scholarly journals PADI4 regulates osteosarcoma proliferation primarily via Wnt/β-catenin and MEK/ERK signaling pathway

2020 ◽  
Author(s):  
Jianping Guo ◽  
Lei Yin ◽  
Xuezhong Zhang ◽  
Peng Su ◽  
Qiaoli Zhai
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Nude Mice ◽  
Colony Formation ◽  
Erk Signaling ◽  
Osteosarcoma Cells ◽  
Rt Pcr ◽  
Normal Tissues ◽  
The Impact

Abstract Background: Peptidylarginine deiminase 4 (PADI4), an important modification enzyme of proteins, has received increased attention for its role in tumorigenesis of several human cancers. However, the effect of PADI4 on osteosarcoma remains largely unknown. Here, we evaluated the impact and mechanism of PADI4 on osteosarcoma proliferation.Methods: Impact of PADI4 on proliferation of osteosarcoma cells is detected by the method of CCK8 and colony formation assay. Expression of PADI4 as well as Wnt/β-catenin and MEK/ERK signaling markers after knocking down or ectopically expressing PADI4 or PADI4 inhibitor treatment is investigated by Western blot and RT-PCR. Then we investigated relevance of the expression level of the PADI4 in osteosarcoma samples and paired normal tissues by Western blot and RT-PCR. To further confirm whether PADI4 affects osteosarcoma tumorigenesis in vivo, we performed tumor formation experiments in nude mice.Results: Firstly, ectopically expressing PADI4 showed positive regulation on colony formation capacity of osteosarcoma cells. Secondly, PADI4 stimulated Wnt/β-catenin and MEK/ERK signaling in osteosarcoma cells. Thirdly, expression of PADI4 is higher in osteosarcoma samples compared with normal tissues. In vivo experiment also verified the positive effect of PADI4 on the growth of transplanted tumors in nude mice.Conclusions: Taken together, our results revealed PADI4 promoted proliferation of osteosarcoma via Wnt/β-catenin and MEK/ERK signaling pathway. This study may expand our understanding of osteosarcoma tumorigenesis and identify PADI4 as a potential target for diagnosis and treatment of osteosarcoma.

scholarly journals PADI4 regulates osteosarcoma proliferation primarily via Wnt/β-catenin and MEK/ERK signaling pathway

2020 ◽  
Author(s):  
Jianping Guo ◽  
Lei Yin ◽  
Xuezhong Zhang ◽  
Peng Su ◽  
Qiaoli Zhai
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Nude Mice ◽  
Colony Formation ◽  
Erk Signaling ◽  
Osteosarcoma Cells ◽  
Rt Pcr ◽  
Normal Tissues ◽  
The Impact

Abstract Background Peptidylarginine deiminase 4 (PADI4), an important modification enzyme of proteins, has received increased attention for its role in tumorigenesis of several human cancers. However, the effect of PADI4 on osteosarcoma remains largely unknown. Here, we evaluated the impact and mechanism of PADI4 on osteosarcoma proliferation. Methods Impact of PADI4 on proliferation of osteosarcoma cells is detected by the method of CCK8 and colony formation assay. Expression of PADI4 as well as Wnt/β-catenin and MEK/ERK signaling markers after knocking down or ectopically expressing PADI4 or PADI4 inhibitor treatment is investigated by Western blot and RT-PCR. Then we investigated relevance of the expression level of the PADI4 in osteosarcoma samples and paired normal tissues by Western blot and RT-PCR. To further confirm whether PADI4 affect osteosarcoma tumorigenesis in vivo, we performed tumor formation experiments in nude mice. Results Firstly, ectopically expressing PADI4 showed positive regulation on colony formation capacity of osteosarcoma cells. Secondly, PADI4 stimulated Wnt/β-catenin and MEK/ERK signaling in osteosarcoma cells. Thirdly, PADI4 is highly expressed in osteosarcoma samples compared with normal tissues. In vivo experiment also verified the positive effect of PADI4 on the growth of transplanted tumors in nude mice. Conclusions Taken together, our results revealed PADI4 promoted proliferation of osteosarcoma via Wnt/β-catenin and MEK/ERK signaling pathway. This study may expand our understanding of osteosarcoma tumorigenesis and identify PADI4 as a potential target for diagnosis and treatment of osteosarcoma.

scholarly journals miR-142-3p Suppresses Cell Growth by Targeting CDK4 in Colorectal Cancer

2018 ◽  
Vol 51 (4) ◽  
pp. 1969-1981 ◽  
Author(s):  
Xiangyu Zhu ◽  
Si-ping Ma ◽  
Dongxiang Yang ◽  
Yanlong Liu ◽  
Yong-peng Wang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Growth ◽  
Tumor Cell ◽  
Nude Mice ◽  
Colony Formation ◽  
Tumor Cell Growth ◽  
Rt Pcr ◽  
Tumor Tissues

Background/Aims: Deregulation of microRNAs (miRNAs) has been associated with a variety of cancers, including colorectal cancer (CRC). Here, we investigated anomalous miR-142-3p expression and its possible functional consequences in primary CRC samples. Methods: The expression of miR-142-3p was measured by quantitative RT-PCR in 116 primary CRC tissues and adjacent non-tumor tissues. The effect of miR-142-3p up- or down-regulation in CRC-derived cells was evaluated in vitro by cell viability and colony formation assays and in vivo by growth assays in xenografted nude mice. Results: Using quantitative RT-PCR, we found that miR-142-3p was down-regulated in 78.4 % (91/116) of the primary CRC tissues tested when compared to the adjacent non-tumor tissues. We also found that the miR-142-3p mimic reduced in vitro cell viability and colony formation by inducing cell cycle arrest in CRC-derived cells, and inhibited in vivo tumor cell growth in xenografted nude mice. Inversely, we found that the miR-142-3p inhibitor increased the viability and colony forming capacity of CRC-derived cells and tumor cell growth in xenografted nude mice. In addition, we identified CDK4 as a potential target of miR-142-3p by predictions and dual-luciferase reporter assays. Concordantly, we found that miR-142-3p mimics and inhibitors could decrease and increase CDK4 protein levels in CRC-derived cells, respectively. Conclusion: From our results we conclude that miR-142-3p may act as a tumor suppressor in CRC and may serve as a tool for miRNA-based CRC therapy.

Tanshinone IIA Inhibits Epithelial-to-mesenchymal Transition Through Regulating β-arrestin1 Mediated β-catenin Signaling Pathway in Colorectal Cancer

2020 ◽  
Author(s):  
Qing Song ◽  
Liu Yang ◽  
Zhifen Han ◽  
Xinnan Wu ◽  
Ruixiao Li ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Western Blot ◽  
Signaling Pathway ◽  
Lung Metastasis ◽  
Nude Mice ◽  
Epithelial To Mesenchymal Transition ◽  
Tanshinone Iia ◽  
Mesenchymal Transition

Abstract Background: Tanshinone IIA (Tan IIA) is a major active ingredient extracted from Salvia miltiorrhiza, which has been proved to inhibit metastasis of various cancers including colorectal cancer (CRC). However, the detailed mechanisms of Tan IIA against CRC metastasis are not well explored. Epithelial-to-mesenchymal transition (EMT) exerts an important regulatory role in CRC metastasis, and our previous mechanism studies demonstrated that β-arrestin1 could regulate CRC EMT partly through β-catenin signaling pathway. Therefore, in this work we investigated whether Tan IIA could regulate CRC EMT through β-arrestin1-mediated β-catenin signaling pathway in vivo and in vitro.Methods: The nude mice tail vein metastasis model was established to observe the effect of Tan IIA on CRC lung metastasis in vivo. The lung metastasis was evaluated by living animal imaging and hemaoxylin-eosin staining. The migratory ability of CRC cells in vitro were measured by transwell and wound healing assays. The protein expression and cellular localization of β-arrestin1 and β-catenin were characterized by immunofluorescence staining and western blot. The β-catenin signaling pathway related proteins and EMT associated proteins in CRC cells were detected by western blot and immunohistochemistry. Results: Our results showed that Tan IIA inhibited the lung metastases of CRC cells in vivo and extended the survival time of nude mice. In vitro, Tan IIA increased the expression of E-cadherin, decreased the secretion of Snail, N-cadherin and Vimentin, thus suppressed EMT and the migratory ability of CRC cells. Further study found the mechanism involving in Tan IIA regulating EMT and metastasis, referring to the suppression of β-arrestin1 expression, reduction of β-catenin nuclear localization, thereby the decreased activity of β-catenin signaling. Conclusion: Our data revealed a new mechanism of Tan IIA on the suppression of EMT and metastasis in CRC via β-arrestin1-mediated β-catenin signaling pathway, and provided support for Tan IIA as anti-metastatic agents in CRC.

scholarly journals LncRNA LIPE-AS1 Predicts Poor Survival of Cervical Cancer and Promotes Its Proliferation and Migration via Modulating miR-195-5p/MAPK Pathway

2021 ◽  
Vol 11 ◽  
Author(s):  
Jie Zhang ◽  
Pinping Jiang ◽  
Shoyu Wang ◽  
Wenjun Cheng ◽  
Shilong Fu
Keyword(s):  
Cervical Cancer ◽  
Western Blot ◽  
Signaling Pathway ◽  
Mapk Pathway ◽  
Mapk Signaling ◽  
Tumor Formation ◽  
Mapk Signaling Pathway ◽  
Rt Pcr ◽  
Mesenchymal Transformation

Aims: A growing number of studies have unveiled that long non-coding RNA (lncRNA) is conductive to cervical cancer (CC) development. However, the effect of LIPE-AS1 is remained to be studied in CC.Main Methods: Reverse transcription-polymerase chain reaction (RT-PCR) was employed to measure LIPE-AS1 expression in CC tissues and the adjacent normal tissues. Additionally, we conducted gain- and loss-of functional experiments of LIPE-AS1 and adopted CCK8 assay, BrdU assay, and in vivo tumor formation experiment to test the proliferation of CC cells (HCC94 and HeLa). Besides, the apoptosis, invasion, and epithelial-mesenchymal transformation (EMT) of CC cells were estimated using flow cytometry, transwell assay, and western blot, respectively. Further, LIPE-AS1 downstream targets were analyzed through bioinformatics, and the binding relationships between LIPE-AS1 and miR-195-5p were verified via dual-luciferase activity experiment and RNA Protein Immunoprecipitation (RIP) assay. Moreover, rescue experiments were conducted to confirm the effects of LIPE-AS1 and miR-195-5p in regulating CC development and the expressions of MAPK signaling pathway related proteins were detected by RT-PCR, western blot, and immunofluorescence.Key Findings: LIPE-AS1 was over-expressed in CC tissues (compared to normal adjacent tissues) and was notably related to tumor volume, distant metastasis. Overexpressing LIPE-AS1 accelerated CC cell proliferation, migration and EMT, inhibited apoptosis; while LIPE-AS1 knockdown had the opposite effects. The mechanism studies confirmed that LIPE-AS1 sponges miR-195-5p as a competitive endogenous RNA (ceRNA), which targets the 3′-untranslated region (3′-UTR) of MAP3K8. LIPE-AS1 promoted the expression of MAP3K8 and enhanced ERK1/2 phosphorylation, which were reversed by miR-195-5p.Significance: LIPE-AS1 regulates CC progression through the miR-195-5p/MAPK signaling pathway, providing new hope for CC diagnosis and treatment.

scholarly journals NF-κB and pSTAT3 synergistically drive G6PD overexpression and facilitate sensitivity to G6PD inhibition in ccRCC

2020 ◽  
Author(s):  
Qiao Zhang ◽  
Zhe Yang ◽  
Yueli Ni ◽  
Honggang Bai ◽  
Qiaoqiao Han ◽  
...  
Keyword(s):  
Western Blot ◽  
Real Time ◽  
Signaling Pathway ◽  
Protein Interaction ◽  
Xenograft Model ◽  
Metabolic Reprogramming ◽  
Regulatory Function ◽  
Rt Pcr ◽  
Repressive Effect ◽  
The Impact

Abstract Background: Glucose 6-phosphate dehydrogenase (G6PD) serves key roles in cancer cell metabolic reprogramming, and has been reported to be involved in certain carcinogenesis. Previous results from our laboratory demonstrated that overexpressed G6PD was a potential prognostic biomarker in clear cell renal cell carcinoma (ccRCC), the most common subtype of kidney cancer. G6PD could stimulate ccRCC growth and invasion through facilitating reactive oxygen species (ROS)-phosphorylated signal transducer and activator of transcription 3 (pSTAT3) activation and ROS-MAPK-MMP2 axis pathway, respectively. However, the reasons for ectopic G6PD overexpression and the proliferation repressive effect of G6PD inhibition in ccRCC are still unclear. Methods: The impact of ROS accumulation on NF-κB signaling pathway and G6PD expression was determined by real-time RT-PCR and Western blot in ccRCC cells following treatment with ROS stimulator or scavenger. The regulatory function of NF-κB signaling pathway in G6PD transcription was analyzed by real-time RT-PCR, Western blot, luciferase and ChIP assay in ccRCC cells following treatment with NF-κB signaling activator/inhibitor or lentivirus infection. ChIP and Co-IP assay was performed to demonstrate protein-DNA and protein-protein interaction of NF-κB and pSTAT3, respectively. MTS assay, human tissue detection and xenograft model were conducted to characterize the association between NF-κB, pSTAT3, G6PD expression level and proliferation functions. Results: ROS-stimulated NF-κB and pSTAT3 signaling over-activation could activate each other, and exhibit cross-talks in G6PD aberrant transcriptional regulation. The underlying mechanism was that NF-κB signaling pathway facilitated G6PD transcription via direct DNA–protein interaction with p65 instead of p50. p65 and pSTAT3 formed a p65/pSTAT3 complex, occupied the pSTAT3-binding site on G6PD promoter, and contributed to ccRCC proliferation following facilitated G6PD overexpression. G6PD, pSTAT3, and p65 were highly expressed and positively correlated with each other in ccRCC tissues, confirming that NF-κB and pSTAT3 synergistically promote G6PD overexpression. Moreover, G6PD inhibitor exhibited tumor-suppressor activities in ccRCC and attenuated the growth of ccRCC cells both in vitro and in vivo . Conclusion: ROS-stimulated aberrations of NF-κB and pSTAT3 signaling pathway synergistically drive G6PD transcription through forming a p65/pSTAT3 complex. Moreover, G6PD activity inhibition may be a promising therapeutic strategy for ccRCC treatment.

scholarly journals PTEN mutant NSCLC require ATM to suppress pro-apoptotic signalling and evade radiotherapy

2021 ◽  
Author(s):  
Thomas Fischer ◽  
Oliver Hartmann ◽  
Michaela Reissland ◽  
Cristian Prieto-Garcia ◽  
Kevin Klann ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Dna Damage ◽  
Western Blot ◽  
Colony Formation ◽  
Ex Vivo ◽  
Radiation Sensitivity ◽  
Genetic Alterations ◽  
Altered Expression ◽  
The Impact

Background: Despite advances in treatment of patients with non-small cell lung cancer, carriers of certain genetic alterations are prone to failure. One such factor frequently mutated, is the tumor suppressor PTEN. These tumors are supposed to be more resistant to radiation, chemo- and immunotherapy. Methods: Using CRISPR genome editing, we deleted PTEN in a human tracheal stem cell-like cell line as well generated primary murine NSCLC, proficient or deficient for Pten, in vivo. These models were used to verify the impact of PTEN loss in vitro and in vivo by immunohistochemical staining, western blot and RNA-Sequencing. Radiation sensitivity was assessed by colony formation and growth assays. To elucidate putative treatment options, identified via the molecular characterisation, PTEN pro- and deficient cells were treated with PI3K/mTOR/DNA-PK-inhibitor PI-103 or the ATM-inhibitors KU-60019 und AZD 1390. Changes in radiation sensitivity were assessed by colony-formation assay, FACS, western-blot, phospho-proteomic mass spectrometry and ex vivo lung slice cultures. Results: We demonstrate that loss of PTEN led to altered expression of transcriptional programs which directly regulate therapy resistance, resulting in establishment of radiation resistance. While PTEN-deficient tumor cells were not dependent on DNA PK for IR resistance nor activated ATR during IR, they showed a significant dependence for the DNA damage kinase ATM. Pharmacologic inhibition of ATM, via KU-60019 and AZD1390 at non-toxic doses, restored and even synergized with IR in PTEN-deficient human and murine NSCLC cells as well in a multicellular organotypic ex vivo tumor model. Conclusion: PTEN tumors are addicted to ATM to detect and repair radiation induced DNA damage. This creates an exploitable bottleneck. At least in cellulo and ex vivo we show that low concentration of ATM inhibitor is able to synergise with IR to treat PTEN-deficient tumors in genetically well-defined IR resistant lung cancer models.

scholarly journals Schisantherin A Inhibits Pulmonary Fibrosis via Regulating ERK Signaling Pathway

2020 ◽  
Vol 15 (8) ◽  
pp. 1934578X2094835
Author(s):  
Wenyue Zhuang ◽  
Na Zhao ◽  
Di Li ◽  
Xiaoming Su ◽  
Yueyang Wang ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Western Blot ◽  
Signaling Pathway ◽  
Inflammatory Cytokines ◽  
Erk Signaling ◽  
Mesenchymal Transition ◽  
Tgf Β1 ◽  
Schisantherin A

There is no effective method for treating pulmonary fibrosis (PF) until now. This study investigated the anti-fibrotic effect of schisantherin A (SCA) extracted from Schisandra chinensis and its potential molecular mechanism in PF. A bleomycin-induced PF mouse model in vivo and transforming growth factor (TGF)-β1-induced A549 epithelial-mesenchymal transition (EMT) cell model in vitro were used for assessing the anti-fibrotic effect of SCA. Histopathological examination was conducted after hematoxylin and eosin and Masson staining. The level of TGF-β1 was tested by ELISA. The expression levels of α-smooth muscle actin, E-cadherin, and inflammatory cytokines (COX2, IL-1β, IL-6, and TNF-α) were determined by quantitative reverse transcription polymerase chain reaction and Western blot. The expression of extracellular signal-regulated kinase (ERK) was tested in lung tissues and cells by Western blot. The in vivo experiments revealed that SCA treatment markedly improved body weight and pulmonary index and reformed the destruction of the lung tissue structure. We observed that SCA inhibited the process of TGF-β1-induced EMT in the in vitro experiments. Inflammatory cytokines were reduced greatly in lung tissues and cells by SCA. Our study also indicated that SCA decreased phosphorylated ERK. It was concluded that SCA can attenuate PF by regulating the ERK signaling pathway, which suggests that SCA may be used as a potential therapeutic drug for PF.

Long Noncoding RNA LINC00707 Accelerates Tumorigenesis and Progression of Bladder Cancer via Targeting miR-145/CDCA3 Regulatory Loop

2021 ◽  
pp. 1-15
Author(s):  
Tongbin Gao ◽  
Yongjian Ji
Keyword(s):  
Bladder Cancer ◽  
Western Blot ◽  
Long Noncoding Rna ◽  
Colony Formation ◽  
Noncoding Rna ◽  
Cell Counting ◽  
In Vitro Assays ◽  
Luciferase Reporter ◽  
Rt Pcr ◽  
The Impact

<b><i>Introduction:</i></b> Growing studies reveal that long noncoding RNA is involved in oncogenesis and progression. Previous studies have demonstrated that long intergenic noncoding RNA 00707 (LINC00707) stimulated tumor progress in numerous neoplasm types; however, the function of LINC00707 in bladder cancer (BC) was not yet clear. Our researches aimed to determine whether LINC00707 was dysregulated in BC and further study its biological functions. <b><i>Methods:</i></b> LINC00707 levels in BC tissues and cells were measured using reverse transcription-PCR (RT-PCR), and the associations between the levels of LINC00707 and clinicopathological features and the months of survival were also examined. Then, Cell Counting Kit-8 assays, flow cytometry, colony formation assays, and Transwell assays were applied for the assessment of the impact of LINC00707 on the abilities of BC cells. The interaction between LINC00707 and miR-145 or cell division cycle associated 3 was determined by luciferase reporter system and RT-PCR. Protein expressions of Wnt/β-catenin signaling were examined using RT-PCR and Western blot. <b><i>Results:</i></b> We found that LINC00707 expressions were notably upregulated in BC samples and cells. Higher expressions of LINC00707 were associated with T stage, grade, and shorter overall survival in BC patients. LINC00707 was also an independent prognostic factor for BC. In vitro assays confirmed that silencing LINC00707 expressions suppressed cell proliferation, colony formation, and metastasis. Mechanistic studies elucidated that LINC00707 was directly targeted to miR-145/CDCA3. Western blot assays revealed that Wnt/β-catenin signaling was inactivated by LINC00707 knockdown. <b><i>Conclusion:</i></b> Our work offers new insight into the function of LINC00707 in the tumorigenesis of BC.

scholarly journals NF-κB and pSTAT3 synergistically drive G6PD overexpression and facilitate sensitivity to G6PD inhibition in ccRCC

2020 ◽  
Author(s):  
Qiao Zhang ◽  
Zhe Yang ◽  
Yueli Ni ◽  
Honggang Bai ◽  
Qiaoqiao Han ◽  
...  
Keyword(s):  
Western Blot ◽  
Binding Site ◽  
Real Time ◽  
Signaling Pathway ◽  
Protein Interaction ◽  
Metabolic Reprogramming ◽  
Regulatory Function ◽  
Rt Pcr ◽  
Repressive Effect ◽  
The Impact

Abstract Background Glucose 6-phosphate dehydrogenase (G6PD) serves key roles in cancer cell metabolic reprogramming, and has been reported to be involved in certain carcinogenesis. Previous results from our laboratory demonstrated that overexpressed G6PD was a potential prognostic biomarker in clear cell renal cell carcinoma (ccRCC), the most common subtype of kidney cancer. G6PD could stimulate ccRCC growth and invasion through facilitating reactive oxygen species (ROS)-phosphorylated signal transducer and activator of transcription 3 (pSTAT3) activation and ROS-MAPK-MMP2 axis pathway, respectively. However, the reasons for ectopic G6PD overexpression and the proliferation repressive effect of G6PD inhibition in ccRCC are still unclear. Methods The impact of ROS accumulation on NK-κB signaling pathway and G6PD expression was determined by real-time RT-PCR and Western blot in ccRCC cells following treatment with ROS stimulator or scavenger. The regulatory function of NK-κB signaling pathway in G6PD transcription was analyzed by real-time RT-PCR, Western blot, luciferase and ChIP assay in ccRCC cells following treatment with NK-κB signaling activator/inhibitor or lentivirus infection. ChIP and Co-IP assay was performed to demonstrate protein-DNA and protein-protein interaction of NK-κB and pSTAT3, respectively. MTS assay, human tissue detection and xenograft model were conducted to characterize the association between NK-κB, pSTAT3, G6PD expression level and proliferation functions. Results ROS-stimulated NF-κB and pSTAT3 signaling over-activation could activate each other, and exhibit cross-talks in G6PD aberrant transcriptional regulation. The underlying mechanism was that NF-κB signaling pathway facilitated G6PD transcription via direct DNA–protein interaction with p65 instead of p50. p65 and pSTAT3 formed a p65/pSTAT3 complex, occupied the pSTAT3-binding site on G6PD promoter, and contributed to ccRCC proliferation following facilitated G6PD overexpression. G6PD, pSTAT3, and p65 were highly expressed and positively correlated with each other in ccRCC tissues, confirming that NF-κB and pSTAT3 synergistically promote G6PD overexpression. Moreover, G6PD inhibitor exhibited tumor-suppressor activities in ccRCC and attenuated the growth of ccRCC cells both in vitro and in vivo . Conclusion ROS-stimulated aberrations of NF-κB and pSTAT3 signaling pathway synergistically drive G6PD transcription through forming a p65/pSTAT3 complex and occupy the pSTAT3-binding site on G6PD promoter. Moreover, G6PD activity inhibition may be a promising therapeutic strategy for ccRCC treatment.

scholarly journals NF-κB and pSTAT3 synergistically drive G6PD overexpression and facilitate sensitivity to G6PD inhibition in ccRCC

2020 ◽  
Author(s):  
Qiao Zhang ◽  
Zhe Yang ◽  
Yueli Ni ◽  
Honggang Bai ◽  
Qiaoqiao Han ◽  
...  
Keyword(s):  
Western Blot ◽  
Real Time ◽  
Signaling Pathway ◽  
Protein Interaction ◽  
Xenograft Model ◽  
Metabolic Reprogramming ◽  
Regulatory Function ◽  
Rt Pcr ◽  
Repressive Effect ◽  
The Impact

Abstract Background: Glucose 6-phosphate dehydrogenase (G6PD) serves key roles in cancer cell metabolic reprogramming, and has been reported to be involved in certain carcinogenesis. Previous results from our laboratory demonstrated that overexpressed G6PD was a potential prognostic biomarker in clear cell renal cell carcinoma (ccRCC), the most common subtype of kidney cancer. G6PD could stimulate ccRCC growth and invasion through facilitating reactive oxygen species (ROS)-phosphorylated signal transducer and activator of transcription 3 (pSTAT3) activation and ROS-MAPK-MMP2 axis pathway, respectively. However, the reasons for ectopic G6PD overexpression and the proliferation repressive effect of G6PD inhibition in ccRCC are still unclear. Methods: The impact of ROS accumulation on NF-κB signaling pathway and G6PD expression was determined by real-time RT-PCR and Western blot in ccRCC cells following treatment with ROS stimulator or scavenger. The regulatory function of NF-κB signaling pathway in G6PD transcription was analyzed by real-time RT-PCR, Western blot, luciferase and ChIP assay in ccRCC cells following treatment with NF-κB signaling activator/inhibitor or lentivirus infection. ChIP and Co-IP assay was performed to demonstrate protein-DNA and protein-protein interaction of NF-κB and pSTAT3, respectively. MTS assay, human tissue detection and xenograft model were conducted to characterize the association between NF-κB, pSTAT3, G6PD expression level and proliferation functions. Results: ROS-stimulated NF-κB and pSTAT3 signaling over-activation could activate each other, and exhibit cross-talks in G6PD aberrant transcriptional regulation. The underlying mechanism was that NF-κB signaling pathway facilitated G6PD transcription via direct DNA–protein interaction with p65 instead of p50. p65 and pSTAT3 formed a p65/pSTAT3 complex, occupied the pSTAT3-binding site on G6PD promoter, and contributed to ccRCC proliferation following facilitated G6PD overexpression. G6PD, pSTAT3, and p65 were highly expressed and positively correlated with each other in ccRCC tissues, confirming that NF-κB and pSTAT3 synergistically promote G6PD overexpression. Moreover, G6PD inhibitor exhibited tumor-suppressor activities in ccRCC and attenuated the growth of ccRCC cells both in vitro and in vivo . Conclusion: ROS-stimulated aberrations of NF-κB and pSTAT3 signaling pathway synergistically drive G6PD transcription through forming a p65/pSTAT3 complex. Moreover, G6PD activity inhibition may be a promising therapeutic strategy for ccRCC treatment.

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