N-myc Downstream Regulated Gene 1 (NDRG1) Promotes Metastasis of Human Scirrhous Gastric Cancer Cells through Epithelial Mesenchymal Transition

Our recent study demonstrated that higher expression of N-myc downregulated gene 1 (NDRG1) is closely correlated with poor prognosis in gastric cancer patients. In this study, we asked whether NDRG1 has pivotal roles in malignant progression including metastasis of gastric cancer cells. By gene expression microarray analysis expression of NDRG1 showed the higher increase among a total of 3691 up-regulated genes in a highly metastatic gastric cancer cell line (58As1) than their parental low metastatic counterpart (HSC-58). The highly metastatic cell lines showed decreased expression of E-cadherin, together with enhanced expression of vimentin and Snail. This decreased expression of E-cadherin was restored by Snail knockdown in highly metastatic cell lines. We next established stable NDRG1 knockdown cell lines (As1/Sic50 and As1/Sic54) from the highly metastatic cell line, and both of these cell lines showed enhanced expression of E-cadherin and decreased expression of vimentin and Snail. And also, E-cadherin promoter-driven luciferase activity was found to be increased by NDRG1 knockdown in the highly metastatic cell line. NDRG1 knockdown in gastric cancer cell showed suppressed invasion of cancer cells into surround tissues, suppressed metastasis to the peritoneum and decreased ascites accumulation in mice with significantly improved survival rates. This is the first study to demonstrate that NDRG1 plays its pivotal role in the malignant progression of gastric cancer through epithelial mesenchymal transition.     

转录因子WSTF对肺癌细胞增殖和侵袭的实验研究

目的:研究转录因子WSTF对肺癌细胞增殖和侵袭作用的影响。方法:采用慢病毒介导的基因转染方法建立A549细胞WSTF高表达细胞系A549-WSTF和空质粒对照细胞系A549-control。细胞增殖实验和克隆形成实验观察ING5过表达对肺癌A549细胞增殖能力的影响;Trans-well迁移实验和侵袭实验观察WSTF对肺癌细胞迁移和侵袭能力的影响。结果:Western blot验证A549-WSTF细胞WSTF蛋白水平显著高于对照细胞A549-control,P=0.0004。WSTF高表达明显促进了肺癌细胞的增殖能力(1-4天P值分别为0.002、0.0004、0.0002和3.21×10-5)和克隆形成能力(P=0.004);WSTF过表达还显著促进了肺癌细胞从trans-well小室迁移到下室的作用,其OD570值分别为0.626±0.013(A549-WSTF)和0.322±0.010(A549-control),P=2.37×10-5;WSTF还促进肺癌细胞穿透基质胶迁移到下室,其OD570值分别为0.600±0.027(A549-WSTF)和0.333±0.017(A549-control),P=0.0004。结论:WSTF可以促进肺癌细胞的增殖和侵袭能力而发挥促癌作用。     

Post-transcriptional Gene Regulation by MicroRNA-194 Promotes Neuroendocrine Transdifferentiation in Prostate Cancer

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The Metastasis Suppressor,N-myc Downstream-regulated Gene 1 (NDRG1), Inhibits Stress-induced Autophagy in Cancer Cells

N-myc downstream regulated gene 1 (NDRG1) is a potent metastasis suppressor with an undefined role in the stress response. Autophagy is a pro-survival pathway and can be regulated via the protein kinase-like endoplasmic reticulum kinase (PERK)/eIF2α-mediated endoplasmic reticulum (ER) stress pathway. Hence, we investigated the role of NDRG1 in stress-induced autophagy as a mechanism of inhibiting metastasis via the induction of apoptosis. As thiosemicarbazone chelators induce stress and up-regulate NDRG1 to inhibit metastasis, we studied their effects on the ER stress response and autophagy. This was important to assess, as little is understood regarding the role of the stress induced by iron depletion and its role in autophagy. We observed that the chelator, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), which forms redox-active iron and copper complexes, effectively induced ER stress as shown by activation of the PERK/eIF2α pathway. Dp44mT also increased the expression of the autophagic marker, LC3-II, and this was dependent on activation of the PERK/eIF2α axis, as silencing PERK prevented LC3-II accumulation. The effect of Dp44mT on LC3-II expression was at least partially due to iron-depletion, as this effect was also demonstrated with the classical iron chelator, desferrioxamine (DFO), and was not observed for the DFO-iron complex. NDRG1 overexpression also inhibited basal autophagic initiation and the ER stress-mediated autophagic pathway via suppression of the PERK/eIF2α axis. Moreover, NDRG1-mediated suppression of the pro-survival autophagic pathway probably plays a role in its anti-metastatic effects by inducing apoptosis. In fact, multiple pro-apoptotic markers were increased, whereas anti-apoptotic Bcl-2 was decreased upon NDRG1 overexpression. This study demonstrates the role of NDRG1 as an autophagic inhibitor that is important for understanding its mechanism of action.     

Ras-PI3K信号负性调节的H3K56乙酰化促进乳腺癌细胞MCF-7的增殖和迁移能力

Ras蛋白常见的第12、13氨基酸残基突变引起的Ras信号通路异常与人类恶性肿瘤发生相关。然而,Ras致瘤信号通路是否涉及表观遗传学因素尚不明了。本研究旨在阐明人乳腺癌MCF-7细胞中组蛋白H3第56位赖氨酸残基乙酰化修饰（H3K56ac）水平是否受Ras信号通路调控,以及H3K56ac水平对MCF-7细胞增殖和迁移能力的影响。点突变结合基因转染揭示,与野生型比较,第12位氨基酸突变的Ras质粒（pEGFP-H-RasG12V）转染导致MCF-7细胞内H3K56ac水平明显降低。采用可特异激活Ras下游3条通路（Ras-Raf、Ras-RalGEF和Ras-PI3K）的3种质粒（pEGFP-H-RasG12V T35S,pEGFP-H-RasG12V E37G和pEGFP-H-RasG12V Y40C）转染证明,只有转染pEGFP-H-RasG12V Y40C的MCF-7细胞内不仅有Ras-PI3K-AKT通路被激活,且与H3K56ac水平下调相伴;而其他两条通路的激活不影响H3K56ac水平。MTT法结合Transwell、软琼脂克隆形成能力实验证明,RasG12V Y40C转染增强细胞增殖、迁移和克隆形成能力。上述结果表明,MCF-7细胞中H3K56ac水平受Ras-PI3K通路的负性调控,但不受Raf和RalGEF通路影响。Ras-PI3K激活导致的H3K56ac水平降低可增强乳腺癌MCF-7细胞的增殖和迁移能力。总之,这些结果提示,组蛋白H3K56ac是Ras-PI3K致瘤信号通路中的重要成员。Ras信号通路与组蛋白修饰相结合研究将会加深对乳腺癌细胞增殖和迁移调控机制的认识。     

Down-Regulating Overexpressed Human Lon in Cervical Cancer Suppresses Cell Proliferation and Bioenergetics

The human mitochondrial ATP-dependent Lon protease functions in regulating the metabolism and quality control of proteins and mitochondrial DNA (mtDNA). However, the role of Lon in cancer is not well understood. Therefore, this study was undertaken to investigate the importance of Lon in cervical cancer cells from patients and in established cell lines. Microarray analysis from 30 cancer and 10 normal cervical tissues were analyzed by immunohistochemistry for Lon protein levels. The expression of Lon was also examined by immunoblotting 16 fresh cervical cancer tissues and their respective non-tumor cervical tissues. In all cases, Lon expression was significantly elevated in cervical carcinomas as compared to normal tissues. Augmented Lon expression in tissue microarrays did not vary between age, tumor-node-metastasis grades, or lymph node metastasis. Knocking down Lon in HeLa cervical cancer cells by lentivrial transduction resulted in a substantial decrease in both mRNA and protein levels. Such down-regulation of Lon expression significantly blocked HeLa cell proliferation. In addition, knocking down Lon resulted in decreased cellular bioenergetics as determined by measuring aerobic respiration and glycolysis using the Seahorse XF24 extracellular flux analyzer. Together, these data demonstrate that Lon plays a potential role in the oncogenesis of cervical cancer, and may be a useful biomarker and target in the treatment of cervical cancer. Lon; immunohistochemistry; cervical cancer; cell proliferation; cellular bioenergetics.     

LncRNA AC009686.2促进乳腺癌细胞增殖和侵袭

长非编码RNAs(long non-coding RNAs,LncRNAs)作为一类基因表达的调控因子,在多种肿瘤的发生发展中发挥关键作用,然而LncRNAs在乳腺癌中的作用及相关机制尚未完全阐明.为了寻找在乳腺癌发生发展中起关键作用的LncRNAs,本研究通过分析TCGA数据库发现,LncRNA AC009686.2...     

A Gly98Val Mutation in the N-Myc Downstream Regulated Gene 1 (NDRG1) in Alaskan Malamutes with Polyneuropathy

The first cases of early-onset progressive polyneuropathy appeared in the Alaskan Malamute population in Norway in the late 1970s. Affected dogs were of both sexes and were ambulatory paraparetic, progressing to non-ambulatory tetraparesis. On neurologic examination, affected dogs displayed predominantly laryngeal paresis, decreased postural reactions, decreased spinal reflexes and muscle atrophy. The disease was considered eradicated through breeding programmes but recently new cases have occurred in the Nordic countries and the USA. The N-myc downstream-regulated gene (NDRG1) is implicated in neuropathies with comparable symptoms or clinical signs both in humans and in Greyhound dogs. This gene was therefore considered a candidate gene for the polyneuropathy in Alaskan Malamutes. The coding sequence of the NDRG1 gene derived from one healthy and one affected Alaskan Malamute revealed a non-synonymous G>T mutation in exon 4 in the affected dog that causes a Gly98Val amino acid substitution. This substitution was categorized to be “probably damaging” to the protein function by PolyPhen2 (score: 1.000). Subsequently, 102 Alaskan Malamutes from the Nordic countries and the USA known to be either affected (n = 22), obligate carriers (n = 7) or healthy (n = 73) were genotyped for the SNP using TaqMan. All affected dogs had the T/T genotype, the obligate carriers had the G/T genotype and the healthy dogs had the G/G genotype except for 13 who had the G/T genotype. A protein alignment showed that residue 98 is conserved in mammals and also that the entire NDRG1 protein is highly conserved (94.7%) in mammals. We conclude that the G>T substitution is most likely the mutation that causes polyneuropathy in Alaskan Malamutes. Our characterization of a novel candidate causative mutation for polyneuropathy offers a new canine model that can provide further insight into pathobiology and therapy of human polyneuropathy. Furthermore, selection against this mutation can now be used to eliminate the disease in Alaskan Malamutes.     

PFTK1 Promotes Gastric Cancer Progression by Regulating Proliferation,Migration and Invasion

PFTK1, also known as PFTAIRE1, CDK14, is a novel member of Cdc2-related serine/threonine protein kinases. Recent studies show that PFTK1 is highly expressed in several malignant tumors such as hepatocellular carcinoma, esophageal cancer, breast cancer, and involved in regulation of cell cycle, tumors proliferation, migration, and invasion that further influence the prognosis of tumors. However, the expression and physiological significance of PFTK1 in gastric cancer remain unclear. In this study, we analyzed the expression and clinical significance of PFTK1 by Western blot in 8 paired fresh gastric cancer tissues, nontumorous gastric mucosal tissues and immunohistochemistry on 161 paraffinembedded slices. High PFTK1 expression was correlated with the tumor grade, lymph node invasion as well as Ki-67. Through Cell Counting Kit (CCK)-8 assay, flow cytometry, colony formation, wound healing and transwell assays, the vitro studies demonstrated that PFTK1 overexpression promoted proliferation, migration and invasion of gastric cancer cells, while PFTK1 knockdown led to the opposite results. Our findings for the first time supported that PFTK1 might play an important role in the regulation of gastric cancer proliferation, migration and would provide a novel promising therapeutic strategy against human gastric cancer.     

Decreased TUSC3 Promotes Pancreatic Cancer Proliferation,Invasion and Metastasis

Pancreatic cancer is an aggressive disease with dismal prognosis. It is of paramount importance to understand the underlying etiological mechanisms and identify novel, consistent, and easy-to-apply prognostic factors for precision therapy. TUSC3 (tumor suppressor candidate 3) was identified as a potential tumor suppressor gene and previous study showed TUSC3 is decreased in pancreatic cancer at mRNA level, but its putative tumor suppressor function remains to be verified. In this study, TUSC3 expression was found to be suppressed both at mRNA and protein levels in cell line models as well as in clinical samples; decreased TUSC3 expression was associated with higher pathological TNM staging and poorer outcome. In three pairs of cell lines with different NF-κB activity, TUSC3 expression was found to be reversely correlated with NF-κB activity. TUSC3-silenced pancreatic cancer cell line exhibited enhanced potential of proliferation, migration and invasion. In an orthotopic implanted mice model, TUSC3 silenced cells exhibited more aggressive phenotype with more liver metastasis. In conclusion, the current study shows that decreased immunological TUSC3 staining is a factor prognostic of poor survival in pancreatic cancer patients and decreased TUSC3 promotes pancreatic cancer cell proliferation, invasion and metastasis. The reverse correlation between NF-κB activity and TUSC3 expression may suggest a novel regulation pattern for this molecule.     

PC-1在前列腺癌细胞系LNCaP中的定位

目的:研究PC-1分子在前列腺癌细胞系LNCaP中的亚细胞定位。方法:利用常规PCR和重叠PCR技术在pEGFP-C1-PC-1上分别扩增PC-1的不同截短体及缺失体基因,PCR产物经酶切后克隆到真核表达载体pEGFP-C1中;经测序确定构建成功的载体在LNCaP细胞中瞬时高表达,在荧光显微镜下观察这些载体表达产物在LNCaP细胞中的定位情况,并通过Western印迹验证这些载体在LNCaP细胞中的表达。结果:构建了多个用于PC-1亚细胞定位研究的、能在LNCaP细胞中表达的载体;同时,还找到了一段对PC-1定位有重要影响的氨基酸序列。结论:为进一步研究PC-1的亚细胞定位及其发挥功能的方式提供了基础。     

Stanniocalcin1 (STC1) Inhibits Cell Proliferation and Invasion of Cervical Cancer Cells

STC1 is a glycoprotein hormone involved in calcium/phosphate (Pi) homeostasis. There is mounting evidence that STC1 is tightly associated with the development of cancer. But the function of STC1 in cancer is not fully understood. Here, we found that STC1 is down-regulated in Clinical tissues of cervical cancer compared to the adjacent normal cervical tissues (15 cases). Subsequently, the expression of STC1 was knocked down by RNA interference in cervical cancer CaSki cells and the low expression promoted cell growth, migration and invasion. We also found that STC1 overexpression inhibited cell proliferation and invasion of cervical cancer cells. Moreover, STC1 overexpression sensitized CaSki cells to drugs. Further, we showed that NF-κB p65 protein directly bound to STC1 promoter and activated the expression of STC1 in cervical cancer cells. Thus, these results provided evidence that STC1 inhibited cell proliferation and invasion through NF-κB p65 activation in cervical cancer.     

Diversin Is Overexpressed in Breast Cancer and Accelerates Cell Proliferation and Invasion

Diversin was recently reported to play roles in Wnt and JNK pathways. However, the expression pattern and biological roles of diversin in human breast cancer have not been reported. In the present study, we found that diversin was overexpressed in breast cancer specimens by immunohistochemistry and western blot. Significant association was observed between diversin overexpression and TNM stage (p = 0.0036), nodal metastasis (p = 0.0033), negative estrogen receptor expression (p = 0.0012) and triple-negative status (p = 0.0017). Furthermore, colony formation assay and matrigel invasion assay showed that knockdown of diversin expression in MDA-MB-231 cell line with high endogenous expression decreased cell proliferation and cell invasion. Transfection of diversin plasmid in MCF-7 cell line increased cell proliferation and invasion. Further analysis showed that diversin depletion downregulated JNK phosphorylation while its overexpression upregulated JNK phosphorylation. In conclusion, our study demonstrated that diversin was overexpressed in human breast cancers. Diversin could contribute to breast cancer cell proliferation and invasion.     

B4GALT3促进肝癌细胞增殖和侵袭

β-1,4-半乳糖基转移酶III（β-1,4-galactosyltransferase III,B4GALT3）在肿瘤的作用正受到关注,但其在肝癌中的表达模式及其作用有待阐明。基于TCGA肿瘤组织数据库和GTEx正常组织数据库进行的生物信息学分析,发现相比于人正常肝组织,B4GALT3在人肝癌组织中的表达显著上调。实时荧光定量PCR结果发现肝癌细胞中B4GALT3的mRNA和Western 印迹检测蛋白质表达水平显著上调。其中肝癌细胞SMMC7721中B4GALT3的mRNA表达水平是正常肝细胞L-02的9.85倍。对TCGA数据库进行分析发现,B4GALT3表达水平与肝癌患者的生存率呈负相关。在内源性高表达B4GALT3的SMMC7721肝癌细胞中,干扰B4GALT3表达,可显著抑制该细胞的增殖能力和侵袭能力。干扰B4GALT3表达能显著上调SMMC7721细胞中p27和E-cadherin的蛋白质表达水平,干扰B4GALT3表达后SMMC7721细胞中,p27和E-cadherin的mRNA水平较对照组上调6.15倍和7.83倍。总之,B4GALT3在肝癌中表达上调,且促进肝癌细胞的增殖和侵袭。     

Overexpression of N-Myc Downstream-Regulated Gene 2 (NDRG2) Regulates the Proliferation and Invasion of Bladder Cancer Cells In Vitro and In Vivo

N-Myc downstream-regulated gene 2 (NDRG2) is a candidate tumor suppressor gene, which plays an important role in controlling tumor growth. The aim of this study was to investigate the expression of NDRG2 gene in bladder cancer (BC) tissues and several bladder cancer cell lines, and to seek its clinical and pathological significance. Ninety-seven bladder carcinoma and 15 normal bladder tissue sections were analyzed retrospectively with immunohistochemistry. The human bladder cancer cell line T24 was infected with LEN-NDRG2 or LEN-LacZ. The effects of NDRG2 overexpression on T24 cells and T24 nude mouse xenografts were measured via cell growth curves, tumor growth curves, flow cytometric analysis, western blot and Transwell assay. NDRG2 was highly expressed in normal bladder tissue, but absent or rarely expressed in cacinomatous tissues (χ²=8.761, p < 0.01). The NDRG2 level was negatively correlated with tumor grade and pathologic stage(r=-0.248, p < 0.05), as well as increased c-myc level (r=-0.454, p< 0.001). The expression of NDRG2 was low in the three BC cell lines. T24 cells infected with LEN-NDRG2 showed inhibition of proliferation both in vitro and in vivo, and NDRG2 overexpression can inhibit tumor growth and invasion in vitro.