scholarly journals A Negative Feedback Loop in Ultraviolet A-Induced Senescence in Human Dermal Fibroblasts Formed by SPCA1 and MAPK

2021 ◽  
Vol 8 ◽  
Author(s):  
Hongfu Xie ◽  
Xiao Xiao ◽  
Yuxin Yi ◽  
Mingxing Deng ◽  
Peihui Li ◽  
...  
Keyword(s):  
Negative Feedback ◽  
Feedback Loop ◽  
Secretory Pathway ◽  
Dermal Fibroblasts ◽  
Mitogen Activated Protein Kinase ◽  
Luciferase Reporter ◽  
Negative Feedback Loop ◽  
Human Dermal Fibroblasts ◽  
Mapk Activity ◽  
Factor C

Secretory pathway calcium ATPase 1 (SPCA1) is a calcium pump localized specifically to the Golgi. Its effects on UVA-induced senescence have never been examined. In our study, expression of SPCA1 was increased in UVA-irradiated human dermal fibroblasts (HDFs) by activating mitogen-activated protein kinase (MAPK) and its downstream transcription factor, c-jun. Dual-luciferase reporter and chromatin immunoprecipitation assays revealed that c-jun regulated SPCA1 by binding to its promoter. Furthermore, downregulating SPCA1 with siRNA transfection aggravated UVA-induced senescence due to an elevation of intracellular calcium concentrations and a subsequent increase in reactive oxygen species (ROS) and MAPK activity. In contrast, overexpression of SPCA1 reduced calcium overload, consequently lowering the ROS level and suppressing MAPK activation. This alleviated the cellular senescence caused by UVA irradiation. These results indicated that SPCA1 might exert a protective effect on UVA-induced senescence in HDFs via forming a negative feedback loop. Specifically, activation of MAPK/c-jun triggered by UVA transcriptionally upregulated SPCA1. In turn, the increased SPCA1 lowered the intracellular Ca2+ level, probably through pumping Ca2+ into the Golgi, leading to a reduction of ROS, eventually decreasing MAPK activity and diminishing UVA-induced senescence.

scholarly journals KLF2 inhibits TGF-β-mediated cancer cell motility in hepatocellular carcinoma

2020 ◽  
Vol 52 (5) ◽  
pp. 485-494 ◽  
Author(s):  
Yining Li ◽  
Shuo Tu ◽  
Yi Zeng ◽  
Cheng Zhang ◽  
Tian Deng ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Motility ◽  
Cancer Cell ◽  
Negative Feedback ◽  
Feedback Loop ◽  
Molecular Mechanisms ◽  
Luciferase Reporter ◽  
Negative Feedback Loop ◽  
Liver Malignancy ◽  
Cancer Cell Motility

Abstract Feedback regulation plays a pivotal role in determining the intensity and duration of TGF-β signaling and subsequently affecting the pathophysiological roles of TGF-β, including those in liver malignancy. KLF2, a member of the Krüppel-like factor (KLF) family transcription factors, has been implicated in impeding hepatocellular carcinoma (HCC) development. However, the underlying molecular mechanisms are not fully understood. In the present study, we found that TGF-β stimulates the expression of KLF2 gene in several HCC cell lines. KLF2 protein is able to inhibit TGF-β/Smad signaling in HCC cells as assessed by luciferase reporter assay. Further studies indicated that KLF2 inhibits the transcriptional activity of Smad2/3 and Smad4 and ameliorates TGF-β-induced target gene expression, therefore creating a novel negative feedback loop in TGF-β signaling. Functionally, stably expression of KLF2 in HCCLM3 cells attenuated TGF-β-induced cancer cell motility in wound-healing and transwell assays by interfering with TGF-β-mediated upregulation of MMP2. Together, our results revealed that KLF2 protein has a tumor-suppressive function in HCC through a negative feedback loop over TGF-β signaling.

scholarly journals MiR-29a Inhibits Glioma Tumorigenesis through a Negative Feedback Loop of TRAF4/Akt Signaling

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Yongjie Liu ◽  
Naiquan Duan ◽  
Shibo Duan
Keyword(s):  
Cell Proliferation ◽  
Negative Feedback ◽  
Feedback Loop ◽  
Akt Signaling ◽  
Luciferase Reporter ◽  
Akt Pathway ◽  
Migration And Invasion ◽  
Negative Feedback Loop ◽  
Expression Correlation ◽  
Qrt Pcr

Background. MiR-29a is known as a repressor of human cancer. However, its relevance in glioma proliferation and invasion remains largely unknown. In this study, we aimed to investigate the function and mechanism of miR-29a in glioma tumorigenesis.Methods. The expression of miR-29a was determined by using qRT-PCR. CCK-8, wound healing, and transwell invasion assays were carried out to analyze the effects of miR-29a in glioblastoma cells. qRT-PCR, luciferase reporter, and western blot experiments were done to validate the targeting of TRAF4/Akt pathway by miR-29a. The expression correlation between levels of TRAF4 and miR-29a was analyzed. Regulation of miR-29a expression by enhanced/reduced TRAF4/Akt expression was finally confirmed by qRT-PCR.Results. MiR-29a was decreased in the glioma tissues, especially in those at higher grades. Following its mimic transfection, we validated that miR-29a inhibited cell proliferation, migration, and invasion. Consistently, miR-29a inhibition induced the opposite effects on cell proliferation, migration, and invasion. We confirmed TRAF4 as a direct target of miR-29a, which might mediate the Akt pathway activation. We showed a significantly negative expression correlation between TRAF4 and miR-29a in normal and glioma tissues. Finally we observed an upregulation of miR-29a in TRAF4/Akt activated cells.Conclusion. MiR-29a is critical tumor suppressor for glioma tumorigenesis by forming a negative feedback loop of TRAF4/Akt signaling and represents a potent therapeutic candidate for treating gliomas.

scholarly journals Snail-Modulated MicroRNA 493 Forms a Negative Feedback Loop with the Insulin-Like Growth Factor 1 Receptor Pathway and Blocks Tumorigenesis

2016 ◽  
Vol 37 (6) ◽  
Author(s):  
Arathy S. Kumar ◽  
Sankar Jagadeeshan ◽  
Ravi Shankar Pitani ◽  
Vijayalakshmi Ramshankar ◽  
Kesavan Venkitasamy ◽  
...  
Keyword(s):  
Growth Factor ◽  
Head And Neck ◽  
Negative Feedback ◽  
Feedback Loop ◽  
Mitogen Activated Protein Kinase ◽  
Negative Feedback Loop ◽  
Insulin Like Growth Factor ◽  
Concomitant Increase ◽  
Nicotine Treatment ◽  
Inhibitory Function

ABSTRACT In this study, we have identified one microRNA, microRNA 493 (miR-493), which could simultaneously and directly regulate multiple genes downstream of the insulin-like growth factor 1 receptor (IGF1R) pathway, including IGF1R, by binding with complementary sequences in the 3′ untranslated region (UTR) of mRNAs of IGF1R, insulin receptor substrate 1 (IRS1), and mitogen-activated protein kinase 1 (MAPK1), thereby potentiating their inhibitory function at multiple levels in development and progression of cancers. This binding was further confirmed by pulldown of miR with AGO-2 antibody. Further, results from head and neck samples showed that miR-493 levels were significantly downregulated in tumors, with a concomitant increase in the expression of IGF1R and key downstream effectors. Functional studies from miR-493 overexpression cells and nude-mouse models revealed the tumor suppressor functions of miR-493. Regulation studies revealed that Snail binds to the miR-493 promoter and represses it. We found the existence of a dynamic negative feedback loop in the regulation of IGF1R and miR-493 mediated via Snail. Our study showed that nicotine treatment significantly decreases the levels of miR-493—with a concomitant increase in the levels of Snail—an indication of progression of cells toward tumorigenesis, reestablishing the role of tobacco as a major risk factor for head and neck cancers and elucidating the mechanism behind nicotine-mediated tumorigenesis.

scholarly journals A Direct Negative Feedback Loop of miR-4721/FOXA1/Nanog Promotes Nasopharyngeal Cell Stem Cell Enrichment and Metastasis 

2021 ◽  
Author(s):  
Mengyang Zhao ◽  
Zibo Tang ◽  
Yijun Wang ◽  
Jiaojiao Ding ◽  
Ying Guo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Negative Feedback ◽  
Feedback Loop ◽  
Side Population ◽  
Nasopharyngeal Cancer ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Negative Feedback Loop ◽  
Linkage Mechanism

Abstract Objective: The recurrence and metastasis of nasopharyngeal cancer (NPC) may be mainly attributed to the persistence of cancer stem cells (CSCs); however, the linkage mechanism has yet to be fully elucidated. Methods: The levels of miR-4721, FOXA1, and Nanog expression in NPC were detected by in situ hybridization and immunohistochemistry. In vivo and in vitro metastasis assays confirmed miR-4721 promotes cell migration and invasion. Tumor spheroid formation assay, side population (SP) assay, and ALDEFLUOR assay verified miR-4721 regulates cancer stem cell-like properties. Luciferase reporter assay showed that miR-4721 directly regulates FOXA1 and FOXA1 effects the promoter activity of miR-4721 and Nanog. Chromatin immunoprecipitation (ChIP) analysis and electrophoresis mobility shift assay (EMSA) revealed that FOXA1 combined the promoter region of human miR-4721 and Nanog and the possible mechanism was also analyzed.Results: In this study, a new mechanism of NPC tumorigenesis related to miR-4721 was verified. We found that miR-4721, FOXA1 and Nanog control their expressions through a negative feedback loop and then activate the downstream regulator of stem cell signaling to promote the enrichment and metastasis of NPC stem cells. Conclusion: These findings elucidate that the feedback loop of miR-4721/FOXA1/Nanog can regulate stemness and metastasis in NPC and may provide an experimental theoretical basis for metastasis and treatment resistance in NPC.

scholarly journals A direct negative feedback loop of miR-4721/FOXA1/Nanog promotes nasopharyngeal cell stem cell enrichment and metastasis

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Mengyang Zhao ◽  
Zibo Tang ◽  
Yijun Wang ◽  
Jiaojiao Ding ◽  
Ying Guo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Negative Feedback ◽  
Feedback Loop ◽  
Side Population ◽  
Nasopharyngeal Cancer ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Negative Feedback Loop ◽  
Linkage Mechanism

Abstract Objective The recurrence and metastasis of nasopharyngeal cancer (NPC) may be mainly attributed to the persistence of cancer stem cells (CSCs); however, the linkage mechanism has yet to be fully elucidated. Methods The levels of miR-4721, FOXA1, and Nanog expression in NPC were detected by in situ hybridization and immunohistochemistry. In vivo and in vitro metastasis assays confirmed miR-4721 promotes cell migration and invasion. Tumor spheroid formation assay, side population (SP) assay, and ALDEFLUOR assay verified miR-4721 regulates cancer stem cell-like properties. Luciferase reporter assay showed that miR-4721 directly regulates FOXA1 and FOXA1 effects the promoter activity of miR-4721 and Nanog. Chromatin immunoprecipitation (ChIP) analysis and electrophoresis mobility shift assay (EMSA) revealed that FOXA1 combined the promoter region of human miR-4721 and Nanog and the possible mechanism was also analyzed. Results In this study, a new mechanism of NPC tumorigenesis related to miR-4721 was verified. We found that miR-4721, FOXA1 and Nanog control their expressions through a negative feedback loop and then activate the downstream regulator of stem cell signaling to promote the enrichment and metastasis of NPC stem cells. Conclusion These findings elucidate that the feedback loop of miR-4721/FOXA1/Nanog can regulate stemness and metastasis in NPC and may provide an experimental theoretical basis for metastasis and treatment resistance in NPC.

scholarly journals SUN-718 Reciprocal Regulation of miR-375 and ICER in Pancreatic Beta Cells

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Stephanye Frias ◽  
Ryan Nielsen ◽  
Isis Perez ◽  
Jesse Garcia ◽  
David M Keller
Keyword(s):  
Insulin Secretion ◽  
Beta Cells ◽  
Negative Feedback ◽  
Feedback Loop ◽  
Luciferase Reporter ◽  
Negative Feedback Loop ◽  
Double Negative ◽  
Reporter Assay ◽  
Recognition Element ◽  
Gfp Reporter

Abstract MicroRNA-375 (miR-375) is overexpressed in people with type 2 diabetes (T2D) and has been linked to decreased insulin secretion and beta cell proliferation. Investigation into the transcription factor inducible cAMP early repressor (ICER) as an intermediate regulator of miR-375 was proposed because both are regulated by the cAMP pathway. This overexpression of miR-375 in T2D led us to hypothesize that beta cells with elevated and reduced levels of miR-375 will result in decreased and increased glucose-stimulated insulin secretion (GSIS), respectively. Results showed that when miR-375 was overexpressed, GSIS decreased by 61% when compared to a control in 25 mM glucose. Results showed that when miR-375 was inhibited, GSIS increased 6% when compared to a control in 25 mM glucose. In human islets, we found that inhibiting miR-375 led to an average 19% increase in GSIS, though due to the variability of human tissue these data were not significant (N=5). To investigate ICER’s binding affinity to the miR-375 promoter, a luciferase reporter assay was conducted. HEK-293 (human embryonic kidney) cells that were transfected with a luciferase reporter plasmid containing a cAMP recognition element (CRE) and a plasmid driving the overexpression of ICER had a 75% decrease when compared to our control (P<0.05). When transiently-expressed ICER was knocked down via siRNA, promoter activity increased by 13.1-fold (P< 0.05). Using a chromatin immunoprecipitation assay we found that an ICER antibody pulled down the rat miR-375 promoter an average of 13-fold compared with a control antibody (N=2). Additionally, because of a sequence alignment showing possible binding of miR-375 to the human ICER transcript we hypothesize that the two are in a negative feedback loop and can regulate each other’s expression. To investigate the double negative feedback loop a plasmid was constructed containing the GFP reporter gene and either the human or rodent ICER 3’UTR predicted miR-375 binding site. The GFP reporter assay was conducted to determine if miR-375 binds to ICER’s microRNA recognition element (MRE) in a species-specific way. In our GFP reporter experiment, data shows there is a 50% reporter gene decrease between our negative control and the human ICER MRE (N=4, P=0.017). Understanding microRNA gene regulation in the pancreas may have important implications for patients with T2D. MiR-375 may have the ability to interact with human ICER in a double negative feedback loop in the cAMP second messenger pathway, which will further clarify cellular mechanisms to potentially improve T2D drugs.

scholarly journals Long Non-Coding RNA LINC00518 Induces Radioresistance by Regulating Glycolysis Through An miR-33a-3p/HIF-1α Negative Feedback Loop in Melanoma

2020 ◽  
Author(s):  
Ke Cao ◽  
Liu yan ◽  
He Dong ◽  
Xiao Mengqin ◽  
Xiang Liang ◽  
...  
Keyword(s):  
Cancer Progression ◽  
Negative Feedback ◽  
Feedback Loop ◽  
Luciferase Reporter ◽  
Negative Feedback Loop ◽  
Reporter System ◽  
Non Coding Rna ◽  
Long Non Coding Rna

Abstract Background: The long non-coding RNA (lncRNA),LINC00518, is highly expressed in many human cancers and is involved in cancer progression. However, the potential function and regulatory mechanism of LINC00518 in cutaneous malignant melanoma (CMM) remain unclear. Methods:Short hairpin RNA (shRNA) was used to silence LINC00518 and HIF-1α, and real-time PCR was performed to determine mRNA expression. Then, cell proliferation, colony formation, flow cytometric, scratch, and transwell assays were to examine the influence of LINC00518 silencing on cellular radiosensitivity. Dual luciferase reporter system,CHIP and COIP was used to verify the target relationship between LINC00518,miR‐33a-5b and HIF-1α,.Glycolysis assays were conducted to exam cell glycolysis process. Western blotting was performed to explore the expression of HIF-1α and LDHA. Finally, animal experiments were performed to demonstrate the effect of LINC00518 silencing on the radiosensitivity of melanoma in vivo.Results: LINC00518 expression was significantly upregulated in CMM samples, and LINC00518 levels were associated with poor prognosis of patients with CMM. Knockdown of LINC00518 in CMM cells significantly inhibited cell invasion, migration, proliferation, and clonogenicity. LINC00518-mediated invasion, migration, proliferation, and clonogenicity were negatively regulated by the microRNA, miR-33a-3p, in vitro, which intensified sensitivity to radiotherapy via inhibition of the hypoxia-induced factor 1α (HIF-1α)/lactate dehydrogenase A (LDHA)-glycolysis axis. Additionally, HIF-1α recognized the miR-33a-3p promoter region and recruited histone deacetylase2 (HDAC2), which decreased the expression of miR-33a-3p and formed an LINC00518/miR-33a-3p/HIF-1α negative feedback loop. Furthermore, signalling initially activated glycolysis and radioresistance in CMM cells was recovered by Santacruzamate A (a histone deacetylase inhibitor) and 2-deoxy-D-glucose (a glycolytic inhibitor). Lastly, knockdown of LINC00518 expression sensitized CMM cancer cells to radiotherapy in an in vivo subcutaneously implanted tumour model. Conclusion: LINC00518 was confirmed to be an oncogene in CMM, which induces radioresistance by regulating glycolysis through an miR-33a-3p/HIF-1α negative feedback loop. Our research may provide a potential strategy to improve the treatment outcome of radiotherapy in CMM.

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