HAX-1 is overexpressed in hepatocellular carcinoma and promotes cell proliferation

Hepatocellular carcinoma (HCC) is the most common primary liver tumor. Due to the asymptomatic nature of early HCC and lack of effective screening strategies, 80% of patients present with advanced HCC at the time of diagnosis. Novel molecular marker identification will be valuable for effective diagnosis and treatment. In this study we reported HCLS1-associated protein X-1 (HAX-1) is overexpression in HCC in human HCC sample. Furthermore, we provided evidence that HAX-1 expression positively correlated with that of Ki67 in patient sample. Statistic analysis indicated that HAX-1 expression level significantly correlated with clinic outcome of HCC. Cell based assay revealed that knockdown of HAX-1 inhibits cell proliferation. This result suggests that HAX-1 can be a novel molecular marker for HCC.     

CCAT1 promotes hepatocellular carcinoma cell proliferation and invasion

It has been reported that CCAT1 is involved in the development of malignancies including colon cancer and gastric cancer. However, the role of CCAT1 in HCC still remains unknown. Real-time PCR was performed to test the relative expression of CCAT1 in HCC tissues and cell lines. We performed Chi-Square Analysis to study the correlation between clinical characteristics and CCAT1 expression. Based on the correlation, cell proliferation assay, cell invasion assay, wound healing assay and cell apoptosis assay were conducted in two HCC cell lines to examine the regulatory effect of CCAT1 on the HCC cells. The results indicated that the expression of CCAT1 was significantly increased in HCC tissues and cells compared with controls. We also found that the abnormally expressed CCAT1 could promote cell proliferation, migration and invasion. Taken together, our findings demonstrated that the aberrant expression of CCAT1 promotes hepatocellular carcinoma in vitro.     

MYH9在人肝癌组织中的表达及其对肝细胞系增殖和凋亡的影响

目的探讨非肌肉肌球蛋白重链(MYH9)在肝癌组织中的表达及通过沉默MYH9基因对肝癌细胞系SMMC-7721的增殖及凋亡的影响。方法收集50组人肝癌组织及癌旁组织,选用人肝癌细胞系SMMC-7721和Hep G2及人正常肝细胞系LO2,免疫组织化学方法及Western blot检测肝癌组织及癌旁组织中MYH9蛋白的表达,Western blot检测SMMC-7721、Hep G2及LO2中MYH9蛋白的表达;将MYH9 siRNA转染SMMC-7721,CKK8法及流式细胞术检测沉默MYH9对肝癌细胞增殖及细胞凋亡的影响。结果 MYH9蛋白在肝癌组织中的表达明显高于癌旁(P0.05);MYH9蛋白在SMMC-7721及Hep G2中的表达均明显高于LO2(P0.05);沉默MYH9基因可抑制细胞增殖(P0.001),促进细胞凋亡(P0.05)。结论 MYH9蛋白在肝癌组织的表达显著高于癌旁组织;MYH9低表达能有效抑制肝癌细胞的增殖,促进其凋亡。     

乙肝病毒促进肝癌细胞系的转移侵袭力

目的探讨乙肝病毒(HBV)对肝癌细胞转移能力的影响及其可能机制。方法以初始汇合度为30%,将3种细胞系HL-7702(人正常肝细胞系)、HepG2(未转染HBV-DNA的人肝癌细胞系)、HepG2.2.15(稳定转染HBV-DNA的人肝癌细胞系)种植于96孔板中,待细胞增殖至70%汇合时,利用划痕器制造划痕伤口,置于活细胞动态成像系统中进行多时间点的显微拍照与数据采集,计算相对伤口密度(RWD),并通过免疫荧光染色与Western blot技术测定细胞中Eph A2蛋白表达,分析其与RWD值的相关性。结果细胞迁移实验中,划痕后24~96 h,HL-7702组RWD显著高于HepG2与HepG2.2.15组(P0.01),划痕后72~144 h,HepG2.2.15组RWD显著高于HepG2组(P0.01);细胞侵袭实验中,HL-7702细胞因不能穿过基质胶,而无RWD值;划痕后72~144 h,HepG2.2.15组RWD显著高于HepG2组(P0.05或P0.01)。Eph A2表达:与HL-7702组比较,HepG2与HepG2.2.15组细胞中Eph A2表达水平显著升高(P0.01),其中HepG2.2.15组中Eph A2表达水平显著高于HepG2组(P0.01),且两组肝癌细胞中Eph A2的表达量与划痕实验的RWD值呈显著正相关(迁移实验:P0.01;侵袭实验:P0.01)。结论乙肝病毒可能促进肝癌细胞的迁移和侵袭能力,其机制可能与上调Eph A2的异常表达有关。     

ITIH4基因沉默对人肝癌细胞HepG2增殖、迁移及侵袭的影响

目的:探讨沉默间-α-胰蛋白酶抑制剂重链H4(inter-alpha-trypsin inhibitor heavy chain H4,ITIH4)基因对人肝癌细胞系HepG2增殖、迁移及侵袭的影响.方法:将4个ITIH4基因沉默载体分别转染HepG2细胞,用2 mg/L嘌呤霉素筛选获得稳定表达细胞株.Western印迹评价沉默效率,并选择效率最高的细胞株(si-ITIH4)用于后续实验.转染无意义序列的细胞株(si-Control)作为对照组.分别用细胞计数及CCK-8细胞计数试剂盒评价两组细胞增殖.用Transwell小室评价两组细胞的迁移及侵袭.结果:成功获得ITIH4基因沉默的HepG2细胞株si-ITIH4,与si-Control组相比,si-ITIH4组沉默效率达到86%.与si-Control组相比,si-ITIH4组细胞增殖显著降低、细胞迁移及侵袭均显著减少,差异有统计学意义(P<0.05).结论:ITIH4基因沉默可显著抑制HepG2的增殖、迁移及侵袭,提示其可能是参与肝细胞癌发生发展的重要基因之一.     

MiR-26b inhibits hepatocellular carcinoma cell proliferation,migration, and invasion by targeting EphA2

Deregulated microRNAs (miRNAs) have been shown to play important roles in cancer progression as a result of changes in expression of their target genes. In this study, we investigated the expression of miR-16b in eight hepatocellular carcinoma (HCC) cell lines, revealed the roles of miR-26b on hepatocellular carcinoma (HCC) cell proliferation, migration, and invasion, and confirmed that EphA2 is a direct target of miR-26b. The miR-26b expression was decreased and EphA2 expression was evaluated in HCC cell lines. Luciferase assays revealed that miR-26b inhibited EphA2 expression by targeting the 3’-untranslated region of EphA2 mRNA. Overexpression of miR-26b dramatically inhibited the proliferation, invasion, and migration of HCC cells by targeting EphA2. Moreover, miR-26b down-regulated c-Myc and CyclinD1 expression, which was reversed by overexpressed EphA2. Taken together, our data demonstrated the mechanism of miR-26b contributed to HCC progression and implicated that miR-26b’s potential in HCC therapy.     

Growth patterns and interstitial invasion of small hepatocellular carcinoma

Twenty-five caw of small hepatocellular carcinoma (HCC; dhmeter ≥30mm) were evaluated for overall morphologic features and growth patterns. The tumors often showed a uelldtfferentlated, normotrabecular histologic pattern and insidious interstitial invasion, which resembled benign hepetocytes scattered in connective tissues. As the tumor grew, B less-differentiated tumor area became predominant. Portal tracts Included in small HCC nodules were quantitatively assessed, revealing that they progressively reduced in number with tumor growth. The tumor margin was often reported to be unclear. The present results indicate that the histologk grade of tumor differentiation, capsular formation, existence of liver cirrhosis and patterns of interstitial invaslon are important factors for determining the nature of the margin. The score of argyrophilic nuclear organizer regions (AgNOR) was examined in 5 cases showing typical interstitial imaslon with the insidious type. In each case, the AgNOR score of the invading tumor cells was lower than that of turnor cells within the HCC nodules, but higher than benign hepatocytes in cirrhotic parenchyma. It clarified that the growth activity of well-differentiated HCC was rather suppressed upon their interstitial invasion.     

Cytohesin-3 is upregulated in hepatocellular carcinoma and contributes to tumor growth and vascular invasion

Hepatocellular carcinoma (HCC) is a malignant tumor with high morbidity and mortality, and is characterized by high potential for metastasis and recurrence. The outcome of it is still poor due to lacking of targeted therapeutic strategies. There is an urgent need to find new therapeutic targets for interventions against HCC metastasis and recurrence. In the present study, we found cytohesin-3, a member of the cytohesin family, was upregulated in HCC tissues, and its expression was negatively correlated with the overall survival and relapse-free survival of HCC patients. Further clinicopathological correlation analysis revealed that cytohesin-3 expression was related with tumor size and vascular invasion. And in vitro studies revealed that knock-down of cytohesin-3 suppressed HCC cells proliferation and migration. These results suggest that cytohesin-3 may act as a novel prognostic factor of HCC, and it might also be useful to exploit targeted therapeutic drugs against HCC growth and metastasis.     

全反式维甲酸对小鼠肝癌细胞Hepa1-6增殖、迁移、侵袭的影响及其机制

目的探讨不同浓度的全反式维甲酸(ATRA)对小鼠肝癌细胞Hepa1-6体外增殖、凋亡、迁移和侵袭的影响及肝癌细胞间质标志蛋白和miR200家族的表达情况。方法以Hepa1-6小鼠肝癌细胞为研究对象,给予0、0.1、1.0和10.0μmol/L终浓度的ATRA处理,结晶紫染色检测细胞增殖,锥虫蓝拒染实验计数活细胞。Hoechst检测细胞凋亡,划痕实验检测迁徙能力,Transwell实验检测侵袭能力,荧光定量PCR(real-time PCR)法检测间质标志蛋白N-cadherin、sail和vimentin和0和10μmol/L ATRA处理后的miR200家族的mRNA表达。结果 ATRA处理后Hepa1-6细胞的增殖、迁移、侵袭能力明显下降(P0.05),凋亡率增高(P0.05),间质标志蛋白N-cadherin、sail和vimentin的表达明显下调(P0.05),ATRA的浓度越高,这些作用越明显。10μmol/L ATRA处理后miRNA200a-3p,200c-3p,141-3p显著上调。结论 ATRA呈浓度依赖性促进肿瘤细胞Hepa1-6凋亡,抑制其增殖、迁移及侵袭能力,这可能与ATRA上调microRNA200家族,抑制细胞的间质表型有关。     

Carboxypeptidase A4 promotes proliferation and stem cell characteristics of hepatocellular carcinoma

Carboxypeptidase A4 (CPA4), a member of the metallo‐carboxypeptidase family, is overexpressed in liver cancer and is associated with cancer progression. The role of CPA4 in hepatocellular carcinoma (HCC) remains unclear. In this study, we aimed to evaluate the relevance of CPA4 to the proliferation and expression of stem cell characteristics of hepatocellular carcinoma cells. Western blot analysis showed high CPA4 expression in the liver cancer cell line Bel7402 and low expression in HepG2 cells. Knock‐down of CPA4 decreased cancer cell proliferation as detected by MTT and clone formation assays. The serum‐free culture system revealed that downregulated CPA4 suppressed the sphere formation capacities of tumour cells. However, upregulated CPA4 increased the proliferation and sphere formation capacity. In addition, the protein expression of CD133, ALDH1 and CD44 also increased in cells with upregulated CPA4. In vivo, the overexpression of CPA4 in tumour cells that were subcutaneously injected into nude mice markedly increased the growth of the tumours. These data suggest that CPA4 expression leads to poor prognoses by regulating tumour proliferation and the expression of stem cell characteristics and may therefore serve as a potential therapeutic target of HCC.     

MDA-7/IL-24真核表达载体的构建和重组腺病毒的制备及其对肝癌细胞生长抑制作用的研究

目的:构建含有MDA-7基因的的真核表达载体并制备重组腺病毒,转染/感染肝癌细胞后,观察其对细胞增殖的影响.方法:构建MDA-7的真核表达载体,将表达质粒转染肝癌细胞,利用平板克隆形成实验观察MDA-7分子对肿瘤细胞系克隆形成能力的影响.利用Adeasy-1腺病毒重组系统构建、包装并扩增含有MDA-7基因的重组腺病毒.利用MTT实验检测Ad-MDA-7对肿瘤细胞增殖的影响.结果:成功构建了MDA-7真核表达载体,利用平板克隆形成实验证实其可抑制肝癌细胞的克隆形成能力.成功地构建并包装了含有MDA-7基因的重组腺病毒,利用MTT实验证明其可抑制肝癌细胞的增殖.结论:MDA-7对肝癌细胞的增殖具有显著的抑制作用,为进一步研究其在肝癌细胞中的功能提供了重要的实验依据.     

肝星状细胞通过SDF-1/CXCR4轴诱导肝癌细胞上皮间质转分化并促进其侵袭

目的 探讨肝星状细胞是否通过SDF-1/CXCR4轴促进肝癌细胞侵袭的作用和可能机制.方法 通过Westernblot和real time RT-PCR,检测肝星状细胞LX02和肝癌细胞系SDF-1、CXCR4表达.Transwell实验检测星状细胞LX02或外源性SDF-1干预对肝癌细胞HepG2以及CXCR4基因沉默后的HepG2侵袭的影响,Westem blot检测上皮标志E-cadherin和间质标志vimentin的表达变化.结果 肝星状细胞LX02中趋化因子SDF-1高表达,4株人肝细胞癌细胞系均有CXCR4高表达,其中HepG2细胞表达最强.星状细胞或SDF-1均能诱导肝癌细胞上皮-间质分化并促进其侵袭.通过RNA干扰技术靶向沉默肝癌细胞CXCR4基因,星状细胞或SDF-1均不能增强其细胞侵袭能力,不能诱导肝癌细胞HepG2发生上皮-间质分化.结论 肝星状细胞通过趋化因子SDF-1/CXCR4轴促进肝癌细胞侵袭,其机制可能与诱导肝癌上皮-间质转化有关.     

溶血磷脂酸对肝癌细胞迁移行为的影响及其相关机制

目的研究溶血磷脂酸(lysophosphatidic acid,LPA)对肝癌细胞MHCC97H迁移行为的影响,并探究其相关的分子机制。方法采用Transwell法检测LPA处理后MHCC97H细胞迁移的变化情况,并通过Rho相关蛋白激酶(ROCK)抑制剂Y-27632检测ROCK信号通路在其中的作用,利用免疫荧光染色和免疫印迹法检测LPA对MHCC97细胞骨架F-actin表达的影响,通过原子力显微镜检测LPA作用后MHCC97H细胞弹性模量的变化。结果LPA显著促进MHCC97H的迁移,ROCK抑制剂Y-27632可阻断LPA诱导的MHCC97H细胞迁移。LPA上调MHCC97H中F-actin的表达,减小MHCC97细胞的弹性模量。结论 LPA可能主要通过ROCK/F-actin通路降低肝癌细胞MHCC97H的硬度,从而促进其迁移行为。     

SOCS-3 is associated with vascular invasion and overall survival in hepatocellular carcinoma

Aims: Alteration of the suppressor of cytokine signalling-3 (SOCS-3) has been observed in certain human cancers. However, the clinical role of this short-lived protein in hepatocellular carcinoma (HCC) is not well established. Therefore, we aimed to explore the potential role of SOCS-3 proteins in HCC. Methods: Paraffin embedded sections from 87 HCC patients were included in this study. The expression patterns of SOCS-3 proteins were analysed using immunohistochemistry and the results were correlated with clinicopathological characteristics and overall survival of the HCC patients. Results: The SOCS-3 expression of HCC lesions and the adjacent non-tumourous liver tissues was significantly correlated (p = 0.035), while the SOCS-3 expression in HCC lesions was significantly and positively correlated with vascular invasion and histological grading (p = 0.034 and 0.032, respectively). The Kaplan-Meier survival curve showed that the HCC patients with high SOCS-3 expression were associated with a poor overall survival rate in the HCC subgroup with positive vascular invasion (p = 0.014). Furthermore, a multivariate Cox regression model showed that SOCS-3 expression was also a significant determinant of the overall survival for HCC (p = 0.006). Conclusions: Our results indicate that altered SOCS-3 expression is associated with the overall survival in a subset of HCC patients with positive vascular invasion. Constitutive and altered SOCS-3 expression may have potential roles in a subset of HCC patients.     

MYCT1-TV对肝癌细胞增殖、凋亡和侵袭能力的影响

目的探讨MYCT1-TV(Myc target 1)基因过表达对肝癌Bel7402细胞增殖、凋亡和侵袭能力的影响。方法将构建的MYCT1-TV-GFP真核表达载体瞬时转染Bel7402细胞,通过RT-PCR和荧光显微镜检测转染效率,分别通过MTT、流式细胞术和Transwell实验检测转染后MYCT1-TV对Bel7402细胞增殖、凋亡和侵袭能力的影响。结果转染MYCT1-TV-GFP后,Bel7402细胞活细胞数显著减少,凋亡细胞数显著增加,穿膜细胞数显著减少。结论 MYCT1-TV过表达能抑制Bel7402细胞生长和侵袭,促进Bel7402细胞凋亡。     

PTPN13在人胃癌组织和胃癌SGC-7901细胞系中的表达及其对细胞增殖和侵袭的影响

目的分析PTPN13在人胃癌组织中及胃癌SGC-7901细胞系中的表达及其对细胞增殖、侵袭的影响。方法收集106例胃癌患者的胃癌组织及癌周正常组织标本。常规培养SGC-7901细胞并分为pc DNA3.1-PTPN13质粒转染组和未转染组。免疫组织化学法检测组织中的PTPN13表达;分析PTPN13表达与患者性别、年龄、肿瘤部位、肿瘤大小、浸润深度及转移的关系;Kaplan-Meier曲线分析不同PTPN13表达患者生存率的差异;CCK-8法检测细胞的增殖能力;Trans-well试验分析侵袭能力;Western blot检测E-cadherin、Snail及MMP9的表达。结果胃癌组织中的PTPN13阳性率低于癌周正常胃组织(31%vs 83%,P0.05);PTPN13的表达与肿瘤直径、浸润深度、淋巴结和远隔器官转移情况有关(P0.05);PTPN13阴性的胃癌患者2生存率较低;PTPN13过表达可以降低SGC-7901细胞的增殖率(P0.05),同时降低其侵袭能力(P0.05);上调PTPN13后SGC-7901细胞上皮化标志物E-cadherin表达增加,而间质化标志物Snail和MMP9表达减少。结论 PTPN13在胃癌组织和胃癌细胞中具有肿瘤抑制作用,较低的PTPN13表达提示患者预后不良。PTPN13具有成为胃癌的治疗诊断或治疗靶点的潜力。     

Silencing SATB1 influences cell invasion,migration, proliferation,and drug resistance in nasopharyngeal carcinoma

Special AT rich sequence binding protein 1 (SATB1) play an important role in many cancers, but the role of SATB1 in nasopharyngeal carcinoma (NPC) is still not full understand. Immunofluorescence staining showed that SATB1 was mainly localized in the nuclei in CNE-2 cell. After successful down-regulation of SABT1 in NPC cell line CNE-2 by shRNA, compared to parental CNE-2 and control shRNA group, the capacity of the proliferation, migration, invasion and drug resistance of CNE-2 cell was reduced, which indicated that SATB1 may be involved in NPC development and progression. SATB1 may be a promising therapeutic target for nasopharyngeal carcinoma.     

Expression of leucine aminopeptidase 3 (LAP3) correlates with prognosis and malignant development of human hepatocellular carcinoma (HCC)

Leucine aminopeptidases (LAPs) were associated with tumor cell proliferation, invasion and/or angiogenesis. LAP3 is one important member of this family. However, its clinical significance and biological function in hepatocellular carcinoma (HCC) remains unknown. In the present study, we demonstrated that LAP3 expression was significantly up-regulated in HCC tissues as well as cells and was closely correlated with lower differentiation, positive lymph node metastasis and high Ki-67 expression, indicating a poor prognosis. Then cell viability assays, flow cytometry assays, wound-healing assays and matrigel invasion assays were performed to demonstrate that LAP3 promoted HCC cells proliferation by regulating G1/S checkpoint in cell cycle and advanced HCC cells migration. Furthermore, we discovered that knockdown LAP3 will enhance the sensitivity of HCC cells to cisplatin, thus promoting the cell death of HCC cells. Collectively, our results indicated that up-regulated expression of LAP3 might contribute to the proliferation and metastasis of HCC. Our data gains greater insight into the cancer-promoting role of LAP3 and its functions in HCC cells, possibly providing potential therapeutic strategies for clinical trials.     

HOXA10 controls proliferation,migration and invasion in oral squamous cell carcinoma

Although HOX genes are best known for acting in the regulation of important events during embryogenesis, including proliferation, differentiation and migration, alterations in their expression patterns have been frequently described in cancers. In previous studies we analyzed the expression profile of the members of the HOX family of homeobox genes in oral samples of normal mucosa and squamous cell carcinoma (OSCC) and identified differently expressed genes such as HOXA10. The present study aimed to validate the increased expression of HOXA10 in OSCCs, and to investigate the effects arising from its knockdown in OSCC cells. The levels of HOXA10 mRNA were determined in human OSCC samples and cell lines by quantitative PCR, and HOXA10-mediated effects on proliferation, apoptosis, adhesion, epithelial-mesenchymal transition (EMT), migration and invasion were studied in HSC-3 tongue carcinoma cells by using retrovirus-mediated RNA interference. Higher expression of HOXA10 mRNA was observed in OSCC cell lines and in tumor tissues compared to normal controls. HOXA10 knockdown significantly reduced the proliferation of the tumor cells which was accompanied by increased levels of p21. HOXA10 silencing also significantly induced the expression of EMT markers and enhanced the adhesion, migration and invasion of HSC-3 cells. No effects on cell death were observed after HOXA10 knockdown. The results of the current study confirm the overexpression of HOXA10 in OSCCs, and further demonstrate that its expression is functionally associated with several important biological processes related to oral tumorigenesis, such as proliferation, migration and invasion.