Effect of lncRNA HULC knockdown on rat secreting pituitary adenoma GH3 cells

Pituitary adenoma is one of the most common tumors in the neuroendocrine system. This study investigated the effects of long non-coding RNAs (lncRNAs) highly up-regulated in liver cancer (HULC) on rat secreting pituitary adenoma GH3 cell viability, migration, invasion, apoptosis, and hormone secretion, as well as the underlying potential mechanisms. Cell transfection and qRT-PCR were used to change and measure the expression levels of HULC, miR-130b, and FOXM1. Cell viability, migration, invasion, and apoptosis were assessed using trypan blue staining assay, MTT assay, two-chamber transwell assay, Guava Nexin assay, and western blotting. The concentrations of prolactin (PRL) and growth hormone (GH) in culture supernatant of GH3 cells were assessed using ELISA. The targeting relationship between miR-130b and FOXM1 was verified using dual luciferase activity. Finally, the expression levels of key factors involved in PI3K/AKT/mTOR and JAK1/STAT3 pathways were evaluated using western blotting. We found that HULC was highly expressed in GH3 cells. Overexpression of HULC promoted GH3 cell viability, migration, invasion, PRL and GH secretion, as well as activated PI3K/AKT/mTOR and JAK1/STAT3 pathways. Knockdown of HULC had opposite effects and induced cell apoptosis. HULC negatively regulated the expression of miR-130b, and miR-130b participated in the effects of HULC on GH3 cells. FOXM1 was a target gene of miR-130b, which was involved in the regulation of GH3 cell viability, migration, invasion, and apoptosis, as well as PI3K/AKT/mTOR and JAK1/STAT3 pathways. In conclusion, HULC tumor-promoting roles in secreting pituitary adenoma might be via down-regulating miR-130b, up-regulating FOXM1, and activating PI3K/AKT/mTOR and JAK1/STAT3 pathways.

Mechanism and Clinical Significance of miR-373 In Elderly Patients with Multiple Myeloma / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To study the mechanism and clinical value of miR-373 in multiple myeloma.</p><p><b>METHODS</b>The expressions of miR-373 in multiple myeloma cells and normal plasma cells were detected by RT-PCR, and the biological function of miR-373 in tumor was analyzed by MTT assay, flow cytometry, luciferase experiment and tumorgenesis experiment.</p><p><b>RESULTS</b>The miR-373 expression levels in MM patients and multiple myeloma cell lines (H929, MM1S and U266) were significantly lower than that in normal plasma cells detected by using RT-PCR (P<0.05). The proliferations of U266 and H929 cells transfected with miR-373 were significantly suppressed (P<0.05); the cell cycle of H929 cell transfected with miR-373 was arrested in the G/G phase(P<0.05) and the cell apoptosis was induced (P<0.05). Luciferase experiment revealed that miR-373 could significantly inhibit the expression of FOXM1 (P<0.05). In mouse tumorigenesis experiments, overexpression of miR-373 significantly inhibited tumor growth by decreasing FOXM1 levels (P<0.05).</p><p><b>CONCLUSION</b>miR-373 inhibits tumor growth in MM by direct targeting FOXM1, thus miR-373 shows an important clinical significance for the treatment of MM.</p>

Relevant coexpression of STMN1, MELK and FOXM1 in glioblastoma and review of the impact of STMN1 in cancer biology / Coexpressão relevante de STMN1, MELK e FOXM1 em glioblastoma e revisão do impacto de stmn1 na biologia do câncer

OBJECTIVE: To analyze the associated expression of STMN1, MELK and FOXM1 in search of alternative drugable target in glioblastoma (GBM) and to review relevant functional roles of STMN1 in cancer biology. METHOD: STMN1, MELK and FOXM1 expressions were studied by quantitative PCR and their coexpressions were analyzed in two independent glioblastoma cohorts. A review of articles in indexed journals that addressed the multiple functional aspects of STMN1 was conducted, focusing on the most recent reports discussing its role in cancer, in chemoresistance and in upstream pathways involving MELK and FOXM1. RESULTS: Significant associated expressions of MELK and FOXM1 were observed with STMN1 in GBM. Additionally, the literature review highlighted the relevance of STMN1 in cancer progression. CONCLUSION: STMN1 is very important to induce events in cancer development and progression, as cellular proliferation, migration, and drug resistance. Therefore, STMN1 can be an important therapeutic target for a large number of human cancers. In glioblastoma, the most aggressive brain tumor, the MELK/FOXM1/STMN1 presented significant associated expressions, thus pointing MELK and FOXM1 as alternative targets for therapy instead of STMN1, which is highly expressed in normal brain tissue. Continuous functional research to understand the STMN1 signaling pathway is worthwhile to improve the therapeutic approaches in cancer.

OBJETIVO: Analisar as expressões associadas de STMN1, MELK e FOXM1 na procura de alvos alternativos de tratamento em glioblastoma (GBM) e revisar os papeis funcionais relevantes de STMN1 na biologia do câncer. MÉTODO: As expressões de STMN1, MELK e FOXM1 foram estudadas por PCR quantitativo e suas coexpressões foram analisadas em dois coortes independentes de GBM. A revisão dos artigos publicados em revistas indexadas na procura dos aspectos funcionais múltiplos de STMN1 foi conduzida focando-se nos estudos mais recentes discutindo o seu papel em câncer, quimiorresistência e vias de sinalização envolvendo MELK e FOXM1. RESULTADOS: Observou-se expressões associadas significantes de MELK e FOXM1 com STMN1. Adicionalmente, a revisão da literatura salientou a relevância do STMN1 na progressão do câncer. CONCLUSÃO: STMN1 é muito importante nos eventos relacionados ao desenvolvimento e progressão do câncer, como proliferação celular, migração e resistência ao tratamento. Desta forma, STMN1 pode ser um forte alvo terapêutico em um grande número de cânceres humanos. Em GBM, o tumor cerebral mais agressivo, MELK/FOXM1/STMN1 apresentaram significativa associação em suas expressões gênicas, indicando, portanto, MELK e FOXM1 como alvos alternativos para terapia em substituição ao STMN1, que apresenta alta expressão no tecido cerebral normal. Perseverar nos estudos funcionais para o entendimento da via de sinalização do STMN1 é relevante para melhorar os esquemas terapêuticos para câncer.

Construction of a lentiviral vector of FoxM1 shRNA and its transfection into human prostate cancer cell lines in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a recombinant lentiviral vector that co-express green fluorescent protein (GFP) and FoxM1 shRNA and establish a prostate cancer cell line with stable FoxM1 down-regulation.</p><p><b>METHODS</b>Three interfering sequences targeting FoxM1 were designed and inserted into the lentiviral vector pHBLV-U6-ZsGreen-Puro. After identification by DNA sequencing, the lentiviral vectors carrying Foxm1 shRNA were packaged in 293 cells. The lentiviral particles were collected to infect human prostate cancer DU-145 cells, and the transfection efficiency was observed under fluorescence microscope; the interference efficiency was assessed using real-time PCR. DU-145 cells with stable FoxM1 down-regulation were screened with puromycin, and the expression level of FoxM1 was detected by Western blotting and the cell growth was observed using MTT assay. The stably transfected cells were examined for cell apoptosis and cell clone formation capacity with flow cytometry and colony formation assay.</p><p><b>RESULTS</b>DNA sequencing demonstrated successful construction of the 3 FoxM1 shRNA lentivirus vectors. Real-time PCR showed a high interference efficiency of FoxM1 shRNA1 vector, which resulted in obvious down-regulation of FoxM1 in DU-145 cells. Western blotting showed that the expression of FoxM1 protein was decreased in FoxM1 shRNA1 lentivirus-transfected cells, which displayed a suppressed cell proliferation, increased apoptosis rate, and attenuated clonogenic ability.</p><p><b>CONCLUSION</b>We have successfully established a prostate cancer cell model with stable FoxM1 down-regulation, which shows lowered proliferative and clonogenic activities with increased cell apoptosis.</p>

Effects of β-catenin on differentially expressed genes in multiple myeloma / 华中科技大学学报（医学）（英德文版）

This study aimed to identify the differentially expressed genes after silencing of β-catenin in multiple myeloma transduced with β-catenin shRNA. The DNA microarray dataset GSE17385 was downloaded from Gene Expression Omnibus, including 3 samples of MM1.S (human multiple myeloma cell lines) cells transduced with control shRNA and 3 samples of MM1.S cells transduced with β-catenin shRNA. Then the differentially expressed genes (DEGs) were screened by using Limma. Their underlying functions were analyzed by employing Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses. Moreover, DEGs annotation was conducted based on the databases of tumor associated genes, tumor suppressed genes and the transcriptional regulation from patterns to profiles. Furthermore, the protein-protein interaction (PPI) relationship was obtained from STRING and the protein-protein interaction network and the functional modules were visualized by Cytoscape. Then, the pathway enrichment for the DEGs in the functional module was performed. A total of 301 DEGs, including 124 up-regulated and 117 down-regulated DEGs, were screened. Functional enrichment showed that CCNB1 and CDK1 were significantly related to the function of cell proliferation. FOS and JUN were related to innate immune response-activating signal transduction. Pathway enrichment analysis indicated that CCNB1 and CDK1 were most significantly enriched in the pathway of cell cycle. Besides, FOS and JUN were significantly enriched in the Toll-like receptor signaling pathway. FOXM1 was identified as a transcription factor. Moreover, there existed interactions among CCNB1, FOXM1 and CDK1 in PPI network. The expression of FOS, JUN, CCNB1, FOXM1 and CDK1 may be affected by β-catenin in multiple myeloma.

Effects of AG1478 on the expression of FOXM1 gene via FOXO3a in non-small cell lung cancer cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To explore the effects of EGFR-TKI AG1478 on the expression of FoxMl and FOXO3a genes in non-small cell cancer (NSCLC) cell lines, and explore the effect on cell proliferation and drug sensitivity to AG1478 after down-regulation of FOXMl and FOXO3a expression by RNAi technique.</p><p><b>METHODS</b>Human lung cancer cells were treated with AG1478 at different concentrations. RT-PCR and Western blot were used to examine the expression of P-EGFR, FOXM1, FOXO3a mRNA and protein. After transient transfection of FOXM1 and FOXO3a siRNA, RT-PCR and Western blot were employed to determine the transfection efficiency and expression of the related proteins. CCK-8 assay, colony formation assay and flow cytometry were performed to evaluate the cell proliferation, colony formation ability and the changes in cell cycle distribution.</p><p><b>RESULTS</b>The expressions of FOXM1 mRNA and protein were inhibited by AG1478 in a dose-dependent manner (both P < 0.05). After transfection with FOXM1 siRNA, the expressions of FOXM1 mRNA and protein, and proteins of cyclin B1, c-Myc, and Bcl-2 were significantly down-regulated, and the expressions of p21 and cleaved-PARP proteins were significantly up-regulated (all P < 0.05). The colony number of FOXM1siRNA transfection group was 37.3 ± 8.6, significantly lower than that of the blank control (135.3 ± 7.0) and negative control group (125.3 ± 7.5, P < 0.05). The colony formation inhibition rate was (7.40 ± 0.94)% in the negative control group and (72.4 ± 6.09)% in the FOXM1 siRNA transfection group. FOXM1siRNA transfection induced cell cycle arrest at G2/M phase with a percentage of (55.6 ± 4.83)%, significantly higher than that of the blank control [(24.30 ± 1.95)%] and negative control group [(21.3 ± 2.06)%, P < 0.05]. Additionally, the FOXM1siRNA transfection significantly increased the chemosensitivity of A549 cells to AG1478 (P < 0.05). Besides, AG1478 induced expression and nuclear relocation of FOXO3a. After the FOXO3a siRNA transfection, the expression of FOXM1 protein was significantly up-regulated, and resulted in a reduction of AG1478-induced inhibition of FOXM1.</p><p><b>CONCLUSIONS</b>The expression of FOXM1 is down-regulated by AG1478 via FOXO3a in the NSCLC cell lines, and then increases the chemosensitivity of A549 cells to AG1478. It suggests that FOXM1 could be a potential target for the therapy and drug exploitation for NSCLC.</p>

Role and regulation of the forkhead transcription factors FOXO3a and FOXM1 in carcinogenesis and drug resistance / 癌症

The FOXO3a and FOXM1 forkhead transcription factors are key players in cancer initiation, progression, and drug resistance. Recent research shows that FOXM1 is a direct transcriptional target of FOXO3a, a vital downstream effector of the PI3K-AKT-FOXO signaling cascade. In addition, FOXM1 and FOXO3a also antagonize each other's activity by competitively binding to the same target genes, which are involved in chemotherapeutic drug sensitivity and resistance. Understanding the role and regulation of the FOXO-FOXM1 axis will provide insight into chemotherapeutic drug action and resistance in patients, and help to identify novel therapeutic approaches as well as diagnostic and predictive biomarkers.

Targeted therapy: tailoring cancer treatment / 癌症

Targeted therapies include small-molecule inhibitors and monoclonal antibodies, have made treatment more tumor-specific and less toxic, and have opened new possibilities for tailoring cancer treatment. Nevertheless, there remain several challenges to targeted therapies, including molecular identification, drug resistance, and exploring reliable biomarkers. Here, we present several selected signaling pathways and molecular targets involved in human cancers including Aurora kinases, PI3K/mTOR signaling, FOXO-FOXM1 axis, and MDM2/MDM4-p53 interaction. Understanding the molecular mechanisms for tumorigenesis and development of drug resistance will provide new insights into drug discovery and design of therapeutic strategies for targeted therapies.

Avaliação de survivina e XIAP e dos fatores de transcrição foxo3a e foxM1 como potenciais biomarcadores de quimiorresistência no câncer de mama / [Evaluation of survivin and XIAP and transcription factors foxo3a and foxM1 as potential chemoresistance biomarkers in breast cancer]

O câncer de mama é a neoplasia que apresenta maior mortalidade entre as mulheres ao redor do mundo, apesar dos avanços na descoberta de novas modalidades terapêuticas e marcadores prognósticos. A resistência ao tratamento quimioterápico é uma das principais causas de falha terapêutica, apontando para a necessidade da identificação de biomarcadores preditivos de resposta. Evidências científicas mostram que a superexpressão de FoxM1 e das proteínas antiapoptóticas Survivina e XIAP, bem como a inativação do fator de transcrição Foxo3a, estão associados a quimioresistência e a um prognóstico desfavorável no câncer de mama. Dessa forma, o objetivo desse estudo é avaliar o papel das proteínas Survivina, XIAP, Foxo3a e FoxM1 como potenciais fatores de resistência à doxorrubicina (doxo), quimioterápico amplamente empregado no tratamento no câncer de mama. Nossos dados mostram que a doxo foi capaz de inibir a viabilidade celular nas linhagens celulares derivadas de carcinoma de mama MCF7 (não-invasiva, positiva para receptores de estrogênio e Her2) e MDA-MB-231 (invasiva, triplo-negativa), como avaliado pelo ensaio de MTT. A droga induziu a perda da adesão celular e fragmentação do DNA, como observado pela análise morfológica com quantificação das células não-aderidas e avaliação do conteúdo de DNA por citometria de fluxo. A análise da ativação das caspases-3, -7 e -9 por Western blotting revelou que a doxo induziu apoptose em células com diferentes status de p53. Em paralelo, o tratamento com a doxo resultou na redução dos níveis proteicos e de RNAm de XIAP e Survivina, como avaliado por Western blotting e PCR em tempo real, respectivamente. Entretanto, a indução da superexpressão da Survivina, por transfecção plasmidial, não foi capaz de conferir resistência ao quimioterápico. Corroborando esses resultados, observamos que o silenciamento gênico por siRNA de XIAP e Survivina, isoladamente ou em combinação, não sensibilizou as células à morte celular induzida pela doxo, indicando que tais proteínas não desempenham papel na resistência à droga. Contrariando dados da literatura, a doxo foi capaz de induzir a fosforilação de Foxo3a e Akt e reduzir a expressão de seu alvo transcricional Bim e dos níveis de RNAm de FOXO3A. De maneira consistente, a droga promoveu a translocação de Foxo3a do núcleo para o citoplasma, como examinado por fracionamento subcelular, apontando para a inativação da sua função. Além disso, a expressão do fator de transcrição FoxM1 foi reduzida, mediante o estímulo apoptótico induzido pela doxo. A indução da superexpressão de FoxM1 foi capaz de reverter a sensibilidade das células MDA-MB-231 à doxo, processo que envolveu a indução dos níveis de Survivina e XIAP. O mesmo efeito não foi observado nas células MCF7 superexpressando FoxM1, uma vez que se mantiveram sensíveis ao quimioterápico e apresentaram inalterados níveis de Survivina e XIAP. O conjunto dos nossos dados indica que a via de sinalização oncogênica mediada pelo fator de transcrição FoxM1 é capaz de promover a resistência à doxo e sugere que a combinação clínica de inibidores de FoxM1 com a doxo tem o potencial de sobrepujar a quimiorresistência no câncer de mama, principalmente em tumores triplo-negativos.

Breast cancer is the leading cause of deaths in women around the world, despite recent advances regarding novel therapeutic options and identification of prognostic factors. Resistance to therapy is the main cause of treatment failure and still there is no predictive biomarker for response to systemic therapy available. Increasing evidence shows that Survivin and XIAP antiapoptotic proteins and FoxM1 overexpression, as well as the inactivation of Foxo3a transcription factor, are closely associated with chemoresistance and poor prognosis in breast cancer. Thus, this study aimed to investigate Survivin, XIAP, Foxo3a and FoxM1 potential role on resistance to doxorubicin (dox), a chemotherapeutic agent widely used to treat breast cancer. Our data demonstrates that dox inhibited cell viability in the breast cancer derived cell lines MCF7 (non-invasive, Her2 and estrogen receptor positive) and MDA-MB- 231 (invasive, triple-negative), as evaluated through the MTT assay. The drug induced loss of cell adhesion and DNA fragmentation, as examined by morphological analysis followed by quantification of non-adherent cells and flow cytometry DNA content analysis. Western blotting evaluation of caspases-3, -7 and -9 activation revealed that dox induced apoptosis in cells with different p53 activation status. In parallel, exposure to dox resulted in reduction in Survivin and XIAP protein and mRNA levels, as evaluated by Western blotting and real time PCR, respectively. However, when we transfected cells with a Survivin-encoding plasmid, we did not observe a cell death-resistant phenotype. Accordingly, XIAP and Survivin silencing through siRNA, individually or in combination, had little effect on breast cancer cells sensitivity towards dox, suggesting that the drug can induce apoptosis independently of their expression. Contrasting data in the literature, dox treatment induced Foxo3a and Akt phosphorylation and reduced the expression of its transcriptional target Bim and FOXO3A mRNA levels. In agreement, dox-exposed cells displayed Foxo3a expression in cytoplasm, differently from predominantly nuclear Foxo3a observed in untreated cells, as examined through subcellular localization. These data point to dox-induced Foxo3a inactivation in breast cancer cells. In addition, we observed that FoxM1 transcription factor expression was inhibited upon dox-mediated apoptotic stimuli. Importantly, FoxM1 overexpression could counteract apoptosis in MDA-MB-231 cells, along with induction of Survivin and XIAP expression. This effect was not observed in MCF7 cells, which remained similarly sensitive to dox and displayed Survivin and XIAP levels unaltered. Altogether, our results demonstrate that FoxM1 signaling pathway can promote dox resistance and suggest that combining FoxM1 inhibitors with dox has the potential to circumvent chemoresistance in breast cancer, specially in triple negative tumors.

Expression and clinicopathological significance of foxM1 gene in laryngeal squamous cell carcinoma / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To investigate the expression of Forkhead Box M1 transcription factor (FoxM1) mRNA and protein in laryngeal squamous cell carcinoma (LSCC) and its clinical significance.@*METHOD@#Immunohistochemistry was used to examine the expression of FoxM1 in 89 LSCC tissues, 89 adjacent normal tissues and 20 laryngeal papilloma (LP) tissues. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to examine the expression of FoxM1 in 20 LSCC tissues, 20 LP tissues and 20 adjacent normal tissues. The relationship between FoxM1 expression and the clinicopathological parameters of LSCC were analyzed.@*RESULT@#FoxM1 mRNA and protein expression were gradually decreased from LSCC to LP and normal tissues. The difference was significant (P0.05).@*CONCLUSION@#The up-regulation of FoxM1 may play an important role in the invasion and metastasis of LSCC.

Correlation between expression of forkhead box M1 (FOXM1) and clinicopathological features and prognosis in patients with non-small cell lung cancer (NSCLC) / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the expression of forkhead box M1 (FOXM1) and its correlation with clinicopathological features and prognosis in patients with non-small cell lung cancer (NSCLC).</p><p><b>METHODS</b>The expression of FOXM1 in 68 cases of NSCLC was detected by immunohistochemistry. The FOXM1 expression in 6 tumor tissues (3 cases with negative and 3 cases with positive expression of FOXM1) was analyzed by Western blotting to confirm the immunohistochemical results. The correlation of the expression of FOXM1 with clinicopathalogical features and overall survival of the NSCLC patients was analyzed.</p><p><b>RESULTS</b>The expression of FOXM1 protein was detected in the nuclei or cytoplasms of the tumor cells. The positive expression rate of FOXM1 was 36.8% (25/68). Western blotting confirmed the immunohistochemical results. The expression level of FOXM1 in advanced stage cancer was significantly higher than that in early stage NSCLC (P = 0.001). The median OS was 23.0 months in patients with negative expression of FOXM1 and 13.0 months in those with positive expression (P = 0.001). Univariate analysis revealed that histological grade, lymph nodes status, TNM stage and FOXM1 expression were significantly associated with prognosis in the NSCLC patients (P < 0.05). The Cox multivariate analysis demonstrated that lymph nodes status, TNM stage and FOXM1 expression were independent poor prognostic factors (P < 0.05).</p><p><b>CONCLUSION</b>The expression status of FOXM1 in NSCLC is an independent prognostic factor and negatively correlated with prognosis.</p>