干扰素诱导的跨膜蛋白1在宫颈鳞癌中的表达及其生物学作用

目的:探讨干扰素诱导的跨膜蛋白1(IFITM1)在宫颈鳞癌中的表达及其生物学作用。方法:通过免疫组化、RT-PCR和Western blot检测宫颈鳞癌组织和癌旁组织中IFITM1的表达。使用靶向IFITM1的si RNA(si-IFITM1组)和高表达IFITM1基因的重组pcDNA3.1质粒(pcDNA3.1-si-IFITM1组)转染Si Ha细胞下调或上调IFITM1的表达。通过伤口愈合实验、Transwells迁移实验和基质胶侵袭实验检测细胞迁移和侵袭能力。细胞计数试剂盒-8 (CCK-8)检测细胞增殖;流式细胞术测定细胞凋亡。Western blot检测PTEN、PI3K和AKT的表达。通过对BALB/c Nude裸鼠接种Si Ha细胞来考察IFITM1在体外对肿瘤生长的影响。结果:癌组织中IFITM1的表达水平明显高于癌旁组织(P<0.05)。与对照组比较,si-IFITM1组的迁移和侵袭能力明显增强,而pcDNA3.1-si-IFITM1组明显降低(P<0.05)。细胞转染48 h和72 h后,与对照组比较,si-IFITM1组的细胞增殖明显增强,而pcDNA3.1-si-IFITM1组明显减弱(P<0.05)。与对照组比较,si-IFITM1组的细胞凋亡率明显降低,而pcDNA3.1-si-IFITM1组明显升高(P<0.05)。与对照组比较,si-IFITM1组的PTEN被下调,而PI3K和AKT被上调(P<0.05);pcDNA3.1-si-IFITM1组的PTEN的被上调,而PI3K和AKT被下调(P<0.05)。与对照组比较,si-IFITM1组裸鼠的肿瘤体积显著增大,而pcDNA3.1-si-IFITM1组显著减小(P<0.05)。结论:IFITM1过表达抑制人宫颈鳞癌细胞Si Ha的生长和转移能力,并在体外抑制肿瘤的形成,从而发挥抗癌作用。IFITM1可能通过调控PTEN/PI3K/AKT信号通路发挥抗癌作用。     

MiR-425对血管平滑肌细胞增殖迁移的作用及分子机制

目的探讨miR-425对人主动脉平滑肌细胞增殖迁移的作用及潜在的分子机制。方法实时定量RT-PCR(qRT-PCR)检测人主动脉平滑肌细胞(human aortic smooth muscle cells, HASMCs)中miR-425的表达水平。转染miR-342inhibitor改变HASMCs中内源性miR-425的表达,采用CCK-8法和流式细胞术检测细胞增殖及周期的变化,Transwell检测细胞迁移能力;Western blot检测PTEN、PI3K、p-AKT/AKT、MMP-9的蛋白表达水平。结果血管紧张素Ⅱ(angiotensionⅡ,AngⅡ)可时间及剂量依赖性地促进HASMCs中miR-425的表达。转染miR-425 inhibitor可抑制AngⅡ对HASMCs的促增殖作用,并抑制细胞迁移,同时细胞中PI3K、p-AKT/AKT及MMP-9的蛋白水平显著降低,PTEN水平显著升高。结论沉默miR-425可通过调控PTEN/PI3K/AKT信号通路抑制人主动脉平滑肌细胞的增殖及迁移,提示miR-425可作为血管重构的一个潜在诊疗靶点。     

miR-20a靶向CCND1作用PI3K/AKT信号通路抑制皮肤鳞状细胞癌的发展

摘要 目的：探究miR-20a与CCND1蛋白在皮肤鳞状细胞癌（CSCC）中的作用关系,以及其可能涉及的信号通路分子机制。方法：分别收集皮肤鳞状细胞癌患者的皮肤癌组织及其邻近正常皮肤组织,采用qRT-PCR分析组织中miR-20a和CCND1基因表达水平。为探究miR-20a对CSCC细胞的影响,将SCL-1细胞分为对照组（不转染）、miR-NC组（转染miR-NC）和miR-20a mimics组（转染miR-20a mimics）;为探究CCND1与PI3K/AKT信号通路的关系,将SCL-1细胞分为对照组（不转染）、si-NC组（转染si-NC）和si-CCND1组（转染si-CCND1）;为探究miR-20a与CCND1间的作用关系及对CSCC细胞的影响,将SCL-1细胞分为miR-NC组（转染miR-NC）、miR-20a mimics组（转染miR-20a mimics）、mimics+pcDNA组（共转染miR-20a mimics和pcDNA）和mimics+CCND1组（共转染miR-20a mimics和pcDNA-CCND1）。采用Western blot分析p-AKT、AKT、p-PI3K、PI3K和GSK-3β蛋白表达水平;采用MTT检测细胞增殖情况;采用流式细胞术检测细胞凋亡情况;采用Transwell分析细胞迁移和侵袭情况;采用双荧光素酶报告基因检测分析miR-20a与CCND1的靶向关系。结果：CSCC癌组织和SCL-1中miR-20a均低表达,CCND1高表达。与对照组和miR-NC组比较,miR-20a mimics组SCL-1细胞增殖水平以及侵袭和迁移数量均降低（P<0.05）,SCL-1细胞凋亡水平升高（P<0.05）,PI3K和AKT蛋白磷酸化水平降低（P<0.05）。TargetScanHuman数据库分析和双荧光素酶报告基因检测结果显示miR-20a与CCND1存在靶向作用关系。与对照组和si-NC组比较,si-CCND1组SCL-1细胞中CCND1和GSK-3β蛋白表达水平以及PI3K和AKT蛋白磷酸化水平均降低（P<0.01）。与miR-20a mimics组或mimics+pcDNA组比较,mimics+CCND1组SCL-1细胞增殖水平以及侵袭和迁移数量均升高（P<0.05）,SCL-1细胞凋亡水平降低（P<0.05）,PI3K和AKT蛋白磷酸化水平均升高（P<0.05）。结论：过表达miR-20a可能通过靶向抑制CCND1的表达而抑制PI3K/AKT信号通路的激活,从而抑制CSCC细胞的增殖、侵袭和迁移,并促进癌细胞凋亡。     

miR-21通过PTEN/PI3K/AKT信号通路调控胶质瘤细胞的增殖

探讨miR-21对胶质瘤细胞U87中人第10号染色体缺失的磷酸酶及张力蛋白同源的基因/磷脂酰肌醇3激酶/蛋白激酶B(PTEN/PI3K/AKT)信号通路相关影响作用研究。脂质体Lipofectamine~(TM)2000将miR-21 inhibitor和miR-21 NC转入U87细胞中,48 h后,RT-PCR检测miR-21表达,MTT法检测细胞活力,Annexin V/PI流式双染检测细胞凋亡情况,Western blotting检测PTEN/PI3K/AKT激活情况及程序性细胞死亡因子4(PDCD4),Bax及Bcl-2的表达。与miR-21 NC组比较,miR-21 inhibitor组miR-21表达量显著降低,U87细胞活力下降,细胞凋亡率提高,PI3K、Bcl-2及p-AKT表达量下调,PTEN、PDCD4及Bax表达量上调,差异均具有统计学意义(p0.01)。miR-21inhibitor能显著的抑制U87细胞的增殖,与PTEN/PI3K/AKT信号通路有关。     

MiR-1297下调PTEN的表达参与子宫内膜癌变

已有研究证明,抑癌子PTEN（phosphatase and tensin homolog）缺失或表达下调可引起子宫内膜癌变;在喉鳞状细胞癌中miR-1297可下调PTEN的表达。然而,miR-1297下调PTEN的机制,以及是否miR-1297靶向抑制PTEN参与子宫内膜癌变尚未见报道。本研究证明,miR-1297靶向抑制PTEN基因的表达参与子宫内膜癌变。收集临床病理检查确诊为子宫内膜腺癌的癌组织及癌旁组织18例。实时定量PCR（RT-qPCR）及Western印迹结果显示,与癌旁子宫内膜比较,子宫内膜癌组织miR-1297高表达,而PTEN则呈低表达或缺失。内膜癌和癌旁内膜组织中,miR-1297与PTEN表达水平的散点图分析揭示,miR-1297的表达水平与PTEN的表达水平呈明显的负相关（内膜癌：miR-1297与PTEN的相关系数r=-0.6928,P=0.0438;癌旁组织：相关系数r=-0.4085,P < 0.0001)。报告酶活性测定显示,与对照比较,pGL3-PTEN-3′-UTR-1/2/3加miR-1297模拟物共转染后,荧光素酶活性在转染的Ishikawa和ECC-1细胞中分别降低了约21%（P=0.0196）和17%（P=0.0306）;相反,转染miR-1297抑制物后,两种细胞的荧光素酶活性分别升高约59%（Ishikawa：P=0.0014）和50%（ECC-1： P=0.0025）。结果提示,miR-1297可通过特异识别PTEN mRNA的3′-UTR,抑制PTEN的表达。基因转染结合Western印迹结果证明,在原代子宫内膜细胞中,过表达miR-1297模拟物导致PTEN蛋白表达下调,而其下游底物分子--磷酸化的AKT（p-AKT）表达上调;而过表达miR-1297抑制物后,PTEN表达上调,p-AKT表达下调。Connexin V/PI双染结合流式分析证明,与对照细胞（3%细胞凋亡）比较,在子宫内膜癌细胞系Ishikawa中,敲减miR-1297表达后细胞调亡明显增加（至10%）,这可能与抑制miR-1297后PTEN的表达上调有关。上述结果提示,miR-1297在子宫内膜癌高表达,而PTEN低表达。上述结果还提示,miR-1297作为一个促癌子可通过特异识别PTEN mRNA的3′-UTR,抑制PTEN的表达,促进AKT磷酸化/激活,从而促进内膜癌的发生,这可能是子宫内膜癌变的发生原因之一。     

姜黄素作用PTEN/PI3K/AKT通路诱导食管癌EC109细胞凋亡

目的:研究姜黄素作用食管癌的分子机制。方法:体外培养人食管癌细胞(EC109),15~120μmol/L姜黄素处理后,采用CCK-8法检测姜黄素对细胞的增殖抑制作用,透射电镜观察细胞超微结构,Annexin V-FITC/PI双染激光共聚焦显微镜观察细胞凋亡,流式细胞术分析15~120μmol/L姜黄素作用EC109细胞后PTEN/PI3K/AKT通路相关蛋白PTEN、AKT、GSK3β和Caspase 3的表达水平。结果:CCK-8检测结果姜黄素能显著抑制EC109细胞的增殖,呈剂量和时间效应关系;透射电镜和激光共聚集显微镜观察发现姜黄素能使EC109细胞发生凋亡;流式细胞仪蛋白水平分析显示姜黄素可增强细胞中PTEN、GSK3β和Caspase 3的表达,抑制AKT的表达。结论:姜黄素抑制EC109细胞的增殖并促进其凋亡的生物学效应与增强PTEN的表达抑制PI3K/AKT信号通路有关。     

CXCR4/Akt信号通路对食管癌细胞侵袭和肿瘤转移的调控作用

多项研究发现CXCR4在各种类型的癌症中高表达,然而尚不清楚CXCR4在食管癌细胞生长和转移中的作用。本研究检测了CXCR4在食管癌组织和细胞系(TE-1)中的表达,并通过转染CXCR4-短发夹RNA(CXCR4-sh RNA)慢病毒来敲低TE-1细胞中CXCR4的表达。应用PI3K/AKT抑制剂LY294002(50μmol/L)处理TE-1细胞12 h来考察AKT信号在食管癌细胞生长和转移中的作用;应用蛋白质印迹分析检测AKT和Rho家族蛋白(RhoA,Rac-1和Cdc42)的表达;应用CCK-8实验检测细胞增殖;Transwell实验检测细胞侵袭;对雄性BALB/c-nu/nu裸鼠皮下注射转染CXCR4-shRNA的TE-1细胞建立肿瘤异种移植模型。研究显示,CXCR4在食管癌组织中的表达水平明显高于癌旁组织,并且与TNM分期和淋巴结转移有关。CXCR4在人食管鳞状细胞癌细胞系(TE-1)中的表达水平明显高于人正常食管上皮细胞系(human normal esophageal epithelial cell line,HEEC)。敲低CXCR4能抑制食管鳞状细胞癌细胞的增殖和侵袭能力,并抑制肿瘤异种移植裸鼠的肿瘤形成。敲低CXCR4抑制了AKT的磷酸化及RhoA、Rac-1和Cdc42的表达。此外,PI3K/AKT抑制剂LY294002处理显著降低了TE-1细胞中AKT的磷酸化,并降低了RhoA、Rac-1和Cdc42的表达。本研究表明,CXCR4在食管癌患者中上调,与不良预后相关。下调CXCR4的表达可在体内和体外抑制食管癌肿瘤的生长和转移。下调CXCR4可通过抑制AKT信号的激活来抑制Rho家族粘附/侵袭相关蛋白的表达,从而抑制肿瘤转移。     

抑制miR-130b-3p通过上调PTEN和灭活PI3K-AKT及整合素β1/FAK信号通路抑制膀胱癌细胞增殖

miR-130家族在癌的进展中发挥作用;然而,其家族成员在膀胱癌中的作用尚罕见报告。本研究证明,抑制miR-130家族成员miR-130b-3p表达促进PTEN表达,诱导细胞凋亡,抑制膀胱癌细胞增殖。首先,我们采用微阵列对来自膀胱癌患者的4对膀胱癌和癌旁组织进行了miRNA和mRNA组学分析,发现miR-130b-3p和miR-106b-3p在膀胱癌组织高表达,而miR-99a-3p、miR-199a-5p和miR-145-3p低表达。RT-q PCR检测30对膀胱癌组织5种miRNA的表达与微阵列中的表达状况一致。此外,我们在mRNA组学分析中还发现,miR-130b-3p在膀胱癌组织的高表达与PTEN表达呈负相关。为此,在后续研究中集中探索了miR-130b-3p在膀胱癌中的作用及其作用机制。生物信息学及荧光素酶报告结果证明,miR-130b-3p可直接结合PTEN的3'-UTR,靶向抑制PTEN的表达。转染结合CCK-8、EDU、流式分析、划痕及Transwell小室实验显示,转染miR-130b-3p模拟物可明显促进膀胱癌T24细胞的增殖、迁移及侵袭能力;相反,转染miR-130b-3p抑制物可明显诱导T24细胞凋亡。鬼笔环肽染色揭示,转染miR-130b-3p模拟物可促进细胞骨架形成,而转染miR-130b-3p抑制物抑制细胞骨架形成。Western印迹证明,转染miR-130b-3p可下调PTEN在T24细胞的表达,上调p-PI3K、p-AKT、p-FAK和整合素β1的表达;而转染miR-130b-3p抑制物上调PTEN的表达,下调p-PI3K、p-AKT、p-FAK和整合素β1的表达。上述结果提示,miR-130b-3p通过抑制PTEN表达,激活PI3K-AKT及整合素β1/FAK信号通路,在膀胱癌中发挥癌基因样作用;相反,抑制miR-130b-3p可上调PTEN表达,抑制PI3K-AKT及整合素β1/FAK信号通路的激活,诱导凋亡,抑制膀胱癌细胞的增殖、迁移和侵袭能力。我们的结果还提示,miR-130b-3p作为可能的临床标志物,对膀胱癌的诊断、靶向治疗具有潜在的应用价值。     

miRNA-181a在胃癌细胞增殖和迁移中的作用

miRNA与恶性肿瘤患者的诊断和预后密切相关,为了考察miRNA-181a在胃癌细胞增殖和迁移中的作用,本研究检测了miRNA-181a在胃癌组织中的表达,并通过对人胃癌细胞系MGC-803转染miR-181a模拟物或抑制剂来考察miR-181a对细胞迁移和增殖的影响。RT-PCR显示,miRNA-181a在胃癌组织中的表达水平显著高于癌旁组织(p<0.05)。伤口愈合实验和Transwell实验显示,转染miR-181a抑制剂或TGF-β受体2(TGFβR2)过表达的pcDNA3.1质粒均可抑制MGC-803细胞的迁移。EdU实验和CCK-8实验显示,转染miR-181a抑制剂或TGFβR2过表达的pcDNA3.1质粒均可抑制MGC-803细胞的增殖。此外,miR-181a抑制剂处理可使TGFβR2蛋白表达明显升高。然而,miR-181a模拟物或抑制剂处理后TGFβR2mRNA水平没有显著变化。总之,本研究表明高表达的miR-181a通过在转录后抑制TGFβR2蛋白表达来促进胃癌细胞的迁移和增殖。miR-181a有望成为胃癌的潜在治疗靶点。     

C20orf54抑制食管癌细胞增殖并促进其凋亡

目的观察过表达C20orf54对食管癌细胞周期和凋亡的影响,探讨C20orf54基因在食管癌发生、发展中的作用。方法应用免疫组织化学染色方法检测C20orf54蛋白在食管癌组织和癌旁正常组织中的表达;应用MTT法和流式细胞术检测过表达C20orf54对食管癌细胞系EC9706细胞增殖能力、细胞凋亡与细胞周期的影响。结果 C20orf54在食管癌组织中免疫反应性显著低于癌旁正常组织。稳定转染表达C20orf54的EC9706细胞生长明显受抑制,且G0/G1期细胞积聚,周期阻滞,细胞凋亡增多。结论 C20orf54基因可能参与食管癌的生长调控,食管癌的发生与C20orf54基因的表达有关。     

Simvastatin inhibits the development of radioresistant esophageal cancer cells by increasing the radiosensitivity and reversing EMT process via the PTEN-PI3K/AKT pathway

Acquired radioresistance compromises the efficacy of radiotherapy for carcinomas including esophageal cancer (EC), thus resulting in recurrence and poor survival. Recent research corroborated radiosensitive function of simvastatin in stem-like breast cancer cells. However, its role in EC radioresistance remains poorly elucidated. Here, we developed a radioresistant EC cell line Ec9706-R with higher resistance to irradiation relative to control Ec9706 cells. Intriguingly, Ec9706-R cells exhibited epithelial-mesenchymal transition (EMT) characteristics with high invasion and migration ability. Simvastatin sensitized radioresistance of Ec9706-R cells and suppressed cell proliferation, but aggravated radiation-induced apoptosis and caspase-3 activity. Furthermore, simvastatin reversed EMT and inhibited cell invasion and migration of Ec9706-R cells. Mechanism assay confirmed the activation of PI3K/AKT pathway after radiation, which was inhibited by simvastatin. After restoring this pathway by its activator, IGF-1, simvastatin-mediated radiosensitivity and EMT reversion were abrogated. Further assay substantiated the PTEN suppression after irradiation, which was elevated following simvastatin pre-treatment. Moreover, PTEN cessation attenuated the inhibitory effect of simvastatin on PI3K/AKT activation, and subsequently antagonized simvastatin-induced radiosensitivity and EMT reversion. Additionally, simvastatin aggravated radiation-mediated Ec9706-R tumor growth inhibition. Together, simvastatin inhibits the development of Ec9706-R cells by increasing radiosensitivity and reversing EMT via PTEN-PI3K/AKT pathway, implying a promising strategy against EC radioresistance.     

miR-21 promotes proliferation and inhibits apoptosis of hepatic stellate cells through targeting PTEN/PI3K/AKT pathway

Abstract

To investigate the effect of microRNA 21 (miR-21) on hepatic stellate cells (HSCs) proliferation and apoptosis, and further to study its potential mechanisms. LX-2 cells were divided into miR-21 mimic group (Mimic), miR-21 mimic negative control group (NM), miR-21 inhibitor group (Inhibitor), miR-21 inhibitor negative control group (NC), and blank control group (Control). The cell proliferation was detected by CCK-8 assay and the cell migration and invasion were detected by scratch and transwell assay. Cell cycle and apoptosis were detected by flow cytometry. The levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α, and transforming growth factor (TGF)-β1 were detected by enzyme-linked immunosorbent assay (ELISA). Proliferation, apoptosis, and phosphatase and tensin homolog (PTEN)/phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway related genes and proteins were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot, respectively. The cells proliferation, migration, and invasion were promoted in Mimic group. The levels of IL-6, TNF-α, and TGF-β1 were increased after miR-21 administration. The expression of α-smooth muscle actin (SMA) and collagen 1 (Colla1) were increased, while Bax/B-cell lymphoma (Bcl)-2 ratio and programed cell death 4 (PDCD4) were reduced after miR?21 treatment. Meanwhile, the mRNA and protein expression of PTEN were reduced and PI3K/AKT pathway been promoted. Our study demonstrated that miR-21 could promote proliferation and inhibit apoptosis of HSCs, and its mechanism may be related to PTEN/PI3K/AKT pathway.     

Retracted: MicroRNA-99b suppresses human cervical cancer cell activity by inhibiting the PI3K/AKT/mTOR signaling pathway

Cervical cancer is common cancer among women with high morbidity. MicroRNAs (miRs) are involved in the progression and development of cervical cancer. This study aimed to explore the effect of miR-99b-5p (miR-99b) on invasion and migration in cervical cancer through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) signaling pathway. The microarray-based analysis was used to screen out differentially expressed miRNAs. Expression of miR-99b, PI3K, AKT, mTOR, and ribosomal protein S6 kinase (p70S6K) was determined in both cervical cancer tissues and paracancerous tissues. Next, alteration of miR-99b expression in cervical cancer was conducted to evaluate levels of PI3K, AKT, mTOR, p70S6K matrix metallopeptidase 2, epithelial cell adhesion molecule, and intercellular adhesion molecule 1, as well as the effect of miR-99b on cell proliferation, invasion, migration, cell cycle distribution, and apoptosis. The results demonstrated that miR-99b expression was decreased and levels of PI3K, AKT, mTOR, and p70S6K were elevated in cervical cancer tissues. More important, overexpressed miR-99b repressed the PI3K/AKT/mTOR signaling pathway, inhibited cell proliferation, invasion, and migration, blocked cell cycle entry, and promoted apoptosis in cervical cancer. These results indicate that miR-99b attenuates the migration and invasion of human cervical cancer cells through downregulation of the PI3K/AKT/mTOR signaling pathway, which provides a therapeutic approach for cervical cancer treatment.     

Retracted: Downregulation of microRNA-1469 promotes the development of breast cancer via targeting HOXA1 and activating PTEN/PI3K/AKT and Wnt/β-catenin pathways

Breast cancer (BC) is a common malignancy which is the most frequently diagnosed cancer in women all over the worldwide. This study aimed to investigate the roles of miR-1469 in the development of BC, as well as its regulatory mechanism. The expression levels of miR-1469 in BC tissues, serum, and cell lines were determined. Effects of overexpression of miR-1469 on MCF7 cell viability, colony-forming ability, apoptosis, migration, and invasion were then investigated. Furthermore, the potential target of miR-1469 in MCF7 cells was explored. Besides, the association between miR-1469, PTEN/PI3K/AKT, and Wnt/β-catenin pathways was elucidated. Notably, confirmatory experiments by downregulation of miR-1469 in SK-BR-3 cells were further performed. The miR-1469 expression was significantly downregulated in BC tissues, serum, and cell lines. The overexpression of miR-1469 significantly inhibited the proliferation, arrested cell-cycle at G2/M phase, increased apoptosis, suppressed migration, and invasion of MCF-7 cells. In addition, HOXA1 was verified as a direct target of miR-1469, and the effects of overexpression of miR-1469 on the malignant behaviors of MCF7 cells were significantly counteracted by overexpression of HOXA1 concurrently. Furthermore, the overexpression of miR-1469 suppressed the activation of PTEN/PI3K/AKT and Wnt/β-catenin pathways, which was reversed overexpression of HOXA1 concurrently. Besides, confirmatory experiments showed that the inhibition of miR-1469 promoted the malignant behaviors of SK-BR-3 cells, which was inversed after miR-1469 inhibition and HOXA1 knockdown at the same time. Our findings reveal that downregulation of miR-1469 may promote the development of BC by targeting HOXA1 and activating PTEN/PI3K/AKT and Wnt/β-catenin pathways. MiR-1469 may serve as a promising target for BC therapy.     

臭椿酮抑制急性骨髓性白血病细胞恶性生物学行为的研究

为了探讨臭椿酮（ailanthone,AIL）对急性骨髓性白血病（acute myelogenous leukemia,AML）细胞恶性生物学行为的影响,用不同浓度（0.2、0.4、0.8、1.6、3.2 μmol·L^-1）的AIL处理对数生长期的HL-60细胞,将miR-449a mimic质粒、mimic对照质粒、miR-449a inhibitor质粒、inhibitor对照质粒分别转染至未经任何处理的HL-60细胞,并用1.0 μmol·L^-1浓度的AIL处理细胞24 h。采用CCK-8法检测细胞增殖水平,细胞划痕实验检测细胞迁移水平,Transwell小室法检测细胞侵袭水平,Annexin V-FITC/PI双染法检测细胞凋亡水平,qRT-PCR法检测miR-449a mRNA表达水平,Western blot法检测磷脂酰肌醇3-激酶（PI3K）、磷酸化PI3K（p-PI3K）、蛋白激酶B（AKT）、磷酸化AKT（p-AKT）蛋白表达水平。结果显示,AIL干预后HL-60细胞增殖抑制率、凋亡率升高,细胞迁移率及细胞侵袭数降低（P<0.05）,miR-449a mRNA表达量升高（P<0.05）。过表达miR-449a可以抑制HL-60细胞增殖、迁移和侵袭,并诱导细胞凋亡（P<0.05）,抑制miR-449a的表达可以起到逆转AIL抑制HL-60细胞增殖、迁移和侵袭,诱导细胞凋亡的作用（P<0.05）。AIL能够显著降低HL-60细胞中p-PI3K/PI3K和p-AKT/AKT比值（P<0.05）,抑制miR-449a表达可以逆转AIL对HL-60细胞p-PI3K/PI3K和p-AKT/AKT比值的下调作用（P<0.05）。结果表明,AIL可通过上调miR-449a抑制AML细胞的增殖、迁移和侵袭,并诱导细胞凋亡,其作用机制可能与抑制PI3K/AKT信号通路有关。结果表明,AIL有望成为AML治疗的候选药物。     

MiR-210 in exosomes derived from CAFs promotes non-small cell lung cancer migration and invasion through PTEN/PI3K/AKT pathway

ObjectiveCancer-associated fibroblasts (CAFs) function as a crucial factor in tumor progression by carrying exosomes to neighboring cells. This study was assigned to expound the underlying mechanism of CAFs-derived exosomal miR-210 in non-small cell lung cancer (NSCLC) progression.MethodCAFs and normal fibroblasts (NFs) were isolated and identified. Exosomes secreted from CAFs and NFs were isolated to analyze their effects on tumor volume and epithelial-mesenchymal transition (EMT). Exosomal miR-210 expression level was measured. The effects of exosomal miR-210 and UPF1 on cell viability, EMT, PTEN/PI3K/AKT signal pathway were determined. Dual-luciferase reporter gene assay was utilized to validate the binding of UPF1 to miR-210.ResultsCAFs-derived exosomes (CAFs-exo) were successfully extracted and proven to be uptake by lung cancer cells. Up-regulated expression level of miR-210 was found in CAFs-exo, which was then proved to enhance cell migration, proliferation, invasion abilities and EMT in NSCLC cells. Overexpression of miR-210 can also inhibit UPF1 and PTEN, but activate the PTEN/PI3K/AKT pathway. UPF1 was a target gene of miR-210. MiR-210 can up-regulate UPF1 expression level to activate PTEN/PI3K/AKT pathway.ConclusionMiR-210 secreted by CAFs-exo could promote EMT by targeting UPF1 and activating PTEN/PI3K/AKT pathway, thereby promoting NSCLC migration and invasion.