MicroRNA-144 inhibits hepatocellular carcinoma cell proliferation,invasion and migration by targeting ZFX

MicroRNA 144 (miR-144), a small non-coding RNA, is frequently dysregulated in human several tumour progression, but its role and the underlying mechanisms in hepatocellular carcinoma (HCC) is poorly investigated. In the present study, the expression of miR-144 was firstly analysed in datasets derived from GSE21362 and TCGA, and then detected in HCC tissues and cell lines by quantitative RT-PCR (qRT-PCR) analysis. MiR-144 was shown to be significantly down-regulated in HCC tissues and cell lines. Subsequently, overexpression of miR-144 was transfected into HCC cell lines so as to investigate its biological function, including MTT, colony formation, and transwell assays. Gain of function assay revealed miR-144 remarkably inhibited cell proliferation, migration and invasion. In addition, bioinformatical analysis and luciferase reporter assay identified ZFX as a novel target of miR-144 in HCC cells, as confirmed by qRT-PCR and Western blot. Furthermore, ZFX was found to be significantly up-regulated using Oncomine database analysis. Loss of function assay further indicated knockdown of ZFX had similar effects of miR-144-mediated HCC cell proliferation and invasion. Therefore, miR-144 has been demonstrated to act as a tumour suppressor in HCC cell growth and motility by directly targeting ZFX, which implicates its potential applications in the development of HCC treatment.     

MicroRNA‐301b‐3p contributes to tumour growth of human hepatocellular carcinoma by repressing vestigial like family member 4

MicroRNAs (miRNAs) are key regulators in the tumour growth and metastasis of human hepatocellular carcinoma (HCC). Increasing evidence suggests that miR‐301b‐3p functions as a driver in various types of human cancer. However, the expression pattern of miR‐301b‐3p and its functional role as well as underlying molecular mechanism in HCC remain poorly known. Our study found that miR‐301b‐3p expression was significantly up‐regulated in HCC tissues compared to adjacent non‐tumour tissues. Clinical association analysis revealed that the high level of miR‐301b‐3p closely correlated with large tumour size and advanced tumour‐node‐metastasis stages. Importantly, the high miR‐301b‐3p level predicted a prominent poorer overall survival of HCC patients. Knockdown of miR‐301b‐3p suppressed cell proliferation, led to cell cycle arrest at G2/M phase and induced apoptosis of Huh7 and Hep3B cells. Furthermore, miR‐301b‐3p knockdown suppressed tumour growth of HCC in mice. Mechanistically, miR‐301b‐3p directly bond to 3′UTR of vestigial like family member 4 (VGLL4) and negatively regulated its expression. The expression of VGLL4 mRNA was down‐regulated and inversely correlated with miR‐301b‐3p level in HCC tissues. Notably, VGLL4 knockdown markedly repressed cell proliferation, resulted in G2/M phase arrest and promoted apoptosis of HCC cells. Accordingly, VGLL4 silencing rescued miR‐301b‐3p knockdown attenuated HCC cell proliferation, cell cycle progression and apoptosis resistance. Collectively, our results suggest that miR‐301b‐3p is highly expressed in HCC. miR‐301b‐3p facilitates cell proliferation, promotes cell cycle progression and inhibits apoptosis of HCC cells by repressing VGLL4.     

PKM2-regulated STAT3 promotes esophageal squamous cell carcinoma progression via TGF-β1-induced EMT

Recent studies have demonstrated pleiotropic roles of pyruvate kinase isoenzyme type M2 (PKM2) in tumor progression. However, the precise mechanisms underlying the effects of PKM2 on esophageal squamous cell carcinoma (ESCC) metastasis and transforming growth factor β1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) remain to be established. In this study, we observed upregulation of PKM2 in ESCC tissues that was markedly associated with lymph node metastasis and poor prognosis. High PKM2 expression in tumor tissues frequently coincided with the high pSTAT3Tyr705 expression and low E-cadherin expression. Furthermore, altered PKM2 expression was significantly associated with proliferation, migration, and invasion of ESCC cells, in addition to expression patterns of EMT markers (Snail, E-cadherin, and vimentin) and pSTAT3^Tyr705/STAT3 ratio. Overexpression of STAT3 significantly attenuated the effects of PKM2 knockdown on cell proliferation and motility as well as expression of pSTAT3 ^Tyr705 and EMT markers. Consistently, stable short hairpin RNA (shRNA)-mediated silencing of PKM2 reversed the effects of TGF-β1 treatment, specifically, upregulation of PKM2, phosphorylation of STAT3 at Tyr705, and increased EMT, migration, and invasion. We propose that PKM2 regulates cell proliferation, migration, and invasion via phosphorylation of STAT3 through TGF-β1-induced EMT. Our findings collectively provide mechanistic insights into the tumor-promoting role of PKM2, supporting its prognostic value and the therapeutic utility of PKM2 inhibitors as potential antitumor agents in ESCC.     

Long non‐coding RNA deleted in lymphocytic leukaemia 1 promotes hepatocellular carcinoma progression by sponging miR‐133a to regulate IGF‐1R expression

Long non‐coding RNA (lncRNA) deleted in lymphocytic leukaemia 1 (DLEU1) was reported to be involved in the occurrence and development of multiple cancers. However, the exact expression, biological function and underlying mechanism of DLEU1 in hepatocellular carcinoma (HCC) remain unclear. In this study, real‐time quantitative polymerase chain reaction (qRT‐PCR) in HCC tissues and cell lines revealed that DLEU1 expression was up‐regulated, and the increased DLEU1 was closely associated with advanced tumour‐node‐metastasis stage, vascular metastasis and poor overall survival. Function experiments showed that knockdown of DLEU1 significantly inhibited HCC cell proliferation, colony formation, migration and invasion, and suppressed epithelial to mesenchymal transition (EMT) process via increasing the expression of E‐cadherin and decreasing the expression of N‐cadherin and Vimentin. Luciferase reporter gene assay and RNA immunoprecipitation (RIP) assay demonstrated that DLEU1 could sponge miR‐133a. Moreover, miR‐133a inhibition significantly reversed the suppression effects of DLEU1 knockdown on HCC cells. Besides, we found that silenced DLEU1 significantly decreased insulin‐like growth factor 1 receptor (IGF‐1R) expression (a target of miR‐133a) and its downstream signal PI3K/AKT pathway in HCC cells, while miR‐133a inhibitor partially reversed this trend. Furthermore, DLEU1 knockdown impaired tumour growth in vivo by regulating miR‐133a/IGF‐1R axis. Collectively, these findings indicate that DLEU1 promoted HCC progression by sponging miR‐133a to regulate IGF‐1R expression. Deleted in lymphocytic leukaemia 1/miR‐133a/IGF‐1R axis may be a novel target for treatment of HCC.     

LncRNA CRNDE promotes the epithelial-mesenchymal transition of hepatocellular carcinoma cells via enhancing the Wnt/β-catenin signaling pathway

Colorectal neoplasia differentially expressed (CRNDE) is a significantly upregulated long noncoding RNA in hepatocellular carcinoma (HCC). CRNDE could promote cell proliferation, migration, and invasion, while its molecular mechanisms were still largely unclear. In this study, we investigated the expression and function of CRNDE. CRNDE was significantly upregulated in tumor tissues compared with adjacent normal tissues. In vitro, we revealed that knockdown of CRNDE inhibited cell proliferation, migration, and cell invasion capacities in HCC. Animal studies indicated that CRNDE knockdown represses both growth and metastasis of HCC tumors in vivo. Moreover, knockdown of CRNDE suppressed the cell epithelial-mesenchymal transition (EMT) process by increasing the expression of E-cadherin and ZO-1, whereas, decreasing the expression of N-cadherin, slug, twist, and vimentin in HCC cells. We also revealed that knockdown of CRNDE suppressed the Wnt/β-catenin signaling in HCC. Thus, CRNDE could modulate EMT of HCC cells and knockdown of CRNDE impaired the mesenchymal properties. CRNDE increased invasion of HCC cells might be through activating the Wnt/β-catenin signaling pathway.     

Vaccinia virus expressing IL‐37 promotes antitumor immune responses in hepatocellular carcinoma

The aim of this study was to investigate the effect of vaccinia virus expressing IL‐37 (VV‐IL‐37) on cell proliferation, migration and invasion of hepatocellular carcinoma (HCC) and its possible underlying molecular mechanisms. In this study, we constructed a cancer‐targeted vaccinia virus carrying the IL‐37 gene knocked in the region of the viral thymidine kinase (TK) gene. Human HCC cell lines were assayed in vitro for cell proliferation, migration and invasion. Serum level, relative mRNA level and protein level of IL‐37 in HCC cell lines SMMC7721 and Bel7402 were tested by ELISA assay, qRT‐PCR and western blot, respectively. The levels of IL‐2, IFN‐γ and TNF‐α in HCC tumor tissues were also analyzed by ELISA. STAT3 and p‐STAT3 expression in tumor tissues were determined by western blot. Our results showed that VV‐IL‐37 efficiently infected and inhibited HCC cells proliferation, migration and invasion via decreasing STAT3 phosphorylation. In vivo, VV‐IL‐37 expressed IL‐37 at a high level in the transplanted tumor, reduced STAT3 activity, and eventually inhibited tumor growth. In conclusion, we demonstrate that VV‐IL‐37 promotes antitumor immune responses in HCC.     

LncRNA SNHG1 regulates immune escape of renal cell carcinoma by targeting miR-129-3p to activate STAT3 and PD-L1

Immune escape of renal cell carcinoma (RCC) impacts patient survival. However, the molecular mechanism of long noncoding RNA (lncRNA) small nucleolar RNA host gene 1 (SNHG1) in RCC immune escape remains unclear. Quantitative real-time PCR and western blotting results revealed that the expression of lncRNA SNHG1 and STAT3 were upregulated in RCC tissues and cells and that the expression of miR-129-3p was downregulated. Enzyme-linked immunosorbent assay results revealed the increased levels of immune-related factors (interferon-γ, tumour necrosis factor α, and interleukin-2) in RCC tissues. SNHG1 knockdown or miR-129-3p overexpression inhibited the proliferation and invasion of A498 and 786-O cells, while the proliferation and cytotoxicity of CD8⁺ T cells increased, which promoted the secretion of immune-related factors. STAT3 overexpression decreased the protective effect of miR-129-3p overexpression on RCC cell immune escape. In addition, miR-129-3p knockdown and STAT3 overexpression decreased the protective effect of lncRNA SNHG1 knockdown on RCC cell immune escape. In addition, PD-L1 expression was downregulated after lncRNA SNHG1 knockdown but upregulated after miR-129-3p knockdown and STAT3 overexpression. Dual-luciferase assays showed that lncRNA SNHG1 targets miR-129-3p, and miR-129-3p targets STAT3. RNA pull-down and RNA immunoprecipitation assays verified the regulatory relationship between SNHG1 and STAT3. In vivo, shSNHG1 prolonged the overall survival of RCC tumour model mice and inhibited RCC tumour growth and immune escape but increased CD8⁺ T cell infiltration in mice. Our findings provide an experimental basis for elucidating the molecular mechanisms of immune escape by RCC and reveal a novel target to treat this disease.     

Inflammatory microenvironment of fibrotic liver promotes hepatocellular carcinoma growth,metastasis and sorafenib resistance through STAT3 activation

The pro-inflammatory and pro-fibrotic liver microenvironment facilitates hepatocarcinogenesis. However, the effects and mechanisms by which the hepatic fibroinflammatory microenvironment modulates intrahepatic hepatocellular carcinoma (HCC) progression and its response to systematic therapy remain largely unexplored. We established a syngeneic orthotopic HCC mouse model with a series of persistent liver injury induced by CCl₄ gavage, which mimic the dynamic effect of hepatic pathology microenvironment on intrahepatic HCC growth and metastasis. Non-invasive bioluminescence imaging was applied to follow tumour progression over time. The effect of the liver microenvironment modulated by hepatic injury on sorafenib resistance was investigated in vivo and in vitro. We found that the persistent liver injury facilitated HCC growth and metastasis, which was positively correlated with the degree of liver inflammation rather than the extent of liver fibrosis. The inflammatory cytokines in liver tissue were clearly increased after liver injury. The two indicated cytokines, tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6), both promoted intrahepatic HCC progression via STAT3 activation. In addition, the hepatic inflammatory microenvironment contributed to sorafenib resistance through the anti-apoptotic protein mediated by STAT3, and STAT3 inhibitor S3I-201 significantly improved sorafenib efficacy impaired by liver inflammation. Clinically, the increased inflammation of liver tissues was accompanied with the up-regulated STAT3 activation in HCC. Above all, we concluded that the hepatic inflammatory microenvironment promotes intrahepatic HCC growth, metastasis and sorafenib resistance through activation of STAT3.     

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.     

CBX4 promotes the proliferation and metastasis via regulating BMI‐1 in lung cancer

Proliferation and metastasis are significantly malignant characteristics of human lung cancer, but the underlying molecular mechanisms are poorly understood. Chromobox 4 (CBX4), a member of the Polycomb group (PcG) family of epigenetic regulatory factors, enhances cellular proliferation and promotes cancer cell migration. However, the effect of CBX4 in the progression of lung cancer is not fully understood. We found that CBX4 is highly expressed in lung tumours compared with adjacent normal tissues. Overexpression of CBX4 significantly promotes cell proliferation and migration in human lung cancer cell lines. The knockdown of CBX4 obviously suppresses the cell growth and migration of human lung cancer cells in vitro. Also, the proliferation and metastasis in vivo are blocked by CBX4 knockdown. Furthermore, CBX4 knockdown effectively arrests cell cycle at the G0/G1 phase through suppressing the expression of CDK2 and Cyclin E and decreases the formation of filopodia through suppressing MMP2, MMP9 and CXCR4. Additionally, CBX4 promotes proliferation and metastasis via regulating the expression of BMI‐1 which is a significant regulator of proliferation and migration in lung cancer cells. Taken together, these data suggest that CBX4 is not only a novel prognostic marker but also may be a potential therapeutic target in lung cancer.     

Overexpression of RhoGDI,a novel predictor of distant metastasis,promotes cell proliferation and migration in hepatocellular carcinoma

RhoGDI (Rho GDP-dissociation inhibitor alpha, or RhoGDIα) was identified as a regulator of Rho GTPases, but its role in cancer remains controversial. In this study, increased expression of RhoGDI was detected in hepatocellular carcinoma (HCC) cell lines and tissues with highly metastatic potential. RhoGDI overexpression correlated with postoperative distant metastasis. Enforced expression of RhoGDI in HCC cells significantly enhanced cell proliferation and migration. Conversely, knockdown of RhoGDI caused an inhibition of the aggressive phenotypes of HCC cells. Furthermore, RhoGDI up-regulated Rho, but not Rac, and enhanced PI3K/AKT and MAPK pathway activity. Our findings suggest that RhoGDI overexpression is a predictor of distant metastasis and plays an important role in the progression of HCC.     

The enhanced expression of estrogen-related receptor α in human bladder cancer tissues and the effects of estrogen-related receptor α knockdown on bladder cancer cells

Estrogen-related receptor α (ERRα) belongs to the superfamily of nuclear orphan receptors. However, the role of ERRα in bladder cancer remains unknown. This study examined the expression of ERRα in bladder cancer tissues and explored the molecular mechanisms of ERRα in bladder cancer progression. The expression of ERRα in bladder cancer tissues from 61 patients was determined by immunohistochemistry. We performed quantitative real-time polymerase chain reaction assay to detect the gene expression levels and carried out Western blot assay to measure protein levels. In vitro functional assays, including colony formation, Cell Counting Kit-8, Transwell invasion, and migration assays, were performed to detect bladder cancer cell growth, proliferation, invasion, and migration, respectively. Flow cytometry was used to determine the cell apoptotic rate of bladder cancer cells. Among the 61 detected bladder cancer tissues, 39 bladder cancer tissues showed positive ERRα immunoreactivity. Higher ERRα immunoreactivity score was significantly associated with TNM stage, tumor grade, distant metastasis, and poor survival in patients with bladder cancer. Univariate and multivariate analyses showed that ERRα immunoreactivity was an independent prognostic factor for overall survival in patients with bladder cancer. ERRα was found to be upregulated in bladder cancer cell lines, and knockdown of ERRα suppressed bladder cancer cell growth, proliferation, invasion, and migration; promoted bladder cancer cell apoptosis; and inhibited the epithelial-mesenchymal transition of bladder cancer cells. On the other hand, bladder cancer cell proliferation, invasion, and migration were significantly enhanced after cells were transfected with an ERRα-overexpressing vector. In vivo tumor growth and metastasis assays showed that ERRα knockdown resulted in remarkable inhibition of tumor growth and tumor metastasis in nude mice. Collectively, our results suggest that the enhanced expression of ERRα may play a key role in the development and progression of bladder cancer and ERRα may serve as an important prognostic factor for bladder cancer.     

Expression levels of the metalloproteinase ADAM8 critically regulate proliferation,migration and malignant signalling events in hepatoma cells

Hepatocellular carcinoma (HCC) is one of the most common metastatic tumours. Tumour growth and metastasis depend on the induction of cell proliferation and migration by various mediators. Here, we report that the A Disintegrin and Metalloproteinase (ADAM) 8 is highly expressed in murine HCC tissues as well as in murine and human hepatoma cell lines Hepa1-6 and HepG2, respectively. To establish a dose-dependent role of different ADAM8 expression levels for HCC progression, ADAM8 expression was either reduced via shRNA- or siRNA-mediated knockdown or increased by using a retroviral overexpression vector. These two complementary approaches revealed that ADAM8 expression levels correlated positively with proliferation, clonogenicity, migration and matrix invasion and negatively with apoptosis of hepatoma cells. Furthermore, the analysis of pro-migratory and proliferative signalling pathways revealed that ADAM8 expression level was positively associated with expression of β1 integrin as well as with the activation of focal adhesion kinase (FAK), mitogen-activated protein kinase (MAPK), Src kinase and Rho A GTPase. Finally, up-regulation of promigatory signalling and cell migration was also seen with a proteolytically inactive ADAM8 mutant. These findings reveal that ADAM8 is critically up-regulated in hepatoma cells contributes to cell proliferation and survival and furthermore induces pro-migratory signalling pathways independently of its proteolytic activity. By this, ADAM8 can promote cell functions most relevant for HCC growth and metastasis.     

Identification of SLAMF3 (CD229) as an Inhibitor of Hepatocellular Carcinoma Cell Proliferation and Tumour Progression

Although hepatocellular carcinoma (HCC) is one of the most common malignancies and constitutes the third leading cause of cancer-related deaths, the underlying molecular mechanisms are not fully understood. In the present study, we demonstrate for the first time that hepatocytes express signalling lymphocytic activation molecule family member 3 (SLAMF3/CD229) but not other SLAMF members. We provide evidence to show that SLAMF3 is involved in the control of hepatocyte proliferation and in hepatocellular carcinogenesis. SLAMF3 expression is significantly lower in primary human HCC samples and HCC cell lines than in human healthy primary hepatocytes. In HCC cell lines, the restoration of high levels of SLAMF3 expression inhibited cell proliferation and migration and enhanced apoptosis. Furthermore, SLAMF3 expression was associated with inhibition of HCC xenograft progression in the nude mouse model. The restoration of SLAMF3 expression levels also decreased the phosphorylation of MAPK ERK1/2, JNK and mTOR. In samples from resected HCC patients, SLAMF3 expression levels were significantly lower in tumorous tissues than in peritumoral tissues. Our results identify SLAMF3 as a specific marker of normal hepatocytes and provide evidence for its potential role in the control of proliferation of HCC cells.     

SNHG16 as the miRNA let-7b-5p sponge facilitates the G2/M and epithelial-mesenchymal transition by regulating CDC25B and HMGA2 expression in hepatocellular carcinoma

Long noncoding RNAs (lncRNAs) play crucial roles in hepatocellular carcinoma (HCC). However, the underlying molecular mechanisms of small nucleolar RNA host gene 16 (SNHG16) for regulating the cell cycle and epithelial to mesenchymal transition (EMT) remain elusive. In this study, SNHG16 expression profiles of HCC tissues or cell lines were compared with those of normal tissues or hepatocyte cell line. The effect of SNHG16 knockdown in HCC cell lines was investigated by using in vitro loss-of-function experiments and in vivo nude mouse experiments. The potential molecular regulatory mechanism of SNHG16 in HCC progression was investigated by using mechanistic experiments and rescue assays. The results revealed that SNHG16 was highly expressed in HCC tissues and cell lines, which predicted poor prognosis of HCC patients. On one hand, the downregulation of SNHG16 induced G2/M cell cycle arrest, inducing cell apoptosis and suppression of cell proliferation. On the other hand, it inhibited cell metastasis and EMT progression demonstrated by in vitro loss-of-function cell experiments. Besides, knockdown of SNHG16 increased the sensitivity of HCC cells to cisplatin. For the detailed mechanism, SNHG16 was demonstrated to act as a let-7b-5p sponge in HCC. SNHG16 facilitated the G2/M cell cycle transition by directly acting on the let-7b-5p/CDC25B/CDK1 axis, and promoted cell metastasis and EMT progression by regulating the let-7b-5p/HMGA2 axis in HCC. In addition, the mechanism of SNHG16 for regulating HCC cell proliferation and metastasis was further confirmed in vivo by mouse experiments. Furthermore, these results can provide new insights into HCC treatment and its molecular pathogenesis, which may enlighten the further research of the molecular pathogenesis of HCC.