| Since1993, the first microRNA was found, more new miRNAs weredetected.miRNAs are a class of small, regulatory, non-coding RNAs and can bind with the3’-untranslated region (3’UTR) of the target mRNAs. In different human cells,miRNAs have abundant expression. A large number of studies show that, aberrantexpression of specific miRNAs has been investigated in all human cancers, andmiRNAs can be important roles in tumorigenesis, such as apoptosis, cell proliferation,differentiation and metastasis. Functional studies performed on animal models ofvarious cancers or in cancer cell lines suggest that, miRNAs can function as tumorsuppressors, oncogenes, and regulate the cancer pathways.Many studys have revealed the aberrant expression of miRNAs in colorectalcancer cell lines by deep sequencing, real-time quantitative PCR or microarray. Theresults are confirmed the stability in different papers. The aberrant expressions ofmiRNAs in colorectal cancer cell lines suggest that, miRNAs may be a vital factor inprocess of colorectal cancer development.At present, the selection of treatment strategy and predicting of therapeuticclinical prognosis are based on clinical factors like clinical stage or TNM stage, tumorinvasion, lymph node metastasis or distant metastasis. The therapeutic strategy of nonmetastasis colorectal cancer is operation combined with postoperative chemotherapy.But patients with high preoperative stage have much higher recurrence rate. From themacro perspective, the low degree of differentiation, high clinical grading orlymphatic invasion and other histological factors indicate the high risk of tumorrecurrence, but the possible molecular mechanism is still unknown. Therefore it isnecessary to find a stable, effective biomarker and new therapeutic target forcolorectal cancer. Besides the mechanism study in cell lines, research on clinicalsamples has become a meaningful way for miRNAs screening. Cancer tissue andparacancerous tissue, peripheral blood, stool are common clinical materials.Quantitative detection of miRNAs expression in tissue samples can help find theaberrant expression miRNAs between colorectal cancer tissue and paracanceroustissue for searching the potential markers. The abnormal expression of miRNAs may be associated with tumor occurrence and development or other clinical features ofcancer patients. When the abnormal expression miRNAs were found, the special cellmodel or animal model could be setted by transfecting the mimics, inhibitor oragomiR, antagomiR.Then, through the up-regulation and down-regulation of theaberrant expression miRNAs, we can investigate the cell biological behavior andspeculate miRNAs function. Using the various target genes database，predict thetarget of miRNA and verify it by dual-luciferase reporter assay system. The result mayreveal that, the miRNAs can regulate tumor’s pathways. Afterall, clinical samplesstudies are still necessary. To assess the clinical value of altered miRNAs expresson incolorectal cancer, the relationship between miRNAs expression andclinicopathological factors should be evaluated, including age, gender, tumor size,location, histological grade, clinical stage, lymph node metastasis, venous invasion,and liver metastasis. Analyzing the survival data and miRNAs can investigate therelationship between miRNA expression levels and patients’prognosis.According to earlier microarray sequencing test, miR-376a/-301b/-196b werefound to be abnormal expression in colorectal cancer. But those are not verified bydeep sequencing or real-time quantitative PCR. In other kinds of human cancers, thethree miRNAs of miR-376a, miR-301b and miR-196b were reported and proved that,they may play important roles in malignant tumors development. The expression levelof miR-376a is associated with tumor metastasis and patients’ prognosis in prostatecancer. MiR-301b can promote the drug resistance of pancreatic carcinoma. Theexpression of miR-196b in lung cancer is helpful to predict the prognosis of patients.This study will measure the differential expression levels ofmiR-376a/-301b/-196b between colorectal cancer tissues and paired adjacent normaltissues by real-time quantitative PCR. Evaluate the relationship between miRNAsexpression and clinicopathological factors, predict prognosis. Set up abnormalexpression cell model to investigate the influence of miR-376a, miR-301b andmiR-196b on cell biological behavior. Thus,our study may suggest a meaningfulprognostic biomarker and potential therapeutic target in colorectal cancer..1. Materials and Method1.1Sample and cell cultureSurgical specimens of cancer tissue and paired adjacent normal mucosa wereobtained from53patients with colorectal cancer. Colorectal cancer cell lines: SW480 SW116and HCT116were cultured in H-DMEM medium.1.2Information collectionInformation of clinical pathology was collected from53patients as well aspostoperative follow-up data.1.3Isolation of total RNAsTotal RNAs of colorectal tissues and cell lines were isolated,and qRT-PCR wasperformed using the All-in-OneTMmiRNA qRT-PCR Detection Kit (tail mathod). U6was selected to be the endogenous control.1.4Immunohistochemistry (IHC)HC staining was carried out on4μm thick sections of paraffin-embedded tissue.After deparaffinization, rehydration and antigen retrieval, VEGF IHC was performed.1.5Cell transfectionUsing the Lipofectamine2000transfection reagent to transfect the mimics,inhibitor, inhibitor control and mimics control into the SW480cell line. qRT-PCR canconfirm the model’s establishment.1.6MTS assayCellTiter96AQueous One Solution Cell Proliferation Assay (MTS）can detectthe cell proliferation ability.1.7Colony formation assayWe seed500cells per hole with low serum culture medium in6well plate andculture for14to21days. When the colony can be observed by naked eye, terminatethe culture.After dyeing, tell the cell models’difference of cell colony forming ability.1.8The scratch test1.9Statistical analysisAll statistical analyses were performed using SPSS13.0software (SPSS). The figureswere maked by GraphPad Prism5software. We used Wilcoxon matched pairs tests,2test,Fisher’s test and log-rank test. Kaplan–Meier curves were used to analyze the survival dataand a Cox proportional hazards model was used for multivariate analysis of survival data.Differences were considered to be significant when p<0.05.2Results2.1Expression levels of miRNAs in colorectalcancer, normal colorectal tissueand cell linesMiRNAs expression levels in53pairs of cancer, normal colorectal tissues and cell lines were detected by qRT-PCR. The results showed miR-376a and miR-301bwere significantly reduced (p<0.01) while the expression level of miR-196b wasincreased in cancer tissue and cell lines.As the altered expression of miR-376a and miR-301b, they might be associatedwith colorectal cancer progression.2.2Clinicopathological significance and prognosis of miRNAs expressionAltered high miR-376a expression tends to be associated with lymph nodemetastasis, VEGF positive and have a shorter survival. Moreover, multivariateanalysis indicates that high expression of miR-376a is an independent, significantprognostic factor for colorectal cancer patients’survival.There is no obvious relationship between miR-301b and patients’ Clinicopathologicalsignificance or prognosis datas.MiR-196b expression was correlated with tumor differentiation.2.3Cell biological behavior after miR-376a, miR-301b transfectionIn the biological behavior of experiment on SW480cells, we found inhibition ofmiR-301b expression can enhance cell viability, colony forming ability, acceleratewound healing, but there was no difference in the up-regulation of miR-301b.Up-regulation of miR-376a expression can significantly improve the celslproliferation, colony formation ability and migration ability. When transfectingSW480cells with miR-376a inhibitor, reducing the expression of miR-376a resultedin inhibit cell’s corresponding abilities. Further，miR-376a may play an important rolein colorectal cancer progression.3Conclusions3.1A positive correlation was observed between high miR-376a expression andlymph node metastasis. And miR-376a expression might relate to patients survival incolorectal cancer.3.2MiR-376a was associated with proliferation and migration of colon cancer cells.3.3MiR-196b expression was correlated with tumor differentiation.3.4MiR-376a may be a meaningful prognostic biomarker and potential therapeutictarget in colorectal cancer. |
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