Effects Of MiR-143 On Biological Behavior Of Hep-2 Cells

Background:MicroRNAs(miRNAs),a class of endogenous small(19-25nt)noncoding single-stranded RNAs,have recently emerged as novel regulators of various fundamental biological processes.They regulate gene expression via binding to the 3'-untranslated regions(3'-UTR)of target expression,thereby resulting in mRNA degradation or translational inhibition.MiRNAs play different roles in the development of different tumors,miR-143 is a common anti-oncogene.To confirm the role of miR-143 in laryngeal carcinoma,take the miR-143 transfecte into Hep-2 cell to observe its effects on the biological characteristics and the mechanism.Methods:The experiment was divided into three groups: miR-143-mimics transfection group,negative control transfection group and blank group.Firstly,the synthesis of miR-143-mimics and negative control sequence.The miR-143 mimics and negative control sequence were transfected in Hep-2 cells with Lipofectamine 2000 liposome,and the blank group was only cultured with Opti-MEM I low serum culture medium,without any sequence.The transfection efficiency was observed under fluorescence microscope.Then,the cell growth and apoptosis were detected by flow cytometry and MTT assay.By using gene prediction software,we can find that HK-2 may be the target gene of miR-143.Finally,RT-PCR and Western blot test were used to detect the expression of mRNA and protein of the target gene HK-2 of miR-143.Results:1.The transfection efficiency of the cells was observed under fluorescence microscope,reaching up to 90%.2.The MTT results show that the proliferation rate of miR-143-mimics transfected cells were slowly.At 0?24?48 and 72 h,the survival rate of experimental group was(98.8±2.82)%,(89.2±2.14)%,(80.3±1.98)%,(76.7±1.26)%,compared with another two group,the difference was statistically significant.(P<0.05).3.The flow cytometry results reveal that the apoptosis rate of transfected miR-143-mimics cells was significantly increased.The apoptosis rates of the three groups were respectively(11.86±1.12)%,(3.24±0.63)%,(3.21±0.32)%.The difference was statistically significant.(P<0.05).4.RT-PCR results showed that the expression of HK-2 gene of blank group,negative control group and miR-143-mimics transfection group were(5.09±1.49),(4.99±1.17),(1.03±0.03).Compared with another two group,the difference was statistically significant.(P<0.05).5.Western blot results showed that the expression of HK-2 protein of blank group,negative control group and miR-143-mimics transfection group were(0.57±0.03)?(0.56±0.03)and(0.26±0.02),the difference was statistically significant.(P<0.05).Conclusions:1.MiR-143 can inhibit the proliferation of Hep-2 cells and induce apoptosis;2.MiR-143 may affect the biological behaviour of Hep-2 cells by regulating the target gene HK-2.