Mechanism Of Action Of Connexin26 On Radiosensitivity Of Liver Cancer Cells

Objective:Liver cancer is one of the cancers with the highest morbidity and mortality in China.Appropriate irradiation and the application of protons and heavy ions in radiotherapy have made radiotherapy a great development in the treatment of liver cancer.It is a key factor to expand the application of radiotherapy in the treatment of liver cancer by studying the influence of liver cancer cell sensitivity and the transmission mechanism of radiation damage signal in liver cancer cells and reducing the damage of irradiation to normal liver tissue.Connexin26（Cx26）is a protein that constitutes gap junction and is widely expressed in the liver.Down-regulation of Cx26expression is one of the important mechanisms of hepatocarcinogenesis,and the gap junction composed of Cx26 is closely related to the transmission of radiation damage signals between cells.In present study,hepatoma cells（HepG2,SK-hep1）with different Cx26 expression levels were used to study the mechanisms of Cx26 in radiosensitivity in liver cancer cells.Methods:In this study,HepG2 hepatoma cells with low expression of Cx26 and SK-hep1 hepatoma cells with high expression of Cx26 were used.After irradiated with0,1,2,3 or 5Gy X-ray,the survival rate were tested by clone formation assay.The micronucleus formation rates of tumor cells 6h after irradiation were detectedto determine the effects of Cx26 expression on the radiation sensitivity of hepatoma cells.The expression of Cx26,p-ERK/ERK,p-p38/p38 and NF-κB protein in irradiated or control cells was detected by western blot.To conform the result,the CRISPR/Cas9plasmid and lentivirus with Cx26 plasmids were used to knock-out or over-express of Cx26.Four cell lines of HepG2 N-,SK-Hep-1 N-,HepG2 A+,SK-Hep-1 A+)were made as experimental subjects.Results:HepG2 and SK-hep1 cells were irradiated with different doses of X-rays.Western blot experiments showed that the expression level of Cx26 in HepG2 cells was lower than that of SK-hep1,and the expression level of Cx26 in both cells was not affected by the irradiation dose.The expression levels of p-ERK/ERK,p-p38/p38 and NF-κB in both cells increased with the increase of X-ray irradiation dose.The expression level of p-ERK/ERK,p-p38/p38 and NF-κB protein in SK-hep1 cells was higher than that of HepG2 cells,and the difference was significant when the dose of X-ray irradiation was 3Gy and 5Gy.Western Blot experiments with reduced expression of Cx26 by CRISPR/Cas9 plasmid showed that,the expression level of protein p-ERK/ERK,p-p38/p38 and NF-κB in four kinds of cells increased with the increase of X-ray irradiation dose,but the expression level of protein p-ERK/ERK,p-p38/p38 and NF-κB of HepG2 N-、SK-hep-1 N-was closer to that of HepG2.The expressions of protein p-ERK/ERK,p-p38/p38 and NF-κB of HepG2 A+、SK-hep-1 A+was closer to that of SK-hep-1.The results of colony formation experiments showed that the D₀ value of SK-hep1A+cells(D_{0（SK-hep1A+）}=2.3878)was lower than that of other cells(D_{0（SK-hep1）}=2.85814;D_0（HepG2）=3.35501;D_{0（SK-hep1N-）}=3.34465;D_{0（HepG2 N-）}=3.66632;D_{0（HepG2 A+）}=2.77888).The micronucleus formation detected by CB method showed that the micronucleus formation rates of HepG2 N-and SK-hep-1 N-were significantly lower than other cells.Conclusion:The HepG2 cells showed higher survival rates and less micronucleus formation than SK-hep1 cells after radiation（P<0.01）.The phosphorylation of p38 and ERK1/2 were lower in HepG2 cells than SK-hep1 cells after radiation（P<0.01）.And also the expression of NF-κB were less in HepG2 cells than SK-hep1 cells after radiation（P<0.01）.These results were conformed in the Cx26 knocking out and overexpressing cells.These results suggested that the cells with higher expression of Cx26 showed higher radiosensitivity.