Function Of Hepatocellular Carcinoma Cells Research Articles

Role of miRNA-99a-5p in Modulating the Function of Hepatocellular Carcinoma Cells: Bioinformatics Analysis and In Vitro Assay.

This study aimed to investigate the biological functions of miRNAs in hepatobiliary tumors as the focus of targeted therapy research. Hepatobiliary tumors are among the leading causes of cancer-related deaths worldwide. Many microRNAs (miRNAs) play an important regulatory role in tumor progression. Our study aims to explore some biologically functional miRNAs from different datasets of hepatobiliary tumors for disease diagnosis or treatment. In this study, we tried to filter out differentially expressed miRNAs in different tumor datasets from the GEO database. In this study, we first perform analyses in different GEO data sets. After taking the intersection, the initial scope is limited to several differential RNAs. Then, combined with the existing research results from Kaplan-Meier survival analysis and literature, the candidate molecule was finally identified to be studied. Furthermore, the biological characteristics analysis of the candidate molecule was performed on the basis of Cancermirnome online tool, including expression levels in tumors, KEGG and GO analysis, ROC analysis, and target gene prediction. Furthermore, the effect of the candidate molecule on the biological functions of liver cancer was verified by In Vitro assay. The preliminary analysis of bioinformatics shows that 16 differentially expressed miRNAs may play an important role in HCC or ICC. Ultimately, we identified miRNA-99a-5p as the only molecule to study. The results showed that miRNA-99a-5p is abnormally expressed in many tumors, and in liver cancer, its level of expression in tumor tissue is significantly lower than that in normal tissue. Then, the KEGG and GO analysis found that it functions in multiple pathways. At the same time, the ROC analysis found that it showed great potential for prognostic prediction in HCC and we also predicted that RUNDC3B is the most likely target to which it binds. Finally, the experimental results of overexpression and knockdown confirmed that miRNA-99a-5p could inhibit cell proliferation in HCC, which also suggested that it may be an important tumor suppressor in HCC. MiRNA-99a-5p was negatively correlated with HCC progression and could act as a novel therapeutic target for HCC.

CCT2 Gene Expression in Hepatocellular Carcinoma and its Effect on the Biological Function of Hepatocellular Carcinoma Cells

The main objective of the study is to investigate the expression level of chaperonin containing t-complex polypeptide 1 subunit 2 in hepatocellular carcinoma tissues and cell lines, and its effect on the proliferation, invasion and migration of hepatocellular carcinoma cells. Immunohistochemistry was used to detect the protein expression levels of chaperonin containing t-complex polypeptide 1 subunit 2 in 89 hepatocellular carcinoma tissues and tumor-adjacent tissues. Cell migration and invasion were detected by transwell assay. A subcutaneous xenograft model was constructed in nude mice to investigate the effect of chaperonin containing t-complex polypeptide 1 subunit 2 gene knockdown on tumor growth. Immunohistochemistry results indicated that 79.78 % of hepatocellular carcinoma patients had highly expressed chaperonin containing t-complex polypeptide 1 subunit 2 proteins and the expression in hepatocellular carcinoma tissues was significantly higher than that in tumor-adjacent tissues. The proliferation, invasion and migration of hepatoblastoma cell line HepG2 cells with chaperonin containing t-complex polypeptide 1 subunit 2 gene knockdown were inhibited, while those of human hepatoma-derived Huh-7 cells with chaperonin containing t-complex polypeptide 1 subunit 2 gene overexpression were enhanced. In vivo experiments also confirmed that the tumor growth rate in the chaperonin containing t-complex polypeptide 1 subunit 2 knockdown group was significantly slower than that in the vector group. Chaperonin containing t-complex polypeptide 1 subunit 2 is an independent poor prognostic factor of hepatocellular carcinoma and its expression is significantly upregulated in hepatocellular carcinoma tissues and cell lines. Chaperonin containing t-complex polypeptide 1 subunit 2 can significantly promote the proliferation, invasion and migration of hepatocellular carcinoma cells, suggesting that it can be an important therapeutic target for hepatocellular carcinoma.

Propofol Inhibits Biological Function of Hepatocellular Carcinoma Cells through LINC00475-Mediated Sonic Hedgehog Pathway

Introduction: Hepatocellular carcinoma (HCC) is the most deadly cancer. Many signal pathways are implicated in HCC development, including sonic hedgehog (SHH). Propofol is an anesthetic commonly used in surgery. Recent studies have reported that propofol inhibits tumorigenesis and the development of HCC in a dose-dependent manner. The study aimed to identify the mechanism of how the propofol-mediated SHH-signaling molecule works in HCC. Methods: Cell proliferation, apoptosis, and invasion were examined, respectively, through colony formation, TUNEL, caspase-3 activity, and transwell assays. Protein levels of SHH, Ptch1, Smo, and Gli1 were determined via Western blot. Results: Propofol could inhibit cell proliferation, migration, and invasion and induce apoptosis via suppression on SHH to inactivate the SHH pathway. By mechanistic assays, miR-340-5p was identified to target SHH and negatively regulate SHH. Long intergenic non-protein coding RNA 475 (LINC00475) was the endogenous sponge of miR-340-5p to upregulate SHH. Finally, the rescue assays were implemented. The activator of the SHH pathway completely rescued the effects of LINC00475 and SHH in propofol-induced HCC cells. Conclusion: Propofol inhibits HCC cell malignant behaviors via repressing LINC00475 to suppress SHH, thus inactivating the SHH pathway. These new findings might contribute to the understanding and application of propofol in HCC.

Molecular docking and in vitro experiments verified that kaempferol induced apoptosis and inhibited human HepG2 cell proliferation by targeting BAX, CDK1, and JUN.

Hepatocellular carcinoma, as a common liver cirrhosis complication, has become the sixth most common cancer worldwide, and its increasing incidence has resulted in considerable medical and economic burdens. As a natural polyphenolic compound, kaempferol has exhibits a wide range of antitumor activities against multiple cancer targets. In this study, the Autodock software was used for molecular docking to simulate the interaction process between kaempferol and HCC targets and the PyMOL software was used for visualization. Proliferation of kaempferol HepG2 cells under the effect of kaempferol was detected using Cell Counting Kit-8 (CCK-8) assay, and the apoptosis rate of HepG2 cells was detected using flow cytometry. The expressions of proteins BAX, CDK1, and JUN protein expressions were detected by Western blot. Molecular docking found that the kaempferol ligand has 3 rotatable bonds, 6 nonpolar hydrogen atoms, and 12 aromatic carbon atoms, and can form complexes with the kaempferol targets P53, BAX, AR, CDK1, and JUN through electrostatic energy. GO (Gene Ontology) enrichment analysis suggests that kaempferol regulates the biological function of hepatocellular carcinoma cells and is related to apoptosis. Cell Counting Kit-8 assay suggested that Kaempferol can significantly inhibited HepG2 cell proliferation, and the inhibition rate increased with the increase in drug concentration and incubation time. Moreover, kaempferol can promoted HepG2 cell apoptosis in a dose-dependent manner. This compound upregulated BAX and JUN expression and downregulated CDK1 expression. Thus, Kaempferol can promote HepG2 cell apoptosis, and the regulatory mechanism may be related to the regulation of the expression levels of the apoptosis-related proteins BAX, CDK1, and JUN.

Long non-coding RNA FOXP4-AS1 facilitates the biological functions of hepatocellular carcinoma cells via downregulating ZC3H12D by mediating H3K27me3 through recruitment of EZH2

BackgroundSome studies have reported the effect of long non-coding RNA forkhead box P4 antisense RNA 1 (lncRNA FOXP4-AS1) on hepatocellular carcinoma (HCC). Here, we aimed to discuss the effects of FOXP4-AS1/enhancer of zeste homolog 2 (EZH2)/trimethylation of lysine 27 on histone H3 (H3K27me3)/zinc finger CCCH-type containing 12D (ZC3H12D) axis on HCC.MethodsThe expression of FOXP4-AS1, EZH2, and ZC3H12D, and abundance of H3K27me3 in HCC tissues and cells were tested. The relationship between FOXP4-AS1 expression and prognosis of HCC patients was analyzed. The biological functions of HCC cells were detected via loss- and gain-of-function assays. The tumor weight and volume in vivo were tested. The interaction between FOXP4-AS1 and EZH2 as well as that between EZH2 and H3K27me3 was verified.ResultsFOXP4-AS1 and EZH2 expression and H3K27me3 abundance were enhanced while ZC3H12D expression was depressed in HCC tissues and cells. Knockdown of FOXP4-AS1 suppressed biological functions of HCC cells as well as the weight and volume of HCC transplanted tumor. Depleting ZC3H12D reversed the effect of downregulated FOXP4-AS1 on HCC cells. FOXP4-AS1 suppressed ZC3H12D expression via mediating H3K27me3 by recruitment of EZH2.ConclusionThe key findings of the present study demonstrate that FOXP4-AS1 suppresses ZC3H12D expression via mediating H3K27me3 by recruitment of EZH2, thus promoting the progression of HCC.Graphical abstract

Lack of MOF Decreases Susceptibility to Hypoxia and Promotes Multidrug Resistance in Hepatocellular Carcinoma via HIF-1α

Hypoxia-inducible factor-1α (HIF-1α) promotes oncogenesis in hepatocellular carcinoma and is functionally linked to cell proliferation, chemoresistance, metastasis and angiogenesis. It has been confirmed that the low expression level of Males absent on the first (MOF) in hepatocellular carcinoma leads to poor prognosis of patients. However, potential regulatory mechanisms of MOF in response to hypoxia remain elusive. Our results demonstrate that MOF expression is negatively associated with HIF-1α expression in hepatocellular carcinoma tissues and in response to chloride-mimicked hypoxia in hepatocellular carcinoma cell lines. MOF regulates HIF-1α mRNA expression and also directly binds to HIF-1α to mediate HIF-1α N-terminal lysine acetylation, ubiquitination and degradation, with downstream effects on MDR1 levels. Functional inactivation of MOF enhances HIF-1α stability and causes cell tolerance to hypoxia that is insensitive to histone deacetylase inhibitor treatment. Dysfunction of MOF in hepatocellular carcinoma cells also results in chemoresistance to trichostatin A, sorafenib and 5-fluorouracil via HIF-1α. Our results suggest that MOF regulates hypoxia tolerance and drug resistance in hepatocellular carcinoma cells by modulating both HIF-1α mRNA expression and N-terminal acetylation of HIF-1α, providing molecular insight into MOF-dependent oncogenic function of hepatocellular carcinoma cells.

The expression profiles and prognostic values of HSP70s in hepatocellular carcinoma

BackgroundThe HSP70 family of heat shock protein plays a critical role in protein synthesis and transport to maintain protein homeostasis. Several studies have indicated that HSP70s are related to the development and occurrence of various cancers.MethodsThe relationship between the overall survival rate of hepatocellular carcinoma patients and the expression of 14 HSP70s from multiple databases, such as TCGA, ONCOMINE, cBioPortal was investigated. Western Blot and PCR were used to evaluate HSPA4 and HSPA14 expressions in various HCC cells to identify suitable cell lines for further experiments .Wound-healing assays, Transwell assays and EdU assays were used to verify the effects of HSPA4 and HSPA14 on the function of hepatocellular carcinoma cells, and statistical analysis was performed.ResultsHepatocellular carcinoma tissues significantly expressed the 14 HSP70s compared to the normal samples. Besides, the high HSPA1A, HSPA1B, HSPA4, HSPA5, HSPA8, HSPA13, and HSPA14 expressions were inversely associated with the overall survival rate of patients, tumor grade, and cancer stage. A PPI regulatory network was constructed using the 14 HSP70s proteins with HSPA5 and HSPA8 at the network center. Univariate and multivariate analyses showed that HSPA4 and HSPA14 could be independent risk factors for the prognosis of hepatocellular carcinoma patients. Cell experiments have also confirmed that reducing HSPA4 and HSPA14 expressions can inhibit the invasion, metastasis, and proliferation of hepatocellular carcinoma cells.ConclusionsTherefore, the HSP70s significantly influence the occurrence and development of hepatocellular carcinoma. For instance, HSPA4 and HSPA14 can be novel therapeutic targets and prognostic biomarkers for hepatocellular carcinoma.

ADORA1 Promotes Hepatocellular Carcinoma Progression via PI3K/AKT Pathway.

ObjectiveHepatocellular carcinoma (HCC) is a common malignancy worldwide. Although the contradictory role of ADORA1 has been explored in certain types of cancers, its clinical significance and function in hepatocellular carcinoma cells are largely unknown.Materials and MethodsThe level of ADORA1 in HCC tissues and cells was evaluated by RT-PCR. The function of ADORA1 overexpression on HCC cell proliferation and invasion was assessed by MTS, transwell analysis, and colony formation assay. In addition, a mouse subcutaneous xenograft model was used to study in vivo effects. The efficacy of knockdown of ADORA1 sensitizes to chemotherapy was assessed by staining with Annexin V/propidium iodide followed with flow cytometry and nuclei fragmentation.ResultsIn this study, ADORA1 was identified to be up-regulated in HCC tissues compared with adjacent normal tissue. High ADORA1 mRNA expression predicted poor survival in hepatocellular carcinoma patients. Ectopic expression of ADORA1 increased hepatocellular carcinoma cell proliferation and invasion. ADORA1 knockdown inhibited HCC cell growth and sensitized to chemotherapy. Furthermore, ADORA1 activated PI3K/AKT oncogenic signaling pathways. Treatment with PI3K inhibitor LY294002 blocked the effects of ADORA1 on tumor growth in either ADORA1-overexpressing or -deficiency cells. Finally, overexpression of ADORA1 stimulates HCC tumor growth in vivo. Treatment of ADORA1 antagonist oppositely suppressed HCC xenograft tumor growth.ConclusionADORA1 serves as an important oncoprotein and a promoter of cell proliferation through PI3K/AKT signaling pathway in hepatocellular carcinoma.

Lnc-ATG9B-4 aggravates progress of hepatocellular carcinoma through cell proliferation and migration by upregulating CDK5.

Long noncoding RNAs play an important role in the occurrence, invasion, as well as metastasis of various human cancers, including hepatocellular carcinoma. Long noncoding RNAs can affect the biological functions of hepatocellular carcinoma cells by regulating various genes; however, only a small fraction of molecular mechanisms of long noncoding RNAs have been elucidated. In the present study, lnc AC010973.1 (lnc-ATG9B-4) was first identified by microarray analysis from 8 patients with hepatocellular carcinoma and confirmed by quantitative PCR in 176 patients with hepatocellular carcinoma. We demonstrated that lnc-ATG9B-4 was tightly relative to the tumorous size, TNM stages, portal vein tumor thrombus (PVTT), the tumor capsule, metastasis, degree of differentiation, and poor prognosis of hepatocellular carcinoma according to long-term follow-up data. In hepatocellular carcinoma cells, overexpression of lnc-ATG9B-4 promoted proliferation, invasion, as well as migration, while inhibiting lnc-ATG9B-4 by siRNA significantly attenuated the proliferation, invasion, as well as migration. Interestingly, lnc-ATG9B-4 increased the expression of cyclin-dependent kinase 5 (CDK5), which was closely related to the development and chemotherapy sensitivity of hepatocellular carcinoma. In summary, our results revealed that lnc-ATG9B-4 suggests an unfavorable prognosis of hepatocellular carcinoma and facilitates the proliferation, invasion, as well as migration of hepatocellular carcinoma cells by upregulating CDK5. This research suggests that lnc-ATG9B-4 may be a new biomarker for predicting the prognosis of hepatocellular carcinoma; meanwhile, targeting lnc-ATG9B-4 might serve as a potential strategy for the treatment hepatocellular carcinoma.

Circ_0000064 adsorption of microRNA-143 promotes malignant progression of hepatocellular carcinoma.

The aim of this study was to investigate the expression characteristics of circ_0000064 in hepatocellular carcinoma, and to further explore the underlying mechanism. Real time quantitative-Polymerase Chain Reaction (qPCR) was used to detect the expression of circ_0000064 in 42 hepatocellular carcinoma tissues and adjacent normal tissues. Meanwhile, the relationship between circ_0000064 expression and clinical indicators, as well as the prognosis of patients with hepatocellular carcinoma, were detected. QPCR was applied to measure circ_0000064 level in hepatocellular carcinoma cell lines as well. Subsequently, the circ_0000064 knockdown model was successfully constructed using lentivirus in hepatocellular carcinoma cell lines. Cell counting kit-8 (CCK-8), colony formation assay, and flow apoptosis were performed to analyze the influence of circ_0000064 on the biological functions of hepatocellular carcinoma cells. The potential mechanism was explored using cell recovery experiments. In addition, the relationship between circ_0000064 and microRNA-143 was finally explored. QPCR results showed that the expression level of circ_0000064 in hepatocellular carcinoma tissues was remarkably higher than that of adjacent normal tissues, and the difference was statistically significant. Compared with patients with lower expression of circ_0000064, patients with higher expression of circ_0000064 exhibited remarkably higher pathological stage and lower overall survival rate. In vitro experiments demonstrated that the proliferation ability of the cells was remarkably reduced after the transfection of si-circ_0000064, while cell apoptosis ability significantly increased when compared with the NC group. Meanwhile, qPCR results indicated that microRNA-143 expression was negatively correlated with circ_0000064 expression in hepatocellular carcinoma. Luciferase reporter gene assay indicated that circ_0000064 could be targeted by microRNA-143 through their binding site. In addition, the cell recovery experiment confirmed that circ_0000064 and microRNA-143 could be mutually regulated, which affected the malignant progression of hepatocellular carcinoma together. Circ_0000064 level was remarkably upregulated in hepatocellular carcinoma and was associated with high pathological stage and poor prognosis of patients. In addition, circ_0000064 significantly promoted proliferation and inhibited apoptosis of hepatocellular carcinoma cells via modulating microRNA-143.

Role of serine-threonine kinase receptor-related protein in the biological function of hepatocellular carcinoma cells

Objective To detect the expression of serine-threonine kinase receptor-related protein (STRAP) in hepatocellular carcinoma (HCC) cells and normal liver tissues, and to explore its biological functions. Methods The relative expression levels of STRAP in 10 HCC cell lines and normal liver tissues were detected, and STRAP overexpression and knockdown cell lines were constructed in HepG2 and SNU449, respectively. The real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting were used to determine the expression of STRAP after the construction of cell lines. The migration and invasion ability of HCC cells was detected by Transwell chamber and Transwell chamber containing Matrigel. Cell proliferation was examined by cell counting kit-8 (CCK-8) and cell clone formation assay, and cell apoptosis was verified by flow cytometry. Results The expression level of STRAP in HCC cell lines was significantly higher than that in normal liver tissues. After STRAP knockdown, the relative expression of mRNA in knockdown group and control group was (0.17±0.19) and (2.22±1.02), respectively. The expression level of the knockdown group was significantly lower than that in the control group (P<0.05), while overexpressing STRAP, the relative expression levels of mRNA in overexpression group and control group were (3.25±0.39) and (1.05±0.37) (P<0.01). After the STRAP gene knockdown, the number of cell clones in control group and knockdown group was (221.30±9.60) and (58.30±4.49) respectively. The number of cells passing through the membrane in control group and knockdown group was (0.76±0.04) and (0.44±0.06) in cell migration assay, and (1.11±0.05) and (0.60±0.05) in cell invasion assay, respectively. In the cell proliferation assay, the number of cell proliferation in the knockdown group decreased as compared with that in the control group at 72, 96 and 120 h. The apoptosis rate in the control group and knockdown group was (4.11±0.12) and (11.73±0.40), respectively. The colony forming ability, migration invasive ability and proliferation ability of SNU449 were significantly inhibited (P<0.05), while the apoptosis was significantly increased (P<0.01). After overexpression of SRTAP gene, the proliferation of HepG2 cells increased at 48, 72, 96 and 120 h as compared with the control group (P<0.001). The cell proliferation assay indicated that the apoptosis rate in the control group and overexpression group was (8.87±0.12) and (8.34±0.06) respectively (P<0.05). Conclusion STRAP is highly expressed in HCC cells, and promotes proliferation, enhances migration and invasion, and inhibits apoptosis of HCC cells. Key words: Carcinoma, hepatocellular; Serine-threonine kinase receptor-related protein; Proliferation; Apoptosis; Metastasis

Effect of liver kinase B1 on biological function of hepatocellular carcinoma cells and its relationship with postoperative prognosis

Objective To investigate the effect of liver kinase B1 (LKB1) on the biological function of hepatocellular carcinoma cells and its relationship with prognosis. Methods LKB1 overexpression lentiviral vector was constructed by molecular biology technology, and the MHCC97H cells with high metastasis were infected by lentiviruses. LKB1 control MHCC97H cell lines (control group) and LKB1 overexpression MHCC97H cell lines (observation group) were established. The proliferation ability of cells was analyzed by cell counting kit-8 (CCK-8) assay in control group and observation group. The invasion ability of cells was analyzed by Transwell in control group and observation group. 142 patients with liver cancer from June 2012 to June 2018 in our hospital were selected as subjects. The expression of LKB1 was analyzed by immunohistochemistry and the recurrence rate of patients was analyzed in LKB1 high expression and low expression groups. Results The LKB1 overexpression MHCC97H cell line was successfully constructed in this study. As compared with control group, the proliferative capacity of MHCC97H cells in observation group was significantly decreased (P<0.05). As compared with control group, the invasive ability of cells in the observation group [(28.32±7.32) cells] was significantly decreased (P<0.05). Immunohistochemical analysis showed there were 98 cases (69.01%) in the LKB1 low expression group and 44 cases (30.99%) in the LKB1 high expression group. The 2- and 3-year recurrence rate was 27.27% and 47.62% in the high-expression group, and 40.82% and 68.37% in the low-expression group, respectively. There was significant difference between the two groups (Log-rank=2.013, P<0.05). Conclusion LKB1 is related to the proliferation and migration of hepatocellular carcinoma cells, and the recurrence rate of patients with high LKB1 expression is significantly increased after 5 years. Key words: Liver kinase B1; Carcinoma, hepatocellular; Proliferation; Invasion; Recurrence

Rottlerin upregulates DDX3 expression in hepatocellular carcinoma

Rottlerin has been reported to exert its anti-tumor activity in various types of human cancers. However, the underlying molecular mechanism has not been fully elucidated. In the current study, we explored whether rottlerin exhibits its tumor suppressive function in hepatocellular carcinoma cells. Our MTT assay results showed that rottlerin inhibited cell growth in hepatocellular carcinoma cells. Moreover, we found that rottlerin induced cell apoptosis and caused cell cycle arrest at G1 phase. Furthermore, our wound healing assay result demonstrated that rottlerin retarded cell migration in hepatocellular carcinoma cells. Additionally, rottlerin suppressed cell migration and invasion. Notably, we found that rottlerin upregulated DDX3 expression and subsequently downregulated Cyclin D1 expression and increased p21 level. Importantly, down-regulation of DDX3 abrogated the rottlerin-mediated tumor suppressive function, whereas overexpression of DDX3 promoted the anti-tumor activity of rottlerin. Our study suggests that rottlerin exhibits its anti-cancer activity partly due to upregulation of DDX3 in hepatocellular carcinoma cells.

Isolation, culture and identification of hepatocellular carcinoma-associated fibroblast and its effect on the biological function of hepatocellular carcinoma cells

Objective To establish an in vitro isolation and culture system for hepatocellular carcinoma (HCC)-associated fibroblast (CAF) and identify based on its specific markers of CAF. Methods CAF was isolated from the tumor tissue of the HCC patients after hepatectomy, by digesting with collagenase enzyme, centrifugation and resuspension. The morphology of CAF was observed by electron microscopy. Immunofluorescence staining was performed for detecting α-SMA and fibronectin as well as other specific markers such as AFP on the surface of HCC cells and CD31 on the surface of vascular endothelial cells. On the basis of the study, the function work between CAF and HCC cell line Huh7, HepG2 was detected under co-culture system. Meanwhile, CCK-8 was used to observe the effect of CAF on the proliferation of Huh7 and HepG2 cells, and Transwell assay was used to analyze CAF effect on the invasion of Huh7 and HepG2 cells. Results Compared with the other cells, the morphological analysis showed that CAF was more elongated or spindle-shaped. Moreover, the cell size and the nucleus were larger than normal epithelial cells. Immunofluorescence staining revealed that the CAF surface specific markers including α-SMA and fibronectin were positive, and mainly were the cell membrane staining. The proliferation and invasion of Huh7 and HepG2 were significantly increased by CAF. The results show that the increasing percentage of cells in 24 hours between blank group and the experimental group was (63 ± 4)%, (78 ± 5)% and (69 ± 5)%, (81 ± 3)% respectively after co-culture with CAF, the difference was statistically significant (P < 0.05). In transwell model, the number of cells in the blank group and the experimental group was (59.4 ± 3.1), (162.9 ± 3.9) and (104.8 ± 2.6), (166.4 ± 4.2), and the difference was also statistically significant (P < 0.05). Conclusion The isolated CAF from HCC enhances the ability of tumor's proliferation and invasion. Key words: Fibroblasts; Liver neoplasms; Smooth muscle myosins; Cell proliferation; Neoplasm invasiveness

N-linked glycosylation at Asn152 on CD147 affects protein folding and stability: promoting tumour metastasis in hepatocellular carcinoma.

Cluster of differentiation 147 (CD147), also known as extracellular matrix metalloproteinase inducer, is a transmembrane glycoprotein that mediates oncogenic processes partly through N-glycosylation modifications. N-glycosylation has been demonstrated to be instrumental for the regulation of CD147 function during malignant transformation. However, the role that site-specific glycosylation of CD147 plays in its defective function in hepatocellular carcinomacells needs to be determined. Here, we demonstrate that the modification of N-glycosylation at Asn152 on CD147 strongly promotes hepatocellular carcinoma (HCC) invasion and migration. After the removal of N-glycans at Asn152, CD147 was more susceptible to degradation by ER-localized ubiquitin ligase-mediated endoplasmic reticulum-associated degradation (ERAD). Furthermore, N-linked glycans at Asn152 were required for CD147 to acquire and maintain proper folding in the ER. Moreover, N-linked glycans at Asn152 functioned as a recognition motif that was directly mediated by the CNX quality control system. Two phases in the retention-based ER chaperones system drove ER-localized CD147 trafficking to degradation. Deletion of N-linked glycosylation at Asn152 on CD147 significantly suppressed in situ tumour metastasis. These data could potentially shed light on the molecular regulation of CD147 through glycosylation and provide a valuable means of developing drugs that target N-glycans at Asn152 on CD147.

Knockdown of Histone Methyltransferase hSETD1A Inhibits Progression, Migration, and Invasion in Human Hepatocellular Carcinoma.

Our aim was to study the expression of human SET domain containing protein 1A (hSETD1A) in hepatocellular carcinoma patients and its relationship with human hepatocellular carcinoma cell function. A total of 30 patients with hepatocellular carcinoma were enrolled in this study. The expression of hSETD1A was detected by real-time polymerase chain reaction (PCR) and Western blotting. The immortalized normal human liver cell line including SMMC-7721 was subjected to real-time PCR for hSETD1A mRNA. Furthermore, hSETD1A-small hairpin RNA (shRNA) was used to knock down hSETD1A expression in SMMC-7721 cells. Cell proliferation, cell apoptosis, and cell migration were determined by CCK8, flow cytometry, and Transwell assays. The positive expression rate level of hSETD1A mRNA and protein in liver carcinoma tissues was 73.33%. hSETD1A knockdown using a specific hSETD1A-shRNA inhibited cell proliferation and promoted cell apoptosis in SMMC-7721 cells. It was also found that downregulation of hSETD1A inhibited cell migration ability but did not affect cell invasion. In conclusion, the expression of hSETD1A occurs at a high rate in hepatocellular carcinoma patients. The expression state of hSETD1A may be a prognostic factor in hepatocellular carcinoma.

Caffeic acid attenuates the angiogenic function of hepatocellular carcinoma cells via reduction in JNK-1-mediated HIF-1α stabilization in hypoxia

Our study revealed a novel mechanism in the CaA-attenuated angiogenic ability in HCC cells possiblyviareducing the JNK-1-mediated HIF-1α stabilization and inducing the ubiquitination-mediated HIF1α degradation.

Knockdown of TMEM16A suppressed MAPK and inhibited cell proliferation and migration in hepatocellular carcinoma.

TMEM16A plays an important role in cell proliferation in various cancers. However, less was known about the expression and role of TMEM16A in hepatocellular carcinoma. We screened the expression of TMEM16A in patients’ hepatocellular carcinoma tissues, and also analyzed the biological function of hepatocellular carcinoma cells by knockdown of TMEM16A, as well as the expression of MAPK signaling proteins, including p38, p-p38, ERK1/2, p-ERK1/2, JNK, and p-JNK, and cell cycle regulatory protein cyclin D1 in TMEM16A siRNA-transfected SMMC-7721 cells by Western blot. Our results showed that TMEM16A was overexpressed in hepatocellular carcinoma tissues. Inhibition of TMEM16A suppressed the cell proliferation, migration, and invasion, and cell cycle progression but did not influence the cell apoptosis. TMEM16A siRNA-suppressed cancer cell proliferation and tumor growth were accompanied by a reduction of p38 and ERK1/2 activation and cyclin D1 induction, and were not influenced by other tested MAPK signaling proteins. In addition, inhibition of TMEM16A suppressed tumorigenicity in vivo. TMEM16A is overexpressed in hepatocellular carcinoma, and that inhibition of TMEM16A suppressed MAPK and growth of hepatocellular carcinoma. TMEM16A could be a potentially novel therapeutic target for human cancers, including hepatocellular carcinoma.

Quantitative Proteomics Reveals That the Inhibition of Na(+)/K(+)-ATPase Activity Affects S-Phase Progression Leading to a Chromosome Segregation Disorder by Attenuating the Aurora A Function in Hepatocellular Carcinoma Cells.

Many studies have shown the Na(+)/K(+)-ATPase (NKA) might be a potential target for anticancer therapy. Cardiac glycosides (CGs), as a family of naturally compounds, inhibited the NKA activity. The present study investigates the antitumor effect of ouabain and elucidates the pharmacological mechanisms of CG activity in liver cancer HepG2 cell using SILAC coupled to LC-MS/MS method. Bioinformatics analysis of 330 proteins that were changed in cells under treatment with 0.5 μmol/L ouabain showed that the biological processes are associated with an acute inflammatory response, cell cycle, oxidation reduction, chromosome segregation, and DNA metabolism. We confirmed that ouabain induced chromosome segregation disorder and S-cell cycle block by decreasing the expression of AURKA, SMC2, Cyclin D, and p-CDK1 as well as increasing the expression of p53. We found that the overexpression or inhibition of AURKA significantly reduced or enhanced the ouabain-mediated the anticancer effects. Our findings suggest that AURKA is involved in the anticancer mechanisms of ouabain in HepG2 cells.

Single-Vehicular Delivery of Antagomir and Small Molecules to Inhibit miR-122 Function in Hepatocellular Carcinoma Cells by using "Smart" Mesoporous Silica Nanoparticles.

MicroRNAs (miRNAs) regulate a variety of biological processes. The liver-specific, highly abundant miR-122 is implicated in many human diseases including cancer. Its inhibition has been found to result in a dramatic loss in the ability of Hepatitis C virus (HCV) to infect host cells. Both antisense technology and small molecules have been used to independently inhibit endogenous miR-122 function, but not in combination. Intracellular stability, efficient delivery, hydrophobicity, and controlled release are some of the current challenges associated with these novel therapeutic methods. Reported herein is the first single-vehicular system, based on mesoporous silica nanoparticles (MSNs), for simultaneous cellular delivery of miR-122 antagomir and small molecule inhibitors. The controlled release of both types of inhibitors depends on the expression levels of endogenous miR-122, thus enabling these drug-loaded MSNs to achieve combination inhibition of its targeted mRNAs in Huh7 cells.