The Effect Of Silencing HK2 Gene On The Radio Sensitivity Of Breast Cancer MDA-MB231 Cell In Vitro

Objective In this study,the cell model of breast cancer was used to investigate whether the short hairpin RNA(sh RNA)mediated hexokinase 2(HK2)gene silencing has an effect on radiotherapy sensitivity and radiotherapy of breast cancer cells.Lentivirus-mediated RNA interference technique was used to construct stable breast cancer MDA-MB231 cell line with low expression of hexokinase-2(HK2)to observe the relationship between HK2 gene silencing and radiation sensitivity for Breast Cancer.Methods To design three gene sequence of silencing HK2(h-HK2 sh RNA1?h-HK2 sh RNA2?h-HK2 sh RNA3)and a negative control sequence by using lentivirus-mediated short hairpin RNA(sh RNA)interference,then the sequences were transfected into 293 T cell afer connected with plasmid.Then the target genes were implant into lentiviral vector,subsequently transfected into breast cancer cells.The stable breast cancer MDA-MB231 cell lines with low expression of HK2 were screened out by antibiotic puromycin.RT-PCR and western blotting were used to detect the silencing effect after transfection from m RNA and protein levels.The stable cells with lower level were selected for the follow-up experiments.The experiments were divided into three groups: control group,negative control group and silent experimental group(HK2 sh RNA).1?the three groups of different cells were placed at 0,2,4,6,8Gy dose of X-ray irradiation,then CCK-8 experimental used to observe of the proliferation of three groups cells in the above different doses.2?The three groups of cells were exposed to 6Gy dose of X-ray irradiation,and then flow cytometry(FCM)were used to detect the apoptosis of three groups of cells.Through above,the relationship between silencing HK2 and the radiation therapy sensitivity of breast cancer cells MDA-MB231 were explored.Results 1.Successfully build up double-marked plasmid with silent HK2 gene sequence.2.The best silent effect of HK2 gene sequence(h-HK2-sh RNA1)in m RNA and protein lever was selected by RT-PCR and western blotting tests and the expression of HK2 m RNA was decreased by 89% after silencing.3.After three groups of cells being exposed to different doses of X-ray.The survival rate of the three groups was significantly lower than that of the control group and the Negative group(P <0.05).Moreover,with the increase of irradiation dose,the survival rate of the three groups of cells gradually decreased,and h-HK2-sh RNA1 group decreased more(P <0.05).4.The results of flow cytometry showed that the apoptotic rates in h-HK2-sh RNA1 group under the 6Gy X-ray irradiation was significantly higher than that of the other two groups with control group(29.1%),Negative group(29.4%)and h-HK2-sh RNA1 group(59.9%),and P <0.05.Conclusion We successfully designed the RNA sequences that could achieve the result of reducing HK2 gene expression and then infected MDA-MB231 breast cancer cell lines to get cell lines with HK2 gene transfected permanently.And silencing HK2 gene through sh RNA can significantly sensitize MDA-MB231 breast carcinoma cells to X radiation.