Mirnas Expression Profiling Of Gastric Carcinoma And Function Of Significantly Down-regulated MIR-9 And MIR-433

Objective To detect miRNAs expression profiling of gastric carcinoma , screen miRNA specific signatures, identify the targets of the miRNAs associated with gastric cancer, establish a novel diagnostic and therapeutic method for gastric carcinoma.Methods 328 miRNAs expression of 3 normal gastric tissues, 24 malignant tissues(2 in early phase and 22 in late phase of gastric cancer), gastric cancer cell SGC7901 and Gastric epithelial cell line 1(GES-1) were detected by miRNA gene chips. We chose the interest miRNAs, and identified them by Quantitative Real-Time polymerase chain reaction(qRT-PCR).Then, we predicted the targets of the interest miRNAs via bioinformation platform. The 3'-untranslated region(3'-UTR)of the interest miRNAs binding site were synthesized and cloned in the downstream of the luciferase gene of pGL3-control.The recombinant plasmid was called pGL3-miRNA. To examine the luciferase activity, 4 groups were set up for miR-9 and miR433:â‘ SGC7901 adding liposome(black control) were transfected;â‘¡pGL3-control was transfected; â‘¢pGL3-miRNA was transfected;â‘£pGL3-miRNA adding the interest miRNA were cotransfected. After the 48 h transfection, luciferase activity was assayed and analyzed by relative light unit(RLU). To evaluate the regulation of the targets of the interest miRNAs, SGC7901 was transfected with the interest miRNAs.There were 3 groups for them:â‘ black control: SGC7901 adding liposome were transfected;â‘¡group 1: 50 pmol of interest miRNA was transfected;â‘¢group 2: 100 pmol of interest miRNA was transfected. After 48 h transfection, the cells were harvested and total protein and total RNAwere extracted. The predicted targets expression levels were detected by Western blot(WB). The interest miRNAs level were detected by qRT-PCR.Results Compared with that in the normal gastric samples, 26 miRNAs expressed abnormally in gastric carcinoma samples(including 24 malignant tissues and SGC7901). 19 miRNAs of these were down-regulated (the expression in carcinoma group was at least 2.2 times lower than that in the normal group). 7 miRNAs were up-regulated (the expression in the carcinoma group was at least 2.1 times higher than that in the normal group).We chose the remarkably down-regulated miR-9 and miR-433 as the interest miRNAs. qRT-PCR was used to detect the expressive level of miR-9 and miR-433.We found that miR-9 was down-regulated 75.4% in 75% of carcinoma tissues ,compared with normal gastric tissues(P<0.01).And it was down-regulated 76.2% in SGC7901, compared with GES-1(P<0.01); miR-433 was down-regulated 83.3% in 83% of carcinoma tissues ,compared with normal gastric tissues(P<0.01).And it was down-regulated 77.3% in SGC7901, compared with GES-1(P<0.01). The results were consistent to the microarray analysis. we predicted the targets of miR-9 and miR-433 might be RAB34(the member of RAS oncogene family) and GRB2(growth factor receptor-bound protein 2) via bioinformation platform. After recombinant plasmid pGL3-miR-9 and pGL3-miR-433 were transfected , respectively, we assayed the luciferase activity and found that luciferase activity of SGC7901 cotransfected pGL3-miR-9 and miR-9 decreased 50% compared with pGL3-miR-9(P<0.01); Luciferase activity of SGC7901cotransfected pGL3-miR-433 and miR-433 decreased by 54% compared with pGL3-miR-433(P<0.01). After miR-9 was transfected into SGC7901,the expression of RAB34 decreased 45% in group 1 (P<0.01)and 72% in group 2(P<0.01)compared with control group; After miR-433 was transfected into SGC7901,the expression of GRB2 decreased 53% in group 1 (P<0.01)and 89% in group 2 (P<0.01)compared with control group. Meanwhile, we measured the level of miR-9 and miR-433 by qRT-PCR. miR-9 level increased 1.3-fold in group 1 and 2.8-fold in group 2 compared with control group(P<0.01). miR-433 level increased 1.6-fold in group 1 and 3.0-fold in group 2 compared with control group(P<0.01).Conclusions Preliminarily, we screened 26 miRNAs associated with gastric carcinoma. miR-9 and miR-433 significantly down-regulated might be used as a special marker for the advanced gastric carcinoma. The both participated in the pathogenesy of gastric carcinoma via negative regulation of RAB34 and GRB2, tumor associated protein. Simultaneously, our results provided related information for establishing a novel diagnostic and therapeutic method based on miRNAs for gastric carcinoma.