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NUAK1 and NUAK2 belong to a family of kinases related to the catalytic α-subunits of the AMP-activated protein kinase (AMPK) complexes. Despite canonical activation by the tumour suppressor kinase LKB1, both NUAKs exhibit a spectrum of activities that favour tumour development and progression. Here, we review similarities in structure and function of the NUAKs, their regulation at gene, transcript and protein level, and discuss their phosphorylation of specific downstream targets in the context of the signal transduction pathways and biological activities regulated by each or both NUAKs.

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BACKGROUND: Globally Renal Cell Carcinoma (RCC) represents 3% of malignant tumours in adults and 1.78% in Egypt. AMPK-related protein kinase 5 (ARK5) is mainly associated with a hypoxic microenvironment which is a feature of the major RCC subtypes. Additionally, it displays decreased mitochondrial respiration. SIRT3 is a mitochondrial deacetylase that modifies multiple mitochondrial proteins. MATERIAL AND METHODS: Fifty eight cases of RCC, and 30 non-neoplastic cases (of End-Stage Kidney Disease (ESKD) were subjected to immunohistochemistry by ARK5 and SIRT3. The results of IHC were correlated together and correlated with the available clinicopathologic and survival data. RESULTS: Although no significant difference was detected between RCC and ESKD groups regarding ARK5 expression, there was a significant association with RCC regarding H-score and nucleocytoplasmic expression (both P = 0.001). Also, SIRT3 was highly expressed in RCC in comparison to the ESKD group (H-score: P = 0.001). There were significant associations between nucleocytoplasmic ARK5 expression and higher tumour grade, low apoptotic and high mitotic indices, tumour extent, advanced tumour stage, and impaired response of tumours to chemotherapeutic drugs (P = 0.039, P = 0.001, P = 0.027, P = 0.011, P = 0.009, and P = 0.014 respectively). Moreover, the H score of ARK5 expression showed significant associations with tumour grade, apoptotic and mitotic indices, tumour extension, tumour stage, and response to therapy (P = 0.01, 0.035, 0.001, 0.004. 0.003 and 0.013). Regarding SIRT3 expression, it showed significant associations with apoptotic and mitotic indices, tumour extent, tumour stage and response to therapy (P = 0.022, 0.02, 0.042, 0.039 and 0.027). Interestingly, there was a highly significant correlation between the expression of ARK5 and SIRT3 (P = 0.009). Univariate survival analysis revealed a significant association between short survival duration and both nucleocytoplasmic expression of ARK5 and positive SIRT3 expression (P = 0.014 and 0.035). CONCLUSION: ARK5 and SIRT3 are overexpressed in RCC and associated with parameters of poor prognosis as well as short survival. Both seem to influence response to therapy in RCC. So, they could be new targets for therapy that may improve tumour response and patients' survival. There is a postulated relationship that needs more extensive investigation.

Asunto(s)

Carcinoma de Células Renales , Neoplasias Renales , Sirtuina 3 , Adulto , Humanos , Carcinoma de Células Renales/patología , Relevancia Clínica , Neoplasias Renales/patología , Pronóstico , Proteínas Quinasas , Microambiente Tumoral

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Objective:To detect the expression of ARK5 in peripheral blood circulating tumor cells (CTCs) from pancreatic cancer patients and explore its correlation with the efficacy and prognosis for gemcitabine chemotherapy.Methods:A total of 175 peripheral blood samples of pancreatic cancer patients who were treated in the Department of Hepatobiliary Surgery, Second Affiliated Hospital of Jiaxing University from January 2016 to June 2021 were collected. CTCs were enriched by nano-microfluidic chip technology. The expression of ARK5 in CTCs was detected by immunofluorescence. According to the expression of ARK5, the patients were divided into two groups: positive group and negative group. The differences on clinicopathological features, the efficacy of chemotherapy, median survival and progression-free survival time between the two groups were compared.Results:CTCs were enriched in 98 of 175 patients (55.6%), including 70 ARK5 positive and 28 ARK5 negative patients. There were no significant differences on clinical features between the two groups, and the two groups were comparable. In the 70 ARK5 positive patients, 64 patients (91.4%) were resistant to gemcitabine, while only 12 of the 28 ARK5 negative patients (42.8%) were resistant to gemcitabine. The incidence of gemcitabine resistance in ARK5 positive patients was significantly higher than that in ARK5 negative patients, and the difference was statistically significant ( P<0.001). The median survival time was 11.5 months in ARK5 positive group and 14 months in ARK5 negative expression group, and the progression-free survival time was 6 months in ARK5 positive expression group and 8 months in negative expression group. The survival time of ARK5 positive group was significantly shorter than that of ARK5 negative group, and the difference was statistically significant ( P<0.05). Conclusions:The pancreatic cancer patients with ARK5 positive CTCs have significantly higher incidence of gemcitabine resistance and shorter survival time than those with ARK5 negative CTCs. Detection of ARK5 expression in CTCs may be a new method to judge chemotherapy efficacy and prognosis for pancreatic cancer patients.

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AMPK-related protein kinase 5 (ARK5) is involved in a broad spectrum of physiological and cell events, and aberrant expression of ARK5 has been observed in a wide variety of solid tumors, including liver cancer. However, the role of ARK5 in liver fibrosis remains largely unexplored. We found that ARK5 expression was elevated in mouse fibrotic livers, and showed a positive correlation with the progression of liver fibrosis. ARK5 was highly expressed not only in activated hepatic stellate cells (HSCs), but also in hepatocytes. In HSCs, ARK5 prevents the degradation of transforming growth factor ß type I receptor (TßRI) and mothers against decapentaplegic homolog 4 (Smad4) proteins by inhibiting the expression of Smad ubiquitin regulatory factor 2 (Smurf2), thus maintaining the continuous transduction of the transforming growth factor ß (TGF-ß) signaling pathway, which is essential for cell activation, proliferation and survival. In hepatocytes, ARK5 induces the occurrence of epithelial-mesenchymal transition (EMT), and also promotes the secretion of inflammatory factors. Inflammatory factors, in turn, further enhance the activation of HSCs and deepen the degree of liver fibrosis. Notably, we demonstrated in a mouse model that targeting ARK5 with the selective inhibitor HTH-01-015 attenuates CCl4-induced liver fibrosis in mice. Taken together, the results indicate that ARK5 is a critical driver of liver fibrosis, and promotes liver fibrosis by synergy between HSCs and hepatocytes.

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Tetracloruro de Carbono , Células Estrelladas Hepáticas , Ratones , Animales , Células Estrelladas Hepáticas/metabolismo , Tetracloruro de Carbono/toxicidad , Hepatocitos/metabolismo , Cirrosis Hepática/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta/genética , Receptor Tipo I de Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Hígado/metabolismo

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Objective To explore the effect of miR-145-5p on the proliferation and apoptosis of human ovarian cancer cells and the possible molecular mechanisms involved.Methods Real-time quantitative PCR was performed to detect the expression of miR-145-5p in ovarian epithelial cells and ovarian cancer cells.CCK-8 and flow cytometry were used to detect the effects of miR-145-5p overexpression on the proliferation and apoptosis of ovarian cancer cells.TargetScan was employed to predict the target genes of miR-145-5p.Western blotting,dual luciferase reporter assay and rescue experiment were employed to predict and verify the underlying molecular mechanism of miR-145-5p function.Results The expression of miR-145-5p in ovarian cancer cells was significantly lower than that in normal ovarian epithelial cells(t=4.345,P=0.049).Compared with the control group,the overexpression of miR-145-5p reduced the proliferation rate(t=-15.790,P<0.001)and increased the apoptosis rate(t=5.433,P=0.032)of ovarian cancer cells.ARK5 was predicted as the direct target gene of miR-145-5p(t=4.583,P=0.010).The cells with ARK5 overexpression showed increased proliferation rate(t=27.290,P<0.001)and decreased apoptosis rate(t=-8.241,P=0.001).The overexpression of miR-145-5p can down-regulate the mRNA(t=-12.824,P<0.001)and protein(t=-4.792,P=0.001)levels of ARK5.The rescuing expression of ARK5 significantly offset the inhibitory effects of miR-145-5p on cell proliferation(t=15.580,P=0.004)and apoptosis(t=-12.470,P=0.006).Conclusion miR-145-5p may inhibit the proliferation and promote the apoptosis of ovarian cancer cells by targeting ARK5.

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MicroARNs , Neoplasias Ováricas , Apoptosis/genética , Carcinoma Epitelial de Ovario/genética , Línea Celular Tumoral , Proliferación Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/genética , Neoplasias Ováricas/genética

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Objective To explore the effect of miR-145-5p on the proliferation and apoptosis of human ovarian cancer cells and the possible molecular mechanisms involved.Methods Real-time quantitative PCR was performed to detect the expression of miR-145-5p in ovarian epithelial cells and ovarian cancer cells.CCK-8 and flow cytometry were used to detect the effects of miR-145-5p overexpression on the proliferation and apoptosis of ovarian cancer cells.TargetScan was employed to predict the target genes of miR-145-5p.Western blotting,dual luciferase reporter assay and rescue experiment were employed to predict and verify the underlying molecular mechanism of miR-145-5p function.Results The expression of miR-145-5p in ovarian cancer cells was significantly lower than that in normal ovarian epithelial cells(

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Femenino , Humanos , Apoptosis/genética , Carcinoma Epitelial de Ovario/genética , Línea Celular Tumoral , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Neoplasias Ováricas/genética

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For several years, the multidrug resistance (MDR) of gastric cancer cells has been a thorny issue worldwide regarding the chemotherapy process and needs to be solved. Here, we report that the ARK5 gene could promote the multidrug resistance of gastric cancer cells in vitro and in vivo. In this study, LV-ARK5-RNAi lentivirus was used to transfect the parental cell line SGC7901 and MDR cell line SGC7901/DDP to construct a stable model of ARK5 interference. Subsequently, the cells were treated with four chemotherapeutic drugs, cisplatin (DDP), adriamycin (ADR), 5-fluorouracil (5-FU) and docetaxel (DR) and were subjected to the CCK8, colony formation, adriamycin accumulation and retention, cell apoptosis and other assays. The study found that, in vitro, the expression of ARK5 in MDR gastric cancer cells was significantly higher than that in parental cells. Additionally, when treated with different chemotherapeutic drugs, compared with parental cells, MDR cells also had a higher cell survival rate, higher colony formation number, higher drug pump rate, and lower cell apoptosis rate. Additionally, in xenograft mouse models, MDR cells with high ARK5 expression showed higher resistance to chemotherapeutic drugs than parental cells. Overall, this study revealed that silencing the ARK5 gene can effectively reverse the drug resistance of MDR gastric cancer cells to chemotherapeutic drugs, providing insights into the mechanism of this process related to its inhibition of the active pump-out ability of MDR cells.

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Deregulated expression of MYC induces a dependence on the NUAK1 kinase, but the molecular mechanisms underlying this dependence have not been fully clarified. Here, we show that NUAK1 is a predominantly nuclear protein that associates with a network of nuclear protein phosphatase 1 (PP1) interactors and that PNUTS, a nuclear regulatory subunit of PP1, is phosphorylated by NUAK1. Both NUAK1 and PNUTS associate with the splicing machinery. Inhibition of NUAK1 abolishes chromatin association of PNUTS, reduces spliceosome activity, and suppresses nascent RNA synthesis. Activation of MYC does not bypass the requirement for NUAK1 for spliceosome activity but significantly attenuates transcription inhibition. Consequently, NUAK1 inhibition in MYC-transformed cells induces global accumulation of RNAPII both at the pause site and at the first exon-intron boundary but does not increase mRNA synthesis. We suggest that NUAK1 inhibition in the presence of deregulated MYC traps non-productive RNAPII because of the absence of correctly assembled spliceosomes.

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Núcleo Celular/metabolismo , Cromatina/metabolismo , Proteínas Quinasas/metabolismo , Proteína Fosfatasa 1/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Represoras/metabolismo , Empalmosomas/metabolismo , Transcripción Genética , Animales , Núcleo Celular/genética , Cromatina/genética , Regulación de la Expresión Génica , Células HeLa , Humanos , Ratones , Células 3T3 NIH , Fosforilación , Proteínas Quinasas/genética , Proteína Fosfatasa 1/genética , Proteína Fosfatasa 1/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Empalme del ARN , Proteínas Represoras/genética , Empalmosomas/genética

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MicroRNAs (miRNAs) have been validated to play prominent roles in the occurrence and development of many kinds of malignant cancer. MiR-424-5p has been reported to participate in various tumors proliferation and metastasis as a suppressor. On the contrary, miR-424-5p would promote cell proliferation in some tumors. However, the expression of miR-424-5p in intrahepatic cholangiocarcinoma (ICC) is rarely reported and its mechanism remains unclear. Here, we discover that miR-424-5p is frequently downregulated in ICC tissues compared with adjacent normal tissues and in ICC cells. Over-expression of miR-424-5p significantly inhibits the invasion and migration of ICC cells in vitro. Importantly, miR-424-5p is found to be a suppressor of ARK5, by binding to 3'-UTR of ARK5 mRNA and then inhibiting mTOR phosphorylated, thus deregulating epithelial-mesenchymal transition (EMT) of ICC. Furthermore, ARK5 is found to play a role in ICC metastasis and regulating EMT. Knockdown of ARK5 inhibits invasion and migration of ICC, while the over-expression gives an opposite effect. Besides, high-expression of ARK5 is also associated with poor prognosis. In conclusion, our study reveals that miR-424-5p is critical to the invasion, migration and EMT progression in ICC cells. Targeting the pathway described here may be a novel approach to inhibit metastasis of ICC and the restoration of miR-424-5p expression may be a promising strategy for ICC therapy.

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Movimiento Celular/fisiología , Proliferación Celular/fisiología , Supervivencia Celular/fisiología , Colangiocarcinoma/metabolismo , Neoplasias Hepáticas/metabolismo , MicroARNs/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Represoras/metabolismo , Western Blotting , Línea Celular , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Supervivencia Celular/genética , Colangiocarcinoma/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Neoplasias Hepáticas/genética , MicroARNs/genética , Proteínas Quinasas/genética , Proteínas Represoras/genética

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AMPK-related kinase 5 (ARK5) is a member of the human AMP-activated protein kinase (AMPK) family, which is associated with increased tumor survival and drug resistance in many cancers. However, the function of ARK5 in pancreatic carcinoma (PC) is unclear. Our study investigated the role of ARK5 in the chemo-resistance of PC and its underlying mechanism. PC cell lines that displayed high expression levels of ARK5 had low sensitivity to gemcitabine (GEM). Suppression of ARK5 increased sensitivity to GEM in PC cell lines. Western blotting and immunofluorescence showed that suppression of ARK5 upregulated expression of E-cadherin and downregulated vimentin expression. Suppression of ARK5 also inhibited the epithelial-mesenchymal transition (EMT) efficiency associated with GEM in PC cell lines and upregulation of ARK5 expression enhanced GEM resistance in PC cell lines by inducing Twist-mediated EMT. In addition, we found that suppression of ARK5 increased GEM sensitivity in PC cell lines under hypoxic conditions. ARK5 increases GEM resistance in PC cell lines via EMT, and suppression of ARK5 increases sensitivity to GEM under both normoxic and hypoxic conditions.

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BACKGROUND/AIMS: Gastric cancer (GC) is a common and lethal malignancy, and AMP-activated protein kinase-related kinase 5 (ARK5) has been discovered to promote cancer metastasis in certain types of cancer. In this study, we explored the role of ARK5 in GC invasion and metastasis. METHODS: ARK5 and epithelial-mesenchymal transition (EMT)-related markers were determined by immunohistochemistry and western blot in GC specimens. Other methods including stably transfected against ARK5 into SGC7901 and AGS cells, western blot, migration and invasion assays in vitro and nude mice tumorigenicity in vivo were also employed. RESULTS: The results demonstrated that ARK5 expression was increased and positively correlated with metastasis, EMT-related markers and poor prognosis in patients with GC. Knockdown of ARK5 expression remarkably suppressed GC cells invasion and metastasis via regulating EMT, rather than proliferation in vitro and in vivo. And knockdown of ARK5 expression in GC cells resulted in the down-regulation of the mTOR/p70S6k signals, Slug and SIP1. CONCLUSION: The elevated ARK5 expression was closely associated with cancer metastasis and patient survival, and it seemed to function in GC cells migration and invasion via EMT alteration, together with the alteration of the mTOR/p70S6k signals, Slug and SIP1, thus providing a potential therapeutic target for GC.

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Regulación Neoplásica de la Expresión Génica , Invasividad Neoplásica/genética , Proteínas Quinasas/genética , Interferencia de ARN , Proteínas Represoras/genética , Neoplasias Gástricas/genética , Estómago/patología , Animales , Línea Celular Tumoral , Transición Epitelial-Mesenquimal , Femenino , Mucosa Gástrica/metabolismo , Humanos , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Invasividad Neoplásica/patología , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/patología , ARN Interferente Pequeño/genética , Transducción de Señal , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Serina-Treonina Quinasas TOR/metabolismo

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Lung cancer incidence and mortality rates are amongst the highest of all malignant tumors worldwide. ARK5 is a member of the human AMP-activated protein kinase (AMPK) family which is implicated in tumor survival and progression. The current study was designed to explore the role of ARK5 in resistance of non-small cell lung cancer (NSCLC) to cisplatin. We studied the sensitivity of two NSCLC cell lines, NCI-H1229 and A549, to cisplatin by using proliferation and cell viability assays. We then examined expression of ARK5, Twist, and the epithelial to mesenchymal transition (EMT) biomarkers, E-cadherin and Vimentin, by Western blot and immunofluorescence. We found that ARK5 downregulation significantly increased the cisplatin chemosensitivity of NSCLC cells, and that NCI-H1299 cells, which express high levels of ARK5 and possess a mesenchymal phenotype, were more resistant to cisplatin than A549 cells, which show low expression ARK5. Furthermore, siRNA-mediated silencing of ARK5 resulted in altered EMT patterns in NSCLC cells. These data support a role for ARK5 in regulating EMT in NSCLC cells. Together, our findings suggest that ARK5 is a potential drug target for combating drug resistance and regulating EMT in NSCLC cells.

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BACKGROUND AND OBJECTIVE: Nua kinase 1 (NUAK1) was identified in multigene signatures of survival and suboptimal debulking in high-grade serous ovarian cancer (HGSOC). This study investigates the individual clinical and biologic contributions of NUAK1 in HGSOC patients and cell lines. METHODS: Public transcript expression, clinical, and outcome data were used to interrogate the relationship between NUAK1 and clinicopathologic factors and patient outcomes including progression-free survival (PFS) and molecular subtypes using logistic and Cox modeling. Analysis of NUAK1 transcript expression was performed in primary tumors from 34 HGSOC patients with < or ≥2 years PFS. The impact of silencing NUAK1 by RNA interference (RNAi) on the migratory potential and chemosensitivity of SOC cells was assessed in vitro. RESULTS: Elevated NUAK1 transcript expression was associated with worse PFS (hazard ratio = 1.134), advanced stage (odds ratio, OR = 1.7), any residual disease (OR = 1.58), and mesenchymal disease subtype (OR = 7.79 ± 5.89). Elevated NUAK1 transcript expression was observed in HGSOC patients with < vs. ≥2 years PFS (p < 0.045). RNAi-mediated silencing of NUAK1 expression attenuated migration of OV90 and E3 HGSOC cells in vitro, but did not modulate sensitivity to cisplatin or paclitaxel. CONCLUSION: Elevated NUAK1 was associated with poor survival as well as advanced stage, residual disease after cytoreductive surgery and mesenchymal molecular subtype. NUAK1 impacted migration, but not chemosensitivity, in vitro. Additional studies are needed to further develop the concept of NUAK1 as a clinically deployable biomarker and therapeutic target in HGSOC.

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AMP-activated protein kinase family member 5 (ARK5) overexpression has been reported in many human cancers, and ARK5 is associated with poor prognosis in hepatocellular carcinoma (HCC). However, whether ARK5 is involved in HCC chemoresistance is unclear. The present study aimed to investigate the role of ARK5 in HCC chemoresistance and the underlying mechanism. In this study, we found that SNU387 and SNU449 HCC cell lines overexpressing ARK5 displayed low doxorubicin sensitivity compared to Huh7 and Hep3B HCC cell lines with ARK5 low-expression. And knockdown of ARK5 increased the doxorubicin sensitivity in all HCC cell lines in the manner of inhibiting cell proliferation. Western blotting and immunofluorescence both showed that ARK5 knockdown upregulated E-cadherin and downregulated vimentin, which was consistent with the knockdown of TWIST, indicating ARK5 was involved in epithelial-mesenchymal transition (EMT). Moreover, suppressing ARK5 by siRNA restored E-cadherin and vimentin expression induced by doxorubicin treatment or hypoxia culture. Our results indicated ARK5 confers doxorubicin resistance in HCC via inducing EMT.

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Antibióticos Antineoplásicos/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Doxorrubicina/farmacología , Resistencia a Antineoplásicos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Neoplasias Hepáticas/tratamiento farmacológico , Proteínas Quinasas/metabolismo , Proteínas Represoras/metabolismo , Antígenos CD , Cadherinas/genética , Cadherinas/metabolismo , Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Concentración 50 Inhibidora , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Quinasas/genética , Interferencia de ARN , Proteínas Represoras/genética , Transducción de Señal/efectos de los fármacos , Transfección , Hipoxia Tumoral , Proteína 1 Relacionada con Twist/genética , Proteína 1 Relacionada con Twist/metabolismo , Vimentina/genética , Vimentina/metabolismo

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Aim To investigate the molecular mecha-nism of Gab2 in the invasion and metastasis of breast cancer and provide a theoretical basis for clinical pre-vention of breast cancer invasion and metastasis. Methods The Gab2 , MMP-2 and MMP-9 expressions in 80 cases of breast cancer were detected by immuno-histochemistry . Western blot was used to detect the ex-pression of Gab2 protein in MDA-MB-231 cells and MCF-7 cells. The siRNA plasmid was used to transfect MDA-MB-231 cells. Western blot was used to detect the proteins expression of Gab2 , MMP-2 and MMP-9 . Transwell in vitro experiment was used to detect the in-vasion ability of each group transfected MDA-MB-231 cells, Western blot was used to analyze phosphorylation of Akt and ARK5 induced by epithelial growth factor ( EGF ) in transfected cells ( SiGab2/MDA-MB-231 and Scr/MDA-MB-231 ) . Results The expression of Gab2 protein in invasive ductal carcinoma was signifi-cantly higher than in normal breast tissue ( P<0. 01 ) . The expression of Gab2 was dramatically correlated with lymph node metastasis, ER expression, tumor his-tological grade, MMP-2 and MMP-9 (P<0. 05). The expression of Gab2 protein in MDA-MB-231 cells was higher than in MCF-7 cells. The expression of Gab2, MMP-2 and MMP-9 decreased in SiGab2/MDA-MB-231 cells and the invasion ability of SiGab2/MDA-MB-231 cells was significantly decreased ( P<0. 05 ) , and after 5 minutes’ stimulating by EGF, the phosphoryla-tion of Akt and ARK5 was significantly reduced. Con-clusion Gab2 can promote the invasion and metasta-sis of breast cancer by effecting the expression of MMP-2 and MMP-9 through PI3 K/ Akt /ARK5 signal path-way.

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Objective To detect the expression of ARK5 in hepatocellular carcinoma (HCC)tissue and hepatoma SMMC-7721 cells,and to investigate its effect on the growth of hepatoma cells.Methods The expression levels of ARK5 mRNA and protein were determined by RT-PCR and Western blotting in 30 cases of HCC tissue, paracarcinoma tissue,SMMC-7721 cells,and hepatic cells LO2.The SiRNA of ARK5 and negative control (NC) siRNA were constructed and transfected into the SMMC-7721 cells,and used as experimental group and negative control group;at the same time blank control group was set up. The proliferation activity and apoptotic rate of transfected cells were detected by MTT assay and flow cytometry (FCM).Results The PCR and Western blotting results showed that the expression levels of ARK5 mRNA and protein in HCC tissue and SMMC-7721 cells were significantly higher than those in paracarcinoma tissue and LO2 cells (P<0.05 ). The MTT assay results demonstrated that the inhibitory rates of growth of transfected cells in experimental group at 24,48 and 72 h were (19.39±5.42)%, (23.19±0.53)%,and (20.74±1.23)%;there were significant differences compared with blank control group and negative control group (P<0.01).The FCM results indicated that the apoptotic rate of the transfected cells in experimental group was (15.017±0.945)%,there were significant differences compared with blank control group (8.770%± 0.656 )% and negative control group (8.763%± 1.201%) (P<0.05 ). Conclusion The ARK5 expression level is significantly increased in HCC tissue and hepatoma SMMC-7721 cells;the inhibition of ARK5 expression could suppress the growth of hepatoma cells and induce apoptosis. So ARK5 maybe act as a cancer-promoting gene and induce hepatocellular carcinogenesis.

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Background and purpose:Numerous researches indicated that the expression of pituitary tumor transforming gene1 (PTTG1) was correlated with the severity of glioma tumors. However the specific mechanism of PTTG1 is not clear in glioma. In this study, we explored the role and significance of PTTG1 in the invasion of glioma cells. Methods:Western blot was used to detect the expression of PTTG1 protein in various glioma cell lines. siRNA plasmid was used to transfect U87 cells. Western blot was used to analyze the expression of PTTG1 protein in transfected U87 cells. Matrigel invasion assay was used to detect the invasive ability in the cells being transfected in vitro. Western blot was used to analyze epithelial growth factor (EGF) induced protein phosphorylation of ARK5 and Akt in the cells being transfected PTTG1 plasmid (siPTTG1/U87) and scrambled siRNA (Scr/U87). Results:The expression of PTTG1 protein was higher in all glioma cell lines. After transfection, the invasion of siPTTG1/U87 was obviously decreased after 5 min with EGF stimulation than the Scr/U87, the phosphorylation of ARK5 and Akt was significantly enhanced. However, whether or not the existence of EGF, the phosphorylation of ARK5 and Akt had no differences in siPTTG1/U87. Conclusion:In glioma cells, PTTG1 protein is high expressed and maybe have an important function in glioma cells invasion through Akt-ARK5 signaling pathway.

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Objective:This study aimed to investigate the effect and significance of a binding protein-2 (Gab2)-Akt-ARK5 signaling pathway on the invasion of glioma cells. Methods:Immunohistochemical methods were used to detect the expressions of Gab2 and ARK5 in 45 cases of glioma tissue. siRNA plasmid was used to transfect LN-229 cells, and western blot was performed to analyze the protein expressions of Gab2 and ARK5. In vitro Matrigel invasion assay was conducted to detect variations in the invasiveness of transfected cells. Western blot was also conducted to analyze the protein phosphorylation of Akt and ARK5 in the cells transfected with Gab2 plasmid. Results:Immunohistochemical assay revealed that the expressions of ARK5 and Gab2 in glioma cells were positively correlated, and both expressions were higher in high-grade glioma (WHO gradeⅢ,Ⅳ) than in low-grade glioma (WHO gradeⅠ,Ⅱ). LN-229 cells transfected with ARK5 plasmid, Gab2 plasmid, ARK5 and Gab2 plasmid, and control plasmid were named siARK5/LN-229, siGab2/LN-229, siARK5 and siGab2/LN-229, and SCR/LN-229, respectively. After transfection was performed, the protein expressions of ARK5 and Gab2 were respectively decreased in siARK5/LN-229 and siGab2/LN-229. The protein expressions of ARK5 and Gab2 in siARK5 and siGab2/LN-229 were also respectively decreased. After ARK5 or Gab2 was downregulated, the number of glioma cells, which invaded and penetrated Matrigel, was decreased (P<0.01). The number of glioma cells also decreased significantly after ARK5 and Gab2 were downregulated. The phosphorylation of Akt and ARK5 in siGab2/LN-229 cells was decreased after these cells were stimulated by insulin-like growth factor-1. Conclusion:The silencing of ARK5 or Gab2 impaired glioma cell invasiveness. The decreased protein expression of Gab2 inhibited the phosphorylation of Akt and ARK5. These results suggested that the Gab2-Akt-ARK5 signaling pathway could be relevantly involved in glioma cell invasion.

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AMP-activated protein kinase (AMPK) is a critical regulator of cellular and whole-body energy homeostasis. Twelve AMPK-related kinases (ARKs; BRSK1, BRSK2, NUAK1, NUAK2, QIK, QSK, SIK, MARK1, MARK2, MARK3, MARK4, and MELK) have been identified recently. These kinases show a similar structural organization, including an N-terminal catalytic domain, followed by a ubiquitin-associated domain and a C-terminal spacer sequence, which in some cases also contains a kinase-associated domain 1. Eleven of the ARKs are phosphorylated and activated by the master upstream kinase liver kinase B1. However, most of these ARKs are largely unknown, and the NUAK family seems to have different regulations and functions. This review contains a brief discussion of the NUAK family including the specific characteristics of NUAK1 and NUAK2.

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Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Quinasas Activadas por AMP/química , Proteínas Quinasas Activadas por AMP/genética , Animales , Humanos , Proteínas Quinasas/química , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo