Role And Mechanism Of M6A-promoted And MBNL3-induced CLSTN1 Gene Alternative Splicing In Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is one of the common malignant tumors in China,Its incidence rate ranks fourth in all malignant tumors,and the mortality rate is third in all tumors.Therefore,HCC is a kind of malignant disease that seriously threatens our national health.So far,the most effective treatment for liver cancer is still surgical resection or liver transplantation.However,a considerable number of patients with HCC have lost the opportunity of operation at the time of diagnosis,and some patients with HCC will might undergo recurrence and metastasis after operation or transplantation.With the development of next-generation sequencing technology,several research groups have carried out whole genome and transcriptome sequencing for HCC,many mutated or differentially expressed genes and corresponding signal pathways in hepatocellular carcinoma were also identified from the perspective of genetics and epigenetics.Recent studies have also revealed the important role of post-transcriptional regulation of RNA in gene expression.for example,alternative splicing of RNA in pathophysiological processes has been gradually recognized.As a post-transcriptional regulatory mechanism,alternative splicing of RNA can make the one gene produce different transcripts,then translate different proteins,or completely abolish its translation ability to produce lnc RNA.Different transcripts or proteins produced by alternative splicing of the one gene can have different effects,or even completely opposite effects.However,the understanding of the mechanism of RNA alternative splicing is still in its infancy.In previous studies,we found splicing factor MBNL3 high expressed in fetal liver,low expressed in adult liver and high expressed in liver cancer.Further functional studies showed that MBNL3 had a strong cancer promoting effect in HCC.m6 A plays important biological functions,such as pre-mi RNA processing,regulating RNA structure,RNA-protein interaction,RNA translation efficiency,nuclear output,splicing,stability and so on.The abnormal level of m6 A can also promote the occurrence,development and prognosis of cancer.M6A can regulate the alternative splicing of pre-m RNA through YTHDC1.So,in this study,we explored the effect of m6 A on MBNL3 alternative splicing and the biological function of different transcripts of downstream target genes.We found the decreased level of m6 A lead to the decreased binding ability of MBNL3 to RNA,the increased level of m6 A lead to the increased binding ability of MBNL3 to RNA,in addition,it is YTHDC1-dependent.Co-mmunocoprecipitation showed the interaction between MBNL3 and YTHDC1,and cellular immunocolocalization also showed both MBNL3 and YTHDC1 were distributed in the nucleus,and interacted with each other.Then,we screened the downstream target gene CLSTN1 regulated by m6 A,YTHDC1 and MBNL3 through the database and transcriptome sequencing data.ENCORI database shows that YTHDC1 binds to intron 11 of CLSTN1 gene,and according to the previous reported motif YGCY bound by MBNL3,it also shows that intron 11 of CLSTN1 gene also contains the binding motif of MBNL3.Ed U proliferation assays,TUNEL apoptosis assays,migration assays and invasion assays showed that CLSTN1-Long promoted proliferation,migration and invasion of SNU-398 and Hu H7 cells,repressed apoptosis of SNU-398 and Hu H7 cells,CLSTN1-Short repressed proliferation,migration and invasion of SNU-398 and Hu H7 cells,promoted apoptosis of SNU-398 and Hu H7 cells.At the cellular level,CLSTN1-Long and CLSTN1-Short exerted opposite functions.The possible mechanism was that CLSTN1-Long can specifically bind Galectin-7,reside Galectin-7 on the cell membrane,and repress Galectin-7 to enter the cell.Furthermore,Galectin-7 was a secretory protein that regulated its expression through autocrine.Therefore,when CLSTN1-Long was overexpressed,it affected Galectin-7 autocrine,and caused the down-regulation expression of Galectin-7.In addition,Overexpression of Galectin-7 can reverse the roles of CLSTN1-Long overexpression in HCC.Therefore,overexpression of CLSTN1-Long exerted tumour oncogenic roles in HCC.CLSTN1-Short can specifically interact with β-Catenin,a ranscriptional activator,inhibit β-Catenin enter to nuclear,and repressed WNT signaling pathway.In addition,ICG-001 can reverse the roles of CLSTN1-Short knockdown in HCC.Therefore,overexpression of CLSTN1-Short exerted tumour suppressive roles in HCC.We first explored the effect of m6 A on MBNL3 alternative splicing and the biological function of different transcripts of downstream target genes.Our study might help to deepen the understanding of the molecular mechanism of m6 A in the occurrence and development of HCC,and construct the m6A-YTHDC1-MBNL3-alternative splicing of downstream target gene pathway,aiming to provide possible molecular markers and possible drug targets for the prevention and treatment of HCC.