| Objective:The purpose of this study was to investigate the expression level of miR-504 in colorectal cancer,to analyze the correlation between miR-504 and clinicopathological parameters and clinical prognosis of patients with colorectal cancer.To further study the effect of miR-504 on proliferation,apoptosis,migration and invasion of colorectal cancer.This study aimed to explore the inhibitory effect of miR-504 on the progression of colorectal cancer by targeting GDF11 possibly,in order to provide a new diagnostic method or potential therapeutic target for colorectal cancer.Methods:1.The cancer and corresponding paracancerous tissues of 40 patients with colorectal cancer were collected,and the expression level of miR-504 in the cancer and adjacent tissues of 40 patients with colorectal cancer was detected by qRT-PCR method.Then the expression of miR-504 in normal intestinal epithelial cells FHC and several colorectal cancer cell lines was detected by qRT-PCR method.2.The relationship between the expression of miR-504 and the clinicopathological features of patients with colorectal cancer was analyzed.Kaplan-Meier survival curves were used to analyze the relationship between the expression of miR-504 and patient survival characteristics.Multivariate analysis was used to analyze the expression of miR-504 and the prognosis of patients with with CRC.3.Transfecting miR-504 inhibitor in HCT116 cells with relatively high miR-504 expression and miR-504 mimics in SW480 cells with relatively low miR-504 expression.After verifying the transfection efficiency with qRT-PCR,CCK8 and clone formation experiments were used to detect the effects of miR-504 on cell proliferation.Flow cytometry was used to detect the apoptotic rate of cells in HCT116 and SW480 cells.Next,the effects of miR-504 on cells migration and invasion ability were investigated by using wound healing and Transwell assays respectively.4.The databases were used to analyze whether the target of miR-504 is GDF11,and the relationship between miR-504 and GDF11 is further verified by WB experiments.Results:1.The qRT-PCR results showed that the expression of miR-504 in colorectal cancer tissues was lower than that in the corresponding adjacent tissues,and the expression level of miR-504 in colorectal cancer cell lines was significantly lower than that of normal intestinal epithelial cells FHC.2.According to the expression level of miR-504 in colorectal cancer samples,they were divided into high expression group and low expression group.Statistics show that the expression of miR-504 is significantly related to TNM stage(P<0.001),lymph node metastasis(P=0.002)and distant metastasis(P=0.008)in colorectal cancer patients.Kaplan-Meier survival curve analysis further showed that patients with high miR-504 levels of colorectal cancer had higher overall survival rates.Multivariate analysis showed that miR-504,TNM stage,lymph node metastasis and distant metastasis were independent prognostic factors affecting the overall survival rate of patients with CRC.3.The results of CCK8 experiments showed that,after transfection of miR-504 inhibitor in HCT116 cells,compared with the NC group,the cell viability was significantly increased.After miR-504 mimics transfection in SW480 cells,compared with NC group,cell viability was significantly decreased.The results of the cloning experiment were consistent with the results of the CCK8 experiments.Flow cytometry results showed that apoptosis was significantly reduced after transfection with miR-504 inhibitor,while apoptosis was significantly increased after transfection with miR-504 mimics.The results of wound healing and Transwell tests showed that the cell migration distance and the invaded cell number were significantly increased.in miR-504 inhibitor-transfected cells compared with cells transfected with the NCs.In contrast,transfecting the cells with the miR-504 mimics significantly diminished the cell migration distance and the invaded cell number.They were further shown that miR-504 inhibits the progression of colorectal cancer.4.The results of database analysis showed that GDF11 is a target of miR-504.Western blot was used to verify the changes of GDF11 expression in CRC cell lines upon miR-504 modification.Further,WB results showed that the expression of GDF11 was significantly increased after miR-504 inhibitor was transfected into HCT116 cells.After transfection of miR-504 mimics into SW480 cells,the expression of GDF11 decreased significantly,which was negatively correlated with the expression of miR-504.This showed that miR-504 inhibits the progression of colorectal cancer by targeting GDF11 possibly.Conclusions:1.miR-504 is low-expressed in colorectal cancer.2.miR-504 is positively correlated with survival rate and is an independent prognostic factor for colorectal cancer.3.miR-504 inhibits the proliferation,migration and invasion of colorectal cancer,and promotes apoptosis of colorectal cancer cells by targeting GDF11 possibly. |
PDF Full Text Request