scholarly journals Inhibiting Effects of Down-regulating of Fascin 1 on Proliferation and Migration in Hepatoma Cells

2021 ◽  
Author(s):  
Wanglu Gu ◽  
Guilan Wang ◽  
Xinyang Zhang ◽  
Li Chen ◽  
Jiaming Zhou
Keyword(s):  
Protein Expression ◽  
Western Blotting ◽  
Hepatoma Cell ◽  
Hepatoma Cells ◽  
Human Hepatoma Cell ◽  
Specific Sequence ◽  
Knock Down ◽  
Huh7 Cells ◽  
And Migration ◽  
Proliferation And Migration

Abstract Objective: To investigate the inhibiting effects of fascin 1 gene knock-down on the proliferation and migration of hepatoma cells by means of small interfering RNA (siRNA).Methods: SiRNA targeting fascin 1 gene (si-fascin) and non-specific sequence siRNA (si-NC）were constructed and transfected into human hepatoma cell lines (HepG2 and Huh7) to down-regulate the expression of fascin 1. RT-qPCR, Western blotting, and Immunofluorescence technique were used to evaluate the efficiency of si-fascin. The proliferation and migration of cells were detected by MTT method and Transwell experiments, and the protein expression of genes related to proliferation and migration in cells were detected by Western blotting. The apoptosis and pseudopodia formation of cells were observed under scanning electron microscope (SEM).Results: Compared with human normal liver cells (LO2), the expressions of fascin 1 mRNA and protein were significantly higher in HepG2 and Huh7 cells. The expression of fascin 1 was overall inhibited in HepG2 and Huh7 cells transfected by the constructed four si-fascins, among which, fascin_siR3 had the highest inhibitory efficiency, therefore was selected in this study. In HepG2 and Huh7 cells transfected by si-fascin significant knock-down target gene expression, while reducing cell proliferation, migration and the formation of pseudopods, and causes reduced protein expression associated with proliferation and migration. Conclusion: This study further confirmed that fascin 1 gene has the function of promoting hepatoma cells proliferation and migration, suggesting that downregulating the expression of fascin 1 in hepatoma cells may be one of the strategies to intervene in liver cancer.

Inhibitory Effects of Lycopene on the Adhesion, Invasion, and Migration of SK-Hep1 Human Hepatoma Cells

2006 ◽  
Vol 231 (3) ◽  
pp. 322-327 ◽  
Author(s):  
Eun-Sun Hwang ◽  
Hyong Joo Lee
Keyword(s):  
Cell Growth ◽  
Hepatoma Cell ◽  
Hepatoma Cells ◽  
Hepatoma Cell Line ◽  
Dependent Manner ◽  
Human Hepatoma ◽  
Human Hepatoma Cells ◽  
Human Hepatoma Cell ◽  
Invasion And Migration ◽  
And Migration

Lycopene, which is the predominant carotenoid in tomatoes and tomato-based foods, may protect humans against various cancers. Effects of lycopene on the adhesion, invasion, migration, and growth of the SK-Hep1 human hepatoma cell line were investigated. Lycopene inhibited cell growth in dose-dependent manners, with growth inhibition rates of 5% and 40% at 0.1 μM and 50 μM lycopene, respectively, after 24 hrs of incubation. Similarly, after 48 hrs of incubation, lycopene at 5 μM and 10 μM decreased the cell numbers by 30% and 40%, respectively. Lycopene decreased the gelatinolytic activities of both matrix metalloproteinase (MMP)-2 and MMP-9, which were secreted from the SK-Hep1 cells. Incubation of SK-Hep1 cells with 110 μM of lycopene for 60 mins significantly inhibited cell adhesion to the Matrigel-coated substrate in a concentration-dependent manner. To study invasion, SK-Hep1 cells were grown either on Matrigel-coated Transwell membranes or in 24-well plates. The cells were treated sequentially for 24 hrs with lycopene before the start of the invasion assays. Cell growth and death were assessed under the same conditions. The invasion of SK-Hep1 cells treated with lycopene was significantly reduced to 28.3% and 61.9% of the control levels at 5 μM and 10 μM lycopene, respectively (P < 0.05). In the migration assay, lycopene-treated cells showed lower levels of migration than untreated cells. These results demonstrate the antimetastatic properties of lycopene in inhibiting the adhesion, invasion, and migration of SK-Hep1 human hepatoma cells.

scholarly journals S100A6 promotes proliferation and migration of HepG2 cells via increased ubiquitin-dependent degradation of p53

Open Medicine ◽  
2020 ◽  
Vol 15 (1) ◽  
pp. 317-326
Author(s):  
Dongqiang Song ◽  
Beili Xu ◽  
Dongmin Shi ◽  
Shuyu Li ◽  
Yu Cai
Keyword(s):  
Protein Expression ◽  
Calcium Binding ◽  
Hepg2 Cells ◽  
Expression Level ◽  
Luciferase Assay ◽  
Transcription Activator ◽  
And Migration ◽  
S100a6 Protein ◽  
Hcc Cells ◽  
Proliferation And Migration

AbstractPurposeS100A6 protein (calcyclin), a small calcium-binding protein of the S100 family, is often upregulated in various types of cancers, including hepatocellular carcinoma (HCC). The aim of this study was to illustrate the molecular mechanism of S100A6 in regulating the proliferation and migration of HCC cells.MethodsThe expressions of S100A6 in human HCC and adjacent non-tumor liver specimens were detected using immunoblotting and quantitative PCR (qPCR). The recombinant glutathione S-transferase (GST)-tagged human S100A6 protein was purified and identified. After treatment with S100A6, the proliferation of HepG2 cells was detected by the MTT and colony formation assay, and the migration of HepG2 cells was investigated by the transwell migration assay; the protein levels of cyclin D1 (CCND1), E-cadherin, and vimentin were also tested by immunoblotting. The effect of S100A6 on p21 and nuclear factor-κB pathway was verified by performing the dual luciferase assay. Then, the expression of p21 and its transcription activator, p53, was examined using immunoblotting and qPCR, the ubiquitination of which was investigated through co-immunoprecipitation.ResultsIt was found that the level of S100A6 was higher in the HCC tissues than in the adjacent non-tumor liver specimens. Exogenous overexpression of S100A6 promoted the proliferation and migration of HepG2 cells. S100A6 was observed to regulate p21 mRNA and protein expression levels and decrease p53 protein expression level, not mRNA level, by promoting the ubiquitination of p53 via the proteasome-dependent degradation pathway.ConclusionOur study indicated that S100A6 overexpression could promote the proliferation and migration of HCC cells by enhancing p53 ubiquitin-dependent proteasome degradation, ultimately regulating the p21 expression level.

scholarly journals miR-636 inhibits EMT, cell proliferation and cell cycle of ovarian cancer by directly targeting transcription factor Gli2 involved in Hedgehog pathway

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Jiong Ma ◽  
Chunxia Zhou ◽  
Xuejun Chen
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Protein Expression ◽  
Signaling Pathway ◽  
Luciferase Reporter ◽  
Cell Proliferation And Migration ◽  
Hh Signaling ◽  
And Migration ◽  
Proliferation And Migration

Abstract Background Hedgehog (Hh) signaling pathway, which is essential for cell proliferation and differentiation, is noted to be aberrantly activated in tumor from increasing studies in recent years. MicroRNAs (miRNAs) as an important non-coding RNA in cells have been proven to possess a regulatory role specific to the Hh signaling pathway. Here, in vitro and in vivo cellular/molecular experiments were adopted to clarify the regulatory mechanism linking miR-636 to the Hh signaling pathway in ovarian cancer (OVC). Methods Protein–protein interaction analysis was performed to identify the hub gene in the Hh pathway. TargetScan database was used to predict the potential upstream regulators for Gli2. qRT-PCR was performed to test the expression of miR-636, while Western blot was conducted to detect the expression of proteins related to the Hh pathway and epithelial-mesenchymal transition (EMT). For cell functional experiments, HO-8910PM OVC cell line was used. MTT assay and wound healing assay were used to measure the effect of miR-636 on cell proliferation and migration. Flow cytometry was carried out to examine the effect of miR-636 on cell cycle, and Western blot was used to identify the change in expression of Hh and EMT-related proteins. Dual-luciferase reporter gene assay was implemented to detect the targeting relationship between miR-636 and Gli2. Xenotransplantation models were established for in vivo examination. Results Gli2 was identified as the hub gene of the Hh pathway and it was validated to be regulated by miR-636 based on the data from TargetScan and GEO databases. In vitro experiments discovered that miR-636 was significantly lowly expressed in OVC cell lines, and overexpressing miR-636 significantly inhibited HO-8910PM cell proliferation, migration and induced cell cycle arrest in G0/G1 phase, while the inhibition of miR-636 caused opposite results. Dual-luciferase reporter gene assay revealed that Gli2 was the target gene of miR-636 in OVC. Besides, overexpressed miR-636 decreased protein expression of Gli2, and affected the expression of proteins related to the Hh signaling pathway and EMT. Rescue experiments verified that overexpression of Gli2 reversed the inhibitory effect of miR-636 on HO-8910PM cell proliferation and migration, and attenuated the blocking effect of miR-636 on cell cycle. The xenotransplantation experiment suggested that miR-636 inhibited cell growth of OVC by decreasing Gli2 expression. Besides, overexpressing Gli2 potentiated the EMT process of OVC cells via decreasing E-cadherin protein expression and increasing Vimentin protein expression, and it reversed the inhibitory effect of miR-636 on OVC cell proliferation in vivo. Conclusion miR-636 mediates the activation of the Hh pathway via binding to Gli2, thus inhibiting EMT, suppressing cell proliferation and migration of OVC. Trial registration: The experimental protocol was established, according to the ethical guidelines of the Helsinki Declaration and was approved by the Human Ethics Committee of The Second Affiliated hospital of Zhejiang University School of Medicine (IR2019001235). Written informed consent was obtained from individual or guardian participants.

Abstract 234: Interleukin 2 Promotes Proliferation and Migration in Vascular Smooth Muscle Cells

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Spencer Barnhill ◽  
Prakash Arumugam ◽  
John Matsuura ◽  
Scott Berceli ◽  
Katie Carroll ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Expression ◽  
Western Blot ◽  
Vascular Smooth Muscle Cells ◽  
Blot Analysis ◽  
Intimal Hyperplasia ◽  
Western Blot Analysis ◽  
Interleukin 2 ◽  
And Migration ◽  
Proliferation And Migration

Interleukin-2 (IL-2) is primarily known as a soluble cytokine that regulates T cell responses. We previously reported, however, that IL-2 is retained in the extracellular matrix by association with perlecan, a heparan sulfate proteoglycan (HSPG). Perlecan is the main HSPG in vascular basement membranes, and previous studies from our laboratory demonstrated that, in human arteries, vascular smooth muscle cells (VSMC) are surrounded by perlecan-bound IL-2. We also noted that IL-2 deficient mice lose SMCs with age, leading to widened esophagi and aortic aneurysms. Given this information, we hypothesized that IL-2 has a direct impact on VSMC, and that VSMC express functional IL-2 receptors (IL-2R). We therefore examined both protein and mRNA expression of each of the three IL-2R subunits (alpha, beta, gamma) on human VSMC grown from arterial explants. These VSMC expressed SMC actin, smooth muscle myosin heavy chain, and when quiescent, smoothelin. Protein expression was assessed by in cell Western and by Western blot analysis. Receptor expression was evaluated under distinct culture conditions, which yielded highly proliferative, intermediate, or quiescent VSMC. Contractile protein expression was low, intermediate, or high, respectively, consistent with the characteristics of proliferating vs quiescent SMCs. Each phenotype expressed all 3 subunits of the IL-2R. IL-2 subunits appeared to follow a cytoskeletal pattern in cells expressing high levels of contractile proteins. Western blot analysis of VSMC lysates revealed expression of all 3 receptors at molecular weights identical to lysates from a T cell line. VSMCs also expressed mRNA for each receptor subunit. Functionally, IL-2 promoted migration (using a Boyden chamber assay) and proliferation in a dose dependent fashion. Because excess proliferation and migration are critical to intimal hyperplasia, we asked whether IL-2 levels change under conditions known to generate intimal hyperplasia. In a rabbit model, IL-2 mRNA increased in venous grafts exposed to high flow for 2h. IL-2 levels, by Western blot, were also increased in human hyperplastic veins. In conclusion, these data show that VSMC have functional IL-2R, and suggest that IL-2 may contribute to the development of intimal hyperplasia.

scholarly journals KLF4-Mediated CDH3 Upregulation Suppresses Human Hepatoma Cell Growth and Migration via GSK-3β Signaling

2019 ◽  
Vol 15 (5) ◽  
pp. 953-961 ◽  
Author(s):  
Li Li ◽  
Shijun Yu ◽  
Qiong Wu ◽  
Ning Dou ◽  
Yandong Li ◽  
...  
Keyword(s):  
Cell Growth ◽  
Hepatoma Cell ◽  
Human Hepatoma ◽  
Human Hepatoma Cell ◽  
Cell Growth And Migration ◽  
And Migration ◽  
Gsk 3Β

scholarly journals YTHDF2 mediates the mRNA degradation of the tumor suppressors to induce AKT phosphorylation in N6-methyladenosine-dependent way in prostate cancer

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Jiangfeng Li ◽  
Haiyun Xie ◽  
Yufan Ying ◽  
Hong Chen ◽  
Huaqing Yan ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Tumor Suppressors ◽  
Mrna Degradation ◽  
Akt Phosphorylation ◽  
Knock Down ◽  
Phosphorylated Akt ◽  
And Migration ◽  
Proliferation And Migration

Abstract Background N6-methyladenosine (m6A) is the most abundant modification in mRNA of humans. Emerging evidence has supported the fact that m6A is comprehensively involved in various diseases especially cancers. As a crucial reader, YTHDF2 usually mediates the degradation of m6A-modified mRNAs in m6A-dependent way. However, the function and mechanisms of m6A especially YTHDF2 in prostate cancer (PCa) still remain elusive. Methods To investigate the functions and mechanisms of YTHDF2 in PCa, in vitro, in vivo biofunctional assays and epigenetics experiments were performed. Endogenous expression silencing of YTHDF2 and METTL3 was established with lentivirus-based shRNA technique. Colony formation, flow cytometry and trans-well assays were performed for cell function identifications. Subcutaneous xenografts and metastatic mice models were combined with in vivo imaging system to investigate the phenotypes when knocking down YTHDF2 and METTL3. m6A RNA immunoprecipitation (MeRIP) sequencing, mRNA sequencing, RIP-RT-qPCR and bioinformatics analysis were mainly used to screen and validate the direct common targets of YTHDF2 and METTL3. In addition, TCGA database was also used to analyze the expression pattern of YTHDF2, METTL3 and the common target LHPP in PCa, and their correlation with clinical prognosis. Results The upregulated YTHDF2 and METTL3 in PCa predicted a worse overall survival rate. Knocking down YTHDF2 or METTL3 markedly inhibited the proliferation and migration of PCa in vivo and in vitro. LHPP and NKX3–1 were identified as the direct targets of both YTHDF2 and METTL3. YTHDF2 directly bound to the m6A modification sites of LHPP and NKX3–1 to mediate the mRNA degradation. Knock-down of YTHDF2 or METTL3 significantly induced the expression of LHPP and NKX3–1 at both mRNA and protein level with inhibited phosphorylated AKT. Overexpression of LHPP and NKX3–1 presented the consistent phenotypes and AKT phosphorylation inhibition with knock-down of YTHDF2 or METTL3. Phosphorylated AKT was consequently confirmed as the downstream of METTL3/YTHDF2/LHPP/NKX3–1 to induce tumor proliferation and migration. Conclusion We propose a novel regulatory mechanism in which YTHDF2 mediates the mRNA degradation of the tumor suppressors LHPP and NKX3–1 in m6A-dependent way to regulate AKT phosphorylation-induced tumor progression in prostate cancer. We hope our findings may provide new concepts of PCa biology.

scholarly journals Cell Clones Selected from the Huh7 Human Hepatoma Cell Line Support Efficient Replication of a Subgenomic GB Virus B Replicon

2002 ◽  
Vol 76 (15) ◽  
pp. 7736-7746 ◽  
Author(s):  
Amedeo De Tomassi ◽  
Maura Pizzuti ◽  
Rita Graziani ◽  
Andrea Sbardellati ◽  
Sergio Altamura ◽  
...  
Keyword(s):  
Host Cell ◽  
Cell Line ◽  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
Human Hepatoma ◽  
Human Hepatoma Cell ◽  
Subgenomic Rna ◽  
Human Hepatoma Cell Line ◽  
Huh7 Cells

ABSTRACT Tamarins (Saguinus species) infected by GB virus B (GBV-B) have recently been proposed as an acceptable surrogate model for hepatitis C virus (HCV) infection. The availability of infectious genomic molecular clones of both viruses will permit chimeric constructs to be tested for viability in animals. Studies in cells with parental and chimeric constructs would also be very useful for both basic research and drug discovery. For this purpose, a convenient host cell type supporting replication of in vitro-transcribed GBV-B RNA should be identified. We constructed a GBV-B subgenomic selectable replicon based on the sequence of a genomic molecular clone proved to sustain infection in tamarins. The corresponding in vitro-transcribed RNA was used to transfect the Huh7 human hepatoma cell line, and intracellular replication of transfected RNA was shown to occur, even though in a small percentage of transfected cells, giving rise to antibiotic-resistant clones. Sequence analysis of GBV-B RNA from some of those clones showed no adaptive mutations with respect to the input sequence, whereas the host cells sustained higher GBV-B RNA replication than the original Huh7 cells. The enhancement of replication depending on host cell was shown to be a feature common to the majority of clones selected. The replication of GBV-B subgenomic RNA was susceptible to inhibition by known inhibitors of HCV to a level similar to that of HCV subgenomic RNA.

scholarly journals FLT3 Inhibitor Quizartinib Facilitates Proliferation and CXCL12-Induced Chemotaxis in Quizartinib Resistant FLT3/ITD + Hematopoietic Cells By Increasing RUNX1

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3324-3324
Author(s):  
Seiji Fukuda ◽  
Nozomi Matsuda
Keyword(s):  
Protein Expression ◽  
Acquired Resistance ◽  
Cxcr4 Expression ◽  
Expression Level ◽  
Expression Levels ◽  
Flt3 Inhibitor ◽  
Flt3 Inhibitors ◽  
And Migration ◽  
Runx1 Expression ◽  
Proliferation And Migration

Abstract RUNX1 generally functions as a tumor suppressor in the hematopoietic system. However, RUNX1 expression is significantly elevated in human AML cells with FLT3/ITD mutations, promotes leukemogenesis induced by FLT3/ITD (Behrens et al. JEM 2017) and enhances the resistance of FLT3/ITD + cells to type-II FLT3 inhibitor quizartinib (Hirade et al IJH 2016). We previously reported that RUNX1 expression is higher in CXCR4-low FLT3/ITD + cells compared to Cxcr4-high FLT3/ITD + cells, even though Cxcr4 expression is trans-activated by RUNX1. This difference in RUNX1 expression level was associated with divergent response to CXCL12 in FLT3/ITD + cells harboring different CXCR4 expression levels that were exposed to quizartinib (Fukuda S. et al. ASH 2019). Our data also demonstrated that RUNX1 expression is down-regulated following withdrawal of quizartinib in FLT3/ITD + cells that became refractory to quizartinib (Hirade et al. IJH 2016), suggesting that RUNX1 expression may be up-regulated by quizartinib in FLT3/ITD + cells. Since RUNX1 regulates proliferation of FLT3/ITD + AML cells, the present study investigated association between RUNX1 expression levels and proliferation of quizartinib resistant FLT3/ITD + cells that are exposed to quizartinib. In the sensitive FLT3/ITD + Ba/F3 cells, RUNX1 protein expression was transiently up-regulated but eventually down-regulated by 5 nM quizartinib, coincident with decline in the viable cells. In contrast, RUNX1 expression was up-regulated by quizartinib and remained elevated in the resistant FLT3/ITD + Ba/F3 cells. Since RUNX1 enhances proliferation of FLT3/ITD + cells, we next examined whether proliferation FLT3/ITD + cells that acquired resistance to quizartinib is facilitated by quizaritinib as a result from quizartinib-mediated up-regulation of RUNX1, using the Cxcr4-low and Cxcr4-high FLT3/ITD + cells that acquired resistance to quizartinib. Although CXCL12 barely enhanced the proliferation of refractory FLT3/ITD + Ba/F3 cells, 5 nM quizartinib significantly increased the proliferation of both Cxcr4-low and Cxcr4-high FLT3/ITD + Ba/F3 cells that acquired resistance to quizartinib compared to those without quizartinib. This increase in the proliferation of Cxcr4-low and Cxcr4-high FLT3/ITD + Ba/F3 cells coincided with the elevation in RUNX1 and CXCR4 protein expression. Moreover, the resistant Cxcr4-low FLT3/ITD + Ba/F3 cells proliferated significantly faster than Cxcr4-high FLT3/ITD + cells, with concomitant higher expression of RUNX1 in Cxcr4-low FLT3/ITD + cells than in Cxcr4-high FLT3/ITD + cells. Likewise, type-I FLT3 inhibitor gilteritinib significantly enhanced proliferation of Cxcr4-low and Cxcr4-high FLT3/ITD + Ba/F3 cells that acquired resistance to gilteritinib. Knocking down Runx1 using shRNAs significantly decreased the enhanced proliferation induced by quizartinib in refractory FLT3/ITD + Ba/F3 cells, coincident with reduction in CXCR4 expression. Since CXCR4 expression level was elevated by quizartinib in the FLT3/ITD + cells refractory to quizartinib, we next examined CXCL12-induced migration in quizartinib-resistant FLT3/ITD + cells following exposure to quzartinib. Pre-incubating the quizartinib resistant Cxcr4-low or Cxcr4-high FLT3/ITD + Ba/F3 cells with 5 nM quizartinib for 72 hours significantly enhanced their migration to 100 ng/ml of Cxcl12 compared to those without quizartinib, coincident with elevation in RUNX1 levels. Surprisingly, migration of CXCR4-low FLT3/ITD + cells to CXCL12 was significantly elevated compared to CXCR4-high cells, with concomitant higher expression of RUNX1 in Cxcr4-low FLT3/ITD + cells than in Cxcr4-high FLT3/ITD + cells. Silencing Runx1 using shRNAs significantly decreased migration to CXCL12 in refractory Cxcr4-low FLT3/ITD + Ba/F3 cells. These data indicate that the FLT3 inhibitor itself can facilitate the proliferation and migration to CXCL12 in FLT3/ITD + cells that are refractory to FLT3 inhibitors by up-regulating RUNX1. The results implicate that FLT3 inhibitors may worsen the disease progression in the patients that became refractory to FLT3 inhibitors by facilitating proliferation and migration to CXCL12 of the resistant FLT3/ITD + AML cells. In this regard, targeting RUNX1 may represent additional strategy to eradicate resistant FLT3/ITD + AML cells, in which their proliferation and migration are supported by FLT3 inhibitors. Disclosures No relevant conflicts of interest to declare.

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