The Expression And Function Study Of MicroRNA-133b In Colorectal Carcinoma

Colorectal cancer (CRC) is one of the leading causes of cancer related mortality worldwide. Analysis of the CRC genome and proteome has demonstrated that focusing on molecular heterogeneity within CRC may be a viable approach to identify and develop novel therapeutics. Recent data suggest that dysregulation of microRNAs is an important step in the development of many cancers, including CRC. MicroRNAs consist of-22 nucleotides and regulate gene expression in a posttranscriptional manner by pairing with complementary nucleotide sequences in 3'-UTRs of target mRNAs. Although at present the precise biological effects are not well understood, miRNAs appear to be crucial factors in diverse regulation pathways, including development, tumorigenesis, cell differentiation, proliferation, and apoptosis. Recent studies have shown that some miRNAs play roles as tumor suppressors or oncogenes in cancers, depending on whether they specifically target tumor suppressor genes or oncogenes, respectively.miR-133b was initially considered to be a muscle-specific miRNA, Through targeting critical genes involved in cardiac development and cardiac channel expression, miR-133b is implicated in the regulation of cardiac myogenesis and development and cardiac ion channel expression. Recently, investigators determined that miR-133b harbored anti-tumorigenic properties in tongue squamous cell carcinoma through direct targeting of pyruvate kinase type M2. Studies have demonstrated that miR-133b was significantly downregulated in several CRC cell lines, as well as in colorectal tumors, compared with nonneoplastic tissues. However, the precise role of miR-133b in CRC remains largely unknown.This article intended to study "The relationship between miR-133b and colorectal cancer". Inspired by the phenomenon that "miR-133b was significantly downregulated in CRC tissues compared with nonneoplastic tissues", we assumed that whether miR-133b as a tumor suppressor or potential cancer therapeutic target in colorectal cancer? Accordingly, the design of this experiment was as follows:1. To investigate the expression levels of miR-133b in normal human colon cells (CCD-18Co) and four colorectal cancer cell lines with different malignant potential, and exploring the relationships between the expression level of miR-133b and tumor differentiation and metastasis.2. miR-133b genomic sequence would be constructed into the eukaryotic expression vector. CRC cells with low miR-133b expression level were stable tansfected with the identified plasmid by Effectene, and then, the cell line with stable expression of miR-133b would be obtained.3. To explore the effects of miR-133b on proliferation, apoptosis, migration and invasion activities in colorectal cancer cells, and then the targets genes of miR-133b would be predicted by a web-based computer program.4. Establishing the subcutaneous tumor model of colorectal cancer in nude mice. Research on the therapeutic effects of miR-133b on bearing-tumor node mice of human coloretal cancer, which can further confirm the role of miR-133b on biological behaviors of colorectal cancer in vivo.ObjectiveTo explore the expression pattern of miR-133b in colorectal carcinoma cell lines with different malignant potential.MethodsThe expression of miR-133b in normal human colon cells (CCD-18Co) and four colorectal cancer cell lines (SW-480,HT-29,LoVo,SW-620) with different malignant potential were examined by real-time PCR. ResultsThe miR-133b expression level in CCD-18Co was much higher than in the four cancer cells (SW-480, HT-29, LoVo, and SW-620), with the lowest showing a 4-fold increase in expression (p<0.05). This downregulation of miR-133b is associated with malignant potential of colorectal cancer cell.Conclusion1. miR-133b expression shown to be greatly downregulated in human colorectal cancer cells compared to normal colon cells and this downregulation is associated with malignant potential of colorectal cancer.2. The expression of miR-133b was lowest in SW-620 cells, and this cell line was selected for further experiment.ObjectiveTo construct eukaryotic expression vector of miR-133b and stable tansfection into human colorectal cancer cell SW-620.Methods1. According to the sequence of pre-miR-133b, design and synthesis the double-stranded nucleotide sequence of Pre-miR-133b. To construct the recombinant plasmid pGenesi 1-1-miR-133b, then get plasmid sequencing.2. SW-620 cells were transfected with pGenesil-1-HK or pGenesil-l-miR-133b plasmid, stable transfectants were then selected by incubation with geneticin G418 and maintained in medium containing 800μg/mL G418, SW-620 cell lines with stable expression of miR-133b or control were obtained, designated SW-620-133b and SW-620-HK, respectively. 3. The expression of miR-133b in SW-620,SW-620-HK and SW-620-133b were detected by real-time quantitative PCR.Results1. Construction of miR-133b eukaryotic expression vector pGenesil-l-miR-133b, sequencing result is correct.2. After a 4-week selection period in medium supplemented with G418 (800μg/mL), SW-620 cell lines with stable expression of miR-133b or control were obtained, designated SW-620-133b and SW-620-HK, respectively.3. The expression of miR-133b was dramatically greater in SW-620-133b compared with SW-620-HK and SW-620. Since miR-133b was not detected in either SW-620 or SW-620-HK, LoVo cells were selected as a standard, in which case a 39.4-fold greater expression was observed in SW-620-133b compared with this standard (p<0.05).ConclusionThe eukaryotic expression vector of miR-133b was successfully constructed. SW-620 cell lines with high expression of miR-133b or control were obtained, designated SW-620-133b and SW-620-HK, respectively.ObjectiveResearch on the effects of high expression miR-133b on the cell proliferation, apoptosis, migration and invasion ability in SW-620 cells and predict the possible targets of miR-133b.Methods1. The experiments were consists of three groups:SW-620 cell group, SW-620-HK cell group and SW-620-133b cell group. 2. MTT assay and plat clone formation assay were employed to measure cell proliferation of SW-620, SW-620-HK and SW-620-133b cells.3. We assessed for the induction of apoptosis in each group through the measurement of flow cytometry for Annexin/PI.4. The effect of miR-133b on migration of SW-620 cells was investigated using a wound-healing assay to evaluate cell migration.5. Matrigel invasion assay in Transwell culture chambers to determine the effect of miR-133b on in vitro SW-620 invasion.6. The possible targets of miR-133b were predicted by a web-based computer program and initially verified by RT-PCR and Western blot.Results1. MTT assay shown that markedly retarded cell proliferation rates were observed in SW-620-133b cells compared to SW-620 and SW-620-HK (p<0.05). Plat clone formation assay was performed to confirm this observation. The SW-620 and SW-620-HK cells grew and formed large colonies than SW-620-133b within 4 wks. The number of colonies in SW-620-133b (106.9±13.6) cells was significantly lower than SW-620 (396.6±43.1) and SW-620-HK (385.4±40.8) cells (p<0.05).2. Fluorescence-activated cell sorting assay:the result showed that as comparing to the SW-620 (4.26±0.65)% and SW-620-HK (4.98±0.71)% group, the percentage of apoptosis cells in SW-620-133b (16.75±2.35)% was markedly increased (P＜0.05), there was no significant difference between SW-620 and SW-620-HK group.3. The wound-healing assay:SW-620-133b (42.1±3.7)%cells exhibited a notable decrease in closure or migrating ability compared with SW-620-HK (74.6±5.2)% or SW-620 (77.9±5.6)% cells (p<0.05), suggesting a role for miR-133b in the migration of SW-620 cells.4. Tanswell invasion assay:the average invasion cells of each group was as follows:SW-620 group, (420±31.5)/HP; SW-620-HK group, (396±30.2)/HP; SW-620-133b group, (108±12.5)/HP. The number of SW-620-133b cells that passed through Matrigel was much lower compared with SW-620 and SW-620-HK cells (p<0.05).5. MET and MMP9 are listed as targets candidate for miR-133b interaction according to a prediction by the web-based microRNA targets prediction program. As confirmed by Western blot and RT-PCR, MET and MMP9 expression was significantly less in SW-620-133b cells compared with SW-620-HK or SW-620 cells (p<0.05); but MET and MMP9 mRNA concentrations were unaffected in these three groups.Conclusion1. In cancer SW-620 cells, ectopic expression of miR-133b potently inhibits tumor cell proliferation, migration and invasion, meanwhile promotes apoptosis in vitro.2. MET and MMP9 are the candidate targets of miR-133b. miR-133b might have negatively regulated MET and MMP9 at the posttranscriptional level.ObjectiveTo explore the growth inhibition effects of miR-133b on planted subcutaneous tumors in human colorectal tumor-bearing nude mice. Further validation of miR-133b to be a new tumor suppressor in vivo.Methods1.30 nude mice were randomized into 3 groups and each group contained 10:SW-620 cell group, SW-620-HK cell group and SW-620-133b cell group.2. To establish the animal model of human colorectal tumor-bearing mice and calculate the tumor-formation rate.3. The volumes of planted subcutaneous tumors were measured per 5 days after tumorigenesis for mapping tumors growth curves.4. Nude mice were sacrificed to obtain tumor 30 days after injection, recorded and compared the tumor weight in each group.5. The cell apoptosis of planted tumor were detected by TUNEL assay.6. The expression levels of MET and MMP9 in tumor were measured by immunohistochemisty.Results1. The model animals of human colorectal tumor-bearing mice were successfully established, the tumor-formation rate was 100%.2. The growth curves of planted tumor were drawed, as compared to SW-620 and SW-620-HK group, the tumor growth rate of SW-620-133b group was much slower (p<0.05). At the end point time, the average tumor volume of SW-620-133b group (0.85±0.14)cm3 was lower than SW-620 (3.56±0.79)cm3 and SW-620-HK group (3.24±0.68)cm3 (p<0.05); the average tumor weight of SW-620-133b group (0.92±0.16)g was lower than SW-620 (4.12±0.64)g and SW-620-HK group (3.95±0.61)g (p<0.05);3. As shown by TUNEL assay, the AI value of SW-620-133b group (30.28±3.47)%was significantly higher than that of SW-620 (7.96±0.92)%and SW-620-HK group (7.26±0.84) (p<0.05), but with no significant difference between SW-620 and SW-620-HK group.4. The results of immunohistochemisty showed that the expression of MET and MMP9 protein was significantly down-regulated in SW-620-133b group compared with SW-620 and SW-620-HK group (p<0.05).Conclusion1. The ectopic expression of miR-133b can effectively inhibite the growth of tumor and promote apoptosis in human colorectal tumor-bearing nude mice.2. The expression of MET and MMP9 was significantly down regulated in SW-620-133b group, miR-133b might suppress tumor growth through modulation of the MET and MMP9 signaling pathway.