Phenotype Of Hepatocellular Carcinoma Cells Research Articles

RNA-binding protein RPS3 contributes to hepatocarcinogenesis by post-transcriptionally up-regulating SIRT1.

Although several studies indicate that RNA-binding proteins (RBPs) contribute to key steps in a variety of physiological processes and cancer, the detailed biological functions and mechanisms remain to be determined. By performing bioinformatics analysis using well-established hepatocellular carcinoma (HCC) datasets, we identified a set of HCC progression-associated RBPs (HPARBPs) and found that the global expression of HPARBPs was significantly correlated with patient prognosis. Among the 42 HPARBPs, human ribosomal protein S3 (RPS3) was one of the most abundant genes whose role remains uncharacterized in HCC. Gain- and loss-of-function analyses demonstrated that RPS3 promoted HCC tumorigenesis both in vitro and in vivo. Mechanistically, we revealed that silent information regulator 1 (SIRT1) was a critical target of RPS3 and was essential for sustaining the RPS3-induced malignant phenotypes of HCC cells. RPS3 stabilized SIRT1 mRNA by binding to AUUUA motifs in the 3448–3530 region of the 3′ untranslated region (UTR) of SIRT1 mRNA. In addition, we found that (5-formylfuran-2-yl) methyl 4-hydroxy-2-methylenebutanoate (FMHM) inhibited HCC progression by repressing the RPS3/SIRT1 pathway. Our study unveils a novel extra-ribosomal role of RPS3 in facilitating hepatocarcinogenesis via the post-transcriptional regulation of SIRT1 expression and proposes that the RPS3/SIRT1 pathway serves as a potential therapeutic target in HCC.

HANR promotes hepatocellular carcinoma progression via miR-214/EZH2/TGF-β axis

BackgroundLncRNA has been shown to associates with the initiation and progression of hepatocellular carcinoma (HCC). Recently, some studies showed that HANR function as an oncogene in HCC; however, the detailed mechanism of HANR-regulated HCC tumorigenesis and progression needs to be elucidated. MethodsWe used RT-qPCR method to probe genes expression. MTT assay, wound healing assay and transwell invasion assay were utilized to examine proliferation and migration and invasion abilities of HepG2 cells. Xenograft tumor experiment was used to show the growth of tumors in vivo. ResultsHANR was evidently upregulated in HCC tumors and cells compared to normal tissues and cells. Besides, HANR knockdown induces attenuated cell proliferation, migration, invasion of HCC cells. By bioinformatic analysis and dual luciferase reporter assay, we found that miR-214 was the downstream target of HANR. Furthermore, miR-214 inhibitor largely enhanced tumor phenotypes of HCC cells regulated by HANR knockdown. HANR and miR-214 regulated the EZH2, then affecting TGFBR2 level. Finally, we demonstrated that EZH2 overexpression could greatly rescue HANR knockdown or miR-214 mimic-induced HCC tumorigenesis and progression. ConclusionsIn this study, we report a newly identified regulatory mechanism HANR/miR-214/EZH2/TGF-β axis, which is implicated in tumorigenesis and progression of HCC. Our findings suggest that HANR facilitates the development of therapeutical strategies or diagnostic markers by targeting HANR.

MiR-590-5p suppresses hepatocellular carcinoma chemoresistance by targeting YAP1 expression.

BackgroundResistance to chemotherapeutic treatment is a common phenomenon in cancers, especially in hepatocellular carcinoma (HCC). The Hippo signaling pathway has been demonstrated to play a role in tumor initiation, development, and progression. However, little is known about its roles in the HCC chemoresistance.MethodsIn this study, real-time PCR and western blotting were used to identify the expression profile of key components of Hippo signaling pathway between chemoresistant and chemosensitive HCC cell lines. In vitro and in vivo loss- and gain-of-function studies were performed to reveal the effects and related mechanism of microRNA-590-5p/YAP1 axis in the chemoresistant phenotype of HCC cells.FindingsWe identified yes-associated protein 1 (YAP1) as the major dysregulated molecules in adriamycin (ADR)-resistant HCC cells. YAP1 was profoundly implicated in the chemoresistant phenotype of HCC cells. Furthermore, microRNA-590-5p was revealed as a functional modulator of YAP1. Importantly, YAP1-mediated chemoresistant phenotype was closely related to increased expression of stemness markers and ATP-binding cassette transporters. HCC patients with poor response to transarterial chemoembolization (TACE) treatment had higher protein level of YAP1 than that in the responsive patients.InterpretationThe microRNA-590-5p/YAP axis plays an important role in the chemotherapeutic resistance of HCC cells, suggesting new adjuvant chemotherapeutic directions in HCC.FundNational Natural Science Foundation of China, Zhejiang Province Medical and Health Care Key Project, Experimental Animal Science and Technology Projects of Zhejiang Province, Public Welfare Technology Application Research Project of Lishui, Chinese Medicine Science and Technology Projects of Zhejiang Province.

LINC01287/miR-298/STAT3 feedback loop regulates growth and the epithelial-to-mesenchymal transition phenotype in hepatocellular carcinoma cells

BackgroundThe long non-coding RNAs (lncRNAs) have participated in the promotion of hepatocellular carcinoma (HCC) initiation and progression. Nevertheless, the biological role and underlying mechanism of LINC01287 in HCC has never been reported.MethodsThe TGCA database was used to explore the abnormal expression of lncRNAs in HCC. Real-time PCR and in situ hybridization assays were used to examine the expression of LINC01287 in HCC tissues. The clinicopathological characteristics of HCC patients in relation to LINC01287 expression were then analyzed. Infection of cells with the si-LINC01287 lentiviral vector was performed to down-regulate LINC01287 expression in HCC cells. MTT and colony formation assays were performed to examine cell growth ability, and FACS analysis was performed to examine the cell cycle and apoptosis. A Boyden assay was used to examine HCC cell invasion ability, and RNA immunoprecipitation tested the interaction between LINC01287 and miR-298. A luciferase reporter assay was used to examine whether STAT3 was a direct target of miR-298, and chromatin immunoprecipitation (ChIP) was used to examine the potential binding of c-jun to the miR-298 promoter.ResultsWe revealed that the expression of LINC01287 was increased in HCC cell lines, as well as tissues. Knockdown of LINC01287 decreased HCC cell growth and invasion both in vitro and in vivo. LINC01287 can negatively regulate miR-298 expression by acting as a ceRNA. miR-298 directly targeted STAT3 and inhibited its expression. LINC01287 exerted its function via the miR-298/STAT3 axis in HCC. Interestingly, STAT3 elevated LINC01287 expression via c-jun, which bound to the LINC01287 promoter. A feedback loop was also discovered between LINC01287 and the miR-298/STAT3 axis.ConclusionsOur data indicated that LINC01287 played an oncogenic role in HCC growth and metastasis and that this lncRNA might serve as a novel molecular target for the treatment of HCC.

Abstract 1402: Silencing of B4GALT1 inversely regulates malignant phenotypes of hepatocellular carcinoma cells

Abstract Identifying molecular targets for hepatocellular carcinoma (HCC) is an urgent need to overcome the treatment failure and death from cancer cell invasion and metastasis. Beta-1,4-galactosyltransferase 1 (B4GALT1) has been documented as the key enzyme in morphogenesis and cellular adhesion, for it adds galactose to N-acetylglucosamine residues of glycoprotein carbohydrate chains; whereas abnormal B4GALT1 expression is associated with tumor progress in many cancers. However, functional roles of B4GALT1 in HCC remains unclear. With clinical correlation, the immunohistochemistry confirmed that lower expression of B4GALT1 in cancer tissue is associated with higher ratio of vascular invasion and worse overall survival. Consistently, stable knockdown of B4GALT1 regarding HCC cell lines promoted cell migration, and invasion; as the overexpressed B4GALT1 inhibited cell migration and invasion of HCC cell lines. Moreover, our data suggested that down-regulated B4GALT1 modified LEL lectin, and interfered hepatocyte growth factor receptor (HGFR), Epidermal growth factor receptor (EGFR), and Axl signaling. Given that, B4GALT1 is sufficient to inversely regulate malignant behaviors of HCC cells with a significant correlation between gene expression and clinical prognosis, implying that B4GALT1 played a crucial role in cancer progress of HCC. Citation Format: Po-Da Chen, Ai-An Chang, Yao-Ming Wu, Min-Chuan Huang. Silencing of B4GALT1 inversely regulates malignant phenotypes of hepatocellular carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1402.

Neuropilin 1 (NRP1) is a novel tumor marker in hepatocellular carcinoma

BackgroundTEA domain transcription factor (TEAD) has an oncogenic role in hepatocellular carcinoma (HCC). However, whether a membrane protein can serve not only as a tumor marker that reflects TEAD function but also as a therapeutic target that stimulates tumorigenesis in HCC remains unknown. MethodsTissue NRP1 was measured using immunohistochemistry. Cell viability, colony formation and caspase3/7 activity were assessed using MTT, soft agar and caspase 3/7 Glo assays, respectively. Serum NRP1 was examined using ELISA and Western blotting. ResultsNRP1 expression was up-regulated by TEAD. We also identified a conserved TEAD-binding motif in the NRP1 promoters, which was essential for the TEAD-NRP1 interaction. NRP1 was upregulated in HCC tissues and cell lines, and knockdown of NRP1 inhibited the transformative phenotypes of HCC cells. Notably, the concentrations of serum NRP1 in the HCC patients were much higher than those of hepatitis B, hepatitis C, cirrhosis, breast cancer, colon cancer, gastric cancer and lung cancer patients. Moreover, serum NRP1 was significantly associated with AFP, γ-GT, Alb, bile acid, ALT, AST, ALP and pre-Alb. The area under the receiver operating characteristic curve (AUC-ROC) for serum NRP1 was 0.971, presenting better diagnostic performance compared to AFP. ConclusionsNRP1 is a novel tumor marker in HCC.

Mir-1287 suppresses the proliferation, invasion, and migration in hepatocellular carcinoma by targeting PIK3R3.

Mature microRNAs (miRNAs) are a class of small noncoding RNA molecules involved in regulation of post-translational gene expression. Although aberrant levels of miRNAs have been found in various tumor tissues, their importance in tumor development and the molecular basis of their regulatory role remain unclear. Our bioinformatic analysis on The Cancer Genome Atlas database and microarray-based comparison of miRNA in different cell lines revealed that the level of mir-1287 is suppressed in hepatocellular carcinoma (HCC) cells. When upregulated, mir-1287 can reduce the tumorigenesis phenotypes of HCC cells in several in vitro models. We further found that mir-1287 directly targets messenger RNA encoding PIK3R3, which is a tumor-promoting factor acting in several pathways linked to tumorigenesis. Our study suggests that aberrant suppression of mir-1287 is potentially responsible for the development of HCC, and miRNA-based strategies may be developed for efficient detection and treatment of HCC.

Galunisertib suppresses the staminal phenotype in hepatocellular carcinoma by modulating CD44 expression

Cancer stem cells (CSCs) niche in the tumor microenvironment is responsible for cancer recurrence and therapy failure. To better understand its molecular and biological involvement in hepatocellular carcinoma (HCC) progression, one can design more effective therapies and tailored then to individual patients. While sorafenib is currently the only approved drug for first-line treatment of advanced stage HCC, its role in modulating the CSC niche is estimated to be small. By contrast, transforming growth factor (TGF)-β pathway seems to influence the CSC and thus may impact hallmarks of HCC, such as liver fibrosis, cirrhosis, and tumor progression. Therefore, blocking this pathway may offer an appealing and druggable target. In our study, we have used galunisertib (LY2157299), a selective ATP-mimetic inhibitor of TGF-β receptor I (TGFβI/ALK5) activation, currently under clinical investigation in HCC patients. Because the drug resistance is mainly mediated by CSCs, we tested the effects of galunisertib on stemness phenotype in HCC cells to determine whether TGF-β signaling modulates CSC niche and drug resistance. Galunisertib modulated the expression of stemness-related genes only in the invasive (HLE and HLF) HCC cells inducing a decreased expression of CD44 and THY1. Furthermore, galunisertib also reduced the stemness-related functions of invasive HCC cells decreasing the formation of colonies, liver spheroids and invasive growth ability. Interestingly, CD44 loss of function mimicked the galunisertib effects on HCC stemness-related functions. Galunisertib treatment also reduced the expression of stemness-related genes in ex vivo human HCC specimens. Our observations are the first evidence that galunisertib effectiveness overcomes stemness-derived aggressiveness via decreased expression CD44 and THY1.

MiR-564 inhibits hepatocellular carcinoma cell proliferation and invasion by targeting the GRB2-ERK1/2-AKT axis.

Recent studies have shown that miR-564 is closely related to the development of various tumors, including breast cancer, lung cancer and glioma. However, few studies have examined miR-564 in hepatocellular carcinoma (HCC). Here, we demonstrated that miR-564 expression in HCC tissues was lower than that in adjacent noncancerous tissues and that miR-564 expression was associated with tumor size, tumor number and vein invasion. Bioinformatics analyses showed that low levels of miR-564 were correlated with poor prognosis. Furthermore, upregulation of miR-564 impaired SMCC7721 and MHCC97H cell proliferation, migration and invasion in vitro and reduced tumorigenesis in vivo. Next, we found that GRB2 was a direct target gene of miR-564 in the HCC cell lines. GRB2 was highly expressed in HCC tissues and negatively correlated with miR-564 expression levels. When GRB2 was downregulated by GRB2-siRNA, HCC cell proliferation, invasion and metastasis were impaired, and restoring GRB2 expression partially reversed the inhibitory effects of miR-564. Western blot analysis showed that miR-564 overexpression reduced GRB2 expression in HCC cell lines and inhibited ERK1/2 and AKT phosphorylation. miR-564 overexpression also upregulated the epithelial-like cell marker E-cadherin and downregulated the interstitial cell-like markers N-cadherin and vimentin. These results suggest that miR-564 inhibits the malignant phenotype of HCC cells by targeting the GRB2-ERK1/2-AKT axis. Consequently, miR-564 may be used as a prognostic marker and therapeutic target for HCC.

Immature colon carcinoma transcript-1 promotes cell growth of hepatocellular carcinoma via facilitating cell cycle progression and apoptosis resistance.

Immature colon carcinoma transcript-1 (ICT1) is a newly identified oncogene, which regulates proliferation, cell cycle progression and apoptosis of cancer cells. However, the clinical significance, biological function and underlying mechanisms of ICT1 in hepatocellular carcinoma (HCC) remain poorly known. In the present study, we showed that the expression of ICT1 in HCC tissues were notably overexpressed compared to corresponding non-tumor tissues. Accordingly, the relative levels of ICT1 were upregulated in HCC cell lines compared with LO2 cells. The positive expression of ICT1 was correlated with large tumor size and advanced TNM tumor stage. Kaplan-Meier plots indicated that ICT1-positive expression in HCC patients showed a prominent shorter survival. In addition, ICT1 knockdown inhibited proliferation and cell cycle progression, and induced apoptosis in HepG2 cells. While, ICT1 overexpression showed opposite effects on these cellular processes of Hep3B cells. Invivo experiments demonstrated that ICT1 deficiency reduced the growth of subcutaneous HCC in nude mice. Notably, ICT1 knockdown reduced the levels of CDK1, cyclinB1 and Bcl-2 and increased the expression of Bax in HepG2 cells. ICT1 overexpression resulted in upregulation of CDK1, cyclinB1 and Bcl-2, and downregulation of Bax in Hep3B cells. Furthermore, microRNA-134 (miR-134) was recognized as a direct upstream regulator and inversely modulated ICT1 abundance in HCC cells. Altogether, our data support that miR-134 regulation of ICT1 facilitates malignant phenotype of HCC cells probably via cell cycle and apoptosis-associated proteins including CDK1, cyclinB1, Bcl-2 and Bax.

Role of the Transforming Growth Factor-\u03b2 in regulating hepatocellular carcinoma oxidative metabolism

Transforming Growth Factor beta (TGF-β) induces tumor cell migration and invasion. However, its role in inducing metabolic reprogramming is poorly understood. Here we analyzed the metabolic profile of hepatocellular carcinoma (HCC) cells that show differences in TGF-β expression. Oxygen consumption rate (OCR), extracellular acidification rate (ECAR), metabolomics and transcriptomics were performed. Results indicated that the switch from an epithelial to a mesenchymal/migratory phenotype in HCC cells is characterized by reduced mitochondrial respiration, without significant differences in glycolytic activity. Concomitantly, enhanced glutamine anaplerosis and biosynthetic use of TCA metabolites were proved through analysis of metabolite levels, as well as metabolic fluxes from U-13C6-Glucose and U-13C5-Glutamine. This correlated with increase in glutaminase 1 (GLS1) expression, whose inhibition reduced cell migration. Experiments where TGF-β function was activated with extracellular TGF-β1 or inhibited through TGF-β receptor I silencing showed that TGF-β induces a switch from oxidative metabolism, coincident with a decrease in OCR and the upregulation of glutamine transporter Solute Carrier Family 7 Member 5 (SLC7A5) and GLS1. TGF-β also regulated the expression of key genes involved in the flux of glycolytic intermediates and fatty acid metabolism. Together, these results indicate that autocrine activation of the TGF-β pathway regulates oxidative metabolism in HCC cells.

A Novel Three-Dimensional Culture System: Decellularized-Tissue Obtained from Cirrhotic Livers Enhances an Epithelial-Mesenchymal Transition Phenotype in Hepatocellular Carcinoma Cells

Miyauchi, Yuya MD; Yasuchika, Kentaro MD, PhD; Fukumitsu, Ken; Ishii, Takamichi MD, PhD; Ogiso, Satoshi MD; Kojima, Hidenobu MD; Yamaoka, Ryoya MD; Katayama, Hokahiro; Kawai, Takayuki MD; Uemoto, Shinji MD, PhD Author Information

Maintenance of stemness is associated with the interation of LRP6 and heparin-binding protein CCN2 autocrined by hepatocellular carcinoma

BackgroundThe overall response rate of hepatocellular carcinoma (HCC) to chemotherapy is poor. In our previous study, oxaliplatin-resistant HCC is found to exhibit an enhanced stemness, and increased levels of CCN2 and LRP6, while the role of CCN2 and LRP6 in the prognosis of HCC patients, and the interaction regulation mechanism between CCN2 and LRP6 are still unclear. MethodsThe expression levels of CCN2 and LRP6 were detected in large cohorts of HCCs, and functional analyses of CCN2 and LRP6 were performed both in vitro and in vivo. The roles of cell surface heparin sulfate proteoglycans (HSPGs) in the mutual regulatory between CCN2 and LRP6 were verified in HCC, and the interventions of low molecular weight heparin sodium (LMWH) were explored. ResultsCCN2 and LRP6 were overexpressed in HCCs, and the CCN2 and LRP6 levels were positively associated with the malignant phenotypes and poor prognosis of HCCs. LRP6 could significantly upregulate the expression of CCN2. Meanwhile, CCN2 was able to enhance malignant phenotype of HCC cells in a dose-dependent manner through binding with LRP6; and knock-down of LRP6 expression, perturbation of HSPGs, co-incubation of CCN2 with LMWH could significantly block the adhesion of CCN2 to LRP6. LMWH enhanced the therapeutic effect of oxaliplatin on HCC with a high CCN2 expression. Conclusions CCN2 plays a promoting role in HCC progression through activating LRP6 in a HSPGs-dependent manner. Heparin in combination with chemotherapy has a synergic effect and could be a treatment choice for HCCs with a high CCN2 expression.

CHSY1 promotes aggressive phenotypes of hepatocellular carcinoma cells via activation of the hedgehog signaling pathway

Abnormal expression of chondroitin sulfate has been found in many types of cancer, while its biological functions in hepatocellular carcinoma (HCC) progression remain uninvestigated. Here, we report that chondroitin sulfate synthase 1 (CHSY1), the enzyme that mediates the polymerization step of chondroitin sulfate, is a critical mediator of malignant character in HCC that acts via modulating the activity of the hedgehog signaling. CHSY1 was up-regulated frequently in HCC where these events were associated with worse histologic grade and poor survival. Enforced expression of CHSY1 was sufficient to enhance cell growth, migration, invasion, and epithelial-mesenchymal transition, whereas silencing of CHSY1 suppressed these malignant phenotypes. Mechanistic investigations revealed that the increase of cell surface chondroitin sulfate by CHSY1 promoted sonic hedgehog binding and signaling. Inhibiting hedgehog pathway with vismodegib decreased CHSY1-induced migration, invasion, and lung metastasis of HCC cells, establishing the critical role of hedgehog signaling in mediating the effects of CHSY1 expression. Together, our results indicate that CHSY1 overexpression in HCC contributes to the malignant behaviors in cancer cells, we provide novel insights into the significance of chondroitin sulfate in hedgehog signaling and HCC pathogenesis.

Mammalian-enabled (MENA) protein enhances oncogenic potential and cancer stem cell-like phenotype in hepatocellular carcinoma cells.

Mammalian‐enabled (MENA) protein is an actin‐regulatory protein that influences cell motility and adhesion. It is known to play a role in tumorigenicity of hepatocellular carcinoma (HCC) but the underlying molecular mechanism remains unknown. This study aimed to investigate the oncogenic potential of MENA and its capacity to regulate cancer stem cell (CSC)‐like phenotypes in HCC cells. Real‐time‐PCR and western blot were used to assess mRNA and protein levels of target genes in human HCC tissue specimens and HCC cell lines, respectively. Stable MENA‐overexpressing HCC cells were generated from HCC cell lines. Transwell cell migration and colony formation assays were employed to evaluate tumorigenicity. Ectopic expression of MENA significantly enhanced cell migration and colony‐forming ability in HCC cells. Overexpression of MENA upregulated several hepatic progenitor/stem cell markers in HCC cells. A high MENA protein level was associated with high mRNA levels of MENA, CD133, cytokeratin 19 (CK19), and epithelial cell adhesion molecule (EpCAM) in human HCC tissues. Overexpression of MENA enhanced epithelial‐to‐mesenchymal transition (EMT) markers, extracellular signal‐regulated kinases (ERK) phosphorylation, and the level of β‐catenin in HCC cells. This study demonstrated that overexpression of MENA in HCC cells promoted stem cell markers, EMT markers, and tumorigenicity. These effects may involve, at least partially, the ERK and β‐catenin signaling pathways.

MiR-217 targeting DKK1 promotes cancer stem cell properties via activation of the Wnt signaling pathway in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common malignancies and exhibits heterogeneity in terms of clinical outcomes and biological activities. Emerging evidence has demonstrated that cancer stem cells (CSCs) play important roles in the tumorigenesis and progression of HCC. However, the molecular mechanisms underlying the stemness maintenance of CSCs remain largely unknown. In the present study, through real-time PCR, western blotting, luciferase assays, RNA immunoprecipitation, and invitro and invivo assays, we demonstrated that miR-217 expression was markedly increased in HCC tissues and cells. Overexpression of miR-217 promoted, while silencing miR-217 suppressed, the fraction of the side population and the expression of cancer stem cell factors invitro and tumorigenicity invivo in HCC cells. Our findings further demonstrated that miR-217 promoted the CSC-like phenotype via dickkopf-1(DKK1) targeting, resulting in constitutive activation of Wnt signaling. Moreover, the stimulatory effects of miR-217 on stem cell properties and Wnt signaling were antagonized by the upregulation of DKK1 in miR-217-overexpressing cells. Conversely, the inhibitory effects of silencing miR-217 on stem cell properties and Wnt signaling were reversed by the downregulation of DKK1 in miR-217-downregulated cells. Therefore, our results indicate that miR-217 plays a vital role in the CSC-like phenotypes of HCC cells and may be used as a potential therapeutic target against HCC.

MicroRNA-187 inhibits tumor growth and metastasis via targeting of IGF-1R in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the primary and most frequently occurring type of malignant liver cancer, accounting for 70-85% of total liver cancer cases worldwide. It has previously been demonstrated that the aberrant expression of microRNAs (miR) contributes to carcinogenesis and progression of various human malignancies, including HCC. However, mechanisms underlying the differential expression and specific roles of miR‑187 in HCC remain to be elucidated, particularly regarding how the modulation of malignant phenotypes in HCC cells occurs. The present study demonstrated that miR‑187 was significantly downregulated in HCC tissues and cell lines. Restoration of miR‑187 expression inhibited cell proliferation, migration and invasion in HCC. Furthermore, insulin‑like growth factor 1 receptor (IGF‑1R) was demonstrated to act as a direct target gene of miR‑187 in HCC. IGF‑1R knockdown mimicked the effects of miR‑187 overexpression in HCC, resulting in a significant inhibition of cell proliferation, migration and invasion. The results of the present study demonstrated that miR‑187 acted as a tumor suppressor in HCC progression via direct targeting of IGF‑1R. miR‑187 may therefore exhibit the potential to act as a novel and therapeutic target for HCC treatment in the future.

MiR-101 and doxorubicin codelivered by liposomes suppressing malignant properties of hepatocellular carcinoma.

MiR‐101, an important tumor‐suppressive microRNA (miRNA) in hepatocellular carcinoma (HCC), has been affirmed significantly downregulated in HCC and participated in promoting apoptosis, decreasing proliferation and invasiveness of HCC cells, as well as increasing sensitivity of chemotherapeutic drug. However, miR‐101‐based combination therapies with doxorubicin (DOX) are not reported yet. Recently, nanomaterials‐based approaches, especially liposome formulations, have been approved for clinical use and seem to provide a great opportunity to codeliver therapeutic agents for cancer therapy. In this study, we have successfully prepared liposome (L) nanoparticles to efficiently deliver miR‐101 and DOX to HCC cells simultaneously. The effects of codelivery system miR‐101/doxorubicin liposome (miR‐101/DOX‐L) on tumor malignant phenotypes of HCC cells were evaluated through analyzing cell proliferation, colony formation, cell migration, cell invasion, cell apoptosis assay, and the expression of related genes. In subcutaneous xenografts developed by HCC cells, the inhibition of tumor growth was analyzed through gross morphology, growth curve, proliferation marker Ki‐67, apoptosis signals, and the expression of related genes. These experiments demonstrated that miR‐101/DOX‐L inhibited tumor properties of liver cancer cells in vitro and in vivo through targeting correlative genes by combinatory role of miR‐101 and DOX. In conclusion, our results indicated that liposome nanoparticle is a reliable delivery strategy to codeliver miR‐101 and DOX simultaneously, and miR‐101‐ and DOX‐based combination therapy can result in significant synergetic antitumor effects in vivo and vitro.

HCRP1 downregulation promotes hepatocellular carcinoma cell migration and invasion through the induction of EGFR activation and epithelial-mesenchymal transition.

Hepatocellular carcinoma related protein 1 (HCRP1), which is essential for internalization and degradation of ubiquitinated membrane receptors, is downregulated in several tumors and strongly affects the outcomes of cancer patients. It is reported the expression of HCRP1 is inversely related to epidermal growth factor receptor (EGFR) in breast cancer and lead to resistance to cetuximab in ovarian cancer. However, its exact mechanism in the progression of Hepatocellular carcinoma (HCC) remains unknown. Herein, HCRP1 expression and its clinical significance were examined in 101 HCC patients using immunohistochemistry. Cell proliferation, migration and invasion assays were conducted in HCC cell lines. EGFR activation and degradation were then observed after EGF inducing in HCRP1 knockdown HepG2 cells. In addition, we also detected whether epithelial-to-mesenchymal transition (EMT) was involved in the malignancy promoted by HCRP1. The results showed that 59 of the 101 HCC cases exhibited downregulation of HCRP1 expression (P<0.01) as compared to 30 benign liver lesions and 20 normal liver tissues, all of which showed a high level of HCRP1. HCRP1 expression was significantly related to age (P=0.017), pathological grade (P=0.003), tumor encapsulation (P=0.037), recurrence (P=0.039) and death (P=0.015), but unrelated to cirrhosis (P=0.216), tumor size (P=0.273), and distant metastasis (P=0.554). Lower HCRP1 expression was correlated with shorter RFS and OS (P<0.001), and decreased HCRP1 level is an independent prognostic marker in HCC patients (P<0.05). Overexpression of HCRP1 decreased and knockdown increased HCC cell proliferation, migration and invasion. HCRP1 depletion increased EGFR activation and inhibited EGFR degradation. EMT phenotype was promoted after HCRP1 downregulation via increase of Snail and Twist1 and activation of Akt phosphorylation in HepG2 cells. Conversely, upregulation of HCRP1 in SMMC-7721 cells led to the opposite effect. In conclusion, our study indicated that downregulation of HCRP1 is a valuable prognostic factor involved in EGFR regulation and acquisition of the mesenchymal phenotype of HCC cells.

Roles of microRNA-330 and Its Target Gene ING4 in the Development of Aggressive Phenotype in Hepatocellular Carcinoma Cells.

Aberrant expression of microRNAs contributes to tumor growth and progression. This study was designed to explore the prognostic and biological significance of miR-330 in hepatocellular carcinoma (HCC). The expression of miR-330 and its associations with tumor parameters and overall survival were analyzed in HCC patients. The biological functions of miR-330 in HCC cell growth, invasion, and tumorigenesis were investigated. Bioinformatic analysis and luciferase reporter assays were performed to search for potential targets of miR-330. The miR-330 level was significantly higher in HCCs than in adjacent normal tissues (P=0.0085). High expression of miR-330 was significantly associated with more aggressive phenotypes and shorter overall survival in HCC. Loss- and gain-of-function studies indicated the favorable effect of miR-330 on tumor cell growth, invasion, and tumorigenesis. Inhibitor of growth 4 (ING4) was identified to be a direct target of miR-330. Overexpression of miR-330 reduced the expression of ING4 in HCC cells. Importantly, restoration of ING4 almost completely reversed the promotion of HCC cell proliferation and invasion by miR-330. Altogether, this study demonstrates that upregulation of miR-330 is associated with poor prognosis and contributes to more aggressive phenotypes of HCC. The oncogenic role of miR-330 in HCC is linked to downregulation of ING4.