miR-375 靶向调控YAP表达对肝癌细胞增殖和侵袭能力的影响

[摘要] 目的：探讨miR-375 靶向调控Yes 相关蛋白（Yes-associated protein, YAP）的表达对肝癌细胞增殖和侵袭能力的影响。方法：收集2015 年1 月至2016 年12 月河南中医药大学第二附属医院普外二科行手术切除的70 例肝细胞癌（hepatocellular carcinoma,HCC）患者的癌组织及对应的癌旁组织标本,以及肝癌细胞系SMMC-7721、Hb611、HepG2 和BEL-7405,用qPCR 法检测HCC癌组织和肝癌细胞中miR-375 的表达水平。双荧光素酶报告基因检测miR-375 与YAP的相互作用;分析miR-375 和HCC患者的临床病理特征之间的关系,MTT法检测miR-375 对肝癌细胞增殖能力的影响,Transwell 小室法检测抑制miR-375 表达后HCC细胞侵袭能力的变化;Western blotting 检测HepG2 细胞中YAP的表达。建立裸鼠肝癌皮下移植瘤模型,观察抑制miR-375表达后移植瘤体积和质量的变化,免疫组化法和Western blotting 检测裸鼠移植瘤中YAP的表达情况。结果：HCC组织中miR-375 和YAP表达水平明显高于癌旁组织（均P<0.05）,肝癌HepG2 细胞中miR-375 的表达水平最高（P<0.05）。miR-375 能与YAP的3’UTR特异性结合,调控YAP的表达活性。抑制miR-375 的表达后,HepG2 细胞增殖、侵袭的能力显著减弱（均P<0.05）,裸鼠移植瘤体积和质量都明显减小（均P<0.05）,移植瘤组织细胞中YAP的表达水平相应下调（P<0.05）。结论：miR-375 在肝癌的发生发展过程中起重要作用,可以通过靶向调控YAP的表达影响肝癌细胞的恶性生物学行为。     

血清miR-375、miR-155与原发性肝癌临床分期及预后的关系

目的 探讨原发性肝癌患者血清miR-375、miR-155与其临床分期及预后的关系。方法 回顾性分析86例原发性肝癌患者资料,比较不同临床分期患者血清miR-375、miR-155表达水平,应用Spearman相关系数分析相关性;将患者以肝外转移分成转移组、非转移组,比较两组患者血清miR-375、miR-155差异,应用受试者曲线(ROC)分析两者诊断临床分期效能;随访1年内生存情况,应用Kaplan-Meier法、COX比例风险模型进行生存分析。结果 不同临床分期患者血清miR-375、miR-155表达水平比较差异有统计学意义(P<0.05);血清miR-375表达水平与肿瘤临床分期呈负相关(P<0.05),miR-155表达水平与临床分期呈正相关(P<0.05);转移组血清miR-375表达水平低于非转移组,miR-155表达水平高于非转移组,差异有统计学意义(P<0.05);血清miR-375、miR-155及联合诊断肿瘤转移的AUC分为0.898、0.847、0.941;miR-375高表达患者平均生存时间均长于miR-375低表达患者,miR-15...     

肝细胞癌血管生成调控与抗血管治疗进展

肝细胞癌（HCC）的血管生成受血管生成因子与血管生成抑制因子的调控。血管生成抑制剂治疗HCC可抑制癌的生长与转移,试验性治疗应用已显示出一定效性。     

血管生成抑制剂TNP-470抑制肝癌生长和转移的实验研究

目的　研究血管生成抑制剂TNP 470对肝细胞癌生长和转移的抑制作用。方法　把高转移人肝癌模型(LCI D2 0 )的肿瘤组织小块种植于裸鼠皮下 ,将 2 4只裸鼠随机分成对照组、治疗组。第 2天起分别给予溶剂 ( 3%酒精 )、TNP 470 ( 30mg kg)隔天皮下注射 ,共 8次。 结果　对照组、治疗组皮下瘤重分别为 ( 2 0 4± 0 34)g、( 0 98± 0 34) g(P<0 0 0 1) ;两组AFP分别为 ( 76 8 6± 2 82 3) μg L ,( 93 4±5 8 6 ) μg L (P <0 0 0 1) ,两组肺转移率分为5 0 % ( 6 12 )、8 3 % ( 1 12 ) (P <0 0 5 )。结论　血管生成抑制剂TNP 470能显著抑制肝细胞癌的生长和转移     

肝癌中血管生成VEGF及其受体水平与预后的关系

目的:探讨原发性肝细胞癌(PHCC)中血管生成、血管内皮生长因子(VEGF)及其受体(VEGFR:Flt-1、Flk-1)表达水平以及血清VEGF水平与预后的关系.方法:采用免疫组化通用型两步法检测微血管密度(MVD)、VEGF表达;采用免疫组化SABC法检测两种VEGFR的表达;采用ELISA法检测患者术前血清VEGF水平,并分析其相关性及与预后的关系.结果:1)MVD、VEGF、Flt-1、Flk-1和血清VEGF分别与肿瘤直径、分期、分化、癌栓、肿瘤数目、肿瘤包膜完整情况有关(P＜0.05).2)复发组的MVD、VEGF、Flt-1、Flk-1和血清VEGF水平均高于未复发组(P＜0.05;0.003,0.013,0.03l,0.026,0.015).3)VEGF表达与MVD表达相关(P＜0.001),Flt-1、Flk-1、血清VEGF与组织中VEGF相关(P＜0.05).结论:PHCC中MVD、VEGF、Flt-1、Flk-1和血清VEGF均可作为估计无瘤生存的独立预后因素,有利于选择术后预防性治疗的方式;各指标在诊断病情和选择治疗方面均有意义;血清VEGF可做为简便有效的独立预后指标,具有临床应用潜力.     

人肝癌血管内皮细胞表型及功能特性的研究

目的分析人肝癌血管内皮细胞(T3A)在表型和功能上的特性。方法从人原发性肝癌组织分离、纯化和培养血管内皮细胞,并分析比较其与盱窦内皮细胞(LSEC)在形态、表型和功能上的差别。用电子显微镜,检查细胞表面窗孔样结构;用流式细胞仪和荧光定量PCR,分析细胞表型的差别;用噻唑盐(MTT)比色法,分析比较TNFα诱导的细胞毒作用;用酶谱法和体外血管生成模型,评价细胞的血管生成能力;用酶联免疫吸附试验(ELISA),比较凝血和纤溶凶子的释放。结果人T3A细胞表达经典的血管内皮细胞标志vWF和CD31,并摄取乙酰化低密度脂蛋白,细胞表面存在大量无隔膜的窗孔样结构。表型分析发现,与肝窦内皮细胞相比,T3A细胞表达TNF受体p75增加,而表达TNF受体p55降低;ICAM-1的表达明显下调,而αvβ3和αvβ5的表达明显上调,这些改变与荧光定量PCR分析的结果一致。功能学研究发现,T3A细胞对TNFα诱导的细胞毒作用明显耐受,血管生成能力明显增强并具有高表达的凝血和纤溶活性。结论人T3A细胞在表型和功能上都具有一定的特殊性。     

姜黄素对化学诱导大鼠肝癌缺氧模型血管生成的影响

目的:探讨姜黄素对二乙基亚硝胺( diethylnitrosamine, DEN)诱发大鼠肝细胞癌（hepatocellular carcinoma, HCC）缺氧后血管形成的影响。方法: 采用DEN诱发大鼠HCC,结扎肝动脉并构建大鼠HCC缺氧模型。将HCC模型大鼠按照数字表法随机分为单纯碘化油栓塞组（A组）、碘化油联合姜黄素栓塞组（B组）、碘化油联合肝周包膜组（C组）、碘化油联合姜黄素及肝周包膜组（D组）,每组10 只。比较各组大鼠相应治疗后HCC细胞及组织VEGF、微血管密度（microvessel density,MVD）及大鼠中位生存时间（median survival time,MST）。结果：B组与D组的VEGF蛋白表达及MVD均比A组显著降低（P<0.01）,而C组上述指标则与A组无显著变化（P>0.05）。B、C、D组比A组大鼠MST均显著延长（P<0.05）,D组大鼠的MST高于B、C组（P<0.05）。结论：姜黄素可抑制HCC缺氧大鼠肿瘤血管的生成,可降低VEGF表达及MVD,起到延长大鼠生存期的作用。     

抗血管生成药治疗肝细胞癌的研究进展

肝细胞癌（HCC）是一种血管丰富的癌症,抗血管生成药可通过阻断血管生成因子,抑制内皮细胞扩增及预防细胞外基质和血管基底膜降解等机制而发挥治疗肝癌的作用,加上其临床相对优良的安全性可能为肝癌治疗提供了治疗新途径。     

PPARγ基因沉默抑制裸鼠肝癌血管生成的初步研究

目的:探讨过氧化物酶体增殖物活化受体γ(PPARγ)基因沉默对肝癌裸鼠模型瘤内血管生成的影响.方法:构建PPARγ短发夹状RNA表达质粒并转染HCCLM3细胞(pshPPARγ组),以空质粒转染为对照组.观察两组皮下种植瘤生长曲线及体积,肺转移灶数量及分级.免疫组织化学法检测抗-CD34、血管内皮生长因子(VEGF)、血小板反应蛋白(TSP1)表达;RT-PCR检测MMP-2和TIMP2表达.结果: pshPPARγ组种植瘤体积为(1.86±0.65) cm3,微血管密度(MVD)为20.84±6. 38,肺转移灶数量为(37.2±0.7)个/肺,分级为1～2级,对照组体积为(4.86±1.15) cm3,MVD为39.48±9.01,肺转移灶数量为(107.8±6.1)个/肺,分级多为3～4级,两组种植瘤体积、MVD和转移灶数量、分级的差异有统计学意义,t值分别为5.082、8.441和21.83, P值均＜0.05.pshPPARγ组TSP1蛋白阳性表达,而VEGF蛋白为弱阳性或阴性表达,MMP-2 mRNA表达下调,而TIMP2 mRNA表达下调,与对照组比较差异有统计学意义,t值分别为11.34、 8.44, P值均＜0.01.结论: PPARγ基因沉默可降低肝癌种植瘤血管生成,与调节MMP-2/TIMP2表达,影响VEGF/TSP1平衡有关.     

肝癌新生血管的形态学异常及其临床意义

 目的 研究肝细胞癌（HCC）新生血管的形态学异常。方法 采用免疫组化SABC法检测56例HCC组织及其相应癌旁肝组织和6例正常肝组织中CD34和Endoglin的表达,并采用透射电镜观察微血管超微结构的改变。结果 56例HCC组织中CD34和Endoglin表示的MVD值分别是127.1±5.9和64.6±11.1,而癌旁肝组织的MVD值分别是7.1±1.3和6.1±1.2。HCC组织的MVD值高于相应的癌旁肝组织,差异具有显著意义（P〈0.01）。HCC组织中微血管结构松散,通透性明显增加,可见微血管内微癌栓形成。结论 HCC组织中新生血管生成明显增加,而且这些新生的微血管在结构上是有缺陷的,这为癌细胞的转移提供了门户。     

MiR-497 suppresses angiogenesis and metastasis of hepatocellular carcinoma by inhibiting VEGFA and AEG-1

Hepatocellular carcinoma (HCC) is a worldwide malignance and displays marked vascular abnormalities and active metastasis. MicroRNAs (miRNAs) have been shown to play important roles in regulating tumor properties in cancer, however, whether miR-497 contributes to HCC angiogenesis or metastasis remains unclear. In this study, we found that miR-497 was significantly down-regulated in HCC tissue samples and cell lines. Gain-of-function and loss-of-function studies revealed that miR-497 could repress both the pro-angiogenic and metastatic ability of HCC cells. Subsequent investigations disclosed that miR-497 directly inhibited the 3′-untranslated regions (UTRs) of vascular endothelial growth factor A (VEGFA) and astrocyte elevated gene-1 (AEG-1). Furthermore, overexpression of these targets antagonized the function of miR-497. Based on nude mouse models, we demonstrated that overexpression of miR-497 significantly repressed microvessel densities in xenograft tumors and reduced pulmonary metastasis. In conclusion, our findings indicate that miR-497 downregulation contributes to angiogenesis and metastasis in HCC.     

miR-211 suppresses hepatocellular carcinoma by downregulating SATB2

Dysregulation of microRNAs (miRs) is involved in carcinogenesis. Deregulation of miR-211 has recently been observed in many tumors, but its function in hepatocellular carcinoma (HCC) is still unknown. Here we found that miR-211 was decreased in HCC cancer tissues compared with adjacent normal tissues. We also found that overexpression of miR-211 repressed proliferation and invasion in HepG2 and SMMC7721 cells. Luciferase reporter assays and western blot indicated that special AT-rich sequence-binding protein-2 (SATB2), is a direct target of miR-211. The expression of SATB2 was upregulated in HCC cancer tissues and cell lines and miR-211 levels inversely correlated with SATB2 levels in HCC. Importantly, SATB2 rescued the miR-211-mediated inhibition of cell invasion and proliferation. Finally, reintroduction of miR-211 repressed tumor formation of HCC in xenograft mice. This study provides insights into molecular mechanisms that miR-211 contributed to HCC.     

MicroRNA-212 suppresses tumor growth of human hepatocellular carcinoma by targeting FOXA1

MicroRNA-212 (miR-212) has been reported to play oncogenic or tumor suppressive role in different human malignancies. Here, we demonstrated that the mean level of miR-212 in hepatocellular carcinoma (HCC) tissues was significantly lower than that in matched tumor-adjacent tissues. Similarly, the expression of miR-212 was obviously reduced in HCC cell lines as compared with a nontransformed hepatic cell line. Ectopic expression of miR-212 inhibited cell viability and proliferation, and induced apoptosis in HepG2 cells. In contrast, down-regulation of miR-212 increased cell viability and proliferation, and suppressed apoptosis in Bel-7402 cells. In vivo studies showed that miR-212 inhibited tumor growth of HCC via suppressing proliferation and inducing apoptosis. Furthermore, we confirmed that Forkhead box protein A1 (FOXA1) was a direct target of miR-212, and it abrogated the function of miR-212 in HCC. Finally, we disclosed that the aberrant expression of miR-212 and FOXA1 was evidently correlated with poor prognostic features of HCC. MiR-212, FOXA1 and their combination were valuable prognostic markers for predicting survival of HCC patients. In conclusion, miR-212 may serve as a prognostic indicator for HCC patients and exerts tumor suppressive role, at least in part, by inhibiting FOXA1.     

MicroRNA-124靶向调控caveolin-1介导肝癌细胞恶性行为的研究

目的:检测miR-124、caveolin-1在肝癌组织及细胞系中的表达,探讨miR-124靶向调控caveolin-1对肝癌细胞增殖和侵袭的影响。方法:回顾性分析2012年8月至2014年7月32例于大连医科大学附属第一医院收治的行肝癌切除术患者的临床资料和标本,通过实时定量逆转录聚合酶链反应(qRT-PCR)检测肝癌、癌旁组织以及肝癌细胞系中miR-124、caveolin-1的表达;通过靶基因预测及双荧光素酶报告分析miR-124与caveolin-1的靶向关系;通过qRT-PCR、Western blot检测miR-124对caveolin-1表达的调控;分别用CCK8、平板克隆及Transwell检测细胞增殖和侵袭能力;通过绘制生存曲线,分析miR-124及caveolin-1表达与临床肝癌患者预后的相关性。结果:miR-124在肝癌组织中的表达低于癌旁组织,在高转移肝癌细胞MHCC97H中的表达低于低转移肝癌细胞MHCC97L,而caveolin-1呈现相反的趋势;caveolin-1为miR-124的靶基因,调控miR-124可影响caveolin-1水平;MHCC97H...     

The copper-chelating agent, trientine, suppresses tumor development and angiogenesis in the murine hepatocellular carcinoma cells.

Angiogenesis is now recognized as a crucial process in tumor development, including hepatocellular carcinoma (HCC). Since HCC is known as a hypervascular tumor, anti-angiogenesis is a promising approach to inhibit the HCC development. Trientine dihydrochloride (trientine) is used in clinical practice as an alternative copper (Cu)-chelating agent for patients with Wilson's disease of penicillamine intolerance. In our study, we examined the effect of Cu-chelating agents on tumor development and angiogenesis in the murine HCC xenograft model. Although both trientine and penicillamine in the drinking water suppressed the tumor development, trientine exerted a more potent inhibitory effect than penicillamine. In combination with a Cu-deficient diet, both trientine and penicillamine almost abolished the HCC development. Trientine treatment resulted in a marked suppression of neovascularization and increase of apoptosis in the tumor, whereas tumor cell proliferation itself was not altered. In vitro studies also exhibited that trientine is not cytotoxic for the tumor cells. On the other hand, it significantly suppressed the endothelial cell proliferation. These results suggested that Cu plays a pivotal role in tumor development and angiogenesis in the murine HCC cells, and Cu-chelators, especially trientine, could inhibit angiogenesis and enhance apoptosis in the tumor with consequent suppression of the tumor growth in vivo. Since trientine is already used in clinical practice without any serious side effects as compared to penicillamine, it may be an effective new strategy for future HCC therapy.     

miR-375靶向SHOX2调控人食管鳞癌细胞侵袭迁移的初步研究

目的 探讨miR-375/SHOX2轴对食管鳞癌细胞侵袭迁移能力的影响,为食管鳞癌的靶向治疗寻找潜在新靶点。方法 选取对数生长期的人食管鳞癌细胞（kyse-70、kyse-30）,分别过表达以及干扰miR-375和SHOX2基因,采用克隆形成、MTT、划痕、Transwell等体外细胞功能学实验检测miR-375、SHOX2对食管鳞癌细胞增殖、迁移和侵袭能力的影响,通过拯救实验验证miR-375对SHOX2的靶向调控作用。结果 与对照组的kyse-70、kyse-30细胞相比,过表达miR-375或干扰SHOX2基因后的细胞增殖率、克隆形成率和Transwell细胞穿透数均降低,差异均有统计学意义（P＜0.01）;与对照组的kyse-70、kyse-30细胞相比,干扰miR-375或过表达SHOX2后的细胞增殖率、克隆形成率和Transwell细胞穿透数均升高,差异均有统计学意义（P＜0.01）;miR-375过表达后的食管鳞癌细胞SHOX2基因表达减少,而抑制miR-375后SHOX2基因表达增强。拯救实验结果显示,共转染miR-375和SHOX2后细胞增殖和侵袭能力较对照组又有所增强。结论 miR-375抑制食管鳞癌细胞增殖和侵袭;SHOX2促进食管鳞癌细胞增殖和侵袭。miR-375靶向负调控SHOX2表达,对食管鳞癌细胞的增殖和侵袭起调控作用。     

MicroRNA-543 acts as an oncogene by targeting PAQR3 in hepatocellular carcinoma

MicroRNAs (miRNAs) are small, non-coding RNAs that can act as oncogenes or tumor suppressor genes in human cancer. Increasing evidences indicate that deregulation of miRNAs contributes to the hepatocarcinogenesis. In this study, we demonstrated that the levels of miR-543 were dramatically increased in clinical hepatocellular carcinoma (HCC) tissues and cell lines. Moreover, forced expression of miR-543 promoted the proliferative and invasive potential of HepG2. We also identified PAQR3 as a direct target gene for miR-543 using a fluorescent reporter assay and western blot. The levels of PAQR3 were dramatically decreased in clinical hepatocellular carcinoma (HCC) tissues and cell lines. The mRNA levels of PAQR3 were inversely correlated with the miR-543 expression level.Thus, our finding provides a new insight into the mechanism of hepatocarcinogenesis, indicating a therapeutic potential of miR-543 in the treatment of HCC.