Exosome-mediated miR-4660 delivery inhibits OPN promoted hepatoma cells aggression through targeting LGALS3BP.

Integrin is important in migration and metastasis of tumor cells. Changes of integrin expression and distribution will cause an alteration of cellular adhesion and migration behaviors. In this study, we investigated sulfatide regulation of the integrin αV subunit expression in hepatoma cells and observed that either exogenous or endogenous sulfatide elicited a robust upregulation of integrin αV subunit mRNA and protein expression in hepatoma cells. This regulatory effect occurred with a corresponding phosphorylation (T739) of the transcription factor Sp1. Based on the electrophoretic mobility shift assay, sulfatide enhanced the integrin αV promoter activity and strengthened the Sp1 complex super-shift. The results of chromatin immunoprecipitation analysis also indicated that sulfatide enhanced Sp1 binding to the integrin αV promoter in vivo. Silence of Sp1 diminished the stimulation of integrin αV expression by sulfatide. In the early stage of sulfatide stimulation, phosphorylation of Erk as well as c-Src was noted, and inhibition of Erk activation with either U0126 or PD98059 significantly suppressed Sp1 phosphorylation and integrin αV expression. We demonstrated that sulfatide regulated integrin αV expression and cell adhesion, which was associated with Erk activation.

Osteopontin (OPN) is over-expressed in a variety of cancers, but its role in hepatocellular carcinoma (HCC) progression has not been clarified. In this study, weakly tumorigenic, non-metastastic human HCC cell line SMMC-7721 cells were forced to over-express OPN via stable transfection. A series of functional assays were performed to assess the effects of OPN on tumor cell behaviors and cDNA microarray was used to identify the genes regulated by OPN. The results showed that OPN significantly enhanced the migration and invasion of SMMC-7721 cells in vitro. In addition, CD44v6 antibody could significantly inhibit the invasion of OPN over-expressing SMMC-7721 cells. Moreover, MMP-2 and uPA expressions were significantly up-regulated in OPN over-expressing SMMC-7721 cells. Together, these findings indicate that OPN enhanced HCC cells invasion through interaction with its receptor CD44v6 and increased MMP-2 and uPA expressions, providing at least one mechanism for OPN-mediated HCC progression and metastasis.

Objective To explore the effect and mechanism of microRNA (miR)-17-5p on the invasion and metastasis of hepatocellular carcinoma (HCC) cells. Methods Expression of miR-17-5p in the normal human L-02 hepatocyte and QGY-7703 HCC cells was detected by RT-PCR. QGY-7703 HCC cells were transfected by miR-17-5p mimic and mimic control respectively. Influence of miR-17-5p on the invasion and metastasis ability of HCC cells was detected using Transwell assay and scratch test. Target gene of miR-17-5p was confirmed by bioinformatic analysis, and its expression in HCC cells was detected by Western blot. After siRNA silenced by target gene, the invasion and metastasis ability of HCC cells were observed. Comparison of microRNA in the two kinds of cells was conducted by t test. Results Expression level of miR-17-5p in HCC cells was 0.16±0.04, significantly lower than 1.01±0.19 in normal L-02 hepatocytes (t=-9.67, P<0.05). Number of trans-membrane cells and metastasis rate of HCC cells transfected by miR-17-5p mimic were respectively 36±4 and (5.37±0.15) mm/d, significantly lower than 62±7 and (7.50±0.01) mm/d of control group (t=-15.40, -32.00; P<0.05). Bioinformatic analysis showed that AKT3 was the key target gene of miR-17-5p, and the expression of AKT3 in HCC cells was obviously higher than that of normal hepatocyte. Number of trans-membrane cells and metastasis rate of HCC cells transfected by siRNA-AKT3 were respectively 13±3 and (4.13±0.15) mm/d, significantly lower than 58±3 and (7.23±0.25) mm/d of control group (t=-17.88, -53.69; P<0.05). Conclusion miR-17-5p inhibits the invasion and metastasis ability of HCC cells through targeting effect on AKT3. Key words: Carcinoma, hepatocellular; miR-17-5p; AKT3; Neoplasm invasiveness; Cell migration

Prickle-1 Negatively Regulates Wnt/β-Catenin Pathway by Promoting Dishevelled Ubiquitination/Degradation in Liver Cancer

Objective To study the effect of recombinant adenovirus with high expression of osteopontin (OPN) gene on malignant biological behaviors of hepatocellular carcinoma (HCC) cells. Methods HCC (HepG2) cells were cultured and divided into rAd-OPN group and rad NC group. HCC cells in rAd-OPN group were transfected with recombinant adenovirus with high expression of OPN gene, and thsose in NC group were transfected with recombinant adenovirus containing invalid fragment. The expression of OPN, and cell migration, invasion and metastasis associated genes was detected. Results At 12, 24, 36, and 48 h after adenovirus transfection, mRNA expression of OPN in rAd-OPN group was 1.93±0.25, 2.58±0.30, 3.61±0.57 and 3.88±0.63, cell migration rate was (18.20±2.76)%, (43.76±7.17)%, (62.48±9.25)% and (77.33±10.24)%, and transmembrane cell number was (25.24±4.26), (32.13±4.97), (40.27±6.24) and (47.95±7.25) cells respectively, significantly higher than in rAd-NC group (P<0.05). At 48 h after transfection of adenovirus, mRNA expression of matrix metalloproteinase (MMP)-2, MMP-9, urokinase-type plasminogen activator (uPA) in rAd-OPN group was respectively 2.32±0.49, 1.78±0.28, 3.14±0.51, significantly higher than that in rAd NC group (P<0.05). Conclusion Recombinant adenovirus with high expression of OPN gene can promote the migration, invasion and up-regulate the metastasis-related gene expression. Key words: Carcinoma, hepatocellular; Osteopontin; Migration; Invasion

To evaluate the expression pattern of non-SMC condensin II complex subunit D3 (NCAPD3) in hepatocellular carcinoma (HCC) tissues, assess its association with clinical characteristics, and explore the effects of NCAPD3 on HCC cells and the potential underlying mechanisms. NCAPD3 expression in HCC tumors and adjacent noncancerous tissues was quantified via quantitative PCR. Patients were divided into high- and low-expression groups on the basis of NCAPD3 levels, and associations with clinical parameters were assessed. The effects of NCAPD3 knockdown and the phosphatidylinositol-3-kinase (PI3K) agonist Y-P 740 on cell functions were examined via cell proliferation, Transwell migration, and invasion assays. Differentially expressed genes following NCAPD3 knockdown in SMMC-7721 cells were identified via mRNA sequencing. Western blotting was performed to measure NCAPD3, AKT serine/threonine kinase 1 (AKT1), and phosphorylated AKT1 levels. NCAPD3 mRNA expression was notably upregulated in HCC tissues as compared with that in adjacent noncancer tissues. A positive correlation was observed between NCAPD3 expression and both lymphatic and distant metastases in patients with HCC. NCAPD3 knockdown reduced the proliferation and metastasis of SMMC-7721 and Huh-7 cells. mRNA sequencing revealed 140 downregulated genes and 125 upregulated genes. Further validation experiments confirmed that NCAPD3 modulated the PI3K-AKT signalling pathway and that the PI3K agonist Y-P 740 counteracted the effects of NCAPD3 knockdown. Elevated NCAPD3 expression was strongly correlated with HCC metastasis. NCAPD3 inhibition impedes HCC cell growth and metastatic potential by suppressing the PI3K-AKT signalling pathway.

miR-16 is known to be abnormally expressed in hepatocellular carcinoma (HCC) cells, and the overexpression of miR-16 inhibits the proliferation, invasion and metastasis of various cancer cells. MicroRNAs (miRNAs or miRs) are closely related to the proliferation, invasion and metastasis of HCC. The present study aimed to explore the effects of miR-16 on the proliferation, invasion and metastasis of HCC cells, and to elucidate the mechanisms involved. A cell line with moderate levels of miR‑16 expression was selected from the SMMC-7721, HepG2, SK-Hep-1 and Huh‑7 HCC cells and validated by reverse transcription-PCR (RT-PCR). The effects of miR‑16 on HCC cell viability were determined by MTT assay; cell migration and invasion were determined by Transwell cell invasion assay, and apoptosis was determined by flow cytometery (FCM). Western blot analysis was used to measure the expression levels of the apoptosis-related proteins, Bax, Bcl-2, matrix metalloproteinase (MMP)-2, MMP-9, as well as to examine epithelial-mesenchymal transition (EMT), and E-cadherin, vimentin, and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway-related protein expression. The mRNA expression levels of miR‑16 were highest in the SMMC-7721 cells and lowest in the SK-Hep‑1 and Huh‑7 cells; moderate levels were observed in the HepG2 cells. The HepG2 cell line was selected as the cell line for use in the follow-up experiments, where we measured cell viability, and the expression of PI3k/Akt, Bax, Bcl-2, MMP-2 and MMP-9, and E-cadherin and vimentin. miR‑16 overexpression significantly inhibited the proliferation, invasion and metastasis of the HepG2 cells, as shown by western blot analysis. This was achieved through the upregulation of Bax expression, the downregulation of Bcl-2 expression and the decrease in the expression of MMP-2 and MMP-9. In addition the expression of E-cadherin increased and vimentin expression decreased. miR‑16 overexpression inhibited PI3K expression and Akt phosphorylation. The results of this study suggest that the overexpression of miR‑16 inhibits the proliferation, invasion and metastasis of HepG2 HCC cells, and that these effects are associated with the PI3K/Akt signaling pathway.

Recent evidence has indicated that inhalational anesthetics may affect the growth and malignant potential of tumor cells and ultimately influence tumor recurrence after surgery. Sevoflurane, a volatile anesthetic, is used extensively in hepatectomy. However, the effect of sevoflurane on the growth of hepatocellular carcinoma (HCC) cells remains unknown. The aim of this study was to explore the effects of sevoflurane on HCC metastasis and its potential mechanisms in the human HCC cell lines, HepG2 and SMMC7721. HepG2 and SMMC7721 cells were treated with 1.7%, 3.4%, and 5.1 % sevoflurane for 6 h. Cell migration was analyzed using invasion, migration, and scratch assays. Based on previous literature, several microRNAs (miRNAs) were screened to determine regulatory miRNA targets of sevoflurane in HepG2 and SMMC7721 cells; miR-665 was detected as a potential target and overexpressed or inhibited in HepG2 and SMMC7721 cells by a lentiviral system. The p-ERK/MMP pathway was also measured by western blotting. Sevoflurane inhibited the migration and invasion of HCC cells in a dose-dependent manner. It also inhibited miR-665 expression in HCC cells. We further observed that sevoflurane inhibited HCC metastasis via miR-665. Sevoflurane-induced downregulation of miRNA-665 led to phosphorylation of ERK and matrix metalloproteinase (MMP-9) via suppression of SPRED1. These results demonstrated that sevoflurane may inhibit invasion and migration via the p-ERK/MMP-9 signaling pathway in HCC cells.

&lt;div&gt;Abstract&lt;p&gt;Radiotherapy has played a limited role in the treatment of hepatocellular carcinoma (HCC) due to the risk of tumor radioresistance. A previous study in our laboratory confirmed that CD147 interacts with integrin β1 and plays an important role in modulating the malignant properties of HCC cells. In this study, we further evaluated the role of CD147 in the radioresistance of HCC and as a potential target for improving radiosensitivity. Upon irradiation, the colony formation, apoptosis, cell-cycle distribution, migration, and invasion of SMMC-7721, CD147-knockout SMMC-7721, HepG2, and CD147-knockdown HepG2 cells were determined. A nude mouse xenograft model and a metastatic model of HCC were used to detect the role of CD147 in radioresistance &lt;i&gt;in vivo&lt;/i&gt;. Deletion of HAb18G/CD147 significantly enhanced the radiosensitivity of SMMC-7721 and HepG2 cells, and knocking out HAb18G/CD147 in SMMC-7721 cells attenuated irradiation-enhanced migration and invasion. The knockout and antibody blockade of CD147 decreased the tumor growth and metastatic potentials of HCC cells under irradiation. CD147-deleted SMMC-7721 cells showed diminished levels of calpain, cleaved talin, active integrin β1, and decreased p-FAK (Tyr397) and p-Akt (Ser473) levels. FAK and PI3K inhibitors, as well as integrin β1 antibodies, increased the radiation-induced apoptosis of SMMC-7721 cells. Our data provide evidence for CD147 as an important determinant of radioresistance via the regulation of integrin β1 signaling. Inhibition of the HAb18G/CD147 integrin interaction may improve the efficiency of radiosensitivity and provide a potential new approach for HCC therapy. &lt;i&gt;Mol Cancer Ther; 14(2); 553–63. ©2014 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;Radiotherapy has played a limited role in the treatment of hepatocellular carcinoma (HCC) due to the risk of tumor radioresistance. A previous study in our laboratory confirmed that CD147 interacts with integrin β1 and plays an important role in modulating the malignant properties of HCC cells. In this study, we further evaluated the role of CD147 in the radioresistance of HCC and as a potential target for improving radiosensitivity. Upon irradiation, the colony formation, apoptosis, cell-cycle distribution, migration, and invasion of SMMC-7721, CD147-knockout SMMC-7721, HepG2, and CD147-knockdown HepG2 cells were determined. A nude mouse xenograft model and a metastatic model of HCC were used to detect the role of CD147 in radioresistance &lt;i&gt;in vivo&lt;/i&gt;. Deletion of HAb18G/CD147 significantly enhanced the radiosensitivity of SMMC-7721 and HepG2 cells, and knocking out HAb18G/CD147 in SMMC-7721 cells attenuated irradiation-enhanced migration and invasion. The knockout and antibody blockade of CD147 decreased the tumor growth and metastatic potentials of HCC cells under irradiation. CD147-deleted SMMC-7721 cells showed diminished levels of calpain, cleaved talin, active integrin β1, and decreased p-FAK (Tyr397) and p-Akt (Ser473) levels. FAK and PI3K inhibitors, as well as integrin β1 antibodies, increased the radiation-induced apoptosis of SMMC-7721 cells. Our data provide evidence for CD147 as an important determinant of radioresistance via the regulation of integrin β1 signaling. Inhibition of the HAb18G/CD147 integrin interaction may improve the efficiency of radiosensitivity and provide a potential new approach for HCC therapy. &lt;i&gt;Mol Cancer Ther; 14(2); 553–63. ©2014 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. The purpose of this study was to determine the effects of daucosterol on HCC by investigating Wnt/β-catenin signaling. In this study, HepG2 and SMMC-7721 cells were treated with varying concentrations of daucosterol, and the corresponding inhibitory effects on HCC cells were examined via CCK-8 assays. Cell migration and invasion abilities were detected via transwell assays. β-Catenin and phospho (p)-β-catenin levels were analyzed via western blotting. Our results showed that daucosterol reduced the proliferation, migration, and invasion capacities of HCC cells in a concentration-dependent manner. In addition, daucosterol reduced the levels of β-catenin and p-β-catenin in HepG2 and SMMC-7721 cells. Furthermore, the Wnt signaling pathway inhibitor SB-216763 was used to treat HepG2 and SMMC-7721 cells with daucosterol. Our results showed that co-treatment with daucosterol and SB-216763 abolished the effects of daucosterol on cell inhibition ratios, cell migration, and cell invasion. These findings indicated that daucosterol inhibited cell migration and invasion in HCC cells via the Wnt/β-catenin signaling pathway. Therefore, our study highlights the use of daucosterol as a promising therapeutic strategy for HCC treatment.

Objective To explore the role of miR-137 in the proliferation and migration of hepatocellular carcinoma (HCC) cells by regulating Notch1 and mediating autophagy. Methods The human SMMC7721 hepatoma cell line was transfected with miR-137 mimics, miR-137 inhibitor and Notch1 interfe-ring RNA (siRNA), and divided into normal control group (NC group), miR-137 mimics group, miR-137 inhibitor group, Notch1 siRNA group.The expression levels of miR-137 and Notch1 mRNA after the transfection were detected by RT-PCR in SMMC7721 cells. Transwell experiments were performed to analyze the effect of miR-137 and Notch1 on the migration and invasion of SMMC7721 cells. The expression levels of β-catenin and vimentin in SMMC7721 cells were detected by immunohistochemistry. The number of autophagosomes was detected by double labeled adenovirus. Western blot was utilized to detect the expression of Notch1, E-Cadherin, N-Cadherin, vimentin, P62, and LC3. Results The results of RT-PCR showed that the relative expression level of Notch1 in miR-137 inhibitor group (5.71±0.45) was significantly higher than that in miR-137 mimics group (0.21±0.06) with statistical significance (P<0.05). The Transwell experiments showed that there were fewer invasive metastatic hepatoma cells in miR-137 mimics group (66.00±4.55) and Notch1 siRNA group (88.00±6.78) than that in the miR-137 inhibitor group (515.00±35.12) (P<0.05). The expression levels of β-catenin in miR-137 mimics group and Notch1 siRNA group were significantly increased and the expression level of vimentin was decreased (P<0.05). The results of autophagy double labeled adenovirus test showed that the number of autophagosomes in miR-137 mimics group (5.50±3.70) was significantly fewer than that in miR-137 inhibitor group (32.75±4.11), and the difference was statistically significant (P<0.05). The expression levels of Notch1, N-cadherin, vimentin, and LC3 protein in miR-137 mimics group were much lower than that in miR-137 inhibitor group and NC group, and the expression levels of E-Cadherin and P62 protein were greatly increased. The expression level of Notch1, N-cadherin, and LC3 protein in Notch1 siRNA group were significantly lower than that in NC group, and the expression levels of E-cadherin and P62 protein were much higher than that in NC group. Conclusion MiR-137 can inhibit the proliferation, migration and invasion of HCC cells by inhibiting the expression of Notch1 and autophagy, which may become a new target for the treatment of HCC. Key words: Hepatocellular carcinoma; MiR-137; Notch1; Invasion and metastasis; Autophagy

BackgroundCD147 plays a critical role in the invasive and metastatic activity of hepatocellular carcinoma (HCC) cells by stimulating the surrounding fibroblasts to express matrix metalloproteinases (MMPs). Tumor cells adhesion to extracellular matrix (ECM) proteins is the first step to the tumor metastasis. MMPs degrade the ECM to promote tumor metastasis. The aim of this study is to investigate the effects of small interfering RNA (siRNA) against CD147 (si-CD147) on hepatocellular carcinoma cells' (SMMC-7721) architecture and functions.MethodsFlow cytometry and western blot assays were employed to detect the transfection efficiency of si-CD147. Confocal microscopy was used to determine the effects of si-CD147 on SMMC-7721 cells' cytoskeleton. Invasion assay, gelatin zymography and cell adhesion assay were employed to investigate the effects of si-CD147 on SMMC-7721 cells' invasion, gelatinase production and cell adhesive abilities. Western blot assay was utilized to detect the effects of si-CD147 on focal adhesion kinase (FAK), vinculiln and mitogen-activated protein kinase (MAPK) expression in SMMC-7721 cells.ResultsDownregulation of CD147 gene induced the alteration of SMMC-7721 cell cytoskeleton including actin, microtubule and vimentin filaments, and inhibited gelatinase production and expression, cells invasion, FAK and vinculin expression. si-CD147 also blocked SMMC-7721 cells adhesion to collagen IV and phosphorylation level of SAPK/JNKs. SAPK/JNKs inhibitor SP600125 inhibited gelatinase production and expression.ConclusionCD147 is required for normal tumor cell architecture and cell invasion. Downregulation of CD147 affects HCC cell structure and function. Moreover, the alteration of cell behavior may be related to SAPK/JNK Pathway. siRNA against CD147 may be a possible new approach for HCC gene therapy.

Background: Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world and contributes to a high cancer mortality globally. The multistep progression of HCC is highly related to the activation of oncogenes and the deactivation of cancer suppressor genes. The cyclin-dependent kinase 7 ( CDK7 ) gene is responsible for maintaining a normal cell cycle, acting as a gatekeeper, and plays an important part in RNA transcription. Although previous studies demonstrate that CDK7 is highly expressed in multiple cancers and is associated with the progression and prognosis of these cancers, the potential role of CDK7 in HCC still needs to be explored. Methods: Data consisting of CDK7 expression levels, patient phenotypes and their matched survival were acquired from The Cancer Genome Atlas (TCGA) database. The associations of the CDK7 expression level with clinicopathological factors and cell proliferation were analyzed. The HCC transcriptome data in the GEO database were inquired and analyzed using GEO2R. Two representative HCC cell models were built to observe the proliferation capacity of HCC cells when CDK7 expression was inhibited by either sh CDK7 or THZ1. Results: Based on the transcriptome and survival data in the TCGA database, the CDK7 expression level was inversely proportional to the overall survival of the HCC patients (pooled HR =1.51, 95% CI =1.06–2.15). For further investigation, the clinical features of HCC were integrated with the expression level of CDK7 . A high expression of CDK7 was proportional to the survival time of the HCC patients (P=2.38×10 −3 ), the neoplasm histologic grade (P −4 ) and cell proliferation (P −3 ). Moreover, CDK7 expression in the liver tumor was higher than that in non-tumor tissues (t-test =6.04, P=2.27×10 −8 ). For HCC patients, CDK7 expression was higher in the tumor tissues than in the para-carcinoma tissues, based on five GEO datasets (Z score =−6.20, P=5.64×10 −10 ). In the SMMC-7721 and Huh7 cell lines, CDK7 shRNA-transfected cells showed significantly lower capabilities of cell growth and proliferation than those of the shRNA control group after 48 hours of cell culture. Furthermore, the cell proliferation capacity was also inhibited when the SMMC-7721 cells (P=6.47×10 −2 ) or Huh7 cells (P=2.52×10 −2 ) were exposed to THZ1. Conclusions: The CDK7 expression level in HCC is associated with HCC progression, prognosis and the cell proliferation capacity. CDK7 may act as a potential target for HCC, and THZ1 might be a potent medicine for HCC treatment.

Periostin contributes to arsenic trioxide resistance in hepatocellular carcinoma cells under hypoxia.