Hepatocellular Carcinoma Cell Colony Formation Research Articles

E3 ubiquitin ligase CHIP interacts with transferrin receptor 1 for degradation and promotes cell proliferation through inhibiting ferroptosis in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the major form of liver malignancy with high incidence and mortality. Identifying novel biomarkers and understanding regulatory mechanisms underlying the development and progression of HCC are critical for improving diagnosis, treatment and patient outcomes. Carboxyl terminus of Hsc-70-interacting protein (CHIP) is a well-described U-box-type E3 ubiquitin ligase which promotes the ubiquitination and degradation of numerous tumor-associated proteins. Recent studies have shown that CHIP can play as a tumor-suppressor gene or an oncogene in different kinds of malignancies. To date, the function and mechanism of CHIP in hepatocellular carcinoma remains largely unknown. Based on TCGA data, we found that compared with high CHIP expression, the overall survival of HCC patients with low expression of CHIP was better. In addition, CHIP overexpression markedly enhanced HCC cell proliferation and colony formation. Conversely, knockdown of CHIP restrained the proliferation and colony formation of HCC cells. Meanwhile, knockdown of CHIP decreased mitochondrial cristae or ruptured outer mitochondrial membrane, promoted the accumulation of Fe2+ and ferroptosis of HCC cells. Further research for the first time confirmed that CHIP interacts and degrades transferrin receptor 1 (TfR1) by ubiquitin-proteasome pathway, which leads to the inhibition of ferroptosis and promotes the proliferation of HCC cells. The analysis of proteomics data from CPTAC revealed a negative correlation between CHIP and TfR1 protein expression levels in HCC. These findings indicate that CHIP acts as a negative modulator of ferroptosis and functions as an oncogene in HCC.

Rho GTPase-activating protein 1 promotes hepatocellular carcinoma progression via modulation by CircPIP5K1A/MiR-101-3p.

There has been an increased focus on regulating cell function with Rho family GTPases, including proliferation, migration/invasion, polarity, and adhesion. Due to the challenges involved in targeting Rho family GTPases directly, it may be more effective to target their regulators, such as Rho GTPase-activating protein 1 (ARHGAP1). This present research was performed to define the clinical significance of ARHGAP1 expression, as well as its regulatory mechanisms in hepatocellular carcinoma. ARHGAP1 and miR-101-3p expression of liver cancer patients, and their relevance with clinicopathological characteristics and prognosis were analyzed by the Cancer Genome Atlas sequencing data, and verified using samples of hepatocellular carcinoma patients. The interactions between miR-101-3p and ARHGAP1 or circPIP5K1A were validated by bioinformatic analyses, as well as confirmed by quantitative reverse transcription polymerase chain reaction, western blotting, and dual-luciferase reporter analysis. Plate clonality assays, cell adhesion and migration experiments, and proliferation experiments were used for assessing the participation of the circPIP5K1A/miR-101-3p/ARHGAP1 pathway in cell proliferation and motility. Elevated ARHGAP1 and reduced miR-101-3p expression are related to poorer survival. MiR-101-3p targets ARHGAP1 to suppress hepatocellular carcinoma cell colony formation and invasion, whereas miR-101-3p inhibitor reverses liver cancer proliferation and metastasis suppression caused by ARHGAP1 knockdown. In addition, circPIP5K1A, which is mainly distributed in the cytosol, showedcarcinogenic effects by sponging miR-101-3p, thus regulating ARHGAP1 expression. ARHGAP1 serves as an oncogenic gene in liver cancer, and the expression thereof is regulated by circPIP5K1A through sponging miR-101-3p.

Hsa_circ_0097009 regulates proliferation, apoptosis, migration and invasion of hepatocellular carcinoma cells via miR-568/RNF38 axis.

Circular RNAs (circRNAs), a class of non-coding RNAs, has been proved as an essential driver of hepatocellular carcinoma (HCC) progression. However, the aim of this study is to explore the role of hsa_circ_0097009 (circ_0097009) in HCC progression. Ki67 expression in HCC tissues was labeled and examined by IHC assay. Quantitative real-time PCR (qRT-PCR) was applied to assess the expression of circ_0097009, miR-568 and RING finger protein 38 (RNF38). Western blot assay was conducted to analyze protein expression. HCC cell colony formation, proliferation, migration, invasion, angiogenesis ability and apoptosis were determined by colony formation assay, 5-ethynyl-29-deoxyuridine (EdU) assay, wound healing assay, trans well assay, angiogenesis assay and flow cytometry. The interaction between circ_0097009 and miR-568 as well as miR-568 and RNF38 was probed by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. We found that circ_0097009 and RNF38 expressions were markedly higher, but miR-568 expression was significantly lower in HCC. Circ_0097009 knockdown blocked HCC cell cloning, proliferation, migration, invasion, angiogenesis and facilitated apoptosis. MiR-568 was a target of circ_0097009 in HCC cells. MiR-568 interference could attenuate the circ_0097009 knockdown-induced inhibition on HCC cells progress. MiR-568 depressed cell clonal formation, proliferation, migration, invasion, angiogenesis and promotion of apoptosis, by targeting RNF38, and RNF38 overexpression reversed the suppression of miR-568 in HCC cells. Also, circ_0097009 knockdown blocked tumor growth in vivoCONCLUSION: Together, this study indicated that the oncogenic function of circ_0097009 in HCC, and circ_0097009/miR-568/RNF38 axis was expected to be a novel therapeutic option for HCC patients (Tab. 1, Fig. 7, Ref. 33). Text in PDF www.elis.sk Keywords: circ_0097009, miR-568, RNF38, hepatocellular carcinoma.

Modulation of the p38 MAPK Pathway by Anisomycin Promotes Ferroptosis of Hepatocellular Carcinoma through Phosphorylation of H3S10.

Hepatocellular carcinoma (HCC) is a prevalent malignant tumor worldwide. Ferroptosis is emerging as an effective target for tumor treatment as it has been shown to potentiate cell death in some malignancies. However, it remains unclear whether histone phosphorylation events, an epigenetic mechanism that regulates transcriptional expression, are involved in ferroptosis. Our study found that supplementation with anisomycin, an agonist of p38 mitogen-activated protein kinase (MAPK), induced ferroptosis in HCC cells, and the phosphorylation of histone H3 on serine 10 (p-H3S10) was participated in anisomycin-induced ferroptosis. To investigate the anticancer effects of anisomycin-activated p38 MAPK in HCC, we analyzed cell viability, colony formation, cell death, and cell migration in Hep3B and HCCLM3 cells. The results showed that anisomycin could significantly suppress HCC cell colony formation and migration and induce HCC cell death. The hallmarks of ferroptosis, such as abnormal accumulation of iron and elevated levels of lipid peroxidation and malondialdehyde, were detected to confirm the ability of anisomycin to promote ferroptosis. Furthermore, coincubation with SB203580, an inhibitor of activated p38 MAPK, partially rescued anisomycin-induced ferroptosis. And the levels of p-p38 MAPK and p-H3S10 were successively increased by anisomycin treatment. The relationship between p-H3S10 and ferroptosis was revealed by ChIP sequencing. The reverse transcription PCR and immunofluorescence results showed that NCOA4 was upregulated both in mRNA and protein levels after anisomycin treatment. And by C11-BODIPY staining, we found that anisomycin-induced lipid reactive oxygen species was reduced after NCOA4 knockdown. In conclusion, the anisomycin-activated p38 MAPK promoted ferroptosis of HCC cells through H3S10 phosphorylation.

CD44 potentiates hepatocellular carcinoma migration and extrahepatic metastases via the AKT/ERK signaling CXCR4 axis.

BackgroundCell adhesion molecule cluster of differentiation 44 (CD44) plays a significant role in cancer cell local invasion, intravasation, migration, and the establishment of metastatic lesions. However, little is known about the underlying mechanism of how CD44 regulates hepatocellular carcinoma (HCC) extrahepatic metastasis (EHM).MethodsThe expression of CD44 in HCC tissues and cell lines was detected through western blot and immunohistochemistry (IHC). Through gain- and loss-of-function assays, we examined the oncogenic roles of CD44 in regulating HCC cell growth and metastasis in vitro and in vivo. To identify the potential mechanism, we employed quantitative real-time polymerase chain reaction, and western blot.ResultsIn this study, CD44 was highly expressed in HCC cells and HCC-patient specimens that exhibited high malignancy potential. The overall survival (OS) was worse and the cumulative recurrence rate was higher in HCC patients with CD44 overexpression than those with low levels of CD44 expression. Our in-vitro and in-vivo experiments showed that CD44 downregulation reduced HCC cell colony formation, migration, and invasion, and HCC tumor growth and metastasis, and that the pro-metastasis effect of CD44 was mediated by the protein kinase B (AKT)/extracellular signal-regulated kinase (ERK) signaling-chemokine receptor C-X-C chemokine receptor type 4 (CXCR4) axis. The reported capacity of CD44 to induce CXCR4 expression and increase the propensity of tumors to invade and metastasize to distant organs is consistent with the aggressive clinical characteristics of HCCs.ConclusionsCD44 could represent a future therapeutic target for EHM.

ARV-771 Acts as an Inducer of Cell Cycle Arrest and Apoptosis to Suppress Hepatocellular Carcinoma Progression.

Hepatocellular carcinoma (HCC) is the most commonly diagnosed liver cancer with limited treatment options and extremely poor prognosis worldwide. Recently, the proteolysis targeting chimeras (PROTACs), which aim to induce proteasome-mediated degradation of interesting proteins via recruiting E3 ligases, have become the advanced tools and attractive molecules for cancer treatment. However, the anticancer effects of PROTACs in HCC remain to be clarified. Here, we evaluate the anticancer activity of ARV-771, a previously reported PROTAC compound designed for bromodomain and extra-terminal domain (BET) proteins, in HCC. We show that ARV-771 suppresses the cell viability and colony formation of HCC cells via arresting cell cycle progression and triggering apoptosis. Further investigations reveal that ARV-771 notably downregulates multiple non-proteasomal deubiquitinases which are critical to the development of cancers. Additionally, HCC cells can decrease their sensitivity to ARV-771 via activating the MEK/ERK and p38 MAPKs. ARV-771 also inhibits HCC progression in vivo. Moreover, we show that ARV-771 and sorafenib, a Raf inhibitor that clinically used for targeted therapy of liver cancer, can synergistically inhibit the growth of HCC cells. Overall, this study not only explores the anticancer activity of ARV-771 and its underlying mechanisms in HCC, but also deepens our understanding of deubiquitinases, MAPKs, cell cycle, and apoptosis induction in cancer therapy.

RPL15 promotes hepatocellular carcinoma progression via regulation of RPs-MDM2-p53 signaling pathway

BackroundRPL15 has been found to participate in human tumorigenesis. However, its function and regulatory mechanism in hepatocellular carcinoma (HCC) development are still unclear. Current study investigated the effects of RPL15 in HCC.MethodsThe expression of RPL15 in clinical tissues and cell lines of HCC was detected by RT-qPCR, Western blotting, and Immunohistochemistry (IHC). Colony formation, CCK-8, flow cytometry, Wound healing and Transwell invasion assays, were used to detect the carcinoma progression of HCC cells with RPL15 overexpression or knockdown in vitro. A xenograft model was constructed to assess the effect of RPL15 knockdown on HCC cells in vivo. The expression of CDK2 and Cyclin E1 related to cell cycles, Bax and Bcl-2 related to cell apoptosis, E-cadherin, N-cadherin and Vimentin related to epithelial–mesenchymal transition (EMT), p53 and p21 related to p53 signaling pathway, were detected by Western blotting. The connection between p53, MDM2 and RPL5/11 affected by RPL15 was analyzed using immunoprecipitation and Cycloheximide (CHX) chase assay.ResultsElevated RPL15 was identified in HCC tissues, which was not only a prediction for the poor prognosis of HCC patients, but also associated with the malignant progression of HCC. RPL15 silencing arrested HCC cell cycle, suppressed HCC cell colony formation, proliferation, invasion, and migration, and induce cell apoptosis. On the contrary, RPL15 upregulation exerted opposite effects. Results also indicated that HCC cell invasion and migration were associated with EMT, and that the RPs-MDM2-p53 pathway was implicated in RPL15-mediated oncogenic transformation. In addition, RPL15 knockdown significantly suppressed HCC xenografts growth.ConclusionsRPL15 played crucial roles in HCC progression and metastasis, serving as a promising candidate for targeted therapies.

Matrix stiffness-induced upregulation of histone acetyltransferase KAT6A promotes hepatocellular carcinoma progression through regulating SOX2 expression.

Lysine acetyltransferase 6 A (KAT6A) is a MYST-type histone acetyltransferase (HAT) enzyme, which contributes to histone modification and cancer development. However, its biological functions and molecular mechanisms, which respect to hepatocellular carcinoma (HCC), are still largely unknown. Immunohistochemical, western blot and qRT-PCR analysis of KAT6A were performed. A series of in vitro and in vivo experiments were conducted to reveal the role of KAT6A in the progression of HCC. We demonstrated that KAT6A expression was upregulated in HCC tissues and cell lines. Clinical analysis showed that increased KAT6A was significantly associated with malignant prognostic features and shorter survival. Gain- and loss-of-function experiments indicated that KAT6A promoted cell viability, proliferation and colony formation of HCC cells in vitro and in vivo. We confirmed that KAT6A acetylates lysine 23 of histone H3 (H3K23), and then enhances the association of the nuclear receptor binding protein TRIM24 and H3K23ac. Consequently, TRIM24 functions as a transcriptional activator to activate SOX2 transcription and expression, leading to HCC tumorigenesis. Restoration of SOX2 at least partially abolished the biological effects of KAT6A on HCC cells. Overexpression of KAT6A acetyltransferase activity-deficient mutants or TRIM24 mutants lacking H3K23ac binding sites did not affect SOX2 expression and HCC biological function. Moreover, matrix stiffness can upregulate the expression of KAT6A in HCC cells. Our data support the first evidence that KAT6A plays an oncogenic role in HCC through H3K23ac/TRIM24-SOX2 pathway, and represents a promising therapeutic strategy for HCC patients.

Anti-cancer effects of 3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one derivatives on hepatocellular carcinoma harboring FGFR4 activation.

Hepatocellular carcinoma (HCC) is disease with a high mortality rate and limited treatment options. Alterations of fibroblast growth factor receptor 4 (FGFR4) has been regarded as an oncogenic driver for HCC and a promising target for HCC therapeutics. Herein, we report that GNF-7, a multi-targeted kinase inhibitor, and its derivatives including SIJ1263 (IC50 < 1 nM against FGFR4) are highly potent FGFR4 inhibitors and are capable of strongly suppressing proliferation of HCC cells and Ba/F3 cells transformed with wtFGFR4 or mtFGFR4. Compared with known FGFR4 inhibitors, both GNF-7 and SIJ1263 possess much higher (up to 100-fold) anti-proliferative activities via FGFR signaling blockade and apoptosis on HCC cells. Especially, SIJ1263 is 80-fold more potent (GI50=24 nM) on TEL-FGFR4 V550E Ba/F3 cells than BLU9931, which suggests that SIJ1263 would be effective for overriding drug resistance. In addition, both substances strongly suppress migration/invasion and colony formation of HCC cells. It is worth noting that SIJ1263 is superior to GNF-7 with regards to the fact that activities of SIJ1263 are higher than those of GNF-7 in all assays performed in this study. Collectively, this study provides insight into designing highly potent FGFR4 inhibitors capable of potentially overcoming drug-resistance for the treatment of HCC patients.

Expression of lncRNA MSC-AS1 in hepatocellular carcinoma cell lines and its effect on proliferation, apoptosis, and migration.

This study aimed to explore the expression of long non-coding RNA MSC-AS1 in hepatocellular carcinoma (HCC) cells and its effect on the proliferation, migration, and apoptosis of HCC cells. The expression of MSC-AS1 in HCC cell lines BEL7402, SMMC7721, Huh7, HepG2, MHCC97-H, and normal hepatocyte line L02 was detected by reverse transcriptase polymerase chain reaction. The HCC cells were divided into blank, negative control (NC)-small interfering RNA (siRNA) (transfected with negative siRNA), and MSC-AS1 siRNA (transfected with MSC-AS1 siRNA) groups. Cell counting kit-8 and colony formation assays were used to determine the proliferation, and cell apoptosis, migration, and invasion were detected by flow cytometry, wound healing, and transwell assays, respectively. Western blot was used to detect the expression of related proteins. The expression of MSC-AS1 in HCC cell lines was significantly higher than that in L02. In the MSC-AS1 siRNA group, the proliferation and colony formation of HCC cells were inhibited, whereas the apoptosis rate was significantly higher than that in the blank and NC-siRNA groups. The rate of wound healing and the number of invasion cells in the MSC-AS1 siRNA group were significantly lower than that in the blank and NC-siRNA groups. MSC-AS1 was upregulated in HCC cells, and the downregulation of MSC-AS1 could inhibit cell proliferation, migration, and invasion and promote apoptosis of HCC cells.

Self-Activated Cascade-Responsive Sorafenib and USP22 shRNA Co-Delivery System for Synergetic Hepatocellular Carcinoma Therapy.

Resistance to sorafenib severely hinders its effectiveness against hepatocellular carcinoma (HCC). Cancer stemness is closely connected with resistance to sorafenib. Methods for reversing the cancer stemness remains one of the largest concerns in research and the lack of such methods obstructs current HCC therapeutics. Ubiquitin‐specific protease 22 (USP22) is reported to play a pivotal role in HCC stemness and multidrug resistance (MDR). Herein, a galactose‐decorated lipopolyplex (Gal‐SLP) is developed as an HCC‐targeting self‐activated cascade‐responsive nanoplatform to co‐delivery sorafenib and USP22 shRNA (shUSP22) for synergetic HCC therapy. Sorafenib, entrapped in the Gal‐SLPs, induced a reactive oxygen species (ROS) cascade and triggered rapid shUSP22 release. Thus, Gal‐SLPs dramatically suppressed the expression of USP22. The downregulation of USP22 suppresses multidrug resistance‐associated protein 1 (MRP1) to induce intracellular sorafenib accumulation and hampers glycolysis of HCC cells. As a result, Gal‐SLPs efficiently inhibit the viability, proliferation, and colony formation of HCC cells. A sorafenib‐insensitive patient‐derived xenograft (PDX) model is established and adopted to evaluate in vivo antitumor effect of Gal‐SLPs. Gal‐SLPs exhibit potent antitumor efficiency and biosafety. Therefore, Gal‐SLPs are expected to have great potential in the clinical treatment of HCC.

BAP31 Promotes Tumor Cell Proliferation by Stabilizing SERPINE2 in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) patients are mostly diagnosed at an advanced stage, resulting in systemic therapy and poor prognosis. Therefore, the identification of a novel treatment target for HCC is important. B-cell receptor-associated protein 31 (BAP31) has been identified as a cancer/testis antigen; however, BAP31 function and mechanism of action in HCC remain unclear. In this study, BAP31 was demonstrated to be upregulated in HCC and correlated with the clinical stage. BAP31 overexpression promoted HCC cell proliferation and colony formation in vitro and tumor growth in vivo. RNA-sequence (RNA-seq) analysis demonstrated that serpin family E member 2 (SERPINE2) was downregulated in BAP31-knockdown HCC cells. Coimmunoprecipitation and immunofluorescence assays demonstrated that BAP31 directly binds to SERPINE2. The inhibition of SERPINE2 significantly decreased the BAP31-induced cell proliferation and colony formation of HCC cells and phosphorylation of Erk1/2 and p38. Moreover, multiplex immunohistochemistry staining of the HCC tissue microarray showed positive associations between the expression levels of BAP31, SERPINE2, its downstream gene LRP1, and a tumor proliferation marker, Ki-67. The administration of anti-BAP31 antibody significantly inhibited HCC cell xenograft tumor growth in vivo. Thus, these findings suggest that BAP31 promotes tumor cell proliferation by stabilizing SERPINE2 and can serve as a promising candidate therapeutic target for HCC.

The Mutational and Transcriptional Landscapes of Hepatocarcinogenesis in a Rat Model.

SummaryHepatocellular carcinoma (HCC) initiation is characterized by stepwise accumulation of molecular alterations, during which the early events are largely unknown. Here, we presented a comprehensive genomic and transcriptomic landscape at stages of hepatitis, cirrhosis, and HCC by using a diethylnitrosamine-induced rat HCC model. We observed the early occurrence of gene instability and aberrant cancer associated signaling pathways in liver hepatitis. We further characterized the progressive molecular changes during hepatocarcinogenesis, wherein the intense rivalry between tumor-suppressive and oncogenic strengths occurred in cirrhosis stage. Despite the significant pathological difference, mutation signatures and expression landscape are highly similar between hepatitis and cirrhosis stages. Furthermore, we identified PI3K-Akt signaling pathway as a key pathway in the process of hepatocarcinogenesis through integrative analysis, and PIK3CD is a potential biomarker indicating HCC recurrence. The dynamic immune response during hepatocarcinogenesis, such as continuous decline of monocytes, suggests an immunological intervention strategy beyond chemoprevention for liver cancer.

CBL0137 administration suppresses human hepatocellular carcinoma cells proliferation and induces apoptosis associated with multiple cell death related proteins.

Hepatocellular carcinoma (HCC) is one of the deadliest cancers worldwide due to the lack of effective therapy methods. Therefore, there is an urgent need to develop novel therapies for HCC. CBL0137 is a small molecule that affects p53 and nuclear factor-kappa B (NF-κB). The expression of p53 was measured by using immunohistochemistry (IHC) in tumor and adjacent tissues. Western blotting (WB) and quantitative real-time polymerase chain reaction (qRT-PCR) were employed to detect the level of p-p53, p53, Bax, and PUMA after CBL0137 administration. CCK-8 and immunofluorescence staining (IF) assays were performed to evaluate the proliferation and viability of HCC cells. Flow cytometry was used to detect the apoptosis of HCC cells. Xenograft model was established to determine the effect of CBL0137 treatment on HCC tumor growth in vivo. HE staining was used to monitor HCC cell morphology and IHC staining for Ki-67 was performed to determine the tumor cell proliferation following CBL0137 treatment. Results showed that the expression of p-p53, p53, Bax, and PUMA was upregulated after CBL0137 administration. The viability, growth, and colony formation of HCC cells were significantly inhibited by CBL0137 in the CBL group compared with the NC group (p<0.05). Meanwhile, the results revealed that the proportions of apoptotic and necrotic cells were significantly elevated in the CBL group compared to the NC group (p<0.05). And the apoptosis-related proteins including PARP, caspase-3, caspase-7, caspase-8, and caspase-9 were increased in the CBL group compared with the NC group (p<0.05), while the NF-κB, p-NF-κB and p-AKT expression levels were significantly downregulated following CBL0137 treatment (p<0.05). Additionally, the tumor volume and weight were significantly reduced in the CBL group compared with the NC group (p<0.05). Moreover, HE staining and IHC staining for Ki-67 indicated that CBL0137 treatment could obviously induce cell apoptosis and suppress cell proliferation. CBL0137 treatment could effectively inhibit HCC cell proliferation and induce cell apoptosis associated with multiple factors expression.

The Roles of GSK-3β in Regulation of Retinoid Signaling and Sorafenib Treatment Response in Hepatocellular Carcinoma.

Rationale: Glycogen synthase kinase-3β (GSK-3β) plays key roles in metabolism and many cellular processes. It was recently demonstrated that overexpression of GSK-3β can confer tumor growth. However, the expression and function of GSK-3β in hepatocellular carcinoma (HCC) remain largely unexplored. This study is aimed at investigating the role and therapeutic target value of GSK-3β in HCC.Methods: We firstly clarified the expression of GSK-3β in human HCC samples. Given that deviated retinoid signalling is critical for HCC development, we studied whether GSK-3β could be involved in the regulation. Since sorafenib is currently used to treat HCC, the involvement of GSK-3β in sorafenib treatment response was determined. Co-immunoprecipitation, GST pull down, in vitro kinase assay, luciferase reporter and chromatin immunoprecipitation were used to explore the molecular mechanism. The biological readouts were examined with MTT, flow cytometry and animal experiments.Results: We demonstrated that GSK-3β is highly expressed in HCC and associated with shorter overall survival (OS). Overexpression of GSK-3β confers HCC cell colony formation and xenograft tumor growth. Tumor-associated GSK-3β is correlated with reduced expression of retinoic acid receptor-β (RARβ), which is caused by GSK-3β-mediated phosphorylation and heterodimerization abrogation of retinoid X receptor (RXRα) with RARα on RARβ promoter. Overexpression of functional GSK-3β impairs retinoid response and represses sorafenib anti-HCC effect. Inactivation of GSK-3β by tideglusib can potentiate 9-cis-RA enhancement of sorafenib sensitivity (tumor inhibition from 48.3% to 93.4%). Efficient induction of RARβ by tideglusib/9-cis-RA is required for enhanced therapeutic outcome of sorafenib, which effect is greatly inhibited by knocking down RARβ.Conclusions: Our findings demonstrate that GSK-3β is a disruptor of retinoid signalling and a new resistant factor of sorafenib in HCC. Targeting GSK-3β may be a promising strategy for HCC treatment in clinic.

Sanguinarine inhibits epithelial\u2013mesenchymal transition via targeting HIF-1\u03b1/TGF-\u03b2 feed-forward loop in hepatocellular carcinoma

Epithelial–mesenchymal transition (EMT) plays a crucial role in hepatocellular carcinoma (HCC) progression. Hypoxia and excessive transforming growth factor-β (TGF-β) have been identified as inducers and target for EMT in HCC. Here, we show hypoxia inducible factor-1α (HIF-1α) and TGF-β form a feed-forward loop to induce EMT in HCC cells. Further mechanistic study indicates under both hypoxia and TGF-β stimulation, Smad and PI3K-AKT pathways are activated. We show sanguinarine, a natural benzophenanthridine alkaloid, impairs the proliferation of nine kinds of HCC cell lines and the colony formation of HCC cells. In hypoxic and TGF-β cell models, sanguinarine inhibits HIF-1α signaling and the expression of EMT markers, translocation of Snail and activation of both Smad and PI3K-AKT pathways. Sanguinarine could also inhibit TGF-β-induced cell migration in HCC cells. In vivo studies reveal that the administration of sanguinarine inhibits tumor growth and HIF-1α signaling, inhibits the expression changes of EMT markers as well as Smad and PI3K-AKT pathway proteins. Our findings suggest that sanguinarine is a promising candidate targeting HIF-1α/TGF-β signaling to improve the treatment for HCC patients.

LINC00473 promotes hepatocellular carcinoma progression via acting as a ceRNA for microRNA-195 and increasing HMGA2 expression

Hepatocellular carcinoma (HCC) is a most aggressive malignant tumor. Nevertheless, the molecular mechanisms underlying HCC are still completely unclear. LINC00473 is identified as a tumor promoter in many cancers. In this investigation, the function of LINC00473 was specifically focused on. We exhibited that LINC00473 was obviously elevated in HCC cells compared to QSG-7701 cells. Functionally, down-regulation of LINC00473 could prevent HCC cell viability and cell proliferation. For another, HCC cell colony formation capacity was greatly restrained while cell apoptosis was triggered by loss of LINC00473. Meanwhile, would-healing assay and transwell invasion experiments were employed in our present study. As demonstrated, we observed that HCC cell migratory and invasive ability were obviously suppressed by the silence of LINC00473. Apart from these, mechanistic investigations implied miR-195 was a sponge target of LINC00473. It is widely established miR-195 is a famous tumor inhibitory gene regulator in various cancers. Here, we confirmed the binding correlation between LINC00473 and miR-195 using RIP assay. Subsequently, in vivo experiments were employed and it was manifested that LINC00473 was able to promote HCC tumor growth via acting as a ceRNA to inhibit miR-195. HMGA2 is a kind of nuclear-binding protein and it is involved in various cancers. We predicted it as a target of miR-195 and we confirmed their correlation. In addition, HMGA2 was repressed by loss of LINC00473, which was rescued by miR-195 inhibitors. Then, we found that angiogenic fator vascular endothelial growth factor (VEGF) was inhibited by loss of LINC00473 whereas anti-angiogenic factor EPN2 was induced in vivo. Taken all these together, our study revealed the significance of LINC00473/miR-195/HMGA2 signaling axis for the first time in HCC progression. It was suggested the potential possibility of LINC00473 as an indicator for HCC.

LINC00857 contributes to hepatocellular carcinoma malignancy via enhancing epithelial-mesenchymal transition.

Hepatocellular carcinoma (HCC) remains the fifth most frequent cancer with high mortality rate worldwide. However, the underlying molecular mechanisms of HCC progression are still barely known. Long noncoding RNAs (lncRNAs) have been recognized as significant therapeutic targets for HCC. Recently, the biological role of LINC00857 in several cancer types has been reported. Our present study was aimed to investigate the role of LINC00857 in HCC progression. We observed that LINC00857 was overexpressed in HCC cell lines (Huh7, Hep3B, HepG2, MHCC-97H, and SNU449). Knockdown of LINC00857 significantly repressed Hep-3B and SNU449 cell proliferation and inhibited the HCC cell colony formation. In addition, cell apoptosis was induced by the silence of LINC00857 and cell cycle progression was blocked in G1 phase. Besides these, downregulation of LINC00857 was able to restrain HCC cell migration and invasion capacity via enhancing epithelial-mesenchymal transition (EMT) process. As displayed, E-cadherin protein expression was increased by LINC00857 silence, while N-cadherin protein level was repressed by LV-shLINC00857 in HCC cells. Finally, the in vivo assays were used and the data indicated that LINC00857 could also obviously suppress the HCC tumor growth in vivo. In conclusion, our study revealed that LINC00857 might provide a novel perspective for the HCC treatment.

LINC00707 promotes hepatocellular carcinoma progression through activating ERK/JNK/AKT pathway signaling pathway.

Increasing evidence has demonstrated that abnormal expression of lncRNA is correlated with various malignant tumors, including hepatocellular carcinoma (HCC). Our current study was aimed to investigate the role of LINC00707 in HCC development. We observed that LINC00707 was upregulated in HCC cell lines compared with normal liver cell lines. Then, Hep3B cells and SNU449 cells were infected with LV-shLINC00707 and LV-LINC00707. LINC00707 silencing could greatly repress the proliferation and colony formation of HCC cells in vitro. On the contrary, overexpression of LINC00707 induced HCC cell proliferation and colony formation. In addition, HCC cell apoptosis was significantly enhanced and HCC cell cycle was blocked in G1 phase by LV-shLINC00707. Hep3B cells and SNU449 cell invasion capacity was restrained by the knockdown of LINC00707, whereas upregulation of LINC00707 exhibited an opposite phenomenon. Accumulating evidence has reported that ERK/JNK/AKT signaling is involved in multiple cancers, including HCC. Here, in our study, we identified that ERK/JNK/AKT signaling was dramatically restrained by silencing of LINC00707 while activated by LV-LINC00707 in HCC cells. Subsequently, an in vivo experiment was conducted, and it demonstrated that LINC00707 could modulate HCC development through activating ERK/JNK/AKT signaling. Taking the above results together, it was implied in our study that LINC00707 contributed to HCC progression through modulating the ERK/JNK/AKT pathway.

Expression of MFF and its biological effects in hepatocellular carcinoma

Objective To evaluate the expression of mitochondrial fission factor (MFF) and its biolo-gical effects in the progression of hepatocellular carcinoma (HCC). Methods ①Quantitative real-time PCR (qPCR), Western blotting and immunohistochemistry analysis were used to detect the expression levels of MFF in HCC tumor tissues and cell lines. ②The effect of MFF knockdown on proliferation of HCC cells was analyzed by methyl thiazolyl tetrazolium (MTT) and colony formation assays in siCtrl, si-MFF#1, si-MFF#2 groups. ③The effect of MFF knockdown on apoptosis of HCC cells was analyzed by apoptosis assay with Annexin Ⅴ-FITC and PI. Results ①The MFF expression was higher in tumor tissues compared with tumor-adjacent normal tissues [mRNA level M(QR): 0.292(0.443) vs. 0.235(0.333), Z=-4.166, P<0.001; protein level M(QR): 5.414 (4.545) vs. 3.120 (3.955), Z=-3.961, P<0.001)]. The MFF expression was higher in HCC cell lines compared with normal liver cell line. ②RNA interference-mediated knockdown of MFF inhibited proliferation of HCC cells (siCtrl vs. si-MFF#1: 5.29±0.34 vs. 3.34±0.37, P=0.014; siCtrl vs. si-MFF#2: 5.29±0.34 vs. 3.09±0.40, P=0.010). RNA interference-mediated knockdown of MFF inhibited colony formation of HCC cells (siCtrl vs. si-MFF#1: 95.35±21.20 vs. 37.56±10.61, P=0.003; siCtrl vs. si-MFF#2: 95.35±21.20 vs. 41.23±10.82, P=0.004). ③RNA interference-mediated knockdown of MFF induced apoptosis of HCC cells (siCtrl vs. si-MFF#1: 9.56%±1.70% vs. 20.08%±2.03%, P<0.001; siCtrl vs. si-MFF#2: 9.56%±1.70% vs. 21.14%±1.38%, P<0.001). Conclusion MFF is overexpressed in HCC, which accelerates cell proliferation and suppresses apoptosis, indicating that MFF can serve as a potential oncogene and drug target in HCC treatment. Key words: Liver neoplasms; Cell proliferation; Apoptosis; Mitochondrial fission factor