Effects Of Interfering Rad21 Gene On The Biological Behavior Of Cervical Cancer Cell And Its Carcinogenic Mechanism

IntroductionCervical cancer is one of the most common gynecological malignancies affecting women's health worldwide,while early screening and treatment reduced the incidence and mortality of cervical cancer,but for many developing countries,cervical cancer is still a threat to women's life.The Rad21 gene is a subunit of Cohesin,participation in the process of the mitotic chromosomes separation,double-stranded DNA damage repair of homologous recombination,and the process of transcription regulation of other genes.The study confirmed that the Rad21 gene was overexpression in a variety of tumors.Our previous study found that the expression intensity and mRNA expression level of Rad21 in cervical cancer tissue was significantly higher than normal cervical tissue,Rad21 gene loci polymorphism was associated with the occurrence of cervical cancer in Han nationality,which suggested that the Rad21gene may play a role in the development of cervical cancer.This study used the method of lentivirus infection of cervical cancer cells to observe the biological behavior and the radiosensitivity of cervical cancer cells after lowering Rad21 expression.The role and possible carcinogenic mechanism of Rad21 is analyzed by bioinformatics tools in cervical cancer,and observe the effect on the transplanted tumor after lowering Rad21 expression by using the cervical cancer model of nude mice,and then explore the possible mechanism of Rad21 gene promoting cervical cancer.Part 1 The effect of interference of Rad21 on the biological behavior and radiotherapy sensitivity of cervical cancer.ObjectiveTo investigate the effect of Rad21 gene on the biological behavior and radiotherapy sensitivity of cervical cancer cells.Methods1.The protein expression of Rad21 gene in HeLa and Siha cells of cervical cancer was detected by immunocytochemistry and Western Blot.2.The four Rad21 interfering plasmids and a negative control plasmid were transfected into 293T cells for packaging.3.Silencing the expression of Rad21 in Siha and HeLa cells by lentivirus infection.After puromycin selection,FQ-PCR and Western Blot were used to confirm the interference efficiency of four Rad21shRNA and choose the best one for the subsequent tests.4.CCK8 assay was used to detect the HeLa and Siha cells proliferation after the changes of Rad21 expression.5.Wound healing and transwell assay was used to determine the HeLa and Siha cells migration and invasion.6.Flow cytometry was used to determine cell cycle and apoptosis of HeLa and Siha cells.7.The survival rate of HeLa and Siha cells was detected by colony technique to observe cells sensitivity to radiation.Results1.Rad21 is expressed in both cervical cancer cells and the expression level in HeLa is slightly higher than that in Siha.2.The best interference effect was TL309968B in HeLa and TL309968C in Siha cells,and the interference efficiency was more than 60%.3.Knockdown of Rad21 expression inhibits the proliferation,decreases the migration and invasion of cervical cancer cells and induces apoptosis,and makes the cell block in the G2/M phase and increases the sensitivity to radiotherapy.Summary1.Rad21 is expressed in both HeLa and Siha cells of cervical cancer.2.Rad21 involved in the malignant biological behavior of cervical cancer cell proliferation,apoptosis,cycle,migration and invasion which suggest that Rad21 plays an important role and closely related to the molecular biology mechanism of cervical cancer.3.The radiotherapy sensitivity was increased after lentiviral infection HeLa and Siha cells knockdown of Rad21 expression,which provided a theoretical basis for therapy for cervical cancer.Part 2 The mechanism of Rad21 gene in cervical cancer was explored by TCGA datebaseObjectiveTo explore the possible carcinogenic mechanism of Rad21 gene in cervical cancer.Methods1.The clinical data of 306 patients with cervical cancer were collected from TCGA database.According to the Rad21 gene expression level,patients were divided into three groups:high,middle and low.Kaplan-meier survival analysis was performed to explore the relationship between Rad21 gene and the survival rate of patients.2.The role of Rad21 gene and the expression of related genes were analyzed by bioinformatics tools in cervical cancer to find out the possible carcinogenic mechanism of Rad21 gene.3.Plasmid transfection and lentivirus infection were used to overexpress and down-regulate the expression of Rad21 in HeLa cells,the mRNA and protein expression of XPO1,P53 and cell cycle-related genes were detected by FQ-PCR and Western Blot.Results1.The total survival time of Rad21 high expression group was significantly lower than that in the low expression group.As the clinical stage increased,the expression of Rad21 gradually increased,but there was no significant difference.2.GO analysis shows Rad21 located in chromosome centromere site,nuclear chromatin and cohesin compound in the body.It can be combined with chromatin and has DNA dependent atpase activity.Rad21 plays an important role in DNA recombination,double strand break repair and mitotic sister chromatid polymerization and separation.KEGG analysis shows that Rad21 plays a role in cell cycle and RNA transport in cervical cancer.3.There is a protein interaction between Rad21 and XPO1.4.When knockdown of Rad21,the mRNA and protein expression of XPO1,CyclinBl and CDK1 decreased,in companion with the up-regulation of P21,P27 and tumor suppressor gene P53.When overexpression of Rad21,above gene expression was reversed.Summary1.The survival time of the patients with Rad21 high expression was shorter than that low expression.2.There is a close functional relationship between Rad21 and XPO1 RNA transshipment.3.Rad21 gene may promote the development of cervical cancer by participating in cell cycle regulation and XPO1's nuclear output of tumor suppressor genes.Part 3 The effect of the down-regulation of Rad21 protein expression on the growth and invasion of cervical cancer nude mice.ObjectiveTo explore the effect of the reduction of Rad21 protein on the growth,proliferation and apoptosis of nude mouse transplanted tumor and to provide a new basis for cervical cancer treatment with Rad21 gene.Methods1.To construct a model of the subcutaneous xenograft model of human cervical carcinoma HeLa cell.2.The growth curve and death of the nude mice were recorded.The nude mice were executed at 30d.The tumor were peeled off,the size of the tumor was measured and the pictures were taken.3.The expression of proliferating cell nuclear antigen(PCNA)and vascular endothelial growth factor receptor 2(VEGFR-2)in two groups of nude mice were detected by Immunohistochemistry.4.TUNEL method was used to detect the apoptosis of transplanted tumor tissue cells.5.The protein expression of Rad21 and related genes in the transplanted tumor tissues of each group was detected by Western Blot.Results1.The tumor model was successfully constructed and the tumor rate was 100%.2.In the 9th day after inoculation,the nude mice of the tumor volume Rad21 interference group was significantly smaller than the control group.The difference was statistically significant(P<0.05),and the significant difference persisted until the end of the experiment.3.The positive cell numbers of PCNA and VEGFR-2 in the transplanted tumor cells were significantly decreased showed by Immunohistochemistry and the apoptosis index was significantly increased showed by TUNEL assay of Rad21 interference group,in comparison with the control group,the difference was statistically significant(P<0.05).4.Knockdown of Rad21 expression,the expression of XPO1,CyclinB1 and CDK1 protein decreased,while the P21,P27 and tumor suppressor gene P53 protein increased.Summary1.The model of cervical cancer transplantation in nude mice was successfully constructed by subcutaneous inoculation of HeLa cells.2.Knockdown the expression of Rad21 in HeLa cells can inhibit the growth,proliferation and angiogenesis,and increase the cell apoptosis of the transplanted tumor in nude mice.3.Rad21 may be involved in the development of cervical cancer through cell cycle regulation and XPO1's nuclear output.Conclusions1.In HeLa and Siha cells of cervical cancer,knockdown of Rad21 expression inhibits the proliferation and reduces the migration and invasion of cervical cancer cells,and induces apoptosis,makes the cell block in the G2/M phase and increases the sensitivity to radiotherapy.2.The survival time of the cervical cancer patients with Rad21 high expression was shorter than that low expression.3.After interfering with the expression of Rad21 protein,it can inhibit the growth,reduce the proliferation and the generation of angiogenesis and increase the apoptosis of cervical cancer transplanted tumor in nude mice.4.There is a close functional relationship between Rad21 and XPO1's RNA nuclear transport.Rad21 gene may promote the development of cervical cancer through cell cycle regulation and nuclear output of tumor suppressor genes by XPO1.