Knockdown of terminal differentiation induced ncRNA (TINCR) suppresses proliferation and invasion in hepatocellular carcinoma by targeting the miR-218-5p/DEAD-box helicase 5 (DDX5) axis

Terminal differentiation induced ncRNA (TINCR), a newly identified lncRNA, has been found to be associated with different human cancers including hepatocellular carcinoma (HCC). However, little is known regarding the pathological mechanisms of TINCR in HCC progression. In this study, we confirmed that TINCR expression was upregulated in HCC tumors and cell lines, and high TINCR expression was associated with larger tumor size, advanced tumor node metastasis stage, and poor prognosis. Functionally, knockdown of TINCR facilitated apoptosis and suppressed viability, colony formation and invasion in Huh7 and Hep3B cells. Mechanically, TINCR functioned as competing endogenous RNA (ceRNA) to regulate DEAD-box helicase 5 (DDX5) expression through sponging miR-218-5p. Moreover, the miR-218-5p expression was downregulated and DDX5 expression was upregulated in HCC tumors. The silencing of miR-218-5p or ectopic expression of DDX5 abated the tumor-suppressive effect of TINCR knockdown in vitro. Furthermore, si-TINCR-induced inactivation of AKT signaling was rescued by suppression of miR-218-5p or overexpression of DDX5. Also, the silencing of TINCR resulted in tumor growth inhibition in vivo. In summary, knockdown of TINCR suppressed HCC progression presumably by inactivation of AKT signaling through targeting the miR-218-5p/DDX5 axis, suggesting a novel TINCR/miR-218-5p/DDX5 pathway and therapy target for HCC.     

Retracted: In vivo and in vitro effects of microRNA-221 on hepatocellular carcinoma development and progression through the JAK–STAT3 signaling pathway by targeting SOCS3

Hepatocellular carcinoma (HCC), as the third leading cancer-caused deaths, prevails with high mortality, and affects more than half a million individuals per year worldwide. A former study revealed that microRNA-221 (miR-221) was involved in cell proliferation of liver cancer and HCC development. The current study aims to evaluate whether miR-221 targeting SOCS3 affects HCC through JAK–STAT3 signaling pathway. A series of miR-221 mimic, miR-221 inhibitor, siRNA against SOCS3, and SOCS3 plasmids were introduced to SMMC7721 cells with the highest miR-221 expression assessed. The expression of JAK–STAT3 signaling pathway–related genes and proteins was determined by Western blot analysis. Cell apoptosis, viability, migration, and invasion were evaluated by means of flow cytometry, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide, and transwell assays, respectively. HCC xenograft in nude mice was performed to measure HCC tumor growth. miR-221 was found to be highly expressed but SOCS3 was poorly expressed in HCC tissues. miR-221 expression was correlated with lymph node metastasis (LNM) and tumor node metastasis (TNM) of HCC, and SOCS3 expression was correlated with LNM, differentiation and TNM of HCC. SOCS3 is a target gene of miR-221. MiR-221 mimic or si-SOCS3 exposure was found to induce cell viability, migration, and invasion, and reduce apoptosis. MiR-221 inhibitor was observed to have inhibitory effects on HCC cell proliferation, migration, and invasion. Moreover, the expression of JAK–STAT3 signaling pathway was suppressed by miR-221 inhibitor. Downregulated miR-221 expression could promote its target gene SOCS3 to inhibit the proliferation, invasion and migration of HCC cells by repressing JAK–STAT3 signaling pathway.     

Effects of bufalin on the proliferation of human lung cancer cells and its molecular mechanisms of action

Bufalin, a naturally occurring small-molecule compound from Traditional Chinese Medicine (TCM) Chansu showed inhibitory effects against human prostate, hepatocellular, endometrial and ovarian cancer cells, and leukemia cells. However, whether or not bufalin has inhibitory activity against the proliferation of human non–small cell lung cancer (NSCLC) cells is unclear. The aim of this study is to study the effects of bufalin on the proliferation of NSCLC and its molecular mechanisms of action. The cancer cell proliferation was measured by MTT assay. The apoptosis and cell cycle distribution were analyzed by flow cytometry. The protein expressions and phosphorylation in the cancer cells were detected by Western blot analysis. In the present study, we have demonstrated that bufalin suppressed the proliferation of human NSCLC A549 cell line in time- and dose-dependent manners. Bufalin induced the apoptosis and cell cycle arrest by affecting the protein expressions of Bcl-2/Bax, cytochrome c, caspase-3, PARP, p53, p21WAF1, cyclinD1, and COX-2 in A549 cells. In addition, bufalin reduced the protein levels of receptor expressions and/or phosphorylation of VEGFR1, VEGFR2, EGFR and/or c-Met in A549 cells. Furthermore, bufalin inhibited the protein expressions and phosphorylation of Akt, NF-κB, p44/42 MAPK (ERK1/2) and p38 MAPK in A549 cells. Our results suggest that bufalin inhibits the human lung cancer cell proliferation via VEGFR1/VEGFR2/EGFR/c-Met–Akt/p44/42/p38-NF-κB signaling pathways; bufalin may have a wide therapeutic and/or adjuvant therapeutic application in the treatment of human NSCLC.     

CircRNA hsa_circRNA_104348 promotes hepatocellular carcinoma progression through modulating miR-187-3p/RTKN2 axis and activating Wnt/β-catenin pathway

Circular RNAs (circRNAs) have confirmed to participate in diverse biological functions in cancer. However, the expression patterns of circRNAs on hepatocellular carcinoma (HCC) remains unclear. In the present study, we clarified that hsa_circRNA_104348 was dramatically upregulated in HCC tissues and cells. Patients with HCC displaying high hsa_circRNA_104348 level possessed poor prognosis. Has_circ_104348 facilitated proliferation, migration, and invasion, meanwhile suppressed apoptosis of HCC cell. Furthermore, hsa_circRNA_104348 directly targeted miR-187–3p, could regulate miR-187-3p to affect proliferation, migration, invasion, and apoptosis of HCC cells, and may have effect on Wnt/β-catenin signaling pathway. Moreover, RTKN2 could be a direct target of miR-187-3p. In addition, knockdown of hsa_circRNA_104348 attenuated HCC tumorigenesis and lung metastasis in vivo. Taken together, these findings indicated that circular RNA hsa_circRNA_104348 might function as a competing endogenous RNA (ceRNA) to promotes HCC progression by targeting miR-187–3p/RTKN2 axis and activating Wnt/β-catenin pathway.Subject terms: Biotechnology, Cancer     

Non-canonical Wnt Signaling Induces Ubiquitination and Degradation of Syndecan4

Dynamic regulation of cell adhesion receptors is required for proper cell migration in embryogenesis, tissue repair, and cancer. Integrins and Syndecan4 (SDC4) are the main cell adhesion receptors involved in focal adhesion formation and are required for cell migration. SDC4 interacts biochemically and functionally with components of the Wnt pathway such as Frizzled7 and Dishevelled. Non-canonical Wnt signaling, particularly components of the planar cell polarity branch, controls cell adhesion and migration in embryogenesis and metastasic events. Here, we evaluate the effect of this pathway on SDC4. We have found that Wnt5a reduces cell surface levels and promotes ubiquitination and degradation of SDC4 in cell lines and dorsal mesodermal cells from Xenopus gastrulae. Gain- and loss-of-function experiments demonstrate that Dsh plays a key role in regulating SDC4 steady-state levels. Moreover, a SDC4 deletion construct that interacts inefficiently with Dsh is resistant to Wnt5a-induced degradation. Non-canonical Wnt signaling promotes monoubiquitination of the variable region of SDC4 cytoplasmic domain. Mutation of these specific residues abrogates ubiquitination and results in increased SDC4 steady-state levels. This is the first example of a cell surface protein ubiquitinated and degraded in a Wnt/Dsh-dependent manner.     

mTOR/miR-145-regulated exosomal GOLM1 promotes hepatocellular carcinoma through augmented GSK-3β/MMPs

Golgi membrane protein 1 (GOLM1/GP73) is a serum marker of hepatocellular carcinoma (HCC). We have previously shown that mTOR promoted tumorigenesis of HCC through stimulating GOLM1 expression. In this study, we demonstrated that the mammalian target of rapamycin (mTOR) was a negative regulator of microRNA-145 (miR-145) expression. miR-145 inhibited GOLM1 expression by targeting a coding sequence of GOLM1 gene. GOLM1 and miR-145 were inversely correlated in human HCC tissues. GOLM1-enriched exosomes activated the glycogen synthase kinase-3β/matrix metalloproteinases (GSK-3β/MMPs) signaling axis of recipient cells and accelerated cell proliferation and migration. In contrast, miR-145 suppressed tumorigenesis and metastasis. We suggest that mTOR/miR-145/GOLM1 signaling pathway should be targeted for HCC treatment.     

DDX3 acts as a tumor suppressor in colorectal cancer as loss of DDX3 in advanced cancer promotes tumor progression by activating the MAPK pathway

Objective: The treatment and prognosis of patients with advanced colorectal cancer (CRC) remain a difficult problem. Herein, we investigated the role of DEAD (Asp-Glu-Ala-Asp) box helicase 3 (DDX3) in CRC and proposed potential therapeutic targets for advanced CRC.Methods: The expression of DDX3 in CRC and its effect on prognosis were explored by databases and CRC tissue microarrays. Stable DDX3 knockdown and overexpression cell lines were established with lentiviral vectors. The effects of DDX3 on CRC were investigated by functional experiments in vitro and in vivo. The molecular mechanism of DDX3 in CRC was explored by western blotting. Molecular-specific inhibitors were further used to explore potential therapeutic targets for advanced CRC.Results: The expression of DDX3 was decreased in advanced CRC, and patients with low DDX3 expression had a poor prognosis. In vitro and in vivo experiments showed that low DDX3 expression promoted the proliferation, migration and invasion of CRC. DDX3 loss regulated E-cadherin and β-catenin signaling through the mitogen-activated protein kinase (MAPK) pathway as shown by western blotting. In addition, the MEK inhibitor, PD98059, significantly reduced the increased cell proliferation, migration and invasion caused by knockdown of DDX3.Conclusions: DDX3 acts as a tumor suppressor gene in CRC. DDX3 loss in advanced cancer promotes cancer progression by regulating E-cadherin and β-catenin signaling through the MAPK pathway, and targeting the MAPK pathway may be a therapeutic approach for advanced CRC.     

The role of HMGB1-RAGE axis in migration and invasion of hepatocellular carcinoma cell lines

High mobility group protein box1 (HMGB1) and its receptor—receptor for advanced glycation end products (RAGE) are pivotal factors in the development and progression of many types of tumor, but the role of HMGB1-RAGE axis in hepatocellular carcinoma (HCC) especially its effects on metastasis and recurrence remains obscure. Here, we report the role of HMGB1-RAGE axis in the biological behaviors of HCC cell lines and the underlying molecular mechanism. We show that the expressions of HMGB1, RAGE, and extracellular HMGB1 increase consistently according to cell metastasis potentials, while the concentration of soluble form of RAGE (sRAGE) is inversely related to metastasis potential of HCC cells. Furthermore, our data show that rhHMGB1 promotes cellular proliferation, migration, and invasion, and increases the level of nuclear factor kappa B (NF-κB), while administrations of HMGB1-siRNA, RAGE-siRNA, anti-HMGB1 neutralizing antibody, anti-RAGE neutralizing antibody, and sRAGE inhibit cellular proliferation, migration, and invasion. Moreover, we also demonstrate that the expression of NF-кB is inhibited by knockdown of HMGB1 or RAGE. Collectively, these data demonstrate that HMGB1 activates RAGE signaling pathways and induces NF-кB activation to promote cellular proliferation, invasion, and metastasis, in HCC cell lines. Taken together, HMGB1-RAGE axis may become a potential target in HCC therapy.     

Notch, IL-1 and leptin crosstalk outcome (NILCO) is critical for leptin-induced proliferation, migration and VEGF/VEGFR-2 expression in breast cancer

High levels of pro-angiogenic factors, leptin, IL-1, Notch and VEGF (ligands and receptors), are found in breast cancer, which is commonly correlated with metastasis and lower survival of patients. We have previously reported that leptin induces the growth of breast cancer and the expression of VEGF/VEGFR-2 and IL-1 system. We hypothesized that Notch, IL-1 and leptin crosstalk outcome (NILCO) plays an essential role in the regulation of leptin-mediated induction of proliferation/migration and expression of pro-angiogenic molecules in breast cancer. To test this hypothesis, leptin's effects on the expression and activation of Notch signaling pathway and VEGF/VEGFR-2/IL-1 were determined in mouse (4T1, EMT6 and MMT) breast cancer cells. Remarkably, leptin up-regulated Notch1-4/JAG1/Dll-4, Notch target genes: Hey2 and survivin, together with IL-1 and VEGF/VEGFR-2. RNA knockdown and pharmacological inhibitors of leptin signaling significantly abrogated activity of reporter gene-luciferase CSL (RBP-Jk) promoter, showing that it was linked to leptin-activated JAK2/STAT3, MAPK, PI-3K/mTOR, p38 and JNK signaling pathways. Interestingly, leptin upregulatory effects on cell proliferation/migration and pro-angiogenic factors Notch, IL-1 and VEGF/VEGFR-2 were abrogated by a γ-secretase inhibitor, DAPT, as well as siRNA against CSL. In addition, blockade of IL-1R tI inhibited leptin-induced Notch, Hey2 and survivin as well as VEGF/VEGFR-2 expression. These data suggest leptin is an inducer of Notch (expression/activation) and IL-1 signaling modulates leptin effects on Notch and VEGF/VEGFR-2. We show for the first time that a novel unveiled crosstalk between Notch, IL-1 and leptin (NILCO) occurs in breast cancer. Leptin induction of proliferation/migration and upregulation of VEGF/VEGFR-2 in breast cancer cells were related to an intact Notch signaling axis. NILCO could represent the integration of developmental, pro-inflammatory and pro-angiogenic signals critical for leptin-induced cell proliferation/migration and regulation of VEGF/VEGFR-2 in breast cancer. Targeting NILCO might help to design new pharmacological strategies aimed at controlling breast cancer growth and angiogenesis.     

Retracted: Long noncoding LINC01551 promotes hepatocellular carcinoma cell proliferation,migration, and invasion by acting as a competing endogenous RNA of microRNA-122-5p to regulate ADAM10 expression

Hepatocellular carcinoma (HCC) is a severe disease with high mortality in the world. It has been shown that long noncoding RNA (lncRNA) might play a role in HCC. The aim of the present study was to identify the role of long intergenic noncoding RNA 01551 (LINC01551) in the HCC development and explore the underlying mechanism of LINC01551/miR-122-5p/ADAM10 axis. The differentially expressed lncRNAs associated with HCC were screened out by a microarray analysis. The expression of LINC01551, miR-122-5p, and ADAM10 was determined in HCC tissues and cells. The potential miRNA (miR-122-5p) regulated by LINC01551 was explored, and the target relationship between miR-122-5p and ADAM10 was confirmed. To evaluate the effect of LINC01551 and miR-122-5p on proliferation, migration, invasion, and apoptosis of HCC, different plasmids were delivered into MHCC97-H cells. High expression of LINC01551 and ADAM10 yet low-expression of miR-122-5p were revealed in HCC tissues and cells. Overexpression of miR-122-5p could downregulate ADAM10. Biological prediction websites and fluorescence in situ hybridization assay verified that LINC01551 was mainly expressed in the cytoplasm. Silencing LINC01551 reduced HCC cell viability, proliferation, migration, invasion, and cell cycle entry yet induce cell apoptosis. Upregulation of LINC01551 increased its ability of competitively binding to miR-122-5p, thus reducing miR-122-5p and upregulating ADAM10 expression, as well as promoting the proliferative, migrative, and invasive ability. Taken together the results, it is highly possible that LINC01551 functions as an competing endogenous RNA (ceRNA) to regulate the miRNA target ADAM10 by sponging miR-122-5p and therefore promotes the development of HCC, highlighting a promising competitive new target for the HCC treatment.     

LncRNA FGD5-AS1 functions as an oncogene to upregulate GTPBP4 expression by sponging miR-873-5p in hepatocellular carcinoma

The long non-coding FGD5-AS1 (LncFGD5-AS1) has been reported to be a novel carcinogenic gene and participant in regulating tumor progression by sponging microRNAs (miRNAs). However, the pattern of expression and the biological role of FGD5-AS1 in hepatocellular carcinoma (HCC) remains largely unknown. The expression level of FGD5-AS1 in tumor tissues and cell lines was measured by RT-qPCR. CCK-8, EdU, flow cytometry, wound healing and transwell chamber assays were performed to investigate the role of FGD5-AS1 in cell proliferation, apoptosis, migration, and invasion in HCC. Dual luciferase reporter, and RNA pull-down assays were performed to identify the regulatory interactions among FGD5-AS1, miR-873-5p and GTP-binding protein 4 (GTPBP4). We found that the expression of FGD5-AS1 was upregulated in HCC tissues and cell lines. Moreover, the knockdown of FGD5-AS1 suppressed cell proliferation, migration and invasion, and induced apoptosis in HCC cells. Further studies demonstrated that FGD5-AS1 could function as a competitive RNA by sponging miR-873-5p in HCC cells. Moreover, GTPBP4 was identified as direct downstream target of miR-873-5p in HCC cells and FGD5-AS1mediated the effects of GTPBP4 by competitively binding with miR-873-5p. Taken together, this study demonstrated the regulatory role of FGD5-AS1 in the progression of HCC and identified the miR-873-5p/GTPBP4 axis as the direct downstream pathway. It represents a promising novel therapeutic strategy for HCC patients.Key words: Hepatocellular carcinoma, FGD5-AS1, miR-873-5p, GTPBP4     

Bufalin enhances the anti-proliferative effect of sorafenib on human hepatocellular carcinoma cells through downregulation of ERK

The purpose of this study was to investigate the effect of bufalin on the anti-proliferative activity of sorafenib in the human hepatocellular carcinoma (HCC) cell lines PLC/PRF/5 and Hep G-2 and to determine the relevant molecular mechanism. Concurrent treatment with sorafenib and bufalin at a fixed ratio (25:1) for 48 h resulted in synergistic growth inhibition in HCC cell lines as determined by CCK-8 cell viability assays. Exposure of both PLC/PRF/5 and Hep G-2 cells to this combination of sorafenib (6.25 μM) and bufalin (50 nM) resulted in noticeable increases in apoptotic cell death, as evidenced by the disruption of mitochondria, compared to treatment with either agent alone. Although both sorafenib (6.25 μM) and bufalin (250 nM) alone inhibited the phosphorylation of ERK, the reduction in pERK was more pronounced in the cells treated with a combination of bufalin (50 nM) and sorafenib (250 nM). Furthermore, the inhibitory effect of bufalin on pERK was blocked by the PI3kinase inhibitor LY294002, suggesting that the reduction in pERK induced by bufalin might be mediated by AKT in these two HCC cell lines. Taken together, the results of our study suggest that bufalin enhances the anti-cancer effects of sorafenib on PLC/PRF/5 and Hep G-2 by contributing to the downregulation of ERK.     

CT23 knockdown attenuating malignant behaviors of hepatocellular carcinoma cell is associated with upregulation of metallothionein 1

The cancer-testis antigen 23 (CT23) gene has been reported in association with the pathogenesis and progress of hepatocellular carcinoma (HCC). However, the alterations of gene expression profiling induced by CT23 knockdown in HCC cells remains largely unknown. In this study, the RNA interfering (RNAi) method was used to silence CT23 expression in BEL-7404 cells. Microarray analysis was performed on mRNA extracted from the CT23 knockdown cells and the control cells to determine the alterations of gene expression profiles. The result showed a total of 1051 genes expressed differentially (two-fold change), including 470 genes upregulated and 581 gene downregulated in the CT23 knockdown cells. A bioinformatic analysis showed that the functional differentially expressed genes (DEGs) were linked to cell proliferation, migration, and apoptosis, and metallothionein 1 (MT1) attained the maximum enrichment scores in functional annotation, classification, and pathway analysis of DEGs. Furthermore, Western blot analysis and cell behaviors assays verified that CT23 modulates cell proliferation, migration, and apoptosis by regulating MT1 expression in HCC cells and non-neoplastic hepatocytes. In summary, downregulated CT23 gene in BEL-7404 cells might change the expressions of carcinogenesis and progression related genes in HCC by upregulating MT1 expression, which would provide insight into searching for a novel therapeutic target for HCC.     

The fatty acid receptor CD36 promotes HCC progression through activating Src/PI3K/AKT axis-dependent aerobic glycolysis

Metabolic reprogramming is a new hallmark of cancer but it remains poorly defined in hepatocellular carcinogenesis (HCC). The fatty acid receptor CD36 is associated with both lipid and glucose metabolism in the liver. However, the role of CD36 in metabolic reprogramming in the progression of HCC still remains to be elucidated. In the present study, we found that CD36 is highly expressed in human HCC as compared with non-tumor hepatic tissue. CD36 overexpression promoted the proliferation, migration, invasion, and in vivo tumor growth of HCC cells, whereas silencing CD36 had the opposite effects. By analysis of cell metabolic phenotype, CD36 expression showed a positive association with extracellular acidification rate, a measure of glycolysis, instead of oxygen consumption rate. Further experiments verified that overexpression of CD36 resulted in increased glycolysis flux and lactic acid production. Mechanistically, CD36 induced mTOR-mediated oncogenic glycolysis via activation of Src/PI3K/AKT signaling axis. Pretreatment of HCC cells with PI3K/AKT/mTOR inhibitors largely blocked the tumor-promoting effect of CD36. Our findings suggest that CD36 exerts a stimulatory effect on HCC growth and metastasis, through mediating aerobic glycolysis by the Src/PI3K/AKT/mTOR signaling pathway.Subject terms: Cancer metabolism, Tumour biomarkers     

Bufalin Reverses Resistance to Sorafenib by Inhibiting Akt Activation in Hepatocellular Carcinoma: The Role of Endoplasmic Reticulum Stress

Sorafenib is the standard first-line therapeutic treatment for patients with advanced hepatocellular carcinoma (HCC), but its use is hampered by the development of drug resistance. The activation of Akt by sorafenib is thought to be responsible for this resistance. Bufalin is the major active ingredient of the traditional Chinese medicine Chan su, which inhibits Akt activation; therefore, Chan su is currently used in the clinic to treat cancer. The present study aimed to investigate the ability of bufalin to reverse both inherent and acquired resistance to sorafenib. Bufalin synergized with sorafenib to inhibit tumor cell proliferation and induce apoptosis. This effect was at least partially due to the ability of bufalin to inhibit Akt activation by sorafenib. Moreover, the ability of bufalin to inactivate Akt depended on endoplasmic reticulum (ER) stress mediated by inositol-requiring enzyme 1 (IRE1). Silencing IRE1 with siRNA blocked the bufalin-induced Akt inactivation, but silencing eukaryotic initiation factor 2 (eIF2) or C/EBP-homologous protein (CHOP) did not have the same effect. Additionally, silencing Akt did not influence IRE1, CHOP or phosphorylated eIF2α expression. Two sorafenib-resistant HCC cell lines, which were established from human HCC HepG2 and Huh7 cells, were refractory to sorafenib-induced growth inhibition but were sensitive to bufalin. Thus, Bufalin reversed acquired resistance to sorafenib by downregulating phosphorylated Akt in an ER-stress-dependent manner via the IRE1 pathway. These findings warrant further studies to examine the utility of bufalin alone or in combination with sorafenib as a first- or second-line treatment after sorafenib failure for advanced HCC.     

miR-186 modulates hepatocellular carcinoma cell proliferation and mobility via targeting MCRS1-mediated Wnt/β-catenin signaling

Previous studies have revealed that miR-186 is involved in the pathogenesis of many malignancies. However, the role of miR-186 in hepatocellular carcinoma (HCC) carcinogenesis and its detailed mechanism are poorly understood. This study was to investigate the function of miR-186 in modulating HCC cell proliferation, cell cycle, migration, and invasion. We found that miR-186 was decreased in HCC tissues and cell lines. Loss-of-function experiments showed that reduction of miR-186 dramatically enhanced tumor cell proliferation and metastasis. Besides, miR-186 also participated in the modulation of the cell cycle. In addition, luciferase reporter assays and Western blot analysis showed that MCRS1 was a novel target of miR-186 in HCC cells. Notably, upregulation of miR-186 suppressed the nuclear β-catenin accumulation and blocked the activation of Wnt/β-catenin signaling in HCC cells. Forced MCRS1 expression abrogated the inhibitory effect of miR-186 on cell growth, metastasis and Wnt/β-catenin signaling in HCC cells. Our findings may provide new insight into the pathogenesis of HCC and miR-186/ MCRS1 might function as new therapeutic targets for HCC.