CARMN Restrains Cervical Progression Through MAPK13 And ROS Mediated MAPK Signaling Pathway

BackgroundCervical cancer(CC)is the fourth universal cancer of women in the world.In 2020,there were 660,000 new cases of CC and 340,000 deaths.Genome research which aims at whole genome sequencing and gene function identification has identified substantial long non-coding RNAs(lncRNA)related to various types of cancer.LncRNA is essential for important biological processes such as transcription,splicing,translation,protein localization,cell cycle and apoptosis.LncRNA CARMN(also known as CARMEN,MIR143 HG,NCR143/145)is the host gene of tumor suppressor miR-143/145.CARMN is situateded on human chromosome 5q32,its expression is down-regulated in a variety of cancer tissues,including CC,and may play the role of tumor suppressor gene in tumorigenesis and development.Previous studies of our research group found that CARMN was mainly located in the nucleus.Overexpression of CARMN could significantly inhibit the proliferation,migration and invasion of cervical cancer cells,block the process of cell cycle and induce cancer cell apoptosis,but its specific mechanism has not been fully clarified,which is worthy of further exploration.This study intends to identify the potential gene and pathway in the process of CARMN exerting its anti-cancer effect in CC.At the same time,molecular and cell biological methods are used to deeply explore the interaction between gene expression and signal pathway,so as to provide basis for finding clinical precise treatment targets.Objectives 1.To identify the potential gene and biological pathway of CARMN in CC.2.To explore the molecular mechanism of CARMN regulating gene expression and signal transduction pathway.3.To find the key regulatory factors in the pathogenesis of CC and provide scientific basis for the development of clinical precision medical drugs.Methods and Results1.LncRNA CARMN can affect the development of CC through MAPK signaling pathway(1)RNA-seq is used to screen the differentially expressed genes(DEGs)of CARMN overexpressed HeLa cells(HeLa-CAR)and NC cells(HeLa-NC).Functional enrichment analysis is carried out to find potential tumor regulation signals with the help of bioinformatics analysis software.The results show that the enrichment pathways include MAPK signal transduction pathway.(2)Western Blot showed that CARMN overexpression can significantly inhibit the MAPK signaling pathway in HeLa cells and C33 A cells,the expression of ERK5,JNK,P38 and ERK protein decreased.Honokiol,an activator of ERK signaling pathway,targets the expression level and activation of ERK in HeLa-CAR and C33A-CAR(CARMN overexpressed C33 A cells),and finally promotes cell proliferation.The rescue experiment further verifies that ERK branching pathway mediates the inhibition of CARMN on the phenotype of CC.2.MAPK13 mediates the regulation of CARMN on MAPK signaling pathway in cervical cancer cells(1)The MAPK data set is downloaded from the Gene Set Enrichment Analysis(GSEA)database.The DEGs of HeLa-CAR and HeLa-NC cells are intersected with the MAPK data set to screen the potential regulatory factors of MAPK signal pathway,and finally determine MAPK13 as a candidate target for further research.qRT-PCR and Western blot showed that CARMN overexpression can inhibit the expression of MAPK13 in cervical cancer cells.Nuclear cytoplasmic separation showed that MAPK13 is mainly located in the nucleus.(2)MAPK13 is transiently transfected into HeLa-CAR and C33A-CAR cells,and then the expression of key proteins of MAPK signaling pathway is detected by Western Blot.It is found that MAPK13 overexpression can up regulate the protein levels of ERK5,P38 and p-ERK,ultimately activate MAPK signaling pathway.The results of CCK-8 method and transwell chamber method show that MAPK13 promotes the proliferation and migration of HeLa-CAR and C33A-CAR cells.3.CARMN affects the expression of MAPK13 through transcriptional regulation mechanism(1)Alibaba2 program,PROMO website and JASPAR software screen the binding transcription factors in MAPK13 promoter region,predict their binding sites,and preliminarily identify TFAP2α may be involved in the transcriptional regulation of MAPK13 in cervical cancer cells by CARMN.(2)Plasmid overexpression and antisense oligonucleotides(ASO)technology up-regulate and knock down TFAP2α in HeLa cells and C33 A cells,respectively.The expression of its target gene MAPK13 is observed and TFAP2α is found positively correlated with the expression of MAPK13 in CC(3)Chromatin immunoprecipitation(ChIP)further finds TFAP2α can enrich the promoter region of MAPK13.Dual-Luciferase reporter assay shows that TFAP2α can significantly up regulate the transcriptional activity of MAPK13 promoter.Then,truncated plasmids of MAPK13 promoter are constructed,and the results show that the-320 ~-100 motif fragment of MAPK13 promoter region may be TFAP2α main areas of combination.(4)RNA pulldown experiment shows that TFAP2α and MAPK13 can be significantly enriched in DNA sense strand of HeLa-CAR cells,qRT-PCR shows that CARMN does not affect TFAP2α expression level.4.CARMN regulates the development of CC through ROS mediated MAPK pathway(1)The regulation of CARMN overexpression on the level of ROS in cervical cancer cells is detected by probe method and flow cytometry.It is found that CARMN can promote the increase of ROS level in cervical cancer cells.(2)DPI(diphenyleneiodonium chloride),an inhibitor of ROS,is used to treat HeLa-CAR and C33A-CAR cells,the MAPK signal pathway is detected by Western Blot.The results show that DPI can restore ERK5/P38/ERK/MAPK signal transduction pathway in HeLa-CAR cells and ERK/MAPK signal transduction pathway in C33A-CAR cells.The results of CCK-8 and transwell experiments show that DPI promotes the proliferation and migration of HeLa-CAR and C33A-CAR cells.ConclusionThis study finds that lncRNA CARMN and MAPK13 are co-located in the nucleus,and CARMN can interact with TFAP2α with its sense chain,repressing TFAP2α transcriptional activates MAPK13 and reduces the expression level of MAPK13,resulting in the inactivation of MAPK signaling pathway.In addition,CARMN can inhibit MAPK signal pathway by activating ROS,DPI can rescue the inactivation of ROS dependent MAPK signal pathway caused by CARMN.CARMN negatively regulates MAPK signaling pathway through the above two pathways,so as to affect cell malignant phenotype and play the role of tumor suppressor gene.The results reveal the key genetic regulatory factors in the pathogenesis of CC,and provide some scientific basis for the development of clinical precision medical drugs.