The Mechanism Of XIST M6A Modification Mediated By RBM15 To Regulate Tumorigenesis Of Female Papillary Thyroid Carcinoma

Background:The most prevalent malignant tumor of the endocrine system in women is papillary thyroid carcinoma(PTC),which is one of the main tumors endangering women’s health,quality of life,and survival.However,the greater frequency of PTC in women cannot entirely be attributed to dietary choices,environmental factors,or estrogen levels.The most notable distinction between the sexes is determined by sex chromosomes.The X inactive specific transcript(XIST)is a crucial component of the X chromosome’s control,and its aberrant expression will prevent the female X chromosome from being inactivated,which will further encourage the creation and growth of malignancies.Modification of N6-methyladenosine(m6A)controls the expression of XIST.RBM15 is a crucial functional subunit of m6 A methyltransferase,which is involved in controlling a number of biological processes.Therefore,it is crucial to investigate the function of genes associated to the m6 A methylation-modified chromosome in female PTC.Objective: This study set out to investigate the molecular causes of the greater frequency of PTC in females.to determine the precise method by which the low expression of the m6 A methyltransferase RBM15 in female PTC results in the abnormal m6 A modification of the X chromosome related gene XIST,controlling the expression of XIST and encouraging the growth of female PTC cells.As a result,the theoretical foundation for the creation of RBM15 and XIST as new diagnostic markers and therapeutic targets for female PTC is presented.This further reveals the epigenetic regulatory mechanism of female PTC occurrence.Methods: Then,using the m6 Atarget database and RMBase V2.0,m6 A regulatory molecules and m6 A modification sites that were abnormally expressed in female PTC were screened.By using m6 A colorimetry,the expression of the m6 A complex was discovered.In order to identify the relative enrichment and intermolecular binding of m6 A,me RIP binding specific q RT-PCR was performed.The m6 A methylation modification of XIST is mediated by RBM15,which also controls the production of XIST,according to the actinomycin D assay.Finally,the human androgen receptor gene CAG’s STR polymorphism sites were used to detect the skewed X-chromosome inactivation(SXCI)in both healthy women and female PTC patients.Results:1.The most notable disparities in PTC between the sexes are seen in genes associated to the sex chromosome.(1)The lavenderblush3 module was localized in the sex chromosomerelated functions and pathways(r=0.97,p<0.01);(2)In female PTC tissues,XIST was markedly underexpressed(p<0.05);Shorter disease-free survival(p=0.02)was associated with low expression of XIST,which was also associated with higher age,Grade stage,and T stage(p<0.05).2.Both in vivo and in vitro,low expression of XIST can encourage the growth of female PTC cells.(1)XIST was strongly expressed in BCPAP(p < 0.01)and significantly less expressed in TPC-1(p<0.05)as compared to the normal cell line Nthy-ori3-1;(2)TPC-1 cell proliferation and clonogenesis were markedly inhibited by XIST overexpression,whereas BCPAP cell proliferation and colony formation were stimulated by XIST downregulation.XIST overexpression can cause TPC-1 cells to become more stagnant in the G0/G1 phase,while XIST silencing results in an increase in BCPAP cells in the S phase;(3)After XIST was knocked down,the volume and weight of subcutaneous tumors in the XIST-overexpressed TPC-1 group were significantly lower than those in the control group(p<0.01)and the size and weight of BCPAP cells to form tumors in vivo were significantly higher than those in the control group(p<0.01).3.RBM15 controls the expression of XIST m6 A and facilitates its methylation modification.(1)RBM15 was down-regulated in female PTC and was associated with younger age,higher Grade stage,and T stage(p<0.05),as well as shorter disease-free survival(p=0.04);(2)RBM15 was significantly less expressed in TPC-1(p<0.05)and highly expressed in BCPAP(p<0.01)compared to the normal cell line Nthy-ori3-1;(3)RBM15 down-regulation promoted BCPAP cell proliferation and colony formation,whereas RBM15 overexpression significantly inhibited the proliferation and clonogenesis of TPC-1 cells.Overexpression of RBM15 can cause TPC-1 cells to become more stagnant in the G0/G1 phase,while RBM15 silencing results in an increase in BCPAP in the S phase;(4)The m6 A complex was considerably underexpressed in TPC-1(p<0.05)and strongly expressed in BCPAP(p<0.05)as compared to the normal cell line Nthy-ori3-1;The m6 A complex was underexpressed in female PTC tissue when compared to normal female thyroid tissue(p<0.05);RBM15 interference reduces m6 A complex levels(p<0.001);(5)Female PTC tissue had a reduced relative enrichment of m6 A on XIST compared to paracancer tissue(p < 0.01);RBM15 interference decreased XIST expression levels(p<0.001)and m6 A modification levels on XIST(p<0.01);(6)YTHDC1 knockdown slowed the rate of XIST degradation in BCPAP cells(p<0.05);Losing YTHDC1 prevented the lowered XIST expression level brought on by RBM15 knockdown in BCPAP cells(p<0.01);(7)In vitro functional salvage experiments revealed that the deletion of XIST dramatically accelerated the development of the PTC cell cycle(p<0.001)and eliminated the colony formation and proliferation inhibitory effects of RBM15 on PTC cells(p<0.01).Knocking down XIST could alleviate RBM15’s suppression of cell growth,according to in vivo functional salvage experiments(p<0.01);(8)Human androgen receptor gene CAG STR polymorphism loci study revealed that the total incidence of SXCI in female PTC patients(55.19%,101/183)was substantially greater than that in the healthy female group(12.15%,22/181)(p<0.05).Conclusion:1.The potential reason of the sex difference in PTC may be abnormal genes connected to the sex chromosomes;2.The low expression of XIST can enhance the development and proliferation ability of female PTC cells in vitro and in vivo and increase the advancement of the cell cycle.The low expression of XIST is related with worse clinicopathological characteristics in female PTC;3.YTHDC1 is required for the m6 A alteration of XIST mediated by RBM15;4.The downstream mechanism causing PTC in females may be X chromosomal inactivation and shift brought on by aberrant XIST expression.