HCCLM3 Cells Research Articles

MiR-1307-3p promotes tumor growth and metastasis of hepatocellular carcinoma by repressing DAB2 interacting protein

Increasing studies provide evidence to support that microRNAs (miRNAs) play important roles in regulating hepatocellular carcinoma (HCC) initiation and progression. However, whether miR-1307-3p is aberrantly expressed in HCC and affects malignant behaviors of cancer cells remain unknown. In this study, we found that miR-1307-3p expression was obviously up-regulated in HCC compared to adjacent nontumor tissues. Moreover, miR-1307-3p expression was prominently higher in HCC cells compared with the normal hepatic cell line LO2. Patients with venous infiltration, tumor size ≥5 cm and advanced tumor stages (III + IV) had significant higher levels of miR-1307-3p in HCC tissues. Notably, the high level of miR-1307-3p predicted poor clinical outcomes of HCC patients. Functionally, miR-1307-3p knockdown inhibited the proliferation, migration and invasion of MHCC97H and HCCLM3 cells, and suppressed the in vivo growth and metastasis of HCCLM3 cells. Conversely, overexpression of miR-1307-3p facilitated Hep3B cell proliferation, migration and invasion. Mechanistically, DAB2 interacting protein (DAB2IP) was screened as a direct target of miR-1307-3p. The expression of DAB2IP mRNA was down-regulated and inversely correlated with miR-1307-3p level in HCC tissues. miR-1307-3p knockdown increased the level of DAB2IP in HCC cells. Luciferase reporter assay confirmed the direct interaction between miR-1307-3p and 3′UTR of DAB2IP. Importantly, DAB2IP overexpression significantly suppressed the proliferation, migration and invasion of HCCLM3 cells. DAB2IP knockdown rescued miR-1307-3p silencing-attenuated HCC cell proliferation, migration and invasion. Taken together, our findings suggest that miR-1307-3p plays a driving role in HCC progression by targeting DAB2IP. Our study may provide new therapeutic targets for HCC treatment.

Sublethal heat treatment of hepatocellular carcinoma promotes intrahepatic metastasis and stemness in a VEGFR1-dependent manner.

Incomplete radiofrequency ablation (RFA) of hepatocellular carcinoma (HCC) could initiate malignant transition. Patient-derived xenograft (PDX) mice model was established to investigate the effect of VEGF pathway in incomplete RFA of HCC with high fidelity. Cancer stem cell markers and metastatic markers were increased after incomplete RFA, with increased VEGFR1 and decreased VEGFR2 expression. In vitro experiments revealed sublethal heat treatment promoted migration ability of HepG2, HCCLM3, and SMMC7721 cells, which coincided with enhanced ability of sphere formation and up-regulation of VEGFR1, CD133, CD44, and EpCAM. Moreover, HCC cells secreted more VEGF after heat-treatment. VEGF promoted migration and enhanced stemness of HCC cells, which could not be suppressed by VEGFR2 inhibitor. PIGF, the ligand of VEGFR1, significantly increased migration and stemness of HCC cells. Blocking VEGFR1 reduced heat-induced enhancement of migration and stemness, whereas inhibition of VEGFR2 could not. In conclusion, VEGFR1 plays a critical role in sublethal heat treatment-induced enhancement of migration and stemness in HCC, suggesting that VEGFR1 may serve as a potential and promising therapeutic target for preventing recurrence after RFA.

Low‑intensity ultrasound enhances the chemosensitivity of hepatocellular carcinoma cells to cisplatin via altering the miR‑34a/c‑Met axis.

Recently, the use of low-intensity ultrasound (LIUS) combined with chemotherapeutic agents is widely used in clinical practice, mainly for the treatment of cancer; however, the mechanisms as to how LIUS enhances the antitumor effects of these agents are not fully understood. The aim of the present study was to explore the synergistic antitumor effects and mechanisms of cisplatin (DDP) combined with LIUS (LIUS-DDP) in hepatocellular carcinoma (HCC). We reported that LIUS effectively enhanced Huh7 and HCCLM3 cell sensitivity to a low concentration of DDP. Reverse transcription-quantitative polymerase chain reaction analysis revealed that LIUS could increase the expression of microRNA-34a (miR-34a) in HCC cells following DDP treatment. In addition, LIUS-DDP significantly increased intracellular reactive oxygen species (ROS) levels in vitro, and the upregulation of miR-34a induced by LIUS-DDP was reversed by the ROS scavenger N-acetylcysteine, suggesting that LIUS upregulates the expression of miR-34a via production of ROS. In addition, knockdown of miR-34a in HCC cells significantly suppressed the synergistic effects of LIUS-DDP treatment. Conversely, overexpression of miR-34a enhanced these synergistic effects. The results of a dual-luciferase assay indicated that c-Met, a well-known oncogene, was a target of miR-34a. We also determined that LIUS-DDP treatment inhibited the expression of c-Met, possibly due to increased ROS production, which upregulated miR-34a expression. Furthermore, overexpression of c-Met reversed the synergistic effects of LIUS-DDP treatment. Our findings suggest that LIUS could enhance the chemosensitivity of HCC cells to DDP by altering the miR-34a/c-Met axis. Therefore, DDP combined with LIUS may be a potential therapeutic application for the clinical treatment of patients with HCC.

Upregulation of cystatin SN promotes hepatocellular carcinoma progression and predicts a poor prognosis.

Cystatin SN, a specific cysteine protease inhibitor, is thought to be involved in various malignant tumors. Therefore, we evaluated the role of cystatin SN in hepatocellular carcinoma (HCC). Notably, cystatin SN was elevated in tumorous samples and cells. Moreover, overexpression of cystatin SN was correlated with tumor diameter and TNM stage. Cox multivariate analysis displayed that cystatin SN was an independent prognosis indicator and that high cystatin SN level was associated with a dismal prognosis. Moreover, cystatin SN enhancement facilitated the proliferation, migratory, and invasive potential of Huh7 and HCCLM3 cells, whereas cystatin SN knockdown caused the opposite effect. Cystatin SN also modulated the epithelial‐mesenchymal transition progression through the PI3K/AKT pathway. In vivo cystatin SN promoted HCCLM3 cell growth and metastasis in xenograft mice model. Thus, cystatin SN was involved in HCC progression and could be a latent target for HCC treatment.

Expression of microRNA-chr1011 in hepatocellular carcinoma and its clinical significance

Objective To observe the expression of microRNA (miRNA, miR)-chr1011 in hepatocellular carcinoma (HCC) tissues and analyze its clinical significance in HCC patients. Methods High-throughput deep sequencing was used to analyze the small RNA sequences in 6 pairs of HCC tissues. Data of deep sequencing were compared with the gene database to predict novel miRNAs by miRdeep 2.0.0.5 software. Northern blot was utilized to verify the structure and authenticity of the novel miRNAs. The expression levels of novel miRNAs in 62 pairs of HCC tissues were detected by Real-time quantitative polymerase chain reaction (RT-qPCR) and the clinical data of HCC patients were analyzed. Results MiR-chr1011 was found in human liver tissues and liver cancer cell lines, which has a stable precursor structure and is located on human chromosome chr10.11: 107803675-107803737: + . Northern blot assay confirmed its expression in HCCLM3 and Huh7 liver cancer cells. In 62 pairs of HCC tissues, the expression of miR-chr1011 in tumor adjacent tissues was higher than tumor tissues (4.37±0.31 vs. 2.81±0.21, P< 0.01), and the expression of miR-chr1011 was negatively correlated with the Barcelona stage of tumor (P<0.01), TNM stage (P<0.01) and portal vein thrombosis (PVTT) formation (P<0.05). Conclusion MiR-chr1011 is lowly expressed in liver cancer tissues and is significantly associated with the prognosis of HCC patients. Key words: Carcinoma, hepatocellular; MicroRNA-chr1011

MiR-1-3p suppresses proliferation of hepatocellular carcinoma through targeting SOX9.

BackgroundLiver cancer was the fourth leading cause of cancer-related death in 2015. Hepatocellular carcinoma (HCC) is the most common type of liver cancer. miR-1-3p plays important roles in cancer, including prostate, bladder, lung cancer, and colorectal carcinoma. The function of miR-1-3p in HCC remains poorly understood.MethodsqRT-PCR was performed to detect the miR-1-3p expression in HCC cell lines (HCCLM3, Hep3B, Bel-7404, SMMC-7721) and the normal human hepatic cell line (LO2). HCCLM3 and Bel-7404 cells were transfected with miR-1-3p mimic or scramble control followed by water-soluble tetrazolium salt (WST-1) assay. Western bolt analysis was performed to determine the protein levels. TargetScan7.1 (http://www.targetscan.org/vert_71/) was used to predict the potential targets of miR-1-3p. SRY (sex determining region Y)-box 9 (SOX9), which has been previously shown to play an important role in HCC, was found to be a target of miR-1-3p. Luciferase reporter assay was used to explore the targeting of miR-1-3p on SOX9. For in vivo tumorigenesis assay, HCCLM3 cells with stable overexpression of miR-1-3p or control plasmid were injected subcutaneously into the flank of the SCID mice and animals were monitored for tumor growth.ResultsmiR-1-3p was significantly downregulated in HCC cell lines (HCCLM3, Hep3B, Bel-7404, and SMMC-7721) compared to normal human hepatic cell line (LO2). Overexpression of miR-1-3p significantly inhibited the proliferation and induced apoptosis in HCCLM3 and Bel-7474 cells. SOX9 was a direct target of miR-1-3p in HCC cells. Inhibition of SOX9 significantly inhibited the proliferation of HCCLM3 and Bel-7474 cells. In vivo, overexpression of miR-1-3p decreased tumor volume in a xenograft model.ConclusionThese results highlight the role of miR-1-3p in HCC. Overexpression of miR-1-3P inhibited the proliferation of HCC at least partly due to the regulation of SOX9. miR-1-3p may be a promising therapeutic candidate for HCC.

MiR-503-5p regulates cell epithelial-to-mesenchymal transition, metastasis and prognosis of hepatocellular carcinoma through inhibiting WEE1.

To explore the role and underlying mechanism of MicroRNA-503-5p (miR-503-5p) in the metastasis and prognosis of hepatocellular carcinoma (HCC). Sixty-three pairs of surgical HCC specimens and adjacent tissue samples were obtained, and Huh7, Hep3B, HCCLM3, MHCC-97H, MHCC-97L, LO2, and HEK293T cell lines were used for this study. The transwell assay was used to detect cell migration and invasion. Additionally, Western blot and quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) were used to detect relative protein and mRNA expression levels, respectively. High miR-503-5p expression inhibited cell mobility in HCCLM3 cells, while low miR-503-5p expression promoted cell migration and invasion in HCCLM3 cells. The same effect of miR-503-5p on EMT was also observed in HCC through Western blot. We then performed a dual-luciferase assay to show that WEE1 is a direct target of miR-503-5p in HCC. Furthermore, WEE1 knockdown inhibited EMT and cell metastasis in HCC cells. WEE1 overexpression impaired the inhibitory effect of miR-503-5p in HCC CONCLUSIONS: MiR-503-5p inhibited cell EMT and metastasis through inhibiting WEE1, which predicted prognosis of HCC. MiR-503-5p and WEE1 may be used as potential biomarkers for the prognosis of HCC.

Insufficient radiofrequency ablation promotes the metastasis of residual hepatocellular carcinoma cells via upregulating flotillin proteins

PurposeRadiofrequency ablation (RFA) therapy has proven to be effective and feasible for early-stage hepatocellular carcinoma (HCC); however, rapid progression of residual tumor cells after RFA has been confirmed, but the molecular mechanisms of this phenomenon are poorly understood. This study evaluated the effect of the lipid raft proteins known as flotillins on the invasive and metastatic potential of residual HCC.MethodsThe human HCC cell line HCCLM3 was used to establish insufficient RFA models in vivo and in vitro. Changes in cellular morphology, soft agar colony formation, motility, metastasis, and epithelial–mesenchymal transition (EMT) markers after insufficient RFA intervention in vitro and in vivo were detected by real-time PCR, western blotting, immunohistochemistry and transwell assays.ResultsThe results showed that flotillin-1 and flotillin-2 expression were upregulated in HCCLM3 cells following 45 °C heat treatment and in residual HCCLM3 xenografts cells after insufficient RFA. Knocking down flotillin-1 or flotillin-2 in HCCLM3 cells by shRNA significantly lowered insufficient RFA-induced tumor growth, EMT changes, and metastasis in vitro and in vivo. Furthermore, mechanism studies indicated that flotillins altered the EMT status and metastatic potential of heat-treated HCCLM3 cells by activating the Akt/Wnt/β-catenin signaling pathway.ConclusionsOur findings present new evidence that flotillins play a key role in the aggressive behaviors of residual cancer cells after insufficient RFA and provide new insights into the regulatory mechanism of Wnt/β-catenin signaling.

SHMT1 inhibits the metastasis of HCC by repressing NOX1-mediated ROS production

BackgroundHepatocellular carcinoma (HCC) is the most major type of primary hepatic cancer. Serine hydroxymethyltransferase 1 (SHMT1) is recently found to play critical roles in human cancers including lung cancer, ovarian cancer and intestinal cancer. However, the expression, function and the underlying mechanisms of SHMT1 in HCC remain uncovered.MethodsqRT-PCR, immunohistochemistry and immunoblotting were performed to detect the expression of SHMT1 in HCC tissues and cell lines. HCC cell migration and invasion were determined by Boyden chamber and Transwell assay in vitro, and tumor metastasis was assessed via lung metastasis model in mice. The expression of key factors involved in epithelial-to-mesenchymal transition (EMT) process was evaluated by western blotting.ResultsIn this study, data mining of public databases and analysis of clinical specimens demonstrated that SHMT1 expression was decreased in HCC. Reduced SHMT1 level was correlated with unfavorable clinicopathological features and poor prognosis of HCC patients. Gain- and loss-of-function experiments showed that SHMT1 overexpression inhibited the migration and invasion of HCCLM3 cells while SHMT1 knockdown enhanced the metastatic ability of Hep3B cells. Furthermore, qRT-PCR and western blotting showed that SHMT1 inhibited EMT and matrix metallopeptidase 2 (MMP2) expression. In vivo experiments showed that SHMT1 suppressed the lung metastasis of HCC cells in mice. Mechanistically, SHMT1 knockdown enhanced reactive oxygen species (ROS) production, and thus promoted the motility, EMT and MMP2 expression in Hep3B cells. Furthermore, NADPH oxidase 1 (NOX1) was identified to be the downstream target of SHMT1 in HCC. NOX1 expression was negatively correlated with SHMT1 expression in HCC. Rescue experiments revealed that NOX1 mediated the functional influence of SHMT1 on HCC cells.ConclusionsThese data indicate that SHMT1 inhibits the metastasis of HCC by repressing NOX1 mediated ROS production.

Effect of alanine-glyoxylate aminotransferase 2-like 1 on proliferation, apoptosis and cell-division cycle of human hepatoma cell lines 97H and HCCLM3

Objective To investigate the effect of alanine-glyoxylate aminotransferase 2-like 1 (AGXT2L1) on the proliferation, apoptosis and cell-division cycle of hepatoma cells. Methods Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) was used to detect the expression of AGXT2L1 in 97H, HCCLM3 and LO2 cells. The siRNA targeting AGXT2L1 was designed, synthetized and transfected into 97H and HCCLM3 cells to construct AGXT2L1 knockdown group, and the efficiency of knockdown was validated by RT-qPCR. The cell proliferation of each group at 0, 12, 24, 48, 72 and 96 h was tested by cell counting kit-8 (CCK-8) assay. Cell cycle assay was applied to detect the distribution of cells in G1, S and G2 phases. The TdT-mediated dUTP nick end labeling (TUNEL) assay was used to detect the apoptosis rate of cells in each group. Results The expression of AGXT2L1 in 97H and HCCLM3 cells was significantly higher than that in normal liver cells (1 350.04±91.36, 1 710.64±92.34, t97H=25.574, P<0.01; tHCCLM3=32.068, P<0.01). The AGXT2L1 expression level in 97H and HCCLM3 cells was decreased significantly after transfection with AGXT2L1-siRNA (0.19±0.04 vs. 1.10±0.04, t97H =-28.722, P<0.01; 0.20±0.01 vs. 0.93±0.09, tHCCLM3=-13.887, P<0.01). The CCK-8 assay showed that proliferation rate of 97H and HCCLM3 cells decreased significantly after knocking down AGXT2L1 (0.71±0.01 vs. 0.95±0.12, t97H=-31.243, P<0.01; 0.67±0.13 vs. 0.87±0.17, tHCCLM3=-17.083, P<0.01). After transfection of AGXT2L1-siRAN into cells, the cell cycle assay showed that the ratio of S phase cells was lower than that in the control group [(13.45±1.20)% vs. (18.73±0.28)%, t97H=-7.451, P<0.01; (15.26±0.52)% vs. (20.45±1.08)%, tHCCLM3=-7.483, P<0.01], and the ratio of G2 phase cells was lower than that in the control group [(16.90±0.54)% vs. (19.99±0.54)%, t97H=-6.992, P<0.01; (16.23±0.28)% vs. (23.36±0.39)%, tHCCLM3=-25.720, P<0.01]. The knockdown of AGXT2L1 could inhibit the transformation of 97H and HCCLM3 cells from G1 to S phase and inhibit the proliferation of human hepatoma cells. Compared with the control group, after the knockdown of AGXT2L1, the apoptosis rate of 97H and HCCLM3 cells was increased [(13.06±1.86)% vs. (1.06±0.75)%, t97H=14.673, P<0.01; (14.41±2.21)% vs. (0.80±0.32)%, tHCCLM3=14.947, P<0.01]. Conclusion The knockdown of AGXT2L1 can inhibit the proliferation of 97H and HCCLM3 cells and promote apoptosis. Key words: Carcinoma, hepatocellular; Alanine-glyoxylate aminotransferase 2-like 1; Proliferation; Apoptosis

MiR-424-5p Regulates Hepatoma Cell Proliferation and Apoptosis.

Yes-associated protein (Yes-associated protein 1 [YAP1]) is an important oncogene that is related to the pathogenesis and progression of liver cancer. It was found that miR-424-5p expression was significantly decreased in liver cancer tissues, revealing its anticancer effect. Bioinformatic analysis demonstrated the targeted relationship between miR-424-5p and the 3' untranslated region of YAP1. This study investigated the role of miR-424-5p in regulating YAP1 expression and affecting hepatoma cell proliferation and apoptosis. Tumors and normal liver tissues adjacent to tumors were collected from patients to detect the expression of miR-424-5p and YAP1. A dual-luciferase reporter gene assay was adopted to explore the targeted regulation between miR-424-5p and YAP1. Liver cancer HCCLM3 and MHCC97-L cells and normal liver HL-7702 cells were cultured in vitro to compare expression levels of miR-424-5p and YAP1. HCCLM3 and MHCC97-L cells were divided into the miR-NC group and miR-424-5p mimic group. Cell apoptosis was detected by flow cytometry. Cell proliferation was determined by EdU staining. Compared with normal liver tissue, miR-424-5p expression was significantly decreased, while YAP1 mRNA and protein levels were obviously upregulated in liver cancer tissues, which were related to the clinical stage. A negative correlation was found between miR-424-5p and YAP1 mRNA levels in liver cancer tissues. Dual-luciferase reporter gene assay confirmed the targeted relationship between miR-424-5p and YAP1. miR-424-5p expression in HCCLM3 and MHCC97-L cells decreased compared with L20 cells, which correlated with malignancy. YAP1 level in HCCLM3 and MHCC97-L cells was significantly enhanced, which correlated with malignancy. miR-424-5p mimic transfection significantly downregulated YAP1 expression in HCCLM3 and MHCC97-L cells, resulting in enhanced apoptosis and attenuated cell proliferation. Decreased miR-424-5p expression and increased YAP1 expression are found in patients with liver cancer. Increased miR-424-5p can inhibit YAP1 expression, attenuate hepatoma cell proliferation, and induce cell apoptosis.

Silencing of the TROP2 gene suppresses proliferation and invasion of hepatocellular carcinoma HepG2 cells.

ObjectivesOverexpression of human trophoblast cell surface antigen 2 (Trop2) has been observed in many cancers; however, its roles in proliferation, apoptosis, migration, and invasion of hepatocellular carcinoma (HCC) remain unclear. Thus, this study aimed to characterize the function of Trop2 in HCC.MethodsTrop2 protein expression was detected by immunohistochemistry in HCC tissues. Cell proliferation, apoptosis, and invasion were respectively measured by CCK-8, flow cytometry, Transwell, and wound healing assays. Expression levels of epithelial–mesenchymal transition-related proteins and Trop2 protein in HCC cell lines were detected by western blotting after silencing of the TROP2 gene.ResultsTrop2 protein was highly expressed in HCC tissues and HCC cell lines. Trop2 mRNA and protein expression levels decreased in HepG2 and HCCLM3 cells after transfection with Trop2 siRNA. Silencing of the TROP2 gene in HepG2 and HCCLM3 cells strongly inhibited cell proliferation and migration, while enhancing cell apoptosis. Investigation of the molecular mechanism revealed that silencing of the TROP2 gene suppressed epithelial–mesenchymal transition of HepG2 and HCCLM3 cells.ConclusionsThe results of the present study may improve understanding of the role of Trop2 in regulation of cell proliferation and invasion, and may aid in development of novel therapy for HCC.

MicroRNA‑3653 inhibits the growth and metastasis of hepatocellular carcinoma by inhibiting ITGB1.

microRNAs (miRNAs) play critical roles in hepatocellular carcinoma (HCC). However, the expression and biological function of miR-3653 in HCC remain unknown. The present study demonstrated that miR-3653 expression was significantly decreased in HCC tissues and cells using qRT-PCR. A decreased miR-3653 level was associated with unfavorable clinical features and poor prognosis of HCC patients. MTT, BrdU, Transwell and western blot assays showed that miR-3653 overexpression inhibited the growth, migration, invasion and epithelial-mesenchymal transition (EMT) of HCCLM3 cells while its knockdown promoted the growth and metastatic ability of Hep3B cells. In vivo experiments showed that miR-3653 overexpression inhibited the subcutaneous and the lung metastasis of HCCLM3 cells in nude mice. Mechanistically, integrin-β1 (ITGB1) was identified to be the downstream target of miR-3653 in HCC. ITGB1 overexpression reversed the inhibitory effects of miR-3653 on the growth, metastasis and EMT of HCCLM3 cells.

Chemoenzymatically synthesized ganglioside GM3 analogues with inhibitory effects on tumor cell growth and migration

Ganglioside GM3, belonging to glycosphingolipid family, has been known as tumor-associated carbohydrate antigen on several types of tumor. Many studies have revealed that GM3 plays a role in cell proliferation, adhesion and differentiation, which is crucial in the process of cancer development. In the present study, we firstly synthesized novel mannose-containing GM3 analogues by enzymatic hydrolysis and chemical procedures. Then the antiproliferative activity of the novel analogues along with galactose-containing analogues we prepared previously was investigated and the data demonstrated that these analogues exhibited antiproliferative effect on K562 and HCT116 cells. Finally, the influence of these analogues on tumor cell migration was studied on B16, B16-F10 and HCCLM3 cells by wound healing test, because the migration of tumor cells represents one of the relevant factors in assessing the malignancy of cancer. This study could lay the foundation for optimizing leading compounds and provide valuable information for finding new antitumor drugs for cancer therapy.

Antimetastatic Effect of Fucoidan-Sargassum against Liver Cancer Cell Invadopodia Formation via Targeting Integrin αVβ3 and Mediating αVβ3/Src/E2F1 Signaling.

Background: Fucoidan is a fucose-enriched, sulfated polysaccharide found in brown algae; in recent years, this polysaccharide has been found to exert several biological effects, including antitumor effects, such as antiproliferation, activating apoptosis, and anti-angiogenesis of cancer cells. However, the antimetastatic effect of fucoidan and the related targeting receptors remain unknown. In the present study, we examined the inhibition of invadopodia formation and underlying mechanism of fucoidan on human liver cancer cells.Methods: We used 98% purified fucoidan from Sargassum species to treat the hepatocellular carcinoma (HCC) cells SMMC-7721, Huh7 and HCCLM3 in vitro and the HCCLM3 cell line in vivo. The HCC cells were cultured with various concentrations of Fucoidan-Sargassum (0-30 mg/mL). Migration, invasion and wound healing assays were performed to determine the antimetastatic effect of fucoidan on the HCC cells. Western blot analysis and immunofluorescence staining were conducted to determine the expression levels of invadopodia formation-regulating proteins and the targeting membrane receptor proteins.Results: Fucoidan-Sargassum inhibited the migration and invasion of HCC SMMC-7721, Huh7 and HCCLM3 cells in a dose-dependent manner. In the HCCLM3 cells, Fucoidan-Sargassum also decreased the expression levels of invadopodia-related proteins including Src, Cortactin, N-WASP, ARP3, CDC42, MMP2, MT1-MMP, and the targeting receptors integrin αV and β3 in a dose-dependent manner. Fucoidan-Sargassum also increased the levels of endoplasmic reticulum-related proteins, including GRP78, IRE1, SPARC, and the type IV collagen receptor proteins integrin α1 and β1. In vivo, Fucoidan-Sargassum reduced the size of liver tumors and decreased the number of lung metastatic foci in nude mice with hepatocellular carcinoma xenografts.Conclusion: These findings indicate that Fucoidan-Sargassum has an antimetastatic effect on SMMC-7721, Huh7 and HCCLM3 liver cancer cells, and the underlying mechanism involves targeting ITGαVβ3 and mediating the ITGαVβ3/SRC/E2F1 signaling pathway. These results suggest that Fucoidan-Sargassum may be a promising therapeutic antimetastatic compound in the development of a metastasis-preventive drug for treating liver cancer.

Mortalin stabilizes CD151-depedent tetraspanin-enriched microdomains and implicates in the progression of hepatocellular carcinoma.

Background: Our previous studies showed that tetraspanin CD151 was implicated in the progression of hepatocellular carcinoma (HCC), mainly depending on the formation of functional complexes with molecular partners, including Mortalin. In this study, we investigate the role of mortalin in CD151-depedent progression of HCCs.Methods: Immunofluorescent staining, western blot and quantitative real-time polymerase chain reaction (qRT-PCR) were used to investigate the expression and location of CD151 and Mortalin in four HCC cell lines with different metastatic ability. The relationship between Mortalin and CD151 was investigated in HCCLM3 cells using co-immunoprecipitation. CD151 or Mortalin expression in HCC cells were modified by transfection technology. Wound-healing assay and Transwell assay were used to assay the role of CD151 and Mortalin in cell migration and invasion. The expression and prognostic implication of CD151 and Mortalin in 187 cases of HCCs were analyzed.Results: Expression of Mortalin in HCC cells was positive related to their metastatic ability and its tendency was in line with the expression of CD151. Immunofluorescent staining showed that Mortalin was located in cytoplasm, while positive staining for CD151 was observed in cytoplasm and membrane of HCC cells. co-IP revealed that Mortalin formed a complex with CD151. Down-regulation of Mortalin induced a moderate decreased CD151 protein, but not CD151 mRNA, while inhibition of CD151 did not influence the expression of Mortalin at the level of both protein and mRNA. Interference of Mortalin significantly inhibited the invasion and migration of HCC cells with high CD151 expression and partially restored the invasion and migration of HCC cells induced by CD151 over-expression. Clinically, high Mortalin expression correlated with malignant phenotype of HCC, such as microvascular invasion (p=0.017) and tumor diameter (p=0.001). HCC patients expressing high Mortalin were tend to have higher expression of CD151. HCC patients expressing high level of CD151 showed the poorer prognosis in a Mortalin-dependent manner.Conclusions: Mortalin maybe stabilize of the structure of CD151-dependent tetraspanin-enriched microdomains and implicate in the progression of HCC.

LncRNA A1BG-AS1 suppresses proliferation and invasion of hepatocellular carcinoma cells by targeting miR-216a-5p.

Extensive evidence indicate that long noncoding RNAs (lncRNAs) regulates the tumorigenesis and progression of hepatocellular carcinoma (HCC). However, the expression and biological function of lncRNA A1BG antisense RNA 1 (A1BG-AS1) were poorly known in HCC. Here, we found the underexpression of A1BG-AS1 in HCC via analysis of The Cancer Genome Atlas database. Further analyses confirmed that A1BG-AS1 expression in HCC was markedly lower than that in noncancerous tissues based on our HCC cohort. Clinical association analysis revealed that low A1BG-AS1 expression correlated with poor prognostic features, such as microvascular invasion, high tumor grade, and advanced tumor stage. Follow-up data indicated that low A1BG-AS1 level evidently correlated with poor clinical outcomes of HCC patients. Moreover, forced expression of A1BG-AS1 repressed proliferation, migration, and invasion of HCC cells in vitro. Conversely, A1BG-AS1 knockdown promoted these malignant behaviors in HepG2 cells. Mechanistically, A1BG-AS1 functioned as a competing endogenous RNA by directly sponging miR-216a-5p in HCC cells. Notably, miR-216a-5p restoration rescued A1BG-AS1 attenuated proliferation, migration and invasion of HCCLM3 cells. A1BG-AS1 positively regulated the levels of phosphatase and tensin homolog and SMAD family member 7, which were reduced by miR-216a-5p in HCC cells. Altogether, we conclude that A1BG-AS1 exerts a tumor suppressive role in HCC progression.

Overexpression of PIK3R1 promotes hepatocellular carcinoma progression

BackgroundPhosphoinositide-3-kinase, regulatory subunit 1 (PIK3R1) could regulate cancer cell proliferation important for cancer cell proliferation; however, its role in Hepatocellular carcinoma (HCC) remains largely unknown. Here, we investigated the role of PIK3R1 in HCC and examined the underlying molecular mechanisms.MethodsThe expression of PIK3R1 was evaluated by immunohistochemistry and qRT-PCR in a series of HCC tissues. The mRNA and protein expression of PIK3R1 was used by qRT-PCR and western blot assays in a series of human HCC cell lines, and then we choose MHCC97H and HCCLM3 cells as a model to investigate the effect of PIK3R1 on HCC progression. The effects of PIK3R1 knowdown on cell proliferation, migration, apoptosis of HCC were assessed by the MTT assay, clonogenic assays, wound healing assay and flow cytometry in vitro. Western blot assay was performed to assess the expression changes of PI3K/AKT/mTOR signaling pathway.ResultsOur results found that PIK3R1 was highly expressed in HCC tissues compared with adjacent normal tissues. Knockdown of PIK3R1 inhibited the proliferation, migration and promoted apoptosis of HCC cell lines. In addition, we proved that knockdown of PIK3R1 downregulated p-PI3K, p-AKT, and p-mTOR expressions in MHCC97H and HCCLM3 cells.ConclusionsIn conclusion, PIK3R1 providing potential novel targets for the treatment of HCC.

Shikonin exerts antitumor activity by causing mitochondrial dysfunction in hepatocellular carcinoma through PKM2-AMPK-PGC1α signaling pathway.

Shikonin, a naphthoquinone derivative isolated from the root of Lithospermum erythrorhizon, exhibits broad-spectrum antitumor activity via different molecular mechanisms. In this study, we investigated the effect of shikonin on mitochondrial dysfunction in hepatocellular carcinoma (HCC). Our results showed that shikonin inhibited the proliferation, migration, and invasiveness of HCCLM3 cells, and promoted cell apoptosis in a dose-dependent manner. More importantly, shikonin affected mitochondrial function by disrupting mitochondrial membrane potential and oxidative stress (OS) status. Furthermore, shikonin decreased the oxygen consumption rate of HCCLM3 cells, as well as the levels of ATP and metabolites involved in the tricarboxylic acid cycle (TCA cycle). We also investigated the molecular mechanisms underlying the regulation of mitochondrial function by shikonin as an inhibitor of PKM2. Shikonin decreased the expression of PKM2 in the mitochondria and affected other metabolic pathways (AMPK and PGC1α pathways), which aggravated the oxidative stress and nutrient deficiency. Our results indicate a novel role of shikonin in triggering mitochondria dysfunction via the PKM2-AMPK-PGC1α signaling pathway and provide a promising therapeutic approach for the treatment of HCC.

The SMAC Mimetic APG-1387 Sensitizes Immune-Mediated Cell Apoptosis in Hepatocellular Carcinoma.

The inhibitor of apoptosis protein (IAP) genes are frequently overexpressed in malignancies. Second mitochondria-derived activator of caspase (SMAC) mimetics, which target IAPs, have potential to trigger cancer cell death and sensitize tumor cells to cytotoxic therapy. The aim of this study was to investigate the anti-tumor potential of a novel bivalent SMAC mimetic, APG-1387, in hepatocellular carcinoma (HCC). The mRNA and protein expressions of IAPs, including cellular IAPs (cIAP1 and cIAP2) and X chromosome-linked IAP (XIAP), were increased in HCC tumors compared with normal liver tissue. APG-1387 treatment alone significantly reduced the protein levels of IAPs, but had only a modest effect on the viability and apoptosis of HCC cells in vitro. However, APG-1387 in combination with tumor necrosis factor-alpha (TNF-α) or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) significantly reduced cell viability and proliferation, and induced apoptosis in HepG2 cells, as well as in HCCLM3 cells that harbors cancer stem cell-like properties. These synergistic killing effects were caspase-dependent and partially dependent on RIPK1 kinase activity. Furthermore, APG-1387 also promoted the killing effect of Natural Killer cells on HCC cells in vitro and the combination therapy significantly inhibited tumor growth by inducing cell apoptosis in xenograft mice model. In conclusion, our study clarified that APG-1387 could sensitize HCC cells to cytokines or immune cells mediated cell killing and implied that potential of SMAC mimetic based combination immunotherapy for HCC treatment.