MiRNA326 Mediates Malignant Biological Behaviors Of Lung Adenocarcinoma By Targeting ZEB1

Background and purpose:The high morbidity and mortality of lung cancer acute threat to physical and emotional health of all our Chinese and global people.The main histopathologic type is lung adenocarcinoma(LADC).The 5-year survival rate of lung cancer patients is still less than 20% due to its complex pathogenesis,lacking of early specific characteristics,and most people are diagnosed at terminal-stage of lung cancer.Therefore,the continued study of malignant biological behavior of lung cancer,in-depth exploration of its own mechanism of generation and development and further optimizing its strategy of diagnosis and treatments are of important significance.MicroRNAs(miRNAs)as a kind of small non-coding and highly conserved RNAs,are involved in many malignant biological behaviors like cell proliferation,invasion,migration and apoptosis.MiR-326 functions as an anti-oncogene in the several types of cancer.However,the underling mechanisms through which miRNA-326 regulates the anti-carcinogenesis of LADC have remained elusive.So the aim of this study was to explore the role and regulatory mechanism of miR-326 in cell proliferation,invasion,migration and apoptosis in LADC and provide a new research direction of searching novel targets of molecular targeted therapy of LADC.Methods: qRT-PCR was used to detect the expression pattern of miR-326 in human bronchial epithelial cells(HBES-2B),4 kinds of LADC cell lines(H23?H1975?H2228 ? H2085)and 20 LADC tissues.Then,H23 cells were infected with miR-326 mimics,miR-326 inhibitors and si-ZEB1 to build up-regulated miR-326 cell lines,down-regulated ZEB1(zinc-finger-enhancer binding protein 1)cell lines,simultaneous down-regulated ZEB1 and miR-326 cell lines.The infection efficiencies were verified by western blot and qRT-PCR.Moreover,CCK-8 assay,transwell invasion assay,wound healing assay and flow cytometry assay were employed to examine the effects of miR-326 and ZEB1 on the proliferation,invasion,migration and apoptosis abilities of H23 cells.Western blot was performed to explore the effects of miR-326 and ZEB1 on the expression of invasion and migration related proteins N-cadherin?E-cadherin?MMP7?MMP13?SLUG and apoptotic proteins PARP?BAX.On the mechanism,the target relationship between miR-326 and ZEB1 was measured by a luciferase reporter system.Results:1.MiR-326 expression was significantly downregulated in LADC tissues and cells and upregulated in normal lung tissues and cells.2.CCK-8 assay showed that overexpression of miR-326 and knockdown of ZEB1 decreased the proliferation ability.Both knockdown of miR-326 and ZEB1 could promoted the proliferation ability.3.Transwell invasion assay and wound healing assay showed that overexpression of miR-326 and knockdown of ZEB1 attenuated the number of invading cells on the transwell chamber,slowed down the rate of wound healing.Both knockdown of miR-326 and ZEB1 could increase the number of invading cells on the transwell chamber and accelerate the rate of wound healing.4.Western blot assay showed that overexpression of miR-326 and knockdown of ZEB1 decreased the expression of invasion and migration related proteins N-cadherin?MMP7?MMP13?SLUG and increased E-cadherin,apoptotic proteins PARP?BAX.Both knockdown of miR-326 and ZEB1 increased the expression of invasion and migration related proteins N-cadherin?MMP7?MMP13?SLUG and decreased E-cadherin,apoptotic proteins PARP ? BAX.5.Luciferase reporter assay showed that ZEB1 was a direct target of miR-326.Conclusion: MiR-326 could target ZEB1 to inhibit the proliferation,invasion,and migration of LADC cells and promote apoptosis,which is a potential therapeutic target for LADC.