Preliminary Study Of LncRNA And RNA M6A Methylation In The Pathogenesis Of Acral Lentiginous Melanoma

CMM is a highly malignant skin tumor whose occurrence and development is attributed to independent or combined genetic and epigenetic events.Significant progress has been made in understanding the role of various genetic changes to pathogenesis of CMM.Recent studies have also revealed the importance of RNA-related epigenetic mechanisms,including non-coding RNA and RNA methylation,in the regulation of gene expression,but their roles in melanoma is less well understood.In addition,research on RNA methylation modification has made breakthroughs in the field of oncology,which is involved in the development of various tumors and is closely related to the occurrence of immunotherapy tolerance.Our previous research results of the research group,this study analyzed lncRNA expression profiles of A3 75 cells of THOC2 RNAi,and a series of lncRNAs including Lnc-PKNOX1-1 were found to be differentially expressed in tumor tissues of ALM.The tumor tissue of the subtype ALM was verified by pathological examination.In addition,research on RNA methylation modification has made breakthroughs in the field of oncology,which is involved in the development of various tumors and is closely related to the occurrence of immunotherapy tolerance.To further understand the role of RNA methelyation in melanoma,we analyzed RNA methylation and the expression of methylation-related enzymes in ALM tissues.Results of this study indicate important functions of Inc-PKNOX1-1 and RNA methylation modification in the pathogenesis of ALM,highlighting further functional studies in the future.Part Ⅰ:LncRNA expression profiling in ALMObjective:To investigate the role and mechanisms of lncRNA in ALM.METHODS:A375 melanoma cells were transfected with THOC2 RNAi and non-silencing control.The efficiency of THOC2 RNAi was verified by RT-PCR.The control and TH02 RNAi cells were subject to lncRNA expression profiling via microarray and the differential gene was analyzed by GO and KEGG enrichment;q-RT PCR was used to verify the expression level of some related LncRNA in the chip.The q-RT PCR data was analyzed using the 2-ΔΔCt method.RESULTS:441 LncRNAs associated with ALM were screened by the expression microarray.We found that the LncRNA of lnc-PKNOX1-1 was significantly down-regulated in the A375 cells followingTHOC2 RNAi,which was accompanied by significant attenuation of the PTEN/PI3K/AKT signaling pathway.In 10 cases of ALM tissue,lnc-PKNOX1-1 was highly expressed in cancer tissues compared to adjacent normal tissues.Conclusion:Inc-PKNOX1-1 is upregulated in ALM and may promote the pathogenesis of ALM through the PTEN/PI3K/AKT signaling pathway.Part Ⅱ:Research on RNA m6A methylation modification in ALMObjective:To explore whether RNA methylation exists in ALM and its role in ALM.METHODS:Tumor and adjacent tissues of 40 patients with ALM confirmed by pathology were collected.Total RNA was extracted from the tissue samples using Trizol reagent.The expression levels of RNA methylation-related enzymes such as ALKBHb5,FTO and METTL3 were accessed by q-RT PCR,and the data were analyzed by 2-ΔΔCt method.The expression level of METTL3 in tumor tissues and adjacent tissues was detected by WB;the total RNA methylation level of ALM was determined by RNA methylation quantitative assay.RESULTS:RNA methylation quantitative experiments showed that the level of RNA methylation in tumor tissues was higher than that in adjacent tissues.QPCR results showed that ALKBHb5,FTO,METTL3,METTL14 were differentially expressed in ALM tumor and adjacent tissues.METTL3 was significantly expressed in tumor tissues(P<0.0001),which western blot results showed that METTL3 protein levels were significantly elevated in cancer tissues.In contrast,FTO was significantly expressed in paratumor tissues(P<0.0001),while ALKBHb5 was up regulated in tumor tissues(P<0.0001).Conclusion:RNA methylation may play an important role in the pathogenesis of ALM.High RNA methylation may play an important role in the tumorigenesis of ALM.METTL3 and FTO may play the role of oncogene and tumor suppressor in ALM,respectively.