RNA Interference Rsf-1/HBXAP Gene Targeting Enhances Chemoradiotherapy Sensitivity Of Cervical Adenocarcinoma

Background: cervical cancer is one of the three major gynecological malignant tumors.the main pathological types are cervical squamous cell carcinoma and cervical adenocarcinoma,accounting for the fourth cause of death in female cancer patients.the prognosis of adenocarcinoma is worse than that of squamous cell carcinoma,which is a hot spot in cervical cancer research.In this study,RNA interference technique was used to silence Rsf-1/HBXAP gene efficiently and specifically,and combined with radiotherapy and chemotherapy to kill human cervical adenocarcinoma Hela cells in order to enhance the sensitivity of human cervical adenocarcinoma Hela cell line to radiotherapy and chemotherapy in vitro.It provides experimental basis for gene targeted molecular radiotherapy and chemotherapy as a new treatment model for cervical cancer.Methods: human cervical adenocarcinoma cell line Hela was transfected with small interfering RNA(siRNA),and the expression of Rsf-1/HBXAP mRNA after transient transfection was detected by fluorescence real-time quantitative polymerase chain reaction(qRT-PCR).The expression of Rsf-1/HBXAP protein was detected by Western Blot.CCK-8assay was used to detect the resistance of human cervical adenocarcinoma Hela cell line to paclitaxel and cisplatin after transient transfection,and plate cloning assay was used to detect the radiosensitivity of human cervical cancer Hela cell line.The apoptosis of human cervical cancer Hela cell line after radiotherapy and chemotherapy was detected by flow cytometry.Results: qRT-PCR results showed that the relative expression of Rsf-1/HBXAPmRNA in Hela cells transfected with siRNA-1 was significantly decreased,and the interference was more than 50%.The interference of siRNA-2 and NC on Rsf-1/HBXAP of Hela cells was less than 50%.SiRNA-1 could significantly inhibit the expression of Rsf-1/HBXAP gene at mRNA level.Western Blot results showed that siRNA-1 could significantly inhibit theexpression of Rsf-1/HBXAP gene at the protein level.CCK-8 results showed that 48 hours after Rsf-1/HBXAP siRNA-1 transfection,The cell survival rates were 99%,80%,43%,17%and 7% at paclitaxel concentrations of 0.0,2.5,5.0,7.5 and 10.0nM,respectively,In the control group,the cell survival rates were 100%,90%,78%,40% and 16% at paclitaxel concentrations of 0.0,2.5,5.0,7.5 and 10.0nM,respectively,The difference was statistically significant(P﹤0.05).The IC50 of paclitaxel to siRNA-1 interfering Rsf-1/HBXAP group and non-interfering group were 7.24nM(95% confidence interval: [3.14,18.60])and 3.66nM(95% confidence interval: [1.65,7.86]),respectively.After siRNA-1 transfection,Hela cells were cultured at the concentration of 0,0.1,1 μ g / ml for 24,48,72 hours.the sensitivity of Hela cells to cisplatin was detected by CCK-8 assay.the results showed that compared with the control group,siRNA-NC cells were sensitive to cisplatin.After transfection of siRNA-Rsf-1/HBXAP,the proliferation of Hela cells was slowed down and the sensitivity to cisplatin was enhanced.The cell survival rates of the control group were 99%,97%,94%,75%,36% and 15% respectively when the concentration of cisplatin was 0.5,1.0,2.0,4.0,8.0and 12.0nM,the difference was statistically significant(P < 0.05).The results of plate cloning experiment showed that,the clone formation rates of siRNA-1 cells were 93%,24% and 5%at irradiation doses of 0,2 and 4Gy,respectively,while those of control group were 95%,52%and 13% at irradiation doses of 0,2 and 4Gy,respectively.The difference was statistically significant(P < 0.05).Compared with the control group,radiotherapy significantly inhibited the plate clone formation of Hela cells transfected with siRNA in a dose-dependent manner.When the concentration of cisplatin was 0,0.1,1 μ g / ml,compared with the control group siRNA-NC,the clone forming ability of Hela cells was decreased and the sensitivity to cisplatin was increased after transfection of siRNA-Rsf-1/HBXAP.The results of flow cytometry showed that the apoptosis rate of Hela cells transfected with Rsf-1/HBXAP siRNA-1 was significantly higher than that of Hela cells after 48 hours of radiotherapy and chemotherapy,and siRNA-1 interference with Rsf-1/HBXAP gene could promote apoptosis.Compared with the control group,after transfection of siRNA-Rsf-1/HBXAP,the proportion of S phase in Hela cells was significantly increased,S phase arrest occurred,and the sensitivity to cisplatin was increased.Conclusion: siRNA increases the chemosensitivity of human cervical adenocarcinoma cell line Hela by inhibiting the expression of Rsf-1/HBXAP gene.It is suggested that Rsf-1/HBXAP gene may be one of the causes of radiotherapy resistance and chemotherapy insensitivity of cervical adenocarcinoma,and Rsf-1/HBXAP gene may be a potential target for the treatment of cervical adenocarcinoma.It provides an experimental basis for gene targeted molecular radiotherapy and chemotherapy as a new treatment model for cervical adenocarcinoma,and provides a theoretical basis for Rsf-1/HBXAP gene as a therapeutic target for cervical cancer.