Caveolin-1基因沉默促进乳腺上皮细胞ERα36 介导的PI3K/AKT信号通路的激活

Caveolin-1(窖蛋白-1)单倍不足可以促进正常乳腺细胞的早期转化, 与新型雌激素受体亚型ERα36介导的膜始动雌激素信号通路的激活有关, 但是关于其机制并未被研究清楚. 本文利用siRNA技术建立了Caveolin-1低表达而ERα36高表达的稳定传代细胞模型MCF10ACE, 采用基因芯片技术检测了ERα36高表达时雌激素信号通路基因表达谱, 研究了ERα36在雌激素激活的PI3K/AKT信号通路中的作用及其与乳腺细胞转化的关系. 结果表明: (1) Caveolin-1表达降低时可以以雌激素依赖性方式促进ERα36表达增加; (2) ERα36高表达可介导MCF10ACE细胞雌激素抗凋亡信号通路(PI3K/AKT)的激活, 促进其与增殖信号通路MEK/ERK之间的交叉对话, 从而使人乳腺细胞增殖加快, 逐渐转化. 结果提示, Caveolin-1与ERα36相互作用调节膜起始的雌激素信号通路是控制乳腺细胞转化的重要机制.     

PI3K/AKT途径在大肠埃希菌诱导U937细胞凋亡中的作用

目的探讨PI3K/AKT信号转导通路在大肠埃希菌（Escherichia coli,E.coli）诱导的人巨噬细胞系U937细胞凋亡中的作用。方法利用Western blot分析检测E.coli感染不同时间后磷酸化及非磷酸化AKT的表达;预先用不同浓度的LY294002（PI3K途径抑制剂）处理U937细胞60min,观察E.coli感染30min后U937细胞的凋亡情况。结果随着感染时间的延长,磷酸化AKT的表达逐渐下降。加入PI3K的抑制剂LY294002后,U937细胞的凋亡率逐渐升高。结论PI3K/AKT信号转导通路参与了E. coli诱导的U937细胞凋亡过程。LY294002通过特异性地抑制PI3K/AKT活性增加E.coli诱导的U937细胞凋亡率。     

臭椿酮抑制急性骨髓性白血病细胞恶性生物学行为的研究

为了探讨臭椿酮（ailanthone,AIL）对急性骨髓性白血病（acute myelogenous leukemia,AML）细胞恶性生物学行为的影响,用不同浓度（0.2、0.4、0.8、1.6、3.2 μmol·L^-1）的AIL处理对数生长期的HL-60细胞,将miR-449a mimic质粒、mimic对照质粒、miR-449a inhibitor质粒、inhibitor对照质粒分别转染至未经任何处理的HL-60细胞,并用1.0 μmol·L^-1浓度的AIL处理细胞24 h。采用CCK-8法检测细胞增殖水平,细胞划痕实验检测细胞迁移水平,Transwell小室法检测细胞侵袭水平,Annexin V-FITC/PI双染法检测细胞凋亡水平,qRT-PCR法检测miR-449a mRNA表达水平,Western blot法检测磷脂酰肌醇3-激酶（PI3K）、磷酸化PI3K（p-PI3K）、蛋白激酶B（AKT）、磷酸化AKT（p-AKT）蛋白表达水平。结果显示,AIL干预后HL-60细胞增殖抑制率、凋亡率升高,细胞迁移率及细胞侵袭数降低（P<0.05）,miR-449a mRNA表达量升高（P<0.05）。过表达miR-449a可以抑制HL-60细胞增殖、迁移和侵袭,并诱导细胞凋亡（P<0.05）,抑制miR-449a的表达可以起到逆转AIL抑制HL-60细胞增殖、迁移和侵袭,诱导细胞凋亡的作用（P<0.05）。AIL能够显著降低HL-60细胞中p-PI3K/PI3K和p-AKT/AKT比值（P<0.05）,抑制miR-449a表达可以逆转AIL对HL-60细胞p-PI3K/PI3K和p-AKT/AKT比值的下调作用（P<0.05）。结果表明,AIL可通过上调miR-449a抑制AML细胞的增殖、迁移和侵袭,并诱导细胞凋亡,其作用机制可能与抑制PI3K/AKT信号通路有关。结果表明,AIL有望成为AML治疗的候选药物。     

溶瘤腺病毒ZD55-IL24联合雷帕霉素协同抑制Hep3B细胞生长的研究

PI3K/AKT/m TOR信号通路的激活能引发人体细胞发生癌变,哺乳动物靶标m TOR作为PI3K/AKT信号通路下游的一个效应分子,被视为针对肿瘤发生和形成的关键治疗靶点,因此阻断该信号通路的相关靶点可以作为恶性肿瘤治疗的一个策略。白细胞介素24(interleukin 24,IL24)是一个选择性诱导肿瘤细胞凋亡的重要抑癌基因。该研究分别利用MTT法和实时无标记细胞功能分析仪检测携带IL24基因的溶瘤腺病毒ZD55-IL24与m TOR抑制剂雷帕霉素(rapamycin)单独或联合作用对多种肝癌细胞的体外杀伤效果;倒置显微镜分别观察ZD55-IL24、雷帕霉素单独作用及两者联合作用引起的细胞形态学变化;Hoechst 33342、流式细胞术和TUNEL染色检测各处理组细胞的凋亡情况;Western blot检测IL24、AKT以及凋亡相关蛋白Bax和Bcl-2的蛋白质水平。结果显示,溶瘤腺病毒ZD55-IL24与雷帕霉素联合作用较两者单独作用更显著地抑制了肝癌细胞Hep3B的生长并诱导了肝癌细胞的凋亡;此外,两者联合作用更有效地上调了肝癌细胞Hep3B中的细胞因子IL24和促凋亡蛋白Bax的表达,同时下调了PI3K/AKT/m TOR信号通路关键蛋白AKT和抗凋亡蛋白Bcl-2的表达。该研究结果表明,雷帕霉素很可能在一定程度上促进了ZD55-IL24病毒介导的IL24表达而增强对肝癌细胞的杀伤作用,进而为肝癌治疗研究提供了一条新型有效的方案。     

蛋白激酶AKT 不同亚型影响乳腺癌恶性表型的研究进展

细胞的增殖、转移、存活等细胞生物学过程的异常对人类众多疾病尤其是恶性肿瘤的发生发展至关重要。大量研究表明,PI3K/AKT信号通路的异常激活在肿瘤的恶性转化过程中发挥重要作用并具有普遍意义。但是,目前的研究多集中于探讨AKT总的激酶活性,而往往忽视了AKT不同亚型的特异性功能。近年来在乳腺癌中的研究发现,AKT家族不同亚型的激酶分子在调控肿瘤细胞的存活、生长、增殖、代谢、转移等众多恶性表型方面发挥独特而关键的作用:与Akt1促进肿瘤细胞增殖、抑制肿瘤细胞转移的作用相反,Akt2在促进肿瘤细胞转移、抑制肿瘤细胞增殖方面发挥重要功能;此外,随着对AKT家族研究的深入,人们对Akt3的特异性生物学功能也有了新的认识。本文在此对AKT不同亚型与乳腺癌恶性表型之间关系的研究进展做一总结。     

干扰视黄醇结合蛋白4对猪脂肪细胞PI3K/Akt信号通路的影响

视黄醇结合蛋白4(Retinol binding protein 4,RBP4)是一种脂肪细胞分泌因子,其表达水平的升高与胰岛素抵抗及Ⅱ型糖尿病等疾病密切相关,但具体作用机制尚不清楚。为明确此机制,通过包装RBP4干扰慢病毒并侵染猪前体脂肪细胞。运用胰岛素激活及诱导胰岛素抵抗模型,利用QRT-PCR及Western blotting方法检测RBP4的干扰效率及处理组PI3K/Akt信号通路相关基因的表达。结果显示RBP4的基因及蛋白的干扰效率达到60%(P<0.01)以上。进一步研究发现在胰岛素诱导及胰岛素抵抗的情况下,LH1-shRBP4干扰后可显著提高胰岛素信号通路AKT2、PI3K、GLUT4和IRS1基因mRNA的表达;明显促进AKT2、PI3K和IRS1蛋白的磷酸化;提高AKT2、PI3K和GLUT4基因的总蛋白水平。总之,RBP4干扰通过上调PI3K/Akt胰岛素信号通路相关因子的表达及其磷酸化水平,提高了胰岛素敏感性。此研究将为胰岛素抵抗相关疾病的治疗提供新思路。     

PI3K/AKT信号通路调控Myogenin和MCK基因的表达

骨骼肌分化过程受多个信号通路调控, PI3K/AKT信号通路是其中最重要的信号转导通路之一。PI3K/AKT信号通路可以调控骨骼肌分化, 但在染色质水平上的调控机制还不是很清楚。文章以小鼠成肌细胞(C2C12)为研究材料, 采用免疫印迹、染色质免疫共沉淀(Chromatin immunoprecipitation, ChIP)、定量PCR (Q-PCR)的方法研究PI3K/AKT信号通路调控Myogenin和MCK基因的表达。研究发现, C2C12细胞分化过程中添加PI3K/AKT信号通路激活剂处理24 h, Myogenin和MCK蛋白表达水平显著升高, 组蛋白H3K27me3去甲基化酶UTX的表达也升高, H3K27me3在Myogenin基因启动子区和MCK基因启动子及增强子区的富集与对照组相比显著降低。用PI3K/AKT信号通路抑制剂处理, 结果相反。因此, PI3K/AKT信号通路可能通过调控组蛋白去甲基化酶UTX的表达活性改变靶基因的H3K27me3的富集进而调控骨骼肌分化。     

甲胎蛋白抑制PTEN活性导致肝癌细胞耐受ATRA诱导的凋亡

研究甲胎蛋白(α-fetoprotein,AFP)对肝癌细胞内PTEN/AKT信息通路信号传递的影响.用Western blotting法分析全反式维甲酸(all trans retinoic acid,ATRA)处理人肝癌Bel 7402和HepG2细胞24 h后PTEN表达的变化.免疫共沉淀(Co-IP)技术研究AFP与PTEN相互作用.激光共聚焦显微镜观察AFP与PTEN在细胞共定位.RNA干扰(RNAi)技术抑制AFP表达,再用ATRA处理24 h后检测细胞内PTEN表达的变化,并分析蛋白激酶B(AKT)的磷酸化.用pcDNA3.1质粒和人afp基因连接构建表达AFP的载体(称为pcDNA3.1-afp),然后转染到不表达AFP的人肝癌HLE细胞.结果显示,人肝癌Bel 7402和HepG2细胞均有PTEN的表达,ATRA(160μmol/L)处理24 h后能促进这些细胞的PTEN表达.Co-IP技术研究发现AFP能与PTEN结合.共聚焦显微镜观察显示AFP与PTEN共定位于细胞浆.干扰AFP表达后,PTEN表达明显提高.抑制AFP表达后,ATRA能显著促进Bel 7402细胞内PTEN的表达,并能抑制AKT的磷酸化.转染pcDNA3.1-afp载体后,HLE细胞内有AFP表达,并与PTEN结合,且发现pcDNA3.1-afp载体能增加AKT的磷酸化[p-AKT(Ser473)],对抗ATRA抑制HLE细胞增殖.研究的结论是:肝癌细胞内表达的AFP能与PTEN结合并抑制PTEN对AKT的去磷酸化作用,肝癌细胞内高表达的AFP能激活AKT信息通路.胞浆内的AFP是肝癌细胞耐受ATRA的重要因子.     

靶向干扰TAGLN表达对HBV阳性肝癌细胞生物学行为的影响及机制初探

目的:探究TAGLN对HBV阳性肝癌细胞HepG2. 2. 15生物学行为的影响及可能的作用机制。方法:免疫组化法和Western blot检测TAGLN在HBV阳性和HBV阴性肝癌组织及细胞中的表达差异;用TAGLN干扰慢病毒感染HepG2. 2. 15细胞,通过嘌呤霉素筛选干扰TAGLN表达的稳定表达细胞系,Western blot验证干扰效率; CCK-8法和克隆形成实验检测干扰TAGLN表达对HepG2. 2. 15细胞增殖能力的影响; Transwell实验检测干扰TAGLN表达对HepG2. 2. 15细胞迁移和侵袭的影响; Western blot检测PI3K、p-PI3K、AKT以及p-AKT的表达。结果:TAGLN在HBV阳性肝癌组织及细胞中的表达高于HBV阴性肝癌组织和细胞(P 0. 01);干扰TAGLN表达能抑制HepG2. 2. 15细胞增殖、克隆形成能力、迁移和侵袭(P 0. 01);降低HepG2. 2. 15细胞中PI3K和AKT(P 0. 01)及p-PI3K和p-AKT(P 0. 05)的表达。结论:在肝癌组织中,HBV感染能增加TAGLN的表达;干扰TAGLN表达后HepG2. 2. 15细胞的增殖能力、克隆形成能力、迁移和侵袭的能力减弱,其机制可能与PI3K及AKT的表达减少有关。     

RHCG基因在非小细胞肺癌中的表达及对细胞增殖的影响

为了探讨Rh type C glycoprotein (RHCG)对非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞增殖的影响及可能的作用机制,本研究使用荧光定量PCR法检测12对NSCLC及癌旁组织样本中RHCG mRNA的表达水平及pcDNA3.1-RHCG质粒对A549细胞RHCG m RNA的表达;采用CCK-8法检测细胞增殖能力;运用PI染色法检测细胞周期;使用免疫印迹法检p-PI3K、PI3K、p-AKT以及AKT蛋白表达水平。本研究发现,与癌旁组织比较,NSCLC中RHCG m RNA表达水平明显降低。RHCG过表达能抑制NSCLC细胞系A549细胞增殖能力。此外,RHCG过表达使A549细胞周期G1/S期转化发生阻滞。本研究还发现,RHCG过表达可下调A549细胞p-PI3K/PI3K和p-AKT/AKT水平。本研究表明,RHCG抑制NSCLC细胞增殖的作用与其抑制PI3K/AKT信号通路有关。     

Hepatitis B virus X protein upregulates alpha-fetoprotein to promote hepatocellular carcinoma by targeting miR-1236 and miR-329

Hepatitis B virus (HBV) infection is the most common cause of hepatocellular carcinoma (HCC) worldwide, wherein the expression of alpha-fetoprotein (AFP) is reactivated to promote tumorgenesis. Hepatitis B virus X protein (HBx) protein encoded by the HBV virus X gene has been considered to be oncogenic and implicated in hepatocarcinogenesis. However, the relationship between HBx and abnormal AFP expression in HCC is yet to be fully understood. To explore the potential regulation of HBx on AFP re-expression in HCC, 97 HCC samples of different etiologies were analyzed, and extremely higher serum AFP levels were found in patients with HBsAg⁺. Analyses of HBV-related HCC specimens showed that the expression of AFP was negatively correlated with the levels of miR-1236 and miR-329. Further analyses indicated that HBx promotes the expression of AFP by orchestrating the levels of miR-1236 and miR-329 both in vitro and in vivo. Specifically, miR-1236 and miR-329 bind to the potential target sequences in AFP mRNA 3′-untranslated region to suppress its expression. HBx transfection resulted in the significant decrement of these microRNAs and increment of AFP expression. Moreover, AFP promotes the proliferation of hepatoma cells and attenuates the proapoptotic effect of chemotherapy agents. These findings revealed a novel regulatory mechanism of HBx on the abnormal AFP expression in HCC, which may provide a therapeutic approach for combating HBV-related HCC by targeting the regulation of AFP expression.     

Pattern analysis of serum alpha-fetoprotein in the early diagnosis of hepatocellular carcinoma in liver cirrhosis

In a surveillance program for hepatocellular carcinoma (HCC), serum alpha-fetoprotein (AFP) was determined every 4 months in 164 patients with liver cirrhosis. Ultrasonography (US) was performed yearly or as dictated by abnormal AFP levels. During a follow-up of 32.5 +/- 20.8 months HCC was identified by US in 16 patients. In 9 of them the AFP levels rose steadily over 4 months, increasing 7, 8 and 12 months in 3 cases before the lesion became detectable by US. In 4 patients tumors developed despite persistently normal AFP levels. Nine more patients showed abnormal fluctuations of AFP but HCC was not detected. AFP sensitivity was higher at a low cut-off point (40 ng/ml) while specificity of the test appeared higher at the 200 ng/ml cut-off point. An AFP value rising steeply over a few months appeared more reliable than a fixed preset threshold in indicating carcinomatous transformation. Screening for AFP can be expected to uncover about 3/4 of HCC developing in cirrhotics with few false-positive reactions. The test may have a unique role in identifying a subset of liver tumors whose early expression is AFP production.     

Alpha fetoprotein plays a critical role in promoting metastasis of hepatocellular carcinoma cells

A high level of serum alpha fetoprotein (AFP) is positively associated with human hepatocellular carcinoma (HCC) carcinogenesis and metastasis; however, the function of AFP in HCC metastasis is unknown. This study has explored the effects of AFP on regulating metastatic and invasive capacity of human HCC cells. Forty‐seven clinical patients' liver samples were collected and diagnosed; HCC cells line, Bel 7402 cells (AFP‐producing) and liver cancer cell line cells (non‐AFP‐producing) were selected to analyse the role of AFP in the metastasis of HCC cells. The results indicated that high serum concentration of AFP was positively correlated with HCC intrahepatic, lymph nodes and lung metastasis. Repressed expression of AFP significantly inhibited the capability of migration and invasion of Bel 7402 cells, expression of keratin 19 (K19), epithelial cell adhesion molecule (EpCAM), matrix metalloproteinase 2/9 (MMP2/9) and CXC chemokine receptor 4 (CXCR4) were also down‐regulated in Bel 7402 cells; migration and invasion, expression of K19, EpCAM, MMP2/9 and CXCR4 were significantly enhanced when HLE cells were transfected with AFP‐expressed vector. The results demonstrated that AFP plays a critical role in promoting metastasis of HCC; AFP promoted HCC cell invasion and metastasis via up‐regulating expression of metastasis‐related proteins. Thus, AFP may be used as a novel therapeutic target for treating HCC patients.     

Interaction of aflatoxin and hepatitis B virus in the pathogenesis of hepatocellular carcinoma

Aflatoxin-B1 (AFB) and chronic hepatitis B virus (HBV) infection epidemiologically correlate with the geographic distribution of hepatocellular carcinoma (HCC). Integration of HBV DNA into the cellular genome of HCCs and the in vivo formation of adducts between AFB and nucleic acids lead us to suggest that hepatocytes with integrated HBV DNA preferentially accumulate AFB; the AFB-adducts formed may then initiate cell transformation by modifying the expression of critical host genes. The altered molecular biology of liver cells in HCC is evidenced by the fact that HBV does not replicate in HCC tissues or cell lines. The effect of AFB on the expression of cellular genes such as endogenous retrovirus(es) and possibly cellular oncogene(s) can be analyzed in HCC cell lines with and without integrated HBV DNA. In addition, human HCC tissues can be probed for HBV sequences and AFB-DNA adducts at the single-cell level. The presence of HBV and AFB can be correlated with the expression of putative transforming genes, providing a new insight into the interaction between liver cells, HBV and AFB in the pathogenesis of HCC.     

Immunohistochemical study of the appearance of some markers in liver adjoining hepatocellular carcinoma

The presence and distribution of AFP, AAT and HBsAg in peritumoral non-neoplastic hepatocytes (NNH) of 27 cases and, at the same time, in the neoplastic tissue of 37 liver cell carcinoma (HCC) were studied; AFP and HBsAg were more frequently found in NNH than in HCC cells; no differences were found for AAT. The presence of HBsAg also in normal liver without cirrhosis is probably best explained by its possible role in neoplastic transformation and by the inhibition of replication of the viruses AFP, considered to be expression of dedifferentiated cells, may possible be taken up by NNH for catabolic purposes.     

Quantitative proteomic signature of liver cancer cells: tissue transglutaminase 2 could be a novel protein candidate of human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common diseases worldwide, with extremely poor prognosis due to failure in diagnosing it early. Alpha-fetoprotein (AFP) is the only available biomarker for HCC diagnosis; however, its use in the early detection of HCC is limited, especially because about one-third of patients afflicted with HCC have normal levels of serum AFP. Thus, identifying additional biomarkers that may be used in combination with AFP to improve early detection of HCC is greatly needed. A quantitative proteomic analysis approach using stable isotope labeling with amino acids in cell culture (SILAC) combined with LTQ-FT-MS/MS identification was used to explore differentially expressed protein profiles between normal (HL-7702) and cancer (HepG2 and SK-HEP-1) cells. A total of 116 proteins were recognized as potential markers that could distinguish between HCC and normal liver cells. Certain proteins, such as AFP, intercellular adhesion molecule-1 (ICAM-1), IQ motif containing GTPase activating protein 2 (IQGAP2), claudin-1 (CLDN1) and tissue transglutaminase 2 (TGM2), were validated both in multiple cell lines and in 61 specimens of clinical HCC cases. TGM2 was overexpressed in some of the AFP-deficient HCC cells (SK-HEP-1 and Bel-7402) and in about half of the tumor tissues with low levels of serum AFP (17/32, AFP-negative HCC). Trace amounts of TGM2 were found to be expressed in the samples with high serum AFP (26/29, AFP-positive HCC). Moreover, TGM2 expression in liver tissues showed an inverse correlation with the level of serum AFP in HCC patients. Notably, TGM2 existed in the supernatant of the AFP-deficient SK-HEP-1, SMMC-7721 and HLE cells, and it was found to be induced in AFP-producing cells (HepG2) by specific siRNA silence assay. Serum TGM2 levels of 109 HCC patients and 42 healthy controls were further measured by an established ELISA assay; the levels were significantly higher in HCC patients, and they correlated with the histological grade and tumor size. These data suggest that TGM2 may serve as a novel histological/serologic candidate involved in HCC, especially for the individuals with normal serum AFP. These novel findings may provide important clues to identify new biomarkers of HCC and indirectly improve early detection of the disease.     

Genome-Wide and Differential Proteomic Analysis of Hepatitis B Virus and Aflatoxin B1 Related Hepatocellular Carcinoma in Guangxi,China

Both hepatitis B virus (HBV) and aflatoxin B1 (AFB1) exposure can cause liver damage as well as increase the probability of hepatocellular carcinoma (HCC). To investigate the underlying genetic changes that may influence development of HCC associated with HBV infection and AFB1 exposure, HCC patients were subdivided into 4 groups depending upon HBV and AFB1 exposure status: (HBV(+)/AFB1(+), HBV(+)/AFB1(-), HBV(-)/AFB1(+), HBV(-)/AFB1(-)). Genetic abnormalities and protein expression profiles were analyzed by array-based comparative genomic hybridization and isobaric tagging for quantitation. A total of 573 chromosomal aberrations (CNAs) including 184 increased and 389 decreased were detected in our study population. Twenty-five recurrently altered regions (RARs; chromosomal alterations observed in ≥10 patients) in chromosomes were identified. Loss of 4q13.3-q35.2, 13q12.1-q21.2 and gain of 7q11.2-q35 were observed with a higher frequency in the HBV(+)/AFB1(+), HBV(+)/AFB1(-) and HBV(-)/AFB1(+) groups compared to the HBV(-)/AFB(-) group. Loss of 8p12-p23.2 was associated with high TNM stage tumors (P = 0.038) and was an unfavorable prognostic factor for tumor-free survival (P =0.045). A total of 133 differentially expressed proteins were identified in iTRAQ proteomics analysis, 69 (51.8%) of which mapped within identified RARs. The most common biological processes affected by HBV and AFB1 status in HCC tumorigenesis were detoxification and drug metabolism pathways, antigen processing and anti-apoptosis pathways. Expression of AKR1B10 was increased significantly in the HBV(+)/AFB1(+) and HBV(-)/AFB1(+) groups. A significant correlation between the expression of AKR1B10 mRNA and protein levels as well as AKR1B10 copy number was observered, which suggest that AKR1B10 may play a role in AFB1-related hepatocarcinogenesis. In summary, a number of genetic and gene expression alterations were found to be associated with HBV and AFB1- related HCC. The possible synergistic effects of HBV and AFB1 in hepatocarcinogenesis warrant further investigations.