The Function And Mechanism Of TNF-α-induced LOC107984552 And Its M~6A Methylation Modification In Cervical Cancer Cells

【Objective】Cervical cancer(CC)is the most common gynecological malignancy and has a high case fatality rate.Tumor necrosis factor alpha(TNF-α)is widely involved in the occurrence and development of various cancers.Long noncoding RNA(lnc RNA)plays an important role in the regulation of epigenetics,and its abnormal expression is usually related to the occurrence and development of human diseases.The m~6A methylation modification is one of the common RNA internal modifications,which plays an important role in regulating RNA stability.In order to discover the lnc RNA that plays a role in the development of cervical cancer,our laboratory performed deep RNA sequencing of He La cells after TNF-αstimulation.After analyzing the results,an RNA whose expression was significantly down-regulated after TNF-αstimulation was selected as the research object,namely LOC107984552.This study aims to explore the mechanism by which TNF-αdown-regulates the expression of LOC107984552 and the mechanism by which LOC107984552 affects the malignant behavior of cervical cancer cells,thus providing new ideas for the treatment and research of cervical cancer.【Methods】First,the results obtained by deep sequencing were verified by RT-q PCR,that is,the expression difference of LOC107984552 in the presence or absence of TNF-αstimulation.Then the same method was used to detect the differential expression of LOC107984552 in 10 pairs of cervical cancer tissues and adjacent tissues,and its location was detected by nucleoplasmic separation experiment.Through the cell function assays,the biological function of LOC107984552 was determined.The process of TNF-αdown-regulating LOC107984552 was explained by dual luciferase reporter system and RT-q PCR.The m~6A methylation level of LOC107984552 was detected by m~6A-RIP,and the effect of m~6A methylation on its half-life was detected by RT-q PCR.Western blotting and RT-q PCR detected the regulatory effect of TNF-αon YTHDF2.Reg RNA and targetscan were used to predict the mi RNA that could bind to LOC107984552 and YTHDF2 at the same time,and the regulation relationship between the three was detected by Western blotting and RT-q PCR.,and the effects of m~6A methylation on the function of LOC107984552were detected by cell function assays.Reg RNA predicted its target mi RNA,and the target protein of this mi RNA was predicted by targetscan。The regulatory relationship between the three was detected by Western blotting and RT-q PCR.The biological function oftarget protein,target mi RNA and the ability of LOC107984552 to rescue its function were detected through cell function experiments.【Results】TNF-αstimulation down-regulated the expression of LOC107984552 in He La,which is mainly localized in the cytoplasm,and its expression in cervical cancer tissues was significantly lower.Cell function experiments found that LOC107984552 is a tumor suppressor in cervical cancer.TNF-αcan inhibit LOC107984552 by up-regulating YY1.LOC107984552 has a high level of m~6A methylation,and it is mainly achieved by the enzyme activity of METTL3.METTL3up-regulates LOC107984552 and extends its half-life;YTHDF2 does the opposite.TNF-αcan down-regulate LOC107984552 by up-regulating YTHDF2.While LOC107984552 can act as ce RNA of mi R-1260a to negatively regulate YTHDF2,further promoting its own expression.m~6A methylation can enhance the function of LOC107984552.Also LOC107984552 can adsorb mi R-345-5p and negatively regulate its expression,which is a tumor promoting factor,and LOC107984552 can partially rescue its function.TMPRSS4 is the target gene of mi R-345-5p and is positively regulated by it,while negatively regulated by LOC107984552.TMPRSS4promotes cell malignant behavior in cervical cancer.【Conclusions】This study found that TNF-αdownregulates the expression level of LOC107984552 by up-regulating YY1 to inhibit transcription or by up-regulating YTHDF2 to promote RNA degradation.And LOC107984552 exerts ce RNA mechanism to adsorb mi R-345-5p to inhibit the expression of TMPRSS4,and finally inhibit the development of cervical cancer.The m~6A methylation modification further enhances its biological function by up-regulating the expression of LOC107984552.