The roles of REIC gene and its encoding product in gastric carcinoma

REIC is downregulated in immortalized cell lines compared with the parental normal counterparts. It may inhibit colony formation, tumor growth and induce apoptosis. Here, gastric carcinoma or epithelial cells transfected with REIC-expressing plasmid, its siRNA or treated with recombinant REIC were subjected to the phenotypes’ measurement or related molecules’ detection. REIC expression was examined in gastric carcinomas by RT-PCR, western blot and immunohistochemistry. REIC overexpression or treatment resulted in a low karyoplasmic ratio and proliferation, G₁ arrest, high apoptosis, low migration, invasion or lamellipodia formation in AGS cells. REIC knockdown caused the opposite in GES-1 cells. Anti-REIC antibody blocked the effects of REIC overexpression on proliferation, G₁/S progression and apoptosis. Ectopic REIC expression downregulated the expression of β-catenin, phosphorylated S6K (Thr389), phosphorylated Akt1/2/3 (Ser473), cyclin D2 and E, WAVE2 and upregulated phosphorylated mTOR (Ser2448) expression and the mRNA level of Akt1, Akt2, mTOR, Raptor and Rictor in AGS cells. REIC expression was negatively associated with tumor size, lymph node metastasis, dedifferentiation or poor prognosis of carcinoma. The serum REIC level was significantly higher in healthy individuals than the carcinoma patients and inversely linked to tumor size by ELISA. The possible mechanisms underlying the forced REIC overexpression or recombinant REIC mediated the reversal of the aggressive phenotypes of gastric carcinoma cells are to downregulate β-catenin and WAVE2 expression and to alter other related target proteins. Downregulated REIC expression was closely linked to aggressive behaviors and poor prognosis of gastric carcinoma.     

乳腺癌中Rho A蛋白表达及其与Cyclin D1和P21WAF1/CIP1蛋白表达的相关性

目的探讨Rho A蛋白在人乳腺癌中的表达情况,Rho A蛋白与临床病理因素的关系,及其与细胞周期蛋白Cyclin D1,细胞周期抑制蛋白 P21 WAF1/CIP1表达的相关性.方法应用免疫组化S-P法,检测64例乳腺癌组织及20例正常乳腺组织中Rho A蛋白、Cyclin D1和P21 WAF1/CIP1蛋白的表达情况.结果 (1)Rho A、 Cyclin D1和P21 WAF1/CIP1蛋白在正常乳腺组织中的表达率分别为5.00%、25.00%、15.00%,在乳腺癌组织中的表达率分别为73.44%、59.38%、48.44%,三者在乳腺癌组织中的阳性表达分别与正常乳腺组织相比,均差异有显著性意义(P< 0.01).(2)Rho A蛋白表达与病理组织分级,淋巴结转移相关(P< 0.05),与患者年龄、肿瘤大小及临床分期无关.(3)RhoA蛋白与P21 WAF1/CIP1蛋白表达呈负相关(χ2=4.548,P<0.05),与Cyclin D1蛋白表达无关.结论乳腺癌患者RhoA蛋白过表达与预后不良有关.RhoA蛋白通过下调P21 WAF1/CIP1蛋白参与细胞周期调节,进而与乳腺癌发展及侵袭转移相关.     

CARMA3 is overexpressed in colon cancer and regulates NF-κB activity and cyclin D1 expression

CARMA3 was recently reported to be overexpressed in cancers and associated with the malignant behavior of cancer cells. However, the expression of CARMA3 and its biological roles in colon cancer have not been reported. In the present study, we analyzed the expression pattern of CARMA3 in colon cancer tissues and found that CARMA3 was overexpressed in 30.8% of colon cancer specimens. There was a significant association between CARMA3 overexpression and TNM stage (p=0.0383), lymph node metastasis (p=0.0091) and Ki67 proliferation index (p=0.0035). Furthermore, knockdown of CARMA3 expression in HT29 and HCT116 cells with high endogenous expression decreased cell proliferation and cell cycle progression while overexpression of CARMA3 in LoVo cell line promoted cell proliferation and facilitated cell cycle transition. Further analysis showed that CARMA3 knockdown downregulated and its overexpression upregulated cyclin D1 expression and phospho-Rb levels. In addition, we found that CARMA3 depletion inhibited p-IκB levels and NF-κB activity and its overexpression increased p-IκB expression and NF-κB activity. NF-κB inhibitor BAY 11-7082 reversed the role of CARMA3 on cyclin D1 upregulation. In conclusion, our study found that CARMA3 is overexpressed in colon cancers and contributes to malignant cell growth by facilitating cell cycle progression through NF-κB mediated upregulation of cyclin D1.     

miR-133 is a key negative regulator of CDC42–PAK pathway in gastric cancer

Cell division cycle 42 (CDC42), an important member of the Ras homolog (Rho) family, plays a key role in regulating multiple cellular processes such as cell cycle progression, migration, cell cytoskeleton organization, cell fate determination and differentiation. Among the downstream effectors of CDC42, P21-activated kinases (PAKs) obtain the most attention. Although a large body of evidence indicates that CDC42/PAKs pathway plays important role in tumor growth, invasion and metastasis, the mechanism of their negative regulation remains unclear. Here, we identified CDC42, a PAKs activating factor, was a target of miR-133. Ectopic overexpression of miRNAs not only downregulated CDC42 expression and PAKs activation, but also inhibited cancer cell proliferation and migration. We also found that miR-133 was down-regulated in 180 pairs gastric cancer tissues. miR-133 expression was negatively associated with tumor size, invasion depth and peripheral organ metastasis. Besides, dysfunction of miR-133 was an independent prognosis factor for overall survival. Our findings could provide new insights into the molecular mechanisms of gastric carcinogenesis, and may help facilitating development of CDC42/PAK-based therapies for human cancer.     

Overexpression of Eukaryotic Translation Initiation Factor 5A2 (EIF5A2) Correlates with Cell Aggressiveness and Poor Survival in Gastric Cancer

Eukaryotic translation initiation factor 5A2 (EIF5A2) plays an important role in tumor progression and prognosis evaluation. However, little information is available about its potential role in gastric cancer. This study aimed to investigate the function of EIF5A2 in tumor progression and its potential mechanisms. EIF5A2 expression was measured in human gastric cancer cell lines, the immortalized gastric mucosal epithelial cell line (GES-1) and human gastric cancer tissues and knocked down by RNA interference or upregulated by EIF5A2 plasmid transfection. Cell proliferation, migration and invasion were assessed in vitro. The downstream targets of EIF5A2 were examined by western blotting. EIF5A2 and its potential target metastasis-associated protein 1 (MTA1) expression were examined in 160 pairs of human gastric cancer and adjacent non-tumor specimens using immunohistochemistry (IHC) staining, and its correlation with clinicopathological features and survival was investigated. Knockdown of EIF5A2 or MTA1 caused an apparent suppression of HGC27 cell proliferation, migration and invasion. After knockdown of EIF5A2 in HGC27 cells, E-cadherin levels were upregulated and vimentin, cyclin D1, cyclin D3, C-MYC and MTA1 levels were downregulated. Upregulation of EIF5A2 in MKN45 cells resulted in the converse. IHC results showed a positive correlation between EIF5A2 and MTA1 expression in gastric cancers (P<0.001). Both EIF5A2 and MTA1 overexpression were correlated with pT stage (P=0.018 and P=0.042), pN stage (P=0.037 and P=0.020) and lymphovascular invasion (P=0.016 and P=0.044). EIF5A2 or MTA1 overexpression was significantly associated with poor overall survival and disease-free survival (All P<0.05). Multivariate analyses identified EIF5A2 as an independent predictor for both overall survival (P=0.012) and disease-free survival (P=0.008) in gastric cancer patients. Our findings indicate that EIF5A2 upregulation plays an important oncogenic role in gastric cancer. EIF5A2 may represent a new predictor for poor survival and is a potential therapeutic target for gastric cancer.     

Upregulation of the Non-Coding RNA OTUB1-isoform 2 Contributes to Gastric Cancer Cell Proliferation and Invasion and Predicts Poor Gastric Cancer Prognosis

Background: The deubiquitinase OTUB1 plays critical oncogenic roles and facilitates tumor progression in cancer. However, less is known regarding the aberrant expression, clinical significance and biological functions of the non-coding RNA OTUB1-isoform 2. We aimed to evaluate the OTUB1-isoform 2 levels in gastric cancer and their possible correlation with clinicopathologic features and patient survival to reveal its biological effects in gastric cancer progression.Methods: Total RNA extraction was performed on 156 gastric cancer case samples, and RT-qPCR was conducted. Chi-square test analysis was used to calculate the correlation between pathological parameters and the OTUB1-isoform 2 mRNA levels. Kaplan-Meier and Cox proportional hazards analyses were used to analyze the overall survival (OS) and disease-free survival (DFS) rates. Nuclear and cytoplasmic RNAs were isolated to detect the subcellular localization of OTUB1-isoform 2. We also assessed whether overexpression of OTUB1-isoform 2 influenced in vitro cell proliferation, cell cycle progression, tumor cell invasion and migration, as well as in vivo nude mouse xenograft and metastasis models.Results: The OTUB1-isoform 2 expression levels were higher in the gastric cancer samples than in the paratumorous gland samples. OTUB1-isoform 2 expression levels tightly correlated with tumor size, lymph node metastasis and TNM staging. Higher OTUB1-isoform 2 expression levels led to significantly poorer OS and DFS rates, and a multivariate analysis revealed that OTUB1-isoform 2 was an independent risk factor for DFS. OTUB1-isoform 2 was predominantly localized in the cell nucleus. Ectopic overexpression of OTUB1-isoform 2 in gastric cancer cells stimulated proliferation by inducing G₁-S transition, suppression of cell apoptosis and promotion of tumor cell invasion and migration. Finally, OTUB1-isoform 2 overexpression promoted tumor growth and tumor metastasis in nude mice models.Conclusions: Our study suggests that OTUB1-isoform 2 independently predicts poor prognosis and promotes tumor progression in gastric cancer. The non-coding RNA OTUB1-isoform 2 should be targeted in future molecular therapies.     

cyclin D1, cyclin E 在乳腺良、恶性病变中的表达及其与p16,p21waf1及Rb 蛋白的关系

为探讨cyclinD1,cyclinE在乳腺癌发生发展中的作用及其与细胞周期调控相关基因蛋白的关系,采用免疫组化检测17例非增生乳腺导管上皮、19例不同程度增生的导管上皮及59例乳腺癌中cyclinD1,cyclinE,p16,p21waf1及Rb基因蛋白的表达.结果显示1.非增生乳腺导管上皮除1例cyclinE过表达外均无cyclinD1和cyclinE的过表达.乳腺癌的cyclinD1和cyclinE的过表达率均明显高于良性乳腺组织(P<0.05).2.乳腺癌cyclinD1过表达与淋巴结转移呈正相关(P<0.05),瘤体直径大于5cm者cyclinE过表达呈增加趋势,但差异无显著性意义.3.CyclinD1和cyclinE的过表达呈正相关(P<0.05).4.从非增生乳腺导管上皮到增生直至乳腺癌,p16,p21与cyclinD1,cyclinE含量的比值逐渐递减,而p21含量高于cyclinD1的乳腺癌体积小、淋巴结转移率低(P<0.05).p21阳性率与cyclinD1过表达呈正相关(P<0.01),也随cyclinE的过表达呈上升趋势.Rb基因蛋白的强表达与cyclinD1过表达呈正相关(P<0.01).结果表明CyclinD1和cyclinE蛋白过表达频发于乳腺癌早期,它们可能与p16、p21waf1、pRb共同参与了乳腺癌的发生发展.     

LncRNA RP1调控周期蛋白质促进结肠癌细胞增殖

长链非编码RNAs（long non-coding RNAs, lncRNAs）是一类无蛋白质编码功能,长度大于 200 nt的RNAs。qRT-PCR实验证实,lncRNA RP1-506.5（命名为RP1）在人结肠癌细胞株中的表达量明显高于人正常结肠上皮细胞（P<0.01）。RP1在结肠癌组织中的表达量为癌旁组织中表达量的8.5倍。在HCT116中,上调RP1的表达,同时在HCT8中沉默RP1的表达,探讨RP1对结肠癌细胞生物学特性的影响。MTS实验、活细胞工作站增殖实验,结合平板克隆实验发现,过表达RP1能明显促进结肠癌细胞HCT116的增殖能力。而在HCT8细胞中沉默RP1表达后,该细胞的增殖能力明显减弱。流式细胞周期实验结果表明,RP1能促进细胞周期快速通过G1/S检测点,并能加速S期进程。荧光定量PCR、Western印迹实验发现,在HCT116中细胞中,上调RP1的表达后,P21的表达水平下调,细胞周期蛋白D1（cyclinD1）、依赖细胞周期蛋白激酶6(CDK6)表达水平上调;当沉默LncRNA RP1的表达后,能上调P21的表达水平,下调cyclinD1、CDK6的表达水平。这些结果表明,LncRNA RP1可通过调控周期相关蛋白质的表达促进结肠癌细胞增殖。     

KLHL38 involvement in non-small cell lung cancer progression via activation of the Akt signaling pathway

Lung cancer is the leading cause of cancer-related death worldwide. KLHL38 has been reported to be upregulated during diapause but downregulated after androgen treatment during the reversal of androgen-dependent skeletal muscle atrophy. This study aimed to clarify the role of KLHL38 in non-small cell lung cancer (NSCLC). KLHL38 expression was evaluated in tumor and adjacent normal tissues from 241 patients with NSCLC using immunohistochemistry and real-time PCR, and its association with clinicopathological parameters was analyzed. KLHL38 levels positively correlated with tumor size, lymph node metastasis, and pathological tumor-node-metastasis stage (all P < 0.001). In NSCLC cell lines, KLHL38 overexpression promoted PTEN ubiquitination, thereby activating Akt signaling. It also promoted cell proliferation, migration, and invasion by upregulating the expression of genes encoding cyclin D1, cyclin B, c-myc, RhoA, and MMP9, while downregulating the expression of p21 and E-cadherin. In vivo experiments in nude mice further confirmed that KLHL38 promotes NSCLC progression through Akt signaling pathway activation. Together, these results indicate that KLHL38 is a valuable candidate prognostic biomarker and potential therapeutic target for NSCLC.Subject terms: Lung cancer, Oncogenesis     

Ndrg2基因表达对胃癌细胞增殖调控及其机理的研究

为研究Ndrg2基因在人类肿瘤发生发展中的作用,以不表达Ndrg2基因的胃癌细胞系HGC-27和表达Ndrg2基因的胃癌细胞系SGC-7901作为对比材料,以Ndrg2基因转染HGC-27胃癌细胞系,以及用Ndrg2的反义寡核苷酸封闭SGC-7901胃癌细胞系中Ndrg2基因的表达.发现Ndrg2可以抑制HGC-27胃癌细胞的软琼脂集落形成,有一定诱导细胞凋亡的作用,对细胞周期蛋白E的表达有明显下调作用.当封闭了SGC-7901胃癌细胞中Ndrg2基因表达的软琼脂集落形成受到抑制,流式细胞仪检测发现此时的SGC-7901细胞周期被阻滞在G1期,细胞周期蛋白D1和E表达下调.Ndrg2基因对两种肿瘤细胞中的细胞外信号调节激酶(ERK)和P38的表达也有不同的影响.     

Ataxia-Telangiectasia Group D Complementing Gene (ATDC) Promotes Lung Cancer Cell Proliferation by Activating NF-κB Pathway

Previous studies suggested Ataxia-telangiectasia group D complementing gene (ATDC) as an oncogene in many types of cancer. However, its expression and biological functions in non-small cell lung cancer (NSCLC) remain unclear. Herein, we investigated its expression pattern in 109 cases of human NSCLC samples by immunohistochemistry and found that ATDC was overexpressed in 62 of 109 NSCLC samples (56.88%). ATDC overexpression correlated with histological type (p<0.0001), tumor status (p = 0.0227) and histological differentiation (p = 0.0002). Next, we overexpressed ATDC in normal human bronchial epithelial cell line HBE and depleted its expression in NSCLC cell lines A549 and H1299. MTT and colony formation assay showed that ATDC overexpression promoted cell proliferation while its depletion inhibited cell growth. Furthermore, cell cycle analysis showed that ATDC overexpression decreased the percentage of cells in G1 phase and increased the percentage of cells in S phase, while ATDC siRNA treatment increased the G1 phase percentage and decreased the S phase percentage. Further study revealed that ATDC overexpression could up-regulate cyclin D1 and c-Myc expression in HBE cells while its depletion down-regulated cyclin D1 and c-Myc expression in A549 and H1299 cells. In addition, ATDC overexpression was also associated with an increased proliferation index, cyclin D1 and c-Myc expression in human NSCLC samples. Further experiments demonstrated that ATDC up-regulated cyclin D1 and c-Myc expression independent of wnt/β-catenin or p53 signaling pathway. Interestingly, ATDC overexpression increased NF-κB reporter luciferase activity and p-IκB protein level. Correspondingly, NF-κB inhibitor blocked the effect of ATDC on up-regulation of cyclin D1 and c-Myc. In conclusion, we demonstrated that ATDC could promote lung cancer proliferation through NF-κB induced up-regulation of cyclin D1 and c-Myc.