| Background and ObjectiveThe pathological development of cervical cancer is considered to be a complex pathogenesis involving a series of tumor suppressor genes and oncogene disorders.In the past 20 years,the incidence of cervical cancer in China has been increasing year by year,and the age of onset tends to be younger.It has become the second most common malignant tumor in Chinese women after breast cancer.Squamous cell carcinoma is the most common pathological type of cervical cancer,accounting for about 75% of all cases.Although surgery,radiotherapy and chemotherapy can increase the therapeutic effect of cervical cancer,the mortality rate of cervical cancer is still high due to postoperative recurrence and drug resistance.Cervical cancer vaccine is mainly used for primary prevention and cannot cover all human papillomavirus types.Therefore,it is imperative to find a new treatment for cervical cancer.In recent years,with the introduction of "Precision Medicine",research on cervical cancer is focusing on finding new tumor markers for early diagnosis and targeted treatment.Among them,gene therapy is a biomedical treatment that aims at the root cause of disease,that is,the abnormal gene itself.It is the normal human genes or therapeutic genes through a certain way into the human body target cells,to correct the defects of genes or play a therapeutic role,so as to achieve the effect of treatment of diseases.Recent studies have shown that ASRGL1(Asparaginase like 1)is a new candidate tumor marker,as it is highly expressed in breast and ovarian cancer,but also expressed or absent in endometrial cancer.However,there are few reports on the expression and research of ASRGL1 gene in cervical cancer.Therefore,we used immunohistochemistry to detect the expression of ASRGL1 protein in cervical squamous cell carcinoma and RT-PCR to detect the expression of ASRGL1 mRNA in cervical cancer cell lines and analyze its correlation.Then,lentivirus mediated ASRGL1-shRNA was used to infect cervical squamous cell carcinoma cells,silencing the genes,analyzing the biological activity of cervical squamous cell after gene down-regulation,and identifying the function of ASRGL1 gene,so as to evaluate whether ASRGL1 gene is an oncogene for cervical cancer,and providing a theoretical basis for the treatment of cervical cancer,with a view to transforming it into clinical gene therapy.Methods:1.Immunohistochemistry was used to detect the expression of ASRGL1 protein in 32 cases of cervical squamous cell carcinoma and paracancerous tissues,and the correlation between ASRGL1 gene and cervical cancer was analyzed.2.ASRGL1 mRNA expression in HeLa and SiHa cell lines was detected by RT-PCR,and the one with high expression level was selected for the subsequent test.3.Construction and identification of ASRGL1 interfering lentivirus expression vector.4.SiHa cells with down-regulated ASRGL1 gene were constructed by using ASRGL1 interfering lentivirus expression vector.Cell cycle and apoptosis were detected by flow cytometry.5.The expressions of ASRGL1 protein,cyclin-dependent kinase 2(CDK2),cyclin A,b-cell lymphoma-2(bcl-2)and bcl-2-associated X protein(Bax)before and after lentivirus transfection of SiHa cells were detected by Western blot.6.Statistical software SPSS21.0 was used for statistical analysis of the experimental data.The chi-square test was used for the immunohistochemical analysis comparison between the cervical cancer group and the paracancer group,and the difference between the lentivirus infection group and the control group was compared by t test.P < 0.05 was considered statistically significant.Results:1.The expression of ASRGL1 in cervical squamous cell carcinoma was higher than that in normal cervical tissue.(P < 0.01).2.ASRGL1 mRNA was expressed in both strains of cells,but the expression level of human cervical squamous cell line SiHa was higher than that of cervical adenocarcinoma HeLa cell line.3.PCR identification of positive clones showed that ASRGL1-shRNA lentiviral plasmid connections were constructed correctly.The cell transfection efficiency was more than 80% under fluorescence microscope,and both Western blot and RT-PCR showed significant knockdown effect,confirming the successful construction of ASRGL1 gene lentivirus expression vector.4.SiHa cells were infected with the successfully constructed ASRGL1 lentivirus vector,and it was found that the biological activity of the cells was inhibited.5.After the down-regulation of ASRGL1 gene mediated by lviruses,the expressions of CDK2,Cyclin A and bcl-2 decreased and Bax increased.Conclusions:1.The expression of ASRGL1 in cervical squamous cell carcinoma was higher than that in adjacent tissues.2.Lentivirus mediated ASRGL1-shRNA can target and reduce endogenous ASRGL1 expression in cervical squamous cell carcinoma SiHa cell line.3.Lentivirus knockdown of ASRGL1 gene can significantly promote the apoptosis of SiHa cells and inhibit their proliferation,further proving that ASRGL1 is a candidate oncogene for cervical squamous cell carcinoma. |
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