Circ_0002331 Interacts with ELAVL1 to Improve ox-LDL-Induced Vascular Endothelial Cell Dysfunction via Regulating CCND2 mRNA Stability.

Circular RNAs (circRNAs) have been discovered to serve as vital regulators in atherosclerosis (AS). However, the role and mechanism of circ_0002331 in AS process are still unclear. Human umbilical vein endothelial cells (HUVECs) were treated with ox-LDL to establish an in vitro model for AS. The expression levels of circ_0002331, Cyclin D2 (CCND2) and ELAVL1 were analyzed by quantitative real-time PCR. Cell proliferation, apoptosis, migration, invasion and angiogenesis were assessed by EdU assay, flow cytometry, transwell assay and tube formation assay. The protein levels of CCND2, ELAVL1, and autophagy-related markers were detected using western blot analysis. IL-8 level was analyzed by ELISA. The relationship between ELAVL1 and circ_0002331 or CCND2 was analyzed by RIP assay and RNA pull-down assay. Moreover, FISH assay was used to analyze the co-localization of ELAVL1 and CCND2 in HUVECs. Our data showed that circ_0002331 was obviously downregulated in AS patients and ox-LDL-induced HUVECs. Overexpression of circ_0002331 could promote proliferation, migration, invasion and angiogenesis, while inhibit apoptosis, autophagy and inflammation in ox-LDL-induced HUVECs. Furthermore, CCND2 was positively regulated by circ_0002331, and circ_0002331 could bind with ELAVL1 to promote CCND2 mRNA stability. Besides, CCND2 overexpression suppressed ox-LDL-induced HUVECs dysfunction, and its knockdown also reversed the regulation of circ_0002331 on ox-LDL-induced HUVECs dysfunction. In conclusion, circ_0002331 might be a potential target for AS treatment, which could improve ox-LDL-induced dysfunction of HUVECs via regulating CCND2 by binding with ELAVL1.

Sema3A inactivates the ERK/JNK signalling pathways to alleviate inflammation and oxidative stress in lipopolysaccharide-stimulated rat endothelial cells and lung tissues.

Semaphorin 3A (Sema3A) is a secretory member of the semaphorin family of immune response regulators. This research focuses on its effects on inflammation and oxidative stress in acute respiratory distress syndrome (ARDS). By analysing the GEO dataset GSE57011, we obtained Sema3A as the most downregulated gene in ARDS samples. Lipopolysaccharide (LPS) was used to stimulate rat pulmonary microvascular endothelial cells (PMVECs) and rats to induce ARDS-like symptoms in vitro and in vivo, respectively. LPS induced severe damage in rat lung tissues, in which reduced immunohistochemical staining of Sema3A was detected. Sema3A overexpression reduced apoptosis and angiogenesis of LPS-induced PMVECs and alleviated lung injury and pulmonary edoema of rats. Moreover, ELISA results showed that Sema3A overexpression downregulated the levels of inflammatory cytokines and oxidative stress markers both in PMVECs and the rat lung. Activation of ERK/JNK signalling aggravated LPS-induced damage on PMVECs; however, the aggravation was partly blocked by Sema3A, which suppressed phosphorylation of ERK/JNK. Overall, this study demonstrates that Sema3A inactivates the ERK/JNK signalling to ameliorate inflammation and oxidative stress in LPS-induced ARDS models. Sema3A might therefore represent a candidate option for ARDS treatment.

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.

Long-term presence of autoantibodies in plasma of cured leprosy patients.

Autoantibodies have been detected in leprosy patients, indicating that infection with M. leprae may lead to autoimmune disorders. However, whether autoimmune response last until patients are cured is unknown. Knowing the autoimmune response in cured leprosy patients is essential to identify whether symptoms are caused by leprosy itself or by other immune-related diseases. This knowledge is essential for the ongoing health management in cured leprosy patients where autoimmune disorders still exist. In our study, we selected six autoantibodies, including anticardiolipin antibody of IgG (ACA), anti-nuclear antibody (ANA), extractable nuclear antigen antibody (ENA), anti-streptolysin O (ASO), anti-double stranded DNA antibody (dsDNA), and rheumatoid factor (RF), that had been reported in leprosy patients as typical autoantibodies. We tested the six typical autoantibodies combined with LACC1, which encodes a protein associated with autoimmune disease such as Crohn's disease and is also the susceptible gene conferring leprosy risk, in cured leprosy patients through ELISA to assess the cured patient's immune status. We observed high positive rates of autoantibodies in cured leprosy patients, and the average plasma levels of five (ACA, ANA, ENA, ASO, and RF) out of the six autoantibodies were significantly higher in cured leprosy patients than in controls. The positive detection of autoantibodies is independent of the recovery period. Moreover, the level of these autoantibodies showed a strong positive correlation with the level of LACC1 in both controls and cured patients. This study showed that there is long-term autoimmunological activation in leprosy patients, even after decades of recovery. Autoimmune responses may influence the development and prognosis of leprosy. Special care should be given to posttreatment or cured leprosy patients regarding long-term autoimmunological activation.

Alpha1B-adreneroceptor is involved in norepinephrine-induced pulmonary artery smooth muscle cell proliferation via p38 signaling.

The higher norepinephrine (NE) concentration induced by sympathetic nerve hyperactivation participated in pulmonary artery smooth muscle cells (PASMCs) over-proliferation and led to pulmonary vascular remodeling (PVR), which played an important role in pulmonary artery hypertension (PAH). However, the underlying mechanism by which NE induced PASMCs proliferation had not been fully elucidated. In the present study, we found that prazosin, the inhibitor of α1-AR, reversed hypoxia-induced changes in pulmonary circulatory function which were analyzed by echocardiography to measure pulmonary artery acceleration time (PAT) and pulmonary arterial velocity time integral (PAVTI) and right heart catheterization to test right ventricular systolic pressure (RVSP), respectively. Western blotting analysis showed that the expression of alpha1B-adreneroceptor (α1B-AR) increased under hypoxic conditions both in vivo and in vitro. Antagonism of α1B-AR by chloroethylclonidine dihydrochloride (CEC) reversed the NE-induced proliferation in rat PASMCs, which was confirmed by applying Western blotting to test proliferating cell nuclear antigen (PCNA) expression, CCK8 assay to test cell viability, scratch-wounding cellular migration assay to show cell proliferative capacity and immunofluorescence to show Ki67 expression. Furthermore, we revealed that antagonism of α1B-AR alleviated NE-induced acceleration of cell-cycle progression by Western blotting to detect cell-cycle-related protein. In addition, Western blotting results showed that antagonism of α1B-AR reversed NE-promoted phosphorylation of p38 (p-p38) under hypoxia. SB202190, an inhibitor of p38, reversed NE-induced proliferation and acceleration of cell-cycle progression of PASMCs. These results suggested that α1B-AR was involved in NE-induced PASMCs proliferation via p38 signaling pathway, which might be downstream of α1B-AR.

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.

Determination of microRNA-122 in hepatocytes by two-step amplification of duplex-specific nuclease with laser-induced fluorescence detection.

MicroRNAs (miRNAs) play important roles in physiological and pathological processes of cells. To develop a fast, simple and sensitive method to determine miRNAs is significant for miRNA studies. In this work, determination of microRNA-122 (miR-122) was achieved by laser-induced fluorescence (LIF) detection. A vial-LIF interface was first applied for sample analysis. A two-step amplification of the fluorescence signal for miR-122 was designed and realized by applying duplex-specific nuclease in the cleaving of two sensing probes. Under optimized conditions, the analysis of a miR-122 sample could be completed in less than 50 min. Only 10 µL sample was required for each test and the detection limit for the method was 0.60 pM equal to 1.2 amol of miR-122 in 10 µL solution. Lastly, the developed method was successfully applied to determine miR-122 in chicken and duck liver. The developed method was fast, selective, sensitive and sample-saving for the determination of miRNAs.

Circular RNA hsa_circ_101555 promotes hepatocellular carcinoma cell proliferation and migration by sponging miR-145-5p and regulating CDCA3 expression.

Circular RNAs have been reported to play significant roles in regulating pathophysiological processes while also guiding clinical diagnosis and treatment of hepatocellular carcinoma (HCC). However, only a few circRNAs have been identified thus far. Herein, we investigated the role of a specific closed-loop structure of hsa_circ_101555 that was generated by back-splicing of the host gene casein kinase 1 gamma 1 (CSNK1G1) in the development and proliferation of HCC. We investigated the expression of Hsa_circ_101555 in HCC and normal tissues using bioinformatics. The expression level of hsa_circ_101555 was further detected by fluorescence in situ hybridization and qRT-PCR in ten HCC patients. Transwell, migration, WST-1 assays, and colony formation assays were used to evaluate the role of hsa_circ_101555 in HCC development and proliferation. The regulatory mechanisms of hsa_circ_101555 in miR-145-5p and CDCA3 were determined by dual luciferase reporter assay. A mouse xenograft model was also used to determine the effect of hsa_circ_101555 on HCC growth in vivo. hsa_circ_101555 showed greater stability than the linear RNA; while in vitro and in vivo results demonstrated that hsa_circ_101555 silencing significantly suppressed cell proliferation, migration, and invasion of HCC cells. Rescue experiments further demonstrated that suppression of miR-145-5p significantly attenuated the biological effects of hsa_circ_101555 knockdown in HCC cells. We also identified a putative oncogene CDCA3 as a potential miR-145-5p target. Thus, our results demonstrated that hsa_circ_101555 might function as a competing endogenous RNA of miR-145-5p to upregulate CDCA3 expression in HCC. These findings suggest that hsa_circ_101555 may be a potential therapeutic target for patients with HCC.

Study of antagonism between some intestinal bacteria with high-speed micellar electrokinetic chromatography.

In this work, high-speed micellar electrokinetic chromatography with LIF detection was applied to study the antagonism between three intestinal bacteria, Escherichia coli (E. coli), Bacillus licheniformis (B. licheniformis) and Bacillus subtilis (B. subtilis). The fluorescent derivatization for the bacteria was performed by labeling the bacteria with FITC. In a high-speed capillary electrophoresis (HSCE) device, the three bacteria could be completely separated within 4 min under the separation mode MEKC. The BGE was 1 × TBE containing 30 mM SDS and 1.5 × 10-5  g/mL polyethylene oxide. The limits of detection for E. coli, B. licheniformis and B. subtilis were 2.80 × 106 CFU/mL, 1.60 × 106 CFU/mL and 1.90 × 106 CFU/mL respectively. Lastly, the method was applied to investigate the antagonism between the three bacteria. The bacteria were mixed and cultured for 7 days. The samples were separated and determined every day to study the interaction between bacteria. The results showed that B. licheniformis and B. subtilis could not inhibit each other, but they could effectively inhibit the reproduction of E. coli. The method developed in this work was quick, sensitive and convenient, and it had great potential in the application of antagonism study for bacteria.

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.

Application of a microfluidic paper-based bioimmunosensor with laser-induced fluorescence detection in the determination of alpha-fetoprotein from serum of hepatopaths.

As a common plasma protein, alpha-fetoprotein (AFP) is widely applied as the tumor biomarker for the diagnosis of many cancers. To develop a low cost, high sensitive and high-throughput method for the determination of AFP is significant for the disease diagnosis. In this work, an immunoassay with sandwich-type structures was performed on a paper-based chip for the analysis of AFP. AFP could be captured by the primary antibodies which were immobilized on the paper by chitosan. On the secondary antibodies, the modified initiator DNAs could trigger the hybridization chain reaction to amplify the fluorescence signals for AFP. A laser-induced fluorescence detector coupled with an interface was applied to detect the targets on the paper-based chip. Under the optimized conditions, the detection limit for AFP was 1.0 pg/mL. For every test, the sample solution consumption only was 10 µL. Finally, the method was applied to determine the AFP in serum of normal person and hepatopaths with hepatic malignant tumor, chronic hepatitis B and other suspected liver diseases. The AFP could be found from all of the samples and the results were similar to that obtained by chemiluminescence immunoassay. The recoveries for AFP ranged from 93.8% to 106%, which indicated the method was reliable. The method based on paper chip had great potential in the application of AFP determination.

Down-regulation of lncRNA Gas5 promotes hypoxia-induced pulmonary arterial smooth muscle cell proliferation by regulating KCNK3 expression.

Pulmonary hypertension (PH) is a progressive and potentially serious lung disease, defined by an abnormal elevation of pulmonary arterial pressure. PH occurs for many reasons, and hypoxia is considered as an important stimulus for the disease. Proliferation and migration of pulmonary artery smooth muscular cells (PASMCs) in the small peripheral pulmonary arteries are common characteristic features in hypoxia-induced PH (HPH). However, the mechanisms involved in the hypoxia-induced cell proliferation and migration are not clear. The aim of the present study was to investigate the role of lncRNA Gas5 in the hypoxia-stimulated proliferation and migration of human PASMCs (hPASMCs). We found that the expression of Gas5 was down-regulated in a rat model with hypoxia and in cultured hypoxic hPASMCs, and silence of Gas5 significantly promoted hPASMCs proliferation and migration in both normal and hypoxia condition. Subsequent studies revealed that miR-23b-3p interacted with Gas5 by directly targeting the miRNA-binding site in the Gas5 sequence, and qRT-PCR results showed miR-23b-3p and Gas5 could affect each other's expression, respectively. Further study demonstrated that Gas5 acted as a competing endogenous RNA (ceRNA) for miR-23b-3p to modulate the KCNK3 expression, and these interactions led to promotion of hPASMCs proliferation and migration. This study identified that Gas5/miR-23b-3p/KCNK3 axis may be a mechanism that hypoxia-induced PASMCs proliferation and migration, providing a strategy for clinical treatment of HPH in the future.

A microfluidic paper-based laser-induced fluorescence sensor based on duplex-specific nuclease amplification for selective and sensitive detection of miRNAs in cancer cells.

MicroRNAs (miRNAs) are considered as the potential biomarkers for many cancers. To determine miRNAs in cancer cells is significant for realizing these diseases. In this work, a microfluidic paper-based laser-induced fluorescence sensor based on duplex-specific nuclease (DSN) amplification was developed and applied to selectively and sensitively determine miRNAs in cancer cells. An interface for laser-induced fluorescence detection was firstly applied to perform the sample detection on the paper-based chip. Under the optimal conditions, DSN (3 µL 0.10 U) and Taqman probes (2 µL 2.5 × 10-7 M) were preserved on the circles (Diameter 4 mm) of the folded paper chip. When miRNA solution was added, the mixed solution could trigger fluorescence signal amplification by cyclically digesting hybrids of miRNAs and Taqman probes by DSN. The whole determination, including sample heating process, could be accomplished within 40 min. The detection limits for miRNA-21 and miRNA-31 were 0.20 and 0.50 fM respectively, corresponding to only 1.0 and 1.5 zmol consumption of miRNAs. The testing of mismatched miRNAs showed that the method had good specificity. Finally, the method was applied to determine miRNA-21 and miRNA-31 in lysates of cancer cells of A549 and HeLa, and hepatocyte LO2. MiRNA-21 and miRNA-31 could be successfully found from the two cancer cells. The concentrations for miRNA-21 and miRNA-31 were 1.74 × 10-13 M and 6.29 × 10-14 M in HeLa cell lysate (3.75 × 104 cells/mL), 3.07 × 10-15 M and 3.28 × 10-15 M in A549 cell lysate (8.33 × 106 cells/mL) respectively. The recoveries ranged from 87.30% to 111.83%, indicating the results were reliable. The developed method was effective, selective and sensitive in the determination of miRNAs in cancer cells.

The circular RNA circMAST1 promotes hepatocellular carcinoma cell proliferation and migration by sponging miR-1299 and regulating CTNND1 expression.

Circular RNAs (circRNAs) are a class of non-coding RNAs with a loop structure; however, their functions remain largely unknown. Growing evidence suggests that circRNAs play a pivotal role in the progression of malignant diseases. However, the expression profiles and function of circRNAs in hepatocellular carcinoma (HCC) remain unclear. We investigated the expression of microtubule-associated serine/threonine kinase 1 (MAST1) circRNA (circMAST1) in HCC and healthy tissues using bioinformatics, quantitative real-time PCR (qRT-PCR), and fluorescence in situ hybridization. Luciferase reporter assays were performed to assess the interaction between circMAST1 and miR-1299. Proliferation assays, colony formation assays, flow cytometry, transwell assays, and western blotting were also performed. A mouse xenograft model was also used to determine the effect of circMAST1 on HCC growth in vivo. CircMAST1 was upregulated in HCC tissues and cell lines; silencing via small interfering RNA inhibited migration, invasion, and proliferation of HCC cell lines in vitro as well as tumor growth in vivo. Furthermore, the expression of circMAST1 was positively correlated with catenin delta-1 (CTNND1) and negatively correlated with microRNA (miR)-1299 in HCC clinical samples. Importantly, circMAST1 sponged miR-1299 to stabilize the expression of CTNND1 and promoted tumorigenic features in HCC cell lines. We found that circMAST1 may serve as a novel biomarker for HCC. Moreover, circMAST1 elicits HCC progression by sponging miRNA-1299 and stabilizing CTNND1. Our data provide potential options for therapeutic targets in patients with HCC.

piRNA-63076 contributes to pulmonary arterial smooth muscle cell proliferation through acyl-CoA dehydrogenase.

Piwi-interacting RNAs (piRNAs) are thought to be germline-specific and to be involved in maintaining genome stability during development. Recently, piRNA expression has been identified in somatic cells in diverse organisms. However, the roles of piRNAs in pulmonary arterial smooth muscle cell (PASMC) proliferation and the molecular mechanism underlying the hypoxia-regulated pathological process of pulmonary hypertension are not well understood. Using hypoxic animal models, cell and molecular biology, we obtained the first evidence that the expression of piRNA-63076 was up-regulated in hypoxia and was positively correlated with cell proliferation. Subsequently, we showed that acyl-CoA dehydrogenase (Acadm), which is negatively regulated by piRNA-63076 and interacts with Piwi proteins, was involved in hypoxic PASMC proliferation. Finally, Acadm inhibition under hypoxia was partly attributed to DNA methylation of the Acadm promoter region mediated by piRNA-63076. Overall, these findings represent invaluable resources for better understanding the role of epigenetics in pulmonary hypertension associated with piRNAs.

A cytosolic heat shock protein 90 and co-chaperone p23 complex activates RIPK3/MLKL during necroptosis of endothelial cells in acute respiratory distress syndrome.

Necrosis with inflammation plays a crucial role in acute respiratory distress syndrome (ARDS). Receptor-interacting protein 3 (RIPK3) regulates a newly discovered programmed form of necrosis called necroptosis. However, the underlying mechanism of necroptosis in ARDS remains unknown. Thus, the purpose of this study was to examine the possible involvement of RIPK3 in ARDS-associated necroptosis. RIPK3 protein levels were found to be significantly elevated in the plasma and bronchoalveolar lavage fluid of ARDS patients. Next, we utilised a mouse model of severe ARDS induced with high-dose lipopolysaccharide and found that lung injury was mainly due to RIPK3-mixed lineage kinase domain-like pseudokinase (MLKL)-mediated necroptosis and endothelial dysfunction. The activation of RIPK3-MLKL by tumour necrosis factor receptor 1 (TNFR1) and TNFR1-associated death domain protein (TRADD) required catalytically active RIPK1 and the inhibition of Fas-associated protein with death domain (FADD)/caspase-8 catalytic activity. We further showed that the molecular chaperone heat shock protein 90 (Hsp90)/p23, as a novel RIPK3- and MLKL-interacting complex, played an important role in RIP-MLKL-mediated necroptosis, inflammation and endothelial dysfunction in the pulmonary vasculature, which resulted in ARDS. Collectively, the results of our study indicate that necroptosis is an important mechanism of cell death in ARDS and the inhibition of necroptosis may be a therapeutic intervention for ARDS. KEY MESSAGES: Lung injury in high-dose LPS-induced severe ARDS is mainly due to RIP3-MLKL-mediated necroptosis and endothelial dysfunction. Chaperone HSP90/p23 is a novel RIP3- and MLKL-interacting complex in HPAECs. HSP90/p23 is a novel RIP3- and MLKL-interacting complex in RIP-MLKL-mediated necroptosis, inflammation and endothelial dysfunction.

Circ-calm4 Serves as an miR-337-3p Sponge to Regulate Myo10 (Myosin 10) and Promote Pulmonary Artery Smooth Muscle Proliferation.

Pulmonary artery smooth muscle cell proliferation is the pathological basis of pulmonary vascular remodeling in hypoxic pulmonary hypertension. Recent studies suggest that circular RNA (circRNA) can regulate various biological processes, including cell proliferation. Therefore, it is possible that circRNA may have important roles in pulmonary artery smooth muscle cell proliferation in hypoxic pulmonary hypertension. In the present study, we aimed to identify functional circRNAs and clarify their roles and mechanisms in pulmonary artery smooth muscle cell proliferation in pulmonary hypertension. RNA sequencing identified 67 circRNAs that were differentially expressed in hypoxic lung tissues of mice. Screening by bioinformatics and quantitative polymerase chain reaction revealed significant elevation of a circRNA derived from alternative splicing of the calmodulin 4 gene (designated circ-calm4). Notably, this circRNA absorbed miR-337-3p. We further identified Myo10 (myosin 10) as a target protein of miR-337-3p. miR-337-3p bound to the 3'-untranslated region of Myo10 mRNA, thereby attenuating the translation of Myo10. Using loss-of-function and gain-of-function approaches, we found that circ-calm4 regulated cell proliferation by regulating the cell cycle. Additionally, we verified the functions of miR-337-3p and Myo10 in hypoxic pulmonary artery smooth muscle. Our results suggested that the circ-calm4/miR-337-3p/Myo10 signal transduction axis modulated the proliferation of pulmonary artery smooth muscle cells at the molecular level, thus establishing potential targets for the early diagnosis and treatment of pulmonary hypertension.

Dacomitinib, a new pan-EGFR inhibitor, is effective in attenuating pulmonary vascular remodeling and pulmonary hypertension.

Accumulating evidence suggests that epidermal growth factor receptor (EGFR) plays a role in the progression of pulmonary arterial hypertension (PAH). Clinically-approved epidermal growth factor inhibitors such as gefitinib, erlotinib, and lapatinib have been explored for PAH. However, None of them were able to attenuate PAH. So, we explored the role of dacomitinib, a new pan-EGFR inhibitor, in PAH. Adult male Sprague-Dawley rats were used to study hypoxia- or monocrotaline-induced right ventricular remodeling as well as systolic function and hemodynamics using echocardiography and a pressure-volume admittance catheter. Morphometric analyses of lung vasculature and pressure-volume vessels were performed. Immunohistochemical staining, flow cytometry, and viability, as well as scratch-wound, and Boyden chamber migration assays were used to identify the roles of dacomitinib in pulmonary artery smooth muscle cells (PASMCs). The results revealed that dacomitinib has a significant inhibitory effect on the thickening of the media, adventitial collagen increased. Dacomitinib also has a significant role in attenuating pulmonary artery pressure and right ventricular hypertrophy. Additionally, dacomitinib inhibits hypoxia-induced proliferation, migration, autophagy and cell cycle progression through PI3K-AKT-mTOR signaling in PASMCs. Our study indicates that dacomitinib inhibited hypoxia-induced cell cycle progression, proliferation, migration, and autophagy of PASMCs, thereby attenuating pulmonary vascular remodeling and development of PAH via the PI3K-AKT-mTOR signaling pathway. Overall, dacomitinib may serve as new potential therapeutic for the treatment of PAH.

Lipopolysaccharide-induced proliferation and glycolysis in airway smooth muscle cells via activation of Drp1.

Abnormal airway smooth muscle cells (ASMCs) proliferation is an important pathological process in airway remodeling contributes to increased mortality in asthma. Mitochondrial dynamics and metabolism have a central role in the maintenance of the cell function. In this study, lipopolysaccharide (LPS)-induced ASMCs proliferative model was used to investigate the effect of mitochondria on the proliferation of ASMCs and the possible mechanism. We used cell and molecular biology to determine the effect of dynamin-related protein 1 (Drp1) on LPS-mediated ASMCs cell cycle progression and glycolysis. The major findings of the current study are as follows: LPS promoted an increased mitochondrial fission and phosphorylation of Drp1 at Ser616 (p-Drp1 Ser616). LPS-induced ASMCs proliferation and cell cycle progression, which was significantly inhibited application of Drp1 RNA interfering. Glycolysis inhibitor 2-deoxyglucose (2-DG) depressed ASMCs proliferative process induced by LPS stimulation. LPS caused mitochondrial metabolism disorders and aerobic glycolysis in a dependent on Drp1 activation. These results indicated that Drp1 may function as a key factor in asthma airway remodeling by mediating ASMC proliferation and cell cycle acceleration through an effect on mitochondrial metabolic disturbance.

Poly r(C) Binding Protein 1 Regulates Posttranscriptional Expression of the Ubiquitin Ligase TRIM56 in Ovarian Cancer.

Our earlier work has shown that the E3 ligase TRIM56 messenger RNA (mRNA) level and vimentin protein expression followed an inverse correlation in ovarian carcinoma patients; however, the regulatory mechanisms underlying TRIM56 expression is unclear. Steady state expression of TRIM56 mRNA expression in the normal ovarian cell line Moody and ovarian cancer cell lines SKOV-3, A2780, and Caov-3 were not significantly different; however, TRIM56 protein expression was significantly lower in the ovarian cancer cell lines compared to the Moody cell line. Polysome profiling showed significant increase in translation of TRIM56 messenger RNA in the Moody cells compared to the SKOV-3 cells. We performed RNA-affinity pulldown using biotinylated TRIM56 5 'and 3'-UTR and postnuclear extracts from Moody and SKOV-3 cells. Whereas no notable difference was observed in affinity pull-down obtained with the 5'-UTR, there was obvious difference in protein binding patterns with the 3'-UTR. Mass spectrometry was used to determine the most differentially binding protein as poly r (c) binding protein 1 (PCBP1). PCBP1 expression and binding to the 3'-UTR was both higher in SKOV-3 cells compared to the Moody cells. Silencing of TRIM56 in Moody cells cause an increase in in vitro migration and invasion, and a similar effect was mimicked by overexpression of PCBP1. Conversely, silencing of PCBP1 or overexpression of TRIM56 in SKOV-3 cells significantly decreased in vitro migration and invasion. In xenograft assays, SKOV-3 cells stably overexpressing shRNA targeting PCBP1 decreased metastasis, whereas shRNA-targeting TRIM56 potentiated detection of metastatic lesions, compared to the parental SKOV-3 cells themselves. Taken together our results reveal a yet undefined posttranscriptional regulatory mechanism underlying low expression of TRIM56 in ovarian cancer. © 2018 IUBMB Life, 71(1):177-182, 2019.