lncRNA在肝细胞癌索拉非尼耐药中的最新研究进展

肝细胞癌(Hepatocellular carcinoma,HCC)是全球最常见和最致命的癌症之一。大多数中晚期患者接受药物为主的治疗,索拉非尼(Sorafenib)作为晚期肝细胞癌的独特靶向药物,在临床上得到了广泛应用。然而,索拉非尼靶向治疗不仅单药有效率低,且耐药性也是亟待解决的大问题。最近研究显示长链非编码RNA(Long non-coding RNA,lncRNA)与肝细胞癌的发生发展、侵袭转移、预后及耐药有着密不可分的联系,并且在耐药研究方面成为新兴热点。本文就lncRNA在肝细胞癌索拉菲尼耐药中的最新发现及调控耐药性的具体机制展开综述。     

CA3诱导肝癌HepG2细胞凋亡及其机制CSCD

目的探讨CA3(CIL56)对肝癌HepG2细胞增殖及凋亡的影响及其机制。方法体外培养HepG2细胞,分别加入不同浓度CA3(0、0.25、0.5、1.0、2.0和4.0 mmol/L)作用24和48 h。增强型CCK-8试剂检测不同浓度CA3对细胞增殖的影响;流式细胞术检测细胞凋亡率;检测细胞内活性氧(ROS)的变化;Western blot法分析YAP1、Bcl-2、Bax、Caspase-3蛋白的表达。结果增强型CCK-8试剂检测结果显示,0.25、0.5、1.0、2.0、4.0 mmol/L的CA3作用HepG2细胞24和48 h后,细胞增殖率分别为(80.5±0.3)%、(79.4±0.2)%、(76.2±0.2)%、(76.4±0.1)%、(49.3±0.4)%和(75.3±0.2)%、(64.8±0.3)%、(48.4±0.2)%、(32.2±0.4)%、(31.9±0.2)%。流式细胞术结果显示,CA3浓度升高至2 mmol/L时,凋亡率增加到58.48%。CA3处理后,HepG2细胞内活性氧产量增加,YAP1、Bcl-2蛋白表达降低,Bax、Caspase-3表达增高。结论 CA3可抑制HepG2细胞增殖并诱导凋亡,其机制可能与细胞内活性氧产量增加,调节YAP1、Bcl-2、Bax及Caspase-3蛋白的表达有关。     

舒尼替尼促进人肝癌细胞HepG2的凋亡及其分子机制

目的：探讨苹果酸舒尼替尼对人肝癌HepG2细胞凋亡的作用及其机制。方法：常规体外培养HepG2细胞,利用MTT法检测舒尼替尼杀伤HepG2细胞的IC50,Western blotting 检测药物处理前后HepG2细胞分子靶点蛋白表达,膜联蛋白(Annexin-V)/碘化丙啶(PI)双标法和TUNEL染色法检测舒尼替尼处理前后HepG2细胞凋亡情况,实时荧光定量PCR检测药物处理前后HepG2细胞凋亡基因 mRNA的表达情况。结果：舒尼替尼杀伤HepG2细胞的IC50值为（3.22±0.50）μmol/L。以对HepG2细胞无明显抑制作用的剂量1 μmol/L舒尼替尼处理HepG2细胞后,细胞内VEGFR1、VEGFR2、PDGFRα、Kit、FLT3蛋白表达均有不同水平下降（均P<0.05）,HepG2细胞的凋亡率\[(15.18±1.28)% vs (5.90±0.45)%,P<0.05\]、凋亡指数（AI）\[(23.54±4.73) vs (4.17±0.64), P<0.05\]均显著升高。舒尼替尼处理HepG2细胞后,上调促凋亡基因Bax、NOXA、PUMA、P53 mRNA表达水平（均P<0.05),降低抑凋亡基因Bcl-2、X-IAP mRNA表达水平（均P<0.05）。结论： 舒尼替尼能够诱导肝癌HepG2细胞凋亡,其机制可能是通过上调促凋亡基因的表达及降低抑凋亡基因表达水平来实现的。     

lncRNA ZFAS1通过miR-588/HMGA2轴促进肝癌HepG2细胞的增殖、侵袭和迁移

目的：探讨长链非编码RNA锌指结构反义转录本1(lncRNA ZFAS1)调控miR-588/高迁移率蛋白A2(HMGA2)轴对肝癌HepG2细胞增殖、侵袭、迁移的影响。方法：qPCR、WB法检测80例肝癌组织及对应癌旁组织（2018年1月至2019年12月在武汉第三医院首义院区手术切除标本）及人正常肝LO2细胞和肝癌HepG2、Huh7、HCCLM3细胞中ZFAS1、miR-588及HMGA2表达水平;采用Kaplan-Meier进行患者生存曲线分析。将HepG2细胞分为空白组、si-NC组、si-ZFAS1组、si-ZFAS1+inhibitor NC组、si-ZFAS1+miR-588 inhibitor组;qPCR检测各组HepG2细胞中ZFAS1、miR-588表达水平,WB法检测各组HepG2细胞中HMGA2蛋白表达;CCK-8法、Transwell和划痕实验分别检测各组HepG2细胞的增殖、侵袭和迁移能力;双荧光素酶报告基因实验分别验证ZFAS1和miR-588、miR-588和HMGA2的靶向关系。用HepG2细胞移植瘤裸鼠模型检测敲减ZFAS1或/和miR-588对移...     

番茄红素对人肝癌HepG2细胞增殖和凋亡的影响

目的 研究番茄红素(Lycopene,LP)对人肝癌HepG2细胞生长的影响并初探其机制。方法取对数生长期HepG2细胞设空白对照组、LP(5 μg/mL)组、LP(10 μg/mL)组、LP(20 μg/mL)组和顺铂(40 μg/mL)组,每组设10个复孔;各组分别给药干预48 h后,噻唑蓝(MTT)比色法测定细胞增殖抑制率,流式细胞术(FCM)检测细胞周期和细胞凋亡状况,RT-PCR法检测Bax mRNA和Bcl-2 mRNA表达,Western blot法检测Caspase-3蛋白表达。结果 与空白对照组比较,经LP(10 μg/mL、20 μg/mL)或顺铂40 μg/mL干预48 h能够显著提高HepG2细胞增殖抑制率,延长细胞周期中G₀/G₁期而缩短G₂/M期,提高细胞凋亡率,上调促凋亡Bax mRNA表达并下调抑凋亡Bcl-2 mRNA表达,提高Bax/Bcl-2比值,上调Caspase-3蛋白表达,LP上述作用具有一定的剂量依赖性。结论 LP具有抑制人肝癌HepG2细胞增殖并促进其凋亡的作用,其机制可能与LP干预细胞周期分布和调节凋亡相关基因蛋白表达有关。     

lncRNA SNHG5在缺氧诱导肝细胞癌细胞侵袭及迁移中的作用

目的：探讨长链非编码RNA（long non-coding RNA, lncRNA）SNHG5在缺氧诱导肝细胞癌（hepatocellular carcinoma,HCC）细胞侵袭及迁移中的作用。方法：收集2017年1月至2018年6月西安交通大学第一附属医院手术切除的20例HCC患者的癌与癌旁组织标本,以及人HCC细胞系HepG2、MHCC-97L、MHCC-97H、Huh7和永生化人肝细胞LO2。利用生物信息学方法分析缺氧诱导因子1α（hypoxia-inducible factor 1α,HIF-1α）与SNHG5的结合位点,将pCMV-HIF-1α、shRNA-SNHG5（sh-SNHG5）质粒转染进HCC细胞,用qPCR法检测HCC组织和缺氧条件下HCC细胞中SNHG5的表达水平,用Western botting检测HCC细胞中HIF-1α蛋白的表达水平,用Transwell小室法检测沉默SNHG5后常氧和缺氧条件下对HCC细胞侵袭及迁移能力的影响。结果：分别与癌旁组织和LO2细胞比较,HCC组织和各细胞系中SNHG5表达水平显著上调（均P<0.01）。缺氧可上调HCC细胞中SNHG5的表达水平,其机制可能与缺氧活化HIF-1α 与 SNHG5 启动子结合从而促进其转录有关 ;缺氧能够增强 HepG2和 MHCC-97L 细胞的侵袭及迁移能力（均P<0.01）,沉默SNHG5表达能够显著抑制缺氧条件下HepG2和MHCC-97L细胞的侵袭及迁移能力（均 P<0.01）。结论：SNHG5在HCC组织及细胞系中高表达,并在缺氧诱导的HCC细胞侵袭及迁移中发挥重要作用。     

三阴性乳腺癌血清外泌体和组织中SUMO1P3表达及临床意义

目的 探讨三阴性乳腺癌(TNBC)血清外泌体、组织中长链非编码RNA(lncRNA)小泛素样修饰子1伪基因3(SUMO1P3)表达对TNBC患者预后的影响.方法 选取我院2015年1月至2016年3月期间91例TNBC患者、100例非TN-BC乳腺癌患者、50例乳腺良性疾病患者、50例女性健康志愿者进行回顾性分析.比较...     

CAAP1抑制肝癌HepG2细胞凋亡而促进其增殖、迁移和侵袭

目的:探讨CAAP1对肝癌HepG2细胞凋亡、增殖、迁移和侵袭的影响及其作用机制.方法:构建CAAP1过表达载体pcDNA3/CAAP1和敲降载体pSilencer 2.l-U6 neo/shR-CAAPl,转染肝癌HepG2细胞后,qPCR和WB实验分别检测CAAP1mRNA和蛋白的表达水平.实验分为过表达对照组(p...     

Lin28 对肝癌HepG2 细胞5-Fu 敏感性的影响及其分子机制

目的：探讨RNA结合蛋白Lin28 对肝癌HepG2细胞5-氟尿嘧啶（5-Fu）敏感性的影响及其机制。方法：应用pcDNA3.1-Lin28 或si-Lin28 转染HepG2 细胞,采用qPCR及WB法检测转染后HepG2 细胞Lin28 的表达水平,CCK-8 法检测5-Fu 作用后转染细胞增殖活性变化并计算5-Fu 对细胞作用的IC50,流式细胞术检测5-Fu 处理后细胞凋亡情况、WB法检测凋亡相关蛋白的表达变化,qPCR法检测转染后耐药相关miRNA（let-7a 和let-7b）及肿瘤干细胞标记物（Oct4、Nanog和Sox2）的mRNA表达水平。结果：与空载对照组（HepG2/Vector）相比,HepG2/Lin28 组细胞中Lin28 mRNA和蛋白的表达水平均显著升高（P<0.05 或P<0.01）,过表达Lin28 可显著抑制HepG2 细胞对5-Fu 作用的敏感性（IC50明显升高,P<0.05）和增强细胞增殖活性、使细胞凋亡率和凋亡相关蛋白caspase-3 表达明显降低（均P<0.01）。与阴性对照组（si-control）相比,si-Lin28 细胞中Lin28 表达水平显著下降（P<0.01）;敲减Lin28 可使细胞增殖活性及5-Fu 的IC50显著降低（均P<0.01）,凋亡率及凋亡蛋白表达明显增加（P<0.01）。与HepG2/Vector 组比较,HepG2/Lin28 细胞中let-7a、let-7b 及肿瘤干细胞标记物（Oct4、Nanog 和Sox2）的表达水平明显上调（均P<0.01）,而si-Lin28 细胞中let-7a、let-7b 及Oct4、Nanog、Sox2 的表达水平较si-control 组明显下调（均P<0.01）。结论：Lin28 可通过调节miRNAs的表达及肿瘤干细胞的形成从而调节肝癌HepG2细胞对化疗的敏感性,靶向调节Lin28 表达有望成为提高肝癌化疗疗效的手段。     

雷公藤红素上调lncRNA MEG3表达抑制肝癌细胞增殖及促进细胞凋亡

目的:探讨MEG3在雷公藤红素诱导的肝癌HepG2细胞凋亡和细胞增殖抑制过程中的作用。方法:采用CCK-8法检测HepG2细胞增殖情况,流式细胞仪检测细胞凋亡情况,Western blot检测cleaved caspase-3、Bax和Bcl-2蛋白的表达,real-time PCR检测MEG3的表达。结果:雷公藤红素抑制HepG2细胞增殖、促进细胞凋亡并上调MEG3的表达;敲低MEG3的表达后显著逆转了雷公藤红素诱导的HepG2细胞增殖抑制和细胞凋亡。结论:雷公藤红素通过上调MEG3的表达抑制HepG2细胞增殖、促进细胞凋亡。     

Inhibiting CBX4 efficiently protects hepatocellular carcinoma cells against sorafenib resistance

Background This study aimed to investigate the possible role of inhibiting chromobox protein homologue 4 (CBX4) to deregulate of cancer stem cells (CSCs) and to evaluate the contribution of these molecules to sorafenib resistance in advanced hepatocellular carcinoma (HCC).Methods HCC cell lines and a xenograft mouse model with resistance to sorafenib were employed to analyse the effects of miR424 on CSC characteristics. RNA expression was analysed by RT-PCR and next-generation sequencing in a cohort of HCC cancer patients and sorafenib-resistant (SR) cell lines, respectively, to validate the key microRNAs and targets in the network.Results MicroRNA and mRNA profiles of SR cell lines identified miR424 and its direct target CBX4 as significantly associated with stem-cell-like properties, poor survival, and clinical characteristics. Functional experiments demonstrated that miR424 suppressed CBX4 and CBX4 induced nuclear translocation of YAP1 protein but was not associated with protein production. When YAP1 and CBX4 were modulated with CA3 and UNC3866, tumorigenicity and stem-like properties were extremely inhibited, thus indicating that these compounds exerted a strong anti-tumour effect in vivo against SR HCC cells.Conclusions Our results revealed that blocking CBX4 expression is critical in response to sorafenib resistance with advanced HCC.Subject terms: Hepatocellular carcinoma, Nuclear receptors     

氯喹通过激活P38MAPK通路诱导人肝癌HepG2细胞凋亡

目的：研究氯喹对人肝癌HepG2细胞增殖和凋亡的影响,并初步研究其可能的机制。方法：体外培养HepG2细胞,采用MTT法检测不同浓度（0、10、20和40 μmol/L）氯喹作用不同时间（24、48和72 h）后对细胞增殖的影响;用DAPI染色观察氯喹处理细胞24 h后细胞核的变化;流式细胞术检测氯喹对HepG2细胞凋亡的影响;Western blot技术检测氯喹对HepG2细胞中Caspase-3、Bcl-2、Bax、P-P53、P38MAPK和P-P38MAPK蛋白表达的影响。结果：与对照组比较,在上述3个时间点10、20、40 μmol/L氯喹（10 μmol/L氯喹作用24 h组除外）对人肝癌HepG2细胞的增殖均有明显抑制作用（P均 < 0.05）;荧光显微镜下发现,10、20、40 μmol/L氯喹处理24 h后均可引起HepG2细胞不同程度的核浓集、固缩等典型凋亡形态变化;流式细胞术结果提示10、20、40 μmol/L氯喹能诱导HepG2细胞凋亡（P均 < 0.05）;Western blot结果显示20和40 μmol/L氯喹作用HepG2细胞后,P-P53、P-P38MAPK、剪切后活化型Caspase-3和Bax蛋白表达量增加（P均 < 0.05）,Bcl-2表达下降,Caspase-3和P38MAPK表达无明显变化（P > 0.05）。结论：氯喹能够抑制人肝癌HepG2细胞的生长并诱导其凋亡,其机制可能与P38MAPK通路有关。     

By inhibiting PFKFB3, aspirin overcomes sorafenib resistance in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the few cancers with a continuous increase in incidence and mortality. Drug resistance is a major problem in the treatment of HCC. In this study, two sorafenib‐resistant HCC cell lines and a nude mouse subcutaneously tumor model were used to explore the possible mechanisms leading to sorafenib resistance, and to investigate whether aspirin could increase the sensitivity of hepatoma cells to sorafenib. The combination of aspirin and sorafenib resulted in a synergistic antitumor effect against liver tumors both in vitro and in vivo. High glycolysis and PFKFB3 overexpression occupied a dominant position in sorafenib resistance, and can be targeted and overcome by aspirin. Aspirin plus sorafenib induced apoptosis in tumors without inducing weight loss, hepatotoxicity or inflammation. Our results suggest that aspirin overcomes sorafenib resistance and their combination may be an effective treatment approach for HCC.     

Inhibition of SUMO2/3 antagonizes isoflurane-induced cancer-promoting effect in hepatocellular carcinoma Hep3B cells

Surgery for patients with complicated liver cancer often results in a long exposure to anesthesia with an increase in side effects. Continued long-term exposure to isoflurane may promote liver cancer progression. Small ubiquitin-like modifier (SUMO) 2 and 3, also known as SUMO2/3, conjugates to substrate proteins when cells undergo acute stress. However, whether or not SUMO2/3 is involved in isoflurane-mediated liver cancer progression is unknown. In the present study, hepatocellular carcinoma (HCC) cells were exposed to 2% isoflurane for 12 h, followed by 36 h of drug withdrawal, and the formation of SUMO2/3 conjugates and cancer behavioral characteristics were studied. The results demonstrated that the formation of SUMO2/3 conjugates was significantly increased following HCC cells being exposed to isoflurane for 0.5 h, and continued to increase for 48 h, even after the drug had been withdrawn. Furthermore, isoflurane-exposed HCC cells exhibited increased proliferation and invasion activity during the subsequent observation period. SUMO specific protease 3 (SENP3), which inhibits the binding of SUMO2/3 to its target proteins, was overexpressed and it was discovered that isoflurane-induced SUMOylation was significantly inhibited, and accordingly, the proliferation and invasion abilities of HCC cells were decreased to a certain extent. These findings indicated that SUMO2/3 is involved in the progression of HCC cells, at least in the Hep3B cell line, induced by the anesthetic isoflurane, and that inhibition of SUMO2/3 may antagonize the response. These results provided a novel target for decreasing the adverse reactions occurring in patients with HCC during anesthesia, particularly those who are exposed to isoflurane for long periods of time.     

HBsAg基因修饰的树突状细胞体外诱导抗 HepG2.2.15特异性细胞毒作用

背景与目的:目前缺少一种针对乙型肝炎病毒感染相关肝癌的有效免疫治疗手段.以树突状细胞( dendritic cell, DC)为基础的肿瘤特异性免疫治疗方法,为免疫治疗乙型肝炎病毒感染相关的肝癌提供了新方法 .本实验通过体外负载乙型肝炎表面抗原基因( HBsAg)的重组质粒转染 DC,制备 HBsAg-DC瘤苗,评价 HBsAg-DC瘤苗体外诱导抗 HepG2.2.15的细胞毒性 T淋巴细胞反应.方法:将已构建含 HBsAg基因的重组质粒 pCR3.1-S转染培养第 5天的 DC; Western blot和免疫荧光法鉴定转染基因表达;以 MTT法测定 DC诱导的抗 HepG2.2.15特异性细胞毒作用.结果:细胞表型鉴定结果显示诱导 5天的 DC CD1a、 CD11c、 CD86、 CD80和 HLA-DR表达量分别为 55.0%、 98.6%、 86.1%、 66.1% 和 88.9%;采用免疫荧光和 Western blot法研究表明 HBsAg基因能在转染的 DC中表达; MTT法检测特异性细胞毒作用结果显示:不同效靶比, pCR3.1-S转染 DC组均显示对 HepG2.2.15肝癌细胞高效特异的杀伤活性,杀伤率分别为:( 52.3± 2.8)%( E∶ T为 5∶ 1)、( 64.6± 2.4)%( 10∶ 1)、( 78.8± 2.6)%( 20∶ 1)、( 82.1± 2.4)%( 40∶ 1),显著高于 pCR3.1-DC 组和 DC组 (P< 0.05, n=4).结论:重组质粒转染的 DC能够有效表达 HBsAg;并在体外诱导出特异性 CTL反应.     

补骨脂酚通过MAPK途径诱导人肝癌HepG2细胞凋亡的研究

目的:研究补骨脂酚对人肝癌细胞凋亡的影响及其作用机制。方法:应用噻唑蓝法（MTT法）和倒置显微镜技术考察补骨脂酚对HepG2细胞生长的影响;采用荧光显微镜观察补骨脂酚处理后细胞凋亡;利用蛋白免疫印迹法检测补骨脂酚对HepG2细胞中凋亡相关蛋白及MAPK家族蛋白表达的影响;引入MAPK家族蛋白抑制剂考察生长抑制率和凋亡相关蛋白表达的变化。结果:补骨脂酚可以剂量依赖性地抑制人肝癌HepG2细胞增殖,其生长抑制作用明显强于临床上常用的抗肿瘤药5-氟尿嘧啶,并且补骨脂酚对人正常肝L02细胞具有较低的毒性。进一步研究发现,补骨脂酚可以诱导HepG2细胞发生凋亡。此外,MAPK家族参与到补骨脂酚诱导的HepG2细胞凋亡过程中,补骨脂酚可以剂量依赖性激活JNK的表达发挥促凋亡的作用,同时抑制了ERK促存活通路,然而对p38无明显影响。结论:本研究首次阐明了补骨脂酚诱导人肝癌HepG2细胞生长抑制作用机制,为进一步的临床应用提供了理论依据。     

Proteome analysis of the effects of sorafenib on human hepatocellular carcinoma cell line HepG2

Sorafenib is a multi-target oral anticancer drug used as first-line treatment for patients with advanced human hepatocellular carcinoma (HCC). But the exact mechanism of sorafenib involved in HCC treatment is not clear yet. In this study, a comparative proteomic approach was performed to identify novel sorafenib-related proteins in HCC. Proteomes of HepG2 cells treated with sorafenib and the control (without sorafenib) were obtained by two-dimensional differential gel electrophoresis. Comprehensive analysis of proteins was focused on total protein spots to filtrate the different protein spots between the two groups. The differentially expressed proteins were identified by peptide mass fingerprinting with high-performance liquid chromatography-tandem mass spectrometry. Then, Western blot and immunohistochemistry were used to verify the expression of some candidate proteins. Results indicated that 19 protein spots were differentially expressed with significant changes, including 6 up-regulated proteins and 13 down-regulated proteins. It was confirmed by Western blot that expressions of Annexin A1 and cyclophilin A were down-regulated in sorafenib-treated HCC cell lines. Immunohistochemical study revealed their oncogenic role in HCC tissues. These observations might be novel findings leading to bring new insights into the exact mechanism of sorafenib and identify possible therapeutic targets.     

索拉非尼逆转肝癌细胞株HepG2/GEM耐药作用的实验研究

目的:探讨索拉非尼能否逆转人肝癌细胞株HepG2/GEM的耐药性及可能机制.方法:以药物大剂量冲击法建立耐药细胞模型HepG2/GEM,MTT法检测索拉非尼对HepG2、HepG2/GEM的抑制率及索拉非尼处理前后2株细胞对GEM敏感性的变化,RT-PCR法检测MDR-1及ERK、VEGFR-2 mRNA水平的变化,蛋白质印迹法检测P-gP、VEG-FR-2蛋白水平的变化.结果:建立了人肝癌HepG2/GEM耐药细胞模型,耐药倍数11.63倍.经1 mg/L的索拉非尼处理后,GEM对HepG2/GEM细胞株的IC50明显下降,逆转倍数为3.08倍,相对逆转率为73.87%,P=0.019.1 mg/L的索拉非尼能够降低HepG2/GEM细胞MDR-1、ERK、VEGFR-2 mRNA表达(P=0.033),并可明显抑制P-gP、VEGFR-2蛋白的表达,P=0.007.结论:经GEM大剂量间断冲击后,HepG2细胞株对GEM产生了耐药性.索拉非尼具有体外部分逆转HepG2/GEM细胞株耐药性的作用.可能与抑制VEGFR-2、ERK1/2信号传导通路,从而下调MDR-1有关.     

干扰RNA对HepG2肝癌细胞内源性c-myc表达的抑制作用

目的 研究干扰RNA(RNAi)对HepG2细胞的c-myc基因表达的干扰效应。方法 建立装载靶向c-mvc基因小干扰性RNA(siRNA)的表达载体。用脂质体法将此表达载体转染HepG2细胞株,以转染空载细胞作为对照。采用定量PCR和Western blot检测c-myc基因的表达。用流式细胞仪及荧光显微镜检测AnexinV标记的凋亡细胞。结果 转染siRNA的表达载体可以抑制内源c-myc基因在转录和转译上的表达,抑制率达67％。c-myc基因的表达抑制与HepG2细胞的凋亡相关联。结论 转染siRNA的表达载体可明显抑制肝癌细胞株HepG2 c-myc基因的表达,并伴有细胞的凋亡。