Study On MiR-34a Enhances The Radiosensitivity By Targeting LyGDI In Non-small Cell Lung Cancer Cells

Objective: Our previous data showed that LyGDI was upregulated in clinicalsamples of non-small cell lung cancer cell (NSCLC) patients, and its expression wasrelated to the increase of metastasis, chemoresistance and anti-radiation. In the study werestore the expression of miR-34a in the NSCLC A549(p53+/+) and H1299(p53-/-) celllines, and then elucidate the function of exogenous miR-34a involved in growth,senescence and apoptosis. Our further aim is to analyze the expression of key signalingmolecules, and to verify that whether LyGDI was a new target gene of miR-34a and tostudy the function and mechanism of miR-34a in the radiation sensitivity in A549cell.This study provided a scientific and practical basis for the improvement of the clinicalradiation therapy effect of small molecules combined with ionizing radiation.Methods:1. Chemical synthetic miR-34a mimics were used to transfect into NSCLC A549and H1299cells by LipofectamineTM2000.2. MTT assay was used to analyze the effect on growth; Senescence associatedβ-galactosidase kit staining assay was used to observe senescence; Annexin V/PIapoptosis kit was used to detect apoptosis; Erythrosine B staining was used to count therate of cell death; Western blot was used to detect the expression at the level of proteinof CDK4、E2F3、Bcl-2、Puma.3. Target sequence of miR-34a in LyGDI3’-UTR was predicted by bioinformaticsapproaches. Chemical synthetic miR-34a mimics were used to transfect into A549cell,then the expression of LyGDI at the level of mRNA is detected by Real time-PCR; Theexpression of LyGDI at the level of protein is analyzed by western blot; Furthermore,chemical synthetic miR-34a was transfected into A549cells stably expressingGFP-LyGDI fusion protein and then the change in fluorescence intensity of GFP wasobserved by fluorescence inverted microscope.4. A549cell was transfected with miR-34a mimics, treated with2Gy γ-ray and treated with miR-34a combined with2Gy γ-ray respectively. Cell survival curveswere measured using a colony-forming assay; Apoptosis was detected by Annexin Vapoptosis kit; the expression of related proteins was analyzed by western blot.Results：After transfection with miR-34a mimics, the growth of A549and H1299wasinhibited, the rate of senescence, apoptosis and cell death was higher. The expressionlevels of Bcl-2, E2F3and Cdk4were significantly decreased and Puma was increasedwhen transfected with miR-34a. miR-34a decrease the expression of LyGDI at the levelof mRNA and protein in A549cell. miR-34a also weakens the GFP fluorescence ofstably expressed GFP fused LyGDI A549cells. Downregulation of LyGDI induced bymiR-34a promoted Rac1activation and membrane translocation leading to cellapoptosis. Furthermore, restoration of miR-34a indirectly reduced COX-2expression.Restoring expression of miR-34a enhanced IR-induced apoptosis and increased theradiation sensitivity of A549cell.Conclusion：1.miR-34a can inhibit the growth and induce the senescence of A549and H1299by downregulating the expression of E2F3and CDK4. miR-34a can induce apoptosis ofA549and H1299by downregulating the expression of Bcl-2and overregulating theexpression of Puma.2.LyGDI is a target gene of miR-34a. miR-34a can reduce the expression of LyGDIat the level of mRNA and protein in A549cell.3.miR-34a can downregulate the expression of LyGDI, which promoted Rac1activation and membrane translocation leading to cell apoptosis. And restoration ofmiR-34a indirectly reduced COX-2expression. Restoring expression of miR-34aenhanced IR-induced apoptosis and increased the radiation sensitivity of A549cell.