The Cross-regulation Between HPV E6 E7 And The LncRNA TMPOP2 In Cervical Cancer Cells And The Influence On Cell Proliferation

Cervical cancer is the second most common malignant tumor affecting women’s health in the world,causing more than 254,700 deaths each year.The continuous infection of human papillomavirus(HPV)is closely related to the occurrence and development of cervical cancer,especially high-risk HPV(including HPV 16,18,etc.).Among them,cancer proteins E6 and E7 play a major role in carcinogenesis.LncRNA(long non-coding RNA)is a non-coding RNA with a length of more than 200 nt.It is a very important regulatory factor in many biological processes.Previous studies have shown that the abnormal expression of some LncRNAs may be related to the occurrence and development of cervical cancer,but the specific mechanism remains unclear.This study first identified 8 LncRNAs that could affect the progression of cervical cancer,including MALAT1,TMPOP2,TUSC8,H19,UCA1,CRNDE,GAS5 and MEG3.They were detected by qPCR in cervical cancer HeLa cells and human immortality HaCaT cells.Subsequently,HPV 18 E6 and HPV 18 E7 overexpression plasmids were constructed and overexpressed in HeLa and HaCaT.The expression of these 8 LncRNAs was detected.It was found that overexpression of HPV 18 E6 and HPV 18 E7 up-regulated the expression of TMPOP2.Level,TMPOP2 was chose as the research object.In order to further determine the relationship between E6,E7 and TMPOP2,E6 and E7 were knocked down in HeLa and CasKi,respectively,and TMPOP2 was down-regulated at the same time.In order to investigate the mechanisms of E6 and E7 regulating the expression of TMPOP2,we futher understood whether there was a link between p5 3 and TMPOP2 because E6 could act directly on p53.We constructed an overexpression plasmid for p53 and overexpressed p53 in HeLa and CasKi.TMPOP2 expression was downregulated.At the same time knocking down p5 3 in HeLa and CasKi,it was found that TMPOP2 expression was upregulated.The relationship between p53 and TMPOP2 expression was further verified in MCF7 cells.Subsequently,using the bioinformatics software UCSC,GeneCards,and ALGGEN,it was predicted that there was a target binding site for p53 on the TMPOP2 promoter,GGGCAGG and CCTGCCC,which were 48 bp apart.To further verify whether p53 could directly target the binding to the TMPOP2 promoter,it was found by Luciferase assay that p53 inhibited the TMPOP2 promoter activity and that p53 was able to bind to the TMPOP2 promoter by CHIP.Subsequently,in order to further understand the role of TMPOP2 in cervical cancer,TMPOP2 expression was simultaneously interfered in HeLa and CasKi.The expression levels of E6 and E7 were down-regulated by qPCR and Western blot.Luciferase assay results showed that TMPOP2 did not affect E6,E7 promoter activity.We found that TMPOP2 bound multiple regions of miRNA-375 and miR-139 through RNA22v2 software,and these two miRNAs had been reported to inhibit the expression of E6 and E7.By qPCR detection,both miRNAs were up-regulated when TMPOP2 was interfered.At the same time,it was verified by transcriptome sequencing analysis,qPCR and Western blot indicated that TMPOP2 could affect the expression of p21,CDK2 and CyclinE.At the same time,PI staining,CCK8 assay and EdU assay were used in HeLa to find that TMPOP2 could promote cell proliferation.The results of this study clarified the relationship between E6E7 and TMPOP2 and the role of TMPOP2 in promoting cell proliferation.The interaction between viral oncogene and TMPOP2 and their molecular mechanisms were revealed for the first time.The effect of TMPOP2 on the proliferation of cervical cancer cells was clearly defined for the first time.