The Effect Of NQO1 Gene Interference On Radiosensitivity Of Cervical Cancer Si Ha Cells And Its Mechanism

Background:Cervical cancer is a common malignant tumor of women,and radiotherapy is the main treatment for locally advanced cervical cancer.With the development of radiotherapy technology,radical radiotherapy and chemotherapy for cervical cancer has achieved good results.However,due to the resistance of radiotherapy,some patients still have poor treatment effect,which seriously affects the life and health of patients.Therefore,it is particularly urgent to find a new radiosensitizing target for cervical cancer.With the application of gene chip and next-generation sequencing technology,it has become a new trend to use bioinformatics methods to find radiosensitizing targets.Researchers used bioinformatics methods to study the radiosensitivity mechanism of normal and tumor cells,and identified 255 tag genes related to radiosensitivity of malignant tumors.At present,there is no research on these 255 tag genes with the radiosensitivity of cervical cancer at home and abroad.Objective:Using bioinformatics method,the target gene NQO1 involved in the regulation of radiosensitivity of cervical cancer was selected from these 255 tag genes,and the radiosensitivity effect of NQO1 gene interference on cervical cancer Si Ha cells was verified in vivo and in vitro,and the potential mechanism was discussed.Method:1.Using the public database（TCGA,The Cancer Genome Atlas）,the radiosensitive genes differentially expressed between cervical cancer and normal tissues were screened by bioinformatics methods from the 255 tag genes.Then univariate COX regression analysis was used to further screen out differentially expressed radiosensitivity tag genes related to the prognosis of cervical cancer.Meanwhile,caret R package was used to randomly divide the whole data set into test data set and verification data set.Finally,LASSO regression was used to further screen and construct a prognosis prediction model based on radiosensitivity tag genes in the test data set and verify it in the whole data set.We also calculated the risk coefficient of each patient through the constructed prognosis model,and discussed the relationship between the risk coefficient and the prognosis of patients.2.Screening out the target gene NQO1 from the radiosensitivity tag genes involved in constructing the prognosis prediction model,using bioinformatics methods to explore its expression level in cervical cancer tissues and cells,and the relationship between its expression level and the basic clinical characteristics of patients.The NQO1 coexpression gene network was constructed by calculating the correlation coefficient.The function and mechanism of NQO1gene in cell metabolism were discussed by using GO function enrichment and GSEA pathway enrichment,and the feasibility of regulating radiosensitivity of cervical cancer by interfering NQO1 was preliminarily demonstrated.3.In cervical cancer Si Ha cells,two cervical cancer Si Ha cell models（sh NQO1-1,sh NQO1-2）with stable and low expression of NQO1 were constructed by sh RNA,and the constructed cell models were verified by RT-PCR and Western blot.CCK-8 method,mitochondrial potential energy method and flow cytometry were used to detect the effects of interfering NQO1gene expression and radiotherapy on proliferation and apoptosis of cervical cancer Si Ha cells in each group.The clone formation rate was calculated by clone formation experiment,and the cell survival curve of each group was fitted by multi-target click model,and the corresponding radiobiological parameters（D₀,D_q,N,SF₂）and radiosensitization ratio SER were calculated.Furthermore,Western blot was used to study the effect of NQO1 gene interference on PI3K/AKT pathway related protein expression.4.The transplanted tumor model of cervical cancer in nude mice was constructed by using plasmids with good interference effect,and the nude mice were divided into negative control group（sh Cont）,interference group（sh NQO1）,radiotherapy group（IR）and interference group combined with radiotherapy group（sh NQO1+IR）according to different interference factors.The tumor size and survival time of nude mice were compared between radiotherapy alone group and interference combined with radiotherapy group,and the promotion effect of NQO1 gene interference on radiotherapy efficacy was further verified.Finally,Western blot was used to verify the effect of NQO1 gene interference on PI3K/AKT pathway related protein expression in transplanted tumor.Results:1.In this study,we selected 9 different radiosensitivity tag genes related to prognosis from 255 radiosensitivity tag genes by using bioinformatics method,and constructed a prediction model of cervical cancer prognosis risk based on these 9 tag genes using test data set.The model can well predict the prognosis of patients in the test data set,and has been verified in the verification data set and the whole data set.2.The target gene NQO1 was screened out from the above 9 genes for predicting the prognosis risk of cervical cancer.Bioinformation analysis showed that NQO1 was highly expressed in cervical cancer tissues and cells,and was related to the age and T stage of patients,but not related to pathological grade,N stage,M stage and TNM stage.Enrichment of GO function indicates that it is mainly involved in metabolism and redox reaction,while enrichment of GSEA pathway indicates that it may regulate radiotherapy sensitivity of cervical cancer by activating DNA damage repair.3.The high expression of NQO1 was confirmed in cervical cancer Si Ha cells,while RT-PCR and Western blot confirmed that NQO1 expression was down-regulated in lentivirus-mediated sh NQO1 interference cell model.It was confirmed that interfering with NQO1 gene expression enhanced the inhibition of radiotherapy on the proliferation of cervical cancer Si Ha cells,further induced apoptosis and improved the radiosensitivity of the cells.Compared with the control group,sh NQO1-1 and sh NQO1-2 SF₂ in the interference group decreased significantly,and the difference was statistically significant,and the radiosensitizing ratios SER of the two groups were 1.269 and 1.511,respectively.4.At animal level,NQO1 gene interference combined with radiotherapy can also inhibit the proliferation of cervical cancer Si Ha cells.Compared with radiotherapy alone group,the transplanted tumor of nude mice in NQO1combined with radiotherapy group was smaller and survived longer,and the difference was statistically significant,which further verified that NQO1 gene interference could improve the radiotherapy effect and realize radiosensitization.5.In the process of mechanism research,it was found that interfering with NQO1 gene expression can inhibit the expression of PI3K and AKT protein in cervical cancer Si Ha cells,and the inhibitory effect has been further verified in nude mice experiments.Conclusion:1.It is a feasible research method to screen the target genes of radiosensitivity by the bioinformatics method.2.NQO1 gene is highly expressed in cervical cancer Si Ha cells,and interfering with its expression can improve the inhibition of cell proliferation induced by radiotherapy,promote cell apoptosis and improve the radiosensitivity of Si Ha cells.3.Interfering with NQO1 gene expression may play a role in sensitizing radiotherapy by inhibiting the activation of PI3K/AKT pathway.