Expression Of Long Noncoding RNA NONHSAT070341 In Cervical Cancer And Its Effect On Biological Function Of SiHa And Caski Cells

Objective:1.To study the expression of long non-coding RNA(LncRNA)NONHSAT070341in cervical cancer tissues and HPV16-positive cervical cancer cell lines SiHa and Caski.2.NONHSAT070341 siRNA knockdown or plasmid overexpression interventions were performed on SiHa and Caski,respectively,to observe their effects on the biological functions of cervical cancer cells including proliferation,migration,invasion and apoptosis.3.NONHSAT070341 siRNA knockdown or plasmid overexpression intervention was performed on SiHa and Caski,respectively,to detect the changes of HPV16 E7 and pRb mRNA expression in cervical cancer cells and to investigate the possible mechanism of NONHSAT070341 action in cervical cancer.Methods:1.From June 2021 to June 2022,30 patients in different stages of cervical lesions,including low grade squamous intraepithelial lesions(LSIL)、 high grade squamous intraepithelial lesions(HSIL)and cervical squamous carcinoma,who were diagnosed by colposcopy and pathologically confirmed,were selected from the Department of Obstetrics and Gynecology of the Second Hospital of Shanxi Medical University for HPV typing test showing simple HPV16 positivity and 30 patients in each group,HPV16-positive normal cervical tissues were used as controls.The expression of LncRNA NONHSAT070341 in different cervical tissues was detected by quantitative real-time polymerase chain reaction(RT-qPCR),and the expression of NONHSAT070341 in SiHa,Caski and human immortalized epidermal cells HaCaT,respectively.2.Three small interfering RNA(siRNA)fragments were transiently transfected in SiHa,Caski cells to establish the low expression NONHSAT070341 cell line(si-NONHSAT070341 group),with a negative control transfected with siRNA as a control group(si-NC group);transiently transfected with an overexpression plasmid into SiHa,Caski cells to establish the overexpression NONHSAT070341 cell line(pEX-3-NONHSAT070341 group),with the transfected pEX-3 empty vector as the control group(pEX-3-vector group).RT-qPCR was used to verify the transfection efficiency.3.After transfection with si-NONHSAT070341 and pEX-3-NONHSAT070341,cell proliferation was measured by CCK8;cell colony formation was observed by plate clone formation assay;the proportion of SiHa and Caski cells in each cycle was determined by cell cycle assay;the lateral migration ability was assessed by Wound-Healing assay;Transwell assay was performed to determine the longitudinal migration and invasion ability of cells;Flow cytometry was used to analyze apoptosis while Western blot was used to detect the expression of caspase3,a key protein of apoptosis.4.RT-qPCR was used to detect the expression of HPV16 E7 mRNA and pRb mRNA after high and low expression of NONHSAT070341.Results:1.The relative expression of NONHSAT070341 was increased in HSIL and cervical cancer tissues compared to HPV16-positive normal cervical tissues.The expression levels of NONHSAT070341 were significantly higher in both SiHa and Caski cells compared to human immortalized epidermal cell HaCaT.2.RT-qPCR results confirmed that all three NONHSAT070341 siRNAs reduced the expression of NONHSAT070341 in SiHa and Caski cells,with SiHa cells showing the most significant down-regulation of NONHSAT070341 expression by transfection with si-NONHSAT070341-1448 and Caski cells showing the most significant down-regulation of NONHSAT070341 expression by transfection with si-NONHSAT070341-493.Therefore,the most significantly down-regulated siRNA was selected for subsequent functional assays.Functional assays revealed that the proliferation,migration,and invasion ability of cervical cancer cells in the si-NONHSAT070341 group were significantly reduced compared with the si-NC group,and the cells were stagnant in the G1 phase,but the apoptosis rate of SiHa and Caski cells was increased,along with the relative expression of caspase3,a key protein for apoptosis.3.RT-qPCR results showed that the NONHSAT070341 overexpression plasmid elevated the expression of NONHSAT070341 in SiHa and Caski cells.Functional assays showed that the proliferation,migration and invasion ability of cervical cancer cells were significantly enhanced in the pEX-3-NONHSAT070341 group compared with the pEX-3-vector group,and the cells were active in S phase,and the apoptosis rate of SiHa and Caski cells were inhibited,and the expression level of Caspase3 protein was reduced.4.The relative expression of HPV16 E7 mRNA in SiHa and Caski cells in the si-NONHSAT070341 group was decreased and the expression level of pRb mRNA was increased compared with the si-NC group;the relative expression of HPV16 E7 mRNA in SiHa and Caski cells in the pEX-3-NONHSAT070341 group was increased and the relative expression of pRb mRNA was decreased compared with the pEX-3-vector group.Conclusion:1.Cervical carcinogenesis was associated with high expression of LncRNA NONHSAT070341.2.Differential expression of LncRNA NONHSAT070341 affects the biological function of SiHa,Caski cells.3.LncRNA NONHSAT070341 may be involved in the development of cervical cancer through regulating the HPV16 E7-pRb pathway.