siRNA Suppressed Cell Proliferation Research Articles

RPL21 siRNA Blocks Proliferation in Pancreatic Cancer Cells by Inhibiting DNA Replication and Inducing G1 Arrest and Apoptosis.

BackgroundOur previous study showed that the ribosomal protein L21 (RPL21) may play an important role in the development and survival of pancreatic cancer. In this article, RNA interference (RNAi) experiments were performed with RPL21-specific small interfering RNA (siRNA) to elucidate the mechanism by which RPL21 controls PC PANC-1 and BxPC-3 cell proliferation.MethodsIn the present study, PANC-1, BxPC-3 cells, and BALB/c nude mice were used to investigate antitumor effect and mechanism by which RPL21 controls cell proliferation and apoptosis in vitro and in vivo. The effects of RPL21 knockdown on PANC-1 and BxPC-3 cell proliferation, cell cycle and cell apoptosis in vitro were determined using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assays and flow cytometry assay. The mechanism of RPL21 regulating cell proliferation was investigated using transcriptome sequencing analysis and luciferase reporter assay. The effects of RPL21 knockdown on PANC-1 and BxPC-3 cell proliferation in vivo were determined using BALB/c nude mice tumor model.ResultsIn PANC-1 and BxPC-3 cells, the knockdown of RPL21 expression with corresponding siRNA suppressed cell proliferation in vitro and in vivo, inhibited DNA replication, and induced arrests in the G1 phase of the cell cycle. Further results showed that the mini-chromosome maintenance (MCM) protein family (MCM2-7), CCND1 and CCNE1 were down-regulated significantly in PANC-1 and BxPC-3 cells after transfected with RPL21 siRNA, which suggests that the suppression of DNA replication is due to the reduced expression of MCM2-7 family, and the induction of G1 arrest is correlated with the inhibition of CCND1 and CCNE1. Luciferase reporter assay showed that RPL21 controls the DNA replication and G1-S phase progression possibly through the regulation of E2F1 transcription factor in PC cells. Moreover, RPL21 siRNA showed an apoptosis-inducing effect only in BxPC-3 and PANC-1 cells but not in normal HPDE6-C7 cells. The increase of caspase-8 activities and the loss of mitochondrial membrane potential after RPL21 silencing indicates that the RPL21 gene may be involved in caspase-8-related mitochondrial apoptosis.ConclusionOur findings suggest that siRNA against the RPL21 gene possesses a potential anti-cancer activity for PC cells by inhibiting their proliferation and DNA replication, as well as inducing cell cycle G1 arrest and cell apoptosis.

Abstract 4362: Glucose transporter 1 regulates cell glycolysis and proliferation in gastrointestinal stromal tumor and its clinicopathological significance

Abstract Background: The unique features that cancer cells prefer glycolysis rather than mitochondrial respiration despite of the presence of oxygen have been a potential target for cancer treatment. GIST is well-known as to demonstrate intense metabolic activity and avid glucose uptake on 18F-FDG PET scanning. In this study, we focused on glucose transporter 1 (GLUT 1), a key glycolytic enzyme which plays critical roles in several cancer type. The aim of this study was to examine the clinicopathological significance of GLUT1 expression in GIST and to characterize the molecular mechanism of cell glycolysis and proliferation by GIST cell. Methods: We determined the expression level of GLUT1 in 65 GIST cases, correlated those values with clinicopathological features. Furthermore, we investigated that the alteration of GLUT1 can influence on glycolysis, cell proliferation, and cell cycle distribution by extracellular flux analyzer, CCK-8 assay and flow cytometry, respectively in GIST cell in vitro. Results: Fifty-one (78.5%) cases were placed in the high GLUT1 expression group. In the high GLUT1 expression group, tumor size was significantly larger compared to the low GLUT1 expression group (P=0.02). There are significantly more high-risk cases by modified-Fletcher classification in GLUT1 expression group (P< 0.01). The patients with high GLUT1 expression significantly exhibited a higher SUVmax value (P< 0.01). Compared with the low GLUT1 expression group, the high expression group showed a significantly poorer prognosis in terms of recurrence-free survival. In vitro, GLUT1-specific siRNA suppressed cell proliferation and led to increase in G1 phase arrest. Also, knockdown of GLUT1 cells reduced glycolysis as a function of ECAR by extracellular flux analyzer. Conclusion: We showed that GLUT1 plays a critical role in GIST by regulating glycolysis and proliferation. Our study suggests that glucose transporter can be a novel target of anticancer therapeutics mediating glucose metabolism in GIST. Citation Format: Masaaki Iwatsuki, Hiroshi Sawayama, Daisuke Kuroda, Yuki Koga, Kohei Yamashita, Kazuto Harada, Shiro Iwagami, Kojiro Eto, Takatsugu Ishimoto, Yoshifumi Baba, Naoya Yoshida, Jaffer A. Ajani, Hideo Baba. Glucose transporter 1 regulates cell glycolysis and proliferation in gastrointestinal stromal tumor and its clinicopathological significance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4362.

MiR-100 inhibits the migration and invasion of nasopharyngeal carcinoma by targeting IGF1R

Nasopharyngeal carcinoma (NPC) is a cancer pattern that often develops in the epithelial cells of the nasopharynx. miR-100 is a miRNA that has been identified in a number of cancers. The aim of the present study was to investigate whether miR-100 can affect cell migration and proliferation of NPC by targeting insulin-like growth factor 1 receptor (IGF1R). Western blot analysis was used to determine the protein levels of genes. The reverse transcription-quantitative PCR (RT-qPCR) was used to detect the expression level of miR-100 and IGF1R. Transwell assay was used to detect the migration and invasion of cell lines. The luciferase reporter assay was employed to confirm the target gene of miR-100. miR-100 expression was highly reduced in NPC tissues compared with non-cancerous tissues. Overexpression of miR-100 significantly inhibited the migration and invasion of NPC cell lines C666-1 and SUNE1. IGF1R was a downstream target of miR-100 and was downregulated by miR-100. Knockdown of IGF1R by siRNA suppressed cell proliferation of the C666-1 cell line. The newly identified miR-100/IGF1R axis offers novel biomarkers for the therapeutic intervention of NPC treatment. As a result, our findings suggest that miR-100 plays an important role in suppressing migration and invasion of NPC cells and suppresses IGF1R expression by directly targeting its 3′-UTR. It is suggested that miR-100 may be a novel therapeutic target of microRNA-mediated suppression of cell migration and invasion in NPC. However, the role of the miR-100/IGF1R axis in NPC progression needs further investigation.

Folate receptor alpha is associated with cervical carcinogenesis and regulates cervical cancer cells growth by activating ERK1/2/c-Fos/c-Jun

Folate receptor alpha (FRα) is over-expressed in numerous epithelial malignancies, however, the association between FRα and cervical cancer remains unclear. The purpose of this study was to explore the effects of FRα on cervical cancer and its regulation of the ERK signaling pathway. In this case-control study, moderate/strong expression of FRα, phosphorylated ERK1/2 (p-ERK1/2), p-c-Fos, and p-c-Jun proteins was increased with the progressive severity of cervix lesions (P < 0.05). Moreover, the expression levels of p-ERK1/2, p-c-Fos, and p-c-Jun proteins was positively correlated with those of FRα protein in cervical squamous cell carcinoma (SCC) group (P < 0.05). In vitro experiment indicated down-regulation of FRα by siRNA suppressed cell proliferation, promoted cell apoptosis, induced cell cycle arrest at G0/G1 phase, and reduced expression of p-ERK1/2, p-c-Fos, and p-c-Jun proteins. The results suggest that FRα is associated with the progression of cervical cancer and regulates cervical cancer cells growth through phosphorylating ERK1/2, c-Fos, and c-Jun, which are key factors of the ERK signaling pathway. Therefore, FRα may be an effective target for early detection and therapy of cervical cancer.

NIMA-related kinase 2 regulates hepatocellular carcinoma cell growth and proliferation.

NIMA-related kinase 2 (Nek2) is often upregulated in human cancer and is important in regulating the cell cycle and gene expression, and maintaining centrosomal structure and function. The present study aimed to investigate the expression pattern, clinical significance, and biological function of Nek2 in hepatocellular carcinoma (HCC). mRNA and protein levels of Nek2 were examined in HCC and corresponding normal liver tissues. The MTT and soft agar colony formation assays, and flow cytometry were employed to assess the roles of Nek2 in cell proliferation and growth. In addition, western blot analysis was performed to assess the expression of cell cycle- and proliferation-related proteins. The results revealed that Nek2 was upregulated in HCC tissues and cell lines. The clinical significance of Nek2 expression was also analyzed. Inhibiting Nek2 expression by siRNA suppressed cell proliferation, growth, and colony formation in hepatocellular carcinoma cell line HepG2 cells, induced cell cycle arrest in the G2/M phase by retarding the S-phase, and promoted apoptosis. Furthermore, Nek2 depletion downregulated β-catenin expression in HepG2 cells and diminished expression of Myc proto-oncogene protein (c-Myc), cyclins D1, B1, and E and cyclin-dependent kinase 1, whilst increasing protein levels of p27. This demonstrates that overexpression of Nek2 is associated with the malignant evolution of HCC. Targeting Nek2 may inhibit HCC cell growth and proliferation through the regulation of β-catenin by the Wnt/β-catenin pathway and therefore may be developed as a novel therapeutic strategy to treat HCC.

Tumoral gene silencing by receptor-targeted combinatorial siRNA polyplexes

Small interfering RNA (siRNA) promises high efficacy and excellent specificity to silence the target gene expression, which shows potential for cancer treatment. However, systemic delivery of siRNA with selectivity to the tumor site and into the cytosol of tumor cells remains a major limitation. To achieve this, we generated oligoaminoamide-based sequence-defined polycationic oligomers by solid-phase assisted synthesis, which can form polyplexes with anionic siRNA by electrostatic interaction to serve as siRNA carrier. Targeting for folate receptor (FR)-overexpressing tumors, we optimized the physicochemical properties of polyplexes by combinatorial optimization of PEGylated folate-conjugated oligomer (for FR targeting and shielding of surface charges) and 3-arm oligomer (for size modification and particle stability). For uni-directional fast coupling between the two groups of oligomers, we activated the cysteine thiol groups of one of the oligomers with 5,5′-dithio-bis(2-nitrobenzoic acid) to achieve a fast chemical linkage through disulfide formation with the free thiol groups of the other oligomer. These targeted combinatorial polyplexes (TCPs) are homogeneous spherical particles with favorable size and surface charge, which showed strong siRNA binding activity. TCPs were internalized into cells by FR-mediated endocytosis, triggered significant eGFP-luciferase marker gene silencing, and transfection with antitumoral EG5 siRNA suppressed cell proliferation in FR-expressing tumor cells. Moreover, the most promising formulation TCP1 after i.v. administration in tumor-bearing mice exhibited siRNA delivery into the tumor, resulting in EG5 gene silencing at mRNA level. Therefore, by covalent combination of two sequence-defined functional oligomers, we developed a siRNA carrier system with optimized size and surface charge for efficient tumor cell-directed gene silencing and cytotoxicity in vitro and in vivo.

A Zbtb7a proto-oncogene as a novel target for miR-125a.

In our previous study, we showed that miR-125a directly targeted a WT1 oncogene, which was overexpressed in leukemia and various kinds of solid tumors including lung, breast, gastric, and colon cancers, and brain tumors and was deeply involved in leukemogenesis and tumorigenesis and that miR-125a knockout mice overexpressed WT1 and developed myeloproliferative disease. It had been also reported that miR-125a is downregulated in leukemia and various types of solid tumors such as lung cancers, suggesting its tumor suppressor function. Therefore, it is important to elucidate what is target(s) of miR-125a for understandings of such functions although few target genes for it are known. In the present study, Zbtb7a oncogene was identified as a potential target for miR-125a by gene expression profiling in miR-125a knockout mice combined with bioinformatics target prediction. EGFP-3'UTR reporter assay showed that miR-125a suppressed Zbtb7a expression through its direct binding to the Zbtb7a-3'UTR. Zbtb7a knockdown by siRNA suppressed cell proliferation and induced G1 cell cycle arrest and apoptosis in lung cancer cells. Furthermore, miR-125a expression showed a negative correlation with Zbtb7a expression in non-small cell lung cancer tissues. The present study showed for the first time that Zbtb7a was a direct target for miR-125a and was involved in cell cycle progression and apoptosis of lung cancer cells. These results also demonstrated that deregulation of miR-125a-Zbtb7a signaling was associated with the development and progression of lung cancer. © 2015 Wiley Periodicals, Inc.

MP47-01 OVEREXPRESSED MIR-27A-3P IS AS INDEPENDENT PROGNOSTIC FACTOR FOR RECURRENCE IN CLEAR CELL RENAL CELL CARCINOMA

INTRODUCTION AND OBJECTIVES: MicroRNAs (miRNAs) are noncoding RNAs that regulate gene expression and influence tumor development and progression. We previously identified miRNAs upregulated in clear cell renal cell carcinoma (ccRCC) compared to matched-pair normal kidney using microarray analysis. However, such cancer-specific miRNAs are not always associated with patient survival and/or recurrence. This study aims to identify miRNAs that are upregulated in ccRCC and that are also associated with survival and/or recurrence. METHODS: We obtained 183 ccRCC clinical specimens from patients who underwent nephrectomy at our hospital between 2000 and 2012. We selected 24 frozen samples, of which 11 patients had evidence of disease or died from cancer (Group 1) and 13 patients had no evidence of disease (Group 2) within two years after surgery. Quantitative real time PCR (qRT-PCR) was performed on the samples to expression of 22 miRNAs that our microarray results indicated as upregulated in ccRCC, and hits were further validated using another 159 frozen samples. Functional analysis of the miRNA was performed using siRNA-mediated knockdown in RCC cell lines. RESULTS: Five miRNAs (Table.1) were upregulated in Group 1 compared to Group 2, and we validated these results using 159 samples of ccRCC. High miR-21-3p (p <0.01) and miR-193b-5p (p <0.05) levels correlated with worse cancer-specific survival. In ccRCC patients without metastasis at time of diagnosis (n1⁄4140), high miR-27a-3p (p <0.01) levels correlated with worse progression-free survival. Multivariate analysis showed that miR27a-3p was an independent prognostic factor for recurrence (Table.2). Knockdown of miR-27a-3p by siRNA suppressed cell proliferation, migration, invasion and colony formation in RCC cell lines. CONCLUSIONS: These results suggest that miR-27a-3p is a good prognostic biomarker to predict recurrence independent of clinicpathological characteristics and could function as an oncomir in ccRCC. As such, miR-27a-3p may be a candidate for targeted molecular therapy. Source of Funding: none

MP47-02 MICRORNA MIR-200B IS DOWN-REGULATED AND SUPPRESSES METASTASIS BY TARGETING LAMA4 IN RENAL CELL CARCINOMA

INTRODUCTION AND OBJECTIVES: MicroRNAs (miRNAs) are noncoding RNAs that regulate gene expression and influence tumor development and progression. We previously identified miRNAs upregulated in clear cell renal cell carcinoma (ccRCC) compared to matched-pair normal kidney using microarray analysis. However, such cancer-specific miRNAs are not always associated with patient survival and/or recurrence. This study aims to identify miRNAs that are upregulated in ccRCC and that are also associated with survival and/or recurrence. METHODS: We obtained 183 ccRCC clinical specimens from patients who underwent nephrectomy at our hospital between 2000 and 2012. We selected 24 frozen samples, of which 11 patients had evidence of disease or died from cancer (Group 1) and 13 patients had no evidence of disease (Group 2) within two years after surgery. Quantitative real time PCR (qRT-PCR) was performed on the samples to expression of 22 miRNAs that our microarray results indicated as upregulated in ccRCC, and hits were further validated using another 159 frozen samples. Functional analysis of the miRNA was performed using siRNA-mediated knockdown in RCC cell lines. RESULTS: Five miRNAs (Table.1) were upregulated in Group 1 compared to Group 2, and we validated these results using 159 samples of ccRCC. High miR-21-3p (p <0.01) and miR-193b-5p (p <0.05) levels correlated with worse cancer-specific survival. In ccRCC patients without metastasis at time of diagnosis (n1⁄4140), high miR-27a-3p (p <0.01) levels correlated with worse progression-free survival. Multivariate analysis showed that miR27a-3p was an independent prognostic factor for recurrence (Table.2). Knockdown of miR-27a-3p by siRNA suppressed cell proliferation, migration, invasion and colony formation in RCC cell lines. CONCLUSIONS: These results suggest that miR-27a-3p is a good prognostic biomarker to predict recurrence independent of clinicpathological characteristics and could function as an oncomir in ccRCC. As such, miR-27a-3p may be a candidate for targeted molecular therapy. Source of Funding: none

Abstract 3892: Inhibitory effects and regulatory mechanism of nestin in pancreatic cancer

Abstract Introduction: Nestin, a progenitor/stem cell marker, is expressed in human pancreatic cancer, and we have previously shown that its expression positively correlates with invasiveness and metastasis of the cancer (Cancer Biol Ther, 2011; Am J Pathol. in press). Here, we examined the inhibitory effects of nestin expression using nestin-targeted small interfering RNA (siRNA) in pancreatic cancer cells. We also investigated the regulatory mechanism of nestin expression in pancreatic cancer. Methods: siRNA targeting nestin was transfected to the pancreatic cancer cell lines PANC-1 and PK-45H, and we analyzed the effect on cell behaviors including proliferation, migration, invasion, sphere-forming ability, and sensitivity to gemcitabine. Next, to clarify the mechanisms of nestin expression, pancreatic cancer cells were injected into the spleen of NOD.Cg-Prkdcscid Il2rgtm1Sug/Jic (NOG) mice, and pancreatic cancer cells were also collected from metastatic foci of NOG mice. We compared the methylation status of the promoter and the 1st and 2nd introns of the nestin gene in metastatic and parental cancer cells using bisulfite sequencing and pyrosequencing. Results: Nestin siRNA inhibited the proliferation, migration, invasion, and sphere-forming ability of the pancreatic cancer cells in vitro. Co-treatment of pancreatic cancer cells with gemcitabine and nestin siRNA decreased cell viability compared to administration of gemcitabine or nestin siRNA alone. Cells derived from the metastatic nodules of NOG mice showed higher expression of nestin compared with parental cells, and methylation of the nestin promoter and the 1st and 2nd introns was lower in the metastatic cells. Compared to control siRNA-treated mice, nestin siRNA significantly inhibited primary and metastatic tumor formation derived from human pancreatic cancer cells in an orthotopic implantation model using NOG mice. In summary, decreasing nestin expression in pancreatic cancer cells using nestin siRNA suppressed cell proliferation, migration, invasion, and sphere formation in vitro and primary and metastatic tumor formation in vivo. Furthermore, nestin siRNA had synergistic effects with gemcitabine in inhibiting pancreatic cancer cell growth. These findings suggest that nestin plays a key role in pancreatic cancer cell metastasis and stemness, and that administration of nestin siRNA is a novel therapeutic strategy for pancreatic cancer. Citation Format: Yoko Matsuda, Toshiyuki Ishiwata, Hisashi Yoshimura, Satoshi Yamashita, Toshikazu Ushijima, Kaiyo Takubo, Tomio Arai. Inhibitory effects and regulatory mechanism of nestin in pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3892. doi:10.1158/1538-7445.AM2014-3892

RETRACTED: Unique proliferation response in odontoblastic cells derived from human skeletal muscle stem cells by cytokine-induced matrix metalloproteinase-3

A pro-inflammatory cytokine mixture (CM: interleukin (IL)-1β, tumor necrosis factor-α and interferon-γ) and IL-1β-induced matrix metalloproteinase (MMP)-3 activity have been shown to increase the proliferation of rat dental pulp cells and murine stem cell-derived odontoblast-like cells. This suggests that MMP-3 may regulate wound healing and regeneration in the odontoblast-rich dental pulp. Here, we determined whether these results can be extrapolated to human dental pulp by investigating the effects of CM-induced MMP-3 up-regulation on the proliferation and apoptosis of purified odontoblast-like cells derived from human skeletal muscle stem cells. We used siRNA to specifically reduce MMP-3 expression. We found that CM treatment increased MMP-3 mRNA and protein levels as well as MMP-3 activity. Cell proliferation was also markedly increased, with no changes in apoptosis, upon treatment with CM and following the application of exogenous MMP-3. Endogenous tissue inhibitors of metalloproteinases were constitutively expressed during all experiments and unaffected by MMP-3. Although treatment with MMP-3 siRNA suppressed cell proliferation, it also unexpectedly increased apoptosis. This siRNA-mediated increase in apoptosis could be reversed by exogenous MMP-3. These results demonstrate that cytokine-induced MMP-3 activity regulates cell proliferation and suppresses apoptosis in human odontoblast-like cells.

ZEB2 Mediates Multiple Pathways Regulating Cell Proliferation, Migration, Invasion, and Apoptosis in Glioma

BackgroundThe aim of the present study was to analyze the expression of Zinc finger E-box Binding homeobox 2 (ZEB2) in glioma and to explore the molecular mechanisms of ZEB2 that regulate cell proliferation, migration, invasion, and apoptosis.Methodology/Principal FindingsExpression of ZEB2 in 90 clinicopathologically characterized glioma patients was analyzed by immunohistochemistry. Furthermore, siRNA targeting ZEB2 was transfected into U251 and U87 glioma cell lines in vitro and proliferation, migration, invasion, and apoptosis were examined separately by MTT assay, Transwell chamber assay, flow cytometry, and western blot.ResultsThe expression level of ZEB2 protein was significantly increased in glioma tissues compared to normal brain tissues (P<0.001). In addition, high levels of ZEB2 protein were positively correlated with pathology grade classification (P = 0.024) of glioma patients. Knockdown of ZEB2 by siRNA suppressed cell proliferation, migration and invasion, as well as induced cell apoptosis in glioma cells. Furthermore, ZEB2 downregulation was accompanied by decreased expression of CDK4/6, Cyclin D1, Cyclin E, E2F1, and c-myc, while p15 and p21 were upregulated. Lowered expression of ZEB2 enhanced E-cadherin levels but also inhibited β-Catenin, Vimentin, N-cadherin, and Snail expression. Several apoptosis-related regulators such as Caspase-3, Caspase-6, Caspase-9, and Cleaved-PARP were activated while PARP was inhibited after ZEB2 siRNA treatment.ConclusionOverexpression of ZEB2 is an unfavorable factor that may facilitate glioma progression. Knockdown ZEB2 expression by siRNA suppressed cell proliferation, migration, invasion and promoted cell apoptosis in glioma cells.