Downregulation of RPL6 by siRNA inhibits proliferation and cell cycle progression of human gastric cancer cell lines

Our previous study revealed that human ribosomal protein L6 (RPL6) was up-regulated in multidrug-resistant gastric cancer cells and over-expression of RPL6 could protect gastric cancer from drug-induced apoptosis. It was further demonstrated that up-regulation of RPL6 accelerated growth and enhanced in vitro colony forming ability of GES cells while down-regulation of RPL6 exhibited the opposite results. The present study was designed to investigate the potential role of RPL6 in therapy of gastric cancer for clinic. The expression of RPL6 and cyclin E in gastric cancer tissues and normal gastric mucosa was evaluated by immunohistochemisty. It was found that RPL6 and cyclin E were expressed at a higher level in gastric cancer tissues than that in normal gastric mucosa and the two were correlative in gastric cancer. Survival time of postoperative patients was analyzed by Kaplan- Meier analysis and it was found that patients with RPL6 positive expression showed shorter survival time than patients that with RPL6 negative expression. RPL6 was then genetically down-regulated in gastric cancer SGC7901 and AGS cell lines by siRNA. It was demonstrated that down-regulation of RPL6 reduced colony forming ability of gastric cancer cells in vitro and reduced cell growth in vivo. Moreover, down-regulation of RPL6 could suppress G1 to S phase transition in these cells. Further, we evidenced that RPL6 siRNA down-regulated cyclin E expression in SGC7901 and AGS cells. Taken together, these data suggested that RPL6 was over-expressed in human gastric tissues and caused poor prognosis. Down-regulation of RPL6 could suppress cell growth and cell cycle progression at least through down-regulating cyclin E and which might be used as a novel approach to gastric cancer therapy.     

Human ribosomal protein S13 promotes gastric cancer growth through down‐regulating p27Kip1

Our previous works revealed that human ribosomal protein S13 (RPS13) was up‐regulated in multidrug‐resistant gastric cancer cells and overexpression of RPS13 could protect gastric cancer cells from drug‐induced apoptosis. The present study was designed to explore the role of RPS13 in tumorigenesis and development of gastric cancer. The expression of RPS13 in gastric cancer tissues and normal gastric mucosa was evaluated by immunohistochemical staining and Western blot analysis. It was found RPS13 was expressed at a higher level in gastric cancer tissues than that in normal gastric mucosa. RPS13 was then genetically overexpressed in gastric cancer cells or knocked down by RNA interference. It was demonstrated that up‐regulation of RPS13 accelerated the growth, enhanced in vitro colony forming and soft agar cologenic ability and promoted in vivo tumour formation potential of gastric cancer cells. Meanwhile, down‐regulation of RPS13 in gastric cancer cells resulted in complete opposite effects. Moreover, overexpression of RPS13 could promote G1 to S phase transition whereas knocking down of RPS13 led to G1 arrest of gastric cancer cells. It was further demonstrated that RPS13 down‐regulated p27^kip1 expression and CDK2 kinase activity but did not change the expression of cyclin D, cyclin E, CDK2, CDK4 and p16^INK4A. Taken together, these data indicate that RPS13 could promote the growth and cell cycle progression of gastric cancer cells at least through inhibiting p27^kip1 expression.     

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。     

Ribosomal proteins S13 and L23 promote multidrug resistance in gastric cancer cells by suppressing drug-induced apoptosis

Ribosomal proteins (RP) S13 and RPL23 were previously identified as two upregulated genes in a multidrug-resistant gastric cancer cell line SGC7901/VCR compared to its parental cell SGC7901 by differential display PCR. The aim of this study was to explore the roles of RPS13 and RPL23 in multidrug resistance (MDR) in gastric cancer cells. RPS13 and RPL23 were genetically overexpressed in SGC7901 cells, respectively. Either RPS13 or RPL23 enhanced resistance of SGC7901 cells to vincristine, adriamycin, and 5-fludrouracil. RPL23 also enhanced resistance of SGC7901 cells to cisplatin. Overexpression of either RPS13 or RPL23 did not alter the population doubling time, [³H]leucine incorporation, and intracellular adriamycin accumulation of SGC7901 cells. However, either RPS13 or RPL23 could protect SGC7901 cells from undergoing vincristine-induced apoptosis. Western blot analysis revealed that both RPS13 and RPL23 significantly increased the expression level of Bcl-2 and Bcl-2/Bax ratio in SGC7901 cells. In addition, overexpression of RPL23 enhanced glutathione S-transferase (GST) activity and intracellular glutathione content in SGC7901 cells. Together, this work demonstrates that either RPS13 or RPL23 can promote MDR in gastric cancer cells by suppressing drug-induced apoptosis, and that RPL23 may also promote MDR in gastric cancer cells through regulation of glutathione S-transferase-mediated drug-detoxifying system.     

Repression of HIP/RPL29 expression induces differentiation in colon cancer cells

We had previously shown that the expression of heparin/heparan sulfate interacting protein/ribosomal protein L29 (HIP/RPL29) was upregulated in colon cancer tissues. The present study investigated the role of HIP/RPL29 in differentiation in colon cancer cells. Inducing cellular differentiation in HT-29 cells by both sodium butyrate and glucose deprivation resulted in a significant downregulation of HIP/RPL29 expression. The beta-catenin/Tcf-4 pathway is the most important pathway controlling the switch between cellular differentiation and proliferation in intestinal epithelial cells. Inducing differentiation by dominant-negative inhibition of the beta-catenin/Tcf-4 complexes in LS174T cells also resulted in downregulation of HIP/RPL29. To determine whether a lower expression of HIP/RPL29 could induce differentiation in cancer cells, small interfering RNA (siRNA) targeting HIP/RPL29 was transfected into LS174T cells. The resultant knockdown of HIP/RPL29 expression induced cellular differentiation, as shown by the increased expression of two known markers of differentiation in LS174T cells, galectin-4 and mucin-2. In addition, the differentiation process induced by repression of HIP/RPL29 expression was accompanied by the upregulation of p21 and p53. In conclusion, HIP/RPL29 plays a role in the cellular differentiation process in colon cancer cells. The differentiation process is at least partially mediated by the upregulation of p21 and p53 pathways.     

Knockdown of RPL34 inhibits the proliferation and migration of glioma cells through the inactivation of JAK/STAT3 signaling pathway

Ribosomal protein L34 (RPL34), belonging to the L34E family of ribosomal proteins, was reported to be dysregulated in several types of cancers and plays important roles in tumor progression. However, the expression and roles of RPL34 in human glioma remain largely unknown. Thus, the objective of this study was to investigate the expression and role of RPL34 in glioma. We report here that RPL34 is highly expressed in human glioma tissues and cell lines. Knockdown of RPL34 markedly inhibited the proliferation, migration, and invasion, as well as prevented the epithelial-mesenchymal transition phenotype in glioma cells. Further, mechanistic analysis showed that knockdown of RPL34 significantly downregulated the levels of p-JAK and p-STAT3 in glioma cells. Taken together, our findings indicated that knockdown of RPL34 inhibits the proliferation and migration of glioma cells through the inactivation of JAK/STAT3 signaling pathway. Thus, RPL34 may serve as a potential therapeutic target for the treatment of glioma.     

慢性胃炎肠化生CD_(44)、CD_(44V6)及Cyclin D_1、Cyclin E的表达和意义

目的探讨慢性胃炎胃粘膜肠化生CD44、CD44V6及cyclin D1、Cyclin E表达的意义。方法利用免疫细胞化学技术对39例伴有肠化生的慢性胃炎和5例正常人胃窦粘膜的活检组织进行检查。结果正常人胃窦粘膜上皮,腺上皮对CD44、CD44V6、Cyclin D1和Cyclin E均为阴性,但在有神经内分泌样细胞的粘膜,CD44V6和cyclin D1为阳性。慢性胃炎肠化生区和不典型增生区除CD44为阴性外,CD44V6、cyclin D1和cyclin E均呈现不同的阳性反应,但未见有阳性的神经内分泌样细胞。间质细胞大都呈阳性反应。结论CD44V6、cyclin D1和cyclin E可能是胃癌前状态的早期事件,而CD44可能为胃癌晚期的标志物。     

RNA沉默抑制子p19调控细胞周期相关蛋白表达

番茄丛矮病毒的P19蛋白不仅是一个重要的病毒致病因子,而且还可作为RNA干扰(RNAi)的抑制子．这种作用是通过限制细胞内的小RNA,比如小干扰RNA(siRNAs)和微RNA(miRNAs)来实现．但是目前对P19蛋白在哺乳动物细胞上的作用还未见报道．构建了一株p19稳定表达的293细胞系,即293-p19．流式细胞仪分析发现在293细胞中过量表达P19蛋白可显著引发细胞周期的G2/M阻滞．细胞增殖实验显示,293-p19细胞的DNA复制及细胞生长均受到显著的抑制． 此外,研究还发现p19可使人胚肾293细胞内的细胞周期调控子的表达谱发生改变． 其中包括上调cyclin A1,CDK2,CDK4,CDK6,p18,cyclin D2,p19INK4d和E2F1,及下调p15,cyclin A,cyclin B1和cyclin E1的表达．上述研究结果提示,p19有可能靶向多个G2/M调控蛋白从而引发细胞的G2/M阻滞．     

CHPF promotes gastric cancer tumorigenesis through the activation of E2F1

Chondroitin polymerizing factor (CHPF) is an important glycosyltransferase involved in the biosynthesis of chondroitin sulfate. However, the relationship between CHPF and gastric cancer has not been fully investigated. CHPF expression in gastric cancer tissues was detected by immunohistochemistry and correlated with gastric cancer patient prognosis. Cultured gastric cancer cells and human gastric epithelial cell line GES1 were used to investigate the effects of shCHPF and shE2F1 on the development and progression of gastric cancer by MTT, western blotting, flow cytometry analysis of cell apoptosis, colony formation, transwell and gastric cancer xenograft mouse models, in vitro and in vivo. In gastric cancer tissues, CHPF was found to be significantly upregulated, and its expression correlated with tumor infiltration and advanced tumor stage and shorter patient survival in gastric cancer. CHPF may promote gastric cancer development by regulating cell proliferation, colony formation, cell apoptosis and cell migration, while knockdown induced the opposite effects. Moreover, the results from in vivo experiments demonstrated that tumor growth was suppressed by CHPF knockdown. Additionally, E2F1 was identified as a potential downstream target of CHPF in the regulation of gastric cancer, and its knockdown decreased the CHPF-induced promotion of gastric cancer. Mechanistic study revealed that CHPF may regulate E2F1 through affecting UBE2T-mediated E2F1 ubiquitination. This study showed, for the first time, that CHPF is a potential prognostic indicator and tumor promoter in gastric cancer whose function is likely carried out through the regulation of E2F1.Subject terms: Gastric cancer, Cell biology     

MiR-150 promotes gastric cancer proliferation by negatively regulating the pro-apoptotic gene EGR2

Accumulating evidence suggests small non-coding RNAs (microRNAs) play important roles in human cancer progression. In the present study, we found miR-150 was overexpressed in gastric cancer cell lines and tissues. Ectopic expression of miR-150 promoted tumorigenesis and proliferation of gastric cancer cells. Luciferase reporter assay demonstrated that EGR2 was a direct target of miR-150. Collectively, our study demonstrated that overexpression of miR-150 in gastric cancer could promote proliferation and growth of cancer cells at least partially through directly targeting the tumor-suppressor EGR2, suggesting a potential strategy for the development of miRNA-based treatment of gastric cancer.     

AEG‐1 induces gastric cancer metastasis by upregulation of eIF4E expression

Gastric cancer is the third leading cause of cancer‐related deaths worldwide, and patients with lymph node, peritoneal and distant metastasis have a poor prognosis. Overexpression of Astrocyte‐elevated gene‐1 (AEG‐1) has been reported to be correlated with the progression and metastasis of gastric cancer. However, its mechanisms are quite unclear. In this study, we found that elevated expression of AEG‐1 was correlated with metastasis in human gastric cancer tissues. Moreover, gain‐ or loss‐of‐function of AEG‐1, respectively, promoted or suppressed epithelial–mesenchymal transition (EMT), migration and invasion of gastric cancer cells. AEG‐1 positively regulated eIF4E, MMP‐9 and Twist expression. Manipulating eIF4E expression by transfection of overexpression constructs or siRNAs partially eliminated AEG‐1‐regulated EMT, cell migration and invasion. In addition, overexpression or knockdown of eIF4E promoted or suppressed EMT, cell migration and invasion in parallel with upregulation of MMP‐9 and Twist expression, while manipulating eIF4E expression partially abrogated AEG‐1‐induced MMP‐9 and Twist. Finally, silencing of AEG‐1 expression not only inhibited tumour growth in parallel with downregulation of eIF4E, MMP‐9 and Twist expression in a xenograft nude mouse model, but also suppressed lymph node and peritoneal metastasis of gastric cancer in an orthotopic nude mouse model. These findings suggest that AEG‐1 promotes gastric cancer metastasis through upregulation of eIF4E‐mediated MMP‐9 and Twist, which provides new diagnostic markers and therapeutic targets for cancer metastasis.     

Linc01133 contributes to gastric cancer growth by enhancing YES1-dependent YAP1 nuclear translocation via sponging miR-145-5p

The long intergenic non-coding RNA linc01133 is reported to be oncogenic in various malignancies. However, the role and mechanism of linc01133 in regulating gastric cancer growth is still not clear. In the present study, we found that linc01133 was significantly upregulated in gastric cancer tissues compared to non-tumorous gastric tissues. Linc01133 over-expression significantly correlated with tumor size and tumor differentiation in gastric cancer patients. The expression of linc01133 was regulated by c-Jun and c-Fos collaboratively. In both in vitro and in vivo studies, linc01133 was shown to promote gastric cancer cell growth. Linc01133 localized in the cytoplasm and functioned as an endogenous competing RNA of miR-145-5p to upregulate the expression of YES1, which was proved to be the target gene of miR-145-5p. By promoting YES1-dependent YAP1 nuclear translocation, linc01133 upregulated the expression of the key cell cycle regulators CDK4, CDK6 and cyclin D1 to promote G1-S phase transition. Thus, our study unveiled the function and mechanism of linc01133 regulating cell cycle progression in gastric cancer.Subject terms: Gastric cancer, Cell growth     

PTEN inhibits the invasion and metastasis of gastric cancer via downregulation of FAK expression

The tumor suppressor gene phosphatase and tensin homolog (PTEN) is essential in inhibiting tumor growth and metastasis. However, the mechanism by which PTEN restricts gastric cancer progression and metastasis remains largely elusive. Here we demonstrated that PTEN overexpression or knockdown in gastric cancer cells led to the downregulation or upregulation of focal adhesion kinase (FAK), and decreased or increased cell invasion, respectively. Moreover, FAK overexpression could rescue the inhibition of cell invasion by PTEN. These results were further confirmed in orthotropic gastric cancer nude mice model. In addition, in human gastric cancer tissues, PTEN protein level was conversely correlated with FAK protein level. Mechanistically, we found that PTEN inhibited PI3K/NF-κB pathway and inhibited the DNA binding of NF-κB on FAK promoter. Taken together, our data reveal a novel mechanism that PTEN inhibits the growth and invasion of gastric cancer via the downregulation of FAK expression and suggest that exploiting PTEN/PI3K/NF-κB/FAK axis is a promising approach to treat gastric cancer metastasis.     

Alternative splicing variants of human Fbx4 disturb cyclin D1 proteolysis in human cancer

Fbx4 is a specific substrate recognition component of SCF ubiquitin ligases that catalyzes the ubiquitination and subsequent degradation of cyclin D1 and Trx1. Two isoforms of human Fbx4 protein, the full length Fbx4α and the C-terminal truncated Fbx4β have been identified, but their functions remain elusive. In this study, we demonstrated that the mRNA level of Fbx4 was significantly lower in hepatocellular carcinoma tissues than that in the corresponding non-tumor tissues. More importantly, we identified three novel splicing variants of Fbx4: Fbx4γ (missing 168–245nt of exon1), Fbx4δ (missing exon6) and a N-terminal reading frame shift variant (missing exon2). Using cloning sequencing and RT-PCR, we demonstrated these novel splice variants are much more abundant in human cancer tissues and cell lines than that in normal tissues. When expressed in Sk-Hep1 and NIH3T3 cell lines, Fbx4β, Fbx4γ and Fbx4δ could promote cell proliferation and migration in vitro. Concordantly, these isoforms could disrupt cyclin D1 degradation and therefore increase cyclin D1 expression. Moreover, unlike the full-length isoform Fbx4α that mainly exists in cytoplasm, Fbx4β, Fbx4γ, and Fbx4δ locate in both cytoplasm and nucleus. Since cyclin D1 degradation takes place in cytoplasm, the nuclear distribution of these Fbx4 isoforms may not be involved in the down-regulation of cytoplasmic cyclin D1. These results define the impact of alternative splicing on Fbx4 function, and suggest that the attenuated cyclin D1 degradation by these novel Fbx4 isoforms provides a new insight for aberrant cyclin D1 expression in human cancers.     

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.     

TFDP3 was expressed in coordination with E2F1 to inhibit E2F1-mediated apoptosis in prostate cancer

TFDP3 has been previously identified as an inhibitor of E2F molecules. It has been shown to suppress E2F1-induced apoptosis dependent P53 and to play a potential role in carcinogenesis. However, whether it indeed helps cancer cells tolerate apoptosis stress in cancer tissues remains unknown. TFDP3 expression was assessed by RT-PCR, in situ hybridization and immunohistochemistry in normal human tissues, cancer tissues and prostate cancer tissues. The association between TFDP3 and E2F1 in prostate cancer development was analyzed in various stages. Apoptosis was evaluated with annexin-V and propidium iodide staining and flow-cytometry. The results show that, in 96 samples of normal human tissues, TFDP3 could be detected in the cerebrum, esophagus, stomach, small intestine, bronchus, breast, ovary, uterus, and skin, but seldom in the lung, muscles, prostate, and liver. In addition, TFDP3 was highly expressed in numerous cancer tissues, such as brain-keratinous, lung squamous cell carcinoma, testicular seminoma, cervical carcinoma, skin squamous cell carcinoma, gastric adenocarcinoma, liver cancer, and prostate cancer. Moreover, TFDP3 was positive in 23 (62.2%) of 37 prostate cancer samples regardless of stage. Furthermore, immunohistochemistry results show that TFDP3 was always expressed in coordination with E2F1 at equivalent expression levels in prostate cancer tissues, and was highly expressed particularly in samples of high stage. When E2F1 was extrogenously expressed in LNCap cells, TFDP3 could be induced, and the apoptosis induced by E2F1 was significantly decreased. It was demonstrated that TFDP3 was a broadly expressed protein corresponding to E2F1 in human tissues, and suggested that TFDP3 is involved in prostate cancer cell survival by suppressing apoptosis induced by E2F1.     

前列腺癌患者组织中RPL6的表达及临床意义

目的:探讨前列腺癌患者组织中核糖体蛋白L6(RPL6)表达,分析其对肿瘤恶性程度的评估作用。方法:收集2013年12月-2015年12月在本院接受治疗的前列腺疾病患者117例,根据病理结果分为前列腺炎组63例、前列腺癌组54例;另取同期进行健康体检的健康者80例作为正常对照组。采用Western-blot法检测三组研究对象的前列腺组织RPL6表达,采用酶联免疫吸附法(ELISA)测定血清肿瘤标志物含量,采用RT-PCR法检测前列腺组织增殖基因、侵袭基因mRNA表达,采用Pearson检验分析RPL6表达量与肿瘤恶性程度的相关关系。结果:前列腺癌组患者的前列腺组织RPL6表达量高于前列腺炎组及正常对照组(P0.05);前列腺癌组患者的血清前列腺特异性抗原(PSA)、前列腺特异性酸性磷酶(PSAP)、尿激酶型纤溶酶原激活剂(u PA)、硫氧还蛋白(TRX)含量高于前列腺炎组及正常对照组(P0.05);前列腺癌组患者的前列腺组织增殖基因URG11、PTEN mRNA表达量高于前列腺炎组及正常对照组,PRDM5 mRNA表达量低于前列腺炎组及正常对照组,侵袭基因IgGHG1、TMPRSS2-ER、PIK3C2B mRNA表达量高于前列腺炎组及正常对照组(P0.05)。前列腺癌患者的组织RPL6表达量与肿瘤标志物含量、增殖及侵袭基因活性均呈直接相关关系(P0.05)。结论:高表达的RPL6是前列腺癌早期诊断的可靠指标,且与肿瘤恶性程度直接相关,有望成为前列腺癌辅助诊断及预后评估的重要手段。     

OVCA1 inhibits the proliferation of epithelial ovarian cancer cells by decreasing cyclin D1 and increasing p16

OVCA1, a tumor suppressor gene, is deleted or lower expressed in about 80% of ovarian cancer. Over expression of OVCA1 in human ovarian cancer A2780 cells inhibits cell proliferation and arrests cells in G1 stage. However, the fact that the molecular mechanism of OVCA1 inhibits cell growth is presently elusive. Here we investigated the potential signaling pathway induced by over-expression of OVCA1. Our results show that over-expression of human OVCA1 in ovarian cancer cells A2780 leads to down-regulation of cyclin D1, and up-regulation of p16, but no effect on the expression of NF-κB. It indicates that OVCA1 could inhibit the proliferation of ovarian cancer cell A2780 by p16/cyclin D1 pathway, but not by NF-κB.