NF-κB Regulates MicroRNAs Contributing To Esophageal Squamous Cancer Cell Biology

Esophageal cancer is one of the most mortality malignancies in the world, includingtwo major histological forms-esophageal squamous cell carcinoma (ESCC) andesophageal adenocarcinoma, and more than90%of esophageal cancer are ESCC.Epidemiology studies indicate that any factor that causes chronic irritation andinflammation of the esophageal mucosa appears to increase the incidence of ESCC. Theincidence of esophageal cancer is increasing recently, however the overall5-year survivalrate is only20%-30%. It is urgent and important to investigate the mechanism for theinitiation and development of ESCC and find novel target for disease diagnosis and therapy.NF-κB pathway plays a key role in linking inflammation to caner development andprogression. It can be triggered by a variety of stimuli, such as pro-inflammatory cytokines,components of bacterial and virus, genotoxic stress and so on. Activated NF-κB affects notonly tumor cells but also inflammatory cells, and involves in the regulation of cell cycle,apoptosis, angiogenesis, metastasis and inflammation. NF-κB plays a complex role in thedevelopment of epithelial cancer; sometimes functions as tumor-promoter, and sometimestumor-suppressor, which may be related to different genes regulated by NF-κB in differenttissues and cells. There are numerous studies reporting overexpression or abnormalactivation of NF-κB and its target genes in ESCC tissues and their association withtherapeutic responses and prognosis of esophageal cancer, indicating NF-κB plays a role inthe development of esophageal squamous cell cancer.MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate geneexpression at the posttranscriptional level. Emerging evidence suggests that miRNAs playimportant roles in regulation of tumor biology and inflammation. Altered expression ofmiRNA has been reported in a variety of cancers, and their expression profiles can be usedas hallmarks for diagnosis, classification and prognosis of human malignancies. Their targetgenes involve in cell proliferation, apoptosis, differentiation, and metastasis and so on.Recent data suggests that miRNAs also play causal roles in esophageal cancer development. Microarray analyses revealed dysregulated miRNA expressions in ESCC. Several miRNAshave been found in participate with esophageal cancer development too.This project is aimed to investigate the role of NF-κB in ESCC development and findfunctional miRNAs involved in NF-κB network in ESCC. So, in the first section, effects ofNF-κB on ESCC cell growth, apoptosis, cell cycle and proliferation were studied. Then,according to bioinformatic analysis and functional studies of miRNAs in recent papers, wescreened two miRNAs, miR-34a and miR-330, as potential targets for NF-κB regulation inESCC. Roles of miR-34a and miR-330in ESCC cell biology and their relationship withNF-κB were investigated.The major results are presented below:1．NF-κB plays a different role in controlling different ESCC cell growth.CCK8, flow cytometry and Edu cell imaging assay were used to detect the effect ofNF-κB on ESCC cell growth, apoptosis, cell cycle and proliferation. The results showedthat overexpression of p65in EC109cells inhibited cell growth, probably through inducingS phase cell cycle arrest and inhibiting cell proliferation. Also overexpression of p65induced apoptosis of EC109cells. While transfection of p65expression vectors promotecell growth in KYSE450cells, which might be caused by enhanced cell proliferation. Andectopic expression of p65in KYSE450cells seemed to have no effect on apoptosis. Geneexpression studies showed that several CDKI expressions, such as p21and p27, wereincreased by overexpressing p65in EC109cells. p53protein levels were also enhanced byp65overexpression, while expressions of the inhibitor of apoptosis protein, survivin weredownregulated. Gene expression studies in transfected KYSE450cells showed thatincreasing p65levels had no impact on all the above gene levels except p21whose proteinlevels were upregulated. These results indicate that NF-κB might have different roles indistinct ESCC cell.2．MiR-34a is a novel target for NF-κB in ESCC.Effects of miR-34a on ESCC cell growth, apoptosis, cell cycle, migration and invasionwere studied by CCK8, flow cytometry and transwell assays. Results demonstrated thatoverexpression of miR-34a inhibited cell growth, induced G1cell cycle arrest, impairedcell motility and invasiveness. However, ectopic expression of miR-34a did not induce apoptosis. These indicated that miR-34a might function as a tumor suppressor in ESCC.Also the regulationship between NF-κB and miR-34a was investigated.Overexpression of NF-κB p65subunit could increase miR-34a levels in EC109which wasowing to enhanced miR-34a transcriptional activity by p65. Mutation of the κB sites locatedin miR-34a promoter impaired p65induced transcriptional activity. And chromatinimmunoprecipitation (CHIP) and electrophoretic mobility shift assays (EMSA) bothshowed that NF-κB could specifically bind to the third κB site located in miR-34a promoter.In addition, overexpression of NF-κB p65could not successfully induce miR-34aexpression in esophageal cancer cell lines with mutant p53(KYSE450) or decreased p53.Reporter assay further showed that NF-κB-induced miR-34a transcriptional activity wasreduced by p53impairment. Nevertheless, binding of NF-κB to miR-34a promoter was notaffected in cells with mutant p53. The above results demonstrated that NF-κB could inducemiR-34a expression at the transcriptional level and induction of miR-34a need wildtype p53function.3．NF-κB regulates miR-330contributing to ESCC cell growth regulation.Effects of miR-330on ESCC cell growth, cell cycle and proliferation were studied byCCK8, flow cytometry and Edu cell imaging assays. Results showed that overexpression ofmiR-330promoted cell growth. And cell cycle analysis indicated that cells withoverexpressed miR-330had increased S, G2/M phase cells. Edu cell imaging analysis alsoshowed that increasing miR-330enhanced ESCC cell growth. These data indicated thatmiR-330might have a tumor-promoting role in the development of ESCC.QRT-PCR, reporter assay and CHIP analysis were used to study the regulationshipbetween NF-κB and miR-330. Transfection with p65downregulated miR-330expressionsin EC109cells and upregulated miR-330levels in KYSE450cells. Reporter assay showedNF-κB p65could promote transcriptional activity in both ESCC cells. CHIP analysis alsoshowed that NF-κB could bind to the κB site in the regulatory region of miR-330gene.These results indicated that NF-κB has the ability of regulating miR-330expression at thetranscriptional levels, but has different impact on the miR-330expression in differentESCC cells.In conclusion, this study indicates that NF-κB plays different regulatory roles ondifferent ESCC cells. MiR-34a and miR-330are novel targets for NF-κB and contributing to ESCC carcinogenesis. Distinct regulation of these two miRNAs in different cells mightcause the divergent role of NF-κB on ESCC cell growth. And distinct regulation of miR-34aby NF-κB might owe different p53status, while the mechanism of different miR-330regulation by NF-κB needs further study. Our research is helpful for understanding thecomplex role of NF-κB and miRNAs in the carcinogenesis of esophageal squamous cellcancer and provides new evidence for screening targets for ESCC diagnosis and therapy.