Screening The Target Gene Of MicroRNA-133b In Colorectal Cancer Cell Lines

Background:Colorectal carcinoma （CRC） is one of the common malignant tumors of the digestive tract and the incidence of CRC is on the rise worldwide. The development of CRC is related to abnormal expression and functional changes of tumor factors. MicroRNAs is a sort of endogenous non-coding RNA which consists of about22nucleotides and it regulates gene expression in a posttranscriptional manner by inhibiting translation of target mRNAs or even degrading them. In2003, Michael first reported that MicroRNAs are differentially expressed between human colorectal cancer and normal colorectal mucosa, and he detected expression changes in28kinds of miRNAs, among which the expression of miR-143and miR-145in cancerous tissue decreased obviously than that of normal tissue. Subsequent studies found numerous MicroRNAs involved in the development process of CRC and closely related to the diagnosis, staging, treatment and prognosis of CRC. MicroRNAs played a part in tumors as proto-oncogenes or cancer suppressor genes by regulating different target genes. miR-133b was initially considered to be a muscle-specific miRNA which was playing an important role in cardiac development and function maintenance. In recent years many research teams have found miR-133b which especially had a clear tumor inhibition effect expressed disorder in lung cancer, esophageal cancer, tongue carcinoma, gastric cancer, bladder cancer and so on. In China, Hu G confirmed that miR-133b in colorectal cancer tissues and cell lines significantly downgraded which was closely related to the degree of malignant colorectal cancer cells. Functional experiments further confirmed that miR-133b could inhibit the invasion and migration colorectal of cancer cells and had obviously effect in cell cycle blocking. Animal experiments also found that miR-133b had definite inhibitory effect on the tumor of nude mice. However, the precise role of miR-133b in CRC remains largely unknown and needs to be studied further.Objectives:This experiment was on the basis of preliminary experiments and intended to establish the colorectal cancer cell lines which miR-133b expressed highly by cell transfection, then compared with non-transfection of colorectal cancer cells for gene chip of high-throughput sequencing and gene function annotation until the target genes of miR-133b probably related to CRC were selected.Materials and Methods:Two colorectal cancer cell lines SW620and HT29were chosen in our study bought from ATCC cell bank, and divided into four groups to be transfection. They were experimental group for miR-133b mimics transfection group （Lipofectamine2000means Lipo2000transfection miR-133b mimics）, NC transfection group （Lipo2000transfection miR negative control）, Blank group （Blank, add an equal amount with the former two groups of Lipo2000, without miRNAs）, and Control group （Control, routine culture cells, without Lipo2000and miRNAs）. The cells were inverted after transfecting in fluorescence microscope to observe the fluorescent chromomeric condition used the method of real-time PCR to detect the expression of miR-133b in each cell, and evaluated its transfection efficiency. The two kinds of cells which were successful transfected by miR-133b mimics matching to corresponding NC transfection group were taken to do the Agilent SurePrint G3Human Gene Expression8x60K. The differential genes which detected according to the gene microarray were screened by the conditions such as down-regulated expression in miR-133b, P<0.05, FC （abs）≥1.5, and further compared with the miRNA target genes prediction software（miRBase, PicTar, TargetScan）. Finally the screened genes would be taken to GO molecular functions and Pathway signal Pathway enrichment degree of statistical analysis.Results:1. Detection of the expression of miR-133b in colorectal cancer cell line after transfectingThe optimal concentration of miR-133b mimics transfecting CRC cell line SW620and HT29was80nM and the transfecting efficiency could reach about50%. We detected the relative expression rate of miR-133b（RQ） in miR-133b mimics transfection group, NC transfection group, blank group and control group by Real-time PCR. Taken the relative expression rate of miR-133b in the control group to1, and we calculated RQ （RQ=2-△△CT） values of other groups. In cell line SW620, the expression of miR-133b （502.461±16.837） increased significantly in miR-133b mimics transfection group （P=0.000563）, the expression of miR-133b （0.841±0.305） in NC transfection group had no significant change （P=0.537648）, the expression of miR-133b （0.803±0.284） in Blank group had no significant change （P=0.431831）, and the expression of miR-133b had no difference between NC transfection group and Blank group（P=0.910242）. In cell line HT29, the expression of miR-133b （332.386±13.039） increased significantly in miR-133b mimics transfection group （P=0.000773）, the expression of miR-133b （0.870±0.219） in NC transfection group had no significant change （P=0.490357）, the expression of miR-133b （0.767±0.356） Blank group had no significant change （P=0.451923）, and the expression of miR-133b had no difference between NC transfection group and Blank group（P=0.807844）.2. Analysis of Agilent SurePrint G3Human Gene Expression8x60KWe used the Agilent SurePrint G3Human Gene Expression8x60K to detect the number of genetic differences, and34364genes in cell line SW620and39443in cell line HT29were detected. Under the conditions such as gene down-regulated expression in miR-133b, P<0.05, FC（abs）≥1.5, there were346genes in cell line SW620and354in cell line HT29. After using the miRNA target genes prediction software （miRBase, PicTar, TargetScan）, there were finally9genes.7genes in cell line SW620were:latent transforming growth factor beta binding protein1, contactin associated protein1, human immunodeficiency virus type I enhancer binding protein2, dual specificity phosphatase5（NM₀04419）, connective tissue growth factor, yippee-like2（Drosophila）, GABA（A） receptor-associated protein like1.2genes in cell line HT29were:family with sequence similarity19（chemokine （C-C motif）-like）, member A5, beta-1,3-N-acetylgalactosaminyltransferase1（globoside blood group）. The9target genes were analysed by GO molecular functions and Pathway signal Pathway enrichment degree statistically of Molecule Annotation System （MAS3.0）. B3GALNT1mainly located in the golgi membrane, related to magnesium ion binding, joined part of protein amino acid glycosylation and had transferase activity. CNTNAP1mainly located in cell membrane, as a kind of cell adhesion molecules, had receptor activity and signal transduction, and related to SH2/SH3binding. CTGF mainly located in cell membrane or cytoplasm, it was a kind of growth factor, related to integrin, insulin-like growth factor, fibroblast growth factor signaling pathway and cell morphology, motion, adhesion, migration, differentiation, epidermal growth, organization form, angiogenesis, ossification, cartilage condensation and other biological processes. DUSP5located in cell nucleus, mainly joined amino acid dephosphorylation and related to the activity of tyrosine phosphatase, hydrolase and MAP kinase phosphatase, and associated with signal pathway. GABARAPL1existed in everywhere in the cell, it could help regulate autophagy, combined with proteins such as GABA receptors, beta-tubulin and so on. HIVEP2mainly located in cell nucleus, related to DNA binding, metal ions binding and regulated the transcription process. LTBP1located on mesenchymal, cytoplasm and nucleus, related with calcium ion binding, growth factors binding, had the TGF-beta receptor activity, and participated in its signal path. FAM19A5mainly located in mesenchymal or membranes. YPEL2mainly located in the nucleus.Conclusion:1.We successfully transfected miR-133b mimics in colorectal cancer cell line SW620and HT29, and made miR-133b express highly in both of them.2.We screened9target gene of miR-133b by gene chip and target gene prediction software, as B3GALNT1, CNTNAP1, CTGF, DUSP5, GABARAPL1, HIVEP2, LTBP1, FAM19A5and YPEL2. After functional annotation we found some of them might participate in the development, invasion, metastasis and other important process of colorectal cancer.