LncRNA FOXD2‐AS1 as a competitive endogenous RNA against miR‐150‐5p reverses resistance to sorafenib in hepatocellular carcinoma

The current study elucidated the role of a long non‐coding RNA (lncRNA), FOXD2‐AS1, in the pathogenesis of hepatocellular carcinoma (HCC) and the regulatory mechanism underlying FOXD2‐AS1/miR‐150‐5p/transmembrane protein 9 (TMEM9) signalling in HCC. Microarray analysis was used for preliminary screening of candidate lncRNAs in HCC tissues. qRT‐PCR and Western blot analyses were used to detect the expression of FOXD2‐AS1. Cell proliferation assays, luciferase assay and RNA immunoprecipitation were performed to examine the mechanism by which FOXD2‐AS1 mediates sorafenib resistance in HCC cells. FOXD2‐AS1 and TMEM9 were significantly decreased and miR‐150‐5p was increased in SR‐HepG2 and SR‐HUH7 cells compared with control parental cells. Overexpression of FOXD2‐AS1 increased TMEM9 expression and overcame the resistance of SR‐HepG2 and SR‐HUH7 cells. Conversely, knockdown of FOXD2‐AS1 decreased TMEM9 expression and increased the sensitivity of HepG2 and Huh7 cells to sorafenib. Our data also demonstrated that FOXD2‐AS1 functioned as a sponge for miR‐150‐5p to modulate TMEM9 expression. Taken together, our findings revealed that FOXD2‐AS1 is an important regulator of TMEM9 and contributed to sorafenib resistance. Thus, FOXD2‐AS1 may serve as a therapeutic target against sorafenib resistance in HCC.     

FAM225A promotes sorafenib resistance in hepatocarcinoma cells through modulating miR-130a-5p–CCNG1 interaction network

Chemotherapy resistance is still a key hurdle in current hepatocellular carcinoma (HCC) treatment. Therefore, clarifying the molecular mechanisms contributing to this acquired resistance is urgent for the effective treatment of liver cancer. In this research, we observed that lncRNA FAM225A expression is dramatically up-regulated not only in HCC tissues and cell lines but also in sorafenib-resistant HepG2/SOR cells. Moreover, FAM225A knockdown significantly weakened HepG2/SOR cells resistance to sorafenib treatment by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Similar results were obtained from the tumor xenograft model in mice. Further mechanistic researches revealed that the direct interaction between FAM225A and miR-130a-5p, while miR-130a-5p negatively modulated Cyclin G1 (CCNG1) expression by targeting 3′UTR of CCNG1. MiR-130a-5p inhibition or CCNG1 overexpression could partially offset FAM225A knockdown-induced increased viability of HepG2/SOR cells in response to sorafenib challenge. Collectively, our findings provide evidence that FAM225A/miR-130a-5p/CCNG1 interaction network regulates the resistance of HCC cells to sorafenib treatment and could supply a possible strategy for restoring sorafenib sensitivity in HCC therapy.     

XIAP及survivin 在原发性肝细胞癌组织中的表达及其相关性研究

目的：探讨X 连锁凋亡抑制蛋白（XIAP）和survivin 在原发性肝细胞癌中的表达及两者的相关性。方法：选取本院收治的60 例原发性肝细胞癌患者,应用免疫组织化学染色的方法对肝癌组织及癌旁组织中的XIAP及survivin 的表达进行检测。结果：经 比较,肝癌组织中XAIP 及survivin 的阳性率均显著高于癌旁组织,差异有统计学意义。XIAP 和survivin 的表达强度与肿瘤的大 小无关,但随肿瘤的分化程度的降低而升高,且不同分化程度之间差异有统计学意义;XIAP 和survivin 存在正相关关系。结论： XIAP与Survivin 在肿瘤组织中的高表达在促进肿瘤发生、增殖、转移以及耐药,并且能够降低肿瘤的分化程度,增加肝癌的恶性 程度。此外,两者可能存在协同作用,但两者的相关性及作用机制仍需进一步探讨。     

XIAP及survivin在原发性肝细胞癌组织中的表达及其相关性研究

目的：探讨X连锁凋亡抑制蛋白（XIAP）和survivin在原发性肝细胞癌中的表达及两者的相关性。方法：选取本院收治的60例原发性肝细胞癌患者,应用免疫组织化学染色的方法对肝癌组织及癌旁组织中的XIAP及survivin的表达进行检测。结果：经比较,肝癌组织中XAIP及survivin的阳性率均显著高于癌旁组织,差异有统计学意义。XIAP和survivin的表达强度与肿瘤的大小无关,但随肿瘤的分化程度的降低而升高,且不同分化程度之间差异有统计学意义;XIAP和survivin存在正相关关系。结论：XIAP与Survivin在肿瘤组织中的高表达在促进肿瘤发生、增殖、转移以及耐药,并且能够降低肿瘤的分化程度,增加肝癌的恶性程度。此外,两者可能存在协同作用,但两者的相关性及作用机制仍需进一步探讨。     

Enhanced expression of mRNAs of antisecretory factor-1, gp96, DAD1 and CDC34 in human hepatocellular carcinomas

To identify differentially expressed genes in hepatocarcinogenesis, we performed differential display analysis using surgically resected hepatocellular carcinoma (HCC) and adjacent non-tumorous liver tissues. We identified four cDNA fragments upregulated in HCC samples, encoding antisecretory factor-1 (AF), gp96, DAD1 and CDC34. Northern blot analysis demonstrated that these mRNAs were expressed preferentially in HCCs compared with adjacent non-tumorous liver tissues or normal liver tissues from non-HCC patients. The expression of these mRNAs was increased along with the histological grading of HCC tissues. These mRNA levels were also high in three human HCC cell lines (HuH-7, HepG2 and HLF), irrespective of the growth state. We also demonstrate that sodium butyrate, an inducer of differentiation, downregulated the expression of AF and gp96 mRNAs, supporting in part our pathological observation. Immunohistochemical analysis revealed that gp96 and CDC34 proteins were preferentially accumulated in cytoplasm and nuclei of HCC cells, respectively. Overexpression of these genes could be an important manifestation of HCC phenotypes and should provide clues to understand the molecular basis of hepatocellular carcinogenesis.     

Aberrant Expression of the p53-Inducible Antiproliferative Gene BTG2 in Hepatocellular Carcinoma is Associated with Overexpression of the Cell Cycle-Related Proteins

We previously reported that the abnormal BTG2 expression was related to genesis/development of hepatocellular carcinoma (HCC). The aim of this study was to evaluate the BTG2 expression in HCC compared with p53, cyclin D1, and cyclin E. For this purpose, modified diethylnitrosamine (DEN)-induced primary HCC rat model was established. Target proteins and mRNAs were measured by western blot and RT-PCR/northern blot, respectively. In rat liver, expression of BTG2 and other proteins was determined by western blot, and BTG2 mRNA in HCC/normal tissues was detected by high-flux tissue microarray (TMA) and in situ hybridization (ISH). BTG2 mRNA/protein expression was increased in fetal liver, 7701, and LO2 cell lines but decreased in HepG2 cells. BTG2/p53 were expressed early after DEN treatment, peaked at 5?weeks and decreased gradually thereafter. Cyclin-D1/Cyclin-E expression increased significantly with the tumor progression. BTG2 mRNA was expressed in 71.19% HCC by ISH and correlated with differentiation. Expression of p53/cyclin D1/cyclin E was positive in 82.35/94.12/76.47% BTG2 mRNA-negative tissues, respectively. BTG2 protein expression was lost in 32.2% (19/59) HCC tissues, and the mRNA/protein expression correlated significantly with the increasing tumor grade (P?相似文献   

MiR-27a-3p enhances the cisplatin sensitivity in hepatocellular carcinoma cells through inhibiting PI3K/Akt pathway

MicroRNAs (miRNAs) play an important role in drug resistance, and it is reported that miR-27a-3p regulated the sensitivity of cisplatin in breast cancer, lung cancer and ovarian cancer. However, the relationship between miR-27a-3p and chemosensitivity of cisplatin in hepatocellular carcinoma (HCC) was unclear, especially the underlying mechanism was unknown. In the present study, we analyzed miR-27a-3p expression levels in 372 tumor tissues and 49 adjacent tissues in HCC samples from TCGA database, and found that the miR-27a-3p was down-regulated in HCC tissues. The level of miR-27a-3p was associated with metastasis, Child–Pugh grade and race. MiR-27a-3p was regarded as a favorable prognosis indicator for HCC patients. Then, miR-27a-3p was overexpressed in HepG2 cell, and was knocked down in PLC cell. Next, we conducted a series of in vitro assays, including MTT, apoptosis and cell cycle assays to observe the biological changes. Further, inhibitor rate and apoptosis rate were detected with pre- and post-cisplatin treatment in HCC. The results showed that overexpression of miR-27a-3p repressed the cell viability, promoted apoptosis and increased the percentage of cells in G₀/G₁ phase. Importantly, overexpression of miR-27a-3p significantly increased the inhibitor rate and apoptosis rate with cisplatin intervention. Besides, we found that miR-27a-3p added cisplatin sensitivity potentially through regulating PI3K/Akt signaling pathway. Taken together, miR-27a-3p acted as a tumor suppressor gene in HCC cells, and it could be useful for modulating cisplatin sensitivity in chemotherapy.     

miR-26b enhances the sensitivity of hepatocellular carcinoma to Doxorubicin via USP9X-dependent degradation of p53 and regulation of autophagy

Multi-drug resistance is a major challenge to hepatocellular carcinoma (HCC) treatment, and the over-expression or deletion of microRNA (miRNA) expression is closely related to the drug-resistant properties of various cell lines. However, the underlying molecular mechanisms remain unclear. CCK-8, EdU, flow cytometry, and transmission electron microscopy were performed to determine cell viability, proliferation, apoptosis, autophagic flow, and nanoparticle characterization, respectively. In this study, the results showed that the expression of miR-26b was downregulated following doxorubicin treatment in human HCC tissues. An miR-26b mimic enhanced HCC cell doxorubicin sensitivity, except in the absence of p53 in Hep3B cells. Delivery of the proteasome inhibitor, MG132, reversed the inhibitory effect of miR-26b on the level of p53 following doxorubicin treatment. Tenovin-1 (an MDM2 inhibitor) protected p53 from ubiquitination-mediated degradation only in HepG2 cells with wild type p53. Tenovin-1 pretreatment enhanced HCC cell resistance to doxorubicin when transfected with an miR-26b mimic. Moreover, the miR-26b mimic inhibited doxorubicin-induced autophagy and the autophagy inducer, rapamycin, eliminated the differences in the drug sensitivity effect of miR-26b. In vivo, treatment with sp94dr/miR-26b mimic nanoparticles plus doxorubicin inhibited tumor growth. Our current data indicate that miR-26b enhances HCC cell sensitivity to doxorubicin through diminishing USP9X-mediated p53 de-ubiquitination caused by DNA damaging drugs and autophagy regulation. This miRNA-mediated pathway that modulates HCC will help develop novel therapeutic strategies.     

ATP citrate lyase inhibitor triggers endoplasmic reticulum stress to induce hepatocellular carcinoma cell apoptosis via p-eIF2α/ATF4/CHOP axis

ATP citrate lyase (ACLY), a key enzyme in the metabolic reprogramming of many cancers, is widely expressed in various mammalian tissues. This study aimed to evaluate the effects and mechanisms of ACLY and its inhibitor BMS-303141 on hepatocellular carcinoma (HCC). In this study, ACLY was highly expressed in HCC tissues, especially in HepG2 and Huh7 cells, but was down-regulated in Hep3B and HCC-LM3 cells. Besides, ACLY knockdown inhibited HepG2 proliferation and clone formation, while opposite result was noticed in HCC-LM3 cells with ACLY overexpression. Moreover, ACLY knockdown impeded the migration and invasion abilities of HepG2 cells. Similarly, BMS-303141 suppressed HepG2 and Huh-7 cell proliferation. The p-eIF2α, ATF4, CHOP p-IRE1α, sXBP1 and p-PERK were activated in HepG2 cells stimulated by BMS-303141. In cells where ER stress was induced, ATF4 was involved in BMS-303141-mediated cell death procession, and ATF4 knockdown reduced HCC cell apoptosis stimulated by BMS-303141. In a mouse xenograft model, combined treatment with BMS-303141 and sorafenib reduced HepG2 tumour volume and weight. In addition, ACLY expression was associated with HCC metastasis and tumour-node-metastases staging. Survival analysis and Cox proportional hazards regression model showed that overall survival was lower in HCC patients with high ACLY expression; AFP level, TNM staging, tumour size and ACLY expression level were independent risk factors affecting their overall survival. In conclusion, ACLY might represent a promising target in which BMS-303141 could induce ER stress and activate p-eIF2α/ATF4/CHOP axis to promote apoptosis of HCC cells, and synergized with sorafenib to enhance the efficacy of HCC treatment.     

Trichostatin A sensitizes HBx-expressing liver cancer cells to etoposide treatment

Agents commonly used in cancer chemotherapy rely on the induction of cell death via apoptosis, mitotic catastrophe, premature senescence and autophagy. Chemoresistance is the major factor limiting long-term treatment success in patients with hepatocellular carcinoma (HCC). Recent studies have revealed that the hepatitis B virus X protein (HBx) exerts anti-apoptotic effects, resulting in an increased drug resistance in HCC cells. In this study, we showed that etoposide treatment activated caspase-8 and caspase-3, leading to cleavages of p53, Bid and PARP, which subsequently induced apoptosis. Furthermore, p53 and Bid were accumulated in cytoplasm following etoposide treatment. However, HBx significantly attenuated etoposide-induced cell death. In HBx-expressing cells, despite the translocation of p53 and Bid to cytoplasm, the activation of caspases was inhibited. Furthermore, the phosphorylation of extracellular-signal-regulated kinase (ERK) was markedly increased in HBx-expressing cells. Moreover, the pretreatment with trichostatin A (TSA, a histone deacetylase inhibitor) or TSA in combination with etoposide significantly sensitized HCC cells to apoptosis by inhibiting ERK phosphorylation, reactivating caspases and PARP, and inducing translocation of p53 and Bid to cytoplasm. Collectively, HBx reduces the sensitivity of HCC cells to chemotherapy. TSA in combination with etoposide can significantly overcome the increased resistance of HBx-expressing HCC cells to chemotherapy.     

Increase in the nuclear localization of PTEN by the Toxoplasma GRA16 protein and subsequent induction of p53‐dependent apoptosis and anticancer effect

This study investigated the efficacy of Toxoplasma GRA16, which binds to herpes virus‐associated ubiquitin‐specific protease (HAUSP), in anticancer treatment, and whether the expression of GRA16 in genetically modified hepatocellular carcinoma (HCC) cells (GRA16‐p53‐wild HepG2 and GRA16‐p53‐null Hep3B) regulates PTEN because alterations in phosphatase and tensin homologue (PTEN) and p53 are vital in liver carcinogenesis and the abnormal p53 gene appears in HCC. For this purpose, we established the GRA16 cell lines using the pBABE retrovirus system, assessed the detailed mechanism of PTEN regulation in vitro and established the anticancer effect in xenograft mice. Our study showed that cell proliferation, antiapoptotic factors, p‐AKT/AKT ratio, cell migration and invasive activity were decreased in GRA16‐stable HepG2 cells. Conversely, the apoptotic factors PTEN and p53 and apoptotic cells were elevated in GRA16‐stable HepG2 cells but not in Hep3B cells. The change in MDM2 was inconspicuous in both HepG2 and Hep3B; however, the PTEN level was remarkably elevated in HepG2 but not in Hep3B. HAUSP‐bound GRA16 preferentially increased p53 stabilization by the nuclear localization of PTEN rather than MDM2‐dependent mechanisms. These molecular changes appeared to correlate with the decreased tumour mass in GRA16‐stable‐HepG2 cell‐xenograft nude mice. This study establishes that GRA16 is a HAUSP inhibitor that targets the nuclear localization of PTEN and induces the anticancer effect in a p53‐dependent manner. The efficacy of GRA16 could be newly highlighted in HCC treatment in a p53‐dependent manner.     

多药耐药基因在肝细胞癌中的表达及与p53、PCNA表达的相关性研究

目的　研究肝细胞癌 (HCC)组织中多药耐药基因MDR 1与p5 3蛋白及增殖细胞核抗原 (PCNA)表达的关系 ,旨在从基因水平进一步探讨预测化学治疗效果的可行性。方法　利用免疫组织化学方法 (S P法 )研究 30例肝穿活检的肝癌组织中MDR 1、p5 3和PCNA的表达。结果　 30例肝细胞癌中MDR 1的阳性表达率为 5 6 6 7% ,MDR 1阳性表达与组织学分级无关 (P >0 0 5 )。MDR 1表达与 p5 3、PCNA表达间无相关性 (P >0 0 5 )。结论　通过检测MDR 1基因可以对肝细胞癌病人进行化学治疗敏感性预测 ;肝细胞癌中MDR 1基因表达不依赖于 p5 3和细胞增殖这些因素。     

Amplification of DDR2 mediates sorafenib resistance through NF-κB/c-Rel signaling in hepatocellular carcinoma

Sorafenib was the first systemic therapy approved by the Food and Drug Administration to treat advanced hepatocellular carcinoma (HCC). However, sorafenib therapy is frequently accompanied by drug resistance. We aimed to explore the mechanisms of sorafenib resistance and provide feasible solutions to increase the response to sorafenib in patients with advanced HCC. The expression profile of discoidin domain receptor 2 (DDR2) in HCC tissues and cells was detected using quantitative real-time PCR (qPCR) and western blotting assays. The effects of DDR2 on sorafenib resistance were examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony formation, TdT-mediated dUTP nick end labeling, and flow cytometry assays. The effect of DDR2 on the nuclear factor kappa B (NF-κB) signaling pathway was evaluated by luciferase reporter, immunofluorescence, qPCR and flow cytometry assays. We demonstrated that DDR2 expression was dramatically upregulated in sorafenib-resistant HCC tissues relative to sensitive tissues. Downregulation of DDR2 sensitized HCC cell lines to sorafenib cytotoxicity. Further analysis showed that DDR2 could increase the nuclear location of REL proto-oncogene, a NF-κB subunit, to mediate NF-κB signaling. Blocking NF-κB signaling using the NF-κB signaling inhibitor, bardoxolone methyl, increased the response of HCC cells to sorafenib. Further analysis showed that DNA amplification of DDR2 is an important mechanism leading to DDR2 overexpression in HCC. Our results demonstrated that DDR2 is a potential therapeutic target in patients with HCC, and targeting DDR2 represents a promising approach to increase sorafenib sensitivity in patients with HCC.     

RNA干扰肝细胞肝癌中PECAM-1的表达对肿瘤细胞侵袭及增殖的影响

目的:探讨PECAM-1在肝细胞肝癌(Hepatocellular carcinoma,HCC)组织中的表达及意义。方法:选择2013年5月-2015年6月在我院接受治疗的HCC患者100例,收集肝癌患者HCC组织及癌旁组织,另选取100例正常肝脏组织作为对照组。应用免疫组织化学法检测PECAM-1在肝癌组织、癌旁组织以及正常肝脏组织中的阳性表达。利用小分子干扰RNA技术(si RNA)构建低表达的PECAM-1,并转染至肝癌细胞中抑制PECAM-1的表达。应用Transwell小室法检测肝癌细胞的侵袭能力,CCK-8法检测肝癌细胞的增殖能力。结果:PECAM-1在肝癌组织、癌旁组织及正常肝脏组织中呈不同程度阳性表达(P0.05);PECAM-1在肝癌组织及癌旁组织中的表达显著高于正常肝脏组织,差异具有统计学意义(P0.05);PECAM-1在肝癌组织中的表达显著高于癌旁组织,差异具有统计学意义(P0.05);转染si RNA PECAM-1后,肝癌细胞中PECAM-1 m RNA的表达水平明显下降,PECAM-1蛋白表达也明显降低,差异具有统计学意义(P0.05);转染si RNA PECAM-1后,肝癌细胞侵袭及增殖能力明显降低,差异具有统计学意义(P0.001)。结论:PECAM-1在肝癌患者血清中高表达,PECAM-1 si RNA能够抑制肝癌细胞的侵袭及增殖能力,提示PECAM-1可作为预测肝癌发生及发展的临床指标。