| Head and neck squamous cell carcinoma(HNSCC) stands sixth among malignant cancers in the world. Although in recent years, considerable research has focused on the diagnosis and treatment of laryngeal squamous cell carcinoma(LSCC), the 5-year survival rate of LSCC patients remains low. Thus, understanding the basis of occurrence and development of LSCC is of importance in clinical practice. MicroRNAs(miRNAs) are endogenous, small, noncoding RNAs and have specific expression in different cells and tissues. The dysregulation of miRNAs might play pivotal roles in tumorigenesis in cancer. Thus, researching the function and expression of miRNAs will contribute to the understanding of the pathogenesis, diagnosis, treatment, and prognosis of such tumors. To investigate the underlying miRNAs in human laryngeal cancer, we used a miRNA array and identified a candidate biomarker, namely, hsa-mi R-301a-3p(miR-301a-3p). We searched for candidate genes using miRNA databases such as TargetScan or miRBase and found Smad4 was a downstream target gene of miR-301a-3p. We used the dual-luciferase reporter gene system to confirm whether there is a targeted modulation of miR-301a-3p and Smad4. The results showed that miR-301a-3p was able to significantly repress luciferase activity of wild-type Smad4-3?-UTR and Smad4 was a direct target of miR-301a-3p. We investigated expression levels of miR-301a-3p and Smad4 in human laryngeal cancer tissues and cell lines Hep-2 and Tu-177, as well as corresponding adjacent healthy tissues and human bronchial epithelial cell line 16 HBE by qRT-PCR or immunohistochemistry. Mi R-301a-3p expression was significantly higher in humanlaryngeal cancer cells and tissues than in the control group. Smad4 expression was significantly lower in human laryngeal cancer cells and tissues than in the control group. By investigating the relationship between the expression of miR-301a-3p, Smad4 and the clinicopathological parameters in the laryngeal cancer patients, mi R-301a-3p expression was found to be associated with primary tumor and Smad4 was found to be associated with histologic differentiation, as well as cervical lymph node metastasis. To investigate the function of mi R-301a-3p and Smad4 in laryngeal cancer, we used the “loss or gain-of-function”, miR-301a-3p Inhibitor or Smad4 vector, which was transfected into human laryngeal cancer cell lines and compared with the negative and blank control. The MTS assay showed that the miR-301a-3p Inhibitor or Smad4 vector reduced the cell proliferation ability of Hep-2 and Tu-177 cells. The colony-formation rates decreased after transfection with miR-301a-3p Inhibitor or Smad4 vector. The EdU assay revealed that duplication activity was inhibited and the proliferation rate of the experimental group was found to be significantly lower than that of the controls. Transwell assay showed that miR-301a-3p Inhibitor or Smad4 vector-transfected cells had less migration or invasion ability through membranes than the controls. FCM showed that after treatment with the miR-301a-3p Inhibitor or Smad4 vector, cells in which apoptosis occurred at early stages was much higher than the controls. Cell cycle was arrested at the G0/G1 phase. Western blot analysis was used to explore the relationship of miR-301a-3p and Smad4 and the genes involved in epithelial–mesenchymal transition(EMT). The level of E-cadherin was found to increase, while the levels of N-cadherin, vimentin, MMP2, and MMP9 decreased. The aims of this study were to investigate the function and role of miR-301a-3p and Smad4 in LSCC and to determine whether miR-301a-3p regulates Smad4 in a targeted manner and evaluate the clinical significance of Smad4 and miR-301a-3p in the development and progression of LSCC. By directly regulating its target gene Smad4, miR-301a-3p acts as an oncogene to play an important biological role. They may represent a novel pathway to target in relation with the occurrence and development of laryngeal cancer. For instance, testing miR-301a-3p and Smad4 in the blood or biopsied tissue of patients might facilitate early detection of laryngeal cancer. The present findings enhance current knowledge for the treatment and evaluation of laryngeal carcinoma. |
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