HCCLM3 Cells Research Articles

LncRNA ALKBH3-AS1 enhances ALKBH3 mRNA stability to promote hepatocellular carcinoma cell proliferation and invasion.

Long noncoding RNAs (lncRNAs) are confirmed as the key regulators of hepatocellular carcinoma (HCC) occurrence and progression, but the role of AlkB homologue 3 antisense RNA 1 (ALKBH3‐AS1) in HCC is unclear. We revealed the overexpression of ALKBH3‐AS1 in HCC tissues. The upregulated levels of ALKBH3‐AS1 were observed in HCC cells. ALKBH3‐AS1 was expressed in the nucleus and cytoplasm of HCC cells. The high ALKBH3‐AS1 expression was markedly associated with a decreased survival rate of HCC patients. ALKBH3‐AS1 knockdown repressed and ALKBH3‐AS1 overexpression enhanced HCC cell invasion and proliferation. ALKBH3‐AS1 silencing restricted HCC growth in vivo. A significant positive correlation between ALKBH3‐AS1 and ALKBH3 mRNA levels was confirmed in HCC specimens. ALKBH3‐AS1 silencing reduced ALKBH3 expression by stabilizing its mRNA stability in HCC cells. Notably, the impact of ALKBH3 silencing on HCC cells was similar to that of ALKBH3‐AS1 knockdown. ALKBH3 restoration prominently attenuated the suppressive effects resulting from ALKBH3‐AS1 silencing in HCCLM3 cells. Hypoxia‐inducible factor‐1α (HIF‐1α) transcriptionally activated ALKBH3‐AS1 expression in hypoxic HCC cells. ALKBH3‐AS1 knockdown markedly attenuated cell proliferation and invasion in hypoxic Huh7 cells. Collectively, HIF‐1α‐activated ALKBH3‐AS1 exerted an oncogenic role by enhancing ALKBH3 mRNA stability in HCC cells.

TRIM36 inhibits tumorigenesis through the Wnt/β-catenin pathway and promotes caspase-dependent apoptosis in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is the most common type of primary liver cancer and has an extremely poor prognosis. We aimed to determine the latent relationships between TRIM36 regulation of apoptosis and the Wnt/β-catenin pathway in HCC.MethodsImmunohistochemistry and western blotting were used to characterize the aberrant expression of TRIM36 in HCC and adjacent tissues. Clinical information was analyzed using Kaplan–Meier and Cox methods. RNA-seq of potential targets was conducted to detect the regulation of TRIM36. Apoptosis assays and cellular proliferation, invasion and migration were conducted in a loss- and gain-of-function manner in cultured cells to determine the biological functions of TRIM36. A rescue experiment was conducted to confirm the role of Wnt/β-catenin signaling in TRIM36 regulation. Finally, in vivo experiments were conducted using cell line-derived xenografts in nude mice to validate the central role of TRIM36 in HCC.ResultsTRIM36 expression was significantly downregulated in HCC tissues compared to adjacent non-tumor tissues. TRIM36 repressed the proliferation, migration, and invasion of Huh7 and HCCLM3 cells, whereas it stimulated apoptosis. Wnt/β-catenin signaling was inhibited by TRIM36, and rescue experiments highlighted its importance in HCC proliferation, migration, and invasion. In vivo experiments further confirmed the effects of sh-TRIM36 on HCC tumorigenesis, inhibition of apoptosis, and promotion of Wnt/β-catenin signaling.ConclusionOur study is the first to indicate that TRIM36 acts as a tumor suppressor in HCC. TRIM36 activates apoptosis and inhibits cellular proliferation, invasion, and migration via the Wnt/β-catenin pathway, which may serve as an important biomarker and promising therapeutic target for HCC.

Pan-cancer analysis suggests histocompatibility minor 13 is an unfavorable prognostic biomarker promoting cell proliferation, migration, and invasion in hepatocellular carcinoma.

Histocompatibility Minor 13 (HM13) encoding the signal peptide peptidase plays an important role in maintaining protein homeostasis but its role in tumors remains unclear. In this study, 33 tumor RNA-seq datasets were extracted from The Cancer Genome Atlas (TCGA) database, and the pan-cancer expression profile of HM13 was evaluated in combination with The Genotype-Tissue Expression (GTEx) datasets. The prognostic significance of abnormal HM13 pan-cancer expression was evaluated by univariate Cox regression and Kaplan-Meier analyses. Co-expression analysis was performed to examine the correlation between abnormal pan-cancer expression of HM13 and immune cell infiltration, immune checkpoint, molecules related to RNA modification, tumor mutational burden (TMB), microsatellite instability (MSI), and other related molecules. CellMiner database was used to evaluate the relationship between the expression of HM13 and drug sensitivity. The results showed overexpression of HM13 in almost all tumors except kidney chromophobe (KICH). Abnormally high expression of HM13 in adrenocortical carcinoma (ACC), kidney renal papillary cell carcinoma (KIRP), uveal melanoma (UVM), liver hepatocellular carcinoma (LIHC), brain lower grade glioma (LGG), head and neck squamous cell carcinoma (HNSC), and kidney renal clear cell carcinoma (KIRC) was associated with poor prognosis. Expression of HM13 correlated strongly with pan-cancer immune checkpoint gene expression and immune cell infiltration. Drug sensitivity analysis indicated that the expression of HM13 was an excellent predictor of drug sensitivity. We verified that both mRNA and protein levels of HM13 were abnormally upregulated in HCC tissues, and were independent risk factors for poor prognosis. Furthermore, interference with HM13 expression in Huh-7 and HCCLM3 cells significantly inhibited proliferation, migration, and invasion. Therefore, our findings demonstrate that HM13 is a potential pan-cancer prognostic marker, thus providing a new dimension for understanding tumor development.

Connexin32 regulates expansion of liver cancer stem cells via the PI3K/Akt signaling pathway.

Liver cancer stem cells (LCSCs) are responsible for liver cancer recurrence, metastasis, and drug resistance. Previous studies by the authors demonstrated that upregulated expression of connexin 32 (Cx32) reversed doxorubicin resistance and reduced invasion and metastasis of liver cancer cells. However, the role of Cx32 in expansion of LCSCs remains unclear. A total of 85 patients were enrolled in the present study and followed-up for 5 years. The expression of Cx32 in hepatocellular carcinoma (HCC) tissues and corresponding paracancerous tissues were detected by immunohistochemistry (IHC). Cx32 was silenced in HepG2 cells and overexpressed in HCCLM3 cells and the stemness of liver cells was examined by detecting the expression of LCSC markers (EpCAM, CD133, Nanog, Oct4, Sox9, c-Myc), sphere formation, and xenograft tumorigenesis. Finally, the effect of the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway on Cx32-regulated LCSC expansion was investigated. Cx32 was downregulated in LCSCs and HCC tissues, and predicted poor prognosis in patients with HCC. Overexpression of Cx32 in HCCLM3 cells significantly inhibited LCSC expansion, tumorigenesis, and phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathway activity. By contrast, silencing of Cx32 in HepG2 cells upregulated expansion of LCSCs and PI3K/Akt pathway activity. Modulating the activity of the PI3K/Akt pathway by SC-79 and LY294002 in HepG2 and HCCLM3 cells, respectively, confirmed that Cx32 could affect the expansion of LCSCs through PI3K/Akt signaling. In conclusion, the present study demonstrated that Cx32 regulated the expansion of LCSCs, and increased expression of Cx32 significantly inhibited the expansion of LCSCs, suggesting that Cx32 may be an optimal target for intervention of HCC.

Sema3d Restrained Hepatocellular Carcinoma Progression Through Inactivating Pi3k/Akt Signaling via Interaction With FLNA.

Hepatocellular carcinoma (HCC) is one of the most lethal malignant tumors worldwide due to the high incidence rate of metastasis and recurrence. Semaphorin 3d (Sema3d) has been shown to play a critical role in vascular development during early embryogenesis and several forms of cancer progression via regulating cell migration. However, the function of Sema3d in hepatocellular carcinoma (HCC) remains elusive. This study aimed to explore the function and mechanisms of Sema3d in HCC. In our study, Sema3d expression was significantly downregulated in HCC tissues and cell lines. Downregulated Sema3d was closely correlated with aggressive clinicopathological features and poor clinical outcomes in HCC patients. Moreover, overexpression of Sema3d in HCCLM3 cells was significantly inhibited and knockdown of Sema3d in PLC/PRF/5 cells promoted proliferation, migration, invasion, and epithelial–mesenchymal transition (EMT) of HCC cells in vitro and tumor growth, EMT, and metastasis in vivo. Furthermore, the RNA sequencing and gene set enrichment analysis (GSEA) indicated that these phenotypic and functional changes in Sema3d-interfered HCC cells were mediated by the Pi3k/Akt signaling pathway, and co-IP–combined mass spectrometry indicated Sema3d might interact with FLNA. Finally, we proved that Sema3d exerted its tumor-restraining effect by interacting with FLNA to inactivate the Pi3k/Akt signaling pathway and remodel the cytoskeleton. Our data showed that Sema3d restrained hepatocellular carcinoma proliferation, invasion, and metastasis through inactivating Pi3k/Akt via interaction with FLNA, which may serve as a novel prognostic predictor and a potential therapeutic target for HCC patients.

RING finger and WD repeat domain 3 regulates proliferation and metastasis through the Wnt/β-catenin signalling pathways in hepatocellular carcinoma

BACKGROUNDHepatocellular carcinoma (HCC) exhibits high invasiveness and mortality rates, and the molecular mechanisms of HCC have gained increasing research interest. The abnormal DNA damage response has long been recognized as one of the important factors for tumor occurrence and development. Recent studies have shown the potential of the protein RING finger and WD repeat domain 3 (RFWD3) that positively regulates p53 stability in response to DNA damage as a therapeutic target in cancers. AIMTo investigate the relationship between HCC and RFWD3 in vitro and in vivo and explored the underlying molecular signalling transduction pathways. METHODS RFWD3 gene expression was analyzed in HCC tissues and adjacent normal tissues. Lentivirus was used to stably knockdown RFWD3 expression in HCC cell lines. After verifying the silencing efficiency, Celigo/cell cycle/apoptosis and MTT assays were used to evaluate cell proliferation and apoptosis. Subsequently, cell migration and invasion were assessed by wound healing and transwell assays. In addition, transduced cells were implanted subcutaneously and injected into the tail vein of nude mice to observe tumor growth and metastasis. Next, we used lentiviral-mediated rescue of RFWD3 shRNA to verify the phenotype. Finally, the microarray, ingenuity pathway analysis, and western blot analysis were used to analyze the regulatory network underlying HCC. RESULTSCompared with adjacent tissues, RFWD3 expression levels were significantly higher in clinical HCC tissues and correlated with tumor size and TNM stage (P < 0.05), which indicated a poor prognosis state. RFWD3 silencing in BEL-7404 and HCC-LM3 cells increased apoptosis, decreased growth, and inhibited the migration in shRNAi cells compared with those in shCtrl cells (P < 0.05). Furthermore, the in vitro results were supported by the findings of the in vivo experiments with the reduction of tumor cell invasion and migration. Moreover, the rescue of RFWD3 shRNAi resulted in the resumption of invasion and metastasis in HCC cell lines. Finally, gene expression profiling and subsequent experimental verification revealed that RFWD3 might influence the proliferation and metastasis of HCC via the Wnt/β-catenin signalling pathway.CONCLUSIONWe provide evidence for the expression and function of RFWD3 in HCC. RFWD3 affects the prognosis, proliferation, invasion, and metastasis of HCC by regulating the Wnt/β-catenin signalling pathway.

Caryophyllene Oxide Induces Ferritinophagy by Regulating the NCOA4/FTH1/LC3 Pathway in Hepatocellular Carcinoma.

Ferritinophagy is associated with tumor occurrence, development, and therapy effects. Ferritinophagy and ferroptosis are regulated by iron metabolism and are closely connected. LC3 protein is a key protein in autophagy. Following the binding of NCOA4 to FTH1, it links to LC3Ⅱ in lysosomes, a symbol of ferritinophagy. A ferritinophagy’s inducer is likely to open new avenues for anticancer medication research and development. In this study, we discovered that caryophyllene oxide has a substantial inhibitory effect on HCCLM3 and HUH7 cells, by regulating the level of cellular oxidative stress, and the levels of autophagy and iron metabolism in HCCLM3 and HUH7 cells, leading to a ferritinophagy-related phenomenon. Furthermore, the results of T-AOC, DPPH free radical scavenging rate, and hydroxyl radical inhibition indicated that caryophyllene oxide can inhibit cell anti-oxidation. The examination of the ferritinophagy-related process revealed that caryophyllene oxide promotes the production and accumulation of intracellular reactive oxygen species and lipid peroxidation. NCOA4, FTH1, and LC3Ⅱ were found to be targeted regulators of caryophyllene oxide. Caryophyllene oxide regulated NCOA4, LC3 Ⅱ, and FTH1 to promote ferritinophagy. In vivo, we discovered that caryophyllene oxide can lower tumor volume, significantly improve NCOA4 and LC3 protein levels in tumor tissue, and raise Fe2+ and malondialdehyde levels in serum. The compound can also reduce NRF2, GPX4, HO-1, and FTH1 expression levels. The reduction in the expression levels of NRF2, GPX4, HO-1, and FTH1 by caryophyllene oxide also inhibited GSH and hydroxyl radical’s inhibitory capacities in serum, and promoted iron deposition in tumor tissue resulting in the inhibition of tumor growth. In summary, our study revealed that caryophyllene oxide mostly kills liver cancer cells through ferritinophagy-mediated ferroptosis mechanisms. In conclusion, caryophyllene oxide may be used as a ferritinophagy activator in the field of antitumor drug research and development.

LncRNA n339260 functions in hepatocellular carcinoma progression via regulation of miRNA30e-5p/TP53INP1 expression.

Currently, the molecular mechanism of the interaction between lncRNAs and microRNAs (miRNAs) and the target of miRNAs in tumor vasculogenic mimicry (VM) formation have not been clarified. Our aim is to study the interaction between lncRNA n339260 and miRNA30e-5p in the formation of VM. Animal xenografts were established, 104 hepatocellular carcinoma (HCC) patients' frozen tissues were obtained and HCC cells in vitro were used to observe the role of n339260 in HCC progression. In vivo experiment showed lncRNA n339260 promoted tumor growth and VM formation. LncRNA n339260 and miRNA30e-5p were found to be associated with VM formation, metastasis and survival time in HCC patients. In vitro experiment showed that LncRNA n339260 could inhibit miRNA30e-5p expression and TP53INP1 was found to be the downstream targets of miRNA30e-5p. Snail, MMP2, MMP9, VE-cadherin, vimentin and N-cadherin overexpression and the downregulation of TP53INP1 and E-cadherin were observed in HCCLM3 and HepG2 cells overexpressing lncRNA n339260 or in cells with decreased expression of miRNA30e-5p. LncRNA n339260 promotes the development of VM, and lncRNA n339260 may enhance Snail expression by decreasing the expression of miRNA30e-5p, thereby reducing TP53INP1 expression. Therefore, a potential lncRNA n339260- miRNA30e-5p- TP53INP1 regulatory axis was associated with HCC progression.

Metabolomics and integrated network pharmacology analysis reveal SNKAF decoction suppresses cell proliferation and induced cell apoptisis in hepatocellular carcinoma via PI3K/Akt/P53/FoxO signaling axis

BackgroundThere is no comprehensive treatment method for hepatocellular carcinoma (HCC); hence, research and development are still focused on systemic therapies, including drugs. Sinikangai fang (SNKAF) decoction, a classic Chinese herbal prescription, has been widely used to treat liver cancer. However, there is no research on its core active component and target.MethodsMouse models were established to measure the anticancer effect of SNKAF decoction on HCC. Further, we investigated the effect of SNKAF decoction on inhibition of hepatoma cells proliferation using cell viability, cloning and invasion assays in vitro. The components of SNKAF were collected from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and TCM@Taiwan database. Metabolomic analysis was used to identify the potential genes and pathways in HCC treated with SNKAF decoction. Then, the expression of phosphoinositide 3-kinase (PI3K), Akt, P53, FoxO proteins of the potential signal pathways were detected using Western blot.ResultsThe animal experiments showed that SNKAF decoction inhibited tumor growth (P < 0.05) and induced no weight loss in the mice. In vitro data showed that HCCLM3 and MHCC97H cell proliferation was inhibited by SNKAF serum in a time- and concentration dependent manner. Further combined analysis network pharmacology with metabonomics showed that 217 target genes overlapped. The core target genes included BCL2, MCL1, Myc, PTEN, gsk3b, CASP9, CREB1, MDM2, pt53 and CCND1. Cancer-associated pathways were largely involved in SNKAF mechanisms, including P53, FoxO, and PI3K/Akt signaling pathways, which are closely related to induced-tumor cell apoptosis. In addition, Western bolt verified that 10% SNKAF serum significantly affected the main proteins of PI3K/Akt/P53/FoxO signaling pathway in both cell lines.ConclusionSNKAF decoction-containing serum inhibited HCCLM3 and MHCC97H cell proliferation, migration, invasion, and induced-tumor cell apoptosis in-vivo. We confirmed that SNKAF decoction is a promising alternative treatments for HCC patients.

The Prognostic and Immunotherapeutic Significance of AHSA1 in Pan-Cancer, and Its Relationship With the Proliferation and Metastasis of Hepatocellular Carcinoma.

The AHSA1 is a main activator of ATPase of Hsp90. Hsp90 is involved in various metabolic and developmental processes of tumor cells. Although, the role of AHSA1 in tumor cells is still unrecognized. In the current research, the RNA-seq of 33 tumors were downloaded using The Cancer Genome Atlas (TCGA) database for the analysis of AHSA1 expression in tumors. The Kaplan-Meier method was used for the evaluation of the prognostic significance of AHSA1 in patients with pan-cancer. Additionally, the correlation between AHSA1 and immune cell infiltration, immune checkpoint, pyroptosis-related molecules, epithelial cell transformation-related molecules, and autophagy-related molecules were analyzed by co-expression. Furthermore, we examined the effect of AHSA1 knockdown on cell function in Huh7 and HCCLM3 cells of hepatocellular carcinoma (HCC) cell lines.According to the finding of this study, up-regulation of AHSA1 expression was observed in numerous tumor tissues, and its over-expression in liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), and esophageal carcinoma (ESCA) could affect the overall survival and disease-specific survival of the patients. Meanwhile, as per the correlation analysis the expression of AHSA1 was greatly correlated with the expression of various immune cell infiltrates, immune checkpoint inhibitors, tumor mutation load, and microsatellite instability. Moreover, this study focused on analyzing the association of AHSA1 expression with multiple pathological stages in HCC, and confirmed that AHSA1 was an independent prognostic factor of HCC by univariate and multivariate COX regression in TCGA and The International Cancer Genome Consortium (ICGC) cohorts. At the same time, cellular experiments proved that the AHSA1 knockdown could decrease the proliferation activity, cell migration and invasion ability of HCC cells. Therefore, the results of this study indicated that AHSA1 can be used as a potential prognostic biomarker of tumors and it may have a significant role in the proliferation as well as migration of HCC cells.

Ceramide synthase 3 affects invasion and metastasis of hepatocellular carcinoma via the SMAD6 gene.

Patients with hepatocellular carcinoma (HCC) have poor prognosis due to lack of early diagnosis and effective treatment. Therefore, there is an urgent need to better understand the molecular mechanisms associated with HCC and to identify effective targets for early diagnosis and treatment. This study is to explore the expression and biological role of ceramide synthase 3 (CerS3) in HCC. A total of 159 pairs of HCC tissues and adjacent non-tumor tissues were obtained from the patients underwent radical resection in Shenzhen People's Hospital, and the total RNA and proteins from HCC tissues and adjacent non-tumor tissues were obtained. The expression of CerS3 protein and mRNA in HCC was detected by immunohistochemistry, Western blotting and real-time PCR. In vitro experiments, Hep3B cells were divided into a control vector group and a CerS3 vector group, and the cells were transfected with retroviral vector containing control cDNA or CerS3 cDNA, respectively. HCCLM3 cells were divided into a normal control shRNA group and a CerS3 shRNA group, and the cells were transfected with lentiviral vectors containing normal control shRNA or CerS3 shRNA, respectively. MTT, EdU, Transwell and scratch method were used to detect cell proliferation, migration and invasion. RNA sequencing was performed to determine the downstream signal of CerS3. Compared with the corresponding adjacent tissues,the mRNA and protein levels of CerS3 were elevated in the HCC tissues, with significant difference (both P<0.05). The Univariate and multivariate analysis showed that the overall survival rate was significantly correlated with the presence of venous invasion (95% CI 1.8-9.2, P<0.01), TNM stage (95% CI 2.3-5.2, P<0.05), poor histological grade (95% CI 1.4-6.8, P<0.05), and CerS3 (95% CI 1.5-3.9, P<0.05). Furthermore, the high CerS3 expression levels in tumor tissues were significantly associated with shorter overall survival rates compared with the low CerS3 expression (P<0.05). Compared with the vector control group, the Hep3B cell viability, EdU positive cells, and migration and invasion cell numbers in the CerS3 vector group were significantly increased (all P<0.05). Compared with the shRNA normal control group, the HCCLM3 cell viability, EdU positive cells, and numbers of migrating and invasive cells in the CerS3 shRNA group were significantly lower (all P<0.05). The RNA sequencing confirmed that the small mothers against decapentaplegic family member 6 (SMAD6) gene as an oncogenic gene could promote the HCC metastasis. Clinically, the overexpression of CerS3 is closely related to poor clinical features and poor prognosis. Functionally, CerS3 participates in the proliferation, invasion and metastasis of liver cancer cells via activating SMAD6 gene.

Ferroptosis-related long non-coding RNA signature predicts the prognosis of hepatocellular carcinoma.

Background: Hepatocellular Carcinoma (HCC) is a highly heterogeneous malignant tumor, and its prognostic prediction is extremely challenging. Ferroptosis is a cell mechanism dependent on iron, which is very significant for HCC development. Long non-coding RNA (lncRNA) is also linked to HCC progression. This work aimed to establish a prognosis risk model for HCC and to discover a possible biomarker and therapeutic target.Methods: The Cancer Genome Atlas (TCGA) database was used to obtain RNA-seq transcriptome data and clinic information of HCC patients. Firstly, univariate Cox was utilized to identify 66 prognostic ferroptosis-related lncRNAs. Then, the identified lncRNAs were further included in the multivariate Cox analysis to construct the prognostic model. Eventually, we performed quantitative polymerase chain reaction (q-PCR) to validate the risk model.Results: We established a prognostic seventeen-ferroptosis-related lncRNA signature model. The signature could categorize patients into two risk subgroups, with the low-risk subgroup associated with a better prognosis. Additionally, the area under the curve (AUC) of the lncRNAs signature was 0.801, indicating their reliability in forecasting HCC prognosis. Risk score was an independent prognostic factor by regression analyses. Gene set enrichment analysis (GSEA) analyses demonstrated a remarkable enrichment of cancer-related and immune-related pathways in the high-risk group. Besides, the immune status was decreased in the high-risk group. Eventually, three prognostic lncRNAs were validated in human HCCLM3 cell lines.Conclusions: The risk model based on seventeen-ferroptosis-related lncRNA has significant prognostic value for HCC and may be therapeutic targets associated with ferroptosis in clinical ways.

Piezo1 promoted hepatocellular carcinoma progression and EMT through activating TGF-\u03b2 signaling by recruiting Rab5c

BackgroundPiezo1 has been revealed to play a regulatory role in vascular development and progression of variety tumors. However, whether and how the progression of hepatocellular carcinoma (HCC) regulated by Piezo1 remains elusive. This study aimed to elucidate the effect and mechanisms of Piezo1 in HCC.MethodsThe mRNA and protein expression level of Piezo1 in HCC samples and cell lines was determined by qRT-PCR, western blot and immunohistochemistry analyses. Two independent study cohorts containing 280 patients were analyzed to reveal the association between Piezo1 expression and clinicopathological characteristics. Series of in vitro and in vivo experiments were used to validate the function of Piezo1 in HCC. Gene set enrichment analysis (GSEA) was performed to explore the signaling pathway of Piezo1. Immunoprecipitation, immunofluorescence and in vitro and in vivo experiments were used to explore the molecular mechanism of Piezo1 in HCC progression.ResultsOur results demonstrated the Piezo1 expression was significantly upregulated in HCC tissues and cell lines, and upregulation of Piezo1 closely correlated with aggressive clinicopathological features and poor prognosis. Knockdown of Piezo1 in HCCLM3 and Hep3B cells significantly restrained proliferation, migration, invasion and epithelial–mesenchymal transition (EMT) of HCC cells in vitro, and tumor growth, metastasis, EMT in vivo. TGF-β signaling pathway was most significant enriched pathway in GSEA. Finally, tumor promotion effect of Piezo1 was found to exerted through recruiting and combining Rab5c to activating TGF-β signaling pathway.ConclusionsPiezo1 significantly related to poor prognosis and promotes progression of hepatocellular carcinoma via activating TGF-β signaling, which suggesting that Piezo1 may serve as a novel prognostic predictor and the potential therapeutic target for HCC patients.

CircSEC24A (hsa_circ_0003528) interference suppresses epithelial-mesenchymal transition of hepatocellular carcinoma cells via miR-421/MMP3 axis

ABSTRACT Accumulating evidence indicates that circular RNAs (circRNAs) function as conclusive modulators in diverse tumors, including in hepatocellular carcinoma (HCC). Nonetheless, knowledge of the latent mechanisms involving circRNAs in HCC development is insufficient. circSEC24A (hsa_circ_0003528) was discovered by microarray analysis of patients with HCC. Binding sites between circSEC24A, miR-421, miR-421 and matrix metalloproteinase 3 (MMP3) were predicted using online bioinformatics tools. Interactions involving miRNA and target genes or circRNAs were verified by luciferase reporter-gene and RNA pull-down assays. Two HCC cell lines (HCCLM3 and Hep3B) and normal THLE-2 liver cells were used for in vitro experiments. miRNA and mRNA expression levels were detected by RT-qPCR, and protein expression was measured by western blotting. Cell proliferation was evaluated using Cell Counting Kit 8 (CCK-8) assays along with colony formation assays. Cell invasion and migration were determined using the Transwell and wound healing migration assays. A xenograft model was used to evaluate the role of circSEC24A in vivo. circSEC24A expression was significantly upregulated in HCCLM3 and Hep3B cells. Silencing circSEC24A mitigated the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of HCC cells, which was abrogated by downregulation of miR-421. Meanwhile, MMP3 could bind to miR-421 to decrease the functional effects of miR-421 and induce tumor metastasis. Knockdown of cicSEC24A suppressed tumor growth in vivo. circSEC24A interference suppressed HCC cell EMT by sponging miR-421, further regulating MMP3, and inhibiting tumor growth in vivo. Therefore, circSEC24A could represent a potential target for HCC patient treatment.

AZD5153, a Bivalent BRD4 Inhibitor, Suppresses Hepatocarcinogenesis by Altering BRD4 Chromosomal Landscape and Modulating the Transcriptome of HCC Cells.

BRD4, a chromatin modifier frequently upregulated in a variety of neoplasms including hepatocellular cancer (HCC), promotes cancer cell growth by activating oncogenes through its interaction with acetylated histone tails of nucleosomes. Here, we determined the anti-HCC efficacy of AZD5153, a potent bivalent BRD4 inhibitor, and elucidated its underlying molecular mechanism of action. AZD5153 treatment inhibited HCC cell proliferation, clonogenic survival and induced apoptosis in HCC cells. In vivo, AZD5153-formulated lipid nanoemulsions inhibited both orthotopic and subcutaneous HCCLM3 xenograft growth in NSG mice. Mapping of BRD4- chromosomal targets by ChIP-seq analysis identified the occupancy of BRD4 with the promoters, gene bodies, and super-enhancers of both mRNA and noncoding RNA genes, which were disrupted upon AZD5153 treatment. RNA-seq analysis of polyadenylated RNAs showed several BRD4 target genes involved in DNA replication, cell proliferation, and anti-apoptosis were repressed in AZD5153-treated HCC cells. In addition to known tumor-promoting genes, e.g., c-MYC, YAP1, RAD51B, TRIB3, SLC17A9, JADE1, we found that NAPRT, encoding a key enzyme for NAD+ biosynthesis from nicotinic acid, was also suppressed in HCC cells by the BRD4 inhibitor. Interestingly, AZD5153 treatment upregulated NAMPT, whose product is the rate-limiting enzyme for NAD+ synthesis from nicotinamide. This may explain why AZD5153 acted in concert with FK866, a potent NAMPT inhibitor, in reducing HCC cell proliferation and clonogenic survival. In conclusion, our results identified novel targets of BRD4 in the HCCLM3 cell genome and demonstrated anti-HCC efficacy of AZD5153, which was potentiated in combination with an NAMPT inhibitor.

SPC25 promotes hepatocellular carcinoma metastasis via activating the FAK/PI3K/AKT signaling pathway through ITGB4

Hepatocellular carcinoma (HCC) is a malignant tumor with a high metastatic rate. Recent studies have shown that the mitosis-associated spindle-assembly checkpoint regulatory protein spindle pole body component 25 homolog (SPC25) promotes HCC progression, although the underlying mechanism has yet to be fully elucidated. The aim of the present study was to investigate the mechanism through which SPC25 may promote HCC progression in greater detail. First, the expression of SPC25 was analyzed in publicly available databases to explore the association between SPC25 and HCC metastasis. Western blotting was subsequently performed to examine the level of SPC25 expression in different HCC cell lines. SPC25 was then silenced in HCCLM3 and Huh7 cells, and the effects of SPC25 silencing were investigated using cell proliferation, wound-healing, Transwell migration assays and an in vivo mouse model. Finally, the mechanism of SPC25 action with respect to the promotion of HCC metastasis was explored using microarray analysis and rescue experiments. The results obtained demonstrated that SPC25 is highly expressed in HCC, and this high level of expression is associated with poor prognosis and metastasis. Moreover, SPC25 silencing led to a marked inhibition of the invasion and migration of HCC cells both in vitro and in vivo. The gene-expression profiling and mechanistic experiments suggest that SPC25 preferentially influences the expression of genes associated with extracellular matrix (ECM)-integrin interactions, including integrin subunit β4 (ITGB4), an upstream element of the integrin pathway. ITGB4 upregulation partly reversed the decline in cell invasion and migration capacities that resulted from SPC25 silencing. Furthermore, deleting both SPC25 and ITGB4 caused a decrease in the phosphorylation of focal adhesion kinase (FAK), phosphoinositide 3-kinase (PI3K) and AKT, which are downstream elements of the integrin pathway. Taken together, the results of the present study demonstrated the important role of SPC25 as a prognostic indicator and as a promoter of metastasis in HCC, and the underlying mechanism of its action has been partially elucidated, suggesting that SPC25 could be used as a biomarker and as a target for therapeutic intervention in the treatment of HCC.

Piperlongumine synergistically enhances the antitumour activity of sorafenib by mediating ROS-AMPK activation and targeting CPSF7 in liver cancer

Sorafenib, a multikinase inhibitor, is the first-line agent for advanced liver cancer. Sorafenib strongly inhibits both cell proliferation and tumour angiogenesis. However, the development of drug resistance hampers its anticancer efficacy. To improve the antitumour activity of sorafenib, we demonstrate that piperlongumine (PL), an alkaloid isolated from the fruits and roots of Piper longum L., enhances the cytotoxicity of sorafenib in HCCLM3 and SMMC7721 cells using the cell counting kit-8 test. Flow cytometry analysis indicated that PL and sorafenib cotreatment induced robust reactive oxygen species (ROS) generation and mitochondrial dysfunction, thereby increasing the number of apoptotic cells and the ratio of G2/M phase cells in both HCCLM3 and SMMC7721 cells. Furthermore, AMP-protein kinase (AMPK) signalling was activated by excess ROS accumulation and mediated growth inhibition in response to PL and sorafenib cotreatment. RNA-sequencing analysis indicated that PL treatment disrupted RNA processing in HCCLM3 cells. In particular, PL treatment decreased the expression of cleavage and polyadenylation specificity factor 7 (CPSF7), a subunit of cleavage factor I, in a time- and concentration-dependent manner in HCCLM3 and SMMC7721 cells. CPSF7 knockdown using a gene interference strategy promoted growth inhibition of PL or sorafenib monotherapy, whereas CPSF7 overexpression alleviated the cytotoxicity of sorafenib in cultured liver cancer cells. Finally, PL and sorafenib coadministration significantly reduced the weight and volume of HCCLM3 cell xenografts in vivo. Taken together, our data indicate that PL displays potential synergistic antitumour activity in combination with sorafenib in liver cancer.

Jatrorrhizine inhibits liver cancer cell growth by targeting the expressions of miR-221-3p and miR-15b-5p

Purpose: To investigate the effect of jatrorrhizine on hepatic cancer cell proliferation and its mechanism of action.&#x0D; Methods: Jatrorrhizine-mediated changes in cell viability were measured using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, while apoptosis induction was evaluated by flow cytometry. Transwell assay was used for the measurement of cell invasion, whereas cell migration was assessed by wound healing assay. The protein expression of Axin2 was determined with western blotting assay.&#x0D; Results: Jatrorrhizine significantly (p &lt; 0.049) suppressed the viability of HepG2 and HCCLM3 cells in the concentration range of 0.5 to 16.0 μM. Treatment of HepG2 and HCCLM3 cells with 4.0 μM jatrorrhizine markedly suppressed cell invasion, when compared to untreated cells (p &lt; 0.0493). Jatrorrhizine significantly promoted the apoptosis of HepG2 and HCCLM3 cells at 48 h, relative to untreated cells, but 16.0 μM jatrorrhizine markedly suppressed the expressions of miR-221-3p and miR-15b-5p (p &lt; 0.0493). Moreover, jatrorrhizine significantly up-regulated the protein expressions of Axin2 in HepG2 and HCCLM3 cells at 48 h (p &lt; 0.0493).&#x0D; Conclusion: Jatrorrhizine inhibits the proliferation, and suppressed the invasiveness and migration of HepG2 and HCCLM3 liver cancer cells, but increases their apoptosis. Moreover, it down-regulates the expressions of miR-221-3p and miR15b-5p and promotes Axin2 protein expression in HepG2 and HCCLM3 cells. Therefore, jatrorrhizine is a potential drug candidate for the treatment of liver cancer.

Associations of DDX60L With the Clinical Features and Prognosis of Hepatocellular Carcinoma.

ObjectiveAlthough the pathogenesis of hepatocellular carcinoma (HCC) is still unclear, hepatitis C virus (HCV) infection is considered a common cause of HCC. It has been reported that DDX60L can inhibit HCV replication, but its role in HCC is still poorly understood.MethodsThe expression levels of DDX60L in HCC tissues and in tissues adjacent to the tumor and their correlation with the clinicopathological features of patients were analyzed. We also used Kaplan–Meier curves of overall survival (OS) with Cox regression analysis and log-rank test to investigate the prognostic value of DDX60L in HCC. We further performed cell proliferation, Transwell, and wound healing assays to elucidate the role of DDX60L in HCC using the siRNA-DDX60L Hep3B or HCCLM3 cell line.ResultsUnivariate analysis showed that sex, Edmondson grade, microvascular invasion, tumor stage (III–IV/I–II), AFP, and DDX60L expression were strongly associated with the prognosis of HCC patients. The results of multivariate analysis further suggested that DDX60L might be an independent prognostic factor for OS in patients with HCC (Pmoderate/low = 0.015, Phigh/low = 0.011). The low DDX60L expression in HCC patients with no-metastasis, age ≥55 years, tumor size <5 cm, Edmondson grade = I–II, microvascular invasion, no cirrhosis, HBV positivity, tumor stage = III–IV, AFP >20 μg/L, and multiple tumor was associated with poorer prognosis (P <0.05). Moreover, the expression of DDX60L was significantly lower in HCC samples (N = 285) than in the normal tissues adjacent to the tumor (N = 167, P <0.001). There were no HCV-related HCC patients in this study. Additionally, we found that DDX60L knockdown can promote the proliferation of Hep3B cells, migration and invasion ability of Hep3B and HCCLM3 cells.ConclusionWe found that the downregulation of DDX60L expression correlated with poor prognosis in patients with HCC, which may be independent of the HCV-related pathway. Furthermore, DDX60L significantly inhibited the proliferation of Hep3B cells, migration and invasion of Hep3B and HCCLM3 cells. Therefore, DDX60L can serve as a prognostic biomarker and therapeutic target for HCC.

Daidzin inhibits hepatocellular carcinoma survival by interfering with the glycolytic/gluconeogenic pathway through downregulation of TPI1.

Daidzin (DDZ) is a natural brassin-like compound extracted from the soybean, and has been found to have therapeutic potential against tumors in recent years. This study investigates the therapeutic effect of DDZ on hepatocellular carcinoma cells and elucidates the possible mechanisms of action. The viability of HCCLM3 and Hep3B cells was detected by MTT assay. Western blots and qPCR were used to detect the protein and mRNA levels of proliferation and apoptosis related genes. Gas chromatography-mass spectrometry (GC-MS) was used for metabolome analysis. In vivo antitumor effects were assessed in nude mice engrafted with HCC cell lines. Our results show that DDZ treatment dose-dependently inhibited cell viability, migration, and survival. The expressions of CDK1, BCL2, MYC, and survivin were reduced, while the expressions of BAX and PARP were increased in DDZ treated cells. The differentially expressed metabolites detected in DDZ treated cultures are associated with glycolysis/gluconeogenesis pathways. Bioinformatic analysis identified TPI1, a gene in the glycolysis pathway with prognostic value for hepatocellular carcinoma (HCC), and DDZ treatment downregulated this gene. In vivo experiments show that DDZ significantly reduced the tumor volume and weight, and inhibited Ki67 expression within tumors. This study shows that DDZ interfered with the survival and migration of hepatocellular carcinoma cells, likely via TPI1 and the gluconeogenesis pathway.