Experimental Study Of MiR-125a-5p Inhibiting Tumor Growth By Targeting Smurf1 In Colorectal Carcinoma

Colorectal carcinoma is the third largest cancer in the world,causing more than 500,000 deaths each year.A variety of risk factors,including lifestyle,environmental factors,gene mutation are considered to be one of the causes of the development of colorectal carcinoma.Colorectal carcinoma is a continuous process from adenoma to cancer,accompanied by the changes in morbidity and mortality.According to the reports,although the incidence of colorectal carcinoma has declined,the optimal screening strategy has not yet been determined,so it is extremely important to find new biomarkers for colorectal carcinoma.Recently,some studies have pointed out and confirmed that MicroRNA(miRNA)plays a strong role in the diagnosis and prognosis of colorectal carcinoma.miRNA,a short non-coding gene,is an important human gene expression controller.There are no less than 1,500 miRNAs in the miRNA information bank,and in the transcriptional correction system for special gene expression,it plays an important role through the 3' terminal untranslated region(UTR),and thus affects the development of cellular programs and disease states.The abnormal expression of MicroRNA can cause the initiation and development of various human cancers.A variety of miRNAs have been shown having a strong correlation with the occurrence,development,and prognosis of colorectal carcinoma,but the role of miR-125a-5p remains unclear.In this study,we observed the regulation of miR-125a-5p in Smurf1 expression in vitro and in animal experiments.First we confirmed that in CT26 cells,Smurf1 is a downstream target gene of miR-125a-5p,and its expression is regulated by miR-125a-5p.At the cellular level,highly expressed miR-125a-5p can inhibit the proliferation and migration of CT26 and SW620 cells.In order to confirm whether the effect of miR-125a-5p on cell proliferation and migration is based on the down-regulation of Smurf1,considering with related literature,we have added the miR-125a-5p + Smurf1 group,and the results were compared with those of miR-125a-5p group.The comparison confirms that miR-125a-5p regulates cell proliferation and migration through Smurf1.The results of in vitro experiments may lead to different roles of miR-125a-5p in vivo due to the more complicated environment in vivo.Therefore,in the third part,we established a mouse model of xenograft with CT26 cells transfected with miR-125a-5p.The amount of Smurf1 expression was used to evaluate the antitumor effect of miR-125a-5p in vivo.These results showed that miR-125a-5p can be a suppressive factor for colorectal carcinoma through the target of Smurf1,and this study may promote the development of diagnostic and therapeutic technologies for colorectal carcinoma.Part one The study of miR-125a-5p regulating the expression of Smurf1 by targeting 3'UTR of Smurf1Objective: To determine the effect of miR-125a-5p on the expression of Smurf1 in colorectal carcinoma,by determining the site of action of miR-125a-5p and Smurf1 and measuring the transcription and translation levels of Smurf1.Methods: Target Scan was used to screen the Smurf1 locus,and a fluorescent gene-containing plasmid vector containing a wild-type 3'UTR or mutant 3'UTR containing Smurf1 gene was constructed and transferred into cells.The cells were then transfected with negative controls and miR-125a-5p for luciferase experiments to verify that miR-125a-5p acts at the 3?UTR of Smurf1.RT-q PCR experiments and western blot were used to compare the amount of Smurf1 m RNA and the expression of Smurf1 protein transfected with miR-125a-5p and negative.Results: 1.Target Scan predicted that the binding site of miR-125a-5p to Smurf1 is at 3?UTR of Smurf1.2.miR-125a-5p had no significant effect on the fluorescence activity of the mutant Smurf1 gene transfection group.3.miR-125a-5p significantly inhibited the fluorescence activity of the wild-type Smurf1 gene transfection group.4.The acting site of miR-125a-5p is located at 3?UTR of Smurf1.5.Western blotting showed that the experimental group transfected with miR-125a-5p significantly reduced the amount of smurf1 protein compared with the control group.6.RT-q PCR results showed that the smurf1 m RNA in the experimental group transfected with miR-125a-5p was significantly reduced compared with the control group.Conclusion: miR-125a-5p may suppress the expression of Smurf1 by directly targeting the 3'UTR of Smurf1.Part two The study of miR-125a-5p inhibiting the proliferation and migration of CT26 cells and SW620 cells through Smurf1Objective: To analyze whether miR-125a-5p can regulate the proliferation and migration of colorectal carcinoma cells through Smurf1,considering that miR-125a-5p can inhibit the expression of Smurf1.Methods: In CT26 cells and SW620 cells,cell groups transfected with miR-125a-5p,miR-125a-5p + Smurf1,and NC were established.Differences among the above groups were detected by CCK-8 cell counting test and wound healing assays.Results: CCK-8 counting test showed that transfection of miR-125a-5p can significantly reduce the proliferation of CT26 cells compared with the control group.Transfection of miR-125a-5p + Smurf1 can significantly increase the proliferation of CT26 cells compared to the group transfected with miR-125a-5p.CCK-8 counting test showed that transfection of miR-125a-5p can significantly reduce the proliferation of SW620 cells compared with the control group.Transfection with miR-125a-5p + Smurf1 can significantly increase the proliferation of SW620 cells compared with the transfected miR-125a-5p group.Wound healing assays showed that miR-125a-5p can significantly reduce the migration of CT26 cells compared with the control group.miR-125a-5p + Smurf1 can significantly increase the migration of CT26 cells compared with the group transfected with miR-125a-5p.Wound healing assays showed that miR-125a-5p can significantly reduce the migration of SW620 cells compared with the control group.miR-125a-5p + Smurf1 can significantly increase the migration of SW620 cells compared to the miR-125a-5p group.Conclusions: 1.Highly expressed miR-125a-5p can significantly reduce the proliferation and migration capacity of CT26 cells and SW620 cells.2.Smurf1 can significantly increase the proliferation and migration capacity of CT26 cells and SW620 cells.3.miR-125a-5p can inhibit the proliferation and migration of CT26 cells and SW620 cells by down-regulating Smurf1.Part Three The study of miR-125a-5p inhibiting tumor growth in nude miceObjective: To analyze the effect and function of miR-125a-5p on tumor cells in vivo.Methods: CT26 cells were transfected with miR-125a-5p and negative control genes,and nude mice were used to establish a mouse transplant model.The expression of miR-125a-5p in the experimental group and the control group was tested by RT-q PCR technology.The size and weight of the transplanted tumor were observed and compared with the control group;immunohistochemical staining were performed to determine the expression of Ki-67 protein and the results were compared with the control group;HE staining was used to count the cells and the results were compared with the control group;western blot technology was used to compare the expression of Smurf1 protein in the experimental group and the control group.Results: 1.The expression of miR-125a-5p in tumor tissue of experimental group was significantly higher than that of control group.2.Compared with the control group,the experimental group showed reduced size and weight of tumor tissue.3.In immunohistochemical staining,Ki-67 protein in the experimental group was significantly lower than that in the control group.4.Counting results of HE stained cells showed that the experimental group had significantly fewer cells than the control group.5.Western blot showed that the expression of Smurf1 in the experimental group was significantly lower than that in the control group.Conclusion: In animal experiments,miR-125a-5p can inhibit the growth of colorectal carcinoma by down-regulating Smurf1.