| Objective: To examine the expression of microRNA-224（miR-224）incolorectal carcinoma and paired normal mucosa using by Taqman real timePCR and study the relationship between the expression and theclinicopathological characteristics of patients. Then to observe the effect ofmiR-224on proliferation and apoptosis of colon cell lines by colony formation,MTT assay and FCM. To evaluate diagnosis value of miR-224for colorectalcarcinoma as tumour marker, elucidate the biological function of miR-224inthe development of tumor, provide theoretical and experimental basis for earlydiagnosis of colorectal carcinoma.Methods：157paired fresh colorectal tissues and their matched normal mucosatissues from Hebei Medical University Fourth Hospital and Hebei MedicalUniversity First Hospital between October,2011and October,2012werecollected.2Examination the expressions of miR-224in colorectal carcinoma andnormal mucosa using by Taqman real time PCR, and study the relationshipbetween the expressions and the clinicopathological characteristics of patients.3Cell culture was performed for colon cell lines(SW620, SW480,SW1116, HCT116, HT29, DLD1, LOVO, Caco-2). The expression ofmiR-224in colon cell lines were detected by Taqman real time PCR, andcompared with normal tissues.4The expression of miR-224in HCT116p53+/+and HCT116p53-/-wascompared to analyze the expression in relation to p53. Examination theexpression of miR-224after transfection48h in the three groups.5miR-224mimics was transfected into colon cancer cell lines HCT116,paralleled with blank and negative control groups. 6Colony formation assay were performed in blank, control andexperimental groups to analyze the effect of miR-224on proliferation ofHCT116cell.7MTT assay were performed in blank, control and experimental groups,OD value were detected respectively after1,2,3,4,5day of transfection andthen cell growth curve were drew.8Flow Cytometry were performed in blank, control and experimentalgroups to analyze the effect of miR-224on apoptosis of HCT116cell.9All experimental data were analyzed by SPSS13.0statistical software.The results were measured by （meansstandard±deviations） or median（quartile）. Wilcoxon-test or t-test was used to analyze comparison of pairedsample, Mann-Whitney-test to analyze independent sample, two-Way ANOVAto analyze repeated measurement data. P<0.05was regarded as statisticalsignificance.Results:1The expressions of miR-224in colorectal carcinoma tissue and matchednormal mucosa tissue were1.812(0.8685，4.653) and0.4073（0.1870，0.8515）respectively. The miR-224expression in colorectal carcinoma was higher thanthat in normal mucosa tissue, there were significant differences (p<0.0001).2The expression of miR-224was correlated with pathological type. Theexpression of miR-224in rectal cancer, Duke’s C and D，with lymphaticmetastasis were higher than that in colon cancer, Duke’s A and B,withoutlymphatic metastasis, there were significantly differences (P=0.0215, P=0.0239, P=0.0488). But its expression was not correlated with ages, sex,pathological type, the depth of invasion and the degree of differentiation.(P>0.05).3The expression of miR-224in colon cancer cell lines SW620, SW480,SW1116, HCT116, HT29, DLD1, LOVO, Caco-2were higher than that innormal mucosa tissue.4The expressions of miR-224did not show significant differencebetween HCT116p53+/+cell and HCT116p53-/-(P>0.05). 5The expression of miR-224in experimental group higher than that incontrol group and blank group after24h of transfection.6The number of cell colony did not show statistical significance betweenblank group and control group in HCT116cell, but the cell colony number inexperimental group(153.67±13.48) was higher than that in controlgroup(82±6.11), there were significantly differences（P=0.0397）7After1,2,3,4,5day of transfection, the OD value by MTT assay didnot show statistical significance between blank group and control group inHCT116cell line;but the OD value in experimental group were higher thanthat in control group at every interval of24hours, there were significantlydifferences（P=0.022）.8The apoptosis rate by Flow Cytometry did not show significantdifference between blank group and control group in HCT116cell; the earlyapoptosis rate（22.35±0.403）%in experimental group was higher than that incontrol group （26.713±0.745）%, there were significantly differences（P=0.0104）, there was not significant difference between the late apoptosisrate （8.97±0.911）%and control group（12.587±2.000）%.Conclusions:1The expression of miR-224was at a high level in colorectal carcinomaand the expressions in rectal cancer, Dukes C and D, with lymphaticmetastasis were higher than that in colon cancer, Dukes A and B, withoutlymphatic metastasis. miR-224might play an important role as oncogene inthe pathogenesis of colorectal carcinoma.2miR-224could promote proliferation and inhibit apoptosis of coloncarcinoma cells.3The expression of miR-224was upregulated in colorectal carcinomaregardless of the exist of p53. They might act as a oncogene in colorectalcarcinoma through p53-independent pathways.4It is necessary to research into the mechanism of miR-224as oncogenein colorectal cancer. It provided effective help to elucidate the pathogenesis ofcolon cancer, and new idea for early diagnosis and target treatment for colorectal carcinoma patients. |
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