MiR-9, MiR-125b In Laryngeal Squamous Cell Biological Function

Laryngeal carcinoma is the second most common head and neck cancer(HNC). Although most early-stage laryngeal cancer could be cured via surgery or radiotherapy, the five year survival rate for advanced disease has remained around 50%. Of notable concern is the lack of improvement in prognosis despite advances in treatment. This can be attributed to late presentation, failure of advanced lesions to respond to treatment, and a lack of suitable markers for screening or tailored therapy. In order to further improve the prognosis of this disease, it is necessary to better understand the mechanisms of carcmogenesis.MicroRNAs(miRNAs) are a group of 20 to 24 nucleotide, highly conserved endogenous small noncoding RNA. They regulate gene expression at the post-transcriptional level through binding to 3'-untranslated mRNAs, which are then degraded or translationally inhibited. Numerous studies have highlighted the existence of distinct miRNA expression profiles between tumor tissues and their corresponding normal tissue. And it has been suggested that miRNAs can both regulate and act as oncogenes or tumor-suppressor genes. Recently, a few studies have reported the aberrantly expressed miRNAs in HNC, including up-regulated miR-9 and down-regulated miR-125b, however, the role of these specific miRNAs in head and neck tumorigenesis remains largely unknown. In this study, we mainly focused on the biological function and molecular mechanisms of miR-9 and miR-125b in laryngeal carcinoma. And meanwhile we took miR-21, which had been identified up-regulated in laryngeal carcinoma, as our positive control.First, we verified the expression of miR-9, miR-125b and miR-21 in 10 paired fresh laryngeal squamous cell carcinoma(LSCC) tissues and the adjacent normal tissues, human laryngeal carcinoma cell line Hep-2 and normal tissues by Real-time PCR. The results indicated that miR-9 and miR-21 were significantly up-regulated in LSCC tissues and Hep-2 cells, whereas miR-125b was down-regulated obviously. To study the biological significance of aberrant miR-9, miR-21 and miR-125b levels in laryngeal carcinoma cells, we used a loss-of-function approach by tansfecting Hep-2 cells with chemically synthesized miR-9 ASO, miR-21 ASO and miR-125b. Then relative cell growth were determined by MTT assays, which showed knocked down of miR-9 and miR-21 in Hep-2 cells caused suppression of cell proliferation at 72 and 96 hours post-transfection when compared with control groups; while after over expression miR-125b in Hep-2, the differences were not statistically significant.Then, to explore how the suppression of miR-9 and miR-21 inhibit Hep-2 cell growth, we studied the effects of miR-9 and miR-21 on cell cycle and cell apoptosis. When Hep-2 cells were transfected with miR-9 ASO and miR-21 ASO, the number of cells in G1 phase significantly increased, and G1-S arrest was obvious; while the cell cycle distribution were not significantly affected by transfecting miR-125b in Hep-2 cells. However, TUNEL assay showed there were no significant increase of cell apoptosis in miR-9 ASO, miR-21 ASO and miR-125b-transfected cells compared with control groups. In addition, when cultured in serum-free medium, the miR-9 ASO transfected Hep-2 cells showed poor survival ability compared with control group.Finally, we predicted the putative target genes of miR-9 using bioinformatic tools MiRanda, TargetScan and PicTar softwares. This laid a foundation for further studies on exploring the biomolecular mechanism of carcinogensis and the development of LSCC, which may help us searching specific biomolecular markers for early diagnosis and the targets for gene therapy in LSCC.In conclusion, miR-9 which is up-regulated in LSCC tissues compared to adjacent normal tissues may function as an oncomir. Suppression of miR-9 inhibited cell growth, possibly through loss of control of cell cycle inducing Gl-S arrest, and decrease of survival ability, but not through a marked increase in apoptosis. miR-125b, which was down-regulated in LSCC tissues, did not show significant biological function in Hep-2 cell line. While as the positive control, the results of miR-21 were in accordance with the study reported before, and this indirectly proved the reliability of our research.