| Objective: To investigate the effects of different doses of X-ray irradiation on the expression of PIWIL2 protein in cervical carcinoma HeLa cells and HPV virus,apoptosis and cell cycle distribution changes,and to analysis the correlation between the expression of PIWIL2 protein in cervical cancer Hela cells and HPV virus,apoptosis and cell cycle distribution changes after irradiation.Methods:(1)Culture of cervical cancer Hela cells:The DMEM high glucose media containing 10% fetal bovine serum(FBS)were cultured in the constant temperature incubator with CO2 concentration of 5%,temperature of 37 ° C and saturation humidity,the cells were cultured for experimental treatment in logarithmic phase.(2)Grouping and X-ray irradiation: Set up the control group(0Gy group),2Gy group,4Gy group,8Gy group,the 6MV X-ray linear accelerator was used for radiation,irradiation distance was 100 cm,the dose rate was 400cGy/min,the doses of 0Gy,2Gy,4Gy,and 8Gy were given respectively.(3)Clonal colony formation assay: Logarithmic Hela cells were inoculated in petri dishes after count,continued to incubate for 14 days after irradiation with grouping and irradiation conditions and the clonal colony formation rate and survival fraetion was counted by staining.(4)Detection of cell cycle and apoptosis: Hela cells were cultured to logarithmic phase,continued to incubate after irradiation with grouping and irradiation conditions,the cell cycle distribution and the apoptosis rate were detected by flow cytometry at 6,12,24,48 hours after radiotherapy.(5)Western blot and fluorescent quantitative PCR were used to detect the expression of PIWIL2 and HPV E6/E7: Hela cells were cultured to logarithmic phase,continued to incubate after irradiation with grouping and irradiation conditions,the expressions of PIWIL2 and HPV E6/E7 in the control group and experimental groups were detected at 6,12,24,48 hours after radiotherapy.Results:(1)Within 48 hours after irradiation,the expression of PIWIL2 protein increased first and then decreased in experimental groups,the PIWIL2 expression peak in group 2Gy was at 6 hours after irradiation,while 12 hours after irradiation of 4Gy group and 8Gy group;6 hours after Hela cells irradiation,the expression level of PIWIL2 in the experimental groups were up-regulated,but with the increase of irradiation,the up-regulation decreased gradually,the up-regulation was negatively correlated with irradiation dose.the expression of PIWIL2 in cervical cancer Hela cells increased in a dose-dependent manner at 12~48 hours after X-ray irradiation.(2)The survival fraetion of Hela cells after X-ray irradiation was positively correlated with the expression level of PIWIL2 at 6 hours after irradiation(r=0.921,P<0.05),while was no correlation between the expression of PIWIL2 in other time points after irradiation(P>0.05).(3)Compared with the control group,the proportion of G2/M phase after irradiation in all experimental groups increased first and then decreased with time,the G2/M phase block peak in group 2Gy was at 6 hours after irradiation,while 12 hours after irradiation of 4Gy group and 8Gy group,the trend of G2/M phase proportion and PIWIL2 protein expression level after X-ray irradiation were consistent with time,and the two were positively correlated(2Gy group r=0.896,group P<0.05;4Gy group r=0.903,P<0.05;8Gy group r=0.873,P<0.05).(4)6 hours and48 hours after irradiation with different doses,comparedwith the control group,HPV-18 E6 and E7 decreased in a dose-dependent manner;The expression levels of the two in all experimental groups reached the highest level at 12 hours after irradiation,and then decreased gradually with time,at 48 hours after irradiation,they were lower than those in the control group(P<0.05);After X-ray irradiation,the expression level of HPV-18 E6 and E7 in all group were not correlated with the expression level of PIWIL2(P<0.05).(5)The apoptosis rate of 2Gy group and 4Gy group at 24 hours after X-ray irradiation was higher than that of the control group(P<0.05),which was negatively correlated with PIWIL2 expression within 24 hours after X-ray irradiation(2Gy group r=-0.671,P<0.05,4Gy group,r=-0.783,P<0.05);The apoptosis rate of 8Gy group within 48 hours after X-ray irradiation was higher than that in control group(P<0.05),which was negatively correlated with PIWIL2 expression within 48 hours after X-ray irradiation(r=-0.595,P<0.05).Conclusions:(1)Within 48 hours after irradiation,X-ray irradiation could up-regulate the expression of PIWIL2 protein in Hela cells,the expression level of PIWIL2 increased first and then decreased with time and the PIWIL2 expression peak in group 2Gy was at 6 hours after irradiation,while 12 hours after irradiation of4 Gy group and 8Gy group.(2)X-ray irradiation could inhibit the expression of HPV-18 E6/E7 in Hela cells,and the inhibition was dose-dependent,but 12~24 hours after irradiation below 4Gy,the expression of E6/E7 was obviously increased;After X-ray irradiation,the expression levels of HPV-18 E6/E7 were not correlated with the expression level of PIWIL2.(3)After X-ray irradiation,the expression level of PIWIL2 in Hela cells was positively correlated with the blocking ratio of G2/M and the survival fraetion.(4)After X-ray irradiation,the expression of PIWIL2 in Hela cells was negatively correlated with apoptosis. |
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