Circ-SIRT1 Promotes Colorectal Cancer Proliferation And EMT By Recruiting And Binding To EIF4A3

According to global cancer statistics,colorectal cancer(CRC)is already the third most common malignant tumor.More than 900,000 people die from CRC every year.The mortality rate ranks fourth in the world and is the main cause of tumor-related deaths.It is also the second most common cause of death in the United States.The current treatment of CRC is still mainly surgical resection.With the continuous improvement of treatment methods,although the 5-year survival rate of CRC has improved,postoperative recurrence and distant metastasis are still the main causes of death in CRC patients.Tumor invasion and metastasis are closely related to epithelial mesenchymal transition(EMT),which has been considered to be the main cause of CRC metastasis.The EMT process is very complicated.In general,epithelial cells change their morphological structure,migration and adhesion ability,and then obtain mesenchyme through EMT,infiltrate surrounding tissues and transfer to distant places.Finding molecular targets involved in the EMT process is of great significance for improving the prognosis of CRC patients.CircularRNA(circRNA)is a ubiquitous endogenous non-codingRNA.Compared with linearRNA,circRNA lacks a 5’cap structure and a 3’poly A tail,which can resist the hydrolysis of exonuclease.Therefore,its stability in cells is relatively high.The circRNA discovered in the early stage is considered to be the product of erroneous shearing.However,with the development of science and technology and the emergence of high-throughput sequencing technology,people have discovered through research that circRNA plays an important role in the occurrence and development of certain diseases.The gene regulation process of many diseases.Although studies have confirmed that circRNA has miRNA binding sites,it can be used as a molecular sponge of miRNA to competitively bind miRNA,regulate downstream target genes,participate in transcription regulation,and interact withRNA binding proteins to regulate the expression level of circRNA in cells,Can promote or inhibit tumor cell migration,proliferation and invasion,inhibit or promote apoptosis.However,the functions of many circRNAs are still unclear.Sirtuins(SIRT)are mammalian NAD~+-dependent histone deacetylases.SIRT1 is a member of the Sirtuin family and is a human ortholog of yeast sir2(silencing information regulator 2)protein.It is involved in cell energy metabolism and proliferation.Aging,inflammatory response,neuroprotection,tumor formation and many other processes.It has been reported that SIRT1 is related to tumor proliferation,metastasis and invasion in esophageal cancer,CRC,and breast cancer.A recent study showed that circ-sirt1 plays an important role in atherosclerosis and neointima formation.However,the function and mechanism of circRNA derived from the SIRT1 host gene in CRC are still unclear.This study explored the expression,function and mechanism of circ-SIRT1 in CRC through the following three parts.Part Ⅰ:Expression of circ-SIRT1 in colorectal cancer tissues and cell linesObjective:By detecting the expression level of circ-SIRT1 in CRC tissues and cell lines(HCT116,HT29)and matched paracancerous tissues and human colon normal epithelial cell lines(FHC),to analyze whether there is a difference in the expression of circ-SIRT1 and whether it is related to clinical features.Methods:1.Design and entrust biological companies to chemically synthesize circ-SIRT1 amplification primers;2.ExtractRNA from CRC tissues,HCT116,HT29 cell lines and matched paracancerous tissues,FHC cell lines,and detect the difference in the expression of circ-SIRT1 by reverse transcription,qRT-PCR and other methods;3.Through statistical analysis of the relationship between the differential expression of circ-SIRT1 and clinical features.Results:The expression of circ-SIRT1 in CRC tissues and cell lines was confirmed by qRT-PCR method.Then 52 cases of CRC tissues pathologically diagnosed as adenocarcinoma and paracancerous tissues more than 10cm were detected.The results showed that compared with normal tissues adjacent to cancer,the expression of circ-SIRT1 was significantly up-regulated in CRC tissues(P<0.05).The expression of circ-SIRT1 was found in HCT116,HT29and FHC cell lines.Compared with FHC cell lines,circ-SIRT1 was highly expressed in HCT116 and HT29 cell lines,which was consistent with that in CRC tissues.Through statistical analysis of patient information corresponding to clinical samples,it was found that the up-regulation of circ-SIRT1expression was positively correlated with tumor invasion depth(P<0.05),but not with age,sex,tumor size,tumor location,TNM stage,lymph node metastasis and so on(P>0.05).Conclusion:The expression of circ-SIRT1 was up-regulated in CRC tissues and cells,and was positively correlated with the depth of tumor invasion.Part Ⅱ:Effects of circ-SIRT1 expression on the biological function and EMT of colorectal cancer cellsObjective:To explore the effect of low expression of circ-SIRT1 on the biologic behavior of CRC cell lines HCT116 and HT29 and the expression of EMT related protein,and to explore whether knocking down circ-SIRT1affects the expression of SIRT1 mRNA.Methods:1.Commissioned a biological company to design and chemically synthesize si-RNA;2.qRT-PCR was used to detect the knockdown efficiency of si-RNA;3.qRT-PCR was used to detect the changes in SIRT1 mRNA expression level after circ-SIRT1 knockdown;4.The effects of circ-SIRT1 knockdown on cell migration,proliferation and invasion were detected by scratch assay,CCK-8 assay,plate cloning assay and Transwell assay;5.The expression of EMT-related proteins was detected by Western Blot.Results:The knockdown efficiency of si‐RNA was statistically significant by qRT‐PCR(P<0.05),and si‐circ‐SIRT1#2 had the highest knockdown efficiency(P<0.01).Meanwhile,circ‐SIRT1 knockdown did not affect SIRT1 mRNA expression level(P>0.05).Functional experiments of CRC cells showed that circ‐SIRT1 knockdown significantly inhibited the migration ability,proliferation activity,clonogenesis ability and invasion ability of CRC cells(P<0.05).Western Blot showed that circ‐SIRT1knockdown down-regulated the expression of N‐cadherin and vimentin,and up-regulated the expression of E‐cadherin(P<0.05).Conclusion:Circ-SIRT1 knockdown inhibited the migration,proliferation and invasion of CRC cells,down-regulated the expression of N-cadherin and vimentin,up-regulated the expression of E-cadherin,and did not affect SIRT1 mRNA expression.Part Ⅲ:Circ-SIRT1 promotes the mechanism of colorectal cancer EMT by binding to eIF4A3Objective:To investigate the molecular mechanism of circ‐SIRT1promoting proliferation,invasion and EMT.Methods:1.Circ Interactome was used to predict the protein binding to circ-SIRT1;2.RIP andRNA pull-down experiments were used to verify;3.To detect the effect of knocking down circ-SIRT1 on the expression of its binding protein;4.RIP assay was used to detect the abundance of EMT mRNA region binding protein after circ-SIRT1 overexpression;5.Western Blot was used to detect the effect of knock-down binding protein(si-BRPs)on the expression of EMT-related protein;6.Western Blot was used to detect the expression of EMT-related proteins after co-transfection of si-circ-SIRT1#2 and si-BRPs.Results:Circ Interactome prediction results showed that EIF4A3 was the protein with the largest number of circ-SIRT1 binding sites,and the binding of circ-SIRT1 to EIF4A3 was confirmed by RIP andRNA pull-down experiments.The results of Western Blot showed that knocking down circ-SIRT1 did not affect the expression of EIF4A3.After overexpression of circ-SIRT1,rip results showed that the abundance of EIF4A3 in N-cadherin and vimentin immunoprecipitation region decreased,but there was no significant change in E-cadherin region.After knocking down EIF4A3 with si-eIF4A3,the expression of N-cadherin and vimentin was up-regulated,but there was no significant change in E-cadherin.After transfection of HCT116cells with si-circ-SIRT1#2 or/and si-eIF4A3,it was found that the inhibition of si-circ-SIRT1#2 on N-cadherin and vimentin was reversed by co-transfection of si-eIF4A3,but had no effect on E-cadherin.Conclusion:Circ-SIRT1 can combine with EIF4A3,and EIF4A3 is not affected by the change of circ-SIRT1 expression.Overexpression of circ-SIRT1 reduces the abundance of EIF4A3 in EMT mRNA region and promotes EMT.