scholarly journals Integrin α7 knockdown suppresses cell proliferation, migration, invasion and endothelium-mesenchymal transformation in hepatocellular carcinoma

2019 ◽  
Author(s):  
Zhiyong Wu ◽  
Xiaoyu Kong ◽  
Zhihui Wang
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Proliferation ◽  
Western Blot ◽  
Cell Counting ◽  
Control Group ◽  
Epithelial Cell Line ◽  
Huh7 Cells ◽  
Protein Expressions ◽  
Mesenchymal Transformation ◽  
E Cadherin

Abstract Background The aim was to investigate whether integrin α7 (ITGA7) influenced hepatocellular carcinoma (HCC) progression, and explore its effect on regulating endothelium-mesenchymal transformation (EMT).Methods ITGA7 mRNA and protein expressions in human normal liver epithelial cell line and HCC cell lines were determined by reverse transcription polymerase chain reaction (RT-qPCR) and western blot. ITGA7 siRNA (ITGA7-KD group) and nonsense siRNA (control group) were transfected into Huh7 cells and SUN449 cells. After transfection, ITGA7 mRNA and protein expressions (RT-qPCR and western blot), cell proliferation (Cell Counting Kit-8), apoptosis (Annexin V/Propidium Iodide assay), migration (Wound scratch assay) and invasion (Transwell assay) were determined. E-cadherin and α-SMA expressions (RT-qPCR and western blot) were determined.Results ITGA7 mRNA and protein expressions were increased in Li7, Huh7, SKHEP1 and SNU449 cells compared to THLE-3 cells. In both Huh7 and SNU449 cells, ITGA7 mRNA and protein expressions were decreased in ITGA7-KD group than control group after plasmids transfection, indicating the successful transfection. Then, cell proliferation was decreased at 48h and 72h; cell apoptosis rate was increased at 48h; cell migration rate was reduced at 24h; cell invasive count was decreased at 24h in ITGA7-KD group compared to control group. Furthermore, increased E-cadherin but decreased α-SMA mRNA and protein expressions were discovered in ITGA7-KD group than control group at 24h.Conclusions ITGA7 knockdown suppresses HCC progression and inhibits EMT process in HCC, indicating that ITGA7 might be a potential novel treatment target for HCC therapy.

miR-369-3p serves as prognostic factor and regulates cancer progression of hepatocellular carcinoma

2021 ◽  
Author(s):  
Can Chen ◽  
Yi Zong ◽  
Jiaojiao Tang ◽  
Ruisheng Ke ◽  
Lizhi Lv ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Proliferation ◽  
Cancer Progression ◽  
Cell Counting ◽  
Migration And Invasion ◽  
Pcr Analysis ◽  
Reverse Transcription Pcr ◽  
Huh7 Cells

Background: The aim of this study was to investigate the role of miR-369-3p in hepatocellular carcinoma (HCC). Materials & methods: The expression levels of miR-369-3p were detected using the quantitative real-time reverse transcription-PCR analysis. The cell counting kit-8 and transwell assays were used to explore the effects of miR-369-3p on cell proliferation, migration and invasion of HCC cells. Results: The miR-369-3p expression was downregulated in HCC tissues and cell lines, in comparison to the normal controls, respectively. In vitro, overexpression of miR-369-3p in Hep 3B and Huh7 cells inhibited cell proliferation, migration and invasion. SOX4 was a direct target of miR-369-3p. Conclusion: Our results suggested that miR-369-3p may be a tumor suppressor in HCC by targeting SOX4.

RNF185-AS1 Promotes Hepatocellular Carcinoma Progression Through Targeting miR-221-5p/integrin β5 Axis

2020 ◽  
Author(s):  
Chunmei Huang ◽  
Ke Li ◽  
Rongfu Huang ◽  
Jianhua Zhu ◽  
Jiayao Yang
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Proliferation ◽  
Western Blot ◽  
Rna Binding ◽  
Epithelial Mesenchymal Transition ◽  
Cell Counting ◽  
Mesenchymal Transition ◽  
Qrt Pcr ◽  
And Migration

Abstract Background: Recently, long noncoding RNAs (lncRNAs) have been reported to play important role in pathogenesis of various cancers. However, the function of RNF185-AS1 in hepatocellular carcinoma (HCC) metastasis has not been well investigated. The present study aims to explore the role and mechanism of RNF185-AS1 in hepatocellular carcinoma metastasis. Methods: The RNF185-AS1 expression in HCC cells and tissues was measured by quantitative real‐time polymerase chain reaction (qRT-PCR). The functional effects of RNF185-AS1 on tumor cell proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) were assessed by Cell Counting Kit-8 (CCK8) assay, colony formation assay, transwell assay and Western blot. The luciferase reporters assay, RNA-binding protein immunoprecipitation assay, qRT-PCR and Western blot were performed to explore and confirm the interaction between RNF185-AS1 and miR-221-5p and integrin β5. The role of RNF185-AS1 in tumor progression was explored through in vivo experiments.Results: RNF185-AS1 was highly expressed in HCC tissues and cell lines. High levels of RNF185-AS1 was correlated with advanced TNM stage, distant metastasis and a poorer overall survival rate. RNF185-AS1 knockdown inhibited cell proliferation, migration, invasion and EMT. Additionally, RNF185-AS1 acted as a sponge for miR-221-5p and integrin β5 was identified as a target gene of miR-221-5p. Rescue assays showed that miR-221-5p inhibitor or integrin β5 overexpression rescued the function of RNF185-AS1 knockdown on cell proliferation, migration, invasion and EMT. Moreover, we found that RNF185-AS1 knockdown inhibited tumor metastases in xenograft tumor mouse model. Conclusion: Our findings demonstrated that RNF185-AS1 promoted cell EMT and migration by regulating miR-221-5p/integrin β5 axis in HCC.

scholarly journals Knockdown of long noncoding RNA HOTAIR inhibits osteoarthritis chondrocyte injury by miR-107/CXCL12 axis

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Jipeng Lu ◽  
Zhongxiong Wu ◽  
Ying Xiong
Keyword(s):  
Cell Proliferation ◽  
Western Blot ◽  
Cell Apoptosis ◽  
Noncoding Rna ◽  
Underlying Mechanism ◽  
Joint Disease ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Cxcl12 Expression ◽  
Ecm Degradation

Abstract Background Osteoarthritis (OA) is a joint disease characterized via destruction of cartilage. Chondrocyte damage is associated with cartilage destruction during OA. Long noncoding RNAs (lncRNAs) are implicated in the regulation of chondrocyte damage in OA progression. This study aims to investigate the role and underlying mechanism of lncRNA homeobox antisense intergenic RNA (HOTAIR) in OA chondrocyte injury. Methods Twenty-three OA patients and healthy controls without OA were recruited. Chondrocytes were isolated from OA cartilage tissues. HOTAIR, microRNA-107 (miR-107) and C-X-C motif chemokine ligand 12 (CXCL12) levels were measured by quantitative real-time polymerase chain reaction and western blot. Cell proliferation, apoptosis and extracellular matrix (ECM) degradation were measured using cell counting kit-8, flow cytometry and western blot. The target interaction was explored by bioinformatics, luciferase reporter and RNA immunoprecipitation assays. Results HOTAIR expression was enhanced, and miR-107 level was reduced in OA cartilage samples. HOTAIR overexpression inhibited cell proliferation, but induced cell apoptosis and ECM degradation in chondrocytes. HOTAIR knockdown caused an opposite effect. MiR-107 was sponged and inhibited via HOTAIR, and knockdown of miR-107 mitigated the effect of HOTAIR silence on chondrocyte injury. CXCL12 was targeted by miR-107. CXCL12 overexpression attenuated the roles of miR-107 overexpression or HOTAIR knockdown in the proliferation, apoptosis and ECM degradation. CXCL12 expression was decreased by HOTAIR silence, and restored by knockdown of miR-107. Conclusion HOTAIR knockdown promoted chondrocyte proliferation, but inhibited cell apoptosis and ECM degradation in OA chondrocytes by regulating the miR-107/CXCL12 axis.

miR-3156-5p Enhances Hypoxia-Induced Angiogenesis in Hepatocellular Carcinoma via Modulating the SOCS5/HIF-1α/VEGF Pathway

2021 ◽  
Author(s):  
Bin Tie ◽  
Zheng Guo ◽  
Li Li ◽  
Wenhui Wang ◽  
Rong Liu ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Proliferation ◽  
Endothelial Cells ◽  
Colony Formation ◽  
Cell Counting ◽  
Angiogenic Potential ◽  
Reverse Transcription Pcr ◽  
Vegf Pathway ◽  
Formation Experiment ◽  
Hcc Cells

Abstract Background: MicroRNAs (miRNAs) are dysregulated in hypoxia-induced hepatocellular carcinoma (HCC). This study probed the regulatory mechanism of miR-3156-5p on HCC under hypoxia. Methods: HCC cells (HepG2) were exposed to normoxia or hypoxia, and the conditioned medium (CM) of HepG2 was applied. Quantitative reverse transcription PCR (qRT-PCR) was implemented to analyze the miR-3156-5p profile. The cell counting kit-8 (CCK-8) assay and the colony formation experiment were conducted to measure cell proliferation, colony formation, and angiogenesis. Results: The results manifested that miR-3156-5p was up-regulated in HCC cells and endothelial cells under hypoxia, and up-regulating miR-3156-5p boosted HCC cell proliferation, endothelial cell angiogenesis, and HIF-1α/VEGF expression. Conclusions: miR-3156-5p activates the HIF-1α/VEGF pathway by hampering SOCS5, thereby enhancing the angiogenic potential of hypoxia-induced endothelial cells in HCC cells.

scholarly journals Ferulic Acid Attenuates TGF-β1-Induced Renal Cellular Fibrosis in NRK-52E Cells by Inhibiting Smad/ILK/Snail Pathway

2015 ◽  
Vol 2015 ◽  
pp. 1-7 ◽  
Author(s):  
Ming-gang Wei ◽  
Wei Sun ◽  
Wei-ming He ◽  
Li Ni ◽  
Yan-yu Yang
Keyword(s):  
Ferulic Acid ◽  
Western Blotting ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Underlying Mechanism ◽  
Tubular Epithelial Cell ◽  
Epithelial Cell Line ◽  
Proximal Tubular ◽  
Mesenchymal Transformation ◽  
E Cadherin

Renal fibrosis is a common cause of renal dysfunction with chronic kidney disease. Central to this process is epithelial-mesenchymal transformation (EMT) of proximal tubular epithelial cells driven by transforming growth factor-β1 (TGF-β1) signaling. The present study aimed to investigate the effect of Ferulic acid (FA) on EMT of renal proximal tubular epithelial cell line (NRK-52E) induced by TGF-β1 and to elucidate its underlying mechanism against EMT related to TGF-β1/Smads pathway. The NRK-52E cells were treated for 48 h with TGF-β1 (5 ng/mL) in different concentrations of FA (0 to 200 µM). Fibronectin, a mesenchymal marker, was assessed by western blotting. Western blotting was also used to examine the EMT markers (E-cadherin, andα-smooth muscle actin (α-SMA)), signal transducer (p-Smad2/3), and EMT initiator (Snail). ILK was also assayed by western blotting. The results showed that TGF-β1 induced spindle-like morphological transition in NRK-52E cells. Smad2/3 signaling pathway activation, increased fibronectin,α-SMA, ILK, and Snail expression, and decreased E-cadherin expression in TGF-β1-treated NRK-52E cells. FA efficiently blocked P-Smad2/3 activation and attenuated all these EMT changes induced by TGF-β1. These findings suggest that FA may serve as a potential fibrosis antagonist for renal proximal tubule cells by inhibiting EMT process.

scholarly journals LncRNA HOTAIR Promotes Proliferation of Malignant Mela-noma Cells Through NF-κB Pathway

2020 ◽  
Author(s):  
Jun WANG ◽  
Jingxin CHEN ◽  
Gang JING ◽  
Daoquan DONG
Keyword(s):  
Malignant Melanoma ◽  
Western Blotting ◽  
Melanoma Cells ◽  
Cell Counting ◽  
Control Group ◽  
Down Regulation ◽  
Proliferation And Apoptosis ◽  
Protein Expressions ◽  
And Control ◽  
Lncrna Hotair

Background: To study the effects of long non-coding ribonucleic acid (lncRNA) HOX transcript antisense intergenic RNA (HOTAIR) on the proliferation and apoptosis of malignant melanoma cells, and to explore its specific regulatory mechanism through the nuclear factor-κB (NF-κB) signaling pathway. Methods: LncRNA HOTAIR small-interfering RNAs (siRNAs) were designed and synthesized, and the effects of si-HOTAIR transfection on the proliferation and apoptosis of malignant melanoma cells were detected via cell counting kit-8 (CCK-8) assay, 4',6-diamidino-2-phenylindole (DAPI) staining assay and flow cytometry, respectively. The gene expressions were determined using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), the changes in NF-κB pathway-related proteins and apoptosis-associated proteins after interference in lncRNA HOTAIR were detected via Western blotting, and the level of NF-κB in each group was determined via ELISA. Results: The results of CCK-8 assay revealed that the cell proliferation rate significantly declined gradually in si-HOTAIR group compared with that in si-NC group and control group (P<0.05). The results of Western blotting and ELISA showed that the activity of NF-κB in si-HOTAIR group was weakened (P<0.05), suggesting that down-regulation of HOTAIR can suppress the activity of NF-κB. Compared with si-NC group and control group, si-HOTAIR group had remarkably increased gene and protein expressions of pro-apoptotic Bax, and remarkably decreased gene and protein expressions of anti-apoptotic Bcl-2 (P<0.05), demonstrating that down-regulation of HOTAIR can promote apoptosis. Conclusion: Down-regulation of lncRNA HOTAIR can inhibit the proliferation and promote the apoptosis of malignant melanoma cells and suppress the NF-κB pathway.

Regulator of Ribosome Synthesis 1 Influences the Proliferation, Migration, and Invasion of Ovarian Cancer Cells

2021 ◽  
Vol 11 (3) ◽  
pp. 407-411
Author(s):  
Shenhua Zhang ◽  
Ting Yu
Keyword(s):  
Western Blot ◽  
Ovarian Carcinoma ◽  
Carcinoma Cells ◽  
Migration And Invasion ◽  
Ovarian Cancer Cells ◽  
Control Group ◽  
Epithelial Cell Line ◽  
Ribosome Synthesis ◽  
Protein Levels ◽  
Ovarian Carcinoma Cells

We investigated the effects of silencing the regulator of ribosome synthesis 1 (RRS1) gene on the proliferation, migration, and invasion of ovarian carcinoma cells, and its possible role in modulating signal transduction in these cells. Normal ovarian epithelial cell line IOSE80 was used as a control. We examined the mRNA and protein level of RRS1 using qRT-PCR and western blot in control and ovarian carcinoma cells (SKOV-3, SW626, and CAOV3). RNA interference technology was used to knockdown RRS1 expression in CAOV3 cells. MTT was used to examine the proliferation of these cells, while a Transwell assay was used to assay the cells’ migration and invasion abilities. Western blot was used to measure the levels of CyclinD1, P21, MMP-2, MMP-9, p-JAK2 and p-STAT3 proteins. In comparison with normal ovarian epithelial cells (IOSE80), RRS1 mRNA and protein levels were increased in ovarian carcinoma cells (SKOV-3, SW626 and CAOV3) (P < 0.05). Because RRS1 levels were highest in CAOV3 cells, these cells were used for subsequent experiments. RRS1 gene expression was knocked down in CAOV3 cells, and in comparison with the negative control group, siRNA-RRS1 cells exhibited decreased proliferation in the MTT assay after 48 h and 72 h (P < 0.05). These cells also exhibited reduced migration and invasion (P < 0.05). Further, siRNA-RRS1 cells exhibited reduced expression of CyclinD1, MMP-2, MMP-9, P-JAK2 and P-STAT3 proteins (P < 0.05), while P21 protein levels were increased (P < 0.05). Silencing RRS1 expression inhibits the proliferation, migration, and invasion of ovarian carcinoma cells. This effect may be mediated by the inhibition of the STAT3 signaling pathway in these cells.

scholarly journals The effects of c-Src kinase on EMT signaling pathway in human lens epithelial cells associated with lens diseases

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Xingyu Li ◽  
Fang Wang ◽  
Meixia Ren ◽  
Minjuan Du ◽  
Jian Zhou
Keyword(s):  
Cell Proliferation ◽  
Epithelial Cells ◽  
Western Blot ◽  
Src Kinase ◽  
Human Lens ◽  
Lens Epithelial Cells ◽  
Protein Levels ◽  
Human Lens Epithelial Cells ◽  
And Migration ◽  
E Cadherin

Abstract Background The signaling pathway of epithelial to mesenchymal transition (EMT) is regulated by c-Src kinase in many cells. The purpose of this study was to investigate the effects of c-Src kinase on EMT of human lens epithelial cells in vivo stimulated by different factors. Methods Human lens epithelial cells, HLE-B3, were exposed to either an inflammatory factor, specifically IL-1α, IL-6, TNF-α or IL-1β, at 10 ng/mL or high glucose (35.5 mM) for 30 mins. Activity of c-Src kinase was evaluated by the expression of p-Src418 with western blot assay. To investigate the effects of activation of c-Src on EMT, HLE-B3 cells were transfected with pCDNA3.1-SrcY530F to upregulate activity of c-Src kinase, and pSlience4.1-ShSrc to knock it down. The expressions of c-Src kinase and molecular markers of EMT such as E-cadherin, ZO-1, α-SMA, and Vimentin were examined at 48 h by RT-PCR and western blot. At 48 h and 72 h of transfection, cell proliferation was detected by MTT, and cell mobility and migration were determined by scratch and transwell assays. Results Activity of c-Src kinase, which causes the expression of p-Src418, was upregulated by different inflammatory factors and high glucose in HLE-B3 cells. When HLE-B3 cells were transfected with pCDNA3.1-SrcY530F, the expression of c-Src kinase was upregulated on both mRNA and protein levels, and activity of c-Src kinase, expression of p-Src418 increased. The expressions of both E-cadherin and ZO-1 were suppressed, while the expressions of vimentin and α-SMA were elevated on both mRNA and protein levels at the same time. Cell proliferation, mobility and migration increased along with activation of c-Src kinase. Conversely, when HLE-B3 cells were transfected with pSlience4.1-ShSrc, both c-Src kinase and p-Src418 expressions were knocked down. The expressions of E-cadherin and ZO-1 increased, but the expressions of Vimentin and α-SMA decreased; meanwhile, cell proliferation, mobility and migration reduced. Conclusions The c-Src kinase in lens epithelial cells is easily activated by external stimuli, resulting in the induction of cell proliferation, mobility, migration and EMT.

scholarly journals Effect of metformin on the improvement of prostate cancer in diabetic rats

2019 ◽  
Vol 17 ◽  
pp. 205873921985855
Author(s):  
Kaijian Hou ◽  
Wansheng Ke ◽  
Jianping Xiong
Keyword(s):  
Prostate Cancer ◽  
Western Blot ◽  
Intervention Group ◽  
Diabetic Rats ◽  
Pathological Examination ◽  
Control Group ◽  
Nuclear Staining ◽  
Sprague Dawley ◽  
Western Blot Assay ◽  
E Cadherin

This study was designed to investigate the effect of metformin on the improvement of prostate cancer in diabetic rats. A total of 20 Sprague Dawley (SD) rats were equally divided into control and intervention groups. The intervention group received intragastric metformin 200 mg/kg, while the control group was given intragastric drinking water for 4 weeks. Tumor volumes were compared, all tumor specimens underwent routine pathological examination, immunohistochemical detection of E-cadherin and N-cadherin, and western blot assay. The tumor volume of control and intervention group was 462.15 ± 45.67 and 23.46 ± 5.32 mm3, respectively. Hematoxylin and eosin (HE) staining showed partial visible glandular structure with deepened nuclear staining in the intervention group. Immunohistochemistry showed high expression (6.5 ± 0.28 vs 3.8 ± 0.26, P < 0.05) of E-cadherin and low expression (3.4 ± 0.12 vs 7.8 ± 0.34, P < 0.05) of N-cadherin in the intervention group. Western blot assay showed higher expression of E-cadherin, while low N-cadherin in the intervention group. Metformin can effectively alleviate lesion extent of prostate cancer and mechanism may be related to upregulation of E-cadherin and downregulation of N-cadherin expression.

scholarly journals Alpha-Fetoprotein Regulates the Expression of Immune-Related Proteins through the NF-κB (P65) Pathway in Hepatocellular Carcinoma Cells

2020 ◽  
Vol 2020 ◽  
pp. 1-9
Author(s):  
Qiu-ting Li ◽  
Meng-jun Qiu ◽  
Sheng-li Yang ◽  
Xiefan Fang ◽  
Xiao-xiao He ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Western Blot ◽  
Protein A ◽  
Radical Resection ◽  
Alpha Fetoprotein ◽  
Control Group ◽  
Invasion And Migration ◽  
Immune Genes ◽  
And Migration ◽  
Hcc Cells

Background. The prognosis of patients with hepatocellular carcinoma (HCC) is poor, with 60% to 70% of patients developing recurrence and metastasis within five years of radical resection. Alpha-fetoprotein (AFP) plays a significant role in predicting the recurrence and metastasis of HCC after surgery. However, its role in modulating tumor immunity has not been investigated. Our objective was to examine the effect of AFP on the expression of B7 family and activation of the NF-κB (P65) pathway in HCC. Methods. We generated human hepatoma SMMC-7721 cell lines with or without recombinant AFP transfection (AFPup and control groups). Colony formation assay, Transwell invasion assay, and wound healing assay were used to detect the function of AFP. Liver cancer xenografts were made in BALB/c nude male mice (N = 6 per group). After 28 days of inoculation, the expression of immune genes in the HCC tissues, including PD-L (B7-H1), B7-H3, B7-H4, and P65, was evaluated by quantitative real-time PCR (qPCR) and western blot. In addition, immunofluorescence was used to determine the subcellular localization of the P65 protein, a key factor in the NF-κB pathway. An online HCC patients’ dataset was also used to detect the connection between AFP and P65. Results. Overexpression of AFP could enhance proliferation, invasion, and migration of HCC cells. Both qPCR and western blot results demonstrated that the expressions of PD-L1, B7-H4, and P65 were significantly higher in the AFP group compared to the controls (P<0.05). Immunofluorescence results indicated that the majority of the P65 protein was located in the cytoplasm in the control group but was translocated to the nucleus in the AFPup group. The Spearman correlation coefficient confirms that AFP has a positive correlation with P65 in HCC patients (R = 0.33, P=0.05). Conclusion. AFP could enhance proliferation, invasion, and migration in HCC cells. The upregulation of AFP would increase the PD-L1 and B7-H4 mRNA and protein expression in HCC tissues through the upregulation and activation of the P65 protein.

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