RNA干扰K-ras基因对肺腺癌H441细胞EGFR-TKI耐药性的影响

目的探讨RNA干扰K-ras基因对肺腺癌H441细胞株细胞EGFR-TKI耐药性的影响。方法构建靶向K-ras基因的表达载体并转染肺腺癌H441细胞,分别检测转染后细胞中K-ras蛋白及基因表达以及对吉非替尼敏感性的改变。结果实验组中K-ras基因及蛋白的表达量明显低于对照组(P0.05);实验组中细胞凋亡率及细胞增殖抑制率均显著升高(P0.05)。结论靶向突变K-ras基因的si RNA可以诱导肺腺癌H441细胞凋亡,减弱对吉非替尼的耐药性,为肺癌的基因治疗提供了新的思路和方法。     

长链非编码RNA小核仁RNA宿主基因3在肺腺癌组织中的表达及其临床意义

目的 探讨肺腺癌组织中长链非编码RNA小核仁RNA宿主基因3(lncRNA SNHG3)的表达及临床意义.方法 收集120例肺腺癌患者术后癌组织及癌旁组织作为样本,检测样本中SNHG3的表达并分析其与患者临床特征的相关性,利用RNA干扰技术对SNHG3的表达进行干扰,通过噻唑蓝(MTT)实验及Tr-answell实验记...     

吉非替尼-线治疗晚期肺腺癌的临床观察

目的 探讨吉非替尼-线治疗晚期肺腺癌的疗效及不良反应.方法 选择住院及门诊确诊肺腺癌Ⅲb或Ⅳ期17例,每天一次口服吉非替尼250 mg,直至出现PD为止,根据WHO标准评价其疗效及毒性不良反应.结果 17例中1例达到完全缓解(CR),8例部分缓解(PR),3例病情稳定(SD),5例病情进展(PD),总有效率为52.9％,疾病控制率(PR+ CR+ SD)为70.6％.结论 吉非替尼对于晚期非小细胞肺腺癌具有较好的疗效和耐受性.     

自噬在厄洛替尼诱导的肺腺癌细胞A549凋亡中的作用

目的探讨自噬对厄洛替尼(Erlotinib,特罗凯)诱导的肺腺癌细胞A549凋亡中的影响。方法用厄洛替尼和(或)自噬抑制剂3甲级腺嘌呤(3-MA)处理人肺腺癌A549细胞系,AnnexinV-FITC/PI双染色流式细胞术检测细胞凋亡率;吖啶橙(AO)染色荧光显微镜观察细胞酸性自噬泡的变化;Western印迹法检测自噬相关蛋白Beclin1及凋亡相关蛋白Caspase9的表达。结果流式细胞仪检测示厄洛替尼组、3-MA组、厄洛替尼+3-MA组细胞凋亡率为(56.35±0.71)%、(9.47±0.15)%、(78.40±0.52)%。厄洛替尼组经吖啶橙染色后,细胞内酸性自噬泡明显增加,呈明亮的红色荧光,而加入3-MA联合作用后,则上述现象被抑制。Western印迹结果示对照组、厄洛替尼组、3-MA组、厄洛替尼+3-MA组Beclin1和Caspase9相对灰度值分别为0.54±0.03、0.86±0.03、0.37±0.02、0.70±0.04和0.43±0.03、0.77±0.03、0.57±0.04、0.90±0.02。结论厄洛替尼可以诱导肺腺癌细胞A549细胞系凋亡,并可激活其自噬;3-MA可能通过抑制自噬且上调Caspase9的表达增强厄洛替尼对肺腺癌细胞的杀伤作用。     

肺腺癌A549细胞KDR基因沉默后对厄罗替尼敏感性的影响

目的研究激酶功能区受体(KDR)基因沉默对人肺腺癌A549细胞增殖和对化疗药物厄罗替尼敏感性的影响。方法设计合成KDR的si RNA序列,LipofectamineTM2000转染人A549细胞。RT-PCR和Western印迹检测KDR在干扰后m RNA和蛋白的表达情况,通过流式细胞仪检测细胞周期。MTT法和细胞克隆形成试验观察KDR基因沉默后A549细胞对厄罗替尼的敏感性。结果 KDR基因沉默48 h后,A549细胞的KDR基因和蛋白表达明显降低(P<0.05)。细胞周期被阻滞在G0/G1期,S期细胞数明显减少(P<0.05)。KDR基因沉默组细胞对厄罗替尼的敏感性显著性增强。结论 KDR特异性si RNA能显著沉默A549细胞KDR基因,抑制细胞增殖,并增强A549细胞对厄罗替尼的敏感性。     

PVT1通过促进肿瘤干细胞特性介导肺腺癌细胞PC9厄洛替尼抵抗

目的探究浆细胞瘤转化迁移基因(PVT) 1对肺腺癌肿瘤干细胞特性及厄洛替尼抵抗的影响。方法慢病毒介导转染PVT1干扰载体si PVT1-1及si PVT1-2下调肺腺癌细胞PC9中PVT1的表达作为干扰A组及干扰B组细胞,转染对照载体为对照组细胞。实时荧光定量PCR(q PCR)技术检测PVT1、CD133和CD44 mRNA的表达水平,Western印迹检测CD133和CD44蛋白的表达水平。CCK-8法检测细胞对厄洛替尼的半抑制浓度(IC50),方差分析及SNK-q检验比较各组指标间的统计学差异。结果干扰A组及干扰B组PVT1、CD133和CD44 mRNA的表达水平均显著低于对照组,差异有统计学意义(P<0. 05)。干扰A组及干扰B组CD133和CD44蛋白表达水平均显著低于对照组,差异有统计学意义(P<0. 05)。干扰A组及干扰B组厄洛替尼IC50显著低于对照组,差异有统计学意义(P<0. 05)。结论 PVT1可通过诱导肺腺癌细胞干细胞特性的维持促进其厄洛替尼抵抗的发生。     

双靶向吉非替尼磷脂胶束纳米载体靶向肺癌干细胞和肺癌细胞研究

目的研究双靶向吉非替尼磷脂胶束纳米载体靶向肺癌干细胞和肺癌细胞方法制备吉非替尼纳米载体和双靶向吉非替尼磷脂胶束纳米载体,检测吉非替尼载药量、包封率、粒径及zeta电位,培养肺癌细胞和干细胞分选,测定不同载药纳米载体在肺癌细胞中的蓄积效率、对肺癌细胞克隆球生长的影响、对肺癌细胞杀伤作用。结果双靶向吉非替尼纳米载体组包封率、粒径均高于吉非替尼纳米载体组,载药量、zeta电位低于吉非替尼纳米载体组,具有统计学差异(P<0.05)。两组载药纳米载体中的吉非替尼在各时间点的释放量比较,无统计学差异(P>0.05)。双靶向吉非替尼纳米载体组在肺癌A549细胞、A431细胞中的蓄积效率均高于吉非替尼纳米载体组,具有统计学差异(P<0.05)。双靶向吉非替尼纳米载体组肺癌A549细胞、A431细胞克隆球形成率均低于吉非替尼纳米载体组,具有统计学差异(P<0.05)。双靶向吉非替尼纳米载体组肺癌A549细胞、A431细胞在各时间点的存活率均低于吉非替尼纳米载体组,具有统计学差异(P<0.05)。结论双靶向吉非替尼磷脂胶束纳米载体可双靶向杀伤肺癌细胞,具有较强的抗肿瘤作用。     

一代表皮生长因子受体酪氨酸激酶抑制剂一线治疗晚期非小细胞肺癌患者166例无进展生存期分析

目的探讨一代表皮生长因子受体(EGFR)酪氨酸激酶抑制剂(TKI)一线治疗晚期非小细胞肺癌(NSCLC)EGFR基因敏感突变患者的无进展生存(PFS)时间获益分析。方法随访2016—2017年中南大学湘雅医院一代EGFR-TKI一线治疗的166例非小细胞肺癌患者,根据PFS将患者分为PFS≤3个月组、3～12个月组、≥12个月组,分析PFS的获益因素。结果一代EGFR-TKI为影响PFS的独立因素(P0.05),其中厄洛替尼进展的风险是埃克替尼的2.16倍,吉非替尼进展的风险是埃克替尼的4.48倍。PFS≤3个月和PFS≥12个月两组的Logistic回归分析显示,埃克替尼组疗效最佳(埃克替尼对吉非替尼,P=0.009;埃克替尼对厄洛替尼,P=0.006),EGFR 19号外显子del (19del)突变组优于EGFR 21号外显子L858R点突变组(21L858R)(P=0.024),腺癌组优于非腺癌组(P=0.044),中分化组优于低分化组(P=0.022)。多因素分析显示,埃克替尼组疗效优于厄洛替尼和吉非替尼组(P分别为0.018和0.006),腺癌组优于非腺癌(P=0.015),中分化组优于低分化组(P=0.034)。结论组织学类型、分化程度、EGFR基因等基线状态的不同可致不同PFS,一代EGFR-TKI药物为影响PFS的独立因素。     

吉非替尼对老年晚期肺腺癌患者血清中血管内皮生长因子、神经元特异性烯醇化酶和细胞角蛋白19片段的调节作用

肺腺癌进展到晚期时,肿瘤细胞产生大量肿瘤相关蛋白,且在血清中表达增高,加速病变的进展,此机制可能与肿瘤中异常表达的基因和蛋白进入血清有关[1].吉非替尼是针对表皮生长因子受体(EGFR)阳性肺腺癌治疗的靶向药物,是选择性EGFR酪氨酸激酶抑制剂,可以对癌细胞增殖和分化有明显抑制[2].本研究在常规化疗基础上加用吉非替尼治疗老年晚期肺腺癌患者,观察其疗效及对血清中血管内皮生长因子受体(VEGF)、神经元特异性烯醇化酶(NSE)和细胞角蛋白19片段(CYFRA21-1)的作用.     

吉非替尼与替莫唑胺治疗老年女性肺腺癌伴脑转移癌的临床疗效

目的 探讨非血脑屏障通透性药物吉非替尼(gefitinb)和血脑屏障通透性药物替莫唑胺(TMZ)治疗老年女性肺腺癌伴脑转移癌的疗效、不良反应及评价生活质量.方法 将34例患者分为吉非替尼组(18例)和TMZ组(16例).观察组于固定时间用温水送服吉非替尼250 mg,1次/d,直到疾病进展、死亡或发生不可耐受的不良反应;TMZ组给予TMZ胶囊150 mg·m-2·d-1口服,连用5 d,每28 d重复1次,连用2～4个周期.治疗2个月后,观察两组患者近期疗效,探讨该疗法的安全性及评价生活质量,随访2年评价远期疗效.结果 吉非替尼组有效率(CR+PR)16.7%,稍高于TMZ组(12.5%),两组比较无统计学差异(P＞0.05).吉非替尼组皮疹发生率高于TMZ组、白细胞下降发生率低于TMZ组,两组比较有统计学差异(P<0.05),吉非替尼组恶心呕吐、腹泻、肝功能损害等不良反应发生率低于TMZ组,两组比较无统计学差异(P＞0.05).吉非替尼组Karnofsky评分提高+稳定者占83.3%(15/18),高于TMZ组[43.8%(7/16)],两组比较有显著性差异(P<0.05).两组1、2 年生存率比较吉非替尼组稍高于TMZ组,但无统计学差异(P＞0.05).结论 非血脑屏障通透性药物吉非替尼治疗老年女性肺腺癌伴脑转移癌的近、远期疗效优于血脑屏障通透性药物TMZ,吉非替尼组不良反应轻微、生活质量改善明显.     

miR-1226靶向MUC1对非小细胞肺癌细胞吉非替尼敏感性的影响

目的 研究miR-1226对非小细胞肺癌(NSCLC)吉非替尼敏感性的影响,并探讨其作用机制.方法 体外培养NSCLC细胞株人肺癌耐吉非替尼细胞株(PC9GR)和人肺腺癌细胞(PC-9);将PC-9组细胞设置为正常组,PC9GR细胞随机分为对照组、miR-1226 mimic组和miR-1226 NC组,并进行转染;利...     

LncRNA MALAT1 Promotes Lung Cancer Proliferation and Gefitinib Resistance by Acting as a miR-200a Sponge

IntroductionLung cancer is a major public health problem, as the second causes of cancer related death worldwide, with relatively low survival rates, and accessible drug resistance. Long non-coding RNAs (LncRNAs) have been identified as activator in lung cancer with elusive mechanisms. We aimed to detect the regulation of LncRNA MALAT1 in the proliferation and gefitinib resistance in lung cancer cells.MethodsMALAT1 in A549 and HCC 1299 human lung adenocarcinoma cell lines was silenced by shRNA or overexpressed using plasmid, and the cell viability and cell proliferation were evaluated by MTT assay and soft agar colony formation assay. RNA levels were detected by RT-PCR, and the protein expression was measured by western blot. The binding between MALAT1 and miR-200a was validated by luciferase reporter assays using pSi-Chech 2 vectors.ResultsThe cell viability and proliferation of A549 cells transfected with MALAT1 shRNA were significantly lower than the control. The MALAT1 expression in gefitinib resistant A549 cells was upregulated. miR-200a significantly inhibited the fluorescence of pSi-Check 2 vector with MALAT1 gene, suggesting the direct binding between MALAT1 and miR-200a. In addition, LncRNA MALAT1 promotes ZEB1 expression in A549 cells.ConclusionOur study showed that MALAT1 promoted the proliferation and gefitinib resistance of lung cancer cells by sponging miR-200a, which regulates expression of ZEB1 in the A549 cells. This MALAT1/miR-200a axis could serve as new therapeutic target for lung cancer treatment.     

Emodin regulating excision repair cross-complementation group 1 through fibroblast growth factor receptor 2 signaling

AIM: To investigate the molecular mechanisms underlying the reversal effect of emodin on platinum resistance in hepatocellular carcinoma. METHODS: After the addition of 10 μmol/L emodin to HepG2/oxaliplatin (OXA) cells, the inhibition rate (IR), 50% inhibitory concentration (IC 50 ) and reversal index (IC 50 in experimental group/IC 50 in control group) were calculated. For HepG2, HepG2/OXA, HepG2/OXA/T, each cell line was divided into a control group, OXA group, OXA + fibroblast growth factor 7 (FGF7) group and OXA + emodin group, and the final concentrations of FGF7, emodin and OXA in each group were 5 ng/mL, 10 μg/mL and 10 μmol/L, respectively. Single-cell gel electrophoresis was conducted to detect DNA damage, and the fibroblast growth factor receptor 2 (FGFR2), phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) and excision repair cross-complementing gene 1 (ERCC1) protein expression levels in each group were examined by Western blotting. RESULTS: Compared with the IC50 of 120.78 μmol/L in HepG2/OXA cells, the IC 50 decreased to 39.65 μmol/L after treatment with 10 μmol/L emodin; thus, the reversal index was 3.05. Compared with the control group, the tail length and Olive tail length in the OXA group, OXA + FGF7 group and OXA + emodin group were significantly increased, and the differences were statistically significant (P < 0.01). The tail length and Olive tail length were lower in the OXA + FGF7 group than in the OXA group, and this difference was also statistically significant. Compared with the OXA + FGF7 group, the tail extent, the Olive tail moment and the percentage of tail DNA were significantly increased in the OXA + emodin group, and these differences were statistically significant (P < 0.01). In comparison with its parental cell line HepG2, the HepG2/OXA cells demonstrated significantly increased FGFR2, p-ERK1/2 and ERCC1 expression levels, whereas the expression of all three molecules was significantly inhibited in HepG2/ OXA/T cells, in which FGFR2 was silenced by     

低表达的microRNA-145对c-Myc表达的影响

目的:探讨microRNA(miR)-145是否通过调控c-Myc参与肝癌行为的调控.方法:实时定量PCR检测肝组织及细胞系miR-145表达水平:检测不同肝组织c-Myc的mRNA和蛋白表达量:通过转染上调miR-145在HepG2细胞表达后,检测转染前后空白组、实验组、阴性对照组HepG2中c-Myc的mRNA和蛋...     

大鼠microRNA-145慢病毒表达载体的构建及其对血管平滑肌细胞表型转化的影响

目的构建针对Rno-miR-145的慢病毒表达载体并探讨其在血小板源生长因子(PDGF)诱导的血管平滑肌细胞(VSMC)表型转化中的作用。方法人工合成含有酶切位点粘端miR-145 shDNA双链模板序列,克隆于LV3 pGLV/H1/GFP+Puro-miRNA慢病毒穿梭载体中,转染293T细胞,收获并浓缩慢病毒颗粒,感染大鼠原代VSMC,倒置荧光显微镜下观察VSMC感染后的荧光表达情况,实时荧光定量PCR检测miR-145的表达情况;实验分为空白对照组、PDGF组、PDGF+miR-145组和细胞转染阴性慢病毒载体组(miR-NC组);采用实时荧光定量PCR测定miR-145对VSMC增殖相关基因PCNA、c-Jun及分化相关基因SM22αmRNA表达水平的影响。结果成功构建了microRNA-145慢病毒载体,测定病毒滴度为1×109TU/mL。倒置荧光显微镜下观察大鼠microRNA-145慢病毒表达载体感染成功,MOI值为50,感染72 h时感染率最高。实时荧光定量PCR结果显示PDGF可使PCNA、c-Jun表达增加,而使SM22α表达降低;miR-145可使PDGF诱导的去分化型VSMC增殖相关基因PCNA、c-Jun表达降低,分化相关基因SM22α表达增加。结论 miR-145慢病毒载体可高效感染大鼠原代VSMC。感染miR-145慢病毒后可抑制VSMC的表型转化。     

干扰LINC00707对食管癌细胞生物行为的影响

目的 探讨干扰LINC00707对食管癌细胞生物行为的影响及分子机制.方法 选取51例食管癌患者癌组织及癌旁正常组织,用实时荧光定量-聚合酶链反应(RT-qPCR)检测LINC00707和miR-382-5p的表达水平;将食管癌细胞EC9706随机分为对照(con)组、si-LINC00707组、si-NC组、miR-...     

miR-147在肺腺癌中的表达及意义

目的观察miR-147在肺浸润性腺癌中的表达,探讨其与细胞增殖和血管内皮生长因子(VEGF)的关系,分析其临床意义。方法选取2015年1月-2015年07月我院收治的65例肺浸润性腺癌患者作为研究对象,留取肿瘤组织作为观察组,留取正常肺组织作为对照组,应用实时荧光定量PCR法检测二组组织中miR-147的表达,应用免疫组化方法检测观察组中Ki67和VEGF的表达。结果观察组中miR-147的表达明显低于对照组(P<0.05),观察组中miR-147的表达在有无胸膜累犯、不同肿瘤最大径、有无淋巴结转移及不同TNM分期的表达中,差别有统计学意义(P<0.05)。miR-147的表达与增殖指数、miR-147和VEGF均呈负相关性(P<0.05)。生存分析显示miR-147的表达与生存时间相关(P<0.05)。结论肺浸润性腺癌中miR-147低表达是肿瘤形成的重要分子事件,其参与肿瘤的进展,miR-147的作用与对细胞增殖和VEGF的调节有关。miR-147的表达可能与预后有关。     

LINC00370 modulates miR-222-3p-RGS4 axis to protect against osteoporosis progression

BackgroundWe aimed to determine the role of the LINC00370/miR-222-3p/RGS4 axis in modulating the process of adipose-derived stem cell (ADSC) osteogenic differentiation.MethodsWe first evaluated the differential expression of LINC00370, miR-222-3p and RGS4 between normal and osteogenically induced ADSCs. Moreover, we transfected ADSCs with LINC00370 siRNA and an miR-222-3p inhibitor to determine the role of LINC00370 in modulating the process of ADSC osteogenic differentiation. Finally, we analyzed the dual-luciferase reporter gene to identify the relationship between LINC00370 and miR-222-3p. We first created osteoporotic rat models by ovariectomy (OVX) and treated with pcDNA-LINC00370. HE and immunohistochemical staining of OCN were performed to assess the changes in bone microarchitecture.ResultsLINC00370 and RGS4 expression was remarkably upregulated in the osteogenic ADSC group compared with the normal medium group. On the other hand, miR-222-3p expression was remarkably decreased in the osteogenic group compared with the normal medium group. Knockdown of LINC00370 reduced the osteogenic differentiation of ADSCs. Moreover, the inhibitor of miR-222-3p partially reversed the reduction of osteogenic differentiation by LINC00370 knockdown. Knockdown of LINC00370 reduced the expression of p-Akt and p-PI3K. The inhibitor of miR-222-3p partially reversed the reduction of the expression of p-Akt and p-PI3K by LINC00370 knockdown. A dual luciferase reporter assay indicated that LINC00370 can directly bind miR-222-3p. LINC00370 suppressed OP progression in OVX and partially upregulated OCN protein expression.ConclusionCollectively, the above results confirm that LINC00370 promotes the process of ADSC osteogenic differentiation via the miR-222-3p/RGS4 axis. Moreover, LINC00370 could protect against OVX-induced OP.     

干扰STX18-AS1通过靶向miR-204保护心肌细胞

目的 探讨干扰长链非编码RNA STX18-AS1(long non-coding RNA, LncRNA STX18-AS1)是否通过上调微小RNA-204(miR-204)的表达从而影响心肌细胞缺氧损伤。方法 体外培养心肌细胞H9C2,采用二氯化钴(CoCl₂)处理心肌细胞建立细胞缺氧损伤模型,采用qRT-PCR检测CoCl₂处理不同时间点STX18-AS1、miR-204的表达量;实验设置对照组、模型组、si-NC组、si-STX18-AS1组、miR-NC组、miR-204组。采用甲基噻唑基四唑(MTT)检测细胞增殖活性;流式细胞术与TUNEL检测细胞凋亡率;采用生化试剂盒检测乳酸脱氢酶(LDH)活性、丙二醛(MDA)水平、超氧化物歧化酶(SOD)活性、过氧化氢酶(CAT)活性;双荧光素酶报告实验验证STX18-AS1、miR-204的靶向关系。结果 与对照组比较,在CoCl₂处理6 h、12 h、18 h、24 h时模型组和si-NC组STX18-AS1表达水平升高(P<0.01),miR-204表达水平...     

YAP调控肺癌A549/DDP细胞顺铂耐药性的机制分析

目的探讨Yse相关蛋白(Yes-associated protein, YAP)对A549/DDP细胞顺铂耐药性的影响及其机制。 方法采用MTS检测顺铂对肺腺癌A549细胞以及肺腺癌耐顺铂A549/DDP细胞的50%抑制浓度(IC₅₀)。使用qPCR以及Western blot检测A549和A549/DDP细胞中YAP mRNA以及蛋白的表达。Western blot检测维替泊芬(Verteporfin, VP)处理A549/DDP后YAP蛋白表达变化。将A549/DDP细胞设置为DMSO对照组、顺铂组(DDP组)、维替泊芬组(VP组)、顺铂联合维替泊芬组(DDP+VP组),采用MTS检测0、24和48 h各处理组细胞活力的变化。采用qPCR检测VP处理A549/DDP后干细胞标志物ALDHA1、CD133、OCT4、NANOG、SOX2的mRNA表达变化。 结果顺铂对A549和A549/DDP细胞的IC50分别为(4.07±0.03)μg/ml、(23.44±0.98)μg/ml,耐药指数为5.76。A549/DDP细胞中YAP显著高于A549细胞(P<0.05)。VP处理A549/DDP细胞后YAP蛋白表达水平较DMSO组明显降低。DDP+VP组较单独DDP组,其细胞活力在24 h和48 h均明显降低(P<0.05)。qPCR检测发现A549/DDP细胞经VP处理后,干细胞标志物ALDHA1、CD133、OCT4、NANOG、SOX2的mRNA均较DMSO组明显降低(P<0.05)。 结论YAP可能参与了肺癌细胞的顺铂耐药。抑制YAP可通过抑制肿瘤干细胞特性,进而发挥逆转耐药的作用。