The Expression Of MiR-224and MiR-378e In Colorectal Cancer Tissue And Its Clinical Significance

Objective: Colorectal cancerone, one of the common malignant tumor, isof the serious threat to life and health. Its pathogenesis is not fully clear,oncogene activation and tumor-suppressor genes deactivation, changes insignaling pathways, cell microsatellite instability, gene polymorphism mayinduce the occurrence. Previous researches suggest that miRNA plays animportant role in a wide variety of tumor’s occurrence and evolution, invasion,metastasis and angiogenesis, including colorectal cancer.In this study, we use gene chip technology to contrast the different expressionlevel of miR-224and miR-378e in colorectal carcinoma tissues and normalmucosa tissue adjacent,which is demonstrated by Real-Time RT-PCR.Throughthe analysis of the relationship between the pathology and clinical data, in thehope of characteristic to colorectal cancer screening of microRNAs markerand therapeutic target molecules, By analyzing the relationship between theclinical pathological data, with a view to screening to the characteristicmiRNA signs of colorectal cancer and the target molecule, for the furtherstudy of miR-224and miR-378e biological functions in colon cancer andrelated downstream target genes lay the foundation.Methods:1Tissue blocks including the tumor and the distant normal tissues wereobtained from44patients with primary colorectal adenocarcinoma whounderwent surgical resection in the First Hospital of Hebei Medical Universityand Tangshan Workers Hospital in May2011-May2012. The patients’clinicopathologic features, such as sex, age, tumor location, and Dukes’stage,were obtained from surgical and/or pathologic records at these Hospitals. All44patients underwent miRNA-u6、miRNA-224、miRNA-378e detection,29males,15females. The mean age was65years (range from29to83years). The specimens were grouped by positions,including primary tumor,adjacentnormal mucosa.2Total RNA of colorectal cancer tissue and normal tissues were isolatedby Trizol method. Then send3cases RNA of colorectal cancer tissue andnormal mucosa tissue to LC Sciences company for miRNA expression genechip analysis, preliminary screening the different expression miRNAs.Reverse transcription according to retroviruses kit instruction book ofAmerican fermentas company. Use Real-time qPCR technique to detect theexpression of miR-224and miR-378e in colorectal cancer tissues and normalmucosa tissues adjacent, according to the primers instruction book ofguangzhou Ruibo company. Reaction conditions:95℃degeneration10min,and then95℃x10s,60℃x30s, and72℃x10s,45circulation, averagerepeat3times. This study used2-ΔΔCTmethod, including ΔCT for CTdifference of target genes and CTU6, ΔΔ CT for ΔCT carcinoma tissue andΔCT difference of normal tissue.3Experiment data were analyzed using SPSS13.0statistical software,and the skewness distribution data to M (P25, P75), using non-parametric testMann Whitney analysis, P<0.05for the difference was statisticallysignificant.Results:1miR-224expression in colorectal cancer tissue and its clinical significance(Fig.1)1.1Gene chip show that miR-224in the cancer tissue signal intensity is170,while only29in normal mucosa, normalized by the logarithm of miR-2242.52-fold up regulated in cancer tissues.Verification in clinical samples,according to the results of miR-224in44patients with carcinoma tissues andnormal mucosa tissues are3.949×10-（51.495×10-5，9.594×10-5）and9.686×10-6（3.277×10-6，3.274×10-5）.There is no significant differencebetween them (Z=-3.547，P＜0.01).1.2miR-224relationship with clinical pathological characteristics ofcolorectal cancer. miR-224expression are not related to gender. Male group relative expressionis2.760（1.160，14.249）, female group2.938（1.383，12.492）.There is nosignificant difference between them (P=0.921).miR-224expression are not related to age.<65group relative expression is2.833（1.428，7.277）,≥65group4.328（1.077，14.978）. There is nosignificant difference between them (P=0.707).miR-224expression are not related to tumor location.rectal group relativeexpression is6.320(1.337，14.249), colon group2.108（1.132，6.959）. Thereis no significant difference between them (P=0.285).miR-224expression are not related to invasive depth.T1+T2group relativeexpression is2.436(0.794，14.914), T3+T4group2.938(1.383，12.492).Thereis no significant difference between them (P=0.698).miR-224expression are not related to lymph node status.Non-metastasisgroup relative expression is2.744(1.365,12.858), metastasis group4.447(1.165,15.587). There is no significant difference between them(P=0.802).miR-224expression are not related to Dukes’ stage. A+B group relativeexpression is2.744(1.365，12.858), C+D group4.447(1.165，15.587).There isno significant difference between them (P=0.802).miR-224expression are not related to grade of differentiation.worse grouprelative expression is2.050(1.464，5.680), better group3.837(1.146，13.853).There is no significant difference between them (P=0.583).miR-224expression are related to histological type. canalicular adenomagroup relative expression is1.992(1.043，3.081), The non-tubularadenocarcinoma (mucus gland carcinoma, papillary adenocarcinoma) group9.245(1.857，115.853). There is significant difference betweenthem(P=0.013).miR-224was not related to patient’s gender, age, tumor location, invasivedepth, lymph node status, Dukes’stage, grade of differentiation, but related tohistological type.2miR-378expression in colorectal cancer tissue and its clinical significance(Fig.1)2.1Gene chip show that miR-378e in the cancer tissue signal intensity is238,while920normal mucosa, normalized by the logarithm of miR-378e1.98-folddown regulated in cancer tissues.Verification in clinical samples, according tothe results of miR-224in44patients with carcinoma tissues and normalmucosa tissues are3.338×10-7（9.267×10-8，1.434×10-6）and9.094×10-7（2.969×10-7，7.619×10-6）.There is no significant difference between them(Z=-2.892，P＜0.01).2.2miR-378e relationship with clinical pathological characteristics ofcolorectal cancer.miR-378e expression are not related to gender. Male group relative expressionis0.299（0.077，0.799）, female group0.282(0.066，1.122). There is nosignificant difference between them (P=0.931).miR-378e expression are not related to age.<65group relative expression is0.307(0.006，0.965),≥65group0.245(0.085，0.536). There is no significantdifference between them (P=0.707).miR-378e expression are not related to tumor location.rectal group relativeexpression is0.150(0.057，0.967), colon group0.308(0.183，0.888). There isno significant difference between them (P=0.307).miR-378e expression are not related to lymph node status.Non-metastasisgroup relative expression is0.245(0.057，0.793), metastasis group0.299(0.118，2.222). There is no significant difference between them(P=0.334).miR-378e expression are not related to Dukes’ stage.A+B group relativeexpression is0.245(0.057，0.793), C+D group0.299(0.118，2.222). There isno significant difference between them (P=0.334).miR-378e expression are not related to histological type.canalicular adenomagroup relative expression is0.188(0.038，0.780), The non-tubularadenocarcinoma (mucus gland carcinoma, papillary adenocarcinoma) group0.324(0.120，1.553). There is no significant difference between them(P=0.200). miR-378e expression are not related to grade of differentiation.worse grouprelative expression is0.340(0.129，2.645), better group0.276(0.060，0.787).There is no significant difference between them (P=0.282).miR-378e expression are related to invasive depth. T1+T2group relativeexpression is0.012(0.001，0.150), T3+T4group0.298(0.080，1.018). There issignificant difference between them(P=0.027).miR-378e was not related to patient’s gender, age, tumor location, invasivedepth, lymph node status, Dukes’ stage, histological type, grade ofdifferentiation, but related to invasive depth.Conclusion：1miR-224in colorectal cancer tissues increases, and in the highernon-tubular adenocarcinoma, suggesting that it may affect the prognosis ofcolorectal cancer.2miR-378e in colorectal cancer tissues lower, and with the depth ofinvasion, it may play a similar role of tumor suppressor genes.3miR-224and miR-378e can be used as potential colorectal cancermolecular markers.