| BACKGROUND&OBJECTIVEEach year, the American Cancer Society estimates the numbers of new cancer cases and deaths expected in the United States in the current year and compiles the most recent data on cancer incidence, mortality, and survival. Among men, cancers of the prostate, lung and bronchus, and colorectum will account for about half of all newly diagnosed cancers. The3most commonly diagnosed types of cancer among women in2012will be breast, lung and bronchus,and colorectum, accounting for about half of the estimated cancer cases in women.Colorectal cancer(CRC) is the third most commonly diagnosed types of cancer and alone will account for9%of incident cases in both women and men. In our country, with the improvement of living standards and dietary changes, colorectal cancer has been increasing gradually year by year. Metastatic disease is the major cause of death in colorectal cancer patients.MicroRNAs are small19to25nucleotide sequences of RNA found in both prokaryotes and eukaryotes that are intimately involved in cell differentiation, cell cycle progression, and apoptosis. MiRNAs act as endogenous suppressors of gene expression through imperfect binding of RISC to the3’-untranslated region (3’-UTR) of target mRNAs, inducing either translational repression or mRNA degradation.MicroRNAs have been demonstrated recently to potentially play a significant role in tumorigenesis. The study of microRNA has been extended into many types of cancer, including leukemias, lung, breast, and colon cancer. MicroRNAs are involved in the pathogenesis of CRC (Colorectal cancer), partly by regulating the expression of oncogenes and tumour suppressors and partly by functioning as oncogenes or tumour suppressors themselves. Researchers have proposed that specific microRNA expression patterns could help identify human solid tumors, suggest patient prognosis, and even represent a novel molecular target for cancer treatment.In a recent work Tavazoie et al. have reported that endogenous miR-335suppresses breast cancer metastasis and migration through targeting of the progenitor cell transcription factor SOX4and extracellular matrix component tenascin C. Expression of miR-335is lost in the majority of primary breast tumours, and miR-335is massively downregulated in drug-resistant ovarian cancer cell lines. miR-335is a negative regulator of hMSC proliferation, and overexpression of miR-335in hMSCs inhibites their proliferation and migration.Furthermore,miR-335might suppress gastric cancer invasion and metastasis by targeting Bcl-w and specificity protein1(SP1). glioma. Up to now,no functional evidence of miR-335in CRC has been documented. The expression level of miR-335and its possible role in CRC should be fully addressed.Based on the above findings, the aim of this study is to examine the expression of miR-335,identify its possible role in CRC pathogenesis, and elucidate the molecular mechanisms of its suppressive or oncogenic activities on CRC. We hope that this study will improve the better understanding of CRC pathogenesis and the development of novel effective therapies for CRC.Methods1.Identification of miR-335in Colorectal cancer cell linesReal-time RT-PCR was used to detect the expression of miR-335in8CRC cell lines,including SW480, SW620, HT29, LOVO, HCT116, Caco2, LS174T and DLD1.2.Identification of the target gene of miR-335(1). Three commonly used databases including TargetScan,Pictar and microRNA.org were used to forecast the target genes with the search terms of miR-335.mRNAS with high predictive values(at least predicted by three databases)were selected as candidates;(2). In four CRC cell lines, real-time RT-PCR were used to detect the the expression of miR-335and it’s target genes, respectively. Spearman correlation was used to analyze their expressions.(3). Recombined vector psiCHECK-2-RASA1containing RASA13’UTR was constructed, and site-directed mutagenesis vector named psiCHECK-2-RASAl-Mut was also established. Luciferase reporter system was used to validate the direct binding of RASA1and miR-335.3. The function of miR-335in CRC(1). miR-335precursor sequence was amplified from genomic DNA by PCR and was cloned into the lentivims vectors pLVTHM labeled with GFP.(2). The transfer vector and the packaging plasmids were co-transfected into293FT cells. The viral particles were used to infect different CRC cell lines.Flow cytometry was employed for sorting the GFP+cells.(3). CCK8method, colony formation assay, transwell in vitro invasion assay and wound-healing assay were carried out to detect cell proliferation, invasion abilities in vitro after upregulation of miR-335.4. The molecular mechanisms of miR-335involved in CRCWestern blot were used to detect the expression of RASA1,p-RASA1, Ras, ERK1/2and p-ERK1/2in SW620and HCT116overexpressed with miR-335.5. Statistical analysis SPSS13.0soRwarc Was used for statistical analysis. Data were presented as Mean±SEM of at least3independent experiments. Relative quantification value(2-△△Ct) of QPCR, colony formation assay, and transwell in vitro invasion assay were analyzed through One-way ANOVA, with the SNK, LSD,or Dunnett’s T3tests for multiple comparisons. The relationship between RASA1and miR-335expression was explored by Spearman’S correlation. Two-tailed Student’S t test was used for comparisons of relative luciferase activities. experiments. The results of CCK8assay was analyzed by Factorial design analysis of variance. P values of<0.05were considered statistically significant.Result1. Identification of expression characteristics of miR-335in CRC (Colorectal cancer) cell linesQuantitive real-time PCR was used to detect the expression of miR-335in8CRC cell lines. The results of One-Way-ANOVA ananlysis showed that the expression of miR-335in8cell lines was significantly different from each other(F=109.947, P<0.001). Except SW480cell line, mir-335expression was higher than other6cell lines compared with HCT116(P=0.028,0.006,0.023,0.007,0.012,0.008).The expression of mir-335in LOVO cell line was lower than in HT29(P=0.002)2. Bioinformatic prediction of miR-335targets and bioinformatic analysis of target genes(1). The following three bioinformatics algorithms which were utilized to predict miR-335target sites were miRBase, Pictar and TargetScan. The intersection of the3algorithms included more than100targets. Some of them play a significant role in tumorigenesis or migration,such as JAG1,MLLT3,JMJD2C,CD79B, WWP1,RASA1, SP1,SNIP1and MAX.(2).Quantitive real-time PCR was used to detect the expression of WWP1, SNIP1, RASA1and miR-335in4CRC cell lines. Real-time RT-PCR results showed that the expression of miR-335and RASA1had significant difference(F=11.934,P=0.003; F=489.764, P<0.001))in4colorectal cancer cell lines. The expressions of miR-335and RASA1had significant negative correlation in4CRC cell lines by Spearman correlation analysis(r=-0.797, P=0.002).(3). Recombined vector psiCHECK-2-RASA1containing RASA13’UTR was constructed, and mutation expression vector psiCHECK-2-RASA1-Mut was also established. The relative luciferase activity were decreased significantly in mir-335mimics group compared with NC group in293A and SW480cells (P=0.001、P=0.023) after cotransfection with psiCHECK-2/WT-3’UTR plasmid. However, there were no significant difference of luciferase activity after cotransfection with psiCHECK-2/MUT-3’UTR (P=0.379、P=0.953). These results indicated that mir-335inhibits the activity of luciferase through binding to RASA13’UTR.3. Effects of miR-335overexpression on biological behaviors of Colorectal cancer cell lines(1). The recombinant lentivirus plasmid pLVTHM-miR335was successfully constructed.(2)Three CRC-derived cell lines SW620-pLVTHM/miR-335HCT116-pLVTHM/miR-335and HT29-pLVTHM/miR-335were established with stable overexpression of miR-335.(3). Identification of expression characteristics of miR-335and RASA1in cell lines with stable overexpression of miR-335.Real-time PCR results showed that mir-335expression in cell lines overexpression of miR-335were significantly higher than blank group and pLVTHM group(P=0.046, P=0.046; P<0.001, P<0.001; P=0.039, P=0.038).RASA1expressions were significantly different from each other in the three goups of SW620and HCT116cell lines(F=28.362, P=0.001; F=12.251, P=0.008).However, there were no differences in the three goups of HT29cell lines (F=0.818, P=0.485).With further comparation,we found that mir-335 expression was significantly lower than pLVTHM group in SW620cell lines(P=0.002),but higher in HCT116cell lines (P=0.003).(4). The effects of mir-335on CRC cells’ behaviors in vitroa. CCK8assays showed that compared to blank or pLVTHM group, the proliferations of SW620and HT29cells were significantly enhanced in mir-335stable overexpression group(P<0.05). However, there were no differences in the three goups of HCT116cells(P>0.05).b. Colony formation assay showed that the ability of SW620cells to form colony were enhanced after transfection of mir-335(P<0.001,P<0.001). However, there were no differences between group with overexpression of miR-335and pLVTHM group in HCT116and HT29.c. In vitro invasion assays showed that migration of SW620,HCT116and HT29cells were markedly increased as compared with blank group and pLVTHM group by use of BD Transwell Inserts(P=0.020,P=0.012; P=0.004,P<0.001; P<0.001,P<0.001).d. Wound-healing assays showed that cells infected with pLVTHM/miR-335plasmid had significantly increased motility as compared with cells infected with pLVTHM plasmid in SW620,HCT116and HT29.4. The molecular mechanisms of miR-335involved in CRCWestern blot results showed that RASA1were decreased in both SW620/mir-335and HCT116/mir-335group.With the inhibition of RASA1, the expressions of p-RASA1,Ras,ERK1/2and p-ERK1/2were upregulated in SW620/mir-335group.But the expressions of Ras was down-regulated slightly in HVT116/mir-335group.Howrver, the amounts of total ERK1/2and phosphorylated ERK1/2had no significant differences in HVT116/mir-335group and the pLVTHM group. Conclusions:1. RASA1is the direct target gene of miR-335in colorectal cancer;2. Overexpression of miR-335promotes the cell proliferation, motility and colony formation rate in SW620cells, and enhances the cell motility in HT29and HCT116cells. But there are no effects on the ability of proliferation and colony formation in HT29and HCT116cells.3. miR-335can activate the Ras/MAPK signaling pathway by targeting RASA1, followed by the up-regulation of Ras, ERK1/2and p-ERK1/2.New findings:1. RASA1is the direct target gene of miR-335in colorectal cancer;2. Overexpression of miR-335can activate the Ras/MAPK signaling pathway by targeting RASA1, followed by the up-regulation of Ras, ERK1/2and p-ERK1/2. |
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