地西他滨联合丙戊酸钠促进肝癌细胞凋亡的ERK 通路研究

目的:本研究对地西他滨联合丙戊酸钠促进肝癌细胞凋亡的相关机制进行研究,为临床治疗和新药研发提供参考。方法:肝癌细胞HepG2经地西他滨联合丙戊酸钠孵育后,MTT法检测二者对HepG2细胞的抑制率,Real-time PCR及免疫印迹法检测细胞凋亡蛋白Caspase3/9、Bax及Bcl2和ERK信号通路关键蛋白的表达情况。结果:地西他滨联合丙戊酸钠能够有效抑制肝癌细胞的增殖,且二者联用的抑制率明显高于单独应用(P0.05),同时与对照组相比,细胞凋亡蛋白Caspase3、Caspase9及Bax表达明显增强(P0.05),Bcl2表达明显降低(P0.05),且ERK信号通路关键蛋白Ras、Raf及MEK和ERK1/2的表达明显升高,与对照组细胞有显著的统计学差异性(P0.05)。结论:地西他滨联合丙戊酸钠主要通过激活MEK/ERK信号转导通路抑制肝癌细胞HepG2的增殖过程。     

碱性成纤维细胞生长因子经ERK信号通路抑制卵巢癌CAOV3细胞凋亡

目的:研究卵巢癌CAOV3细胞中碱性成纤维细胞生长因子(bFGF)经MEK/ERK1/2信号转导通路对CREB、Bcl-2表达以及对细胞凋亡的影响作用。方法:无血清饥饿诱导细胞凋亡。分为对照组、bFGF组、bFGF PD98059组。Annexin-EGFP/PI荧光双染法检测细胞凋亡。Western印迹法检测ERK1/2、CREB以及Bcl-2蛋白表达。RT-PCR法检测Bcl-2的mRNA表达。结果:bFGF可抑制无血清饥饿诱导的细胞凋亡;时效依赖性地诱导ERK1/2活性增高、刺激CREB磷酸化、促进Bcl-2的mRNA及蛋白表达增加。bFGF作用CAOV3细胞15 min时ERK1/2活性达高峰;45 min时CREB磷酸化达峰值;Bcl-2的mREN表达高峰为6h,8h时蛋白表达最高。MEK1抑制剂PD98059可抑制bFGF的上述作用。结论:bFGF可能通过激活MEK/ERK1/2/CREB/Bcl-2信号转导途径抑制卵巢癌CAOV3细胞凋亡。     

GDF15表达下调促进人胶质母细胞瘤细胞系U87MG增殖

目的探讨生长分化因子15(GDF15)表达下调对人胶质母细胞瘤U87MG细胞系增殖的影响。方法选取稳定下调GDF15的人胶质母细胞瘤U87MG细胞作为shGDF15组,以scramble细胞作为对照组。Western blot检测GDF15蛋白表达;增殖曲线和BrdU掺入实验检测细胞增殖;Western blot检测ERK1/2和p-ERK1/2蛋白表达;CCK-8实验检测细胞增殖。结果与scramble组相比较,shGDF15组细胞增殖明显加快(P0.05);细胞周期S期DNA合成增加(P0.01);ERK通路激活水平明显增加(P0.05);对化疗药物VM-26的耐受性明显增强(P0.05),并且ERK通路抑制剂可降低GDF15表达下调促进细胞增殖作用。结论GDF15可通过下调ERK通路抑制人胶质母细胞瘤U87MG细胞S期DNA合成及细胞增殖,可作为提高胶质瘤临床化疗敏感性的潜在靶点。     

胰岛素受体亚型改变及相关通路活化在糖尿病小鼠肠上皮细胞过度增殖中的作用

 目的:探讨胰岛素受体亚型改变及其相关下游通路活化情况在糖尿病小鼠肠上皮细胞异常增殖中的作用。方法:用腹腔注射链脲霉素的方法制作糖尿病小鼠模型,采用增殖细胞核抗原标记法比较糖尿病小鼠及对照组小鼠肠上皮细胞的增殖情况。利用RT-PCR法测定胰岛素受体亚型表达比例在2组中的差异。采用real-time PCR及Western blot法分别从mRNA和蛋白质水平检测2组之间胰岛素受体相关通路各分子MEK1/2、ERK1/2、PI3K以及Akt的表达情况。结果:糖尿病组小鼠的小肠上皮细胞增殖指数显著升高(P<0.05),且细胞中胰岛素受体亚型IR-A/IR-B的比值也明显升高(P<0.05)。糖尿病小鼠肠上皮细胞中MEK1、MEK2和ERK1/2的mRNA水平及磷酸化蛋白水平均高于对照组(P<0.05)。结论:糖尿病小鼠肠上皮细胞过度增殖可能与其中胰岛素受体亚型IR-A/IR-B的比值增高及其相关MEK/ERK通路的激活有关。     

Claudin-3调节MEK/ERK信号通路对紫杉醇耐药卵巢癌细胞增殖、迁移和侵袭的影响

目的 探讨紧密连接蛋白3(Claudin-3)调节MEK/ERK信号通路对紫杉醇（PTX）耐药卵巢癌（OC）细胞增殖、迁移和侵袭的影响。方法 检测Ovcar-3、Ovcar-3/PTX及IOSE80细胞中Claudin-3表达；将PTX耐药Ovcar细胞（Ovcar-3/PTX）随机分为对照组（Control）、阴性对照组（sh-NC）、转染质粒组（sh-Claudin-3）、MEK/ERK激活剂组（C16-PAF）、sh-Claudin-3+C16-PAF组，分别检测细胞增殖、迁移、侵袭和E-cadherin、N-cadherin、Vimentin及p-MEK、MEK、p-ERK、ERK蛋白表达。结果 Claudin-3在Ovcar-3/PTX细胞中的表达显著高于IOSE80与Ovcar-3(P<0.05)；不同浓度的PTX均降低细胞存活率，且sh-Claudin-3组IC50显著低于sh-NC组（P<0.05）；与sh-NC组比较，sh-Claudin-3组细胞克隆数、迁移、侵袭、N-cadherin、Vimentin、p-MEK/EMK、p-ERK/ERK显著降低，E...     

环氧合酶抑制剂NS-398增强放射诱导的肝癌细胞凋亡

目的：探讨环氧合酶-2（COX-2）选择性抑制剂NS-398对放射诱导的人肝癌细胞HepG2凋亡的影响及可能作用机制。 方法:应用MTT 法检测NS-398对细胞的抑制率;透射电子显微镜观察细胞凋亡的形态学变化,流式细胞术(FCM)定量检测细胞凋亡;实时荧光定量PCR检测凋亡相关基因bcl-2、bax及caspase-3 mRNA表达;Western blotting检测Bc1-2、Bax蛋白表达;比色法分析caspase-3酶活性变化。 结果:NS-398对HepG2细胞的生长抑制作用呈时间与剂量依赖性;电镜下处理组细胞呈现典型的凋亡形态学变化,NS-398显著增加放射诱导的细胞凋亡,上调bax mRNA、Bax蛋白及caspase-3mRNA 表达,并增强caspase-3酶活性,而Bcl-2 表达无明显变化（P>0.05）。 结论:NS-398能增加放射诱导的HepG2细胞凋亡,其机制可能与上调Bax、 caspase-3表达,上升Bax／Bcl-2比例,激活线粒体凋亡通路,活化caspase-3,最终诱导细胞凋亡相关。     

ERK和Smad通路在TGF-β1抑制大鼠血管平滑肌细胞增殖中的作用

目的： 探讨ERK通路是否参与TGF-β/Smad 通路诱导的血管平滑肌细胞增殖及可能的分子机制。方法: 原代培养大鼠胸主动脉平滑肌细胞,细胞分4组：(1)对照组;(2)TGF-β1组;(3)ERK阻断剂(PD98059)组;(4)TGF-β1+ ERK阻断剂(PD98059)组。MTT法测细胞的增殖活性,Western blotting法检测VSMCs中细胞内核增殖抗原(PCNA)、Smad2/3、ERK1/2、Smad7蛋白表达及相关磷酸化蛋白含量,RT-PCR方法测VSMCs中Smad2、3、7mRNA的表达。结果: (1)各组PCNA蛋白表达和MTT法测得A值均低于对照组 (P＜0.05),TGF-β1+ ERK阻断剂组与TGF-β1组相比无显著差异。（2）与对照组相比,TGF-β1组p-Smad2/3、p-ERK1/2蛋白含量和Smad7蛋白表达增高 (P<0.05),ERK阻断剂组则明显降低(P<0.05);与TGF-β1组比,TGF-β1+ERK阻断剂组降低 (P<0.05)。各组间Smad2/3、ERK1/2蛋白表达无显著差异。(3)与对照组相比,TGF-β1组Smad7 mRNA表达增高 (P<0.05),ERK阻断剂组和TGF-β1+ERK阻断剂组降低(P<0.05);与TGF-β1组比,TGF-β1+ERK阻断剂组表达降低(P<0.05)。各组内VSMCs 的Smad2、Smad3 mRNA表达无显著差异。结论: (1)TGF-β1诱导的Smad2/3蛋白磷酸化依赖ERK通路激活,但对TGF-β1的抑制平滑肌细胞增殖的作用无影响,可能有其它信号通路参与此过程。(2)ERK通路可通过蛋白和mRNA水平促进Smad7表达,反过来其又可促进ERK通路激活。(3)ERK通路对Smad2/3蛋白和mRNA水平无影响。     

阻断PD-L1在增强奥沙利铂治疗原发性肝细胞肝癌疗效中的实验研究

目的初步探讨阻断程序性死亡-配体1(programmed death-ligand 1,PD-L1)在增强奥沙利铂(oxaliplatin,L-OHP)治疗原发性肝细胞肝癌(hepatocellular carcinoma,HCC)疗效中的作用及其分子机制。方法 L-OHP处理人HCC细胞系HepG2后,q RT-PCR和Western blot检测PD-L1在m RNA及蛋白水平的表达变化,以及细胞外调节蛋白激酶1/2(extracellular regulated protein kinases,ERK1/2)磷酸化水平的改变;建立Hepa1-6细胞C57BL/6J小鼠HCC移植瘤模型,随机分为对照组、L-OHP组、PD-L1单抗组、L-OHP+PD-L1单抗组,计算处理后各组小鼠肿瘤的质量、抑制率和生存时间;体外刺激或抑制HepG2细胞系ERK1/2的磷酸化,L-OHP处理后q RT-PCR和Western blot检测PD-L1的表达变化。结果 L-OHP能显著抑制HepG2细胞系的生长,其IC50分别为20.94 mg/L;L-OHP处理HepG2细胞后,p-ERK1/2和PD-L1的表达量均高于对照组(P0.05)。小鼠HCC移植瘤模型中,与对照组、L-OHP组和PD-L1单抗组相比较,L-OHP+PD-L1单抗组的肿瘤质量显著减小(P0.05),肿瘤抑制率大幅提升(P0.05),生存时间明显延长(P0.05)。体外刺激ERK1/2的磷酸化后,PD-L1表达显著上调,而抑制HCC细胞株ERK1/2的磷酸化后,L-OHP诱导PD-L1表达升高效应明显受到抑制。结论 L-OHP能通过提高ERK1/2磷酸化水平上调HCC细胞的PD-L1表达,从而导致治疗失败,而联合PD-L1单抗可显著增强L-OHP的抗HCC作用。     

GCS通过MEK/ERK通路调控白血病多药耐药细胞凋亡相关基因bcl-2的表达

目的:探讨葡萄糖神经酰胺合成酶(GCS)是否通过MEK/ERK信号通路调控凋亡相关基因bcl-2的表达,从而诱导人白血病K562/A02细胞多药耐药。方法:用小干扰RNA(siRNA)靶向干扰K562/A02细胞中GCS的表达,real-time PCR、Western blotting检测Bcl-2、磷酸化及总ERK水平;用MEK特异性化学抑制剂U0126抑制MEK/ERK信号通路的活化,real-time PCR与Western blotting技术分别检测Bcl-2 mRNA与蛋白水平;CCK-8试剂盒检测细胞存活情况。结果:与阴性对照组比较,GCS siRNA明显抑制K562/A02细胞GCS和Bcl-2的表达,并抑制MEK/ERK信号通路的活化;U0126使Bcl-2 mRNA及蛋白水平呈浓度依赖性下降,并使K562/A02细胞ADM敏感性增加。结论:GCS通过MEK/ERK信号通路调控K562/A02细胞株中凋亡相关基因bcl-2的表达,从而诱导白血病细胞多药耐药。     

miR-203a-3p抑制人肝癌细胞系增殖

目的 探索miR-203a-3p对人肝癌细胞系增殖和凋亡的分子调节机制。方法 RT-qPCR检测临床肝癌(HCC)组织及肝癌细胞系中miR-203a-3p的表达;建立miR-203a-3p过表达的HepG2细胞模型;MTT法检测细胞增殖;流式细胞测量术检测细胞凋亡;平板集落形成实验检测集落形成;细胞划痕实验检测细胞迁移;表达谱芯片检测mRNA水平;ELISA检测转化生长因子-β1(TGF-β1)的分泌;Western blot检测TGF-β1表达。结果 miR-203a-3p在HCC组织中低表达(P<0.05),与肿瘤分期呈负相关(P<0.05);HepG2细胞中过表达miR-203a-3p可显著降低细胞增殖速率(P<0.05)、迁移能力(P<0.05)、克隆形成能力(P<0.05),促进细胞凋亡(P<0.05);TGF-β1表达降低(P<0.05),而TGFβR1表达升高(P<0.05);细胞培养上清中TGF-β1含量降低(P<0.05);细胞内TGF-β1的蛋白降低(P<0.05);加入TGF-β1重组蛋白可减轻miR-2...     

索拉非尼对敏感人肝癌细胞株MAPK信号通路基因表达的影响

 目的：筛选对索拉非尼敏感的人肝癌细胞株,检测索拉非尼作用于敏感细胞株后MAPK信号通路基因表达谱的变化。方法：索拉非尼作用于人肝癌细胞株Huh7、 MHCC97H、 HepG2 、SMCC7721、 HepG2.2.15和PLC,流式细胞术检测肝癌细胞凋亡,CCK-8测定细胞增殖的抑制率,筛选敏感肝癌细胞株;采用实时定量PCR基因芯片,观察索拉非尼作用于敏感细胞株前后MAPK信号通路基因的表达。结果：索拉非尼作用后,流式细胞术的结果显示,凋亡较明显的细胞株为PLC和HepG2.2.15,相对不明显的细胞株为SMCC7721和MHCC97H;CCK-8测定索拉非尼对PLC、HepG2.2.15、Huh7、HepG2、MHCC97H和SMCC7721细胞的IC50值分别为5.25 μmol/L、5.30 μmol/L、6.80 μmol/L、7.01 μmol/L、11.7 μmol/L和15.0 μmol/L。对索拉非尼相对敏感细胞株和不敏感细胞株分别为PLC和SMCC7721。索拉非尼作用敏感细胞株PLC后,MAPK信号通路表达 >2.0倍的有2个,≤0.5倍的有6个。结论：PLC为对索拉非尼相对敏感的人肝癌细胞株;索拉非尼主要导致MAPK信号通路中与调控细胞周期和活化转录因子相关的基因表达发生变化。     

MicroRNA-92b augments sorafenib resistance in hepatocellular carcinoma via targeting PTEN to activate PI3K/AKT/mTOR signaling

Sorafenib (SOR) resistance is still a significant challenge for the effective treatment of hepatocellular carcinoma (HCC). The mechanism of sorafenib resistance remains unclear. Several microRNAs (miRNAs) have been identified as playing a role in impairing the sensitivity of tumor cells to treatment. We examined the mechanism behind the role of miR-92b in mediating sorafenib resistance in HCC cells. We detected that miR-92b expression was significantly upregulated in SOR-resistant HepG2/SOR cells compared to parental HepG2/WT cells. After transfection with miR-92b inhibitor, the proliferation of HepG2/SOR cells was remarkably weakened and rates of apoptosis significantly increased. PTEN was considered to be a functional target of miR-92b according to a luciferase reporter assay. Knockdown of PTEN significantly impaired the ability of miR-92b inhibitor on increasing sorafenib sensitivity of HepG2/SOR cells. Furthermore, we confirmed by western blotting and immunofluorescence that miR-92b can mediate sorafenib resistance by activating the PI3K/AKT/mTOR pathway in HCC cells by directly targeting PTEN. These findings further validate the mechanism of miR-92b in SOR resistance in HCC treatment.     

LY3214996 relieves acquired resistance to sorafenib in hepatocellular carcinoma cells

Background: Sorafenib, an oral multi-kinase inhibitor of rapidly accelerated fibrosarcoma; vascular endothelial growth factor receptor-2/3, platelet-derived growth factor receptor, c-Kit, and Flt-3 signaling, is approved for treatment of advanced hepatocellular carcinoma (HCC). However, the benefit of sorafenib is often diminished because of acquired resistance through the reactivation of ERK signaling in sorafenib-resistant HCC cells. In this work, we investigated whether adding LY3214996, a selective ERK1/2 inhibitor, to sorafenib would increase the anti-tumor effectiveness of sorafenib to HCC cells.Methods: The Huh7 cell line was used as a cell model for treatment with sorafenib, LY3214996, and their combination. Phosphorylation of the key kinases in the Ras/Raf/MAPK and PI3K/Akt pathways, protein expression of the cell cycle, and apoptosis migration were assessed with western blot. MTT and colony-formation assays were used to evaluate cell proliferation. Wound-healing assay was used to assess cell migration. Cell cycle and apoptosis analyses were conducted with flow cytometry.Results: LY3214996 decreased phosphorylation of the Ras/Raf/MAPK and PI3K/Akt pathways, including p-c-Raf, p-P90RSK, p-S6K and p-eIF4EBP1 activated by sorafenib, despite increased p-ERK1/2 levels. LY3214996 increased the anti-proliferation, anti-migration, cell-cycle progression, and pro-apoptotic effects of sorafenib on Huh7^R cells.Conclusions: Reactivation of ERK1/2 appears to be a molecular mechanism of acquired resistance of HCC to sorafenib. LY3214996 combined with sorafenib enhanced the anti-tumor effects of sorafenib in HCC. These findings form a theoretical basis for trial of LY3214996 combined with sorafenib as second-line treatment of sorafenib-resistant in advanced HCC.     

15-deoxy-delta-12-14-PGJ2 regulates apoptosis induction and nuclear factor-kappaB activation via a peroxisome proliferator-activated receptor-gamma-independent mechanism in hepatocellular carcinoma

The peroxisome proliferator-activated receptor-gamma (PPARgamma) high-affinity ligand, 15-deoxy-Delta-12,14-PGJ(2) (15d-PGJ(2)), is toxic to malignant cells through cell cycle arrest and apoptosis induction. In this study, we investigated the effects of 15d-PGJ(2) on apoptosis induction and expression of apoptosis-related proteins in hepatocellular carcinoma (HCC) cells. 15d-PGJ(2) induced apoptosis in SK-Hep1 and HepG2 cells at a 50 micro M concentration. Pretreatment with the pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (2-VAD-fmk), only partially blocked apoptosis induced by 40 micro M 15d-PGJ(2). This indicated that 15d-PGJ(2) induction of apoptosis was associated with a caspase-3-independent pathway. 15d-PGJ(2) also induced down-regulation of the X chromosome-linked inhibitor of apoptosis (XIAP), Bclx, and apoptotic protease-activating factor-1 in SK-Hep1 cells but not in HepG2 cells. However, 15d-PGJ(2) sensitized both HCC cell lines to TNF-related apoptosis-induced ligand-induced apoptosis. In SK-Hep1 cells, cell toxicity, nuclear factor-kappaB (NF-kappaB) suppression, and XIAP down-regulation were induced by 15d-PGJ(2) treatment under conditions in which PPARgamma was down-regulated. These results suggest that the effect of 15d-PGJ(2) was through a PPARgamma-independent mechanism. Although cell toxicity was induced when PPARgamma was down-regulated in HepG2 cells, NF-kappaB suppression and XIAP down-regulation were not induced. In conclusion, 15d-PGJ(2) induces apoptosis of HCC cell lines via caspase-dependent and -independent pathways. In SK-Hep1 cells, the ability of 15d-PGJ(2) to induce cell toxicity, NF-kappaB suppression, or XIAP down-regulation seemed to occur via a PPARgamma-independent mechanism, but in HepG2 cells, NF-kappaB suppression by 15d-PGJ(2) was dependent on PPARgamma.     

miR-634 在肝癌中的表达及对肝癌细胞生物学行为的影响

目的:检测microRNA-634(miR-634)在肝癌中的表达水平及其对肝癌细胞常见生物学行为的调控作用。方法:采用实时荧光定量PCR(RT鄄qPCR)法检测肝癌细胞系(HepG2、SMMC7721、Bel7402、Bel7404、SNU739)、69 例肝癌组织及匹配癌旁组织中miR-634 的相对定量,分析miR-634 表达与肝癌患者性别、年龄、肿瘤直径、分化程度、Child-Pugh 分级、BCLC 分期、门静脉癌栓及肝外转移的关系,同时构建miR-634 的真核表达载体并转染肝癌细胞系,采用活细胞计数试剂盒CCK-8、流式细胞仪Annexin V/ PI 双染法和Transwell 侵袭实验检测转染miR鄄634 对细胞增殖、凋亡和侵袭能力的影响。结果:与正常人肝细胞系L-02 相比,肝癌细胞的miR-634 水平均降低(P <0.05),表达量依次为HepG2 >SNU739 >Bel7402 > Bel7404 >SMMC7721;69 例肝癌组织的miR鄄634 水平为(0.253±0.019),低于匹配癌旁组织(P<0.05),且与肿瘤直径、分化程度、BCLC分期、门静脉癌栓及肝外转移均有关(P<0.05)。过表达组转染24 ~96 h 后的miR鄄634 水平持续升高,与对照组和空转染组的差异有统计学意义(P<0.05);与对照组和空转染组相比,转染组的增殖抑制率、凋亡率均升高,但穿膜细胞数降低,差异有统计学意义(P<0.05)。结论:miR-634 在肝癌组织和细胞中均为低表达,且与临床病理参数有关,上调其水平可抑制肝癌细胞增殖及侵袭并诱导凋亡,对于肝癌防治有重要借鉴价值。     

RNAi screening with shRNAs against histone methylation-related genes reveals determinants of sorafenib sensitivity in hepatocellular carcinoma cells

Sorafenib is the first drug currently approved to treat advanced hepatocellular carcinoma (HCC). However, very low response rate and acquired drug resistance makes rare patients benefit from sorafenib therapy, therefore it is urgent to find biomarkers for sorafenib sensitivity. Histone modifications, including histone methylation, have been demonstrated to influence the initiation and progression of HCC. It is of great interest to elicit the possibility whether histone methylation plays a role in regulation of sorafenib sensitivity. In present work, a high throughput RNAi screening with 176 shRNA pools against 88 histone methyltransferases (HMTs) and histone demethyltransferases genes was applied to HepG2 cells. Silencing of 3 genes (ASH1L, C17ORF49 and SETD4) was validated to specifically promote HepG2 cells sensitivity to sorafenib. Western blotting results showed that those 3 HMT genes knockdown alone or sorafenib treatments alone both induce AKT/ERK activation. However, combination treatment with sorafenib and silencing of C17ORF49 or SETD4 downregulated AKT phosphorylation and hence induced HCC cells death. Our work may provide potential biomarkers for sorafenib sensitivity and therapeutic combination for sorafenib treatment in HCC patients.     

MicroRNA-539靶向调控E2F转录因子3抑制肝癌的发生与发展

目的 探讨肝癌组织中microRNA-539（miR-539）表达水平及其抑制肝癌细胞增殖的作用机制。方法 收集90例肝癌患者术后肝癌组织及癌旁组织标本,用实时定量聚合酶链反应（Real-time PCR）检测组织中miR-539表达量。在PCL/PRF5、Huh7和QGY-7701肝癌细胞中转染miR-539 模拟物（mimics）,用Real-time PCR检测miR-539表达量。用MTT法检测miR-539对肝癌细胞增殖的影响;用流式细胞仪检测miR-539对肝癌细胞凋亡的影响;用免疫印迹法检测miR-539对肝癌细胞中E2F转录因子3（E2F3）蛋白表达的影响。结果 肝癌组织中miR-539表达量显著低于癌旁组织（P<0.05）;miR-539表达量与肝癌患者性别、年龄及肝癌组织分化程度无关（P>0.05）;与肿瘤直径和淋巴结转移相关（P<0.05）。转染miR-539 mimics组肝癌细胞中miR-539表达量显著高于空白组和miR-539 NC组（P<0.05）。在QGY-7701细胞中,过表达miR-539能显著抑制细胞增殖（P<0.05）,降低E2F3蛋白的表达（P<0.05）,对细胞凋亡无明显作用（P>0.05）。结论 miR-539是肝癌发生中的抑癌因子,可能是通过靶向下调E2F3蛋白水平达到抑制癌细胞的增殖。     

CHOP在索拉菲尼联合辛二酰苯胺异羟肟酸诱导人肝癌细胞MHCC97L凋亡中的作用

目的:探讨CCAAT/增强子结合蛋白同源蛋白(CHOP)在索拉菲尼联合辛二酰苯胺异羟肟酸(SAHA)诱导人肝癌细胞MHCC97L凋亡中的作用。方法:采用CCK-8法检测索拉菲尼联合SAHA对MHCC97L细胞活力的影响;采用流式细胞术检测索拉菲尼联合SAHA对细胞周期及细胞凋亡的影响;应用Western blot法检测索拉菲尼联合SAHA对MHCC97L细胞中内质网应激相关分子葡萄糖调节蛋白78(GRP78)、蛋白激酶R样内质网激酶(PERK)、p-PERK、活化转录因子4(ATF4)及CHOP蛋白表达的影响。此外,应用CHOP siRNA沉默CHOP后,采用流式细胞术观察索拉菲尼联合SAHA对MHCC97L细胞凋亡的影响。结果:索拉菲尼联合SAHA可使MHCC97L细胞活力明显下降(P<0.05),细胞周期结果显示细胞生长被阻滞在G 1期;Western blot结果显示索拉菲尼联合SAHA可显著上调上述内质网应激凋亡通路中相关蛋白的表达,同时流式细胞术检测发现索拉菲尼联合SAHA可显著促进MHCC97L细胞凋亡(P<0.01);CHOP siRNA能显著抑制索拉菲尼联合SAHA诱导的细胞凋亡。结论:索拉菲尼联合SAHA可显著抑制MHCC97L细胞生长并诱导其凋亡,机制可能与上调内质网应激凋亡通路中CHOP的表达有关。     

CRIPTO promotes an aggressive tumour phenotype and resistance to treatment in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the third leading cause of cancer‐related death worldwide. Despite increasing treatment options for this disease, prognosis remains poor. CRIPTO (TDGF1) protein is expressed at high levels in several human tumours and promotes oncogenic phenotype. Its expression has been correlated to poor prognosis in HCC. In this study, we aimed to elucidate the basis for the effects of CRIPTO in HCC. We investigated CRIPTO expression levels in three cohorts of clinical cirrhotic and HCC specimens. We addressed the role of CRIPTO in hepatic tumourigenesis using Cre‐loxP‐controlled lentiviral vectors expressing CRIPTO in cell line‐derived xenografts. Responses to standard treatments (sorafenib, doxorubicin) were assessed directly on xenograft‐derived ex vivo tumour slices. CRIPTO‐overexpressing patient‐derived xenografts were established and used for ex vivo drug response assays. The effects of sorafenib and doxorubicin treatment in combination with a CRIPTO pathway inhibitor were tested in ex vivo cultures of xenograft models and 3D cultures. CRIPTO protein was found highly expressed in human cirrhosis and hepatocellular carcinoma specimens but not in those of healthy participants. Stable overexpression of CRIPTO in human HepG2 cells caused epithelial‐to‐mesenchymal transition, increased expression of cancer stem cell markers, and enhanced cell proliferation and migration. HepG2‐CRIPTO cells formed tumours when injected into immune‐compromised mice, whereas HepG2 cells lacking stable CRIPTO overexpression did not. High‐level CRIPTO expression in xenograft models was associated with resistance to sorafenib, which could be modulated using a CRIPTO pathway inhibitor in ex vivo tumour slices. Our data suggest that a subgroup of CRIPTO‐expressing HCC patients may benefit from a combinatorial treatment scheme and that sorafenib resistance may be circumvented by inhibition of the CRIPTO pathway. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.