| Gastric cancer is one of the most common malignant tumors with high morbidity and mortality. Early diagnosis and new therapeutic targets finding are crucial for better prognosis. MicroRNAs(miRNAs) are a class of single-strand small non-coding RNAs with 19–24 nucleotides in length, which can negatively regulate genes by triggering either miRNAs degradation or translational repression through binding to the3’-untranslated region of their target mRNAs. Emerging evidence has showed the association of aberrantly expressed miRNA with the oncogenesis and development in many kinds of malignant tumors. MiRNA can play a role similar to oncogenes or tumor suppressors, which is involved in tumor growth, differentiation, adhesion, apoptosis,invasion and metastasis. In the study, we focused on looking for differential expression of microRNA in gastric cancer tissue, and revealed their role in carcinogenesis and progressing of the gastric cancer. This study not only would help for deep understanding the pathogenesis of gastric cancer, but also could be informative for discovery of specific and sensitive molecular biomarkers and drug targets of gastric cancer.The first part of this study is to screen the differentially expressed mi RNAs in gastric cancer, and analyze their correlations with clinicopathologic characteristics. To obtain the aberrant expressions of miRNAs between gastric cancer and adjacent normal tissues, we employed miRNA expression chips to study the differences of mi RNA expression profiles in 35 pairs of gastric cancer tissues and matched normal tissues adjacent to the tumor with different clinical stage(stage I to IV). 56 mi RNAs were found to be aberrantly expressed,of which 32 were down-regulated and 24 up-regulated in gastric cancer tissues adjacent normal tissues. We enlarge clinical samples to verify the reliability of chip results by quantitative real-time PCR,and the results show that miR-214 expression is significantly raised, while miR-148 a and miR-1225-5p expression is lower in gastric cancer tissues,which were consistent with the microarray data. We further analyze the correlations ofmiRNAs expression with clinicopathologic characteristics, such as age, gender, tumor stage and lymph node metastasis etc, and results indicate a negative correlation of the miR-1225-5p expression level with TNM stage and lymph node metastasis in gastric cancer. These results suggest that miR-1225-5p may play a role in proliferation, invasion and metastasis of gastric cancer, yet the involved mechanism have not been elucidated.The second part of the study is to investigate the effect of miR-1225-5p on the gastric cancer cells. To address this issue, MiR-1225-5p is over-expressed by transfecting gastric cancer cells with miR-1225-5p precursor molecules(mimics), and the effect of miR-1225-5p on cell proliferation, invasion and metastasis were determined by CCK-8assay, soft agar colony formation assay, transwell migration assay and matrigel invasion assay respectively. Furthermore, MGC803 gastric cell lines over-expressing miR-1225-5p were established by infection of recombinant lentivirus and used to inoculated BALB/c nude mice, and the xerograft growth was measured. The results demonstrated that over-expression of miR-1225-5p inhibits gastric cancer cell proliferation, migration and invasion.The third part of the study is to explore the target genes of miR-1225-5p and regulation of the biological behavior of gastric cancer cells by interreaction between miR-1225-5p and their target mRNAs. Firstly, the prediction of the candidate target genes for miR-1225-5p was performed by the programs of Targetscan, PicTar and MiRanda. Then the luciferase reporter assay and westem blot analysis were adapted to confirm the IRS1 as a target of miR-1225-5p. Secondly, the effect of knockdown of IRS1 on gastric cancer cell growth, proliferation, migration, and invasion is similar to those of over-expression of miR-1225-5p. Furthermore, overexpression of IRS1 can partially rescue the inhibition effect of miR-1225-5p on gastric cancer cell proliferation, migration and invasion. These findings indicated that miR-1225-5p may play important role in carcinigenesis and development of gastric cancer. |
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