Preliminary Study On The Expression And Function Of MiR-17-5p In Serum Exosomes Of Patients With Colorectal Cancer

Part One Differential expression of miR-17-5p in serum exosomes of patients with colorectal cancerObjective:Exosome is a tiny vesicle of lipid bilayer membrane structure secreted by cells.It can be detected in a variety of body fluid samples.Its components include protein,lipid,nucleic acid and metabolites,which can reflect the pathophysiological status of the source cells.MicroRNA?microRNA?is a kind of endogenous non-coding RNA with a length of about 22 nucleotides.It can target and regulate the expression of many oncogenes or tumor suppressor genes,and participate in the regulation of tumorigenesis and development.MiR-17-92 is the first family of microRNAs found to be associated with tumors,which can participate in regulating the progression of digestive system tumors.MiR-17-5p is a core member of the family.Studies have shown that the expression of microRNA-17-5p is high in colorectal cancer.The purpose of this study is to investigate the expression level of microRNA-17-5p in serum exosomes of patients with colorectal cancer and its clinical significance.Method:1.The study included 32 patients with newly diagnosed colorectal cancer,including 16 patients without metastasis,16 patients with metastasis,and 15 volunteers at the same time as healthy controls.Serum exosomes were extracted using the Minute^TM Hi-Efficiency Exosome Precipitation Reagent kit,and the expression of serum exosome-specific surface markers CD9 and CD63 protein levels were detected by Western Blot.2.The exosome microRNA was extracted using the miRNeasy Mini Kit kit.The expression of miR-17-5p in serum exosomes was detected by real-time quantitative PCR?RT-qPCR?,and the correlation between its expression and clinicopathological features was analyzed.Result:1.Western Blot test results showed that the products extracted by the MinuteTM Hi-Efficiency Exosome Precipitation Reagent kit highly expressed exosomal specific surface markers CD9 and CD63 proteins.2.The results of RT-qPCR showed that the expression level of miR-17-5p in the metastatic group was significantly higher than that in the healthy control group and the non-metastatic group?P<0.05?.The expression level of miR-17-5p in the untransferred group was significantly higher than that in the healthy control group?P<0.05?.3.The expression of serum exosome miR-17-5p in colorectal cancer patients has no relationship with age,sex,tumor markers,tumor location,pathological type,differentiation degree,depth of invasion,lymph node metastasis,and tumor staging and far At the place of metastasis,the difference was statistically significant?P<0.05?.Part Two The effect of miR-17-5p on the biological function of colorectal cancer cells and its mechanism of actionObjective:MiRNA can regulate cell differentiation,development and growth.It has been reported that the miRNA-17-92 family can inhibit the expression of tumor suppressor genes and promote cell proliferation.The purpose of this part of the experiment is to study the expression of microRNA-17-5p in colorectal cancer cell lines and its effects on proliferation,invasion,migration,metastasis and apoptosis of colorectal cancer cells,and to explore its mechanism.Method:1.Total RNA was extracted from the cells transfected with microRNAs-17-5p mimics,mimics control,microRNAs-17-5p inhibitor and inhibitor control respectively.RT-qPCR was used to detect the expression level of miR-17-5p in each group.2.CCK-8,matrigel invasion,Transwell migration and scratch healing were used to detect the effects of over-expression/inhibition of microRNA-17-5p on proliferation,invasion,migration and metastasis of colorectal cancer cells.3.Annexin V and PI double staining flow cytometry were used to detect the effect of over-expression/inhibition of microRNA-17-5p on early apoptosis of colorectal cancer cell line HCT116.4.The cells were stained with EDU and PI,and the effect of overexpression/inhibition of miR-17-5p on the cell cycle of colorectal cancer cell line HCT116 was detected by flow cytometry.5.The target gene of miR-17-5p was predicted by bioinformatics software miRTarBase,and the expression level of target gene in colorectal cancer cell HCT116 was detected by RT-qPCR.Result:1.The results of RT-qPCR showed that the expression level of microRNA-17-5p in the cells transfected with microRNA-17-5p mimics was significantly increased?P<0.05?,and the expression level of microRNA-17-5p in the cells transfected with microRNA-17-5p inhibitor was significantly decreased?P<0.05?.2.CCK-8 results showed that over-expression of microRNA-17-5p could promote the proliferation of colorectal cancer cell line HCT116?P<0.05?.Inhibiting the expression of microRNA-17-5p could inhibit the proliferation of HCT116 cells?P<0.05?.3.Transwell chamber and scratch healing experiment showed that over-expression of microRNA-17-5p could promote the invasion and migration of colorectal cancer cell line HCT116?P<0.05?.On the contrary,inhibiting the invasion and migration of HCT116 cells after microRNA-17-5p was also inhibited?P<0.05?.4.Annexin V and PI double staining flow cytometry showed that over-expression of miR-17-5p could inhibit the early apoptosis of colorectal cancer cell line HCT116?P<0.05?.On the contrary,inhibiting the expression of microRNA-17-5p could promote the early apoptosis of colorectal cancer cell line HCT116?P<0.05?.5.Flow cytometry results showed that overexpression of miR-17-5p promoted G1/S phase conversion of colorectal cancer cell HCT116?P<0.05?.Inhibition of miR-17-5p expression resulted in G1/S phase arrest in colorectal cancer cell line HCT116?P<0.05?.6.Predicted by bioinformatics software miRTarBase,PTEN is the target gene of microRNA-17-5p.Overexpression of microRNA-17-5p can inhibit the expression of PTEN,thereby affecting PTEN-PI3K/AKT signaling pathway and promoting cell invasion and metastasis.Conclusion:The expression of microRNA-17-5p in serum exosomes of patients with colorectal cancer is significantly up-regulated,which is positively correlated with tumor stage and distant metastasis.Upregulation of MiR-17-5p expression can promote the invasion,migration and proliferation of colorectal cancer cells,inhibit cell early apoptosis,and inhibit PTEN-PI3K/AKT signaling pathway by targeting PTEN expression.