Study The Mechanisms Of RNA-binding Protein CELF6 In Regulating Cell Cycle And Cell Proliferation

In this study,we constructed CELF6 knockout HCT116 cell lines using CRISPR/Cas9 technology.Transcriptome sequencing and gene enrichment analysis identified that p53 signaling pathway was significantly enriched in CELF6 gene knockout cell lines.It indicates that CELF6 may be involved in the regulation of cell proliferation through p53 signaling.We detected the expression of p53,p27,p21,Wee1,cyclin B1,Gadd45? by q PCR and Western blot.We found that knockout/knockdown or overexpression of CELF6 significantly affected the expression of p21.We used actomycin D to inhibit de novo RNA synthesis,p21 protein was dramatically accymulated in cells overexpressing CELF6.QPCR result showed that overexpressing CELF6 significantly delay the degradation of p21 m RNA,suggesting that CELF6 affected p21 m RNA stability.RIP-q PCR assay revealed that CELF6 could bind to p21 m RNA;In order to determine CELF6 binds to 3'UTR or 5'UTR region of p21 m RNA,we constructed three overexpression plasmids p21(5'UTR+ORF),p21(full length),or p21(ORF+3'UTR)and cotransfected with His-CELF6 into HCT116 cells respectively.The protein expression of p21 in p21(5'UTR+ORF)transfected cells was significantly decreased,indicating that CELF6 may regulate p21 m RNA stability through binding to the 3'UTR region of p21 m RNA.In addition,we also explored the effects of CELF6 on cell proliferation and cell cycle.Flow cytometry analysis showed that the G1 phase popupation decreased in CELF6 knockout/knockdown cells,whereas overexpressing CELF6 caused G1 phase arrest.Ed U immaging assay also demonstrated that S phase cells were largely increased in CELF6 knockout cells.Furthermore,CCK8 cell proliferation and colony formation assay revealed that cell proliferation was significantly faster in CELF6 knockout/knockdown cells comparing with that of the control cells.Taken together,our results demonstrate that CELF6 affects the stability of p21 m RNA by binding to the 3'UTR region of p21 m RNA,and this effect is p53-dependent,CELF6 regulates cell cycle progression and cell proliferation through post-transcriptional regulation of p21.