| Object:Colorectal cancer(CRC)is the most common type of cancers in the current world and 90%of its mortality is accounted by metastasis.epithelial-mesenchymal transition EMT,It refers to the process by which epithelial cells with polar characteristics can be transformed into interstitial cells with active ability through specific procedures and acquire the ability of invasion and migration.Angiogenesis,It refers to the growth of new capillaries derived from existing capillaries and capillaries.Tumor angiogenesis is an extremely complex process,generally including endothelial substrate degradation of endothelial cells,endothelial cell migration,proliferation,endothelial cells,pipelining branches form a loop and form new basement membrane and so on.Both play an important role in the development of colorectal cancer.Connective tissue growth factor(CTGF)is a member of the CCN family,secreting polyfunctional proteins,which contain high levels of cysteine.Connective tissue growth factor CTGF is reported to promote the progression of multiple cancers.MicroRNAs(miRNAs)is a kind of small non-coding RNA,species are widely kept in between,at present,a series of research evidence suggests that miRNAs are closely associated with the occurrence of tumor development,can affect the rights of the cell colonization,invasion and migration and epithelial-interstitial transformation,angiogenesis.MicroRNAs associated with cancer,now discovered-part to promote the development of tumor,can promote the effect of malignant tumor,while others can inhibit the occurrence of tumor development,play the role of inhibition of malignant tumor.The report showed that mir-218 was significantly reduced in the sample of cancer patients and played a role in the development of cancer,which has been identified as a tumor suppressor miRNA.However,little is known about the role of mir-218 in colorectal cancer.Now,we studied the miR-218 for colorectal cancer cells of epithelial-mesenchymal transition and the influence of the angiogenic process,and expounds through targeted CTGF regulation of colorectal cancer epithelial mesenchymal transformation and the mechanism of angiogenesis.Methods:Part 1:1.Cultured colorectal cancer cell line SW620,SW480,HCT8 and HCT116 and human normal colonic epithelial cell line NCM460 w(ATCC USA),The expression of mir-218 in tissue and four colon cancer cell lines was verified by qPCR,and two lower expressions were selected for follow-up experiments.2.After transfection with mir-218mimic which was established in the mimic NC group and mir-218 mimic group.After transfection with mir-218mimic,the expression of mir-218 was incresed that verified by qPCR.;3.After transfection,a blank control group,mimic NC group and mir-218 mimic group were established,and MTT verified the influence of mir-218 mimic on the proliferation of colon cancer cell lines;4.Set up blank control group,mimic NC group,miR-218 mimic,Transwell assay validation of miR-218 mimic effect on migration and invasion of colon cancer cell line;5.A blank control group,mimic NC group,mir-218 mimic group and colony formation assay were established to verify the influence of mir-218 mimic on colon cancer cell line cloning.Part 2:1.In colorectal cancer cell lines(SW620,HCT116),the blank control group,mimic NC group and mir-218 mimic group were established respectively,and the increase of mir-218 expression was verified by rt-qpcr,and the successful transfection was verified.2.The effect of mir-218 on epithelial-mesenchymal transformation was verified by Western blot test(e-cadherinis,a-catenin,Vimentin,fibronectin)3.Colorectal cancer cell line(SW620,HCT116)respectively set up blank control group,mimic the NC group,miR-218 mimic group,by miR-218 transfection or control under the condition of colorectal cancer cell line(SW620,HCTI116)medium(CM)dealing with human umbilical vein endothelial cells(HUVEC)4.With the experimental measurement on the form(the tube formation assay)were determined in colorectal cancer cell line(SW620,HCT116)blank control group,mimic NC group,miR-218 mimic endothelial cells forming the ability of capillary structure5.W620,HCT116 respectively set up blank control group,mimic the NC group,miR-218 mimic group,miR-218 expression by RT-qPCR rise,after the success of the validation of transfection,by Western blot method to detect changes(AngptⅡ,VEGF-A)and RT-qPCR detection(AngptⅡ,VEGF-A))mRNA level change,validation of miR-218 effects on tumor angiogenesis(angiogenesis).Part 3:1.The target gene of mir-218 was predicted by Targetscan software,and we found that mir-218 could target Connective tissue growth factor CTGF gene,and construct CTGF overexpression plasmid and transfection cells.2.In colorectal cancer cell line(SW620,HCT116)respectively set up blank control group,mimic the NC group,miR-218 mimic group,through the RT-qPCR detection of colorectal cancer cell line(SW620,HCT116)CTGFmRNA levels of change and by Western blot method to detect CTGF expression level changes3.Using siRNA silence CTGF in colon cancer cell line,by Western blot method to detect(E-cadherinis,a-catenin,Vimentin and fibronectin)and(AngptⅡ,VEGF-a)),research of colorectal cancer epithelial-interstitial cell transformation and blood vessel growth.Results:Part 1:1.Cultured colorectal cancer cell line SW620,SW480,HCT8 and HCT116 and human normal colonic epithelial cell line NCM460 w(ATCC USA);The expression of mir-218 in tissue and four colon cancer cell lines was verified by Rt-qPCR,and two lower expressions were selected for follow-up experiments.After transfection with mir-218mimic which was established in the mimic NC group and mir-218 mimic group.After transfection with mir-218mimic,the expression of mir-218 was incresed that verified by qPCR.2.After transfection,MTT verified the inhibitory eftfect of mir-218 mimic on colon cancer cell lines.3.Transwell assay results show that mir-218 mimicry reduces the migration and invasion of colon cancer cell lines.4.Colony formation assay results showed that mir-218 mimic inhibited the formation of colon cancer cell line clones.Part 2:1.In colorectal cancer cell lines(SW620,HCT116),a blank control group,mimic NC group,mir-218 mimic group were established,and mir-218 expression was increased by Rt-qPCR,2.After the successful transfection,the expression of e-cadherinis and a-catenin in mir-218 mimic group was up-regulated by Western blot method,while the expression of Vimentin and fibronectin in mir-218 mimic group was down-regulated,indicating the inhibitory effect of mir-218 mimic on the tumor angiogenesis of CRC.3.Angiogenesis also plays important role in tumor development.To investigate whether miR-218 could regulate tumor angiogenesis,the tube formation assay was used,which can measure the capacity of endothelial cells to form capillary-like structures.Human umbilical vein endothelial cells(HUVECs)were treated with conditioned medium(CM)from SW620 or HCT116 transfected with miR-218 mimic or controls.As shown,miR-218 over expression significantly suppressed tube formation ability of HUVECs by decreasing the number of branching point,indicating an inhibition effects on tumor angiogenesis of CRC.4.By Western blot method to detect(AngptⅡ,VEGF-A)and RT-qPCR detection(AngptⅡ,VEGF-A))mRNA level,according to the results in the miR-218 mimic group significantly lowered,it shows that the inhibition of tumor angiogenesis of CRC,through the above experimental results show that miR-218 by adjusting the EMT and angiogenesis related genes to suppress the progress of the CRC.Part 3:1.The CTGF was constructed and transfected into cells,and the transfection gene was proved to be successful by double luciferase assay2.By RT-qPCR detection of colorectal cancer cell line SW620,HCT116)in miR-218 mimic group CTGFmRNA expression decreased obviously,by Western blot method to detect colorectal cancer cell line(SW620,HCT116))in miR-218 mimic group CTGF expression level also decreased obviously3.Using siRNA silence CTGF in colon cancer cell line,by Western blot method to detect E-cadherinis,a-catenin expression in the miR-218 mimic group,Vimentin and fibronectin in miR-218 mimic group expression,and(Angptll,VEGF-a)levels,according to the results in the miR-218 mimic group lowered,It indicates that mir-218 can inhibit the expression of EMT related markers and angiogenesis by targeting CTGF.Statistical analysisGraphPadPrism version 6.0 software(GraphPad,USA)was used to analyze differences between two groups with student’s t-test.A two-tailed P<0.05 was significant,and data were presented as the mean ± SD.Conclusion:miR-218 directly targets CTGF and inhibits its expression,leading to suppression on EMT and angiogenesis of CRC cells.miR-218 might be used as potential therapeutic strategy for CRC treatment. |
