry experiments showed that the SLC26A6 overexpression group of QSG-7701 and SK-Hep1 were compared with the idling group,respectively,cell cycle of the QSG-7701 cell overexpression group was decreased obviously in G0/G1 phase and G2/M phase and cells were increasd significantly in the S phase,cell cycle of the SK-Hep1 cell overexpression group was increased significantly in the G0/G1 phase and cells in S phase and G2/M phase were decreased significantly.Compared with the idling group,cell cycle of the QSG-7701 SLC26A6 knockdown group showed a significant decrease in G0/G1 phase and a obviously increase in G2/M phase and S phase.The cell cycle was blocked obviously in the G0/G1 phase cells and cells in S phase were decreased significantly in the Huh7 knockdown group(P < 0.05,P < 0.01,P < 0.001).10.Flow cytometry(Annexin V-FITC/PI)showed that the Huh7-knockdown group significantly induced early and late apoptosis of HCC cells compared with the idling group(P < 0.001).Conclusion: Upregulated expression of SLC26A6 promotes the malignant transformation of normal hepatocytes and the proliferation of hepatocellular carcinoma cells.Downregulated expression of SLC26A6 inhibits the proliferation of normal liver and liver cancer cells,and promotes apoptosis of liver cancer cells.SLC26A6 may be a new cancer-promoting gene. |
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