小核糖体核蛋白B对肝癌细胞增殖与转移的调控作用

目的研究小核糖体核蛋白B(SNRPB)在肝癌组织及细胞中的表达情况, 以及SNRPB对肝癌细胞增殖与转移的作用。方法通过生物信息数据库starBase v3.0和GEPIA分析SNRPB在肝癌组织和正常肝脏组织中的表达, 并进行肝癌患者预后生存分析;qRT-PCR和蛋白质印迹法(Western blot)分析SNRPB mRNA和蛋白在肝癌细胞系中的表达;利用RNA干扰技术(siRNA)下调SNRPB蛋白的表达, 通过MTT实验观察其对肝癌细胞增殖的作用;Transwell侵袭迁移实验检测下调SNRPB后肝癌细胞转移能力的变化;利用Western blot技术检测下调SNRPB表达后肝癌细胞上皮-间质转化(EMT)标志物的改变。数据两组间比较采用t检验, 多组间比较采用方差分析。结果 SNRPB在肝癌组织中的表达明显高于正常肝脏组织, 且其表达水平并与肝癌患者预后相关。与永生化肝细胞LO2相比, SNRPB在肝癌细胞系中的表达显著升高(P值均< 0.01)。siRNA-SNRPB显著抑制肝癌细胞内SNRPB mRNA和蛋白表达。MTT结果显示SNRPB敲低组的吸光度值较阴性对照组...     

LIM激酶1对肝癌细胞增殖与转移的调控作用

目的:研究LIM激酶1（LIMK1）在肝癌组织及细胞中的表达情况,以及LIMK1对肝癌细胞增殖与转移的调控作用。方法:通过在线数据库starBase v3.0和GEPIA分析LIMK1在肝癌组织和肝脏正常组织中的表达情况,并进行相关生存分析;蛋白质印迹（Western blot）技术分析LIMK1在肝癌细胞株中的表达情...     

上皮间质转化在原发性肝癌侵袭、转移中的作用

上皮间质转化(epithelial-mesenchymal transition,EMT)是指上皮细胞在特定的情况下向间质细胞转分化的现象。在原发性肝细胞癌的侵袭、转移中存在这一现象。因此,本文除介绍EMT及其诱导调控因素之外,还对它在原发性肝癌侵袭、转移中的作用进行综述。     

中医药调控上皮-间质转化抑制肝癌转移的研究进展

转移是影响肝癌复发和死亡的主要原因,而上皮-间质转化(EMT)是肝癌转移的重要机制。EMT受TGFβ、Wnt/β-catenin、Notch等多种信号通路介导的转录因子Snail、Twist、ZEB等的调控。抑制肝癌EMT相关分子和信号通路是抑制肝癌侵袭转移的重要途径。近年来研究表明,多种中药复方或其有效成分具有抑制肝癌侵袭转移的作用,阻滞或逆转肝癌细胞EMT是其重要的作用机制。本文对EMT的作用和调控机制,以及近年来中药及其来源的天然化合物通过调控细胞EMT抑制肝癌侵袭转移的研究进行概述,以期为肝癌转移的中医药防治提供科学依据,为肝癌治疗提供新思路。     

GPCR48通过诱导肝癌细胞上皮间质转化促进肝癌侵袭转移的机制

目的:观察G蛋白偶联受体48 (GPCR48)在不同转移潜能肝癌细胞株中的表达及其对肝癌细胞Huh7上皮间质转化(EMT)特性和侵袭转移的影响。方法:采用蛋白质印迹（Western blot）检测不同转移潜能肝癌细胞GPCR48的蛋白表达水平。构建携带GPCR48基因的慢病毒载体,在肝癌细胞Huh7过表达GPCR48;...     

小干扰RNA沉默甲胎蛋白基因对HepG2细胞迁移及侵袭的影响

目的研究甲胎蛋白(AFP)对肝癌HepG2细胞迁移和侵袭能力的影响及其机制。方法应用合成的靶向沉默AFP小干扰RNA(siRNA)转染肝癌HepG2细胞,分为空白对照、阴性对照组及AFP siRNA组,各组细胞干预48 h后,采用实时定量PCR(qRT-PCR)和ELISA法检测细胞转染后的沉默效率,Transwell小室实验检测沉默AFP基因后HepG2细胞的侵袭、迁移能力,用Western blotting法观察沉默AFP基因的表达对上皮-间质转化(EMT)相关蛋白(N-cadherin、Vimentin和E-cadherin)、AKT和p-AKT蛋白表达的影响。计量资料多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。结果转染沉默后,与空白对照组相比,AFP siRNA组中AFP mRNA的相对表达量明显降低(P 0.01),抑制率达86.440%,同时AFP siR NA组细胞上清液中AFP蛋白表达水平同样明显降低(P 0.01)。与空白对照组相比,AFP siRNA组的迁移细胞数和侵袭细胞数显著下降,差异具有统计学意义(P值均0.01)。沉默HepG2细胞中AFP基因的表达后,与空白对照组比较,AFP siRNA组EMT相关蛋白E-cadherin蛋白的表达水平升高(P 0.01),而N-cadherin及Vimentin表达水平均明显降低(P值均0.05),且PI3K/AKT信号通路相关蛋白p-AKT的表达水平明显降低(P 0.01)。结论沉默AFP可抑制肝癌细胞转移,基于HepG2细胞株的机制研究可能与阻断PI3K/Akt通路抑制EMT相关。     

上皮间质转化及相关信号通路在原发性肝细胞癌转移中的研究进展

上皮间质转化(epithelial-mesenchymal transition,EMT)现象是指上皮细胞失去上皮表型,获得间质细胞表型的过程.存在于胚胎发生、器官纤维化和肿瘤转移过程中.在原发性肝细胞癌的转移中存在这一现象.本文重点介绍了TGF-β通路、Wnt(wingless-type)/β-catenin通路、Hedgehog通路的组成及其在原发性肝细胞癌的上皮间质转化过程中起到的重要调控作用.此外,还谈及了其他调控因子,如NF-κB通路、Notch通路、转录因子、microRNA、CHD1L、ARHGEF9、乙肝病毒X基因(hepatitis B virus X gene,HBX)、细胞外基质等也参与了EMT的发生.研究EMT的分子机制是一个有前景的课题,可以为原发性肝细胞癌的治疗提供新的靶点,更好地理解靶向治疗中的耐药机制.     

肝癌转移相关的微小RNAs在不同转移潜能肝癌细胞系的定量研究

目的 研究与转移密切相关的微小RNAs(miRNAs)在不同转移潜能肝癌细胞系的表达水平,探讨其在肿瘤转移过程中的生物学功能.方法 提取细胞系MHCC97H、MHCC97L、HepG2、L02的总RNA,通过反转录获得特异miRNA(miR-122a、miR-124a、miR-148a、miR-148b、miR-15a、miR-219、miR-30c、miR-338、miR-34a、Let-7g、miR-9)的cDNA,并应用TaqMan MGB探针法对其进行定量检测.采用AB17500系统软件V1.3.1采集Ct值,并使用miRNA内参基因RNU6B校正,相对定量计算公式RQ=2-△Ct,A Ct=CtmiRNAs-CtRNu6B.数据均经SPSS13.0统计软件包处理,采用t检验或非参数检验. 结果 转移相关的miRNAs(miR-124a除外)在MHCC97H与MHCC97L中表达,差异均有统计学意义.HepG2中miR-30c、miR-338、miR-34a和Let-g的表达水平明显高于L02,分别为miR-30c(8.41±0.40比6.82±0.29),miR-338(3.14±0.29比-2.36±0.32),miR-34a(0.71±0.40比-2.95±0.26),Let-7g(-4.07±0.55比-6.98±0.56),t值依次为2.948,12.656,7.484,3.684,P值均＜0.05,差异有统计学意义.而miR-148b,miR-9的表达则显著低于正常肝细胞,分别为miR-148b(1.96±0.51比3.76±0.28),miR-9(-4.38±0.86比-1.10±0.53),t值依次为-3.073,-3.324,P值均＜0.05,差异有统计学意义.miR-148家族中miR-48b在所测细胞系中的表达(5.46±1.21)均显著强于miR-148a的表达(1.29±0.35),Z=-5.097,P=3×10-7,差异有统计学意义.结论 可以利用肝癌细胞系列细胞平台进一步研究肝癌转移相关miRNAs在肿瘤转移过程中的生物学功能.     

上皮-间质转化与肿瘤转移及干细胞的研究进展

肿瘤细胞的侵袭、转移及干性特征是患者治疗失败、预后差的主要原因。为了给肿癌患者治疗提供一种新的治疗方案,从分子水平来解释肿瘤细胞的侵袭、转移及干性特征至关重要。最近的研究表明上皮-间质转化(epithelial-mesenchymal transition,EMT)对肿瘤细胞的侵袭、转移及干性特征起到举足轻重的作用。EMT 可使上皮性肿瘤细胞获得间充质细胞表型,在增强肿瘤细胞的侵袭和转移能力的同时,也使得肿瘤细胞具有自我更新等干细胞特性。本综述阐明 EMT 与肿瘤转移及干细胞的相互关系及其调控机制,有望为肿瘤的靶向治疗开辟新思路。     

肝癌复发与转移的基础研究

肝细胞癌(HCC)无论“早切”、“再切”、“二期切”或“肝移植”均遇到复发和转移的问题。上海医科大学肝癌研究所报道肝癌根治性切除后5年复发率为61．5％，小肝癌为43．5％提示攻克肝癌复发转移的重要意义。     

hnRNP B1及hnRNP B1小干扰RNA对肺癌作用的研究进展

异质性胞核核糖核蛋白(hnRNP)B1是肺癌的早期重要分子标志.RNA干扰是通过特异降解靶基因mRNA从而达到转录后基因沉默的一种全新的基因阻断技术.它广泛存在于动物和植物中,在防御外源基因入侵和自身基因异常表达中起重要作用.hnRNP B1小干扰RNA能抑制肺癌细胞增殖,有望成为早期肺癌基因治疗的合理策略之一.     

hnRNP B1及hnRNP B1小干扰RNA对肺癌作用的研究进展

异质性胞核核糖核蛋白(hnRNP)B1是肺癌的早期重要分子标志.RNA干扰是通过特异降解靶基因mRNA从而达到转录后基因沉默的一种全新的基因阻断技术.它广泛存在于动物和植物中,在防御外源基因入侵和自身基因异常表达中起重要作用.hnRNP B1小干扰RNA能抑制肺癌细胞增殖,有望成为早期肺癌基因治疗的合理策略之一.     

Proteinase-activated receptor 2 promotes tumor cell proliferation and metastasis by inducing epithelial-mesenchymal transition and predicts poor prognosis in hepatocellular carcinoma

AIM To clarify the role of proteinase-activated receptor 2(PAR2) in hepatocellular carcinoma, especially in the process of metastasis.METHODS PAR2 expression levels were assessed by qRT-PCR and immunohistochemistry(IHC) in patient tissues and in hepatocellular carcinoma cell lines SMMC-7721 and Hep G2. Cell proliferation and metastasis were assessed both in vitro and in vitro. Immunoblotting was carried out to monitor the levels of mitogen-activated protein kinase(MAPK) and epithelial-mesenchymal transition markers.RESULTS The prognosis was significantly poorer in patients with high PAR2 levels than in those with low PAR2 levels. Patients with high PAR2 levels had advanced tumor stage(P = 0.001, chi-square test), larger tumor size(P = 0.032, chi-square test), and high microvascular invasion rate(P = 0.037, chi-square test). The proliferation and metastasis ability of SMMC-7721 and Hep G2 cells was increased after PAR2 overexpression, while knockdown of PAR2 decreased the proliferation and metastasis ability of SMMC-7721 and Hep G2 cells. Knockdown of PAR2 also inhibited hepatocellular carcinoma tumor cell growth and liver metastasis in nude mice. Mechanistically, PAR2 increased the proliferation ability of SMMC-7721 and Hep G2 cells via ERK activation. Activated ERK further promoted the epithelial-mesenchymal transition of these cells, which endowed them with enhanced migration and invasion ability. CONCLUSION These data suggest that PAR2 plays an important role in the proliferation and metastasis of hepatocellular carcinoma. Therefore, targeting PAR2 may present a favorable target for treatment of this malignancy.     

Yes-associated protein at the intersection of liver cell fate determination

A recent publication highlights the importance of high yes-associated protein (YAP) expressing cells in liver regeneration following partial hepatectomy. Although the names of the cell populations described in these articles [hybrid periportal hepatocytes (HybHP) or epithelial-mesenchymal transition (EMT)-reprogrammed hepatocytes] are not identical, they all express high levels of YAP. We hypothesize that the HybHP and EMT-reprogrammed hepatocytes might be a similar cell population. Hippo signaling is the primary pathway that regulates YAP activity. According to the contribution of these two types of cells to liver regeneration and the high YAP expression, Hippo-YAP signaling activation may be a common regulatory pathway experienced by cells undergoing dedifferentiation and reactivating proliferative activity during liver regeneration. Although no evidence has shown that HybHP cells contribute to hepatocellular carcinoma in mouse models, we can not rule out the possibility that these highly regenerative cells can further develop into tumor cells when they acquire mutations caused by viral infection or other risk factors like alcohol. The detailed mechanistic insight of the regulation of YAP expression and activity in HybHP (or other types of cells contributing to liver regeneration) is unknown. We hypothesize that liver regeneration under various conditions will eventually lead to divergent consequences, likely due to the duration of YAP activation regulated by Hippo-large tumor suppressor 1 and 2 pathway in a context- and cell type-dependent manner.     

奥曲肽对人肝癌细胞生长增殖、甲胎蛋白合成及细胞凋亡的作用

目的　研究奥曲肽对肝癌细胞生长增殖及细胞凋亡的作用 ,探讨其对肝癌的作用机制 ,为临床应用提供实验依据。方法　采用MTT法、生长曲线观察奥曲肽对肝癌细胞HepG2生长增殖的影响 ,电化学发光法测定培养上清液中甲胎蛋白 (AFP)含量 ,并用荧光染色、透射电镜和流式细胞仪检测细胞凋亡。结果　奥曲肽在 0 .0 0 5～ 80 μg/ml浓度范围内呈剂量依赖性方式抑制肝癌细胞HepG2的生长增殖 ,并能显著减少肝癌细胞AFP合成。奥曲肽 (0 .2 5～ 4 .0 μg/ml)作用 4 8h后 ,荧光染色与透射电镜可见部分HepG2细胞呈典型的凋亡形态学改变。流式细胞仪检测 ,出现凋亡峰 ,与对照组相比 ,细胞的凋亡率显著升高 (P <0 .0 5 )。结论　奥曲肽能够显著抑制肝癌细胞生长 ,并诱导肝癌细胞凋亡 ,有望成为治疗肝细胞癌的一个有效药物     

SNRPN基因在HepG2细胞中的印迹状态研究

目的:研究小核核糖核蛋白多肽N基因(SNRPN)在肝癌肿瘤HepG2细胞株的表达及基因印迹状态.方法:采用RT-PCR方法检测出SNRPN基因在肝癌肿瘤HepG2细胞株中的表达状况,对HepG2细胞株基因组DNA和cDNA中的SNRPN基因外显子4nt1654312位点用RT-PCR为基础的RFLP方法进行基因分型.结果:HepG2细胞稳定表达SNRPN,SNRPN外显子4nt1654312(数字依据NT_026446,SNPrs705)为杂合子(C/T);RT-PCR为基础的RFLP分析表明,SNRPN的双等位基因中只有T等位基因产生mRNA转录本.结论:SNRPN基因在HepG2肝癌细胞株中有表达,其基因印迹状态未丢失.     

Rac1对L02肝细胞株上皮细胞间质转化及增殖和凋亡的影响

目的检测Rac1在TGF-β1诱导的L02细胞上皮间质转化中的作用,及其对细胞增殖和凋亡的影响。方法应用不同活性的Rac1质粒pExRed-NLS Flag（空载体组）、pExRed-NLS Flag Rac1（野生型Rac1组）、pExRed-NLS Flag Rac1T17N（显性负调控Rac1组）、pExRed-NLS Flag Rac1G12V（持续活化型Rac1组）瞬时转染L02细胞,经5 ng/ml TGF-β1处理细胞。采用免疫印迹法检测融合蛋白Flag-Rac1表达,采用细胞免疫荧光及免疫印迹法检测Ck8和Vimentin表达,采用细胞划痕实验及Transwell法检测细胞迁移能力。使用不同浓度的Rac1特异性抑制剂NSC23766处理L02细胞,采用CCK-8法检测细胞增殖,采用Annexin V-FITC/PI双染法检测细胞凋亡。结果四组质粒均成功瞬时转染到L02细胞中;与空载体组和野生型Rac1组比,持续活化型Rac1转染细胞Vimentin蛋白表达水平显著增高,CK8蛋白表达水平降低,细胞迁移能力增加;与空载体组和野生型Rac1组比,显性负调控Rac1转染细胞Vimentin蛋白表达水平降低,CK8蛋白表达水平增高,细胞迁移能力降低（P〈0.05）;在NSC23766处理L02细胞后,细胞增殖被抑制,但各处理组细胞凋亡无明显差异。结论 Rac1可促进TGF-β1诱导的L02肝细胞株上皮间质转化和细胞增殖,但对细胞凋亡无明显影响。     

Comparative study on proliferation activity in small hepatocellular carcinoma related to hepatitis virus B and C

AIM: To compare the proliferation activity of small hepatocellular carcinoma (HCC) related to hepatitis B virus (HBV) and hepatitis C virus (HCV).METHODS: Sixty liver biopsy specimens were obtained from patients with small HCC (≤3 cm in diameter) and examined immunohistochemically using anti-proliferating cell nuclear antigen monoclonal antibody. Of the 60 specimens, 30 were HBV-related and 30 were HCV-related. The 60 patients providing the samples for study were matched by sex and morphologic features of the HCC specimens.RESULTS: The labeling index of proliferating cell nuclear antigen was 7.9% in the HBV-related HCC specimens and 12.5% in the HCV-related HCC specimens. There was no statistically significant difference between the two groups (P > 0.05).CONCLUSION: In the early phase, or small stage, of HCC, HBV-related HCC shows similar proliferating activity to that of HCV-related HCC; this finding suggests that in the early phase, HBV-related HCC has similar malignancy to HCV-related HCC.     

Inhibitory effect of dimeric β peptide on the recurrence and metastasis of hepatocellular carcinoma in vitro and in mice

AIM: To block the adhesion of tumor cells to the extracellular matrix, and prevent tumor metastasis and recurfence, the dimer of the β peptide (DLYYLMDLSYSMKGGDLYYLMDLSYSMK, β2) was designed and synthesized and its anti-adhesion and anti-invasion effects on hepatocellular carcinoma cells were assessed. Additionally,its influence on the metastasis and recurrence of mouse hepatocellular carcinoma was measured.METHODS: The anti-adhesion effect of β2 on the highly metastatic hepatocellular carcinoma cell line HCCLM6 cells and fibronectin (FN) was assayed by the HTT assay. The inhibition of invasion of HCCLM6 cells by β2 was observed using a Transwell (modified Boyden chamber)and matrigel. Using the hepatocellular carcinoma metastasis model and LCI-D20 nude mice, the influence of β2 on the metastasis and recurrence of hepatocellular carcinoma after early resection was investigated.RESULTS: HCCLM6 cells co-incubated with 100 μmol/L,50 μmol/L, 20 μmol/L or 10 μmol/L β2 for 3 h showed an obvious decrease in adhesion to FN. The adhesion inhibition ratios were 11.8%, 21.7%, 29.6% and 48.7%,respectively. Additionally, HCCLM6 cells cultured with 100 μmol/L β2 had a dramatic decrease in cell invasion.β2 was also observed to inhibit the incisal edge recurfence and the distant metastasis of nude mice hepatocellular carcinoma after early resection (P ＜ 0.05).CONCLUSION: The β2 peptide can specifically block the adhesion and invasion of HCCLM6 cells, and can inhibit HCC recurrence and metastasis of LCI-D20 model posthepatectomy in vivo. Thus, β2 should be further studied as a new anti-tumor drug.