The Effect Of MiR-195-5p On Proliferation And Invasion Of Colorectal Cancer And Its Regulation Of NOTCH2 Signaling Pathway

Background Colorectal cancer(CRC)is one of the most common human cancers,which rank third and fourth in morbidity and mortality.The disease is more frequent in Europe,North America,Oceania and South Asia,however it is relatively rare in Central Asia and Africa,and the current incidence of the disease in China showed in a high level.The diagnosis and prognosis of CRC is still a difficult problem,about 20%of the CRC diagnosis has occurred in a distant shift and the current five-year survival rate of CRC in China remains only 31%.It's rather necessary and pivotal to make screening for populations with CRC for high levels of morbidity,long preclinical period,treatable precursor disorders,high treatment costs and high correlation between mortality and disease phase of it.Therefore,by exploring the mechanisms of occurrence and progression of CRC at the molecular level,we will find more specific and sensitive diagnostic markers and provide new targets and strategies for the diagnosis and treatment of CRC.MiRNA is a class of evolutionarily conserved short-chain RNA small molecule,about 22 nucleotides,whose main mode of action is by inducing mRNA degradation or inhibiting the translation of mRNA by binding to the mRNA 3'UTR of the target site resulting in a decrease in the protein level of the target gene.MiRNA is involved in a variety of biological processes,including cell proliferation,differentiation,invasion,migration and disease prognosis.MiRNA plays an important role in different types of cancer.A single miRNA can target multiple mRNAs simultaneously,while a single mRNA can also be regulated by multiple miRNAs at the same time.Because of the large number of miRNA present in the cell,this two-way regulation forms a complex network of intracellular networks that participates in many physiological processes and diseases in the body.In recent years,by studying the distribution of miRNA expression profiles in CRC tissue,it has been found that there are many miRNA in the CRC that are deficient in expression.Studies have shown that these specific miRNA are closely related to the pathogenesis,metastasis,recurrence and prognosis of CRC,and may serve as a potential biomarker for the diagnosis and treatment of tumors.Recently advances have been made in the study of the mechanism of miRNA in CRC,and studies have shown that certain miRNA is significant in terms of CRC carcinogenicity.So far,we have a part of the study of the mechanism of action of miRNA in cancer tissues,and their intrinsic mechanisms of action need to be further studied.Our study focuses on the biological functions of miRNA in CRC cancer cells from the specific miRNA in CRC tissue,and explores their effects on the development and progression of CRC in vivo and in vivo,with a view to early diagnosis and treatment of CRC provide new leads.Part ? The expression of miR-195-5p in colorectal cancer tissues and cell linesObjective To verify the expression of miR-195-5p in cancer tissue and cell lines screened from miRNA chip data of colorectal cancer.Methods The miRNA chip data of colorectal cancer on the common platform were selected and the miRNAs with high expression miRNA and low expression miRNA were screened out.From the low expression group,we further performed cluster analysis,and selected miR-195-5p,which had low expression and statistically significant significance,to explore its important role in colorectal cancer The We collected samples of colorectal cancer tissues and matched normal tissues from colorectal cancer surgery in Zhongnan Hospital of Wuhan University from 2011 to 2015.The selected 30 patients had not treated with radiotherapy before the surgery,and had a pathological diagnosis of relevant information.We used the method of qRT-PCR to detect the expression of miR-195-5p in 30 tissue samples.At the same time,human CRC cancer cells(HCT116 and DLD1)and normal intestinal epithelial cell line NCM460 were purchased from Wuhan University cell bank.The expression of miR-195-5p in human CRC cancer cells and normal intestinal epithelial cell line was detected by qRT-PCR.Results The expression of miR-195-5p in colorectal cancer tissues was significantly different from that in adjacent normal tissues,and the expression level of it in colorectal cancer tissues was significantly lower.The difference was statistically significant(P<0.001).This confirms the results of our preliminary chip synthesis analysis.At the same time,we also selected two colorectal cancer cell lines and a colorectal normal cell line.The results of cell experiments showed that miR-195-5p expression in colorectal cell lines was significantly lower than that in normal colorectal cancer cells.Conclusions The expression of miR-195-5p is low in colorectal cancer tissues and cell lines,and the next step will study the role of miR-195-5p in colorectal cancer.Part ? The function of miR-195-5p in human colorectal cancer cells and in vivoObjective To investigate the biological function of miR-195-5p in human colorectal cancer cells and to explore the effect of miR-195-5p on cancer cells in vivo.Methods To investigate the biological function of miR-195-5p in colorectal cancer cells,we perform transiently transfection of HCT116 and DLD1(human colon cancer cell lines)with the synthesized miR-195-5p mimetic and inhibitor and its control.The effect of miR-195-5p on tumor cell proliferation were measured by CCK8 and EdU assays.The effect of miR-195-5p on the cycle and apoptosis of tumor cells was detected by flow cytometry.The effect of miR-195-5p on the tumorigenesis of colorectal cancer cells was observed by Transwell assay.The effect of miR-195-5p on the tumorigenesis of colorectal cancer cells in vivo was conducted by nude mice tumorigenesis experiment.Results CCK-8 test showed that at 48 h and 72 h,the OD value of the cells overexpressing miR-195-5p group was significantly lower than the control group,and the OD value of the cells with the miR-195-5p inhibitor group was opposite.And EdU assays showed that overexpression of miR-195-5p significantly reduced the DNA synthesis of colon cancer cells.Compared with the control group,the cells with the miR-195-5p inhibitor group were increased and the cell proliferation was stronger than that of the control group the results of flow cytometry showed that there was no significant increase in the proportion of apoptosis cells in the cells group with miR-195-5p mimic or inhibitor with the control group.The Wound assay results showed that the activity of two colorectal cancer cells with overexpressing miR-195-5p was significantly inhibited after 48 hours compared with the control group,while the activity of two colorectal cancer cells with miR-195-5p inhibitor was enhanced(P<0.05).Transwell results showed that the migration and invasion of cells overexpressing miR-195-5p were significantly down-regulated compared with the control group,and the migration ability of cells in the miR-195-5p group was significantly higher than that in the control group Compared to rising.Finally,in nude mice tumor test,we confirmed that miR-195-5p can inhibit the tumorigenicity of subcutaneous colorectal cancer cell lines in nude mice,leading to a significant reduction in the size and weight of tumor tissue,indicating that miR-195-5p also has a significant anti-cancer effect in vivo.Conclusions miR-195-5p can regulate the biological function of colorectal cancer cells,miR-195-5p is a tumor suppressor miRNA,and it can inhibit tumor cell proliferation,invasion and migration ability.miR-195-5p involved in the inhibition of tumor tissue growth showed significant anti-cancer effect in vivo.Part ? The investigation of miR-195-5p targets in CRCObjective Predict the possible target of miR-195-5p and verify its targetMethods The possible targets of miR-195-5p were retrieved by bioinformatics,and the possible target genes were screened to determine their association with the occurrence and development of cancer.Based on the results of function,the binding of miR-195-5p to target gene was determined by double luciferase reporter assay.The regulation of miR-195-5p to target gene was further confirmed by qRT-PCR and Western blot.The effect of miR-195-5p on the predicted target gene was further validated in vivo by qRT-PCR,Western blot and immunohistochemistry.Results In order to explore the mechanism of action of miR-195-5p in cells,we selected several major bioinformatics predictive methods to search and screen miR-195-5p possible targets,and predicted miR-195-5p May be involved in the regulation of the NOTCH2 pathway.The results of double luciferase reporter assay showed that miR-195-5p could specifically bind to the predicted sites in 3'-UTR of NOTCH2 mRNA,and had the potential ability to regulate the expression of NOTCH2.The binding of miR-195-5p to NOTCH2 primitive matched sequence decreased significantly after this mutation.The results of qRT-PCR and Western blot showed that miR-195-5p analogues could decrease the expression level of mRNAs and proteins of NOTCH2 pathway in cells.And the expression level of these was improved after treated with miR-195-5p inhibitor.The expression levels of Jagged1,Jagged2,Heyl,Hesl and Smad3 were consistent with the expression levels of corresponding proteins in NOTCH2 pathway.The miR-195-5p was also able to inhibit the expression of NOTCH2 pathway-related mRNA and protein in vivo by qRT-PCR,Western blot and immunohistochemistry.Conclusions MiR-195-5p is capable of targeting the expression of the NOTCH2 pathway in colorectal cancer cells and in vivo.