Suppressed Hepatocellular Carcinoma Cell Growth Research Articles

CircSNX6 promotes proliferation, metastasis, and angiogenesis in hepatocellular carcinoma via miR-383-5p/VEGFA signaling pathway

The role of circular RNA (circRNAs) in hepatocellular carcinoma (HCC) has been extensively studied. Previous research has highlighted the regulatory role of circSNX6 in HCC cells and tissues. However, the precise mechanism underlying HCC progression still requires comprehensive investigation. The study initially utilized quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to assess circSNX6 expression levels in HCC cell lines and tissues. Subsequently, the stability of circRNA was evaluated through Ribonuclease R and actinomycin D treatment assays. The impact of circSNX6 knockdown on proliferation, migration, invasion, and angiogenesis abilities was determined using various assays including colony formation, Transwell culture system, tube formation assay, and cell counting kit (CCK)-8 assays. Additionally, RNA immunoprecipitation chip and dual-luciferase reporter assays were employed to investigate the interactions between circSNX6 and miR-383-5p. Finally, an HCC xenograft tumor model in mice was established to assess the in vivo expression of circSNX6 and its functional role in HCC. Our findings revealed an elevated circSNX6 expression in HCC tissues, which was correlated with poor patient prognosis. Knockdown of circSNX6 suppressed HCC cell growth, invasion, metastasis, and angiogenesis. The downregulation of miR-383-5p, a target of circSNX6, significantly attenuated the tumor-suppressive effects induced by circSNX6 knockdown. Moreover, circSNX6 was found to modulate VEGFA expression by targeting miR-383-5p. The inhibition of HCC cell proliferation by miR-383-5p could be partially reversed by overexpressing VEGFA. Silencing circSNX6 also suppressed tumor formation and the metastasis of HCC cells in a mouse model. In summary, our findings suggest that circSNX6 promotes cell proliferation, metastasis, and angiogenesis in HCC by regulating the miR-383-5p/VEGFA pathway.

Solamargine Induces Hepatocellular Carcinoma Cell Apoptosis and Ferroptosis via Regulating STAT1/MTCH1 Axis.

Solamargine (SM) has been shown to play anti-tumor role in hepatocellular carcinoma (HCC). However, the underlying molecular mechanisms of SM in HCC progression deserve further exploration. HCC cell proliferation and apoptosis were assessed by cell counting kit 8 assay, colony formation assay and flow cytometry. Ferroptosis was evaluated by detecting the levels of Fe2+, iron, MDA, ROS and GSH in HCC cells. In addition, mitochondrial carrier 1 (MTCH1) mRNA level was detected by quantitative real-time PCR. Western blot was used to test MTCH1 and signal transduction and activation of transcription 1 (STAT1) protein levels. Dual-luciferase reporter assay was employed to analyze the interaction between STAT1 and MTCH1. A mouse xenograft model was also constructed to explore the role of SM in vivo. SM could potentially suppress HCC cell growth by inducing ferroptosis. MTCH1 was highly expressed in HCC tissues and cells, and its silencing inhibited HCC cell proliferation, promoted apoptosis and ferroptosis. MTCH1 expression was reduced by SM, and its overexpression reversed SM-induced HCC cell apoptosis and ferroptosis. Furthermore, STAT1 facilitated MTCH1 transcription and promoted its expression. Besides, STAT1 expression could be reduced by SM, and its overexpression abolished the decreasing effect of SM on MTCH1 expression. In vivo, SM suppressed HCC tumor growth by reducing MTCH1 expression. SM promoted HCC cell apoptosis and ferroptosis via the STAT1/MTCH1 axis.

Inhibition of hepatocellular carcinoma growth via modulation of the miR-221/SOX11 axis by curcumin and berberine

Hepatocellular carcinoma (HCC) is a fatal malignancy that has limited treatment options. This study focused on the potential therapeutic effects of curcumin (CUR) and berberine (BBR) on the miR-221/SRY-box transcription factor 11 (SOX11) axis in HCC. We investigated the combined effects of CUR and BBR on HEPG2 and Huh7 cell survival and miR-221 expression using Cell Counting Kit-8 assays and RT-qPCR, respectively. Western blotting was used to detect changes in the apoptosis-related caspase-3/9 protein levels. We performed bioinformatics analysis and dual-luciferase assays and measured apoptotic protein levels to assess the role of the miR-221/SOX11 axis in mediating the effects of CUR-BBR. Both CUR and BBR suppressed HCC cell growth in a dose-dependent manner, with the most potent combined effect observed at a 2:1 ratio. CUR-BBR treatment significantly downregulated miR-221 expression, and miR-221 overexpression partially reversed the CUR-BBR-mediated decrease in cell survival. In addition, SOX11 was found to be a direct target of miR-221. CUR-BBR treatment upregulated SOX11 expression, and overexpression of SOX11 restored the inhibitory effects of CUR-BBR on cell growth, migration, and invasion and promoted apoptosis in the presence of miR-221. Furthermore, CUR-BBR activated pro-apoptotic proteins caspase-3/9 through the miR-221/SOX11 axis. The combined effect of CUR-BBR played an important role in inhibiting the growth of HCC cells. This combined effect was achieved by regulating the miR-221/SOX11 axis and activating the synthesis of pro-apoptotic proteins. Our findings highlight a promising combined therapeutic approach for HCC and underscore the importance of targeting the miR-221/SOX11 axis.

Lipid droplet turnover at the lysosome inhibits growth of hepatocellular carcinoma in a BNIP3-dependent manner.

Hepatic steatosis is a major etiological factor in hepatocellular carcinoma (HCC), but factors causing lipid accumulation leading to HCC are not understood. We identify BNIP3 (a mitochondrial cargo receptor) as an HCC suppressor that mitigates against lipid accumulation to attenuate tumor cell growth. Targeted deletion of Bnip3 decreased tumor latency and increased tumor burden in a mouse model of HCC. This was associated with increased lipid in bnip3−/− HCC at early stages of disease, while lipid did not accumulate until later in tumorigenesis in wild-type mice, as Bnip3 expression was attenuated. Low BNIP3 expression in human HCC similarly correlated with increased lipid content and worse prognosis than HCC expressing high BNIP3. BNIP3 suppressed HCC cell growth by promoting lipid droplet turnover at the lysosome in a manner dependent on BNIP3 binding LC3. We have termed this process “mitolipophagy” because it involves the coordinated autophagic degradation of lipid droplets with mitochondria.

Scutellaria barbata Inhibits Hepatocellular Carcinoma Tumorigenicity by Inducing Ferroptosis of Hepatocellular Carcinoma Cells.

Ferroptosis is caused by accumulation of iron-dependent lipid peroxidation, which is characterized by reduction in cell volume and increase in mitochondrial membrane density. Studies have shown that ferroptosis contributes to the development and progression of numerous major diseases, including hepatocellular carcinoma (HCC). As a unique biomedical resource, Traditional Chinese Medicine (TCM) has been widely used in the treatment of HCC. In this present study, Scutellaria barbata was used to treat HCC cells in vitro, and the results revealed that S. barbata suppressed HCC cell growth through inducing ferroptosis. Next, the exploration of the molecular mechanism on how S. barbata induced ferroptosis in HCC cells suggested that S. barbata may induce ferroptosis by promoting iron perioxidation and lipid ROS metabolism. Finally, S. barbata also inhibited HCC tumorigenicity in vivo by inducing ferroptosis of HCC cells. These results provided theoretical basis for explaining the mechanism of TCM treatment for HCC and offered therapeutic opportunities for HCC patients.

Extracellular Vesicle-Encapsulated MicroRNA-375 from Bone Marrow-Derived Mesenchymal Stem Cells Inhibits Hepatocellular Carcinoma Progression through Regulating HOXB3-Mediated Wnt/β-Catenin Pathway.

Nowadays, microRNA-375 (miR-375) has been implicated in many types of cancers, including hepatocellular carcinoma (HCC), and the functions of miRNAs encapsulated by extracellular vesicles (EV) in HCC progression have also been extensively investigated. In this research, we aimed to probe into the mechanism of EV-encapsulated miR-375 from bone marrow-derived mesenchymal stem cells (BM-MSCs) in HCC progression. At first, miR-375 expression in HCC tissues and cells was detected using RT-qPCR, and miR-375 was overexpressed to specify the effects of miR-375 on the malignant phenotype of HCC cells. miR-375 was downregulated in HCC, and overexpression of miR-375 suppressed HCC cell growth. Then, BM-MSCs and EV were isolated and identified, and, EV were cocultured with HCC cells for further functional assays. It was found that miR-375 encapsulated by EV could restrict the malignant phenotypes of HCC cells. Furthermore, the downstream genes and signaling cascades involved in HCC growth were investigated. HOXB3 was determined to be a downstream target of miR-375, and upregulation of miR-375 decreased Wnt1 and β-catenin protein expression. Furthermore, HOXB3 blocked the repressive effects of miR-375 on HCC cells and Wnt1 and β-catenin expression. This study highlights that miR-375 encapsulated by EV inhibits HCC development via modulating the HOXB3/Wnt/β-catenin axis.

Methyltransferase-Like 3-Mediated m6A Methylation of Hsa_circ_0058493 Accelerates Hepatocellular Carcinoma Progression by Binding to YTH Domain-Containing Protein 1.

Circular RNAs (circRNAs) are highly correlated with the progression and prognosis of hepatocellular carcinoma (HCC). In addition, mounting evidence has revealed that N6-methyladenosine (m6A) methylation, a common RNA modification, is involved in the progression of malignancies. In this research, a novel circRNA, hsa_circ_0058493, was proven to be upregulated in HCC, which was correlated with the prognosis of HCC patients. Experimentally, hsa_circ_0058493 knockdown suppressed the growth and metastasis of HCC cells in vivo and in vitro. On the contrary, the overexpression of hsa_circ_0058493 in HCC cells had the opposite effect in vitro. Mechanistic experiments revealed that hsa_circ_0058493 contained m6A methylation sites and that methyltransferase-like 3 (METTL3) mediated the degree of methylation modification of hsa_circ_0058493. Furthermore, YTH domain-containing protein 1 (YTHDC1) could bind to hsa_circ_0058493 and promote its intracellular localization from the nucleus to the cytoplasm. In addition, both si-METTL3 and si-YTHDC1 suppressed HCC cell growth and metastasis, whereas rescue experiments confirmed that overexpression of hsa_circ_0058493 inverted the inhibitory effects of si-METTL3 and si-YTHDC1 on HCC cells. Taken together, this study explored the oncogenic role of m6A-modified hsa_circ_0058493 and found to accelerate HCC progression via the METTL3-hsa_circ_0058493-YTHDC1 axis, indicating a potential therapeutic target for this deadly disease.

Alpelisib combination treatment as novel targeted therapy against hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the sixth most common primary cancer with an unsatisfactory long-term survival. Gain of function mutations of PIK3CA occur in a subset of human HCC. Alpelisib, a selective PIK3CA inhibitor, has been approved by the FDA to treat PIK3CA mutant breast cancers. In this manuscript, we evaluated the therapeutic efficacy of alpelisib, either alone or in combination, for the treatment of HCC. We tested alpelisib in mouse HCC induced by hydrodynamic injection of c-Met/PIK3CA(H1047R) (c-Met/H1047R), c-Met/PIK3CA(E545K) (c-Met/E545K), and c-Met/sgPten gene combinations. Alpelisib slowed down the growth of c-Met/H1047R and c-Met/E545K HCC but was ineffective in c-Met/sgPten HCC. Mechanistically, alpelisib inhibited p-ERK and p-AKT in c-Met/H1047R and c-Met/E545K HCC progression but did not affect the mTOR pathway or genes involved in cell proliferation. In human HCC cell lines transfected with PIK3CA(H1047R), alpelisib synergized with the mTOR inhibitor MLN0128 or the CDK4/6 inhibitor palbociclib to suppress HCC cell growth. In c-Met/H1047R mice, alpelisib/MLN0128 or alpelisib/palbociclib combination therapy caused tumor regression. Our study demonstrates that alpelisib is effective for treating PIK3CA-mutated HCC by inhibiting MAPK and AKT cascades. Furthermore, combining alpelisib with mTOR or CDK4/6 inhibitors has a synergistic efficacy against PIK3CA-mutated HCC, providing novel opportunities for precision medicine against HCC.

A Novel Mechanism of Cannabidiol in Suppressing Hepatocellular Carcinoma by Inducing GSDME Dependent Pyroptosis.

Cannabidiol (CBD), a phytochemical derived from Cannabis sativa L., has been demonstrated to exhibit promising anti-tumor properties in multiple cancer types. However, the effects of CBD on hepatocellular carcinoma (HCC) cells remain unknown. We have shown that CBD effectively suppresses HCC cell growth in vivo and in vitro, and induced HCC cell pyroptosis in a caspase-3/GSDME-dependent manner. We further demonstrated that accumulation of integrative stress response (ISR) and mitochondrial stress may contribute to the initiation of pyroptotic signaling by CBD. Simultaneously, CBD can repress aerobic glycolysis through modulation of the ATF4–IGFBP1–Akt axis, due to the depletion of ATP and crucial intermediate metabolites. Collectively, these observations indicate that CBD could be considered as a potential compound for HCC therapy.

3,3′-Diindolylmethane Suppresses the Growth of Hepatocellular Carcinoma by Regulating Its Invasion, Migration, and ER Stress-Mediated Mitochondrial Apoptosis

Hepatocellular carcinoma (HCC) is the leading cause of cancer-related death worldwide with limited treatment options. Biomarker-based active phenolic flavonoids isolated from medicinal plants might shed some light on potential therapeutics for treating HCC. 3,3′-diindolylmethane (DIM) is a unique biologically active dimer of indole-3-carbinol (I3C), a phytochemical compound derived from Brassica species of cruciferous vegetables—such as broccoli, kale, cabbage, and cauliflower. It has anti-cancer effects on various cancers such as breast cancer, prostate cancer, endometrial cancer, and colon cancer. However, the molecular mechanism of DIM involved in reducing cancer risk and/or enhancing therapy remains unknown. The aim of the present study was to evaluate anti-cancer and therapeutic effects of DIM in human hepatoma cell lines Hep3B and HuhCell proliferation was measured with MTT and trypan blue colony formation assays. Migration, invasion, and apoptosis were measured with Transwell assays and flow cytometry analyses. Reactive oxygen species (ROS) intensity and the loss in mitochondrial membrane potential of Hep3B and Huh7 cells were determined using dihydroethidium (DHE) staining and tetramethylrhodamine ethyl ester dye. Results showed that DIM significantly suppressed HCC cell growth, proliferation, migration, and invasion in a concentration-dependent manner. Furthermore, DIM treatment activated caspase-dependent apoptotic pathway and suppressed epithelial–mesenchymal transition (EMT) via ER stress and unfolded protein response (UPR). Taken together, our results suggest that DIM is a potential anticancer drug for HCC therapy by targeting ER-stress/UPR.

MicroRNA-4325 Suppresses Cell Progression in Hepatocellular Carcinoma via GATA-Binding Protein 6

MicroRNAs (miRs) are regulators of the formation and development of hepatocellular carcinoma (HCC). The biological role of miR-4325 in HCC has yet to be determined. This study is aimed at dissecting the role of miR-4325 in HCC and the underlying mechanism. Reverse transcription-quantitative PCR (RT-qPCR) was used to detect miR-4325 expression in HCC tissue specimens and cells. Cell proliferation, migration, and invasion were assessed by using the MTT assay and Transwell assay, respectively. The miR-4325 target was predicted based on bioinformatics analysis and validated using the dual-luciferase reporter assay. Rescue experiments in the cells were utilized to functionally characterize the downstream molecular targets of miR-4325. We observed that miR-4325 expression levels were significantly reduced in both HCC tissue specimens and cell lines. Meanwhile, a lower miR-4325 level was associated with a poorer prognosis. Gain and loss of function assays revealed that miR-4325 markedly downregulated HCC cell growth, migration, and invasion. Moreover, we identified GATA-binding protein 6 (GATA6) as a miR-4325 target and found that GATA6 was abnormally expressed in HCC. Rescue assays demonstrated that the regulatory function of miR-4325 in HCC was mediated by GATA6. Taken together, miR-4325 suppresses HCC cell growth, migration, and invasion by targeting GATA6, suggesting that miR-4325 may potentially serve as a novel therapeutic target for HCC.

An oncolytic adenovirus delivering TSLC1 inhibits Wnt signaling pathway and tumor growth in SMMC-7721 xenograft mice model

Tumor suppressor in lung cancer-1 (TSLC1) was first identified as a tumor suppressor for lung cancer, and frequently downregulated in various types of cancers including hepatocellular carcinoma (HCC). The Wnt pathway plays a critical role in tumorigenesis, migration, and invasion in HCC. However, the function of TSLC1 in modulating Wnt signaling in HCC is unclear. In this study, we evaluated the effect of TSLC1-armed oncolytic adenovirus (S24-TSLC1) on the Wnt/β-catenin pathway, cell viability, invasion and migration abilities of HCC in vitro and the growth of SMMC-7721-xenografted tumor in mice model. We detected the expression of TSLC1 in tumor samples and HCC cell lines. The results showed that TSLC1 expression was low in HCC, but high in pericarcinomatous tissue and normal cells, which implied that TSLC1 is a tumor suppressor of liver cancer. S24-TSLC1 exhibited an antitumor effect on HCC cell growth in vitro, but did little damage to normal liver cells. Overexpression of TSLC1 downregulated the transcriptional activity of TCF4/β-catenin and inhibited the mRNA or protein expression of Wnt target genes cyclinD1 and c-myc. S24-TSLC1 also inhibited the invasion and migration of HCC cells. Animal experiments further confirmed that S24-TSLC1 significantly inhibited tumor growth of the SMMC-7721-xenografted tumor. In conclusion, TSLC1 could downregulate the Wnt signal pathway and suppress HCC cell growth, migration and invasion, suggesting that S24-TSLC1 may be a potent antitumor agent for future clinical trials in liver cancer treatment.

CHPF Regulates the Aggressive Phenotypes of Hepatocellular Carcinoma Cells via the Modulation of the Decorin and TGF-β Pathways.

Simple SummaryAltered extracellular chondroitin sulfate (CS) contributes to tumor progression in many cancers. CHPF is a key enzyme supporting the elongation of CS. Here we showed that CHPF was frequently downregulated in hepatocellular carcinoma (HCC) tumors compared with adjacent non-tumor tissues, and its downregulation was associated with poor overall survival. CHPF regulated aggressive phenotypes of HCC cells in vitro and in vivo, and the TGF-β pathway involved in the phenotypical changes. Mechanistically, CHPF modified CS on decorin (DCN), which could facilitate DCN accumulation surrounding HCC cells, and modulate activation of TGF-β pathway. Indeed, the expression of DCN were positively associated with CHPF levels in primary HCC tissue. The research proposed novel insights into the significance of CHPF, which modified DCN and modulated TGF-β signaling.Aberrant composition of glycans in the tumor microenvironment (TME) and abnormal expression of extracellular matrix proteins are hallmarks of hepatocellular carcinoma (HCC); however, the mechanisms responsible for establishing the TME remain unclear. We demonstrate that the chondroitin polymerizing factor (CHPF), an enzyme that mediates the elongation of chondroitin sulfate (CS), is a critical elicitor of the malignant characteristics of HCC as it modifies the potent tumor suppressor, decorin (DCN). CHPF expression is frequently downregulated in HCC tumors, which is associated with the poor overall survival of HCC patients. We observed that restoring CHPF expression suppressed HCC cell growth, migration, and invasion in vitro and in vivo. Mechanistic investigations revealed that TGF-β signaling is associated with CHPF-induced phenotype changes. We found that DCN, as a TGF-β regulator, is modified by CHPF, and that it affects the distribution of DCN on the surface of HCC cells. Importantly, our results confirm that CHPF and DCN expression levels are positively correlated in primary HCC tissues. Taken together, our results suggest that CHPF dysregulation contributes to the malignancy of HCC cells, and our study provides novel insights into the significance of CS, which affects DCN expression in the TME.

Wnt8B, transcriptionally regulated by ZNF191, promotes cell proliferation of hepatocellular carcinoma via Wnt signaling.

Dysregulation of wingless‐type (Wnt) signaling is implicated in hepatocellular carcinoma (HCC). Wnt family member 8B (Wnt8B), one of the canonical Wnt ligands, is implicated in oncogenesis. However, the role of Wnt8B in human HCCs and its transcriptional regulation mechanism are presently unknown . Here, we report that Wnt8B expression was frequently increased in HCCs and was significantly associated with poorer patient prognosis. Wnt8B knockdown suppresses HCC cell growth both in vitro and in vivo via inhibiting the canonical Wnt signaling. Zinc finger transcription factor 191 (ZNF191) can positively regulate Wnt8B mRNA and protein expression, and promoter luciferase assay indicated that ZNF191 can increase the transcription activity of the 2‐Kbps WNT8B promoter. Chromatin immunoprecipitation‐qPCR and electrophoretic mobility shift assay showed that ZNF191 protein directly binds to the WNT8B promoter, and the binding sites are at nt‐1491(ATTAATT) and nt‐1178(ATTCATT). Moreover, Wnt8B contributes to the effect of ZNF191 on cell proliferation, and Wnt8B expression correlates positively with ZNF191 in human HCCs. Our findings suggested that Wnt8B, directly transcriptionally regulated by ZNF191, plays a pivotal role in HCC proliferation via the canonical Wnt pathway and may serve as a new prognostic biomarker and a potential therapeutic target for HCC patients.

Role and Clinical Significance of miR-144-3p in Primary Hepatocellular Carcinoma

The morbidity of liver cancer (LC) is increasing and its high mortality poses a significant health threat worldwide. Therefore, it is crucial to identify the underlying mechanism of LC development and progression. The expression of miR-144-3p and ZEB1 gene in LC and paracanerous tissues was measured by Polymerase chain reaction (PCR). We transfected miR-144-3p-mimics, miR-NC, si-ZEB1, and si-NC into HepG2, Huh-7, and HL-7702 normal liver cells to establish a cell model. Protein expression was examined by western blot (WB) analysis. Cell proliferation, invasion, and apoptosis were assessed by CCK-8, Transwell, and flow cytometry assays. The relationship between miR-144-3p and ZEB1 was determined using a dual-luciferase reporter assay. We found that miR-144-3p expression decreased dramatically, whereas ZEB1 increased in hepatocellular carcinoma (HCC). Moreover, over-expressing miR-144-3p suppressed cell growth and induced apoptosis. The expression of apoptosis-related proteins was consistent with the induction of apoptosis. The relationship between miR-144-3p and ZEB1 was confirmed using a dual-luciferase reporter assay. Finally, rescue experiments revealed that over-expressing ZEB1 counteracted miR-144-3p inhibition on LC cell growth, invasion, and apoptosis induction. We conclude that miR-144-3p suppresses HCC cell growth and invasion and promotes apoptosis by regulating ZEB1 expression, suggesting that this interaction may represent a target for HCC treatment.

MiR-133a-3p Regulates Hepatocellular Carcinoma Progression Through Targeting CORO1C.

IntroductionMicroRNAs (miRNAs) are key modulators for gene expression via inducing translational repression or target gene degradation. miR-133a-3p was reported to stimulate or inhibit cancer progression but its role in hepatocellular carcinoma (HCC) remains to be explored.MethodsQuantitative real-time PCR (RT-qPCR) was utilized to explore miR-133a-3p expression level in HCC cells. Dual-luciferase activity reporter assay was used to validate the direct interaction between miR-133a-3p and coronin-like actin-binding protein 1C (CORO1C). In addition, we analyzed the expression levels of miR-133a-3p and CORO1C in HCC tissues and normal tissues on the UCALAN website. Functional assays including cell counting kit-8 assay, colony formation assay, flow cytometry analysis and transwell invasion assay were conducted to explore the biological functions of miR-133a-3p in HCC.ResultsmiR-133a-3p was found to have downregulated expression in HCC tissues and cells. Meanwhile, we showed that low miR-133a-3p levels were correlated with poorer overall survival of HCC patients. Overexpression of miR-133a-3p suppressed HCC cell growth and invasion but promoted cell apoptosis via targeting CORO1C.DiscussionOur results revealed a novel mechanism of miR-133a-3p in regulating HCC progression and provided evidence that miR-133a-3p functions as a tumor suppressor in HCC.

LncRNA-HEIH suppresses hepatocellular carcinoma cell growth and metastasis by up-regulating miR-199a-3p.

The aim of this study was to explore the functional changes of long non-coding ribonucleic acid (lncRNA)-HEIH on hepatocellular carcinoma (HCC) Huh7 and Hep3B cells. The expression changes of HEIH in 18 pairs of HCC tissues and adjacent normal tissues were detected by quantitative real time-polymerase chain reaction (qRT-PCR). According to its expression changes in HCC cells silenced by short hairpin ribonucleic acid (shRNA) transfection in vitro, these cells were divided into sh-HEIH group and sh-NC group. The effects of lowly expressed HEIH on the proliferation, migration and apoptosis of HCC cells were examined through functional assays. Western blotting was adopted to determine the expression changes of epithelial-mesenchymal transition (EMT) proteins, vimentin, matrix metalloproteinase (MMP)-2 and MMP-3. In addition, the role of HEIH downstream effector micro RNA (miR)-199a-3p in HCC was explored. Compared with adjacent normal tissues, HEIH was highly expressed in HCC tissues (p<0.01). HEIH silencing significantly inhibited the proliferation and migration, but induced the apoptosis of Huh7 cells (p<0.05). The expressions of vimentin and MMP-2 in sh-HEIH group were remarkably lower than those in sh-NC group (p<0.05). Furthermore, miR-199a-3p was identified as the downstream effector of HEIH. The expression of miR-199a-3p increased markedly in Huh7 and Hep3B cells with silenced HEIH expression (p<0.01). Moreover, when miR-199a-3p expression was inhibited, the effects of HEIH on Huh7 and Hep3B cells were weakened, manifested as notably enhanced cell proliferation and migration capabilities (p<0.05). LncRNA-HEIH suppresses HCC cell growth and metastasis by up-regulating miR-199a-3p. Our findings suggest that HEIH may be a promising target for HCC treatment.

CLK3 Is A Direct Target Of miR-144 And Contributes To Aggressive Progression In Hepatocellular Carcinoma.

BackgroundHepatocellular carcinoma (HCC) is the most common type of primary liver cancer with high incidence. The underlying molecular mechanisms of HCC development have been intensively studied. CLK3 (CDC Like Kinase 3) is a nuclear dual-specificity kinase and regulates gene splicing. We investigated the expression profile and functional role of CLK3 in HCC.MethodsImmunohistochemistry (IHC) and Western blot were performed to determine CLK3 expression in HCC tissues. Bioinformatics analysis using TCGA and GEO database was conducted to evaluate the relationship between CLK3 expression and HCC prognosis. Cell proliferation was assessed by CCK8, EdU and colony formation assays, while transwell and wound-healing assays were performed to investigate the cell migration and invasion in vitro. Xenograft nude mouse model was used to test the function of CLK3 on tumor growth in vivo. Luciferase reporter assay, Western blot and RT-qPCR were conducted to verify the miRNA that directly targeted CLK3.ResultsCLK3 was markedly upregulated in HCC tissues, and the expression levels of CLK3 were closely associated with TNM stages and HCC prognosis. Functional analysis indicated that knockdown of CLK3 could suppress HCC cell growth, invasion and migration in vitro, and inhibit tumor development in vivo. Moreover, CLK3 was demonstrated as a direct target of miR-144 and miR-144 expression was inversely correlated with CLK3 expression in HCC. Enforced overexpression of miR-144 markedly inhibited the CLK3 expression while overexpression of CLK3 partially reversed the inhibitory function of miR-144 on HCC cell growth and metastasis. Mechanistically, we found that miR-144 overexpression inhibited Wnt/β-catenin signaling and the inhibition could be partly abolished by overexpression of CLK3.ConclusionIn summary, we demonstrate tumor suppressor miR-144 suppresses hepatocellular carcinoma development and metastasis via regulating CLK3 and Wnt/β-catenin signaling, indicating that miR-144/CLK3 could be used for HCC diagnosis and treatment.

Artemisinin suppresses hepatocellular carcinoma cell growth, migration and invasion by targeting cellular bioenergetics and Hippo-YAP signaling

The primary liver cancer (PLC) is one of the leading causes of cancer-related death worldwide. The predominant form of PLC is hepatocellular carcinoma (HCC), which accounts for about 85% of all PLC. Artemisinin (ART) was clinically used as anti-malarial agents. Recently, it was demonstrated to inhibit cell growth and migration in multiple cancer types. However, the molecular mechanism underlying these anti-cancer activity remains largely unknown. Herein, it is discovered that ART dramatically suppresses HCC cell growth in vitro through arresting cell cycle progression, and represses cell migration and invasion via regulating N-cadherin-Snail-E-cadherin axis. In addition, the disruption of cellular bioenergetics contributed to ART-caused cell growth, migration and invasion inhibition. Moreover, ART (100mg/kg, intraperitoneally) substantially inhibits HCC xenograft growth in vivo. Importantly, Hippo-YAP signal transduction is remarkably inactivated in HCC cells upon ART administration. Collectively, these data reveal a novel mechanism of ART in regulating HCC cell growth, migration, and invasion, which indicates that ART could be considered as a potential drug for the treatment of HCC.

Ribosome profiling analysis identified a KRAS-interacting microprotein that represses oncogenic signaling in hepatocellular carcinoma cells.

The roles of concealed microproteins encoded by long noncoding RNAs (lncRNAs) are gradually being exposed, but their functions in tumorigenesis are still largely unclear. Here, we identify and characterize a conserved 99-amino acid microprotein named KRASIM that is encoded by the putative lncRNA NCBP2-AS2. KRASIM is differentially expressed in normal hepatocytes and hepatocellular carcinoma (HCC) cells and can suppress HCC cell growth and proliferation. Mechanistically, KRASIM interacts and colocalizes with the KRAS protein in the cytoplasm of human HuH-7 hepatoma cells. More importantly, the overexpression of KRASIM decreases the KRAS protein level, leading to the inhibition of ERK signaling activity in HCC cells. These results demonstrate a novel microprotein repressor of the KRAS pathway for the first time and provide new insights into the regulatory mechanisms of oncogenic signaling and HCC therapy.