Circ-calm4 regulates hypoxia-induced pulmonary artery smooth muscle autophagy by binding Purb.

Pulmonary hypertension (PH) is a serious and fatal disease characterized by pulmonary vasoconstriction and pulmonary vascular remodeling. The excessive autophagy of pulmonary artery smooth muscle cells (PASMCs) is one of the important factors of pulmonary vascular remodeling. A number of studies have shown that circular RNA (circRNA) can participate in the onset of PH. Our previous studies have shown that circRNA calmodulin 4 (circ-calm4) is involved in the progression of hypoxic PH. However, the role of circ-calm4 on regulation of hypoxic PH autophagy has not been reported. In this study, we demonstrated for the first time that hypoxia-mediated upregulated circ-calm4 expression has a key regulatory effect on autophagy in hypoxia-induced PASMCs and hypoxic PH mouse models. Knockdown of circ-calm4 both in vivo and in vitro can inhibit the autophagy in PASMCs induced by hypoxia. We also performed bioinformatics predictions and conducted experiments to verify that circ-calm4 bound to the purine-rich binding protein (Purb) to promote its expression in the nucleus, thereby initiating the transcription of autophagy-related protein Beclin1. Interestingly, we found that Beclin1 transcription initiated by Purb was accompanied by a modification of Beclin1 super-enhancer to improve transcription activity and efficiency. Overall, our results confirm that the circ-calm4/Purb/Beclin1 signal axis is involved in the occurrence of hypoxia-induced PASMCs autophagy, and the novel regulatory mechanisms and signals transduction pathways in PASMC autophagy induced by hypoxia.

RPS4XL encoded by lnc-Rps4l inhibits hypoxia-induced pyroptosis by binding HSC70 glycosylation site.

Pyroptosis is involved in pulmonary hypertension (PH); however, whether this process is regulated by long non-coding RNAs (lncRNAs) is unclear. Some lncRNAs encode peptides; therefore, whether the regulation of pyroptosis in PH depends on lncRNAs themselves or their encoded peptides needs to be explored. We aimed to characterize the role of the peptide RPS4XL encoded by lnc-Rps4l and its regulatory mechanisms during pyroptosis in PH. Transgenic mice overexpression of lnc-Rps4l was established to rescue the inhibition of hypoxia-induced pyroptosis in pulmonary artery smooth muscle cells (PASMCs). An adeno-associated virus 9 construct with a mutation in the open reading frame of lnc-Rps4l was used to verify that it could inhibit hypoxia-induced PASMCs pyroptosis through its encoded peptide RPS4XL. Glutathione S-transferase (GST) pull-down assays revealed that RPS4XL bound to HSC70, and microscale thermophoresis (MST) was performed to determine the HSC70 domain that interacted with RPS4XL. Through glycosylation site mutation, we confirmed that RPS4XL inhibited hypoxia-induced PASMCs pyroptosis by regulating HSC70 glycosylation. Our results showed that RPS4XL inhibits pyroptosis in a PH mouse model and hypoxic PASMCs by regulating HSC70 glycosylation. These results further clarify the important mechanism of vascular remodeling in PH pathology.

Effect of Interleukin-1ß on Gene Expression Signatures in Schwann Cells Associated with Neuropathic Pain.

Interleukin-1ß (IL-1ß) plays a critical role in the development of neuropathic pain through activation of Schwann cells (SCs) after nerve injury. Here, we applied an RNA sequencing (RNA-seq) approach to identify the effect of IL-1ß on gene signatures of a rat SC line (RSC96) and the potential molecular mechanisms underlying the development of neuropathic pain. RNA-seq data demonstrated a total of 57 significantly differentially expressed genes (DEGs) with 35 up-regulated and 22 down-regulated between SCs treated with IL-1ß, and control SCs without treatment. Bioinformatics analysis showed that key upregulated DEGs included those associated with immune and inflammation-related processes, neurotrophin production and SC proliferation. Five proteins encoded by key upregulated DEGs (Ceacam1, Hap1, Irs3, Lgi4 and Mif) were further verified by Western blot. Consistent with the RNA-Seq results, the expression of key genes was confirmed in SCs by immunofluorescence of the chronic constriction injury (CCI) sciatic nerve in rats. Furthermore, we demonstrated that treatment with IL-1ß resulted in an increase in p38/ERK phosphorylation, and activators of p38/ERK enhanced the effect of IL-1ß on the expression some of the key genes, whereas p38/ERK inhibitors reversed these effects. In conclusion, the present study highlights key genes involved in the development of neuropathic pain through activation of SCs after nerve injury. Identification of these genes and subsequent evidence of their mediation by IL-1ß treatment promote our understanding of molecular mechanisms of nerve injury induced neuropathic pain, and highlight potential molecular targets for the treatment of neuropathic pain.

lnc-Rps4l-encoded peptide RPS4XL regulates RPS6 phosphorylation and inhibits the proliferation of PASMCs caused by hypoxia.

Pulmonary artery smooth muscle cells (PASMCs) proliferation caused by hypoxia is an important pathological process of pulmonary hypertension (PH). Prevention of PASMCs proliferation can effectively reduce PH mortality. Long non-coding RNAs (lncRNAs) are involved in the proliferation process. Recent evidence has demonstrated that functional peptides encoded by lncRNAs play important roles in cell pathophysiological process. Our previous study has demonstrated that lnc-Rps4l with high coding ability mediates the PASMCs proliferation under hypoxic conditions. We hypothesize in this study that a lnc-Rps4l-encoded peptide is involved in hypoxic-induced PASMCs proliferation. The presence of peptide 40S ribosomal protein S4 X isoform-like (RPS4XL) encoded by lnc-Rps4l in PASMCs under hypoxic conditions was confirmed by bioinformatics, immunofluorescence, and immunohistochemistry. Inhibition of proliferation by the peptide RPS4XL was demonstrated in hypoxic PASMCs by MTT, bromodeoxyuridine (BrdU) incorporation, and immunofluorescence assays. By using the bioinformatics, coimmunoprecipitation (coIP), and mass spectrometry, RPS6 was identified to interact with RPS4XL. Furthermore, lnc-Rps4l-encoded peptide RPS4XL inhibited the RPS6 process via binding to RPS6 and inhibiting RPS6 phosphorylation at p-RPS6 (Ser240+Ser244) phosphorylation site. These results systematically elucidate the role and regulatory network of Rps4l-encoded peptide RPS4XL in PASMCs proliferation. These discoveries provide potential targets for early diagnosis and a leading compound for treatment of hypoxic PH.

Altered gene expression signatures by calcitonin gene-related peptide promoted mast cell activity in the colon of stress-induced visceral hyperalgesia mice.

BACKGROUND: Calcitonin gene-related peptide (CGRP) is possibly involved in recruitment of mucosal mast cells (MCs) in the gut that may be associated with the development of irritable bowel syndrome (IBS), but the role of CGRP on the activation of MCs is still unknown. METHODS: Using RNA sequencing (RNA-seq), we examined differentially expressed genes (DEGs) in mouse MCs following CGRP treatment. The expression of key genes in colonic MCs and their relationship with CGRP-containing fibers were examined by immunofluorescence in chronic water-avoidance stress (WAS)-induced visceral hyperalgesia mice. KEY RESULTS: A total of 29 DEGs were found significantly changed with 28 upregulated and 1 downregulated following treatment of MCs with CGRP. Bioinformatics analysis showed that key higher DEGs included those associated with response to corticotropin-releasing hormone (CRH), regulation of transcription, MC activation, and proliferation. These processes are enriched for genes associated with stress-induced MC activation in IBS. Western blot verified changes in representative DEGs (Nr4a3, Crem, Gpr35, FosB, Sphlk1) and real-time cell analysis (RTCA) verified the MC proliferation. The vast majority of colonic MCs nearly CGRP-containing fibers in WAS mice overexpressed only Nr4a3 with little to no FosB, Gpr35, Sphlk1, or Crem expression. Nr4a3 knockdown may attenuate the promotion effect of CGRP on MC viability. CONCLUSIONS & INFERENCES: Our results suggest that CGRP is a critical regulator of key expressed genes in MC activation. Nr4a3 as a novel regulator of MC function may have an effect on stress-induced visceral hyperalgesia, and this may represent the novel target for drug development.

Long Noncoding RNA Rps4l Mediates the Proliferation of Hypoxic Pulmonary Artery Smooth Muscle Cells.

Pulmonary hypertension (PH) is a rare and fatal disorder involving the vascular remodeling of pulmonary arteries mediated by the enhanced proliferation of pulmonary artery smooth muscle cells (PASMCs). Long noncoding RNAs are a subclass of regulatory molecules with diverse cellular functions, but their role in PH remains largely unexplored. We aimed to identify and determine the functions of long noncoding RNAs involved in hypoxia-induced PH and PASMC proliferation. RNA sequencing in a hypoxic mouse model identified hypoxia-regulated long noncoding RNAs, including Rps4l. Rps4l expression was significantly reduced in PH-model mice and hypoxic PASMCs. The subcellular localization of Rps4l was detected by RNA fluorescence in situ hybridization and quantification of nuclear/cytoplasmic RNA. Rps4l overexpression rescued pulmonary arterial hypertension features, as demonstrated by right ventricle hypertrophy, right ventricular systolic pressure, hemodynamics, cardiac function, and vascular remodeling. At the cellular level, Rps4l overexpression weakened cell viability and proliferation and suppressed cell cycle progression. Potential Rps4l-binding proteins were identified via RNA pull-down followed by mass spectrometry, RNA immunoprecipitation, and microscale thermophoresis. These results indicated that Rps4l is associated with and affects the stabilization of ILF3 (interleukin enhancer-binding factor 3). Rps41 further regulates the levels of HIF-1α and consequently leads to hypoxia-induced PASMC proliferation and migration. Our results showed that in hypoxic PASMCs, Rps4l expression decreases due to regulation by hypoxia. This decrease affects the proliferation, migration, and cell cycle progression of PASMCs through ILF3/HIF-1α. These results provide a theoretical basis for further investigations into the pathological mechanism of hypoxic PH and may provide insight for the development of novel treatments.

Increased levels of the long noncoding RNA, HOXA-AS3, promote proliferation of A549 cells.

Many long noncoding RNAs (lncRNAs) have been identified as powerful regulators of lung adenocarcinoma (LAD). However, the role of HOXA-AS3, a novel lncRNA, in LAD is largely unknown. In this study, we showed that HOXA-AS3 was significantly upregulated in LAD tissues and A549 cells. After knockdown of HOXA-AS3, cell proliferation, migration, and invasion were inhibited. Xenografts derived from A549 cells transfected with shRNA/HOXA-AS3 had significantly lower tumor weights and smaller tumor volumes. We also demonstrated that HOXA-AS3 increased HOXA6 mRNA stability by forming an RNA duplex. In addition, HOXA6 promoted cell proliferation, migration, and invasion in vitro. Using a RNA pull-down assay, we found that HOXA-AS3 bonded with NF110, which regulated the cell localization of HOXA-AS3. Moreover, histone acetylation was involved in upregulation of HOXA-AS3. These results demonstrate that HOXA-AS3 was activated in LAD and supported cancer cell progression. Therefore, inhibition of HOXA-AS3 could be an effective targeted therapy for patients with LAD.

Bone morphogenetic protein-7 inhibits endothelial-mesenchymal transition in pulmonary artery endothelial cell under hypoxia.

Pulmonary artery hypertension (PAH) is characterized by structural changes in pulmonary arteries. Increased numbers of cells expressing α-smooth muscle actin (α-SMA) is a nearly universal finding in the remodeled artery. It has been confirmed endothelial-to-mesenchymal transition (EndoMT) may be a source of those α-SMA-expressing cells. In addition, the EndoMT is reversible. Here, we show that under hypoxia, the expression of bone morphogenetic protein 7 (BMP-7) was decreased both in vivo and in vitro. We also found that under normoxia, BMP-7 deficiency induced spontaneous EndoMT and cell migration. The hypoxia-induced EndoMT and cell migration were markedly attenuated after pretreatment with rh-BMP-7. Moreover, m-TOR phosphorylation was involved in EndoMT and BMP-7 suppressed hypoxia-induced m-TORC1 phosphorylation in pulmonary artery endothelial cells. Our results demonstrate that BMP-7 attenuates the hypoxia-induced EndoMT and cell migration by suppressing the m-TORC1 signaling pathway. Our study revealed a novel mechanism underlying the hypoxia-induced EndoMT in pulmonary artery endothelial cells and suggested a new therapeutic strategy targeting EndoMT for the treatment of pulmonary arterial hypertension.

Tetrahydrocurcumin induces mesenchymal-epithelial transition and suppresses angiogenesis by targeting HIF-1α and autophagy in human osteosarcoma.

Human osteosarcoma is considered a malignant tumor with poor prognosis that readily metastasizes. Tetrahydrocurcumin (THC) has been reported to have anti-tumor activity in numerous tumors. In addition, hypoxia-inducible factor-1α (HIF-1α) has been demonstrated to be associated with tumor metastasis by regulating epithelial-mesenchymal transition (EMT). However, the role of THC in osteosarcoma remains uncertain. Therefore, this study aimed to elucidate the potential mechanisms. We found that THC significantly reduced the growth of osteosarcoma cells and suppressed migration and invasion, as tested in a nude mouse lung metastasis model. Additionally, the mesenchymal-epithelial transition (MET) process was facilitated by THC. Mechanistically, our study showed that HIF-1α had a pivotal role in the anti-metastatic effect of THC. Importantly, HIF-1α expression was downregulated by THC by inhibiting Akt/mTOR and p38 MAPK pathways. Moreover, THC exhibited a remarkable inhibitory effect on HIF-1α expression and angiogenesis under hypoxic conditions. Furthermore, THC activated autophagy and induced MET and suppressed angiogenesis in a HIF-1α-related manner. Taken together, our findings suggest that THC suppresses metastasis and invasion and this may be associated with HIF-1α and autophagy, which would potentially provide therapeutic strategies for human osteosarcoma.