BTG2 Is Down-Regulated and Inhibits Cancer Stem Cell-Like Features of Side Population Cells in Hepatocellular Carcinoma

Digestive Diseases and Sciences - Our previous study found that B cell translocation gene 2 (BTG2) was hyper-methylated and down-regulated in side population (SP) cells of hepatocellular carcinoma...     

MiR-3910 Promotes the Growth and Migration of Cancer Cells in the Progression of Hepatocellular Carcinoma

Digestive Diseases and Sciences - Previous studies have reported that specific depletion of mammalian sterile-like kinase (MST1) in the mouse liver driven Hepatocellular carcinoma (HCC). However,...     

Analysis of the Genome-Wide DNA Methylation Profile of Side Population Cells in Hepatocellular Carcinoma

Background

DNA methylation plays an important role in maintaining pluripotency and regulating the differentiation of stem cells, but the DNA methylation profile of stem cells in hepatocellular carcinoma (HCC) remains unclear.

Aims

To investigate the genome-wide DNA methylation profile of side population (SP) cells of HCC, a special subpopulation of cells enriched with cancer stem cells, by DNA methylation microarray analysis and to analyze the functions and signal pathways of the aberrantly methylated genes in SP cells.

Methods

Side population cells were isolated from HCC cell lines Huh7 and PLC/PRF/5 using flow cytometry, and the tumorigenicity of these SP cells was assessed in NOD/SCID mice. The genome-wide DNA methylation status of SP cells and non-SP (NSP) cells was detected and compared by DNA methylation microarray analysis. Genes with differential methylation between SP and NSP cells were further analyzed for their functions and roles in related signaling pathways.

Results

Subcutaneous inoculation of 1 × 10³ SP cells yielded tumors in 60 % NOD/SCID mice, whereas no tumor was developed after the inoculation of 1 × 10⁶ NSP cells. Genome-wide DNA methylation microarray analysis showed that 72 and 181 genes were hypermethylated and hypomethylated, respectively, in both Huh7 and PLC/PRF/5 SP cells as compared with their corresponding NSP cells. Analyses of signaling pathways revealed that hypermethylated and hypomethylated genes were related to four and eight pathways, respectively.

Conclusions

Hepatocellular carcinoma SP cells possessed a differential DNA methylation status compared with NSP cells, and the differentially methylated genes in SP cells were involved in 12 signaling pathways. Our results provide valuable clues for further investigations in elucidating the importance of epigenetic regulation in sustaining HCC SP cells and tumorigenesis.     

Dermcidin Enhances the Migration,Invasion, and Metastasis of Hepatocellular Carcinoma Cells In Vitro and In Vivo

Background and AimsHepatocellular carcinoma (HCC) is a common primary liver neoplasm with high mortality. Dermcidin (DCD), an antimicrobial peptide, has been reported to participate in oncogenesis. This study assessed the effects and underlying molecular events of DCD overexpression and knockdown on the regulation of HCC progression in vitro and in vivo.MethodsThe serum DCD level was detected using enzyme-linked immunosorbent assay. DCD overexpression, knockdown, and Ras-related C3 botulinum toxin substrate 1 (Rac1) rescue were performed in SK-HEP-1 cells using plasmids. Immunofluorescence staining, quantitative PCR, and Western blotting were used to detect the expression of different genes and proteins. Differences in HCC cell migration and invasion were detected by Transwell migration and invasion assays. A nude mouse HCC cell orthotopic model was employed to verify the in vitro data.ResultsThe level of serum DCD was higher in patients with HCC and in SK-HEP-1 cells. DCD overexpression caused upregulation of DCD, fibronectin, Rac1, and cell division control protein 42 homologue (Cdc42) mRNA and proteins as well as actin-related protein 2/3 (Arp2/3) protein (but reduced Arp2/3 mRNA levels) and activated Rac1 and Cdc42. Phenotypically, DCD overexpression induced HCC cell migration and invasion in vitro, whereas knockout of DCD expression had the opposite effects. A Rac1 rescue experiment in DCD-knockdown HCC cells increased HCC cell migration and invasion and increased the levels of active Rac1/total Rac1, Wiskott-Aldrich syndrome family protein (WASP), Arp2/3, and fibronectin. DCD overexpression induced HCC cell metastasis to the abdomen and liver in vivo.ConclusionsDCD promotes HCC cell migration, invasion, and metastasis through upregulation of noncatalytic region of tyrosine kinase adaptor protein 1 (Nck1), Rac1, Cdc42, WASP, and Arp2/3, which induce actin cytoskeletal remodeling and fibronectin-mediated cell adhesion in HCC cells.     

The Down-Regulation of Notch1 Inhibits the Invasion and Migration of Hepatocellular Carcinoma Cells by Inactivating the Cyclooxygenase-2/Snail/E-cadherin Pathway In Vitro

Background

The Notch signaling pathway plays an important role in cancer, but the mechanism by which Notch1 participates in invasion and migration of hepatocellular carcinoma (HCC) cells is unclear.

Aims

Our purpose is to confirm the anti-invasion and anti-migration effects of the down-regulation of Notch1 in HCC cells.

Methods

The invasion and migration capacities of HCC cells were detected with Transwell cell culture chambers. The expressions of Notch1, Notch1 intracellular domain (N1ICD), E-cadherin, Snail, and cyclooxygenase-2 (COX-2) were analyzed by RT-PCR and/or western blotting. Notch1 and Snail were down-regulated by RNA interference, and COX-2 was inhibited by NS-398. Cell apoptosis was analyzed by MTT and flow cytometry.

Results

In HCC cells, Snail, Notch1, and COX-2 were up-regulated, and E-cadherin was down-regulated in mRNA and/or protein levels. The down-regulation of Snail or Notch1 or the inhibition of COX-2, respectively, can increase the mRNA and protein expressions of E-cadherin and decrease the invasion and migration capabilities of HCC cell. Down-regulated Notch1 or inhibited COX-2 can reduce the mRNA and protein expressions of Snail. The down-regulation of Notch1 can also reduce the protein expression of COX-2. However, exogenous PGE2 can reverse the role of down-regulated Notch1. The results of MTT and flow cytometry showed that down-regulated Notch1 did not affect HCC cell viability.

Conclusions

Down-regulated Notch1 may be an effective approach to inactivating Snail/E-cadherin by regulating COX-2, which results in inhibiting the invasion and migration of HCC cells. The inhibitory effects of down-regulated Notch1 on cell invasion and migration were independent of apoptosis.     

Lipopolysaccharide-Induced Toll-Like Receptor 4 Signaling in Cancer Cells Promotes Cell Survival and Proliferation in Hepatocellular Carcinoma

Background

Recent studies have shown that toll-like receptor 4 (TLR4) is involved in hepatocarcinogenesis. However, the significance of TLR4 signaling in cancer development and progression remains unclear.

Aim

The purpose of this study was to investigate the role of TLR4 in cancer cell survival and proliferation in hepatocellular carcinoma (HCC).

Methods

Fifty-three HCC and ten normal liver specimens were analyzed by immunohistochemistry, and three cell lines (HL-7702, PLC/PRF/5 and HepG2) were used for in vitro studies. Lipopolysaccharide (LPS), a specific ligand of TLR4, was used to activate TLR4 signaling. The effects of LPS-TLR4 signaling on cell survival, proliferation and invasion were examined. Specific inhibitors of NF-κB and MAPK (JNK, ERK and p38) signaling pathways were used to explore the role of each pathway in LPS-TLR4 signaling.

Results

TLR4 was overexpressed in HCC cell lines and in human HCC tissues, where it correlated with Ki-67 expression. LPS-induced activation of TLR4 signaling promoted cancer cell survival and proliferation. LPS-TLR4 signaling was associated with regulation on the activation of NF-κB and MAPK signaling pathways. LPS-TLR4-induced activation of ERK and JNK signaling promotes cell proliferation through regulating Bax translocation to mitochondria. Activation of NF-κB and p38 mediates cytotoxicity of LPS, and inhibition on these two pathways promotes cell proliferation in HCC cells.

Conclusion

Our results indicate that TLR4 signaling in cancer cells promotes cell survival and proliferation in HCC.     

Long Noncoding RNA GAPLINC Promotes Cells Migration and Invasion in Colorectal Cancer Cell by Regulating miR-34a/c-MET Signal Pathway

Background

Gastric adenocarcinoma predictive long intergenic noncoding RNA (GAPLINC) has been detected in colorectal cancer (CRC) cells and reportedly performs many functions related to tumor proliferation and metastasis. Aim The present study aimed to comprehensively explore the biological functions of GAPLINC and their underlying mechanism in CRC cell.

Methods

The human cancer LncRNA PCR array was used to detect the differentially expressed long noncoding RNAs in human CRC samples. Real-time PCR, dual-luciferase assay, RNA pull-down assay, Transwell assay, and western blot analysis were performed to explore the molecular mechanism underlying GAPLINC functions related to migration and invasion of a human CRC cell line (HCT116).

Results

Compared to the non-cancerous tissues, GAPLINC expression was obviously increased in CRC tissues. In HCT116, silencing of GAPLINC weakened cell migration and invasion, while overexpression of GAPLINC significantly promoted cell migration and invasion. Through dual-luciferase, RNA pull-down, and Transwell assays, we verified that miR-34a was the downstream molecule of GAPLINC and that miR-34a negatively regulated the migration and invasion of HCT116 cell. Furthermore, we found that GAPLINC positively regulated the miR-34a target gene c-MET in CRC tissues.

Conclusions

Our findings revealed that GAPLINC was up-regulated in CRC tissues and was involved in the migration and invasion of CRC cells by regulating miR-34a/c-MET signaling pathway.

     

Inactivation of MYO5B Promotes Invasion and Motility in Gastric Cancer Cells

Background  

Loss of cell polarity and tissue disorganisation are hallmarks of cancer. MYO5B mutations disrupt epithelial cell polarity, suggesting that MYO5B may be involved in tumorigenesis.     

Syndecan-1对结肠癌细胞增殖、迁移和侵袭的影响

背景:Syndecan-1是表达于上皮细胞表面的一种硫酸乙酰肝素蛋白聚糖,在多种肿瘤细胞中的表达下降,与肿瘤的多种生物学行为密切相关,但其在细胞水平上对结直肠癌发展的作用目前尚无深入的研究。目的:评估Syndecan-1对人结肠癌细胞株增殖、迁移和侵袭能力的影响,并初步探讨可能的分子机制。方法:分别将Syndecan-1高表达质粒和Syndecan-1 siRNA转染人结肠癌细胞株SW620和SW480,分别以转染空载质粒或无关序列作为阴性对照。采用CCK-8法检测细胞增殖,Transwell细胞迁移实验和Matrigel细胞侵袭实验分别检测细胞迁移、侵袭能力,蛋白质印迹法检测上皮细胞间质转化主要分子标记物E-cadherin的蛋白表达。结果:与阴性对照组相比,转染Syndecan-1高表达质粒的SW620细胞生长速度、迁移细胞数和侵袭细胞数均明显降低,同时E-cadherin表达升高;转染Syndecan-1 siRNA的SW480细胞生长速度、迁移细胞数和侵袭细胞数均明显升高,同时E-cadherin表达降低。结论:Syndecan-1能抑制结肠癌细胞的增殖、迁移和侵袭能力,其机制可能与上皮细胞间质转化有关。     

百里醌抑制人胰腺癌BxPC-3细胞体外运动和侵袭的研究

背景:胰腺癌是恶性程度最高的消化道肿瘤,目前吉西他滨依赖的化疗对抑制胰腺癌转移的治疗效果欠佳。研究发现百里醌对多种肿瘤细胞具有抑制增殖、促进凋亡的作用。目的:探讨百里醌对人胰腺癌BxP C-3细胞体外运动和侵袭的影响及其作用机制。方法:常规培养人胰腺癌细胞株BxP C-3,加入不同浓度百里醌进行处理。采用Boyden小室法检测细胞体外运动、侵袭情况;蛋白质印迹法检测细胞FAK、Akt蛋白表达和Akt磷酸化水平的改变;免疫荧光技术检测细胞内FAK表达、细胞黏着斑和F-actin的变化。结果:10、25μmol/L百里醌对BxP C-3细胞体外运动的抑制率分别为43.4%、73.8%,对体外侵袭的抑制率分别为60.5%、75.6%,百里醌呈浓度依赖性地抑制胰腺癌BxP C-3细胞的体外运动、侵袭(P0.05)。百里醌能明显下调BxP C-3细胞FAK表达,并抑制细胞磷酸化Akt的激活。百里醌可诱导FAK弥散分布于胞质,明显抑制黏着斑形成和F-actin的聚合集化。结论:百里醌通过抑制FAK/PI3K/Akt通路的信号转导和激酶活性,浓度依赖性地抑制人胰腺癌BxP C-3细胞的体外运动和侵袭。     

Differentiation-Inducing Activity of Hydroxycamptothecin on Cancer Stem-Like Cells Derived from Hepatocellular Carcinoma

Background  

Hydroxycamptothecin (HCPT) is an anti-tumor agent that can induce differentiation in human cancer cells. Recent evidence indicates that side population (SP) cells possess characteristics of stem-like cells, and may be capable of initiating tumor growth.     

Stem Cell Origins and Animal Models of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a common malignant tumor that almost always occurs within a preexisting background of chronic liver disease and cirrhosis. Currently, medical therapy is not effective in treating most HCC, and the only hope of cure is either resection or liver transplantation. A small minority of patients is eligible for these therapies, which entail major morbidity at the very least. In spite of immense scientific advances during the past 3 decades, patient survival has improved very little. In order to reduce morbidity and mortality from HCC, improvements in early diagnosis and development of novel local and systemic therapies for advanced disease are essential, in addition to efforts geared towards primary prevention. Studies with experimental animal models that closely mimic human disease are very valuable in understanding physiological, cellular and molecular mechanisms underlying the disease. Furthermore, appropriate animal models have the potential to increase our understanding of the effects of image-guided minimally invasive therapies and thereby help to improve such therapies. In this review, we examine the evidence for stem cell origins of such tumors, critically evaluate existing models and reflect on how to develop new models for minimally invasive, image-guided treatment of HCC.     

Anti-miR-362-3p Inhibits Migration and Invasion of Human Gastric Cancer Cells by Its Target CD82

Aim

This study was to investigate the effects and mechanisms of miR-362-3p on regulation of gastric cancer (GC) cell metastasis potential.

Methods

We detected miR-362-3p level in GC and adjacent normal tissues and investigated the relationship with clinicopathological factors. Next, we analyzed the level of miR-362-3p expression and CD82 in different differentiated GC cells compared with a normal gastric mucosa cell by RT-PCR and Western blot. Dual-luciferase reporter assay and Western blot confirmed a direct interaction between miR-362-3p and CD82 3′UTR. After miR-362-3p and CD82 were silenced in GC cells, we compared the transfected GC cells migration and invasion capacity by transwell assay. In addition, we detected the effects on cells angiogenesis by tube formation assay. Western blot was used to detect the impact of CD82 and miR-362-3p on epithelial-to-mesenchymal transition markers in treated GC cells.

Results

Level of miR-362-3p expression was much higher in GC cells than in normal gastric mucosa cell, and miR-362-3p expression negatively correlated with CD82 mRNA expression in these cell lines. Furthermore, miR-362-3p expression inhibited GC cell metastasis capacity by suppression of CD82 expression. Level of miR-362-3p may mediate E-cadherin, N-cadherin, and vimentin expression in GC cells.

Conclusion

This study illuminated that downregulation of miR-362-3p along with the upregulation of CD82 in GC cells resulted in the inhibition of GC migration and invasion. Thus, our results suggested that miR-362-3p or CD82 can be exploited as a new potential target for control of GC in the future.