Effects of MLL3 Gene on Radiosensitivity and DNA Damage Repair of Cervical Cancer Cells
Objective:To explore the effects of myeloid/lymphoid or mixed-lineage leukemia 3(MLL3)gene on the growth,metastasis,radiosensitivity and DNA damage repair of cervical cancer cells.Methods:Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the level of MLL3 mRNA in 60 patients with cervical squamous cell carcinoma and their adjacent tissues.SiHa cells were cultured in vitro and divided into the following 5 groups:Control group(no transfection),NC-sh group(transfection of negative control shRNA lentivirus),MLL3-sh group(transfection of MLL3shRNA lentivirus),NC-OE group(transfection of negative control overexpression lentivirus)and MLL3-OE group(transfection of MLL3 overexpression lentivirus).Radio-resistant SiHa cells(Si-HaR)were further established by using 2300EX linear accelerator 9 MeV β irradiation,and then divided into NC-sh group,MLL3-sh group,8Gy+NC-sh group and 8Gy+MLL3-sh group.The cells in NC-sh group and MLL3-sh group were not irradiated,while the cells in 8Gy+NC-sh group and 8Gy+MLL3-sh group were irradiated with 9 MeV β rays for 8 Gy.Cell proliferation was detected by MTT method.Apoptosis was detected by AnnexinV-FITC/PI double staining.Cell invasion was detected by Transwell.The levels of MLL3,ataxia-telangiectasia mutated gene(ATM),ATM-Rad3-Related(ATR),breast cancer susceptibility gene 1(BRCA1)and RAD50 double strand break repair protein(RAD50)mRNA were detected by qRT-PCR.The expression of MLL3,B-cellymphoma-2(Bcl-2),Bcl-2 as-sociated X protein(Bax),cleaved caspase3,matrix metalloproteinase(MMP)2,MMP9 and γ-H2AX was detected by Western blot.The expression level of γ-H2AX was detected by immunofluorescence staining.Results:Compared with the adjacent tissues,the level of MLL3 mRNA in cervical squamous cell carcinoma decreased significantly(P＜0.001).Compared with NC-sh group,the relative expres-sion of MLL3 mRNA and protein decreased,the cell viability increased,the apoptosis rate,the relative expression of Bax and cleaved caspase3 protein decreased,the relative expression of Bcl-2 protein increased,the number of invasive cells and the relative expression of MMP2 and MMP9 protein of SiHa cells increased in MLL3-sh group(P＜0.05).Compared with NC-OE group,the relative expression of MLL3 mRNA and protein increased,the cell viability decreased,the apoptosis rate,the relative expression of Bax and cleaved caspase3 protein increased,the relative expression of Bcl-2 protein decreased,the number of invasive cells and the relative expression of MMP2 and MMP9 protein of SiHa cells decreased in MLL3-OE group(P＜0.05).Compared with SiHa cells,the relative expression of MLL3 mR-NA and protein in SiHaR cells increased(t=18.476,P＜0.001;t=29.126,P＜0.001).Compared with 8Gy+NC-sh group,the cell viability decreased,the relative expression of γ-H2AX protein and the number of γ-H2AX foci increased,and the levels of ATM,ATR,BRCA1 and RAD50 mRNA of SiHaR cells decreased in 8Gy+MLL3-sh group(P＜0.05).Conclusion:The downregulation of MLL3 expression promotes the growth and metastasis of cervical cancer cells,but reduces the ability of DNA damage repair and improves the radiosensi-tivity of cervical cancer cells.
Cervical cancerMyeloid/lymphoid or mixed-lineage leukemia 3GrowthMetastasisRadiosensitivityDNA damage repair
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