Study on the relationship between Methylation of Neuregulin (NRG) Gene and Cervical Carcinoma.

Objective: To investigate the relationship between the DNA methylation state of NRG1 promoter and its expression changes, and to analyze the clinical significance of its regulatory mechanism of DNA methylation in cervical carcinoma. Methods: This was a retrospective study. One-hundred and twenty patients from the Department of Gynecology of Cangzhou People's Hospital from September 2017 to September 2019 were selected, including 40 cases of cervical SCC, 40 cases of high grade squamous intraepithelial lesions(HSIL) and 40 cases of control cervical tissues. RT-qPCR, immunohistochemistry and DNA methylation-specific PCR(MSP) were used to detect the mRNA and protein expression of NRG1 and DNA methylation status in different tissue types. Results: Immunohistochemical results showed that the positive protein expression rate of NRG1 gene in the SCC group was lower than that in both HSIL and Control groups. RT-qPCR results showed that the mRNA gene of NRG1 gradually decreased in expression with the increase of cervical tissue lesions, with a statistically significant difference. Similarly, it also found that the mRNA expression level of NRG1 in the SCC group was independent of patients' age (p>0.05), but significantly correlated with tumor pathological staging, surgical pathology staging and lymphatic metastasis (p<0.05). Furthermore, methylation-specific PCR results revealed a significantly higher DNA methylation rate of NRG1 gene in the SCC group than in both HSIL and Control groups, with a statistically significant difference. Moreover, the methylation degree of NRG1 gene in SCC tissues was negatively correlated with its mRNA expression (p<0.05). Conclusions: Abnormal DNA hypermethylation of NRG1 gene inhibits the expression of mRNA and protein in the progression of cervical tissue from normal to cancerous state, which is involved in the occurrence and development of cervical carcinoma.

Expressions and Functions of Krüppel Like Factor 5 and Tumor Necrosis Factor Receptor Superfamily Member 11a in Cervical Cancer Tissues and Cells.

Objective To investigate the expressions of Krüppel like factor 5 (KLF5) and tumor necrosis factor receptor superfamily member 11a (TNFRSF11a) in cervical cancer tissues and their effect on proliferation,migration,and invasion of HeLa cells. Methods Microarray technology was used to detect the mRNA expression of gene in cytocine stimulusin cervical tissues,and the result was verified by real-time fluorescence quantitative polymerase chain reaction. The expressions of KLF5 and TNFRSF11a in cervical tissues were detected by double immunofluorescence staining. HeLa cells were transfected with specific small interfering RNA to knock down the endogenous TNFRSF11a and KLF5 and were infected with adenovirus containing KLF5 to over-express KLF5,respectively. Protein level was detected by Western blot. The regulatory effect of KLF5 on candidate target gene (TNFRSF11a) was determined by dual-luciferase reporter assay. The activity of the cell proliferation,migration,and invasion was detected by using cell counting kit-8 assay and Transwell assay. Results The results of microarray technology showed that the expressions of KLF5 and TNFRSF11a were significantly higher in cervical squamous cell carcinoma tissues compared with normal cervical tissues (P=0.002,P=0.045),and real-time fluorescence quantitative polymerase chain reaction showed that the mRNA expressions of KLF5 and TNFRSF11a were significantly higher in cervical intraepithelial neoplasia (CIN) Ⅰ,CINⅡ-Ⅲ and cervical squamous cell carcinoma tissues compared with normal cervical tissues (KLF5:F=32.79,P=0.018,P=0.014,and P=0.011;TNFRSF11a:F=36.72,P=0.013,P=0.010,and P=0.009) and double immunofluorescence staining showed that the protein expressions of KLF5 and TNFRSF11a were significantly higher in CIN Ⅰ,CIN Ⅱ-Ⅲ and cervical squamous cell carcinoma tissues compared with normal cervical tissues (KLF5:F=42.38,P=0.014,P=0.008,and P=0.002;TNFRSF11a:F=35.42,P=0.021,P=0.012,and P=0.004) and increased with the carcinogenesis. The experiment in vitro confirmed that KLF5 promotes proliferation,migration,and invasion of HeLa by up-regulating TNFRSF11a expression. Clinical analysis showed that the expression of TNFRSF11a mRNA was positively correlated with tumor pathological grading,clinical stage,depth of invasion,and lymph node metastasis (all P<0.05). Conclusion KLF5 and TNFRSF11a are related to cervical cancer. KLF5 promote the proliferation,migration,and invasion of cervical cancer cells partly by upregulating the transcription of TNFRSF11a.