摘要
AIM:To investigate the function of microRNA-143(miR-143)in gastric cancer and explore the target genes of miR-143.METHODS:A quantitative real-time reverse transcription-polymerase chain reaction(RT-PCR)analysis was performed to evaluate miR-143 expression in gastric cancer cell lines.After transfecting gastric cancer cells with miR-143-5p and miR-143-3p precursors,Alamar blue and apoptosis assays were used to measure the respective proliferation and apoptosis rates.Cyclooxygenase-2(COX-2)expression was determined by realtime RT-PCR and Western blot assays after miR-143transfection.Reporter plasmids were constructed,and a luciferase reporter assay was used to identify the miR-143 binding site on COX-2.RESULTS:Both miR-143-5p and miR-143-3p were significantly downregulated in multiple gastric cancer cell lines.Forced miR-143-5p and miR-143-3p expression in gastric cancer cells produced a profound cytotoxic effect.MiR-145-5p transfection into gastric cancer cells resulted in a greater growth inhibitory effect(61.23%±3.16%vs 46.58%±4.28%,P<0.05 in the MKN-1cell line)and a higher apoptosis rate(28.74%±1.93%vs 22.13%±3.31%,P<0.05 in the MKN-1 cell line)than miR-143-3p transfection.Further analysis indicated that COX-2 expression was potently suppressed by miR-143-5p but not by miR-143-3p.The activity of a luciferase reporter construct that contained the 3’-untranslated region(UTR)of COX-2 was downregulated by miR-143-5p(43.6%±4.86%,P<0.01)but not by miR-143-3p.A mutation in the miR-145-5p binding site completely ablated the regulatory effect on luciferase activity,which suggests that there is a direct miR-145-5p binding site in the 3’-UTR of COX-2.CONCLUSION:Both miR-143-5p and miR-143-3p function as anti-oncomirs in gastric cancer.However,miR-143-5p alone directly targets COX-2,and it exhibits a stronger tumor suppressive effect than miR-143-3p.
AIM: To investigate the function of microRNA-143 (miR-143) in gastric cancer and explore the target genes of miR-143. METHODS: A quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed to evaluate miR-143 expression in gastric cancer cell lines. After transfecting gastric cancer cells with miR-143-5p and miR-143-3p precursors, Alamar blue and apoptosis assays were used to measure the respective proliferation and apoptosis rates. Cyclooxy-genase-2 (COX-2) expression was determined by realtime RT-PCR and Western blot assays after miR-143 transfection. Reporter plasmids were constructed, and a luciferase reporter assay was used to identify the miR-143 binding site on COX-2. RESULTS: Both miR-143-5p and miR-143-3p were significantly downregulated in multiple gastric cancer cell lines. Forced miR-143-5p and miR-143-3p expression in gastric cancer cells produced a profound cytotoxic effect. MiR-145-5p transfection into gastric cancer cells resulted in a greater growth inhibitory effect (61.23% +/- 3.16% vs 46.58% +/- 4.28%, P < 0.05 in the MKN-1 cell line) and a higher apoptosis rate (28.74% +/- 1.93% vs 22.13% +/- 3.31%, P < 0.05 in the MKN-1 cell line) than miR-143-3p transfection. Further analysis indicated that COX-2 expression was potently suppressed by miR-143-5p but not by miR-143-3p. The activity of a luciferase reporter construct that contained the 3'-untranslated region (UTR) of COX-2 was downregulated by miR-143-5p (43.6% +/- 4.86%, P < 0.01) but not by miR-143-3p. A mutation in the miR-145-5p binding site completely ablated the regulatory effect on luciferase activity, which suggests that there is a direct miR-145-5p binding site in the 3'-UTR of COX-2. CONCLUSION: Both miR-143-5p and miR-143-3p function as anti-oncomirs in gastric cancer. However, miR-143-5p alone directly targets COX-2, and it exhibits a stronger tumor suppressive effect than miR-143-3p. (C) 2013 Baishideng Publishing Group Co., Limited. All rights reserved.
