MicroRNA-429 induces tumorigenesis of human non-small cell lung cancer cells and targets multiple tumor suppressor genes

Lung cancer is the major cause of cancer death globally. MicroRNAs are evolutionally conserved small noncoding RNAs that are critical for the regulation of gene expression. Aberrant expression of microRNA (miRNA) has been implicated in cancer initiation and progression. In this study, we demonstrated that the expression of miR-429 are often upregulated in non-small cell lung cancer (NSCLC) compared with normal lung tissues, and its expression level is also increased in NSCLC cell lines compared with normal lung cells. Overexpression of miR-429 in A549 NSCLC cells significantly promoted cell proliferation, migration and invasion, whereas inhibition of miR-429 inhibits these effects. Furthermore, we demonstrated that miR-429 down-regulates PTEN, RASSF8 and TIMP2 expression by directly targeting the 3′-untranslated region of these target genes. Taken together, our results suggest that miR-429 plays an important role in promoting the proliferation and metastasis of NSCLC cells and is a potential target for NSCLC therapy.     

MicroRNA-575靶向抑制BLID促进非小细胞肺癌细胞的增殖和侵袭

目的:通过体外实验探讨miR-575对非小细胞肺癌(NSCLC)细胞增殖与侵袭能力的影响及相关机制。方法:采用实时定量PCR法检测不同非小细胞肺癌细胞系中miR-575、BLID的表达;CCK-8法检测转染miR-575模拟物、抑制因子后不同时间A549细胞增殖情况的变化;Transwell法检测A549细胞的侵袭情况;Targetcan法及双荧光素酶检测miR-575对BLID 3'UTR端的靶向作用;Western blot法检测BLID蛋白的表达。结果:A549、SPC-A1、H1299、H1650等人非小细胞肺癌细胞系中miR-575的表达均显著高于永生化的人支气管上皮细胞系16HBE(P0.001)。MiR-575模拟物转染的A549细胞miR-575的表达明显高于对照组(P0.001),同时细胞的增殖和侵袭力增强(P0.05);反之,miR-575抑制因子转染的A549细胞miR-575的表达显著降低,且细胞的增殖和侵袭力明显降低(P0.01)。Targetscan法预测BLID可能是miR-575的下游靶基因,荧光素酶结果显示miR-575不仅能够有效抑制野生型BLID 3'UTR端的荧光素酶反应(P0.01),而且能够降低BLID的蛋白表达量(P0.01)。实时定量PCR结果显示BLID在NSCLC细胞系中均呈现显著的低表达(P0.001),且转染BLID后,NSCLC细胞的增殖和细胞侵袭被明显抑制(P0.05),而当miR-575与BLID共转染时,miR-575能够逆转BLID所抑制的细胞增殖和侵袭(P0.01)。结论:在NSCLC细胞系中,miR-575的表达上调,且能够通过直接作用于下游靶点抑癌基因BLID从而促非小细胞肺癌细胞增殖及侵袭。     

miR-126 enhances the sensitivity of non-small cell lung cancer cells to anticancer agents by targeting vascular endothelial growth factor A

Increasing evidence suggests that hsa-miR-126 (miR-126) is down-regulated in non-small cell lung cancer (NSCLC) cell lines and the restoration of miR-126 impairs tumor cell proliferation, migration, invasion, and survival by targeting specific molecules. Here, we reported for the first time that miR-126 was involved in regulating the response of NSCLC cells to cancer chemotherapy. After transfected A549 cells with miR-126 mimic or inhibitor, we found that an elevated level of miR-126 was significantly associated with a decreased half maximal inhibitory concentration of adriamycin (ADM) and vincristine, an increased accumulation of ADM, down-regulation of vascular endothelial growth factor A (VEGFA) and multidrug resistance-associated protein 1 (MRP1), and inactivation of the Akt signaling pathway. Furthermore, enhanced expression of miR-126 suppressed the growth of A549 xenograft and inhibited the expression of VEGFA and MRP1. miR-126 could efficiently down-regulate VEGFA expression through the interaction with the VEGFA 3'-untranslated region, whereas restoration of VEGFA could partially attenuate the suppression of MRP1 by miR-126. However, LY294002, an inhibitor of the PI3K/Akt signaling pathway, diminished this effect, suggesting that enhanced expression of miR-126 increased the sensitivity of NSCLC cells to anticancer agents through negative regulation of a VEGF/PI3K/Akt/MRP1 signaling pathway.     

MiR-186-5p通过靶向调控PTTG1抑制肺腺癌细胞的上皮-间质转化

先前研究表明,miR-186-5p在人类许多恶性肿瘤中扮演抑癌基因的作用,但其在肺腺癌上皮-间质转化（epithelial-mesenchymal transition,EMT）中的作用并不明确。本研究旨在证明,miR-186-5p可通过靶向调控PTTG1抑制肺腺癌细胞的上皮-间质转化。我们首先分析了miR-186-5p在人肺癌细胞中的表达。荧光定量PCR（QRT-PCR）结果显示,与人正常肺上皮细胞BEAS-2B相比,肺腺癌细胞SPC-A1、A549中的miR-186-5p表达量明显降低。为研究miR-186-5p在肺腺癌细胞中的功能,利用GV369-miR-186-5p表达载体,实现了在A549细胞中的过表达。基因转染结合Transwell侵袭结果显示,与对照质粒转染的A549细胞相比,过表达miR-186-5p的A549细胞的体外侵袭能力明显下降。Western印迹检测细胞中EMT相关标志物揭示,GV369-miR-186-5p转染的A549细胞中的上皮-钙黏着蛋白（E-cadherin）表达明显上调,而神经-钙黏着蛋白（N-cadherin）和波形蛋白（vimentin）表达明显下调。同时,GV369-miR-186-5p转染引起其靶基因--垂体肿瘤转化基因1（pituitary tumor-transforming gene 1,PTTG1）编码蛋白在A549细胞中明显降低。重要的是,过表达miR-186-5p与敲减PTTG1均可导致上皮-钙黏着蛋白表达上调,而神经-钙黏着蛋白和波形蛋白下调|而miR-186-5p和PTTG1表达载体共转染后,3种EMT相关标志物在A549的表达与对照细胞的表达无明显差异,提示过表达PTTG1可抵消miR-186-5p对EMT相关标志物表达的影响。综上所述,miR-186-5p可通过靶向调控PTTG1抑制EMT的发生,进而抑制肺腺癌细胞的侵袭转移。     

Altered expression of microRNA-365 is related to the occurrence and development of non-small-cell lung cancer by inhibiting TRIM25 expression

The purpose of this current study is to elucidate whether altered microRNA-365 (miR-365) has an association with the initiation and development of non-small-cell lung cancer (NSCLC) by targeting TRIM25 expression. The expression of miR-365 and TRIM25 in NSCLC tissues, adjacent normal tissues, and NSCLC cell lines were detected. The relationship between miR-365 expression and TRIM25 with the clinicopathological characteristics of NSCLC was analyzed. The putative binding site between miR-365 and TRIM25 was determined by luciferase activity assay. miR-365 inhibitors and miR-365 mimics were transfected to human NSCLC A549 cells, and the cell viability was detected by cell counting kit-8 assay; flow cytometry was carried out to determine cell cycle and apoptosis rate. Poorly expressed miR-365 and overexpressed TRIM25 was found in NSCLC tissues. TRIM25 was determined as a target gene of miR-365. The miR-365 and TRIM25 expression were related to the clinicopathological features of NSCLC, such as pathological classification, differentiation degree, TNM stage as well as lymph node metastasis. miR-365 suppressed the expression of TRIM25 and elevated the expression of the proapoptotic protein in NSCLC cells. Our study demonstrates that altered expression of miR-365 has a close association with the occurrence and development of NSCLC by inhibiting TRIM25 expression.     

miR-146b Reverses epithelial-mesenchymal transition via targeting PTP1B in cisplatin-resistance human lung adenocarcinoma cells

Epithelial-mesenchymal transformation (EMT) is associated with drug resistance in human lung adenocarcinoma cells, but its specific mechanism has not been clarified. In this study, we investigated the effect of miRNA-146b on EMT in cisplatin (DDP) resistant human lung adenocarcinoma cells and the corresponding mechanism. Cisplatin resistant (CR) human lung adenocarcinoma cells (A549/DDP and H1299/DDP) were established, and the EMT characteristics and invasion and metastasis ability of CR cells were determined by tumor cell-related biological behavior experiments. The role of miR-146b in EMT of CR cells was determined by in vitro functional test. The targeted binding of miR-146b to protein tyrosine phosphatase 1B (PTP1B) was verified by biological information and double luciferin gene reporting experiments. The effect of miR-146b on tumor growth and EMT phenotype in vivo was investigated by establishing the xenotransplantation mouse model. Compared with the control group, H1299/DDP and A549/DDP cells showed the enhanced EMT phenotypes, invasion and migration ability. Besides, miR-146b was lowly expressed in H1299/DDP and A549/DDP cells. More importantly, overexpressed miR-146b could specifically bind to PTP1B, thus inhibiting the EMT process and ultimately reducing CR in H1299/DDP and A549/DDP cells. Finally, overexpressed miR-146b observably inhibited tumor growth in xenograft model mice and inhibited the EMT phenotype of A549/DDP cells in vivo by regulating the expressions of EMT-related proteins. Overexpressed miR-146b could reverse the EMT phenotype of CR lung adenocarcinoma cells by targeting PTP1B, providing new therapeutic directions for CR of lung adenocarcinoma cells.     

AIMP3 inhibits cell growth and metastasis of lung adenocarcinoma through activating a miR-96-5p-AIMP3-p53 axis

Aminoacyl-tRNA synthetase-interacting multifunctional protein-3 (AIMP3) is a tumour suppressor, however, the roles of AIMP3 in non-small cell lung cancer (NSCLC) are not explored yet. Here, we reported that AIMP3 significantly inhibited the cell growth and metastasis of NSCLC (lung adenocarcinoma) in vitro and in vivo. We have firstly identified that AIMP3 was down-regulated in human NSCLC tissues compared with adjacent normal lung tissues using immunohistochemistry and western blot assays. Overexpression of AIMP3 markedly suppressed the proliferation and migration of cancer cells in a p53-dependent manner. Furthermore, we observed that AIMP3 significantly suppressed tumour growth and metastasis of A549 cells in xenograft nude mice. Mechanically, we identified that AIMP3 was a direct target of miR-96-5p, and we also observed that there was a negative correlation between AIMP3 and miR-96-5p expression in paired NSCLC clinic samples. Ectopic miR-96-5p expression promoted the proliferation and migration of cancer cells in vitro and tumour growth and metastasis in vivo which partially depended on AIMP3. Taken together, our results demonstrated that the axis of miR-96-5p-AIMP3-p53 played an important role in lung adenocarcinoma, which may provide a new strategy for the diagnosis and treatment of NSCLC.     

MicroRNA-567 inhibits cell proliferation and induces cell apoptosis in A549 NSCLC cells by regulating cyclin-dependent kinase 8

MicroRNA-567 (miR-567) plays a decisive role in cancers whereas its role in non-small cell lung cancer (NSCLC) is still unexplored. This study was therefore planned to explore the regulatory function of miR-567 in A549 NSCLC cells and investigate its possible molecular mechanism that may help in NSCLC treatment. In the current study, miR-567 expression was examined by quantitative real time-polymerase chain reaction (qRT-PCR) in different NSCLC cell lines in addition to normal cell line. A549 NSCLC cells were transfected by miR-567 mimic, miR-567 inhibitor, and negative control siRNA. Cell proliferation was evaluated by MTT and 5-bromo-2′deoxyuridine assays. Cell cycle distribution and apoptosis were studied by flow cytometry. Bioinformatics analysis programs were used to expect the putative target of miR-567. The expression of cyclin-dependent kinase 8 (CDK8) gene at mRNA and protein levels were evaluated by using qRT-PCR and western blotting. Our results found that miR-567 expressions decreased in all the studied NSCLC cells as compared to the normal cell line. A549 cell proliferation was suppressed by miR-567 upregulation while cell apoptosis was promoted. Also, miR-567 upregulation induced cell cycle arrest at sub-G1 and S phases. CDK8 was expected as a target gene of miR-567. MiR-567 upregulation decreased CDK8 mRNA and protein expression while the downregulation of miR-567 increased CDK8 gene expression. These findings revealed that miR-567 may be a tumor suppressor in A549 NSCLC cells through regulating CDK8 gene expression and may serve as a novel therapeutic target for NSCLC treatment.     

miR-137 impairs the proliferative and migratory capacity of human non-small cell lung cancer cells by targeting paxillin

Human lung cancer is the leading cause of cancer motility worldwide, with nearly 1.4 million deaths each year, among which non-small cell lung cancer (NSCLC) accounts for almost 85 % of this disease. The discovery of microRNAs (miRNAs) provides a new avenue for NSCLC diagnostic and treatment regiments. Currently, a large number of miRNAs have been reported to be associated with the progression of NSCLC, among which serum miR-137 has been examined to be down-regulated in NSCLC patients. However, the function of miR-137 on NSCLC cells migration and invasion and the relative mechanisms were less known. Here, we found that ectopic expression of miR-137 could inhibit cell proliferation, induce cell apoptosis, and suppress cell migration and invasion in NSCLC cell line A549. Moreover, we found that paxillin (PXN) was a target gene of miR-137 in NSCLC cells and restored expression of PXN abolished the miR-137-mediated suppression of cell migration and invasion. Taken together, our results showed that miR-137 acted as a tumor suppressor in NSCLC by targeting PXN, and it may provide novel diagnostic and therapeutic options for human NSCLC clinical operation in future.     

miR-126 inhibits non-small cell lung cancer cells proliferation by targeting EGFL7

MicroRNAs (miRNAs) represent an abundant group of small non-coding RNAs that regulate gene expression, and have been demonstrated to play roles as tumor suppressor genes (oncogenes), and affect homeostatic processes such as development, cell proliferation, and cell death. Subsequently, epidermal growth factor-like domain 7 (EGFL7), which is confirmed to be involved in cellular responses such as cell migration and blood vessel formation, is identified as a potential miR-126 target by bioinformatics. However, there is still no evidence showing EGFL7’s relationship with miR-126 and the proliferation of lung cancer cells. The aim of this work is to investigate whether miR-126, together with EGFL7, have an effect on non-small cell lung cancer (NSCLC) cells’ proliferation. Therefore, we constructed overexpressed miR-126 plasmid to target EGFL7 and transfected them into NSCLC cell line A549 cells. Then, we used methods like quantitative RT-PCR, Western blot, flow cytometry assay, and immunohistochemistry staining to confirm our findings. The result was that overexpression of miR-126 in A549 cells could increase EGFL7 expression. Furthermore, the most notable finding by cell proliferation related assays is that miR-126 can inhibit A549 cells proliferation in vitro and inhibit tumor growth in vivo by targeting EGFL7. As a result, our study demonstrates that miR-126 can inhibit proliferation of non-small cell lung cancer cells through one of its targets, EGFL7.     

MiR-200a enhances the migrations of A549 and SK-MES-1 cells by regulating the expression of TSPAN1

MicroRNA-200a (miR-200a) has been reported to regulate tumour progression in several tumours; however, little is known about its role in non-small cell lung cancer cells (NSCLCs). Here, we found that miR-200a was up-regulated in A549 and SK-MES-1 cells compared with normal lung cells HELF. By a series of gain-of-function and loss-of-function studies, over-expression of miR-200a was indicated to enhance cells migration, and its knock-down inhibited migration of cells in NSCLC cell lines. Furthermore, miR-200a was identified to induce TSPAN1 expression which was related to migration. TSPAN1 was proved to induce migration, and so up-regulation of TSPAN1 by miR-200a may explain why over-expressing miR-200a promotes NSCLC cells migration.     

MicroRNA-217 Functions as a Tumour Suppressor Gene and Correlates with Cell Resistance to Cisplatin in Lung Cancer

MiR-217 can function as an oncogene or a tumour suppressor gene depending on cell type. However, the function of miR-217 in lung cancer remains unclear to date. This study aims to evaluate the function of miR-217 in lung cancer and investigate its effect on the sensitivity of lung cancer cells to cisplatin. The expression of miR-217 was detected in 100 patients by real-time PCR. The effects of miR-217 overexpression on the proliferation, apoptosis, migration and invasion of SPC-A-1 and A549 cells were investigated. The target gene of miR-217 was predicted by Targetscan online software, screened by dual luciferase reporter gene assay and demonstrated by Western blot. Finally, the effects of miR-217 up-regulation on the sensitivity of A549 cells to cisplatin were determined. The expression of miR-217 was significantly lower in lung cancer tissues than in noncancerous tissues (p < 0.001). The overexpression of miR-217 significantly inhibited the proliferation, migration and invasion as well as promoted the apoptosis of lung cancer cells by targeting KRAS. The up-regulation of miR-217 enhanced the sensitivity of SPC-A-1 and A549 cells to cisplatin. In conclusion, miR-217 suppresses tumour development in lung cancer by targeting KRAS and enhances cell sensitivity to cisplatin. Our results encourage researchers to use cisplatin in combination with miR-217 to treat lung cancer. This regime might lead to low-dose cisplatin application and cisplatin side-effect reduction.     

MicroRNA-193b modulates proliferation, migration, and invasion of non-small cell lung cancer cells

MicroRNAs have been reported to be closely related to the development of human lung cancers. However, the functions of microRNAs in non-small cell lung cancer (NSCLC) remain largely undefined. Here, we investigated the role of microRNA-193b (miR-193b) in NSCLC. Our data showed that miR-193b was markedly down-regulated in NSCLC cancer tissues compared with adjacent normal tissues. The NSCLC cell line (A549) transfected with the miR-193b exhibited significantly decreased proliferation, migration, and invasion capacities when compared with the control cells. In contrast, inhibition of miR-193b increased the proliferation, migration, and invasion of A549 cells. Moreover, miR-193b repressed the expressions of cyclin D1 and urokinase-type plasminogen activator in A549 cells. These data suggest that miR-193b is a tumor suppressor in NSCLC.     

A novel fine tuning scheme of miR-200c in modulating lung cell redox homeostasis

Oxidative stress induces miR-200c, the predominant microRNA (miRNA) in lung tissues; however, the antioxidant role and biochemistry of such induction have not been clearly defined. Therefore, a lung adenocarcinoma cell line (A549) and a normal lung fibroblast (MRC-5) were used as models to determine the effects of miR-200c expression on lung antioxidant response. Hydrogen peroxide (H₂O₂) upregulated miR-200c, whose overexpression exacerbated the decrease in cell proliferation, retarded the progression of cells in the G2/M-phase, and increased oxidative stress upon H₂O₂ stimulation. The expression of three antioxidant proteins, superoxide dismutase (SOD)-2, haem oxygenase (HO)-1, and sirtuin (SIRT) 1, was reduced upon H₂O₂ stimulation in miR-200c-overexpressed A549 cells. This phenomenon of increased oxidative stress and antioxidant protein downregulation also occurs simultaneously in miR-200c overexpressed MRC-5 cells. Molecular analysis revealed that miR-200c inhibited the gene expression of HO-1 by directly targeting its 3′-untranslated region. The downregulation of SOD2 and SIRT1 by miR-200c was mediated through zinc finger E-box-binding homeobox 2 (ZEB2) and extracellular signal-regulated kinase 5 (ERK5) pathways, respectively, where knockdown of ZEB2 or ERK5 decreased the expression of SOD2 or SIRT1 in A549 cells. LNA anti-miR-200c transfection in A549 cells inhibited the endogenous miR-200c expression, resulting in increased expressions of antioxidant proteins, reduced oxidative stress and recovered cell proliferation upon H₂O₂ stimulation. These findings indicate that miR-200c fine-tuned the antioxidant response of the lung cells to oxidative stress through several pathways, and thus this study provides novel information concerning the role of miR-200c in modulating redox homeostasis of lung.     

miR-210 promotes lung adenocarcinoma proliferation,migration, and invasion by targeting lysyl oxidase-like 4

Accumulating evidence has revealed that various microRNAs are deregulated and involved in lung cancer development and metastasis. miR-210 is implicated in several cancer progression. However, the detailed biological function and role of miR-210 in lung adenocarcinoma remains unclear. Our current study was aimed to investigate the mechanism of miR-210 in lung adenocarcinoma progression. We observed that miR-210 was significantly upregulated in lung cancer cell lines (A549 and H1650) in comparison to BEAS-2B cells. In addition, we found that miR-210 was greatly elevated in lung adenocarcinoma tissues. Then, it was shown that overexpression of miR-210 was able to promote lung cancer cell proliferation and colony formation ability while inhibitors of miR-210 exhibited a reversed phenomenon. Subsequently, A549 and H1650 cell migration and invasion capacity were obviously restrained by miR-210 inhibition whereas induced by miR-210 mimics. Lysyl oxidase-like 4 (LOXL4), a member of the secreted copper-dependent amine oxidases has been found to be increased or decreased in different cancer types. Here, we confirmed that LOXL4 could serve as a downstream target of miR-210 and miR-210 promoted lung cancer progression via targeting LOXL4. In A549 and H1650 cells, knockdown of LOXL4 dramatically repressed lung cancer cell proliferation, migration, and invasion. In conclusion, our study implied that miR-210 might indicate a new perspective for lung cancer.     

microRNA-448 inhibits the progression of non-small–cell lung cancer through regulating IRS2

Recently, microRNA-448 (miR-448) has been reported to be a tumor-associated miRNA in many human cancers. In this study, we investigated the function of miR-448 in non-small–cell lung cancer (NSCLC) progression and confirmed the relationship between miR-448 and insulin receptor substrates 2 (IRS2). First, downregulation of miR-448 and upregulation of IRS2 were detected in NSCLC using the quantitative real-time polymerase chain reaction (qRT-PCR) assay. Furthermore, the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay showed that miR-448 inhibited cell viability in NSCLC. Transwell and Western blot assays indicated that the upregulation of miR-448 inhibited cell metastasis and epithelial-to-mesenchymal transition (EMT) in NSCLC. And it was found that overexpression of miR-448 reduced the adhesion of A549 cells to HUVEC cells using the adhesion assay. Furthermore, the dual luciferase assay indicated that miR-448 directly targeted IRS2 in NSCLC. In addition, it was found that IRS2 silencing had an inhibitory effect on the progression of NSCLC, and the upregulation of IRS2 partially impaired the inhibitory effect of miR-448 in NSCLC. Briefly, overexpression of miR-448 inhibited cell proliferation, metastasis, and EMT by suppressing IRS2 expression in NSCLC.     

MiR-1827 functions as a tumor suppressor in lung adenocarcinoma by targeting MYC and FAM83F

The bioactivity of microRNA-1827 (miR-1827) in lung adenocarcinoma cells would be explored. The expression level of gene and miR-1827 in 76 pairs of lung adenocarcinoma tissues and adjacent counterparts were analyzed by a quantitative real-time polymerase chain reaction. Primary lung adenocarcinoma cells were derived from patients’ tissues. These cells were treated with miR-1827 agomir to mimic the upregulation of endogenous miR-1827. The malignant degree of lung adenocarcinoma cells in vitro was evaluated by cell proliferation, colony formation, transwell invasion, and apoptosis assays. Western blot analysis was used to observe the transition of lung adenocarcinoma cells from epithelial-to-mesenchymal. Target genes of miR-1827 were predicted by bioinformatics analysis. In addition, the interaction between miR-1827 and candidate messenger RNAs was verified by dual-luciferase reporter assay and AGO2-RNA immunoprecipitation. Besides, the effect of miR-1827 on tumors was verified by in vivo experiments. Transient gene overexpression was achieved by plasmids transfection. In this study, we found that the expression of miR-1827 was downregulated in lung adenocarcinoma, and its low expression was significantly correlated with the progression of lung adenocarcinoma and poor prognosis of patients. miR-1827 overexpression remarkably reduced the malignancy of primary lung adenocarcinoma cells in vitro. MYC and FAM83F were identified as two targeted genes of miR-1827 in lung adenocarcinoma cells. The levels of these two genes were upregulated in lung adenocarcinoma, and their high expression was significantly associated with the progression of lung adenocarcinoma and poor prognosis of patients. Overexpression of MYC or FAM83F attenuated the effects of miR-1827 on primary lung adenocarcinoma cells in vitro. In addition, in vivo experiments showed that miR-1827 inhibited tumor growth by reducing the levels of MYC and FAM83F. In conclusion, miR-1827 might repress the development of lung adenocarcinoma by targeting oncogenic genes MYC and FAM83F.