MicroRNA-9Regulates Gastric Adenocarcinoma Cell Growth By Targeting NF-κB1

Aims:MicroRNAs (miRNAs) are a class of small non-coding RNAs that post-transcriptionally regulate gene expression. Recent evidences indicate that special miRNAs may function as tumor suppressors or oncogenes and play a critical role in cancer initiation and progression by negatively regulating their target genes. Using real-time RT-PCR assay, we found that microRNA-9(miR-9) was significantly down-regulated in human gastric adenocarcinoma tissue samples. In this study, we focused on the effects of miR-9on the phenotypes of gastric adenocarcinoma cells as well as the identification and effects on the phenotypes of gastric adenocarcinoma cells of the direct target genes of miR-9, in order to illuminate the molecular mechanisms of miR-9in the initiation and progression of human gastric adenocarcinoma.Methods:We detected the differencial expression of miR-9in human gastric adenocarcinoma and adjacent normal tissues by real-time RT-PCR assay. Both level and function of miR-9were enhanced in human gastric adenocarcinoma cell line MGC803and the changes of cell phenotypes were detected with MTT assay, colony formation assay and animal work. Subsequently, we used bioinformation and identified the candidate target genes for miR-9. The reliability of the direct target gene was confirmed by fluorescent reporter experiment. Furthermore, the mRNA levels and protein levels of target gene in miR-9-enhanced gastric cancer cells or tissues were detected with real-time RT-PCR and western blot, in order to confirm the regulating role of miR-9in target gene expression. Finally, the function of target gene was inhibited in human gastric adenocarcinoma cell line MGC803and the changes of cell phenotypes were detected with MTT assay and colony formation assay.Results:Experimental results showed that the expression of miR-9in human gastric adenocarcinoma tissues was lower than in adjacent normal tissues. We found that after overespression of miR-9, the MGC803cell proliferation activity and the colony formation activity were both inhibited. Moreover, the speed of tumor growth was significantly descended. Subsequently, we identified tumor related gene nuclear factor kappa B1(NF-κB1) as one of the candidate target genes for miR-9. The NF-κB1mRNA3’-untranslated region (3’UTR) contains the potential binding site of miR-9. The fluorescent reporter experiment also confirmed that miR-9can directly bind to the NF-κB1mRNA3’UTR and negatively regulate the gene expression. After overexpression of miR-9in gastric cells or tissues, mRNA level and protein level of NF-κB1were both descended. Finally, we found that after knockdown of NF-κB1, the MGC803cell proliferation activity and the colony formation activity were both inhibited.Conclusions:Our results indicate that in gastric adenocarcinoma cells, miR-9functions as a tumor suppressor gene and inhibites cell proliferation. The ability of downexpressed miR-9in gastric adenocarcinoma cells directly down-regulating the expression level of tumor related gene NF-κB1is decreased and result in the enhancement-of-function of NF-κB1, leading to the activation of cell proliferation. The elucidation of common mechanisms of miR-9in gastric adenocarcinoma helps us to further understand cancer initiation and progression, and provides new evidences to cancer diagnosis and therapy.