Positive Feedback Regulatory Loop Research Articles

Positive Feedback Regulation between KLF5 and XPO1 Promotes Cell Cycle Progression of Basal like Breast Cancer.

Basal-like breast cancer (BLBC), overlapping with the subgroup of estrogen receptor (ER), progesterone receptor (PR), and HER2 triple-negative breast cancer, has the worst prognosis and limited therapeutics. The XPO1 gene encodes nuclear export protein 1, a promising anticancer target which mediates nucleus-cytoplasm transport of nuclear export signal containing proteins such as tumor suppressor RB1 and some RNAs. Despite drugs targeting XPO1 are used in clinical, the regulation of XPO1 expression and functional mechanism is poorly understood, especially in BLBC. This study finds that KLF5 is a transcription factor of XPO1, which increases RB1 nuclear export and cell proliferation in BLBC cells. Furthermore, XPO1 interacts with the RNA-binding protein PTBP1 to export FOXO1 mRNA to cytoplasm and thus activates the FOXO1-KLF5 axis as a feedback. This work demonstrates that XPO1 inhibitor KPT-330 in combination with CDK4/6 inhibitor additively suppressed BLBC tumor growth in vivo. These results reveal a novel positive feedback regulation loop between KLF5 and XPO1 and provide a novel treatment strategy for BLBC.

M6A-modified circSTX6 as a key regulator of cervical cancer malignancy via SPI1 and IL6/JAK2/STAT3 pathways.

In recent years, circRNAs have garnered increasing attention for their role in cervical cancer. However, the functions of many newly identified circRNAs remain unclear and require further exploration. In this study, we investigated the expression and oncogenic potential of the novel circRNA circSTX6 in cervical cancer. Our findings revealed that circSTX6 is highly expressed in cervical cancer (CC) and is significantly associated with poor patient prognosis, promoting cell survival, proliferation, invasion, and migration. Mechanistically, circSTX6 enhances the stability of the transcription factor SPI1 by binding to it, thereby upregulating IL6 transcription and activating the JAK2/STAT3 signaling pathway. Additionally, METTL3-mediated N6-methyladenosine (m6A) modification stabilizes circSTX6 through recognition by YTHDC1, forming a positive feedback regulatory loop among METTL3, circSTX6, and SPI1. These findings not only deepen our understanding of the biological mechanisms underlying CC but also highlight circSTX6 as a potential target for molecular therapies.

PRDX2 promotes gastric cancer progression by forming a feedback loop with PKM2/STAT3 axis.

Peroxiredoxin 2 (PRDX2) is an antioxidant enzyme that has been reported to be overexpressed in various cancers. However, the role of PRDX2 in gastric cancer progression and its underlying mechanism remains unclear. Herein, we revealed the function of PRDX2 in gastric cancer progression and explored its molecule mechanism. We identified that PRDX2 was upregulated and associated with poor prognosis in gastric cancer. The knockdown of PRDX2 inhibited the proliferation, migration and invasion of gastric cancer cells in vitro and suppressed tumor growth in vivo. Mechanistically, PRDX2 interacted with PKM2 (pyruvate kinase isozyme type M2) and protected PKM2 from ubiquitination and degradation, which enhanced glycolysis in gastric cancer cells. The interaction between PRDX2 and PKM2 also enhanced the binding affinity between PKM2 and importin α5, which induced PKM2 nuclear translocation and activated STAT3 signaling pathway. In addition, STAT3 (signal transducer and activator of transcription 3) was identified to bind to PRDX2 gene promoter and upregulate PRDX2 expression, which forms a positive regulatory feedback loop in gastric cancer cells. The present study unravels the biological role of PRDX2 in cancer progression and illustrates the underlying molecular mechanism, which may provide a potential therapeutic target for gastric cancer.

The abscisic acid-responsive transcriptional regulatory module CsERF110–CsERF53 orchestrates citrus fruit coloration

Carotenoid biosynthesis is closely associated with abscisic acid (ABA) during the ripening process of non-climacteric fruits, but the regulatory mechanism that links ABA signaling to carotenoid metabolism remains largely unclear. Here, we identified two master regulators of ABA-mediated citrus fruit coloration, CsERF110 and CsERF53, which activate the expression of carotenoid metabolism genes (CsGGPPS, CsPSY, CsPDS, CsCRTISO, CsLCYB2, CsLCYE, CsHYD, CsZEP, and CsNCED2) to facilitate carotenoid accumulation. Further investigations showed that CsERF110 not only activates the expression of CsERF53 by binding to its promoter but also interacts with CsERF53 to form the transcriptional regulatory module CsERF110–CsERF53. We also discovered a positive feedback regulatory loop between the ABA signal and carotenoid metabolism regulated by the transcriptional regulatory module CsERF110–CsERF53. Our results reveal that the CsERF110–CsERF53 module responds to ABA signaling, thereby orchestrating citrus fruit coloration. Considering the importance of carotenoid content for citrus and many other carotenoid-rich crops, the revelation of molecular mechanisms that underlie ABA-mediated carotenoid biosynthesis in plants will facilitate the development of transgenic/gene-editing approaches, further contributing to improving the quality of citrus and other carotenoid-rich crops.

RNA m5C modification upregulates E2F1 expression in a manner dependent on YBX1 phase separation and promotes tumor progression in ovarian cancer

5-Methylcytosine (m5C) is a common RNA modification that modulates gene expression at the posttranscriptional level, but the crosstalk between m5C RNA modification and biomolecule condensation, as well as transcription factor-mediated transcriptional regulation, in ovarian cancer, is poorly understood. In this study, we revealed that the RNA methyltransferase NSUN2 facilitates mRNA m5C modification and forms a positive feedback regulatory loop with the transcription factor E2F1 in ovarian cancer. Specifically, NSUN2 promotes m5C modification of E2F1 mRNA and increases its stability, and E2F1 binds to the NSUN2 promoter, subsequently reciprocally activating NSUN2 transcription. The RNA binding protein YBX1 functions as the m5C reader and is involved in NSUN2-mediated E2F1 regulation. m5C modification promotes YBX1 phase separation, which upregulates E2F1 expression. In ovarian cancer, NSUN2 and YBX1 are amplified and upregulated, and higher expression of NSUN2 and YBX1 predicts a worse prognosis for ovarian cancer patients. Moreover, E2F1 transcriptionally regulates the expression of the oncogenes MYBL2 and RAD54L, driving ovarian cancer progression. Thus, our study delineates a NSUN2-E2F1-NSUN2 loop regulated by m5C modification in a manner dependent on YBX1 phase separation, and this previously unidentified pathway could be a promising target for ovarian cancer treatment.

A novel PDIA3/FTO/USP20 positive feedback regulatory loop induces osteogenic differentiation of preosteoblast in osteoporosis.

Osteoporosis is a chronic skeletal disease and the major source of risk for fractures in aged people. It is urgent to investigate the mechanism regulating osteoporosis for developing potential treatment and prevention strategies. Osteogenic differentiation of preosteoblast enhances bone formation, which might be a promising strategy for treatment and prevention of osteoporosis. Protein disulfide isomerase family A, member 3 (PDIA3) could induce bone formation, yet the role of PDIA3 in osteogenic differentiation of preosteoblast remains unknown. In this study, m6 A RNA methylation was detected by methylated RNA immunoprecipitation (MeRIP), while mRNA stability was identified by RNA decay assay. Besides, protein-protein interaction and protein phosphorylation were determined using co-immunoprecipitation (Co-IP). Herein, results revealed that PDIA3 promoted osteogenic differentiation of preosteoblast MC3T3-E1. Besides, PDIA3 mRNA methylation was suppressed by FTO alpha-ketoglutarate dependent dioxygenase (FTO) as RNA methylation reduced PDIA3 mRNA stability during osteogenic differentiation of MC3T3-E1 cells. Moreover, ubiquitin specific peptidase 20 (USP20) improved FTO level through inhibiting FTO degradation while PDIA3 increased FTO level by enhancing USP20 phosphorylation during osteogenic differentiation of MC3T3-E1 cells, suggesting a positive feedback regulatory loop between PDIA3 and FTO. In summary, these findings indicated the mechanism of PDIA3 regulating osteogenic differentiation of preosteoblast and provided potential therapeutic targets for osteoporosis.

Carboxypeptidase A4 negatively regulates HGS-ETR1/2-induced pyroptosis by forming a positive feedback loop with the AKT signalling pathway

Pyroptosis, a mode of inflammatory cell death, has recently gained significant attention. However, the underlying mechanism remains poorly understood. HGS-ETR1/2 is a humanized monoclonal antibody that can bind to DR4/5 on the cell membrane and induce cell apoptosis by activating the death receptor signalling pathway. In this study, by using morphological observation, fluorescence double staining, LDH release and immunoblot detection, we confirmed for the first time that HGS-ETR1/2 can induce GSDME-mediated pyroptosis in hepatocellular carcinoma cells. Our study found that both inhibition of the AKT signalling pathway and silencing of CPA4 promote pyroptosis, while the overexpression of CPA4 inhibits it. Furthermore, we identified a positive regulatory feedback loop is formed between CPA4 and AKT phosphorylation. Specifically, CPA4 modulates AKT phosphorylation by regulating the expression of the AKT phosphatase PP2A, while inhibition of the AKT signalling pathway leads to a decreased transcription and translation levels of CPA4. Our study reveals a novel mechanism of pyroptosis induced by HGS-ETR1/2, which may provide a crucial foundation for future investigations into cancer immunotherapy.

VPS35 promotes gastric cancer progression through integrin/FAK/SRC signalling-mediated IL-6/STAT3 pathway activation in a YAP-dependent manner

VPS35 is a key subunit of the retromer complex responsible for recognising cytosolic retrieval signals in cargo and is involved in neurodegenerative disease and tumour progression. However, the function and molecular mechanism of VPS35 in gastric cancer (GC) remains largely unknown. Here, we demonstrated that VPS35 was significantly upregulated in GC, which was associated with poor survival. VPS35 promoted GC cell proliferation and metastasis both in vitro and in vivo. Mechanistically, VPS35 activated FAK-SRC kinases through integrin-mediated outside-in signalling, leading to the activation of YAP and subsequent IL-6 expression induction in tumour cells. What’s more, combined mass spectrometry analysis of MGC-803 cell and bioinformatic analysis, we found that phosphorylation of VPS35 was enhanced in GC cells, and phosphorylated VPS35 has enhanced interaction with ITGB3. VPS35 interacted with ITGB3 and affected the recycling of ITGB3 in GC cells. Gain- and loss-of-function experiments revealed that VPS35 promoted tumour proliferation and metastasis via the IL-6/STAT3 pathway. Interestingly, we also found that STAT3 directly bound to the VPS35 promoter and increased VPS35 transcription, thereby establishing a positive regulatory feedback loop. In addition, we demonstrated that VPS35 knockdown sensitised GC cells to 5-FU and cisplatin. These findings provide evidence that VPS35 promotes tumour proliferation and metastasis, and highlight the potential of targeting VPS35- and IL-6/STAT3-mediated tumour interactions as promising therapeutic strategies for GC.

The transcriptional regulatory module CsHB5-CsbZIP44 positively regulates abscisic acid-mediated carotenoid biosynthesis in citrus (Citrus spp.).

Carotenoids contribute to fruit coloration and are valuable sources of provitamin A in the human diet. Abscisic acid (ABA) plays an essential role in fruit coloration during citrus fruit ripening, but little is known about the underlying mechanisms. Here, we identified a novel bZIP transcription activator called CsbZIP44, which serves as a central regulator of ABA-mediated citrus carotenoid biosynthesis. CsbZIP44 directly binds to the promoters of four carotenoid metabolism-related genes (CsDXR, CsGGPPs, CsBCH1 and CsNCED2) and activates their expression. Furthermore, our research indicates that CsHB5, a positive regulator of ABA and carotenoid-driven processes, activates the expression of CsbZIP44 by binding to its promoter. Additionally, CsHB5 interacts with CsbZIP44 to form a transcriptional regulatory module CsHB5-CsbZIP44, which is responsive to ABA induction and promotes carotenoid accumulation in citrus. Interestingly, we also discover a positive feedback regulation loop between the ABA signal and carotenoid biosynthesis mediated by the CsHB5-CsbZIP44 transcriptional regulatory module. Our findings show that CsHB5-CsbZIP44 precisely modulates ABA signal-mediated carotenoid metabolism, providing an effective strategy for quality improvement of citrus fruit and other crops.

Paracrine activin B-NF-κB signaling shapes an inflammatory tumor microenvironment in gastric cancer via fibroblast reprogramming

BackgroundImportant roles of INHBB in various malignancies are increasingly identified. The underlying mechanisms in gastric cancer (GC) microenvironment are still greatly unexplored.MethodsThe clinical significance of INHBB and the correlation between INHBB and p-p65 in GC were assessed through analyzing publicly available databases and human paraffin embedded GC tissues. The biological crosstalk of INHBB between GC cells and fibroblasts was explored both in vitro and in vivo. RNA-seq analyses were performed to determine the mechanisms which regulating fibroblasts reprogramming. Luciferase reporter assay and chromatin immunoprecipitation (CHIP) assay were used to verify the binding relationship of p65 and INHBB in GC cells.ResultsOur study showed that INHBB level was significantly higher in GC, and that increased INHBB was associated with poor survival. INHBB positively regulates the proliferation, migration, and invasion of GC cells in vitro. Also, activin B promotes the occurrence of GC by reprogramming fibroblasts into cancer-associated fibroblasts (CAFs). The high expression of INHBB in GC cells activates the NF-κB pathway of normal gastric fibroblasts by secreting activin B, and promotes fibroblasts proliferation, migration, and invasion. In addition, activin B activates NF-κB pathway by controlling TRAF6 autoubiquitination to induce TAK1 phosphorylation in fibroblasts. Fibroblasts activated by activin B can induce the activation of p65 phosphorylation of GC cells by releasing pro-inflammatory factors IL-1β. p65 can directly bind to the INHBB promoter and increase the INHBB transcription of GC cells, thus establishing a positive regulatory feedback loop to promote the progression of GC.ConclusionsGC cells p65/INHBB/activin B and fibroblasts p65/IL-1β signal loop led to the formation of a whole tumor-promoting inflammatory microenvironment, which might be a promising therapeutic target for GC.

The NONRATT023402.2/rno-miR-3065-5p/NGFR axis affects levodopa-induced dyskinesia in a rat model of Parkinson’s disease

Levodopa-induced dyskinesia (LID) is a common motor complication in Parkinson’s disease. However, few studies have focused on the pathogenesis of LID at the transcriptional level. NONRATT023402.2, a long non-coding RNA (lncRNA) that may be related to LID was discovered in our previous study and characterized in rat models of LID. In the present study, NONRATT023402.2 was overexpressed by injection of adeno-associated virus (AAV) in striatum of LID rats, and 48 potential target genes, including nerve growth factor receptor (NGFR) were screened using next-generation sequencing and target gene predictions. The NONRATT023402.2/rno-miR-3065-5p/NGFR axis was verified using a dual luciferase reporter gene. Overexpression of NONRATT023402.2 significantly increased the abnormal involuntary movements (AIM) score of LID rats, activated the PI3K/Akt signaling pathway, and up-regulated c-Fos in the striatum. NGFR knockdown by injection of ShNGFR-AAV into the striatum of LID rats resulted in a significant decrease in the PI3K/Akt signaling pathway and c-Fos expression. The AIM score of LID rats was positively correlated with the expressions of NONRATT023402.2 and NGFR. A dual luciferase reporter assay showed that c-Fos, as a transcription factor, bound to the NONRATT023402.2 promoter and activated its expression. Together, the results showed that NONRATT023402.2 regulated NGFR expression via a competing endogenous RNA mechanism, which then activated the PI3K/Akt pathway and promoted c-Fos expression. This suggested that c-Fos acted as a transcription factor to activate NONRATT023402.2 expression, and form a positive feedback regulation loop in LID rats, thus, aggravating LID symptoms. NONRATT023402.2 is therefore a possible novel therapeutic target for LID.

SLC26A3/NHERF2-IκB/NFκB/p65 feedback loop suppresses tumorigenesis and metastasis in colorectal cancer

Colorectal cancer (CRC) is a formidable disease due to the intricate mechanisms that drive its proliferation and metastasis. Despite significant progress in cancer research, the integration of these mechanisms that influence cancer cell behavior remains elusive. Therefore, it is imperative to comprehensively elucidate the underlying mechanisms driving CRC proliferation and metastasis. In this study, we reported a novel role of SLC26A3 in suppressing CRC progression. We found that SLC26A3 expression was downregulated in CRC, which was proportionally correlated with survival. Our in vivo and in vitro experiments demonstrated that up-regulation of SLC26A3 inhibited CRC proliferation and metastasis, while down-regulation of SLC26A3 promoted CRC progression by modulating the expression level of IκB. Furthermore, we identified NHERF2 as a novel interacting protein of SLC26A3 responsible for stabilizing the IκB protein and removing ubiquitination modification. Mechanistically, SLC26A3 augmented the interaction between NHERF2 and IκB, subsequently reducing its degradation. This process inhibited the dissociation of p65 from the IκB/p65/p50 complex and reduced the translocation of p65 from the cytoplasm to the nucleus. Moreover, our investigation revealed that NF-κB/p65 directly bound to the promoter of SLC26A3, leading to a decline in its mRNA expression. Thus, SLC26A3 impeded the nuclear translocation of NF-κB/p65, enhancing the transcription of SLC26A3 and establishing a positive regulatory feedback loop in CRC cells. Collectively, these results suggest that a SLC26A3/NHERF2-IκB/NF-κB/p65 signaling loop suppresses proliferation and metastasis in CRC cells. These findings propose a novel SLC26A3-driven signaling loop that regulates proliferation and metastasis in CRC, providing promising therapeutic interventions and prognostic targets for the management of CRC.

MiR‑27a‑3p upregulation by p65 facilitates cervical tumorigenesis by increasing TAB3 expression and is involved in the positive feedback loop of NF‑κB signaling.

An altered microRNA (miRNA/miR)‑27a‑3p expression has been identified in cervical cancer, while the exact regulatory mechanisms responsible for the dysregulation of miR‑27a‑3p remain to be fully elucidated. In the present study, a NF‑κB/p65 binding site was identified upstream of the miR‑23a/27a/24‑2 cluster and p65 binding enhanced the transcription of pri‑miR‑23a/27a/24‑2, as well as the expression levels of mature miRNAs, including miR‑27a‑3p in HeLa cells. Mechanistically, using bioinformatics analyses and experimental validation, TGF‑β activated kinase 1 binding protein 3 (TAB3) was identified as a direct target of miR‑27a‑3p. By binding to the 3'UTR of TAB3, miR‑27a‑3p significantly enhanced TAB3 expression. Functionally, it was found that the overexpression of miR‑27a‑3p and TAB3 promoted the malignant potential of cervical cancer cells, as evaluated using cell growth, migration and invasion assays, and specific cell marker determinations in the epithelial mesenchymal transition progression, and vice versa. Further rescue experiments revealed that the enhanced malignant effects induced by miR‑27a‑3p were mediated via its upregulation of TAB3 expression. Moreover, miR‑27a‑3p and TAB3 also activated the NF‑κB signaling pathway and formed a positive feedback regulatory loop composing of p65/miR‑27a‑3p/TAB3/NF‑κB. On the whole, the findings presented herein may provide novel insight into the underlying cervical tumorigenesis and novel biomarker identification for clinical applications.

Modulation of anthocyanin accumulation in storage roots of sweetpotato by transcription factor IbMYB1-2 through direct binding to anthocyanin biosynthetic gene promoters

The storage roots of purple-fleshed sweetpotato rich in anthocyanins are considered nutrient-rich foods with health effects. However, the molecular mechanism underlying anthocyanin biosynthesis and regulation remains to be revealed. In this study, IbMYB1-2 was isolated from purple-fleshed sweetpotato “Xuzishu8”. The phylogenetic and sequence analysis indicated that IbMYB1-2 belongs to the SG6 subfamily with a conserved bHLH motif. Subcellular localization analysis and transcriptional activity assay revealed that IbMYB1-2 is a key transcriptional activator and is specific to the nucleus. Agrobacterium rhizogenes-mediated overexpression of IbMYB1-2 in sweetpotato through in vivo root transgenic system led to an increase in anthocyanins in the root of sweetpotato. qRT-PCR and transcriptome analysis depicted that the transcript levels of IbMYB1-2, IbbHLH42, and eight structural genes that are associated with the synthesis of anthocyanin were upregulated in overexpressed IbMYB1-2 transgenic roots. Dual-luciferase reporter (DLR) assay and yeast one-hybrid (Y1H) assay demonstrated IbMYB1-2 binding to the promoter regions of IbbHLH42 and other anthocyanin biosynthetic genes, including IbCHS, IbCHI, IbF3H, IbDFR, IbANS, IbGSTF12, IbUGT78D2, and IbUF3GT. Moreover, IbbHLH42 was shown to be an active enhancer for the formation of MYB-bHLH-WD40 (MBW) complex, which strongly supports the promoter activities of the IbCHS, IbANS, IbUGT78D2, and IbGSTF12 genes to induce anthocyanin accumulation. Taken together, our findings not only revealed the underlying regulatory molecular mechanism of IbMYB1-2 for anthocyanin accumulation in the storage roots of sweetpotato but also uncovered a potential mechanism by which IbbHLH42 modulated anthocyanin biosynthesis through a positive feedback regulatory loop.

GhWRKY41 forms a positive feedback regulation loop and increases cotton defence response against Verticillium dahliae by regulating phenylpropanoid metabolism.

Despite the established significance of WRKY proteins and phenylpropanoid metabolism in plant immunity, how WRKY proteins modulate aspects of the phenylpropanoid pathway remains undetermined. To understand better the role of WRKY proteins in plant defence, we identified a cotton (Gossypium hirsutum) protein, GhWRKY41, that is, universally and rapidly induced in three disease-resistant cotton cultivars following inoculation with the plant pathogenic fungus, Verticillium dahliae. We show that overexpression of GhWRKY41 in transgenic cotton and Arabidopsis enhances resistance to V. dahliae, while knock-down increases cotton more susceptibility to the fungus. GhWRKY41 physically interacts with itself and directly activates its own transcription. A genome-wide chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq), in combination with RNA sequencing (RNA-seq) analyses, revealed that 43.1% of GhWRKY41-binding genes were up-regulated in cotton upon inoculation with V. dahliae, including several phenylpropanoid metabolism master switches, receptor kinases, and disease resistance-related proteins. We also show that GhWRKY41 homodimer directly activates the expression of GhC4H and Gh4CL, thereby modulating the accumulation of lignin and flavonoids. This finding expands our understanding of WRKY-WRKY protein interactions and provides important insights into the regulation of the phenylpropanoid pathway in plant immune responses by a WRKY protein.

A Monoclonal Antibody That Provides a Model for C3 Nephritic Factors.

Complement is a major innate defense system that protects the intravascular space from microbial invasion. Complement activation results in the assembly of C3 convertases, serine proteases that cleave complement protein C3, generating bioactive fragments C3a and C3b. The complement response is rapid and robust, largely due to a positive feedback regulatory loop mediated by alternative pathway (AP) C3 convertase. C3 nephritic factors (C3NEFs) are autoantibodies that stabilize AP convertase, resulting in uncontrolled C3 cleavage, which, in principle, can promote critical tissue injury similar to that seen in certain renal conditions. Investigations of C3NEFs are hampered by a challenging issue: each C3NEF is derived from a different donor source, and there is no method to compare one C3NEF to another. We have identified a widely available mouse anti-C3 mAb that, similar to many C3NEFs, can stabilize functional AP convertase in a form resistant to decay acceleration by multiple complement regulators. The antibody requires the presence of properdin to confer convertase stability, and hampers the activity of Salp20, a tic salivary protein that accelerates convertase dissociation by displacing properdin from the convertase complex. This mAb can serve as an urgently needed standard for the investigation of C3NEFs. This study also provides novel insights into the dynamics of AP convertase.

Activation of Piezo1 contributes to matrix stiffness-induced angiogenesis in hepatocellular carcinoma.

Despite integrin being highlighted as a stiffness-sensor molecule in matrix stiffness-driven angiogenesis, other stiffness-sensor molecules and their mechanosensory pathways related to angiogenesis in hepatocellular carcinoma (HCC) remain obscure. Here, we explored the interplay between Piezo1 and integrin β1 in the mechanosensory pathway and their effects on HCC angiogenesis to better understand matrix stiffness-induced angiogenesis. The role of Piezo1 in matrix stiffness-induced angiogenesis was investigated using orthotopic liver cancer SD rat models with high liver stiffness background, and its clinical significance was evaluated in human HCC tissues. Matrix stiffness-mediated Piezo1 upregulation and activation were assayed using an in vitro fibronectin (FN)-coated cell culture system with different stiffness, Western blotting and Ca2+ probe. The effects of shPiezo1-conditioned medium (CM) on angiogenesis were examined by tube formation assay, wound healing assay and angiogenesis array. The underlying mechanism by which Piezo1 participated in matrix stiffness-induced angiogenesis was analyzed by microRNA quantitative real-time polymerase chain reaction (qRT-PCR), matrix stiffness measurement, dual-luciferase reporter assay, ubiquitination assay and co-immunoprecipitation. Increased matrix stiffness significantly upregulated Piezo1 expression at both cellular and tissue levels, and high expression of Piezo1 indicated an unfavorable prognosis. High matrix stiffness also noticeably enhanced the activation level of Piezo1, similar to its expression level. Piezo1 knockdown significantly suppressed tumor growth, angiogenesis, and lung metastasis of HCC rat models with high liver stiffness background. shPiezo1-CM from HCC cells attenuated tube formation and migration abilities of vascular endothelial cells remarkably, and analysis of differentially expressed pro-angiogenic factors revealed that Piezo1 promoted the expression and secretion of vascular endothelial growth factor (VEGF), CXC chemokine ligand 16 (CXCL16) and insulin-like growth factor binding protein 2 (IGFBP2). Matrix stiffness-caused Piezo1 upregulation/activation restrained hypoxia inducible factor-1α (HIF-1α) ubiquitination, subsequently enhanced the expression of downstream pro-angiogenic factors to accelerate HCC angiogenesis. Besides, collagen 1 (COL1)-reinforced tissue stiffening resulted in more expression of Piezo1 via miR-625-5p. This study unravels a new mechanism by which the integrin β1/Piezo1 activation/Ca2+ influx/HIF-1α ubiquitination/VEGF, CXCL16 and IGFBP2 pathway participates in matrix stiffness-driven HCC angiogenesis. Simultaneously, a positive feedback regulation loop as stiff matrix/integrin β1/miR-625-5p/Piezo1 and COL1/stiffer matrix mediates matrix stiffness-caused Piezo1 upregulation.

The positive regulatory loop of TCF4N/p65 promotes glioblastoma tumourigenesis and chemosensitivity.

BackgroundNF‐κB signaling is widely linked to the pathogenesis and treatment resistance in cancers. Increasing attention has been paid to its anti‐oncogenic roles, due to its key functions in cellular senescence and the senescence‐associated secretory phenotype (SASP). Therefore, thoroughly understanding the function and regulation of NF‐κB in cancers is necessary prior to the application of NF‐κB inhibitors.MethodsWe established glioblastoma (GBM) cell lines expressing ectopic TCF4N, an isoform of the β‐catenin interacting transcription factor TCF7L2, and evaluated its functions in GBM tumorigenesis and chemotherapy in vitro and in vivo. In p65 knock‐out or phosphorylation mimic (S536D) cell lines, the dual role and correlation of TCF4N and NF‐κB signaling in promoting tumorigenesis and chemosensitivity was investigated by in vitro and in vivo functional experiments. RNA‐seq and computational analysis, immunoprecipitation and ubiquitination assay, minigene splicing assay and luciferase reporter assay were performed to identify the underlying mechanism of positive feedback regulation loop between TCF4N and the p65 subunit of NF‐κB. A eukaryotic cell‐penetrating peptide targeting TCF4N, 4N, was used to confirm the therapeutic significance.ResultsOur results indicated that p65 subunit phosphorylation at Ser 536 (S536) and nuclear accumulation was a promising prognostic marker for GBM, and endowed the dual functions of NF‐κB in promoting tumorigenesis and chemosensitivity. p65 S536 phosphorylation and nuclear stability in GBM was regulated by TCF4N. TCF4N bound p65, induced p65 phosphorylation and nuclear translocation, inhibited its ubiquitination/degradation, and subsequently promoted NF‐κB activity. p65 S536 phosphorylation was essential for TCF4N‐led senescence‐independent SASP, GBM tumorigenesis, tumor stem‐like cell differentiation and chemosensitivity. Activation of p65 was closely connected to alterative splicing of TCF4N, a likely positive feedback regulation loop between TCF4N and p65 in GBM. 4N increased chemosensitivity, highlighting a novel anti‐cancer strategy.ConclusionOur study defined key roles of TCF4N as a novel regulator of NF‐κB through mutual regulation with p65 and provided a new avenue for GBM inhibition.

A positive feedback regulatory loop, SA-AtNAP-SAG202/SARD1-ICS1-SA, in SA biosynthesis involved in leaf senescence but not defense response

Salicylic acid (SA) is an important plant hormone that regulates defense responses and leaf senescence. It is imperative to understand upstream factors that regulate genes of SA biosynthesis. SAG202/SARD1 is a key regulator for isochorismate synthase 1 (ICS1) induction and SA biosynthesis in defense responses. The regulatory mechanism of SA biosynthesis during leaf senescence is not well understood. Here we show that AtNAP, a senescence-specific NAC family transcription factor, directly regulates a senescence-associated gene named SAG202 as revealed in yeast one-hybrid and in planta assays. Inducible overexpreesion of AtNAP and SAG202 lead to high levels of SA and precocious senescence in leaves. Individual knockout mutants of sag202 and ics1 have markedly reduced SA levels and display a significantly delayed leaf senescence phenotype. Furthermore, SA positively feedback regulates AtNAP and SAG202. Our research has uncovered a unique positive feedback regulatory loop, SA-AtNAP-SAG202-ICS1-SA, that operates to control SA biosynthesis associated with leaf senescence but not defense response.

The lncARSR/PTEN/Akt/nuclear factor-kappa B feedback regulatory loop contributes to doxorubicin resistance in hepatocellular carcinoma.

Chemoresistance is a major obstacle to hepatocellular carcinoma (HCC) chemotherapy. Our previous study found that long noncoding RNA lncARSR (lncRNA Activated in RCC with Sunitinib Resistance) activated Akt signaling via repressing phosphatase and tensin homolog (PTEN) during doxorubicin resistance in HCC. The purpose of this study is to further explore lncARSR-mediated mechanisms and roles during doxorubicin resistance in HCC. The expression of lncARSR was detected by real-time quantitative polymerase chain reaction (qPCR). Nuclear factor-kappa B (NF-κB) activity was detected by NF-κB luciferase reporter assays, western blot, and NF-κB transcription factor assays. The effects of NF-κB on lncARSR were detected by chromatin immunoprecipitation assay, promoter luciferase reporter assay, and real-time qPCR. The effects of lncARSR/Akt/NF-κB on doxorubicin resistance were detected by Cell Counting Kit-8 assay, capsase-3 activity assay, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelingassay. lncARSR activated NF-κB signaling through activation of Akt. NF-κB transactivated lncARSR through directly binding lncARSR promoter and increasing lncARSR promoter activity. Akt transactivated lncARSR via activating NF-κB signaling. Thus, lncARSR, Akt, and NF-κB formed a positive feedback regulatory loop in HCC. Through this feedback loop, lncARSR auto-regulated its transcription. Drug sensitivity assays showed that the lncARSR/Akt/NF-κB feedback regulatory loop promoted doxorubicin resistance in HCC. These findings identified the lncARSR/Akt/NF-κB feedback regulatory loop in HCC, which represent potential therapeutic targets for improving doxorubicin sensitivity in HCC.