外源性p16基因对人肺癌细胞生物学特性的影响

目的：研究外原性p16基因对肺癌细胞生物学特性的影响。方法：利用脂质体介导的基因转染方法将外源性p16基因转入p16基因缺陷的人肺腺癌细胞株A549中,用逆转录聚合酶链反应（RT－PCR）及免疫组化的方法检测p16基因的表达,同时观察转染后细胞恶性生长的变化。结果：外源性p16基因在A549细胞中能稳定表达,转染后A549细胞生长速度明显减慢,在软琼脂上形成克隆的能力降低。流式细胞仪检测显示A549细胞G1期阻滞并发生了凋亡,接种裸鼠后致瘤性降低,肿瘤生长明显减慢。结论：外源性p16基因导入人肺癌细胞株A549中可稳定表达并抑制细胞的恶性生长,同时诱导调亡。     

核转录因子-κB圈套寡核苷酸对环格列酮诱导肺癌细胞分化作用的影响

目的 探讨核转录因子-κB(NF-κB)圈套寡核苷酸在环格列酮对肺癌细胞A549增殖抑制和分化调控过程中的影响。方法 运用基因转染技术将NF-κB圈套寡核苷酸转入人肺癌细胞A549中,凝胶阻滞分析实验(EMSA)检测转染前后NF-κB活性的变化,免疫印迹观察转染前后多药耐药蛋白(mdrl)的变化。进而以。100μmol/L环格列酮处理转染和未转染的细胞1-4d,生长曲线观察A549细胞生长,流式细胞仪进行细胞周期分析,免疫印迹观察处理前后细胞周期素D1的变化。结果 (1)EMSA显示转染后NF-κB活性明显下降,mdrl蛋白表达水平降低。(2)环格列酮能抑制A549细胞增殖。(3)转染NF-κB圈套寡核苷酸增强环格列酮对A549细胞增殖的抑制作用,更多的细胞被阻滞于G1／G0期,细胞中细胞周期素D1的水平更低。结论NF-κB圈套寡核苷酸能增加肺癌细胞A549对环格列酮的敏感性,即NF-κB圈套寡核苷酸能协同环格列酮抑制肺癌细胞A549的恶性增殖及诱导其分化。     

4-氨基-2-三氟甲基苯基维甲酸酯对肺腺癌细胞株A549分化和增殖的影响

目的观察4-氨基-2-三氟甲基苯基维甲酸酯（4-amino-2-trifluoromethyl-phenyl retinoate）对人肺腺癌细胞株A549生长抑制、分化的影响。方法4-氨基-2-三氟甲基苯基维甲酸酯组及全反式维甲酸（ATRA）处理肺腺癌细胞株A549,观察A549细胞生长情况,流式细胞仪进行细胞周期分析,苏木精-伊红（HE）染色观察肺癌细胞形态变化。结果4-氨基-2-三氟甲基苯基维甲酸酯及ATRA具有抑制A549细胞增殖、使细胞周期阻滞于G0／G1期,肺癌A549细胞在,发生了细胞胞体变小、分裂状态的细胞数减少等形态学变化。结论4-氨基-2-三氟甲基苯基维甲酸酯及ATRA使细胞周期阻滞于G0／G1期,从而影响其DNA的合成,抑制其恶性增殖并诱导其分化。     

三叶青黄酮通过调控miR-497表达对肺癌细胞增殖、凋亡的影响

目的 探讨三叶青黄酮(RTHF)对肺癌细胞增殖、凋亡的影响及其对miR-497的调控作用。方法 体外培养人肺癌细胞A549,加入不同剂量的RTHF处理细胞;miR-NC、miR-497 mimics转染至A549细胞,anti-miR-NC、anti-miR-497转染至A549细胞后加入RTHF处理细胞;采用噻唑蓝(MTT)实验与流式细胞术分别检测细胞增殖及细胞凋亡率;采用实时荧光定量聚合酶链反应(qRT-PCR)检测肺癌组织、癌旁组织及各组A549细胞中miR-497的表达量;Western印迹法检测B细胞淋巴瘤(Bcl)-2、Bcl-2相关X蛋白(Bax)蛋白表达量。结果 RTHF可明显提高细胞增殖抑制率、凋亡率及p21、Bax蛋白水平明显降低细胞周期蛋(Cyclin)D1、Bcl-2蛋白水平(均P<0.05);与癌旁组织比较,肺癌组织中miR-497的表达水平明显降低(P<0.05);RTHF可明显提高A549细胞中miR-497的表达水平(P<0.05);转染miR-497 mimics可明显提高细胞增殖抑制率与凋亡率及p21、Bax蛋白水平,明显降低Cyc...     

miR-145-3p在肝癌中的表达及其对肝癌细胞生长的调控作用

背景miR-145-3p在头颈癌、肺癌和胃癌等肿瘤可发挥抑癌的作用,而其在肝癌中的表达和作用并不清楚.目的探讨miR-145-3p在肝癌中的表达及其对肝癌细胞的增殖与凋亡的调控作用.方法用RT-q PCR法检测肝癌组织、癌旁组织、肝细胞株(Hep3B、Huh-7、Hep G2和SMMC-7721)与正常肝细胞株L-02中miR-145-3p的表达水平;将miR-145-3pmimic或miR-145-3pinhibitor转入Hep3B细胞,用CCK-8法、流式细胞术、TUNEL法和Westernblot法分别检测细胞活力、细胞周期、细胞凋亡和4型黏蛋白(mucin4, MUC4)表达.用荧光素酶报告基因实验鉴定miR-145-3p的靶基因.将pc DNA-MUC4转入已转染miR-145-3p mimic的细胞,用CCK-8法和TUNEL法分别检测细胞活力与细胞凋亡.结果miR-145-3p在肝癌组织和细胞中呈低表达.过表达miR-145-3p能抑制细胞增值和G0/G1期到S期转换,促进凋亡和降低MUC4表达;而敲减miR-145-3p则作用相反. MUC4是miR-145-3p的靶基因.过表达MUC4能逆转miR-145-3p mimic对细胞存活的抑制作用.结论miR-145-3p在肝癌低表达,且其表达与T分期呈负相关.过表达miR-145-3p可抑制肝癌细胞存活与生长,而敲减miR-145-3p能促进肝癌细胞存活与生长.     

LncRNA MCM3AP-AS1靶向调控miR-16-5p表达对肺癌细胞增殖、迁移侵袭的影响及机制

目的研究LncRNA MCM3AP-AS1对肺癌细胞增殖、迁移侵袭的影响和潜在机制。方法以BEAS-2B为对照组,实时荧光定量(qRT)-聚合酶链反应(PCR)检测MCM3AP-AS1和miR-16-5p RNA的表达,四氮唑蓝(MTT)法测定A549细胞增殖活性,Transwell实验检测细胞迁移和侵袭能力,Western印迹检测增殖蛋白CyclinD1、p21和p27及迁移蛋白基质金属蛋白酶(MMP)-2、MMP-9和MMP-14,双荧光素酶报告系统验证MCM3AP-AS1和miR-16-5p的关系。结果与对照组相比,肺癌细胞系A549、SPC-A1和NCI-H460中MCM3AP-AS1表达量均显著升高(P0.05),miR-16-5p表达量显著下降(P0.05);抑制MCM3AP-AS1表达和过表达miR-16-5p均可抑制A549细胞增殖、迁移和侵袭;MCM3AP-AS1靶向负向调控miR-16-5p的表达;抑制miR-16-5p表达逆转了下调MCM3AP-AS1表达对A549细胞增殖、迁移、侵袭的作用。结论 LncRNA MCM3AP-AS1通过靶向miR-16-5p调控肺癌A549细胞增殖、迁移和侵袭。MCM3AP-AS1是肺癌潜在分子靶点。     

survivin—siRNA对肺癌细胞A549增殖抑制的影响

目的探讨survivin—siRNA对肺癌细胞A549的增殖抑制作用。方法将针对survivin的siRNA表达载体与脂质体和pSi—scrambled两个对照组一起分别转染肺癌细胞A549后，利用半定量RT—PCR和Western印迹法分别检测细胞survivin mRNA及蛋白表达水平，MTT法测定细胞的增殖能力，流式细胞术检测细胞周期和凋亡。结果转染72h后，实验组细胞的survivin mRNA水平是脂质体对照组的31.1％，蛋白表达水平是脂质体对照组的29．2％。MTT检测实验组细胞的生长速度是脂质体对照组的36．6％，流式细胞术发现实验组G1期细胞比例明显高于两个对照组，而G2期和S期细胞比例减少，细胞出现凋亡。结论利用survivin—siRNA可明显抑制肺癌细胞A549的增殖，细胞受阻于G1期，诱导细胞凋亡。     

凋亡抑制基因XIAP与p53在非小细胞肺癌中表达的相关性

目的 研究凋亡抑制基因XIAP与抑癌基因p53在非小细胞肺癌中的表达及两者的相关性.方法 转染特异短发夹样RNA(XIAP shRNA)序列的表达载体至非小细胞肺癌细胞A549中,应用半定量PT PCR方法测定转染前后细胞中XIAP与p53的表达情况并进行对比.结果 PT-PCR结果显示XIAP在阳性转染组的表达较阴性转染组、未转染组细胞表达明显降低;p53在阳性转染组表达升高,加入顺铂后,阳性转染组XIAP mRNA表达较阴性转染组、未转染组细胞降低,p53在阳性转染加顺铂组的表达明显提高.结论 非小细胞肺癌中XIAP与p53呈负相关,高表达的XIAP抑制野生型p53的表达,反之XIAP受到抑制后野生型p53表达增高,促进非小细胞肺癌细胞凋亡.     

microRNA-21在非小细胞肺癌患者体内外周血和癌组织中的表达水平及其通过调节PTEN蛋白参与抑癌的机制

目的探讨非小细胞肺癌(NSCLC)患者外周血清和癌组织中microRNA-21表达水平及其对肺癌细胞的侵袭和增殖影响。方法 32例NSCLC患者为研究组,30例同期肺部良性病变患者为对照组,采用Real-time RCR检测研究组肿瘤组织、癌旁组织及两组血清中microRNA-21的表达水平;此外,对肺癌细胞株A549转染microRNA-21的抑制物(inhibitor)或模拟物(mimics)后,采用transwell及CCK-8法观察microRNA-21对肺癌细胞株的侵袭及增殖的变化,并采用Western印迹检测A549中张力蛋白同源基因(PTEN)蛋白水平。结果研究组血清及肿瘤组织中microRNA-21的表达水平显著高于对照组及癌旁组织(均P<0.05);A549转染microRNA-21 mimics后细胞的侵袭和增殖能力显著增加(P<0.05),转染microRNA-21 inhibitor后,细胞的侵袭和增殖能力显著降低(P<0.05),而PTEN蛋白转染后,表达水平与上述结果相反。结论 NSCLC患者肿瘤组织和血清中高表达的microRNA-21可能通过抑制PTEN蛋白参与癌细胞侵袭和增殖能力的调节。     

MDR1介导LncRNAFENDRR对非小细胞肺癌细胞特性的影响及机制研究

目的探究多耐药基因1(MDR1)介导的长链非编码RNA FENDRR(LncRNA FENDRR)对非小细胞肺癌细胞特性的影响,并探讨其作用机制。方法选择非小细胞肺癌患者癌组织标本及癌旁组织标本,对比非小细胞肺癌组织及癌旁组织中的FENDRR基因表达差异,选择A549细胞系,分为空白对照组和FENDRR组,对比两组细胞的增殖活力及凋亡情况。结果与癌旁组织相比,FENDRR在非小细胞肺癌组织中呈现明显低表达(P0.05); FENDRR表达与非小细胞肺癌分化程度及T分期具有相关性,癌细胞分化程度越低、T分期越高,FENDRR表达越低(P0.05),与M、N分期无关(P0.05);与空白对照组相比,FENDRR组细胞中mRNA表达明显升高,细胞迁移能力及侵袭能力均明显降低,同时A549细胞凋亡率明显上升(P0.05)。结论 LncRNAFENDRR与非小细胞肺癌的发生、发展密切相关,其过表达可抑制癌细胞增殖,降低癌细胞迁移与侵袭,促进癌细胞凋亡。     

Retinoic acid treatment of human leiomyoma cells transformed the cell phenotype to one strongly resembling myometrial cells

Background  Uterine leiomyomas are clinically significant tumours that may develop due to an altered differentiation pathway. We have previously identified a dysregulated retinoic acid (RA) pathway that reduced retinoic exposure in human leiomyoma surgical specimens, and have shown that the leiomyoma phenotype was characterized by excessive and disorganized extracellular matrix (ECM).
Objective  The goal of this study was to determine the impact of RA exposure on the disrupted ECM phenotype of leiomyomas.
Design and methods  Study of immortalized and molecularly confirmed cells generated from surgical specimens of spontaneous uterine leiomyoma and matched myometrium.
Results  Immortalized leiomyoma and myometrial cells retained the molecular characteristics of their progenitor tissue. Proliferation of leiomyoma cells was inhibited by all -trans retinoic acid (ATRA). Furthermore, there was a dose-dependent decrease in soluble extracellular collagen protein in ATRA-treated leiomyoma cells. Exposure of leiomyoma cells to ATRA resulted in a dose-dependent inhibition of templates for specific ECM protein production including collagen 1, collagen 4, fibronectin and versican. Notably, expression levels in treated leiomyoma cells approached those found in myometrial cells. These mRNA alterations translated into altered protein. Down-regulation was also observed among the RA pathway genes such as CYP26A1 with exposure to ATRA. Finally, ATRA down-regulated TGF-β3 mRNA expression and the TGF-β regulated genes in leiomyoma cells.
Conclusion  Exposure of leiomyomas to ATRA down-regulated cell proliferation, ECM formation, RA metabolism and TGF-β regulation, suggesting that RA exposure can alter the leiomyoma phenotype to one that more closely approximates normal myometrium.     

维甲酸诱导的人食管癌EC—109细胞分化相关基因的克隆

目的：克隆维甲酸诱导的人食管癌ＥＣ－１０９细胞分化相关基因。方法：在全反式维甲酸诱导人食管癌ＥＣ－１０９细胞系分化的基础上，采用ｍＲＮＡ差异显示技术（ｍＲＮＡｄｉｆｆｅｒｅｎｔｉａｌｄｉｓｐｌａｙ，ＤＤ）对ＥＣ－１０９细胞全反式维甲酸诱导前后的基因表达差异情况进行了分析。结果：全反式维甲酸诱导前后的细胞之间存在明显的基因表达差异，经克隆筛选获得了一些经全反式维甲酸诱导激活或抑制的差异表达基因片段。对其中一个片段（ＲＡａｌ２）的序列分析及同源性比较结果表明其与人的ａｌｐｈａ－ｃａｔｅｎｉｎ基因（Ｄ１３８６６，ＨＵＭＡＣＡ，２４３９ｂｐ）１００％同源。结论：全反式维甲酸具有调控分化相关基因表达的重要作用，在全反式维甲酸诱导前后的细胞之间存在明显的基因表达差异     

p16、p53、p21基因对肺癌细胞增殖的影响

目的 观察肿瘤抑制基因ｐ１６,ｐ５３及细胞周期信赖激酶抑制物ｐ２１基因单独或联合应用时对肺癌细胞增殖的影响。方法 应用十八酰基胺阳离子脂质体介导ｐ１６,ｐ５３,ｐ２１基因单独或共转染到非小细胞肺癌细胞系Ａ５４９和小细胞肺癌细胞系ＳＨ７７,观察转染后１,３,５日该细胞增殖的活力。采用四甲基偶氮唑 盐微量酶反应比色法（ＭＴＴ法）测定吸光度（Ａ）,以检测细胞增殖活力,结果 Ａ５４９细胞系：Ａ均值分别为：     

全反式维甲酸对卵巢癌SKOV3细胞增殖、分化的影响

目的 探讨全反式维甲酸（ATRA）对卵巢癌SKOV3细胞增殖、分化的影响.方法 以1×10^-6 mol/L的ATRA处理人卵巢癌SKOV3细胞为实验组,细胞不加ATRA处理为对照组.应用MTT法测定细胞增殖抑制率,流式细胞仪测定细胞周期分布,免疫组化法检测鼠抗人分化相关基因1（NDRG1）表达,显微镜观察细胞形态变化.结果 与对照组比较,实验组细胞增殖抑制率、NDRG1表达升高（P均＜0.05）;随着作用时间延长,实验组G/G0期细胞增多、S期细胞减少,显微镜下可见细胞生长差,增殖缓慢,分裂像少见.结论 ATRA有较强的抑制卵巢癌SKOV3细胞增殖和诱导其分化作用.     

Forced expression of the cyclin-dependent kinase inhibitor p16(INK4A) in leukemic U-937 cells reveals dissociation between cell cycle and differentiation.

OBJECTIVE: The aim of this study was to investigate how the tumor suppressor protein p16(INK4A) interferes with growth and differentiation of leukemic U-937 cells. MATERIALS AND METHODS: U-937 clones constantly overexpressing the cyclin-dependent kinase inhibitor p16(INK4A) were established. Clones transfected with empty vector were used as controls. The effects of high-level expression of p16(INK4A) on proliferation and cell cycle progression were investigated (cell cycle distribution, proliferation rate, analyses of different cell cycle regulatory proteins). The effect of introduction of p16(INK4A) on capacity for induced differentiation, assayed by capacity to reduce nitroblue tetrazolium, was determined. RESULTS: Overexpressed p16(INK4A) protein was active as judged by its ability to bind to CDK-4 in a coimmunoprecipitation assay. Clones overexpressing p16(INK4A) grew slower than controls, without any apparent effects on the phosphorylation status of the retinoblastoma protein (pRb). Instead, p16(INK4A) overexpression affected the phosphorylation status of pRb-related pocket protein p130, which was detected in its growth-restraining hypophosphorylated form. Despite an enhanced tendency to accumulate in G(0)/G(1), p16(INK4A)-overexpressing cells were less sensitive to induction of differentiation with vitamin D(3) or ATRA than control cells. CONCLUSIONS: Constitutive expression of p16(INK4A) in U-937 cells resulted in decreased proliferation as a result of activated p130 rather than pRb. Also, we showed that introduction of p16(INK4A) into U-937 cells impaired their capacity to differentiate. Moreover, the results support the notion that cell differentiation and cell cycle progression are dissociated and independently regulated processes.     

维甲酸抑制人胃癌细胞株层粘素受体基因和原癌基因C—Ha—ras mRNA的表达

研究维甲酸（RA）对人胃癌细胞株（SGC7901）层粘素受体（LNR）基因和原癌基因C－Ha-ras表达的影响。方法：应用原位交方法标记LNR探讨和C－Ha-ras探针，对人胃癌细胞（分为经RA处理及未经RA处理两组）中LNR和C－Ha-ras基因的mRNA表达进行检测。结果：经RA处理的人胃癌细胞LNRmRNA表达较未经RA处理者显著降低（P＜0．01－0．001）；C－Ha-ras转录水平亦显著降低（P＜0．01－0．001）。结论：RA可显著抑制人胃癌细胞LNR和C－Ha-ras基因的表达。，     

Retinoic acid-induced glandular differentiation of the oesophagus

BACKGROUND: Retinoic acid (RA) is a powerful differentiation agent. Barrett's oesophagus occurs when duodeno-gastro-oesophageal reflux causes squamous epithelium (SE) tissue to become columnar epithelium tissue by an unknown mechanism. The bile acid lithocholic acid (LCA) competes for the retinoid X receptor retinoid binding site. Hence, RA pathways may be implicated in Barrett's oesophagus. METHODS: RA activity in tissues and cell lines treated with all-trans retinoic acid (ATRA) with or without LCA was assessed using a reporter. Expression of p21 was determined by real-time PCR in Barrett's oesophagus cell lines with or without LCA. SE and Barrett's oesophagus biopsy specimens were exposed to 100 muM of ATRA or 20 mM of a RA inhibitor, citral, in organ culture for >72 h. Characteristics of treated specimens, compared with untreated controls, were analysed by immunohistochemical analysis (cytokeratins (CKs), vimentin) and RT-PCR (CKs). Confocal microscopy assessed temporal changes in co-localisation of CK8/18 and vimentin. Cell proliferation was assessed by bromo-deoxyuridine incorporation and immunohistochemical analysis for Ki67 and p21. RESULTS: RA biosynthesis was increased in Barrett's oesophagus compared with SE (p<0.001). LCA and ATRA caused a synergistic increase in RA signalling as shown by increased p21 (p<0.01). Morphological and molecular analysis of SE exposed to ATRA showed columnar differentiation independent of proliferation. Metaplasia could be induced from the stromal compartment alone and vimentin expression co-localised with CK8/18 at 24 h, which separated into CK8/18-positive glands and vimentin-positive stroma by 48 h. Citral-treated Barrett's oesophagus led to phenotypic and immunohistochemical characteristics of SE, which was independent of proliferation. CONCLUSION: RA activity is increased in Barrett's oesophagus and is induced by LCA. Under conditions of altered RA activity and an intact stroma, the oesophageal phenotype can be altered independent of proliferation.