| MicroRNAs are important epigenetic regulators of protein expression by triggering degradation of target mRNAs and/or inhibiting their translation.Dysregulation of microRNA expression has been reported in several cancers,including colorectal cancer(CRC).A good deal of researchs show that miRNA is also involved in regulating Wnt signaling pathway,we know that WNT/β-catenin pathway plays an important role in the tumorigenesis and metastasis of colorectal cancer.Research revealed that miRNA can regulate target genes to regulated WNT/β-catenin pathway is also involved in the tumorigenesis and metastasis in oral squamous cell carcinoma,lung cancer,pancreatic cancer,prostate cancer and esophageal cancer.And microRNA-145 can play a role in colorectal cancer cells through targeting catenin delta-1 to regulate WNT/β-catenin signaling pathway.Results:(1)Tissue miRNA microarray profiling:Identification of aberrantly expressed miRNAs is the first step toward elucidating miRNA-mediated oncogenic pathways in colorectal cancer.Based on this point,we performed tissue miRNA microarray to discover the global mi RNA expression.The results showed that miR-24-1-5p low level expressed in AOM+DSS group mice,however after 12 weeks BRB anthocyanins consumption,the expression level was significantly up-regulated,which suggested miR-24-1-5p might play a tumor suppressive role during the tumor formation and chemprevention of BRB anthocyanins.(2)Expression of mi R-24-1-5p is down-regulated in CRC tissues and up-regelated in CRC cells lines treated with BRB anthocyanins:To further evaluate the results in colon tissue from microarray analysis,miR-24-1-5p expression in colon tissue from AOM+DSS group mice with or without BRB anthocyanins was confirmed by qRT-PCR.The data suggested that the expression pattern of miR-24-1-5p was consistent with previously.Meanwhile,the miR-24-1-5p expression was also determined in human colorectal cancer cell lines Caco2,HCT-116,Love,HT29 and SW480 treated with different doses BRB anthocyanins,the miR-24-1-5p level was increased by BRB anthocyanins in vitro,and the pattern of miR-24-1-5p expression were also consistent with the previous findings in colon tissues.(3)The effect of miR-24-1-5p on the biological behavior of colorectal cancer cells:In order to further evaluate the biological function of miR-24-1-5p,we used RT-qPCR to detect the transfection efficiency of CRC cell lines(HCT-116 and Caco2),and the expression of miR-24-1-5p was significantly increased.To prove that our transfection success,to be able to carry out the next test of the standard.MTT cell proliferation assay showed that the viability of miR-24-1-5p treated cells after HCT-116 and Caco2 cells transfected with mi R-24-1-5p plasmid 24 hours later in HCT-116 and Caco2 cells Respectively,56% and 52%,while the control cell viability was 100%,indicating that compared with the control cells miR-24-1-5p can significantly reduce the proliferation rate of CRC cells.The results of cell scratches showed that the scavenging distance was significantly increased in miR-24-1-5p transfected cells,indicating that miR-24-1-5p could significantly inhibit cell migration.Transwell experiments showed that miR-24-1-5p significantly reduced the number of cells observed under fluorescence microscopy in miR-24-1-5p transfected cells,thus demonstrating that miR-24-1-5p significantly inhibited CRC invasion.The number of clone formation of HCT-116-vector was 386.3,the number of clone formation of HCT-116-miR-24-1-5p was 130,the number of Caco2-vector clone was 607.3,Caco2-miR-24-1-5p clone formation number of 31,indicating that miR-24-1-5p can inhibit the formation of CRC cell clone formation rate.(4)β-catenin was predicted to be the target gene regulated by mi R-24-1-5p in CRC :Bioinformatics method mi Randa v3.3a with the default parameters and cutoffs(Score S ≥140 and Energy E ≤ –7.0)to predict the target genes that miR-24-1-5p may bind to.From all of the candidates,we selected β-catenin as a potential target considering the important role that β-catenin plays in colorectal cancer development.Furthermore,the mi R-24-1-5p binding sequence in the β-catenin 3’-UTR was highly conserved across species.Furthermore,we performed quantitative RT-qPCR and western blotting to investigate the influence of miR-24-1-5p over-expression on mRNA and protein levels of β-catenin.The expression levels of the β-catenin protein were significantly decreased by the introduction of miR-24-1-5p in the HCT-116 and Caco2 cells,but not the mRNA level of β-catenin.Next,miR-24-1-5p plasmid or control vector were transfected to HCT-116 and Caco2 cells for 24 hours,then the cells were harvested at different time points(0,10,12,14h)after protein synthesis inhibitor CHX(Cycloheximide,10μm)treated.The protein synthesis rate of β-catenin protein was significantly decreased in transfected miR-24-1-5p plasmids group compared with that in transfected control vector,indicating that miR-24-1-5p could inhibit the synthesis of β-catenin protein.These results demonstrate that miR-24-1-5p positively regulated the protein synthesis of β-catenin by directly binding to the 3’-UTR of β-catenin.(5)Genes associated wnt/β-catenin pathway was regulated by miR-24-1-5p:Further,the effects of over expressed mi R-24-1-5p on the downstream genes of β-catenin include cyclin D1,c-myc and CDK4 were also investigated in HCT-116 and Caco2 cells by using RT-qPCR and western blot,as the data shown that both the mRNA and protein level were decreased followed the β-catenin.Meanwhile,other genes related with Wnt/β-catenin pathway like SFRP2,SFRP5,E-cadherin,GSK3-β,p-GSK3-β,and BCL-2 were also detected,the expression of E-cadherin,p-GSK3-β and SFRP5 was up-regulated,but the expression of BCL-2 was down-regulated,and no significant changes was observed in GSK3-β and SFRP2 expression.Taken together,these results demonstrated that β-catenin might act as a direct target of miR-24-1-5p,besides β-catenin,the genes associated with β-catenin signaling pathway were also could be regulated by miR-24-1-5p. |
PDF Full Text Request