Expressions, Clinical Significance And Roles Of MiR-214and MiR-10a In Colorectal Adenocarcinoma

Objective: To confirm if miR-214and/or miR-10a would be biomarkersfor colorectal adenocarcinoma diagnosis, and understand the role of miR-214in the development of tumor and the pathogenesis of colorectaladenocarcinoma that two miRNAs are involved in, the relationship betweenthe expressions of miR-214or miR-10and pathological characteristics ofcolorectal adenocarcinoma was identified by detecting the expressions ofmiR-214and miR-10a in colorectal adenocarcinoma and normal mucosa weremeasured using real time PCR. In addition, miR-214and miR-10a expressionswere also determined in two colon carcinoma cell lines, HCT116^p53+/+andHCT116^p53-/-in order to confirm if there is a relationship between twomiRNAs and p53. Furthermore, colony formation, MTT assay and FCM wasused to observe the effect of miR-214on proliferation and apoptosis of coloncell lines.Methods：1. Forty four cases of fresh colorectal tissue specimens from Hebei MedicalUniversity Fourth Hospital and Hebei Medical University First Hospitalbetween May2011and May2012were collected, including colorectaladenocarcinomas and colorectal marginal mucosa tissues.2. The expressions of miR-214and miR-10a were examined in colorectaladenocarcinoma and normal mucosa using real time PCR to confirm therelationship between the miRNA expressions and the clinicopathologicalcharacters of colorectal adenocarcinoma.3. The expressions of miR-214and miR-10a were also determined in9colon carcinoma cell line(sSW620、SW480、SW1116、HT29、DLD1、LOVO、Caco-2、HCT116^p53+/+、HCT116^p53-/-).4. The expressions of miR-214and miR-10a were detected in9colon carcinoma cell lines. In addition, the levels of both miRNAsin HCT116^p53+/+and HCT116^p53-/-was compared to determine the relationship between themand p53gene.5. Two transfected colon carcinoma cell lines HCT116and SW1116withmiR-214minic were used as an experimental group; two cell lines transfectedwith only vehicles and non-transfected cell lines were used as a control groupand a blank group, respectively.6. Colony formation assay were performed in blank, control andexperimental groups to identify the effect of miR-214on proliferation ofHCT116cell and SW1116cell.7. MTT assay were conducted in blank, control and experimental groups,OD value were measured at12,24,36,48,60h after transfection to obtaincell growth rates.8. Flow Cytometry were performed in blank, control and experimentalgroups to evaluate the effect of miR-214on apoptosis of HCT116cell.9. All obtained experimental data were analyzed by SPSS13.0statisticalsoftware. Wilcoxon-test or t-test was used to analyze the differences of pairedsamples, while Mann-Whitney-test was applied to compare the differencebetween independent samples, two-way ANOVA to analyze repeatedmeasurement data. A P<0.05was regarded as statistical significant.Results:1. The expressions of miR-214and miR-10a in colorectal adenocarcinomatissue and matched normal mucosa tissueThe expressions of miR-214in colorectal adenocarcinoma tissue andmatched normal mucosa tissue were0.0264（0063,0.0591） and0.0505（0.0250,0.1121）, respectively. The miR-214expression in colorectaladenocarcinoma was markedly enhanced as compared withnormal mucosatissue, there was a significant difference between them （Z=-2.591, P=0.0097）.The expressions of miR-10a in colorectal adenocarcinoma tissue and matchednormal mucosa tissue were0.0264（0.0063,0.0591） and0.0505（0.0250,0.1121）, respectively. The miR-10a expression in colorectal adenocarcinoma was higher than that in normal mucosa tissue, a significant difference waspresent（Z=-2.486, P=0.0131）.2. Relationship between the expressions of miR-214and miR-10a andclinicopathological features of colorectal adenocarcinomaThe expressions of miR-214and miR-10a were correlated with Histologicaltypes. The levels of miR-214and miR-10a in adenocarcinoma were higherthan those in mucinous adenocarcinoma, there were significant differences （P=0.0138and P=0.0224, respectively）. But its expression was not correlatedwith ages, sex, neoplasm location, lymph node metastasis and clinical stages（P>0.05）.3. The expressions of miR-214and miR-10a in colon cellsThe expressions of miR-214and miR-10a in colon carcinoma cell linesSW620、SW480、SW1116、HT29、DLD1、LOVO and Caco-2were higherthan that in normal mucosa tissue. No significant difference in the expressionsof miR-214and miR-10a was present between HCT116^p53+/+cell and HCT116^p53-/-.4. The effect of miR-214on cell colony formation of colon carcinoma cellsThere was no significant difference in the number of cell colony betweenblank group and control group in SW1116cell, but the cell colony number inexperimental group was higher than that in control group, there weresignificant differences（P=0.0006）. Similarly, the number of cell colony didnot show a significant difference between blank group and control group inHCT116cell; however, more cell colonies in experimental group wereproduced than those in control group（P=0.0269）5. The effect of miR-214on cell proliferation of colon carcinoma cells byMTT assayAt12,24,36,48,60h after transfection, the OD values of MTT assay didnot show significant difference between blank group and control group inHCT116and SW1116cell lines; the OD values in experimental group werehigher than those in control group at every interval of12hours, there weresignificant different（P<0.0001）. 6. The effect of miR-214on cell apoptosis of colon carcinoma cell by FCMFlow Cytometry data did not show that the significant difference intheapoptosis rate between blank group and control group in HCT116cell; theearly apoptosis rate in experimental group was higher than that in controlgroup, there were significant different（P=0.0104）, but the late apoptosis ratedid not show significant difference.Conclusions:1. The expressions of miR-214and miR-10a were at a low level incolorectal adenocarcinoma. miR-214and miR-10a might play an importantrole as anti-oncogenes in the pathogenesis of colorectal adenocarcinoma.2. The expressions of miR-214and miR-10a in mucinousadenoadenocarcinoma were lower than that in non-mucinous adenocarcinoma.Their lower expressions was correlated with production of mucus3. miR-214and miR-10a expressions were downregulated in colorectalcarcinoma regardless of the presence of p53. They might act as a tumorsuppressor in colorectal carcinoma through p53-independent pathways.4. miR-214and miR-10a could inhibit proliferation and induce apoptosis ofcolon carcinoma cells5. It is necessary to investigate the mechanism of miR-214and miR-10a astumor suppressors in colorectal adenocarcinoma. It provids effective help toelucidate the pathogenesis of colon adenocarcinoma, and new idea for earlyserodiagnosis for colorectal adenocarcinoma patients.