Effect And Mechanism Of MiR-101 On Radiosensitization Of Cancer Cells

Objective:To study the effect and mechanism of microRNA101?miR-101?on radiosensitization of human uterine cervix cancer HeLa cells cultured in vitro.Overexpression of miR-101 in HeLa cells by transfecting miR-101 mimic?miRNA simulation?in vitro,it is to measure the survival rate of HeLa cells after exposure to miR-101 combined with X-ray radiation and to study the effect of miR-101 on radiosensitization of HeLa cells.The mechanism of miR-101 radiation sensitivity effect on HeLa cells was explored by observing the effection on its DNA damage and the expression of ATM and DNA-PKcs of HeLa cells after exposure to miR-101 combined with X-ray radiation.The basis is provided to develop the new drugs for improving the effect of tumor radiotherapy.Methods:The method of liposome transfecting miR-101 mimic in vitro is used to overexpress the miR-101 in HeLa cells and the method of real-time quantitative PCR?qRT-PCR?is used to monitor the expression of miR-101.CCK-8 assay and colon forming assay were used to detect the survival rate of Hela cells and to study the acute and chronic toxicity effects of miR-101 on Hela Cells,respectively.At the same time,clonogenic survival assay was applied to measure the effect of miR-101overexpression and X-ray radiation on survival rate of HeLa cells and to study the effect of miR-101 on radiosensitization of Hela cells.?-H2AX immunofluorescence technique and Western blot assay were performed to observe the DNA double-strand breaks and the expression of ATM and DNA-PKcs of HeLa cells,respectively,and to study the mechanism of miR-101 on the radiosensitivity of HeLa cells.Results:?qRT-PCR experimental results showed that HeLa cells treated with different conditions for 0h?24h?36h?48h?60h?72h,with the increase of transfection time,the relative expression of miR-101 in the negative control group had no significant change?P>0.05?.However,in the experimental group,the expression of miR-101 increased with the increase of transfection time,reached the highest at 48h?t=19.71,P<0.05?,and then decreased.based on this result,in the following experiments we take 48h that can significantly increase the miR-101 expression of HeLa cells as the transfection time of miR-101 mimic.??1?The CCK-8 experimental results showed that the survival rate of the negative control group was significantly lower than that of the blank group with the increase of transfection time,and it explained that the transfection reagent had some toxic effects on the cells?P<0.05?.Compared with the negative control group,the survival rate of the experimental group was also decreased,which indicated that miR-101 could inhibit the growth of HeLa cells?P<0.05?.?2?The results of colon forming assay showed that the survival rates of HeLa cells in blank control group,negative control group and experimental group were 100%,67.8%and 38.6%,respectively.The results indicated that miR-101 and transfection reagent had obvious toxic effect on HeLa cells.?The results of the survival rate of cells after radiation which was detected by colon forming assay showed that:the cell survival rate of the experimental group was significantly different from that of the control group after irradiation with different doses?0Gy?2Gy?4Gy?6Gy?8Gy?of X-rays?P<0.05?.There was no significant difference in survival rate between control group and negative control group?P>0.05?.There was significant difference between the negative control group and the experimental group?F=7.72,P<0.05?.The cell survival curve was fitted by the multi-target click model,and the values of D₀ and D_q were shown in table 5.The results showed that the D₀ and D_q values of the experimental group were lower than that of the blank control group?p<0.05?,while the D₀ and D_q values of the experimental group had no significant difference compared with the negative control group?P>0.05?.The results showed that over expression of miR-101 could increase the radiosensitivity of Hela cells.The SER_Do and SER_Dq of miR-101 on Hela cells were 1.20 and 1.29,respectively.?DNA damage experimental results showed that:after transfection with miR-101mimic or miR-NC mimic for 48 h,without the use of X-rays,the number of fluorescent foci in the blank control group,negative control group and experimental group cells in the nucleus of DNA double strand breaks generated were 3.10±0.27?2.02±0.31?2.94±0.32.2h after using 4Gy X-ray radiation,the number of focal points of DNA double strand breaks in the cell nucleus of the blank control group,the negative control group and the experimental group were respectively11.80±0.35?12.38±0.39?29.82±0.73.There was no significant difference in the number of fluorescent foci between the unexposed groups?P>0.05?.For the exposed groups,compared with the control group,the number of focus fluorescence in the experimental group was significantly increased?t=22.36,P<0.05?,and the negative control group cell fluorescent focus was no significant difference.?P>0.05?.?The results of Western blot showed that:the expression of ATM and DNA-PKcs in HeLa cells was significantly lower than that in control group after transfection with miR-101 for 48h in vitro.The expression of ATM and DNA-PKcs protein in the negative control group was not significantly different from that in the blank control group.The results indicated that overexpression of miR-101 could inhibit the expression of ATM and DNA-PKcs protein in HeLa cells.Conclusions:?The expression of mi R-101 was highest in HeLa cells transfected with miR-101mimic for 48h in vitro.Based on this result,in the following experiments we take 48h that can significantly increase the miR-101 expression of HeLa cells as the transfection time of miR-101 mimic.?miR-101 mimic has obvious short-term and long-term toxic effects on HeLa cells,indicating that miR-101 mimic can inhibit the growth of He La cells.?The over expression of miR-101 could increase the radiosensitivity of HeLa cells,and the radiosensitivity ratio was 1.29.?miR-101 overexpression of HeLa cells in the nucleus of DNA double strand breaks produced by the number of fluorescent foci was significantly increased,however,the expression of ATM and DNA-PKcs in two proteins related to DNA repair was significantly reduced.It can be concluded that mi R-101 can enhance the radiosensitivity by inhibiting the repair of DNA damage after IR.