Functional And Mechanism Research Of X Chromosome Linked LncRNA In Endometrial Cancer Tumorigenesis And Progression

Part one X chromosome linked lnc-XLEC1 associated with the occurrence and development of endometrial cancerResearch purpose:Usually,more than 95%of the nucleotides in the human genome has transcription phenomenon.However,only 1 to 2%of the entire genome transcript can encode protein,this suggests that the most stable transcription of RNA is non-coding RNA.Non-coding RNA can be divided into mircoRNA and long noncoding RNA(LncRNA)according to its length.LncRNA widely participated in the genomic imprinting,X chromosome inactivation,chromosome modification,transcription regulation and telomere biology.The abnormal expression of LncRNA is associated with the occurrence and development many malignant tumor.The research of the relationship between LncRNA and malignant tumor have been carried out for a period of time and a series of research achievements have been made.However,few stydies focus on the X chromosome linked LncRNA.The best studied X chromosome linked lncRNA is Xist,which is one 15 to 17 kb transcript and expressed before the X chromosome inactivation.Endometrial carcinoma is one of the three female reproductive system tumor and is the fourth most common cancer in women in the world,and the incidence of EC has been increasing rapidly.Therefore,it is necessary to explore the molecular mechanism in the process of endometrial cancer development.Research methods:(1)we sequenced the transcriptome of nine pairs of human EC and matched noncancerous tissues.Based on the standardized RNA-Seq analysis procedure,the tests revealed different expression levels of nine candidate IncRNAs on the X chromosome.(2)Analysis the differential expression of lnc-XLEC1 in total 120 endometrial carcinoma and adjacent tissues from eastern and southern Chinese population.(3)established the stable Inc-XLEC1 overexpression and lower expression endometrial cancer cells.(4)Performed the pull-down assays with biotinylated Inc-XLEC1,followed by mass spectrometry(MS),to search for potential Inc-XLEC1 interacting proteins.(5)Perform Luciferase reporter gene experiment,Chromatin Immunoprecipitation(CHIP)and DNA captured experiment study the regulatory mechanism of lnc-XLEC1.(6)Using CCK 8 experiment,colony forming experiment,cell invasion and migration experiment,wound healing assay,flow cytometry analysis technology,and establish an subcutaneous xenotransplanted tumor model to investigated the effects of Inc-XLEC1 on endometrial cancer cell proliferation and invasive ability.Results and conclusions:In this study,by using RNA-seq we report the identification of a new X chromosome-linked lncRNA(lnc-XLEC1)that is aberrantly downregulated during the development of endometrial carcinoma(EC).The overexpression of lnc-XLEC1 reduces the migration and proliferation of EC cells.Flow cytometry analysis indicated that lnc-XLEC1 overexpression resulted in a substantial accumulation of EC cells in the G1 phase.In addition,lnc-XLEC1 had inhibitive effects that may result from its collaboration with MBP-1 during the suppression of the c-Myc expression and the negative regulating of the Cdk/Rb/E2F pathway.The anti-tumor effects of lnc-XLEC1 on EC progression suggest that Inc-XLECl has some potential value in anti-carcinoma therapies and deserves further investigation.Our study reported for the first time that the lnc-XLEC1 might be related to the incidence and prognosis of EC.Thus,lnc-XLEC1 could find a use as a therapy against endometrial tumors.Part two Using RNA-seq data to study the X chromosome dosage compensation effect and explore the correlation between skewed X-chromosome inactivation and endometrial carcinoma progress.Research purpose:X-chromosome inactivation(XCI)is a process,during which one of the two X chromosomes in females is silenced,leading to equal gene expression as that in males,who have only one X chromosome.Theoretically,the ratio of the inactive paternal X-linked allele to the inactive maternal one should be 1:1,and any significant deviation from the ratio is termed skewed X-chromosome inactivation(SXCI).Previous studies have shown that SXCI is related to the development of multiple tumors;so we analyzed the frequency of SXCI in EC patients.Research methods:(1)Genomic DNA were exacted from peripheral blood cells from total 120 EC patients.Corrected ratio(CR)≥3 and CR>10 were used as criteria of SXCI.SXCI frequencies of patients of were compared between lnc-XLEC1 overexpressed and lower expressed patients.(2)We compared the relative expression of X-chromosome to autosomes utilizing RNA-seq data from human endometrial and prostate carcinoma and their paired adjacent tissues.To study the dosage compensation in human carcinoma.Results and conclusions:We found that the expression of lnc-XLEC1 was significantly downregulated in samples with SXCI compared to that in samples without SXCI.This result showed that the downregulated expression of lnc-XLEC1 in EC might be caused by SXCI.Beside this,there existed dosage compensation insufficiency(DCI)in carcinoma tissues.These results indicated that aneuploidy caused by DCI might facilitate tumor progression.