| Backgroud:Despite improvement in early diagnosis, gastric cancer is still considered to be one of the most common cancers in the world. It is increasingly recognized that gastric carcinogenesis is a complex process in which many environmental and genetic factors are implicated. Studies have shown that abnormalities of various oncogenes, tumor suppressor genes, growth factors and transcription factors may play important roles in tumor pathogenesis. These molecular changes could affect downstream signal pathways which involved in the control of cell growth and differentiation. Among them, miRNA has been considered to have an association with gastric cancer.MicroRNAs (miRNA) are short non-coding RNAs with a length of19-22nucleotides that function as post-transcriptional regulators by directly cleaving target messenger RNA (mRNA) or translational repression. Aberrant miRNAs in gastric cancer could regulate genes involved in proliferation, metastasis and invasion. Recent studies have investigated that miRNAs played an important role in regulating cell survival signal pathways involving Spl in different cancers. However, the mechanism is not completely clear.The transcription factor specificity protein1(Spl) also palyed an important roles in it. Spl has GC-rich promoter sequences and regulates transcription through these GC boxes. The human Spl gene maps to12q13.1and encodes a protein of785amino acids. It is a well-characterized, sequence-specific, DNA-binding protein that is involved in many cellular process. As a transcription factor, Spl can regulate thousands of genes, Thus, it is important for a variety of physiological processes, including cell cycle progression, angiogenesis, cell migration and invasion. Spl can affect tumorigenesis by modulating the expression of its target genes.Objetives:1.To investigate the expression of miR-145, miR-133a and miR-133b in gastric cancer by Real-Time PCR.2.To investigate the biological effects of miR-145, miR-133a and miR-133b in gastric cancer.3.To elucidate underlying mechanisms thatmiR-145, miR-133a and miR-133b played in gastric cancer.Methods:1. The expression level of miR-145, miR-133a and miR-133b in gastric cancer tissues and4human gastric cell lines (SGC7901, MKN45, BCG823and GES-1) were examined by Taqman real-time PCR analysis.2. CCK8(Cell Counting Kit-8) assays were conducted to examine the role of miR-145, miR-133a and miR-133b in the proliferation of gastric cancer cells,3.Transwell assays were performed in gastric cancer cell lines SGC7901, MKN45and BCG823to determine whether miR-145, miR-133a and miR-133b could regulate human gastric cancer cell migration and invasion,4.Flow cytometry was executed to determine the effects of miR-145, miR-133a and miR-133b on cell cycle alterations.5.Luciferase reporter assays were performed to further confirm that Spl is a direct target for these miRNAs.6. Spl expression in gastric cancer cells was analyzed by Western blot and immunofluorescence assay.7. To confirm whether Spl participate in the transcriptional regulation of MMP-9and cyclin D1in gastric cancer, the expression of Spl in SGC7901, MKN45and BCG823cells were knocked down by transfection with small-interfering RNA (siRNA).8.The expression of Spl, MMP-9and Cyclin Dlwereanalyzed by Western blot.Results:1. miR-145, miR-133a and miR-133b were significantly down-regulated in gastric cancer tissues compared with their matched adjacent normal tissues.Down-regulation of these miRNAs was also found in gastric cancer cell lines SGC7901, MKN45and BCG823compared with human gastric mucosal epithelial cell line GES-1.2. Ectopic expression of miR-145, miR-133a and miR-133b significantly suppressed cell proliferation in gastric cancer cell lines SGC7901, MKN45and BCG823.3. Ectopic expression of miR-145, miR-133a and miR-133b significantly decreased the migration and invasion capability in gastric cancer cell lines SGC7901, MKN45and BCG823.4. miR-145, miR-133a and miR-133b mimics induced G1cell cycle arrest in gastric cancer cell lines SGC7901, MKN45and BCG823, and the cells arrested in S phase were highly increased.5. Public database predicted that Sp1was a direct target gene of miR-145, miR-133a and miR-133b and luciferase reporter assays conformed this.6. Spl was over-expressed in SGC7901, MKN45and BCG823compared with GES-1by Western blot. Consistent with this, immunofluorescence staining of Spl was detected in SGC7901, MKN45and BCG823.7. Knock down of Sp1resulted in a decrease in the expression of MMP-9and Cyclin D1. And the expression of MMP-9and Cyclin D1were down-regulated in cells transfected with miR-145, miR-133a and miR-133b. Conclusions:miR-145, miR-133a and miR-133b were down-regulated in gastric cancer tissues and gastric cancer cells. The loss of miR-145, miR-133a and miR-133b promoted cell proliferation, migration, invasion and cell cycle progression in vitro. Sp1contains a binding site for these miRNAs at3’-UTR and was a direct target of them. miR-145, miR-133a and miR-133b inhibited cell malignant behaviors by negatively regulating Spl and its downstream molecules MMP-9, Cyclin Dl. Taken together, miR-145, miR-133a and miR-133b inhibited cell proliferation, migration, invasion and cell cycle progression via or partly through decreasing the Sp1expression and its downstream molecules MMP-9, cyclin D1. Backgroud:Lung cancer was the leading cause of cancer-related death in the world. Patients with non-small-cell lung cancer were mostly treated with platinum-based chemotherapy, often in combination with radiation therapy. However, the development of chemo-resistance, either intrinsic or acquired, was a major hurdle limiting successful treatment. Earlier studies had indicated the cytological mechanisms of drug resistance of cancer cells, such as increased detoxification of anticancer drugs by glutathione system, defective apoptosis pathway, increased levels of DNA repair or DNA tolerance, decreased uptake of water-soluble drugs and enhanced drug efflux from cancer cells mediated by ATP-binding cassette (ABC) transporters. Recently, researchers had found that epigenetic changes including aberrant CpG island methylation, histone modifications, and abnormal expression of microRNAs (miRNAs) might be responsible for the drug resistance of cancer cells. Specially, the epigenetic change opposed to genetic mutations, might play a more important role in acquired drug resistance of cancer cells. Among them, miRNAs, as a class of small non-coding RNAs of18-24nucleotides, were post-transcriptional regulators that binded complementary sequences of target mRNAs, usually resulting in translational repression or target degradation and gene silencing. By regulating gene expression at the post-transcriptional level, miRNAs had been linked to pathways related to cell differentiation, proliferation and survival, and their aberrant expression had been shown widely involved in drug resistance of different types of tumors, such as breast cancer, colorectal carcinoma, gastric cancer and glioblastoma multiforme. Recently, there was increasing interest in understanding the role of miR-503in cancers. miR-503was found down-regulated in oral cancer cells and hepatocellular cancer cells while over-expressed in parathyroid carcinoma, retinoblastoma and adrenocortical carcinoma. The up-regulation of miR-503was associated with shorter overall survival among patients with adrenocortical carcinoma. miR-503was a novel regulator of CUGBP1expression and thus increased the sensitivity of intestinal epithelial cells to apoptosis. Moreover, miR-503was able to reduce S phase cell populations and caused cell growth inhibition via suppressing the endogenous CCND1both at protein and mRNA levels, suggesting that miR-503might be a putative tumor suppressor. However, little was known about the effect of miR-503in the development of drug resistance in lung cancers.Methods:1.The level of miR-503was analyzed in A549/CDDP cells compared with their parent cell line by quantitative real-time PCR.2. MTT assay was conducted to examine the role of miR-503played in the regulation of drug resistance in human lung cancer cell line.3. Dual-luciferase activity assay was performed to further confirm that BCL2is a direct target for miR-503.4. Western blot analysis was executed to analyze the expression of BCL2.5.Flow cytometry was used to detect apoptosis.Results:1.miR-503was down-regulated in cisplatin resistant lung cancer cells A549/CDDP compared with the parental A549cells.2.Overexpression of miR-503sensitized A549/CDDP cells to cisplatin, whereas inhibition of miR-503in A549/CDDP cells increased cisplatin resistance.3.miR-503specifically targeted to BCL2, an anti-apoptotic protein up-regulated in A549/CDDP cells.4. BCL2was over-expressed in A549/CDDP cells compared with A549by Western blot.5.Ectopic expression of miR-503reduced BCL2protein level and sensitized A549/CDDP cells to cisplatin-induced apoptosis.Conclusions:The expression of miR-503was decreased in cisplatin resistant lung cancer cells and it could regulate cell apoptosis at least in part by targeting BCL2, thus modulated cisplatin resistance in non-small-cell lung cancer cells.
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