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.

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

2020 ◽  
Vol 20 (1) ◽  
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 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 High Glucose Enhances the Odonto/Osteogenic Differentiation of Stem Cells from Apical Papilla via NF-KappaB Signaling Pathway

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Yanping Wang ◽  
Yanqiu Wang ◽  
Yadie Lu ◽  
Jinhua Yu
Keyword(s):  
Stem Cells ◽  
Western Blot ◽  
Osteogenic Differentiation ◽  
Real Time ◽  
Signaling Pathway ◽  
Mtt Assay ◽  
High Glucose ◽  
Rt Pcr ◽  
Alizarin Red ◽  
Apical Papilla

Objective. The transport and metabolism of glucose are important during mammalian development. High glucose can mediate the biological characteristics of mesenchymal stem cells (MSCs). However, the role of high glucose in the odonto/osteogenic differentiation of stem cells from apical papilla (SCAPs) is unclear. Materials and Methods. SCAPs were isolated and identified in vitro. Then, SCAPs were cultured in normal α-MEM and high glucose α-MEM separately. MTT assay was applied to observe the proliferation of SCAPs. ALP activity, alizarin red staining, real-time RT-PCR, and western blot were used to detect the odonto/osteogenic capacity of SCAPs as well as the participation of NF-κB pathway. Results. SCAPs in 25mmol/L glucose group expressed the maximum proteins of RUNX2 and ALP as compared with those in 5, 10, and 15 mmol/L groups. MTT assay showed that 25 mmol/L glucose suppressed the proliferation of SCAPs. ALP assay, alizarin red staining, real-time RT-PCR, and western blot showed 25 mmol/L high glucose can obviously enhance the odonto/osteogenic capacity of SCAPs. Moreover, the NF-κB pathway was activated in 25mmol/L glucose-treated SCAPs and the odonto/osteogenic differentiation was inhibited following the inhibition of NF-κB signaling pathway. Conclusions. High glucose can enhance the odonto/osteogenic capacity of SCAPs via NF-κB pathway.

scholarly journals The Suppression of CCL2 Expression Decrease the Proliferation of HL-60 Cells through Downregulation of Cyclin d1 Via Arresting Cells at the G1 Phase

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4809-4809
Author(s):  
Jin Yang ◽  
Dan Hong ◽  
Qi Zhou ◽  
Jun-jie Fan ◽  
Le Li ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Western Blot ◽  
Cyclin D1 ◽  
Real Time ◽  
Signaling Pathway ◽  
G1 Phase ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Ccl2 Expression

Abstract Background and purpose: Chemokine (C-C motif) ligand 2(CCL2) is a member of the CC subfamily which displays chemotactic activity for monocytes and basophils. It has played a very important role in many solid tumors and changes in bone marrow microenvironment. However, its role in acute myeloid leukemia (AML) has not yet been clear. In this point, we established a cell line with CCL2 down-expression to explore the effect of CCL2 gene on leukemogenesis. Methods: Lentivirus with CCL2-knockdown was successfully constructed after screening effective CCL2 shRNA sequence and tranfected into HL-60 cells which was validated on the level of mRNA and protein by real-time PCR and Western blot. The cells coming from parental, sh-Vectors and shCCL2 were detected for cell growth viability by CCK-8 assay, cell cycles and apoptosis by Flow cytometry. We applied exon sequencing technology to identify the gene profiling between the CCL2 knockdown and the control, of which, Cyclin d1 was selected for further experiments as its expression level was significantly downregulated. Then we successfully down regulated cyclin d1 expression in HL-60 by means of RNA interference to detect the cell proliferation through CCK-8 assay, cell cycles and apoptosis through Flow cytometry. Results: HL-60 cell line expressed the highest level of CCL2 among acute leukemia cell lines (Figure 1). Among 4 pairs of CCL2 interference sequences, only pair 2 had the most efficient potential in knockdown CCL2 expression which was constructed into sh-Vector, GV248, and validated by real-time RT-PCR and Western blot(Figure 2). Low expression of CCL2 significantly decreased HL-60 cell growth. Meanwhile, the CCL2-shRNA-mediated HL-60 cells showed about 12% more cells arrested in G1 phase compared with controls (Table 1, Figure 3). The results of expression profiling showed that there were total 159 genes differentially expressed (Figure 4), of which, ten top pathways were illustrated in Table 2. Cyclin D1 was related to cell cycle, NOD-like receptor signaling pathway, TNF signaling pathway and NF-kappa B signaling pathway which was the lowest expression among cell cycle gene related in HL-60 cells transfect with shCCL2(Table 2, highlighted raw) and further validated by real-time RT-PCR and Western blot (Figure 5). After Cyclin D1 was decreased on the level of mRNA and protein of HL-60, the cell proliferation was evidently slow and cell cycle analysis also indicated a similar pattern of CCL2 (Figure 6). Conclusion: CCL2 involved in cell proliferation which was mediated by cyclin D1 via blocking more cells at G1 phase. Figure 3. Knockdown of CCL2 inhibits cell proliferation via G1 phase arrested. A: Down regulation of CCL2 influenced cell proliferation. From day 2 to 5, the proliferation rate of HL-60 cells transfected by shCCL2 grew significantly slower than controls. B: CCL2 played a role in cell cycle process. More cells transfected shCCL2 were arrested in G1 phase compared with controls. *Indicate significant differences with P-values <0.05 Figure 5. Cylin D1 was the most influenced gene among cell proliferation related genes profile. A: quantitative RT-PCR analysis showed that mRNA levels of preliferation related genes: PCNA, cyclin D1, c-jun, surviving, erk1, and erk2. Among them, Cyclin D1 was expressed lowest. B: Western blot analysis confirmed that the protein expression of total cyclin D1 was much lower compared with controls. Figure 6. The effect of Cyclin D1 on cell proliferation. A: Cyclin D1 was successfully knockdowned in HL-60. mRNA levels were determined by quantitative RT-PCR and revealed that Cyclin D1 expression was knockdowned by the vector of shccnd. B: Knockdown of cyclin D1 inhibits cell proliferation. Compared with control, HL-60 cells with low level of Cyclin D grew significant slowly. C: Down regulation of Cyclin D1 influences more cells arrested in G1 phase compared with control. *P-values <0.05 Figure 1. Figure 1. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures No relevant conflicts of interest to declare.

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 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 Astragaloside positively regulated osteogenic differentiation of pre-osteoblast MC3T3-E1 through PI3K/Akt signaling pathway

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Wei Bing Jing ◽  
Hongjuan Ji ◽  
Rui Jiang ◽  
Jinlong Wang
Keyword(s):  
Western Blot ◽  
Osteogenic Differentiation ◽  
Real Time ◽  
Signaling Pathway ◽  
Real Time Pcr ◽  
Akt Signaling ◽  
Calcium Deposition ◽  
Control Group ◽  
Akt Signaling Pathway ◽  
Western Blot Assay

Abstract Background Osteoporosis is a widespread chronic disease characterized by low bone density. There is currently no gold standard treatment for osteoporosis. The aim of this study was to explore the role and mechanism of Astragaloside on osteogenic differentiation of MC3T3-E1 cells. Methods MC3T3-E1 cells were divided into control and different dose of Astragaloside (10, 20, 40, 50, and 60 μg/ml). Then, ALP and ARS staining were performed to identify the effects of Astragaloside for early and late osteogenic capacity of MC3T3-E1 cells, respectively. Real-time PCR and western blot were performed to assess the ALP, OCN, and OSX expression. PI3K/Akt signaling pathway molecules were then assessed by Western blot. Finally, PI3K inhibitor, LY294002, was implemented to assess the mechanism of Astragaloside in promoting osteogenic differentiation of MC3T3-E1 cells. Results Astragaloside significantly increased the cell viability than the control group. Moreover, Astragaloside enhanced the ALP activity and calcium deposition than the control groups. Compared with the control group, Astragaloside increased the ALP, OCN, and OSX expression in a dose-response manner. Western blot assay further confirmed the real-time PCR results. Astragaloside could significantly increase the p-PI3K and p-Akt expression than the control group. LY294002 partially reversed the promotion effects of Astragaloside on osteogenic differentiation of MC3T3-E1 cells. LY294002 partially reversed the promotion effects of Astragaloside on ALP, OCN, and OSX of MC3T3-E1 cells. Conclusion The present study suggested that Astragaloside promoted osteogenic differentiation of MC3T3-E1 cells through regulating PI3K/Akt signaling pathway.

scholarly journals Expression pattern of Wif1 and β-catenin during development of anorectum in fetal rats with anorectal malformations

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4445 ◽  
Author(s):  
Xiao Bing Tang ◽  
Huan Li ◽  
Jin Zhang ◽  
Wei Lin Wang ◽  
Zheng Wei Yuan ◽  
...  
Keyword(s):  
Western Blot ◽  
Real Time ◽  
Expression Pattern ◽  
Immunohistochemical Staining ◽  
Anorectal Malformations ◽  
Rt Pcr ◽  
Rat Embryos ◽  
Fetal Rats ◽  
Normal Rat ◽  
Wnt Inhibitory Factor 1

Purpose This study was performed to investigate the expression pattern of Wnt inhibitory factor 1 (Wif1) and β-catenin during anorectal development in normal and anorectal malformation (ARM) embryos and the possible role of Wif1 and β-catenin in the pathogenesis of ARM. Methods ARM was induced with ethylenethiourea on the 10th gestational day in rat embryos. Cesarean deliveries were performed to harvest the embryos. The expression pattern of Wif1 and β-catenin protein and mRNA was evaluated in normal rat embryos (n = 288) and ARM rat embryos (n = 306) from GD13 to GD16 using immunohistochemical staining, Western blot, and real time RT-PCR. Results Immunohistochemical staining revealed that in normal embryos Wif1 was constantly expressed in the cloaca from GD13 to GD16. On GD13 and GD14, Wif1-immunopositive cells were extensively expressed in the cloaca. On GD15, the expression of Wif1 were mainly detected on the very thin anal membrane. In ARM embryos, the epithelium of the hindgut and urorectal septum demonstrated faint immunostaining for Wif1 from GD14 to GD16. Western blot and real time RT-PCR revealed that Wif1 and β-catenin protein and mRNA expression level was significantly decreased in the ARM groups compared with the normal group on GD14 and GD15 (p < 0.05). Conclusions This study demonstrated that the expression pattern of Wif1 and β-catenin was disrupted in ARM embryos during anorectal morphogenesis, which demonstrated that downregulation of Wif1 and β-catenin at the time of cloacal separation into the primitive rectum and urogenital septum might related to the development of ARM.

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