过表达OMA1通过促进线粒体释放细胞色素C诱导肺腺癌A549细胞凋亡

目的:探讨OMA1在肺腺癌中的表达水平及其对肺腺癌A549细胞凋亡的影响及机制。方法:收集2018年01月至2019年10月在本院保存的42例经病理确诊为肺腺癌患者的癌组织样本及其癌旁组织(距离肿瘤边缘≥5 cm),免疫组化染色法检测癌组织及癌旁组织中OMA1表达情况。体外培养人肺腺癌细胞系NCI-H2009、Calu-3、SPC-A-1、A549及人正常肺上皮细胞BEAS-2B,qRT-PCR和Western blotting检测细胞中OMA1 mRNA和蛋白表达水平。采用OMA1过表达慢病毒及其空载慢病毒感染A549细胞,分为OMA1过表达慢病毒感染组(OMA1组)和空载慢病毒感染组(Vector组),另设置空白对照组(Blank组)。采用MTT检测细胞增殖活性;流式细胞术检测细胞凋亡水平;JC-10染色法检测细胞线粒体膜电位变化;qRT-PCR法检测细胞中OMA1 mRNA表达水平;Western blotting法检测细胞中OMA1、cleaved caspase-3、OPA1以及细胞线粒体和胞浆中Cyt C蛋白表达水平。结果:肺腺癌患者癌组织中OMA1表达水平显著低于癌旁组织...     

人截短型线粒体凋亡诱导因子蛋白的克隆、表达及诱导肝癌细胞凋亡的活性鉴定

背景与目的:线粒体在细胞凋亡中扮演关键的角色,线粒体凋亡诱导因子(apoptosis-inducing factor,AIF)是定位于线粒体中的一种重要的凋亡蛋白,对线粒体蛋白质的研究可深入阐明线粒体在凋亡中的作用.本研究的目的在于克隆、表达重组的人截短型AIF,并对其诱导肿瘤细胞核凋亡的生物学活性进行鉴定.方法:采用RT-PCR技术从人肝癌细胞SMMC-7721中扩增出剪切掉线粒体定位信号的人AIF基因片段,并按阅读框克隆到原核表达载体pET32a( )中.进行酶切与测序鉴定后,以构建的正确重组质粒pET32a-AIF转化大肠杆菌BL21(DE3)菌株,在异丙基-β-D-硫代半乳糖苷(IPTG)诱导下表达AIF蛋白,表达产物用SDS-PAGE和Western blot检测;采用镍柱亲和层析法纯化目的蛋白;采用凝胶滞后实验(EMSA)与Hoechst 33258染色检测AIF蛋白的生物学活性.结果:获得了去掉线粒体定位信号的AIF基因,并克隆到pET32a( )载体中,经酶切与测序鉴定完全正确.此重组质粒转化人大肠杆菌,经IPTG诱导后,在大肠杆菌中可表达相对分子质量约Mr 70 000的目的蛋白;表达量约占菌体蛋白总量的11%,表达的目的蛋白与抗His标签抗体与抗人的AIF蛋白具有良好的反应性;纯化后,AIF的纯度达到95%.经EMSA与Hoechst 33258染色实验证实:获得的AIF蛋白具有良好地与DNA结合并可诱导肿瘤细胞核凋亡的能力,凋亡率为37%.结论:人AIF基因在PET表达系统中得到有效表达:蛋白复性后能有效地在体外与DNA结合,并可诱导细胞凋亡.     

康莱特注射液诱导肝癌细胞凋亡及其对凋亡蛋白procaspase-3和caspase-9表达的影响

目的:探讨康莱特注射液(Kanglaite)对人肝癌BEL-7404细胞的增殖抑制作用、凋亡诱导作用以及对凋亡蛋白procaspase-3和caspase-9表达的影响.方法:采用MTT法检测康莱特注射液和阳性对照顺铂(cisplatin ,DDP)对BEL-7404细胞的增殖抑制作用,Hoechst 33258染色观察凋亡细胞核的情况,FCM检测细胞凋亡率,Western印迹法检测凋亡蛋白procaspase-3和caspase-9的表达.结果:MTT结果显示,经不同体积浓度康莱特注射液(10、20、40、80及160 μL/mL)作用BEL-7404细胞后,80 μL/mL康莱特注射液作用48 h时对肝癌BEL-7404细胞有明显的抑制增殖作用;Hoechst 33258染色可见细胞出现典型的凋亡细胞形态学改变;FCM法检测结果提示,空白对照组、DDP 10 μg/mL组和康莱特注射液80 μL/mL组的凋亡率分别为(1.23±0.40)%、(32.53±0.65)%和(3.13±0.32)% (P＜0.01);Western印迹法检测结果提示,康莱特注射液及DDP作用48 h后 procaspase-3蛋白的表达量明显降低 (P＜0.01),caspase-9蛋白的表达量显著升高(P＜0.01).结论:康莱特注射液可抑制肝癌BEL-7404细胞增殖并诱导其凋亡,其作用机制可能与凋亡相关蛋白procaspase-3表达下调及caspase-9表达上调有关.     

缺氧条件下骨肉瘤MG-63细胞中TG2通过线粒体途径调节细胞凋亡的机制

目的探讨缺氧条件下TG2在骨肉瘤MG-63细胞凋亡中的作用以及TG2通过阻止细胞色素C的释放和调节Caspase-3的表达及活性抑制细胞凋亡的机制。方法建立骨肉瘤细胞体外缺氧培养模型,设立四组:(1)常氧组;(2)单纯缺氧组;(3)对照si RNA缺氧组;(4)TG2 si RNA缺氧组。观察各组在缺氧培养不同时相(6、12、24、48、72 h)骨肉瘤MG-63细胞TG2、Caspase-3表达、胞核和胞质细胞色素C的变化以及细胞凋亡率。结果与常氧组比较,单纯缺氧组及对照si RNA缺氧组的TG2活性、m RNA及蛋白表达水平明显增强(P<0.01),且随缺氧时间延长明显升高;Caspase-3活性未见明显增加,而Caspase-3及细胞质内细胞色素C蛋白的表达水平和细胞凋亡率轻度增加。与前三组比较,在TG2 si RNA缺氧组,当通过转染TG2 si RNA抑制TG2的表达时,Caspase-3的活性及胞质内细胞色素C蛋白表达水平明显增强(P<0.01);细胞凋亡率也显著增加(P<0.01)。结论缺氧条件下,MG-63骨肉瘤细胞TG2表达增强,并且可以阻止细胞核内细胞色素C向胞质释放,从而降低Caspase-3的表达及活性,抑制肿瘤细胞的凋亡。     

棘霉素对肝癌SMMC7721细胞凋亡相关基因影响的研究

目的：探讨棘霉素诱导肝癌SMMC7721细胞的凋亡作用及对转录因子Twist、p53、Survivin和hTERT（人端粒酶）基因的影响。方法：采用MTT法测定棘霉素对肝癌细胞的生长抑制率；半定量逆转录聚合酶链反应（RT-PCR）检测与肝癌的发生和转移密切相关的转录因子Twist，与细胞增殖和凋亡联系紧密的p53、Survivin和hTERT的mRNA表达水平的改变；利用Westernblot方法分析棘霉素治疗后上述基因的蛋白表达情况。结果：体外实验证实，棘霉素浓度〉10．0ng／mL各组对细胞的生长抑制均显著增加，P〈0．05，随棘霉素药物浓度增加，细胞抑制率明显增大。棘霉素抑制肝癌细胞株SMMC7721的作用呈剂量一时间依赖性。RT-PCR方法证实，随着棘霉素剂量的增加，肝癌SMMC7721细胞株中Twist、Survivin和hTERTmRNA表达水平逐渐下调，P〈0．01，p53mRNA表达水平微弱上调，P〈0．05。Westernblot检测显示，Twist、Survivin和hTERT蛋白的表达量呈递减趋势，P〈0．01，p53蛋白的表达微弱上调，P〈0．01。结论：随着棘霉素剂量的增加，细胞凋亡率显著增加。棘霉素能够抑制与肝癌生长密切相关基因的Twist、Survivin和hTERTmRNA表达，促进p53mRNA表达上调，相应蛋白的表达呈下调和上调。棘霉素可能通过抑制肝癌细胞的生长、转移和促进肝癌细胞的凋亡起到抑制肿瘤生长的作用。     

棘霉素对肝癌SMMC7721细胞凋亡相关基因影响的研究

目的:探讨棘霉素诱导肝癌SMMC7721细胞的凋亡作用及对转录因子Twist、p53、Survivin和hTERT(人端粒酶)基因的影响.方法:采用MTT法测定棘霉素对肝癌细胞的生长抑制率;半定量逆转录聚合酶链反应(RT-PCR)检测与肝癌的发生和转移密切相关的转录因子Twist,与细胞增殖和凋亡联系紧密的p53、Survivin和hTERT的mRNA表达水平的改变;利用Western blot方法分析棘霉素治疗后上述基因的蛋白表达情况.结果:体外实验证实,棘霉素浓度＞10.0 ng/mL各组对细胞的生长抑制均显著增加,P<0.05,随棘霉素药物浓度增加,细胞抑制率明显增大.棘霉素抑制肝癌细胞株SMMC7721的作用呈剂量-时间依赖性. RT-PCR 方法证实,随着棘霉素剂量的增加,肝癌SMMC7721细胞株中Twist、Survivin和hTERT mRNA表达水平逐渐下调,P<0.01,p53 mRNA表达水平微弱上调,P<0.05.Western blot检测显示,Twist、 Survivin和hTERT蛋白的表达量呈递减趋势,P<0.01,p53蛋白的表达微弱上调,P<0.01.结论:随着棘霉素剂量的增加,细胞凋亡率显著增加.棘霉素能够抑制与肝癌生长密切相关基因的Twist、Survivin和hTERT mRNA表达,促进p53 mRNA表达上调,相应蛋白的表达呈下调和上调.棘霉素可能通过抑制肝癌细胞的生长、转移和促进肝癌细胞的凋亡起到抑制肿瘤生长的作用.     

姜黄素损伤线粒体诱导食管癌Ec-109细胞凋亡的研究

目的:探讨姜黄素损伤线粒体诱导食管癌Ec-109细胞凋亡的作用机制。方法:80μmol/mL姜黄素作用于食管癌Ec-109细胞不同时间后,流式细胞仪检测细胞亚二倍体凋亡峰变化及线粒体膜电位变化;蛋白质印迹法检测细胞色素C释放及Caspase-9表达。结果:姜黄素呈时间依赖性诱导食管癌Ec-109细胞凋亡,12、24和48 h细胞亚二倍体凋亡峰分别为(15.89±2.12)%、(26.80±1.87)%和(36.97±1.80)%,对照组为(3.23±0.24)%,总体比较差异有统计学意义,F=6.75,P<0.01,各组间比较差异均有统计学意义,P<0.01。姜黄素作用3、6和12 h后线粒体膜电位分别为84.78%、67.03%和63.16%,对照组为97.17%,差异有统计学意义,F=5.12,P<0.05,各组间比较,6 h组与12 h组差异无统计学意义,P=0.062,余各组间有差别;6 h出现细胞色素C表达,12 h表达最高;12 h检测到Caspase-9表达,24 h表达最高。结论:姜黄素呈时间依赖性诱导食管癌细胞凋亡是通过损伤细胞线粒体、使之释放出细胞色素C以及激活Caspase-9实现的。     

树突状细胞体外诱导抗肿瘤免疫通过诱导肿瘤细胞凋亡抑制裸鼠 …

研究突状细胞体外诱导的细胞免疫制裸鼠移植瘤生长作用及其机理。方法联合应用粒／巨噬细胞集落刺激因子及白介素－４直接从肝癌患者外周血中培养出ＤＣ,以人肝癌细胞系ＨｅｐＧ２肿瘤细胞的肿瘤抗原粗提物刺激ＤＣ,ＤＣ激活同源的Ｔ淋巴细胞,     

顺铂诱导人肝癌细胞凋亡机理研究

顺铂是多种化疗方案的核心药物,在对人卵巢癌细胞和大鼠肝癌细胞的研究中,发现其具有诱导凋亡的作用。本实验从细胞形态、ＤＮＡ裂解片段、细胞周期、ＤＮＡ断裂点末端标记,凋亡相关基因观察顺铂诱导人肝癌细胞凋亡的作用机理。1　材料与方法1.1　细胞培养及药物人肝癌细胞ＳＭＭＣ-7721,引自中国科学院上海细胞生物学研究所细胞库。在含10%小牛血清的ＲＰＭＩ-1640培养基(Ｇｉｂｃｏ产品),37℃,5%ＣＯ2条件下培养。顺氯氨铂(ｃｉｓｐｌａｔｉｎ,ＤＤＰ齐鲁制药厂出品,批号:960601),用含10%正常大鼠血清的ＲＰＭＩ-1640培养液配制,ＤＤＰ终…     

辣椒素通过氧化应激诱导HepG2肝癌细胞凋亡的研究

摘 要：［目的］ 观察辣椒素对HepG2细胞增殖和凋亡的影响及其分子机制。［方法］采用CCK8法观察辣椒素对HepG2细胞增殖的影响,流式细胞术和JC-1探针检测辣椒素对HepG2细胞凋亡率和线粒体膜电位的影响,采用DCFH-DA探针联合高内涵分析仪检测氧化应激表达水平的变化。［结果］ 辣椒素可剂量依赖性抑制HepG2细胞增殖、诱导细胞凋亡。200μmol/L辣椒素作用24h可引起HepG2 细胞明显坏死、脱落和漂浮。相比对照组,200μmol/L辣椒素明显降低HepG2细胞线粒体膜电位红绿荧光表达量比值（8.39±0.12 vs 4.23±0.04）。200μmol/L辣椒素可引起HepG2细胞内氧化应激水平上调1.78倍。使用还原剂维生素C抗氧化可恢复细胞活力,减轻凋亡。［结论］ 辣椒素剂量依赖性诱导人肝癌HepG2细胞凋亡,其机制与氧化应激水平过激活相关。     

Mda-7/IL-24基因转染对肝癌细胞生长的影响

目的研究Mda-7/IL-24基因对肝癌细胞系Hep3B生长的影响。方法构建Mda-7/IL-24基因的真核表达载体pcDNA3.1/Mda-7/IL-24,用脂质体介导的基因转染法分别将真核重组体pcD-NA3.1/Mda-7/IL-24和pcDNA3.1空载体质粒导入人肝癌细胞系Hep3B细胞中,经G418筛选获得细胞克隆。通过聚合酶链式反应（PCR）、免疫组织化学等方法检测基因和蛋白的表达、细胞生长增殖情况。结果将pcDNA3.1载体和pcDNA3.1/Mda-7/IL-24重组体转染肝癌细胞系Hep3B中,RT-PCR结果显示pcDNA3.1/Mda-7/IL-24克隆中有680bp的扩增条带,而pcDNA3.1空载体转染的克隆未见的扩增条带,未转染的Hep3B细胞亦未见680bp的条带;转染Mda-7/IL-24的Hep3B细胞与空白质粒载体转染的Hep3B细胞和Hep3B细胞的生长速度相比,后两者生长速度较快。结论将外源性Mda-7/IL-24基因导入Hep3B细胞后,可以抑制细胞生长。     

mda-7/IL-24对裸鼠肝癌移植瘤的生长抑制和促凋亡作用

目的: 探讨mda-7/IL-24对裸鼠肝癌细胞移植瘤的生长抑制和促凋亡作用及其相关机制.方法:构建携带mda-7基因的重组腺病毒载体Ad.mda-7.以HepG2细胞皮下接种建立裸鼠肝癌移植瘤模型,采用瘤内单点注射的方法分别给予Ad.GFP、Ad.mda-7和ALLN(毒胡萝卜素,N-Ac-L-L-norleucinal)+Ad.mda-7,观察瘤质量和瘤体积的变化,通过免疫组化和TUNEL法检测肿瘤组织内凋亡相关蛋白caspase-3活化、细胞增殖相关抗原ki-67和微血管密度、细胞凋亡率,并通过Western blotting检测caspase-12、caspase-3和Bax的表达.结果:Ad.mda-7治疗组和Ad.GFP对照组肿瘤体积分别为(312.6±30.2)mm3和(520.6±30.0)mm3(P<0.01),两组的肿瘤质量分别为(0.321±0.031)g和(0.534±0.030)g(P<0.01);Ad.mda-7治疗后瘤细胞的凋亡率显著高于对照组(P<0.01);Ad.mda-7可抑制肝癌组织ki-67表达、微血管密度和促进caspase-3的表达. 经 ALLN 处理的裸鼠,明显抑制Ad.mda-7对肝癌细胞的致凋亡作用(P<0.05),并且下调Ad.mda-7诱导的caspase-12、caspase-3和Bax的表达.结论:Ad.mda-7可显著抑制裸鼠肝癌移植瘤的生长和新生血管的形成,并通过内质网应激通路显著诱导肿瘤细胞的凋亡.     

Suppression of Her2/Neu mammary tumor development in mda-7/IL-24 transgenic mice

Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) encodes a tumor suppressor gene implicated in the growth of various tumor types including breast cancer. We previously demonstrated that recombinant adenovirus-mediated mda-7/IL-24 expression in the mammary glands of carcinogen-treated (methylnitrosourea, MNU) rats suppressed mammary tumor development. Since most MNU-induced tumors in rats contain activating mutations in Ha-ras, which arenot frequently detected in humans, we presently examined the effect of MDA-7/IL-24 on Her2/Neu-induced mammary tumors, in which the RAS pathway is induced. We generated tet-inducible MDA-7/IL-24 transgenic mice and crossed them with Her2/Neu transgenic mice. Triple compound transgenic mice treated with doxycycline exhibited a strong inhibition of tumor development, demonstrating tumor suppressor activity by MDA-7/IL-24 in immune-competent mice. MDA-7/IL-24 induction also inhibited growth of tumors generated following injection of Her2/Neu tumor cells isolated from triple compound transgenic mice that had not been treated with doxycycline, into the mammary fat pads of isogenic FVB mice. Despite initial growth suppression, tumors in triple compound transgenic mice lost mda-7/IL-24 expression and grew, albeit after longer latency, indicating that continuous presence of this cytokine within tumor microenvironment is crucial to sustain tumor inhibitory activity. Mechanistically, MDA-7/IL-24 exerted its tumor suppression effect on HER2⁺ breast cancer cells, at least in part, through PERP, a member of PMP-22 family with growth arrest and apoptosis-inducing capacity. Overall, our results establish mda-7/IL-24 as a suppressor of mammary tumor development and provide a rationale for using this cytokine in the prevention/treatment of human breast cancer.     

过表达MDA-7/IL-24蛋白的亚细胞定位

目的了解标签蛋白GFP的位置对过表达的MDA-7/IL-24融合蛋白亚细胞定位的影响。方法分别构建在MDA-7/IL-24 cDNA的5'端和3'端带有GFP-tag的重组表达质粒pEGFP-C1-mda-7/IL-24和pEGFP-N3-mda-7/IL-24,瞬时转染猴胚肾Cos7细胞,48h后用Hoechst 33258染核,在荧光显微镜下观察GFP-MDA-7/IL-24融合蛋白在Cos7细胞的亚细胞分布。结果测序表明重组质粒pEGFP-C1-mda-7/IL-24和pEGFP-N3-mda-7/IL-24构建正确;分别转染Cos7细胞后,在氨基端带有GFP-tag的MDA-7/IL-24融合蛋白表达阳性细胞中,绿色荧光均匀分布于胞浆和胞核,但在羧基端带有GFP-tag的MDA-7/IL-24融合蛋白表达阳性细胞中,绿色荧光则分布于核膜外侧和胞浆。结论GFP-tag位置的不同对过表达融合蛋白MDA-7/IL-24的亚细胞定位有显著的影响。     

mda7-/IL2-4和IL2-4delE5诱导急性髓系白血病细胞向单核细胞分化

目的:研究人黑素瘤分化相关基因-7(melanoma differentiation associated gene-7,mda-7;又称IL-24)及其剪接体IL-24 delE5与急性髓系白血病(acute myeloid leukemia,AML)细胞分化的关系。方法:十四烷酰佛波醇-乙酯(12-O-tetrade-canoylphorbol-13-acetate,TPA)处理人急性髓系白血病细胞系U937、HL-60,real-time PCR及Western blotting检测细胞中mda-7/IL-24和IL-24 delE5的表达,FACS检测细胞表面CD11b、CD14和CD115的表达。siRNA干扰U937、HL-60细胞中mda-7/IL-24和IL-24 delE5的表达后,再以TPA诱导细胞分化,FACS检测CD11b、CD14和CD115的表达。用前期构建的mda-7/IL-24和IL-24 delE5载体转染U937、HL-60及AML-M5患者的原代白血病细胞,观察异位过表达mda-7/IL-24和IL-24 delE5能否诱导白血病细胞向单核细胞分化。结果:TPA在诱导U9...     

BI-69A11 enhances susceptibility of colon cancer cells to mda-7/IL-24-induced growth inhibition by targeting Akt

Background:

Akt and its downstream signalling pathways contribute to the aetiology and progression of colorectal carcinoma (CRC). Targeting the Akt pathway is an attractive strategy but few chemotherapeutic drugs have been used to treat CRC with only limited success. BI-69A11, a small molecule inhibitor of Akt, efficiently inhibits growth in melanoma cells. Melanoma differentiation associated gene-7 (mda-7)/interleukin-24 promotes cancer-selective apoptosis when delivered by a tropism-modified replication incompetent adenovirus (Ad.5/3-mda-7). However, Ad.5/3-mda-7 displays diminished antitumour efficacy in several CRC cell lines, which correlates with the expression of K-RAS.

Methods:

The individual and combinatorial effect of BI-69A11 and Ad.5/3-mda-7 in vitro was studied by cell viability, cell cycle, apoptosis and invasion assays in HT29 and HCT116 cells containing wild type or mutant K-ras, respectively. In vivo HT29 tumour xenografts were used to test the efficacy of the combination treatment.

Results:

BI-69A11 inhibited growth and induced apoptosis in CRC. However, combinatorial treatment was more effective compared with single treatment. This combination showed profound antitumour and anti angiogenic effects in vitro and in vivo by downregulating Akt activity.

Conclusions:

BI-69A11 enhances the antitumour efficacy of Ad.5/3-mda-7 on CRC overexpressing K-RAS by inducing apoptosis and regulating Akt activity thereby warranting further evaluation in treating CRC.     

Combining histone deacetylase inhibitors with MDA-7/IL-24 enhances killing of renal carcinoma cells

In the present study we show that histone deacetylase inhibitors (HDACIs) enhance the anti-tumor effects of melanoma differentiation associated gene-7/interleukin 24 (mda-7/IL-24) in human renal carcinoma cells. Similar data were obtained in other GU tumor cells. Combination of these two agents resulted in increased autophagy that was dependent on expression of ceramide synthase 6, with HDACIs enhancing MDA-7/IL-24 toxicity by increasing generation of ROS and Ca²⁺. Knock down of CD95 protected cells from HDACI and MDA-7/IL-24 lethality. Sorafenib treatment further enhanced (HDACI + MDA-7/IL-24) lethality. Anoikis resistant renal carcinoma cells were more sensitive to MDA-7/IL-24 that correlated with elevated SRC activity and tyrosine phosphorylation of CD95. We employed a recently constructed serotype 5/3 adenovirus, which is more effective than a serotype 5 virus in delivering mda-7/IL-24 to renal carcinoma cells and which conditionally replicates (CR) in tumor cells expressing MDA-7/IL-24 by virtue of placing the adenoviral E1A gene under the control of the cancer-specific promoter progression elevated gene-3 (Ad.5/3-PEG-E1A-mda-7; CRAd.5/3-mda-7, Ad.5/3-CTV), to define efficacy in renal carcinoma cells. Ad.5/3-CTV decreased the growth of renal carcinoma tumors to a significantly greater extent than did a non-replicative virus Ad.5/3-mda-7. In contralateral uninfected renal carcinoma tumors Ad.5/3-CTV also decreased the growth of tumors to a greater extent than did Ad.5/3-mda-7. In summation, our data demonstrates that HDACIs enhance MDA-7/IL-24-mediated toxicity and tumor specific adenoviral delivery and viral replication of mda-7/IL-24 is an effective pre-clinical renal carcinoma therapeutic.     

Caspase-8和Bcl-2在PIG11诱导HepG2细胞凋亡中的作用

目的：利用本实验室已建立PIG11蛋白稳定低表达的HepG2细胞株和PIG11蛋白稳定高表达HepG2细胞株,研究Caspase-8和Bcl-2在PIG11诱导HepG2细胞凋亡中的作用,进一步探讨PIG11基因表达对HepG2细胞凋亡的机制。方法：细胞培养,分组：HepG2细胞、pLXSN-PIG11-HepG2（pLXSN-PIG11转染建立PIG11蛋白高表达HepG2细胞）、pLXSN-HepG2（pLXSN空载体转染HepG2细胞）、miR-PIG11-HepG2（miRNA干扰PIG11蛋白低表达HepG2细胞）、miR-HepG2（干扰空载体HepG2细胞）。Hoechst33342染色荧光和PI染色流式细胞仪检测各组细胞凋亡情况。Western blot检测Caspase-8蛋白、Bcl-2蛋白的表达情况。结果：Hoechst 33342染色荧光显微镜下pLXSN-PIG11-HepG2细胞组可见核染色质浓缩,核碎片,荧光增强,以及凋亡小体。PI染色流式细胞仪检测结果显示：HepG2细胞、miR-PIG11-HepG2细胞、miR-HepG2细胞、pLXSN-PIG11-HepG2细胞、pLXSN-HepG2细胞的凋亡率分别为5.72%±0.81%、1.34%±0.71%、5.10%±0.40%、34.83%±2.29%、5.34%±0.60%。PIG11高表达的pLXSN-PIG11-HepG2细胞组凋亡率比其它各组增高（P〈0.01）,PIG11低表达的miR-PIG11-HepG2细胞组凋亡率降低（P〈0.01）。Western blot检测结果显示PIG11高表达的pLXSN-PIG11-HepG2细胞Caspase-8蛋白表达上调,Bcl-2蛋白表达下调（P〈0.01）,PIG11低表达的miR-PIG11-HepG2细胞Caspase-8蛋白的表达下调,Bcl-2蛋白表达上调（P〈0.01）。结论：PIG11蛋白高表达能诱导HepG2细胞凋亡,其机制可能与Caspase-8蛋白及Bcl-2蛋白表达相关。     

A novel oncogenic pathway by TLS-CHOP involving repression of MDA-7/IL-24 expression

Background:

Translocated in liposarcoma-CCAAT/enhancer binding protein homologous protein (TLS–CHOP) (also known as FUS-DDIT3) chimeric oncoprotein is found in the majority of human myxoid liposarcoma (MLS), but its molecular function remains unclear.

Methods:

We knockdowned TLS–CHOP expression in MLS-derived cell lines by a specific small interfering RNA, and analysed the gene expression profiles with microarray.

Results:

TLS-CHOP knockdown inhibited growth of MLS cells, and induced an anticancer cytokine, melanoma differentiation-associated gene 7 (MDA-7)/interleukin-24 (IL-24) expression. However, double knockdown of TLS–CHOP and MDA-7/IL-24 did not inhibit MLS cell growth.

Conclusion:

Repression of MDA-7/IL-24 expression by TLS–CHOP is required for MLS tumour growth, and TLS–CHOP may become a promising therapeutic target for MLS treatment.