Effects Of MiR-193a-3P On Proliferation,Apoptosis And Invasion Of Laryngeal Carcinoma Cell Line Hep-2

Background:Laryngeal carcinoma is one of the most common head and neck cancers and constitutes approximately 2-3% of all malignant tumors, constituting the second main upper respiratory tract tumor in incidence and mortality rates. Despite many advances in the diagnosis and treatment of this disease, laryngeal carcinoma patient survival remained unchanged over recent years due to its uncontrolled recurrence and lymph node metastasis.Thus, the current urgent problems at the genetic level is to find new treatments for laryngeal cancer.MicroRNAs (miRNAs) are a group of non-coding, single-stranded RNAs that are approximately 20-23 nucleotides in length. miRNAs bind to the 3’untranslated region (UTR) of their target messenger RNAs via complete or partial base-pair complementarity, which leads to mRNA degradation or translational repression, respectively. Aberrant expression of miRNAs has been demonstrated to play a critical role in the initiation and progression of various cancers. A large number of miRNAs which are aberrantly expressed in cancer tissues have been functionally identified as tumor-suppressive miRNAs or oncomirs using gain-of-function and loss-of-function experiments. Full understanding of the biological functions and molecular mechanisms of miRNA-mediated processes may provide significant advances in the diagnosis and treatment of malignant disease. The expression patterns and biological functions of miRNAs in laryngeal carcinoma have been investigated in recent years. For example, let-7a and miR-34c are downregulated in laryngeal carcinoma and act as tumor suppressors by directly targeting oncogenes, such as ras, c-myc and c-met. In contrast, miR-21 functions as an oncomir by negatively regulating the expression of BTG2, resulting in the increased proliferation of laryngeal cancer cells in vitro and in vivo. Recent miRNA profiling studies have revealed that the expression of miR-193a was frequently down-regulated in head and neck squamous cell carcinomas.However, the function and molecular mechanism of miR-193a in laryngeal carcinoma cells remains unknown.miR-193a-3p have been shown to repress the expression of important cancer-related genes and might prove useful in the diagnosis and treatment of laryngeal carcinoma.Objective:Recent miRNA profiling studies have revealed that the expression of miR-193a was frequently down-regulated in head and neck squamous cell carcinomas. However, there has been no report about the function in human laryngeal cancer. This research is to study the effect and mechanism of miR-193a-3p in the proliferation, apoptosis and invasion of laryngeal carcinoma cell line Hep-2.Methods:1、The laryngeal carcinoma cell line Hep-2 was cultured at 37℃ at 5%CO2 in RPMI-1640 media supplemented with 10% fetal bovine serum (FBS).2、The optimal conditions were detemined by transfecting diffrent concentrations of NC-FAM. The efficiency of the transfection was detected by fluorescence microscope. Hep-2 cells were divided into three groups:The Hep-2 cells were transfected with miR-193a-3p mimics(group A) or negative control RNA(group B) by Lipofectamine 2000,and the non-transfected cells were taken as the blank controls(group C).3、Real-time fluorescence quantitative PCR (qRT-PCR) method was used to detect the miR-193a-3p level in Hep-2 cells.4、Methyl thiazolyl tetrazolium(MTT) were used to determine the proliferation.5、The apoptosis evaluated in Hep-2 cells at 48h after transfected miR-193a-3p and analyzed by fluorescence microscopy using chromatin stain Hoechst33258.The Hep-2 cells were stained by using Annexin V-FITC and propidium iodide (PI) double stain detection kit. The apoptosis rate was detected by flow cytometry.6、Forty-eight hours after transfection, transwell assay were used to determine the invasion abilities.7、The miRNA targets were predicted using the algorithms Target-Scan, PicTar, and miRBase Targets.8、The expressions of myeloid cell leukemin-1(Mcl-1) were detected by Western Blot.Results:1、The miR-193a-3p mimics at a concentration of 60nM were used for the transfection through employing Lipofection2000 according to the optimal condition.2、After transfected 48 hours, the expression level of miR-193a-3p in group A was significantly higher than that in group B and C.3、After 24h transfection, an MTT assay demonstrated that cell growth was decreased upon transfection with miR-193a-3p (p<0.05), and the proliferation was significantly decreased at 48h post-transfection.4、Apoptotic cells in group A was significantly more than those in other groups(p<0.01) by Annexin V staining and Hoechst 33258.5、Decreased cells via the transwell member in group A (7±2)were detected as compared with group B(45±9) and group C(49±7,p<0.01).6、A search of the Target-Scan, PicTar, and miRBase Database revealed that Mcl-1 was a potential target of miR-193a-3p.7、The relative expression level of mcl-1 protein in group A was lower than those in other control groups.Conclusion:miR-193a-3p is able to inhibit the proliferation and invasion abilities of Hep-2 cells and promote their apoptosis probably through downregulation of the expression of Mcl-1 gene.