MicroRNA-144 regulates proliferation,invasion, and apoptosis of cells in malignant solitary pulmonary nodule via zinc finger E-box-binding homeobox 1

Aims: This study was to investigate the expression of microRNA (miR)-144 in malignant solitary pulmonary nodule (SPN) tissues and peripheral blood, as well as the biological function of miR-144 in the occurrence and development of lung cancer. Methods: In this study, 39 malignant and 30 benign SPN patients were included. The expression of miR-144 was examined using quantitative real-time polymerase chain reaction. Receiver operating characteristic (ROC) curve was used to identify the clinical value of miR-144 in the early diagnosis of malignant SPN. MTT assay was performed to determine A549 cell proliferation and Transwell assay was used to detect changes in A549 cell invasion and migration ability. Flow cytometry was performed to monitor cell apoptosis, while Western blotting assay was used to measure protein expression levels. At last, dual-luciferase reporter assay was employed to test whether miR-144 regulates zinc finger E-box-binding homeobox 1 (ZEB1) gene expression. Results: Expression of miR-144 was reduced in patients with malignant SPN. miR-144 had diagnostic value for malignant SPN. Proliferation of A549 cells was inhibited by miR-144. Invasion ability of A549 cells was reduced by miR-144. Apoptosis of A549 cells was promoted by miR-144. miR-144 induced A549 cell apoptosis by targeting ZEB1 protein. miR-144 regulated the expression of ZEB1 by interacting with its 3’-UTR region. Conclusions: Expression of miR-144 is reduced in malignant SPN tissues and peripheral blood, being of clinical value in the diagnosis of malignant SPN. miR-144 promotes the apoptosis of lung cancer cells, and inhibits the proliferation, invasion and migration of lung cancer by regulating ZEB1 gene.     

miR-425 regulates cell proliferation,migration and apoptosis by targeting AMPH-1 in non-small-cell lung cancer

BackgroundMicroRNAs (miRNAs) have been proved to act as vital roles on non-small-cell lung cancer (NSCLC), and miR-425 has been proven to serve an important function in several tumors. However, the functional role of miR-425 on NSCLC is still unclear.MethodsThe mRNA and protein expression of miR-425 and AMPH-1 were determined by qRT-PCR and western blot analysis, respectively. NSCLC cells (SK-MES-1 and A549) proliferation and migration were measured by CCK-8 and transwell assay, respectively. Cell apoptosis was assessed by flow cytometry and western blotting, In addition, luciferase reporter assay was carried out to confirm the direct targeting of AMPH-1 by miR-425. Xenograft experiments were performed to observe the tumorigenesis of miR-425 in vivo.ResultsThe results showed that miR-425 was overexpressed and AMPH-1 expression was downregulated in SK-MES-1 and A549 cells. Silencing miR-425 inhibited proliferation, migration and promoted apoptosis of NSCLC cells. Moreover, we proved that miR-425 could target AMPH-1. The expression of AMPH-1was upregulated in A549 with miR-425 inhibitor. Moreover, miR-425 knockdown were less tumorigenic than the control in vivo.ConclusionsTaken together, miR-425 could promote the proliferation, invasion and suppress apoptosis by targeting AMPH-1 in NSCLC cells. miR-425/AMPH-1 axis may represent a potential therapeutic strategy or novel prognostic biomarkers to NSCLC.     

miR-107 regulates cisplatin chemosensitivity of A549 non small cell lung cancer cell line by targeting cyclin dependent kinase 8

Previous studies demonstrated that the acquired drug resistance of non-small cell lung cancer (NSCLC) was related to deregulation of miRNAs. However, the effects of miR-107 and the mechanism through which miR-107 affects the cisplatin chemoresistance in NSCLC have not been reported. TaqMan RT-PCR or Western blot assay was performed to detect the expression of mature miR-107 and cyclin dependent kinase 8 (CDK8) protein. The viabilities of treated cells were analyzed using MTT assay. We found that the expression level of miR-107 in A549 cells was significantly lower than that in normal human bronchial epithelial cells (0.45 ± 0.26 vs. 1.00 ± 0.29, P = 0.032). The MTT assay showed that the A549 cells transfected with miR-107 mimics were significantly more sensitive to the therapy of cisplatin than control cells. A549 cells transfected with miR-107 mimics showed a decreased CDK8 protein expression. Downregulation of CDK8 expression by siRNAs, A549 cells became more sensitive to the therapy of cisplatin. In addition, the enhanced growth-inhibitory effect by the miR-107 mimic transfection was enhanced after the addition of CDK8 siRNA. In conclusion, the present study provides the first evidence that miR-107 plays a key role in cisplatin resistance by targeting the CDK8 protein in NSCLC cell lines, suggesting that miR-107 can be used to predict a patient’s response to chemotherapy as well as serve as a novel potential maker for NSCLC therapy.     

MiR-1260b promotes the migration and invasion in non-small cell lung cancer via targeting PTPRK

Objective

Non-small cell lung cancer (NSCLC) accounts for 80–85% of lung cancer cases which cause most of cancer-related deaths globally. As our previous study discovered miR-1260b can be regarded as a specific signature for metastasis in NSCLC patients. However, the molecular mechanisms of miR-1260b underlying NSCLC progression and metastasis remain dismal.

LncRNA NCK1-AS1 promotes non-small cell lung cancer progression via regulating miR-512-5p/p21 axis

ObjectiveThis study aimed at probing into the effect of lncRNA NCK1-AS1 on proliferation, migration and invasion of non-small cell lung cancer (NSCLC) cells and its regulatory function on miR-512-5p/p21 molecular axis.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) was used to assess the expressions of NCK1-AS1 and miR-512-5p in NSCLC tissues and cell lines. The alterations of cell proliferation, migration, invasion and cell cycle were examined by cell counting kit-8 (CCK-8) assay, BrdU experiment, Transwell experiment and flow cytometry, respectively. The dual-luciferase reporter assay and RNA immunoprecipitation experiment were performed to validate the binding relationships between miR-512-5p and NCK1-AS1, and miR-512-5p the 3'UTR of p21 mRNA. Western blot was used to determine the effects of NCK1-AS1 and miR-512-5p on p21 protein expression.ResultsNCK1-AS1 expression was up-regulated in NSCLC tissues and cells, and its high expression was correlated with shorter overall survival time and faster progression of patients. Overexpression of NCK1-AS1 promoted NSCLC cell proliferation, migration and invasion, and accelerated the cell cycle, whereas NCK1-AS1 siRNA inhibited these malignant biological behaviors, and arrested cell cycle. NCK1-AS1 could bind to miR-512-5p, p21 was verified as a target gene of miR-512-5p, and NCK1-AS1 could up-regulate the expression of p21 in NSCLC cells via repressing miR-512-5p expression.ConclusionNCK1-AS1 promotes NSCLC progression by regulating miR-512-5p/p21 molecular axis.     

MicroRNA-137 inhibits cell migration and invasion by targeting bone morphogenetic protein-7 (BMP7) in non-small cell lung cancer cells

MicroRNA-137 (miR-137) was reported to be dysregulated in several human cancers. However, the function and mechanism of miR-137 in non-small cell lung cancer (NSCLC) is still unclear. In the current study, we explored the role of miR-137 in NSCLC progression. Using qRT-PCR, our data showed that miR-137 was significantly down-regulated in NSCLC tissues and cell lines. In vitro functional assay, we found that over-expression of miR-137 suppressed NSCLC cells proliferation, migration and invasion, indicating that miR-137 could act as a tumor suppressor in NSCLC progression. In addition, bone morphogenetic protein-7 (BMP7) was identified as a target of miR-137 in NSCLC cells, Luciferase reporter assay suggested that miR-137 directly targeted 3’-UTR of BMP7, and correlation analysis revealed that BMP7 inversely correlated with miR-137 in NSCLC tissues. Furthermore, Restoration of BMP7 remarkably reversed the tumor suppressive effects of miR-137 on NSCLC cell proliferation, migration, and invasion. Taken together, our findings suggested that miR-137/BMP7 axis could contribute to the progression of NSCLC, suggesting miR-137 as a potential therapeutic target for the treatment of NSCLC.     

过表达miR-10b促进肺癌细胞系A549增殖

目的探讨非小细胞肺癌(NSCLC)患者肺癌及癌旁组织中miR-10b(miR-10b)表达水平及miR-10b是否通过调控锌指转录蛋白基因(KLF4)对肺癌细胞系A549恶性化的影响。方法 40例NSCLC患者病理切片,原位杂交检测肺癌及癌旁组织中miR-10b的表达量;对肺癌细胞系A549转染miR-10b mimics后,CCK-8法检测肺癌细胞增殖;real-time PCR及Western blot检测KLF4 mRNA及蛋白水平;软琼脂克隆形成实验检测过表达miR-10b对A549细胞的肿瘤恶性化程度的影响。结果肺癌细胞A549及肺癌组织中miR-10b的表达量分别高于正常肺上皮细胞16HBE及癌旁组织;过表达miR-10b模拟物的A549细胞中,KLF4蛋白水平显著下降(P0.05);过表达的miR-10b可显著增加A549细胞的增殖速度及在软琼脂内的成瘤性。结论 miR-10b在不同类型细胞及组织中具有分布差异性、可能是通过抑制KLF4的表达促进肺癌细胞增殖及恶性化。     

LINC00662 Promotes Oral Squamous Cell Carcinoma Cell Growth and Metastasis through miR-144-3p/EZH2 Axis

PurposeLong non-coding RNA (lncRNA) is identified as an important regulator involved in oral squamous cell carcinoma (OSCC) tumorigenesis. This study aimed to investigate the functional role and underlying mechanism of LINC00662 in OSCC.Materials and MethodsThe expression levels of LINC00662, miR-144-3p, and enhancer of zeste homolog 2 (EZH2) mRNA were quantified with quantitative real-time polymerase chain reaction in OSCC tissues and cell lines. Western blot analysis was used to assay the expression levels of E-cadherin, Vimentin, and EZH2. Cell proliferation, migration, and invasion were monitored by cell counting kit-8 and Transwell assays. Dual-luciferase reporter and RNA immunoprecipitation assays were employed to verify the regulatory relationship between LINC00662 and miR-144-3p.ResultsThe expression of LINC00662, positively associated with the increased TNM stage and lymph node metastasis of the patients, was up-regulated in OSCC tissues and cells. The overexpression of LINC00662 facilitated the proliferation, migration, and invasion of OSCC cells. MiR-144-3p could bind to LINC00662, and the promoting effect of LINC00662 overexpression was counteracted by miR-144-3p mimic. Moreover, EZH2 expression was negatively regulated by miR-144-3p and positively regulated by LINC00662. The silencing of EZH2 attenuated the promoting effects of overexpression of LINC00662 on cell proliferation, migration, invasion, and epithelial-mesenchymal transition.ConclusionLINC00662, as an oncogenic lncRNA of OSCC, accelerates OSCC progression by repressing miR-144-3p expression and increasing EZH2 expression.     

miR-34b抑制非小细胞肺癌A549的侵袭和迁移

目的：本研究旨在探索miR-34b对非小细胞肺癌A549侵袭和迁移的影响及HIF1α在其中所起的作用。方法：A549细胞转染miR-34b mimic和pc-HIF1α过表达载体以过表达miR-34b和缺氧诱导因子-1α (HIF1α)。A549细胞分为4组：A549(空对照)组,miR-34b mimic组,pc-HIF1α组和miR-34b mimic+pc-HIF1α组。实时定量PCR(qRT-PCR)检测miR-34b表达和HIF1α的mRNA水平。荧光素酶分析验证miR-34b和HIF1α的靶向关系。蛋白印迹检测HIF1α、基质金属蛋白酶2 (MMP-2),Snail和血管内皮细胞生长因子(VEGF)的蛋白水平。Transwell分析细胞侵袭。划痕实验检测细胞迁移。结果：转染miR-34b mimic可提高A549细胞miR-34b的表达水平,降低HIF1α的mRNA水平(P<0.001)。miR-34b可直接靶向HIF1α。miR-34b 过表达可抑制 pc-HIF1α诱导的HIF1α蛋白水平升高(P<0.01)。与对照组相比,miR-34b 过表达可显著降低细胞侵袭数目和愈合率(P<0.01)。此外,miR-34b 过表达可显著抑制 HIF1α过表达导致的细胞侵袭和愈合率增加(P<0.01)。miR-34b mimic组MMP-2、Snail和VEGF相对蛋白水平低于对照组(P<0.01)。pc-HIF1α组MMP-2、Snail和VEGF相对蛋白水平高于对照组(P<0.01)。与pc-HIF1α组相比,miR-34b mimic+pc-HIF1α组MMP-2、Snail和VEGF相对蛋白水平下降(P<0.01)。结论：miR-34b-5p过表达通过靶向HIF1α抑制非小细胞肺癌A549的侵袭和迁移。     

MiR-520b inhibits proliferation,migration and invasion in gallbladder carcinoma by targeting RAB22A

IntroductionPrevious studies have reported that miR-520b exhibited inhibitory effects on various human tumors, whereas the effects of miR-520b on gallbladder carcinoma (GBC) have remained unclear. To investigate the effects of miR-520b on GBC progression and reveal the underlying mechanisms, this study was performed.Material and methodsMiR-520b and RAB22A mRNA levels were analyzed by quantitative real-time PCR (qPCR). RAB22A protein level was analyzed via Western blot and immunohistochemical (IHC) analysis. The proliferation, colony formation ability, migration and invasion of NOZ cells were measured via MTT, colony formation, wound healing and transwell invasion assay respectively.ResultsMiR-520b expression level was lower in human GBC tissues than that in neighboring normal tissues. MiR-520b mimic repressed NOZ cell proliferation, colony formation ability, migration and invasion, whereas miR-520b inhibitor exhibited opposite effects. Dual luciferase reporter assay confirmed that miR-520b could bind to the 3′-untranslated regions of RAB22A mRNA. Moreover, RAB22A overexpression significantly abolished the anti-tumor effects of miR-520b in a NOZ cell model. Western blot, qPCR and IHC analysis proved that human GBC tissues showed a higher RAB22A expression level than neighboring normal tissues. Additionally, there was a negative association between miR-520b and RAB22A expression.ConclusionsMiR-520b had suppressive effects on GBC via targeting RAB22A in vitro.     

miR-106a promotes growth and metastasis of non-small cell lung cancer by targeting PTEN

MicroRNAs are a class of small non-coding RNAs that play essential roles in cancer development and progression. Recent studies suggested that abnormal expression of miRNAs occurs frequently in non-small cell lung cancer (NSCLC) tissues compared to adjacent normal tissues. In this study, we investigated the expression and the biological roles of miR-106a in non-small cell lung cancer. Our results showed that miR-106a was up-regulated in NSCLC tissues and cell lines. Inhibition of miR-106a in NSCLC cells substantially inhibited cell proliferation, migration, and invasion. Phosphatase and tensin homolog (PTEN) was identified as a direct target of miR-106a, and over-expression of miR-106a suppressed PTEN by direct binding to its 3’-untranslated region (3’-UTR). Furthermore, the presence of miR-106a was inversely correlated with PTEN in NSCLC tissues. Overall, this study suggested that miR-106a inhibited the growth and metastasis of NSCLC cells by decreasing PTEN expression. These data provide novel insights with potential therapeutic applications for the treatment of NSCLC.     

MiR-26b inhibits hepatocellular carcinoma cell proliferation,migration, and invasion by targeting EphA2

Deregulated microRNAs (miRNAs) have been shown to play important roles in cancer progression as a result of changes in expression of their target genes. In this study, we investigated the expression of miR-16b in eight hepatocellular carcinoma (HCC) cell lines, revealed the roles of miR-26b on hepatocellular carcinoma (HCC) cell proliferation, migration, and invasion, and confirmed that EphA2 is a direct target of miR-26b. The miR-26b expression was decreased and EphA2 expression was evaluated in HCC cell lines. Luciferase assays revealed that miR-26b inhibited EphA2 expression by targeting the 3’-untranslated region of EphA2 mRNA. Overexpression of miR-26b dramatically inhibited the proliferation, invasion, and migration of HCC cells by targeting EphA2. Moreover, miR-26b down-regulated c-Myc and CyclinD1 expression, which was reversed by overexpressed EphA2. Taken together, our data demonstrated the mechanism of miR-26b contributed to HCC progression and implicated that miR-26b’s potential in HCC therapy.     

p21活化激酶6削弱非小细胞肺癌A549细胞侵袭及迁移能力的分子机制

 目的：探讨调控p21活化激酶6(PAK6)的微小RNA（miRNA）与非小细胞肺癌侵袭及迁移的关系,并了解PAK6下游分子信号机制。方法：通过生物信息学数据库预测靶向PAK6的miRNA,转染miRNA模拟物及抑制剂,Western blotting及实时定量荧光PCR（real-time PCR）检测A549细胞中PAK6的表达量,萤光素报告酶实验检测预测miRNA对PAK6的调控,并行体外迁移及侵袭实验,检测转染miRNA对PC-3细胞迁移及侵袭能力的影响。转染目标miRNA及siPAK6,Western blotting检测A549细胞基质金属蛋白酶9（MMP-9）表达量。结果：生物信息学预测miR-23a可能是调控PAK6的靶向miRNA。Western blotting检测转染miR-23a组PAK6表达量下降69％,而转染miR-23a抑制剂组PAK6表达量上升52％,差异均有统计学意义。萤光素报告酶实验结果显示,转染miR-23a后野生型PAK6组萤光素酶活性下降52％,而突变组差异无统计学意义（P＞0.05）。Real-time PCR检测3组PAK6 mRNA的表达差异无统计学意义（P＞0.05）。体外迁移及侵袭实验示,转染miR-23a组迁移细胞数减少73％,侵袭的细胞数减少59％。Western blotting检测发现,转染siPAK6组MMP-9的表达下降85％（P＜0.01）,转染miR-23a组MMP-9的表达下降76％（P＜0.01）。结论：miR-23a通过PAK6-MMP-9信号途径引起非小细胞肺癌细胞迁移及侵袭能力的下降。     

长链非编码MALAT-1 调控miR-205 的表达影响非小细胞肺癌细胞的生长和迁移

目的:探讨在非小细胞肺癌中,LncRNA MALAT-1 与miR-205 的相互关系,以及影响肺癌细胞生物学行为的机制。方法:qPCR 检测不同非小细胞肺癌中LncRNA MALAT-1 的表达情况;双荧光素酶报告基因检测MALAT-1 与miR-205的相互作用;Transwell 侵袭实验和划痕实验检测抑制MALAT-1 后肺癌细胞侵袭能力的变化,以及抑制miR-205 的表达后肺癌细胞迁移和侵袭能力的恢复情况;裸鼠皮下成瘤检测抑制LncRNA MALAT-1 后肺癌细胞体外成瘤体积和质量变化。结果:与其他肺癌细胞株相比,A549 细胞中MALAT-1 表达最高,miR-205 的表达水平最低;双荧光素酶实验证实MALAT-1 能与miR-205 的3忆UTR 特异性结合,可以调控miR-205 的表达与活性;抑制MALAT-1 的表达后可以降低肺癌细胞的迁移和侵袭能力;抑制miR-205 的表达水平过后,肺癌细胞的迁移和侵袭能力相对增强;抑制MALAT-1 的表达后,荷瘤小鼠的肿瘤体积和重量都明显减小。结论:MALAT-1 可以调控miR-205 的表达影响肺癌细胞A549 的侵袭和迁移能力。     

miR-144 regulates transforming growth factor-β1 iduced hepatic stellate cell activation in human fibrotic liver

Objectives: Activation of hepatic stellate cells (HSCs) into collagen producing myofibroblasts is critical for pathogenesis of liver fibrosis. Transforming growth factor-β1 (TGF-β1) is one of the main profibrogenic mediators for HSC transdifferentiation. Recent studies have shown effect of microRNAs (miRNAs) on regulating TGF-β1-induced HSC activation during liver fibrosis. Here, we aimed to explore the roles of miR-144 and miR-200c in human liver fibrosis. Methods: Expression of TGF-β1 was detected in 42 fibrotic and 18 normal human liver tissues by quantitative real time polymerase chain reaction (qRT-PCR) and immunohistochemistry, and its correlation with α-smooth muscle actin (α-SMA) was calculated. miR-144 and miR-200c expression level in fibrotic liver tissues were also detected by qRT-PCR. The correlation of TGF-β1 expression with miR-200c and miR-144 in the fibrotic liver was analyzed. Results: The results showed that TGF-β1 expression was much higher in fibrotic liver than that in normal liver tissues (P<0.05). TGF-β1 protein high expressing liver fibrosis showed α-SMA positive cells in the liver parenchyma indicating activated HSCs. Expression of TGF-β1 in fibrotic liver was significantly correlated with α-SMA expression (R=0.633, P<0.001). Furthermore, miR-144 was less expressed in liver fibrosis (P<0.05) and was significantly correlated with expression of TGF-β1 in fibrotic liver tissues (R=-0.442, P<0.01). However, miR-200c did not show significant difference between normal and fibrotic liver (P=0.48) and correlation with TGF-β1 expression (R=0.106, P=0.51). Conclusion: All the results indicate that miR-144 can be a novel regulator of TGF-β1-induced HSC activation during liver fibrosis.     

Melittin suppresses growth and induces apoptosis of non-small-cell lung cancer cells via down-regulation of TGF-β-mediated ERK signal pathway

The purpose of this study was to investigate the anti-cancer effect of melittin on growth, migration, invasion, and apoptosis of non-small-cell lung cancer (NSCLC) cells. This study also explored the potential anti-cancer mechanism of melittin in NSCLC cells. The results demonstrated that melittin suppressed growth, migration, and invasion, and induced apoptosis of NSCLC cells in vitro. Melittin increased pro-apoptotic caspase-3 and Apaf-1 gene expression. Melittin inhibited tumor growth factor (TGF)-β expression and phosphorylated ERK/total ERK (pERK/tERK) in NSCLC cells. However, TGF-β overexpression (pTGF-β) abolished melittin-decreased TGF-β expression and pERK/tERK in NSCLC cells. Treatment with melittin suppressed tumor growth and prolonged mouse survival during the 120-day observation in vivo. Treatment with melittin increased TUNEL-positive cells and decreased expression levels of TGF-β and ERK in tumor tissue compared to the control group. In conclusion, the findings of this study indicated that melittin inhibited growth, migration, and invasion, and induced apoptosis of NSCLC cells through down-regulation of TGF-β-mediated ERK signaling pathway, suggesting melittin may be a promising anti-cancer agent for NSCLC therapy.     

miR-140-3p通过靶向PD-L1抑制非小细胞肺癌A549细胞的活力、迁移和侵袭

目的:探究微小RNA-140-3p(mi R-140-3p)对程序性细胞死亡配体1(PD-L1)的靶向关系以及对非小细胞肺癌A549细胞的活力、迁移和侵袭的影响。方法:使用RT-qPCR检测HLF-1、A549和H1299不同细胞株中mi R-140-3p的表达水平,选择差异最显著的A549细胞用作后续研究对象; Target Scan软件预测和双萤光素酶报告基因实验验证mi R-140-3p和PD-L1之间的靶向关系; RT-qPCR和Western blot检测转染mi R-140-3p模拟物和抑制剂对PD-L1表达水平的影响; MTT法检测mi R-140-3p和PD-L1对A549细胞活力的影响; Transwell实验检测mi R-140-3p和PD-L1对A549细胞迁移和侵袭的影响。结果:mi R-140-3p在人肺癌A549和H1299细胞的表达中显著下调(P 0. 05); mi R-140-3p高表达能够使PD-L1表达下调,对A549细胞的活力、迁移和侵袭具有抑制作用;转染pc DNA3. 0-PD-L1能够阻断mi R-140-3p对A549细胞活力、迁移和侵袭的抑制作用。结论:mi R-140-3p可通过靶向负调控PD-L1抑制A549细胞的活力、迁移和侵袭。