MicroRNA-378Inhibits Cell Growthand Enhances L-OHP-induced Apoptosisin Human Colorectal Cancer

Colorectal cancer is one of the most common diseases in the digestive system.MiRNA shows significant importance in development and progress of cancer.MiRNAcan bind to3’UTR of target gene mRNA and be involved in disease development andprogress.In the treatment of colorectal cancer, drug resistance is a key factor to affectthe disease healing. To now, many miRNAs were found to be involved in the sensitivityand mechanism of drug usage. The aim of this research was to investigate the biologicalfunction of miR-378in colorectal cancer.This research investigated the function of miR-378in colorectal cancer from fourpart. PartⅠ: We aimed to detect of miR-378expression in colorectal cancer tissues andcancer cell lines, and biological function in cell lines. PartⅡ: Through biologicalbi-west and report, we aimed to found the target gene of miR-378. PartIII: We aimed toinvestigate the miR-378function in L-OHP-induced apoptosis. PartIV: We aimed toexplore miR-378function in nude mice xenograft model.Methods: PartⅠ: Through real-time quantative PCR, we detected the expressionof miR-378in20colorectal cancer tissues and its adjacent normal tissues;It wasdetected in six kind of colorectal cancer cells:HCT116, HT29, DLD-1, LOVO, SW620,SW480and two kind normal colorectal epithelial cells FHC, NCM460; It also wasdetected in the serum of51patient. We also analyzed the correlation between miR-378expression level and survival curve. In colorectal cancer cells HCT116and HT29,transfection of miR-control or miR-378mimics was done to over-express miR-378.Then with the assays of MTT, clone formation and cell cytometry, we detected thechange of cell clone formation、cell growth curve and cell cycle.PartⅡ: Thorough thebioinformatics blast, we aimed to find the target of miR-378. Through the plasmidconstruction assay, we constructed plasmids containing CDC40mRNA3’UTR widetype and mutation type sequence. Through luciferase reporter assay，we detected thebinding activity of miR-378to CDC403’UTR. Through real-time quantative PCR andwestern blotting, we detected CDC40mRNA and protein level with overexpression ofmiR-378. We transfected cells of HT29and HCT116with si-control and CDC40 siRNA-1/2to detect CDC40function in colorectal cancer cells. Through theknockdown of CDC40, we detected its function in cell clone formation, growth and cellcycle. In the rescue assay, we transfected HT29and HCT116cell with mimics control，pCMV6vector+miR-378mimics, pCMV6/CDC40+miR-378mimics respectively.Through the overexpress of CDC40, we detected the rescue efficiency underoverexpress of miR-378. Then with the assay of clone formation, we detected therescue efficiency. PartIII: In colorectal cancer cells HCT116and HT29, we transfectedwith miRNA mimics and miR-378mimics. Then detect the apoptosis rate throughAnnexin-V/7-AAD and cell cytometry. After transfected with miRNA mimics andmiR-378mimics, dealing the cells with different concentration of L-OHP and detectedthe cell activity by MTT assay. Dealing the cells with4μM L-OHP for72h and thendetected the cell apoptosis rate by Annexin-V/7-AAD and cell cytometry. TUNNELwas also used to detect cell apoptosis. PartIV: We transfected miR-control and miR-378mimics into HCT116. We injected cell with100Ul cell suspensionsin right dorsal flankper mouse. Tumors were examined every3days; length, width, and thicknessmeasurements were obtained with calipers and tumor volumes were calculated. Tumorvolume was calculated using the equation (L*W2)/2.On day27after cells inoculation,the animals were killed, tumors were excised and weighed.Results:PartⅠ:Expression and Function of MiR-544in Colorectal CancermiR-378was showed significant lower level in colorectal cancer tissues comparedto its adjacent normal tissue and cell lines（P＜0.05）. MiR-378showed significantlower level in the serum of patients with colorectal cancer compared to the serum ofvolunteer（P＜0.05）. Analysis of correlation between miR-378expression level andpatient survival curve showed that they had no correlation. The results of MTT assayshowed that overexpress of miR-378inhibit cell growth significantly compared tocontrol cells. The results of cytometry showed that overexpression of miR-378inhibited cell cycle at G0/G1.PartⅡ: MiR-378Regulate Cell Growth and Cell Cycle by Targeting CDC40In the database of PicTar and TargetScan, we found miR-378can bind to sequenceof CDC40mRNA3’UTR which is AGUCCAG.The results of luciferase assay showsthat miR-378can target CDC40mRNA3’UTR. In the colorectal cancer cells of HT29and HCT116, overexpress of miR-378can inhibit the expression of CDC40mRNA and protein（P＜0.05）.In colorectal cancer cells, transfection of CDC40siRNA-1/2inhibited the expression of CDC40.Knockdown of CDC40inhibited cell growth andprohibited cell cycle to G0/G1. In the rescue assay, transfection of CDC40expressplasmid enhanced the CDC40protein level.CDC40overexpression in miR-378overexpress cells can rescue cell proliferation ability.PartIII: Overexpression of MiR-378Enhanced L-OHP Induced Apoptosis.In colorectal cancer cells,overexpression of miR-378cannot affect cellapoptosis.In colorectal cancer cells HCT116and HT29, we analyze the IC50afterdifferent concentration of L-OHP treatment. Overexpression of miR-378enhancedL-OHP induced inhibitory effect. After4μM L-OHP treatment, overexpression ofmiR-378enhanced L-OHP-induced apoptosis. The results of TUNNEL showed thesimilar function.PartIV: Detection of MiR-378Effect on Tumor Formation in vivoOverexpress of miR-378inhibited tumor growth in nude mice. It showed slowervolume growth compared to control. The P value is lower than0.01. Overexpression ofmiR-378inhibited tumor weight.Conclusion:(1): miR-378was showed low-expression in colorectal cancer tissues and celllines, and high-expression in patient serum. Expression level of serum miR-378wasshowed no correlation with survival curve.(2): In vivo and in vitro test showed miR-378can regulate colorectal cancer cellgrowth.(3): miR-378can regulate colorectal cancer cell growth by targeting CDC40.(4):Overexpression of miR-378enhanced L-OHP-induced apoptosis.