| Osteosarcoma(OS)is a mesenchymal-derived tumor that primarily affects the long bone metaphysis and is the most common primary skeletal sarcoma in children and young adults.Despite significant advances in OS treatment in the 1970 s and 1980 s,the prognosis of OS has not improved obviously in recent decades,which is related to the heterogeneity of OS and the lack of in-depth study of its molecular mechanisms.PRMT5 has previously been shown to overexpress in a series of cancers.Recent studies have shown that PRMT5 mediates DNA damage signaling pathway by regulating the expression of various DNA repair enzymes.Since DNA damage is the most important predisposing factor for cell senescence,It is speculated that PRMT5 regulates OS cell senescence by participating in the DNA damage pathway.In order to confirm the above hypothesis,this study explored the role and molecular mechanism of PRMT5 in OS through clinical pathology and in vitro experiments,in order to provide new guidance and research basis for the prevention and treatment of OS.We first determined the expression of PRMT5 using tissue-array by immunohistochemistry,the PRMT5 expression was significantly increased as disease progress.Next,we sought to investigate how PRMT5 may affect cell senescence of OS.Of note,PRMT5 depletion induced a pronounced cellular senescence,as evidence by β-galactosidase(β-gal)staining assay.We herein demonstrated that PRMT5 depletion-induced senescence of OS is mainly through the induction of p21 protein expression.In support of this,knockdown of PRMT5 also up-regulated the m RNA expression of SASP genes including CXCL-1,CXCL-2,CXCL-3,IL-6,IL-8,TNF-a,ICAM-1,and CCL2.Altogether,these results consistently support the fact that PRMT5 plays a role in the regulation of cellular senescence of OS.We next investigated whether PRMT5 regulates OS cell senescence correlated with DNA damage.Knockdown of PRMT5 elicits DNA damage accumulation as visualized by comet assay.In support of this,Western blot showed that knockdown of PRMT5 did elicit a severe double strand breaks,along with a significant induction of p21 expression.We then assessed the regulation of PRMT5 in DNA repair signaling in OS.Knockdown of PRMT5 significantly enhanced the γH2AX foci after treatment of Cisplatin for 3 h and then release for 24 h,this reflect the block of DNA repair with PRMT5 depletion.It has been reported recently that TXNIP plays critical role in DNA damage,we therefore aiming to determine whether TXNIP is involved in PRMT5-mediated cellular senescence.Knockdown of PRMT5 significantly elevated TXNIP protein expression,but not m RNA level.Next,we sought to determine how PRMT5-regulated TXNIP in cellular senescence.As expected,knockdown of TXNIP attenuated the induction of p21 and senescence elicited by PRMT5 depletion.Taken together,these results suggest that PRMT5 regulates TXNIP/p21 axis and is essential for cellular senescence.In addition,we then confirm the mechanism by which PRMT5 regulates TXNIP,as we had demonstrated that knockdown of PRMT5post-transcriptional enhance the expression of TXNIP.PRMT5 and TRIM21 were found to co-localized in the cytoplasm,and that the endogenous interaction between PRMT5 and TRIM21 was also validated using Co-IP and Bi Fc.To confirm the functional relationships between PRMT5 and TRIM21 in regulating cellular senescence,we did demonstrate that TRIM21 plays the similar role as PRMT5 in regulating TXNIP/p21 expression.Taken together,our results suggest that TRIM21 functional cooperates to PRMT5-regulated TXNIP/p21 expression and cellular senescence. |
