Serum exosomal miR-7977 as a novel biomarker for lung adenocarcinoma

Exosomal microRNAs (miRNAs) have great potentials as a novel biomarker to predict lung cancer. We applied a miRNA microarray to identify aberrantly expressed serum exosomal miRNAs as candidate biomarkers for patients with lung adenocarcinoma (LUAD). Compared with the normal control, 31 exosomal miRNAs were found to be upregulated and 29 exosomal miRNAs were downregulated in the serum of LUAD respectively. Then, 10 dysregulated exosomal miRNAs expression levels in serum were further validated via qRT-polymerase chain reaction. Notably, exosomal miR-7977 was highest expressed and miR-98-3p was lowest expressed in the patients with LUAD, and exosomal miR-7977 showed significant correlation with the N stage and TNM stage with patients with LUAD (P < .05). Receiver operating characteristic curve showed that the abundant level of exosomal miR-7977 may predict LUAD with an area of under the curve (AUC) of 0.787. In comparison with exosomal miR-7977, exosomal miR-98-3p had a smaller area (0.719). The combination of exosomal miR-7977 and miR-98-3p improved the AUC to 0.816. Furthermore, in vitro experiments revealed that inhibition of miR-7977 enhanced the proliferation, invasion, and inhibited apoptosis in A549 cells, the opposite results were performed by miR-7977 mimics. In conclusion, exosomal miR-7977 was identified as a novel biomarker for patients with LUAD and may play as a tumor suppressor in lung cancer.     

表达miR-192的重组腺病毒的构建及其在肝细胞中的表达分析

目的：构建表达miR-192的重组腺病毒,感染HepG2细胞建立miR-192高表达的细胞模型,以研究miR-192在肝细胞中的功能。方法：将miR-192前体基因插入腺病毒穿梭载体pAdTrack,构建miR-192穿梭载体pAdTrack-miR-192,经线性化和同源重组,构建重组腺病毒载体pAd-miR-192;线性化后,在QBI-293A细胞中进行病毒包装;用包装成功的重组腺病毒感染HepG2细胞,72h后收集细胞,提取总RNA,逆转录后进行定量PCR,检测miR-192和潜在靶分子视网膜母细胞瘤基因1（RBl）mRNA的变化。结果：获得670bp的miR-192前体基因,经过穿梭载体后重组构建表达miR-192的腺病毒载体pAd-miR-192;将pAd-miR-192转染QBI-293A细胞,第8d细胞病变及荧光的表达结果表明重组腺病毒成功包装;感染HepG2细胞后,定量PCR检测表明miR-192的表达较对照增加了835．87倍,同时检测到细胞中RBlmRNA的表达显著降低。结论：构建了高效表达miR-192的腺病毒,建立了miR-192高表达的肝细胞模型,证明在肝细胞中抑制的RBl是miR-192靶分子,为后续miR-192在肝细胞中功能的研究奠定了基础。     

非小细胞肺癌组织中miR-19b与miR-20a的表达及临床意义

目的:检测mi R-19b与mi R-20a在非小细胞肺癌(NSCLC)组织中的表达,探讨两者与肺癌临床病理的关系。方法:选择我院NSCLC肺癌患者50例,使用Real-time RT-PCR法对其癌组织及癌旁正常组织中mi R-19b和mi R-20a的含量进行检测,并利用2(-△△CT)法处理结果,分析与临床病理资料的关系。结果:相对于内参U6,mi R-19b基因在NSCLC组织中的表达量明显低于癌旁正常组织,而mi R-20a基因在NSCLC组织中的表达量明显高于癌旁正常组织,差异具有统计学意义(P0.05);在NSCLC组织标本中,mi R-19b基因表达量在临床分期I-II的标本中明显高于临床分期III,而mi R-20a基因表达量在临床分期I-II的标本中明显低于临床分期III,差异均就有统计学意义(P0.05);mi R-19b基因表达量在肿瘤病理低分化组织中明显低于肿瘤病理高分化组织,而mi R-20a基因表达量在肿瘤病理低分化组织中明显高于肿瘤病理高分化组织,差异均具有统计学意义(均P0.05)。结论:mi R-19b低表达和mi R-20a高表达与NSCLC的临床分期、病理分级具有密切关系,早期检测有助于NSCLC诊断和治疗。     

非小细胞肺癌患者血清miR-146a、miR-141的表达及临床意义

目的:研究非小细胞肺癌(NSCLC)患者血清微小RNA-146a(mi R-146a)、mi R-141的表达及临床意义。方法:选取2015年1月至2018年1月我院收治的106例NSCLC患者记为NSCLC组,另选取同期于我院进行体检的40例健康体检者记为健康对照组。采用荧光实时定量PCR(qRT-PCR)检测两组血清mi R-146a、mi R-141的表达水平,分析NSCLC患者血清mi R-146a、mi R-141表达水平与临床病理参数的关系,比较血清mi R-146a、mi R-141单一诊断及联合诊断NSCLC的曲线下面积(AUC)、灵敏度、特异度。结果:NSCLC组患者的血清mi R-146a、mi R-141表达水平高于健康对照组(P0.05)。NSCLC患者血清mi R-146a表达水平与病理分期有关(P0.05),与年龄、性别、吸烟史、病理类型、病理分级、肿瘤直径及是否伴有淋巴结转移无关(P0.05)。NSCLC患者血清mi R-141表达水平与病理分期、病理分级及是否伴有淋巴结转移有关(P0.05),与年龄、性别、吸烟史、病理类型、肿瘤直径无关(P0.05)。血清mi R-146a、mi R-141单一诊断及两者联合诊断NSCLC的AUC分别为0.841、0.772、0.921,敏感度分别为85.1%、80.5%、93.5%,特异度分别为84.2%、75.3%、89.8%。结论:NSCLC患者血清mi R-146a、mi R-141表达水平升高,且与部分临床病理参数有关,两者联合检测对NSCLC诊断具有较高的诊断价值。     

miR-191在非小细胞肺癌中的表达及其临床意义

目的:探讨miR-191在非小细胞肺癌(non-small cell lung cancer,NSCLC)中的表达及其临床意义。方法:采用实时荧光相对定量聚合酶链式反应(quantitative realtime polymerase chain reaction,qRT-PCR)技术检测非小细胞肺癌患者的癌组织及癌旁组织中miR-191的表达水平,并分析其与患者临床病理特征及生存期的相关性。结果:与癌旁组织比较,癌组织中miR-191的表达显著上调。组织分化程度低或有淋巴结转移的NSCLC患者癌组织中miR-191表达明显高于组织分化程度高或无淋巴结转移的NSCLC患者(P0.05),癌组织高表达miR-191的NSCLC患者生存期明显短于癌组织低表达miR-191的NSCLC患者(P0.05)。结论:miR-191在非小细胞肺癌中表达上调,与组织分化程度、淋巴结转移和患者生存期有关。     

非小细胞肺癌患者血浆miR-17和miR-204水平表达的临床意义及其预测价值分析

摘要 目的：检测非小细胞肺癌（NSCLC）患者血浆miR-17和miR-204表达,分析其表达与临床病理参数的关系及其对NSCLC预后的预测价值。方法：选择我院2015年6月至2017年6月期间收治的117例NSCLC患者（观察组）和同期于我院进行体检的100例健康者（对照组）作为研究对象,采用实时荧光定量PCR (qRT-PCR)检测血浆中miR-17和miR-204表达,分析血浆miR-17、 miR-204表达与NSCLC患者临床病理参数的关系。采用受试者工作特征（ROC）曲线分析血浆miR-17、 miR-204对NSCLC患者预后的预测价值。结果：观察组患者血浆miR-17表达高于对照组（P＜0.05）、miR-204表达低于对照组（P＜0.05）,miR-17表达与淋巴结转移、远处转移有关（P＜0.05）,miR-204表达与TNM分期、远处转移有关（P＜0.05）。ROC曲线分析结果显示miR-17、 miR-204预测 NSCLC患者预后的曲线下面积（AUC）分别为0.821、0.836。miR-17、miR-204两指标的联合检测对NSCLC患者预后的预测价值更高：敏感度和特异度分别为89.36%(42/47)、92.86%（65/70)。结论：NSCLC患者血浆miR-17、miR-204均存在异常表达,且与患者临床病理参数有关,可能作为NSCLC患者预后预测的潜在生物学指标。     

Downregulation of miR-16 promotes growth and motility by targeting HDGF in non-small cell lung cancer cells

MicroRNAs play important roles in the development and progression of non-small cell lung cancer (NSCLC). miR-16 functions as a tumor-suppressor and is inhibited in several malignancies. Herein, we validated that miR-16 is downregulated in NSCLC tissue samples and cell lines. Ectopic expression of miR-16 significantly inhibited cell proliferation and colony formation. Moreover, miR-16 suppressed cell migration and invasion in NSCLC cells. Hepatoma-derived growth factor (HDGF) was found to be a direct target of miR-16 in NSCLC cell lines. Rescue experiments showed that the suppressive effect of miR-16 on cell proliferation, colony formation, migration, and invasion is partially mediated by inhibiting HDGF expression. This study indicates that miR-16 might be associated with NSCLC progression, and suggests an essential role for miR-16 in NSCLC.     

Lung CSC-derived exosomal miR-210-3p contributes to a pro-metastatic phenotype in lung cancer by targeting FGFRL1

Lung cancer has the highest mortality rate among human cancers, and the majority of deaths can be attributed to metastatic spread. Lung cancer stem cells (CSCs) are a component of the tumour microenvironment that contributes to this process. Exosomes are small membrane vesicles secreted by all types of cells that mediate cell interactions, including cancer metastasis. Here, we show that lung CSC-derived exosomes promote the migration and invasion of lung cancer cells, up-regulate expression levels of N-cadherin, vimentin, MMP-9 and MMP-1, and down-regulate E-cadherin expression. Moreover, we verified that these exosomes contribute to a pro-metastatic phenotype in lung cancer cells via miR-210-3p transfer. The results of bioinformatics analysis and dual-luciferase reporter assays further indicated that miR-210-3p may bind to fibroblast growth factor receptor-like 1 (FGFRL1); silencing FGFRL1 enhanced the metastatic ability of lung cancer cells, whereas overexpressing FGFRL1 suppressed metastasis. Taken together, our results provide new insights into a potential molecular mechanism whereby lung CSC-derived exosomal miR-210-3p targets FGFRL1 to promote lung cancer metastasis. FGFRL1 may be a promising therapeutic target in lung cancer.     

Promising renoprotective effect of gold nanoparticles and dapagliflozin in diabetic nephropathy via targeting miR-192 and miR-21

Diabetic nephropathy (DN) is a worldwide issue that eventually leads to end-stage renal failure, with limited therapeutic options. Prior research has revealed that gold nanoparticles (AuNPs) have a substantial antidiabetic impact. In addition, sodium-glucose cotransporter2 (SGLT2) inhibitors, including dapagliflozin (DAPA), had renoprotective impact on DN. Therefore, this research attempted to determine the potential AuNPs and DAPA impacts in ameliorating experimentally DN induction and the underlying mechanisms focusing on miR-192 and miR-21, correlating them with autophagy, apoptosis, fibrosis, and oxidative stress. Diabetes induction was through a single intraperitoneal streptozotocin (55 mg/kg) injection, and rats with diabetes received AuNPs (2.5 mg/kg/day) as well as DAPA (2 mg/kg/day) for 7 weeks as a treatment. AuNPs and DAPA treatment for 7 weeks substantially alleviated DN. AuNPs and DAPA significantly increased catalase (CAT) activity as well as serum total antioxidant capacity (TAC), along with a substantial decline in malondialdehyde (MDA). AuNPs and DAPA treatment alleviated renal fibrosis as they decreased transforming growth factorß1(TGF-ß1) as well as matrix metalloproteinase-2 (MMP-2) renal expression, decreased apoptosis through alleviating the proapoptotic gene (caspase-3) renal expression and increased the antiapoptotic gene (Bcl-2) renal expression, and increased autophagy as they increased LC-3 as well as Beclin-1 renal expression. Autophagy activation, inhibition of apoptosis, and renal fibrosis could be due to their inhibitory impact on miR-192 and miR-21 renal expression. AuNPs and DAPA have a protective effect on DN in rats by targeting miR-192 and miR-21 and their downstream pathways, including fibrosis, apoptosis, autophagy, and oxidative stress.     

miR-194 relieve neuropathic pain and prevent neuroinflammation via targeting FOXA1

The emerging role of microRNAs (miRNAs) have been deeply explored in multiple diseases including neuropathic pain. miR-194 was widely reported to be a tumor suppressor and was related to the inflammatory response. The critical role of neuroinflammation on neuropathic pain leads to a thinking about the relationship between miR-194 and neuropathic pain. However, the function of miR-194 in neuropathic pain remains unknown. This study was aimed to explore the relationship between miR-194 and neuropathic pain progression by chronic sciatic nerve injury (CCI). miR-194 abnormally downregulated in the CCI model rat and its overexpression significantly alleviates neuroinflammation in vivo. We predict Forkhead box protein A1 (FOXA1) as a direct target of miR-194, whose restoration can markedly reverse the effects of miR-194 on neuropathic pain. Overall, our study demonstrated a novel mechanism of neuropathic pain progression that miR-194 alleviates neuropathic pain via targeting FOXA1 and preventing neuroinflammation by downregulating inflammatory cytokines containing cyclooxygenase 2, interleukin 6 (IL-6), and IL-10 in vivo, which can be reversed by the overexpression of FOXA1.     

非小细胞肺癌组织miR-1179、miR-1182表达水平与Notch信号通路、临床病理特征和预后的关系

摘要 目的：探讨非小细胞肺癌（NSCLC）组织微小核糖核酸（miRNA）-1179、miR-1182表达与缺口(Notch)信号通路、临床病理特征和预后的关系。方法：选取2018年1月～2019年12月武汉市中医医院收治的118例NSCLC患者,收集手术切除的癌组织和癌旁组织标本,采用实时荧光定量聚合酶链式反应检测miR-1179、miR-1182和Notch信号通路相关分子表达。分析miR-1179、miR-1182表达与Notch信号通路相关分子和NSCLC患者临床病理特征的关系。根据NSCLC组织中miR-1179、miR-1182表达均值分为高、低表达组,采用K-M法绘制不同miR-1179、miR-1182表达NSCLC患者生存曲线,多因素Cox回归分析NSCLC患者预后的影响因素。结果：与癌旁组织比较,NSCLC组织中miR-1179、miR-1182表达降低,Notch受体1（Notch1） 信使核糖核酸（mRNA）、Notch2 mRNA、Notch3 mRNA、Notch4 mRNA表达升高（P＜0.05）。Pearson相关性分析显示,NSCLC组织中miR-1179、miR-1182表达与Notch1 mRNA、Notch2 mRNA、Notch3 mRNA、Notch4 mRNA表达均呈负相关（P＜0.05）。不同分化程度、TNM分期、淋巴结转移NSCLC患者miR-1179、miR-1182表达比较有统计学差异（P＜0.05）。118例NSCLC患者随访3年,失访5例,3年总生存率为55.75%。K-M生存曲线分析显示,miR-1179、miR-1182高表达组总生存率高于低表达组（P＜0.05）。多因素Cox回归分析显示,低分化、TNM分期Ⅲ期、淋巴结转移为NSCLC患者预后的独立危险因素,miR-1179、miR-1182升高为其独立保护因素（P＜0.05）。结论：NSCLC组织中miR-1179、miR-1182低表达,与Notch信号通路、分化程度、TNM分期、淋巴结转移和预后有关,miR-1179、miR-1182表达可能通过抑制Notch信号通路发挥抑癌作用。     

Programmed cell death ligand-1 expression and survival in a cohort of patients with non-small cell lung cancer receiving first-line through third-line therapy in Denmark

BackgroundPD-L1 expression on tumor cells (TCs) or immune cells (ICs) may be used as a prognostic marker for survival in patients with NSCLC. We characterized PD-L1 expression on TCs or ICs in a patient cohort with NSCLC to determine associations between PD-L1 expression and overall survival (OS), according to EGFR and KRAS mutation status.MethodsDanish patients aged >18 years diagnosed with NSCLC before 2014 on first- (N = 491), second- (N = 368), or third-line (N = 498) therapy were included. Data were extracted from population-based medical registries. Tumor samples from pathology archives were tested for biomarkers. High PD-L1 expression was defined as expression on ≥25 % of TCs or ICs based on first diagnostic biopsy or surgical resection. KRAS and EGFR mutation status were tested using PCR-based assays. Cox regression analysis was used to compute adjusted HRs and associated 95 % CIs.ResultsPD-L1 TC and IC ≥ 25 % were observed in 24.3 %–31.0 % and 11.7–14.7 % of patients, respectively. EGFR and KRAS mutations were detected in 4.7 %–8.8 % and 26.5 %–30.7 % of patients, respectively. PD-L1 TC ≥ 25 % was not associated with survival advantage in first- (HR = 0.96, 95 % CI: 0.75–1.22), second- (1.08, 0.81–1.42), or third-line (0.94, 0.74–1.20) therapy. PD-L1 IC ≥ 25 % was associated with survival advantage in second-line (HR = 0.56, 95 % CI: 0.36–0.86) and third-line (0.69, 0.49–0.97) but not first-line (1.00, 0.70–1.41) therapy.ConclusionNo association was observed between PD-L1 TC ≥ 25 % and OS in any therapy line. PD-L1 IC ≥ 25 % may confer survival benefit among some patients who reach second-line therapy.     

Defining lung cancer stem cells exosomal payload of miRNAs in clinical perspective

Since the first publication regarding the existence of stem cells in cancer [cancer stem cells(CSCs)] in 1994, many studies have been published providing in-depth information about their biology and function. This research has paved the way in terms of appreciating the role of CSCs in tumour aggressiveness, progression,recurrence and resistance to cancer therapy. Targeting CSCs for cancer therapy has still not progressed to a sufficient degree, particularly in terms of exploring the mechanism of dynamic interconversion between CSCs and non-CSCs. Besides the CSC scenario, the problem of cancer dissemination has been analyzed indepth with the identification and isolation of microRNAs(miRs), which are now considered to be compelling molecular markers in the diagnosis and prognosis of tumours in general and specifically in patients with non-small cell lung cancer.Paracrine release of miRs via "exosomes"(small membrane vesicles(30-100 nm),the derivation of which lies in the luminal membranes of multi-vesicular bodies)released by fusion with the cell membrane is gaining popularity. Whether exosomes play a significant role in maintaining a dynamic equilibrium state between CSCs and non-CSCs and their mechanism of activity is as yet unknown.Future studies on CSC-related exosomes will provide new perspectives for precision-targeted treatment strategies.     

MiR-491在非小细胞肺癌发生发展过程中的作用机制研究

人类miR-491 (has-miR-491)是一段长度为84 bp的微小RNA,可以通过与靶mRNA的3'端非翻译区(3'-untranslated region,3'-UTR)结合,降解靶mRNA或抑制其翻译,在转录后水平对肿瘤细胞基因表达进行调控,影响肿瘤发展进程.非小细胞肺癌(non-small lang ca...     

Circular RNA hsa_circ_0004015 regulates the proliferation,invasion, and TKI drug resistance of non-small cell lung cancer by miR-1183/PDPK1 signaling pathway

Circular RNAs (circRNAs) were recently reported to be involved in the pathogenesis of Non-small cell lung cancer (NSCLC), however, the molecular mechanisms of circRNAs in cell proliferation, invasion and TKI drug resistance remain largely undetermined. Here, we identified hsa_circ_0004015 was upregulated in NSCLC tissues, and was associated with the poor overall survival rate of NSCLC patients. Knockdown of hsa_circ_0004015 significantly decreased cell viability, proliferation, and invasion, whereas overexpression exhibited opposed effects in vivo and in vitro. Furthermore, hsa_circ_0004015 could enhance the resistance of HCC827 to gefitinib. In mechanism, hsa_circ_0004015 acted as a sponge for miR-1183, and PDPK1 was revealed to be target gene of miR-1183. Subsequently, functional assays illustrated that the oncogenic effects of hsa_circ_0004015 was attributed to the regulation of miR-1183/PDPK1 axis. In conclusion, circ_0016760/miR-1183/PDPK1 signaling pathway might play vital roles in the tumorigenesis of NSCLC.     

Up-regulation of long noncoding RNA MINCR promotes non-small cell of lung cancer growth by negatively regulating miR-126/SLC7A5 axis

A growing body of evidence suggests that MYC induced long noncoding RNA (MINCR) is involved in the initiation and progression of various tumors. However, little is known about the biological function and clinical value of MINCR in non-small cell lung cancer (NSCLC). In the present study, results found that MINCR over expression in NSCLC tissue and cell lines was closely related to poor survival in NSCLC. Functional experiments found that decreased MINCR expression inhibits NSCLC cell proliferation and migration and promotes cells apoptosis. Tumor formation assay found that knockdown of MINCR significantly inhibited tumor growth. Results also found that MINCR functions as an oncogene in the metastasis of NSCLC, in part, by acting as a competing endogenous RNA to modulate the miR-126/SLC7A5 axis. Dysfunction of MINCR, miR-126 and SLC7A5 predicted poor prognosis of patients with NSCLC. In conclusion, results suggest that the MINCR-miR-126-SLC7A5 axis plays an important role in the progression of NSCLC and may serve as a potential target for lung cancer diagnosis and treatment.     

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.     

miR-32在非小细胞肺癌中的表达

目的:基于芯片数据库分析的基础上,探讨miR-32在非小细胞肺癌患者中的表达水平。方法:在GEO和Array Expression数据库中搜索关键词microRNA和肺癌检索出两个样本量最大的芯片数据,分别是GEO数据库中编码为GSE61741和Array Expression数据库中编码为E-TABM-22的芯片数据,对两组数据进行分析找出有差异的microRNA。另外收集2010.02-2012.09期间在吉林大学附属中日联谊医院胸外科进行肺癌手术切除的32名患者的肺癌组织及配对的癌旁正常肺组织标本,检测标本中miR-32的表达水平。结果:综合分析GSE61741和E-TABM-22的芯片数据发现有共同差异的microRNA共有8个,其中上调的有2个,分别是hsa-miR-192与hsa-miR-197,下调的有6个,分别是hsa-miR-126、hsa-miR-199b-3p、hsa-miR-219-1-3p、hsa-miR-26a、hsa-miR-32与hsa-miR-9。为了进一步探讨miR-32在癌症中的作用,我们对GSE61741中其他癌症患者及健康者血浆中miR-32的芯片数据进行分析,发现miR-32在结肠癌、神经胶质瘤、肾癌、前列腺癌、Wilm's瘤中的表达水平均显著低于健康者(P0.01)。对32例及E-TABM-22芯片数据中65例NSCLC患者癌及配对的癌旁正常肺组织中miR-32的表达水平分析发现癌组织中miR-32的表达水平显著下调(P0.001)。结论:miR-32在非小细胞肺癌组织中低表达,提示miR-32可能为新的NSCLC诊断标记分子。