The Role And Mechanisms Of MicroRNA-185-5p-YWHAZ Axis In Invasion,metastasis And EMT In Colorectal Cancer

Colorectal cancer is the third most common cancer and the third leading cause of cancer death in the world.It has high morbidity and mortality rates,seriously endangering the health of people and the quality of life.The occurrence of metastasis to distant organ is the main cause of failure in colorectal cancer treatment.Latest research shows that the epithelial-to-mesenchymal transition(EMT)is a key step in the metastasis of tumors.To understand EMT-specific molecular mechanisms in colorectal cancer and to utilize the key molecular target genes to reverse EMT is an effective intervention strategy in the treatment of colorectal cancer.It has been found some molecules are closely associated with EMT and invasion in colorectal cancer,which give us some clues in understanding the mechanisms of EMT.However,the occurrence of EMT involves multiple molecules and signaling pathways.There is a need to find more molecular targets and pathways to better comprehend the underlying mechanisms of EMT and metastasis.microRNAs are important endogenous non-coding small RNAs and play an important role in the development of tumor.Current research suggests that a single microRNA plays a regulatory role by negative control of target genes in tumor invasion and EMT.SinglemicroRNA can regulate the expression of multiple target genes at the same time.So far only a few microRNAs were confirmed to participate in the EMT of tumor development.Our previous study indicates that microRNA-185-5p was down-regulated in SW620 cell line compared with SW480 cell line.The results suggest microRNA-185-5p inhibits invasion and EMT in CRC cells.However the molecular mechanisms of microRNA-185-5p and its target gene in colorectal cancer remain to be elucidated.【Objective】 To understand the role and mechanisms of microRNA-185-5p-target gene axis in invasion,metastasis and EMT in colorectal cancer.【Methods】 1.Bioinformatics prediction,m RNA chip and i-TRAQ protein spectrum technology were used to screen important functional target genes of microRNA-185-5p.Dual luciferase reporter gene experiment was used to verify the target gene of microRNA-185-5p.2.q RT-PCR and Western blotting were used to further validate the expression of the target genes in CRC cell line SW480 and SW620.3.Immunohistochemical staining was used to test the expression of target gene YWHAZ in colorectal cancer specimens,and its correlation with clinical pathological parameters were analyzed.4.Transwell experiment,cell immunofluorescence staining,and Western blotting were used to verify the regulatory effect of target gene YWHAZ on invasion,migration and EMT phenotype of colorectal cancer.The function of target gene YWHAZ in vivo was verified through tumor formation experiment in nude mice via tail vein.5.Transwell experiment and Western blotting experiments were used to confirm that microRNA-185-5p inhibit invasion,migration and EMT through binding to the target gene YWHAZ.【Results】 1.By using four bioinformatic prediction softwares(Taget Scan,mi Randa,CLIP-Seq,mi RDB)to predict the functional target genes of microRNA-185-5p,111 molecules were preliminarily ascertained as the candidate of functional target genes after analysis.m RNA chip and i-TRAQ protein spectrum technology results suggested that YWHAZ is the potential target gene of microRNA-185-5p.Dual luciferase reporter gene results indicated the luciferase activity can be inhibited by microRNA-185-5p via binding with 3’-UTR of YWHAZ m RNA directly.2.Western blotting and q RT-PCR experiment results suggested that compared with SW480 cell line,YWHAZ expression was significantly increased in SW620 cell line.3.Immunohistochemical staining suggested YWHAZ expression was increased significantly compared with adjacent normal tissue in the specimens of 90 colorectal cancer cases.Statistical analysis showed the expression level of YWHAZ was significant correlated with lymph node metastasis,TNM stages and survival.4.Transwell experiments suggested that cell invasion and migration ability was significantly enhangced after down-regulating YWHAZ expression by using small interference RNA in SW620 cell line.Cellular immune fluorescence experiment,western blotting and q RT-PCR results suggested that EMT phenotype could also be reversed by YWHAZ.To demonstrate the function of YWHAZ in vivo,colorectal cancer cells were injected into nude mouses via tail vein.The results suggested that the liver metastasis was decreased significantly in mice injected with SW480 cells overexpressing YWHAZ.5.The expression of YWHAZ was increased significantly by down-regulating the expression of microRNA-185-5p in the SW480 cell line.In SW620 cell line,YWHAZ expression was significantly decreased by up-regulating the expression of microRNA-185-5p.Binding assays showed microRNA-185-5p inhibited invasion,metastasis and EMT of colorectal cancer cell through binding to the target gene YWHAZ.【Conclusion】 YWHAZ participates in promoting the invasion and metastasis and regulating the phenotype of EMT in colorectal cancer cells.So it could be a new biomarker in prognosis and treatment of colorectal cancer.The expression level of YWHAZ in the tissue of colorectal cancer was significantly correlated with poor prognosis which provides a strong evidence for predicting survival in patients with colorectal cancer.Micro RNA-185-5p regulates invasion and metastasis and EMT of colorectal cancer cell through binding to the target gene YWHAZ.