Aberrant splicing of CaV1.2 calcium channel induced by decreased Rbfox1 enhances arterial constriction during diabetic hyperglycemia.

Diabetic hyperglycemia induces dysfunctions of arterial smooth muscle, leading to diabetic vascular complications. The CaV1.2 calcium channel is one primary pathway for Ca2+ influx, which initiates vasoconstriction. However, the long-term regulation mechanism(s) for vascular CaV1.2 functions under hyperglycemic condition remains unknown. Here, Sprague-Dawley rats fed with high-fat diet in combination with low dose streptozotocin and Goto-Kakizaki (GK) rats were used as diabetic models. Isolated mesenteric arteries (MAs) and vascular smooth muscle cells (VSMCs) from rat models were used to assess K+-induced arterial constriction and CaV1.2 channel functions using vascular myograph and whole-cell patch clamp, respectively. K+-induced vasoconstriction is persistently enhanced in the MAs from diabetic rats, and CaV1.2 alternative spliced exon 9* is increased, while exon 33 is decreased in rat diabetic arteries. Furthermore, CaV1.2 channels exhibit hyperpolarized current-voltage and activation curve in VSMCs from diabetic rats, which facilitates the channel function. Unexpectedly, the application of glycated serum (GS), mimicking advanced glycation end-products (AGEs), but not glucose, downregulates the expression of the splicing factor Rbfox1 in VSMCs. Moreover, GS application or Rbfox1 knockdown dynamically regulates alternative exons 9* and 33, leading to facilitated functions of CaV1.2 channels in VSMCs and MAs. Notably, GS increases K+-induced intracellular calcium concentration of VSMCs and the vasoconstriction of MAs. These results reveal that AGEs, not glucose, long-termly regulates CaV1.2 alternative splicing events by decreasing Rbfox1 expression, thereby enhancing channel functions and increasing vasoconstriction under diabetic hyperglycemia. This study identifies the specific molecular mechanism for enhanced vasoconstriction under hyperglycemia, providing a potential target for managing diabetic vascular complications.

Dysregulated Rbfox2 produces aberrant splicing of CaV1.2 calcium channel in diabetes-induced cardiac hypertrophy.

BACKGROUND: L-type Ca2+ channel CaV1.2 is essential for cardiomyocyte excitation, contraction and gene transcription in the heart, and abnormal functions of cardiac CaV1.2 channels are presented in diabetic cardiomyopathy. However, the underlying mechanisms are largely unclear. The functions of CaV1.2 channels are subtly modulated by splicing factor-mediated alternative splicing (AS), but whether and how CaV1.2 channels are alternatively spliced in diabetic heart remains unknown. METHODS: Diabetic rat models were established by using high-fat diet in combination with low dose streptozotocin. Cardiac function and morphology were assessed by echocardiography and HE staining, respectively. Isolated neonatal rat ventricular myocytes (NRVMs) were used as a cell-based model. Cardiac CaV1.2 channel functions were measured by whole-cell patch clamp, and intracellular Ca2+ concentration was monitored by using Fluo-4 AM. RESULTS: We find that diabetic rats develop diastolic dysfunction and cardiac hypertrophy accompanied by an increased CaV1.2 channel with alternative exon 9* (CaV1.2E9*), but unchanged that with alternative exon 8/8a or exon 33. The splicing factor Rbfox2 expression is also increased in diabetic heart, presumably because of dominate-negative (DN) isoform. Unexpectedly, high glucose cannot induce the aberrant expressions of CaV1.2 exon 9* and Rbfox2. But glycated serum (GS), the mimic of advanced glycation end-products (AGEs), upregulates CaV1.2E9* channels proportion and downregulates Rbfox2 expression in NRVMs. By whole-cell patch clamp, we find GS application hyperpolarizes the current-voltage curve and window currents of cardiac CaV1.2 channels. Moreover, GS treatment raises K+-triggered intracellular Ca2+ concentration ([Ca2+]i), enlarges cell surface area of NRVMs and induces hypertrophic genes transcription. Consistently, siRNA-mediated knockdown of Rbfox2 in NRVMs upregulates CaV1.2E9* channel, shifts CaV1.2 window currents to hyperpolarization, increases [Ca2+]i and induces cardiomyocyte hypertrophy. CONCLUSIONS: AGEs, not glucose, dysregulates Rbfox2 which thereby increases CaV1.2E9* channels and hyperpolarizes channel window currents. These make the channels open at greater negative potentials and lead to increased [Ca2+]i in cardiomyocytes, and finally induce cardiomyocyte hypertrophy in diabetes. Our work elucidates the underlying mechanisms for CaV1.2 channel regulation in diabetic heart, and targeting Rbfox2 to reset the aberrantly spliced CaV1.2 channel might be a promising therapeutic approach in diabetes-induced cardiac hypertrophy.

Association of gut microbiome and primary liver cancer: A two-sample Mendelian randomization and case-control study.

BACKGROUND AND AIMS: Observational epidemiology studies suggested a relationship between the gut microbiome and primary liver cancer. However, the causal relationship remains unclear because of confounding factors and reverse causality. We aimed to explore the causal role of the gut microbiome in the development of primary liver cancer, including hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). METHODS: Mendelian randomization (MR) study was conducted using summary statistics from genome-wide association studies (GWAS) of the gut microbiome and liver cancer, and sequencing data from a case-control study validated the findings. A 5-cohort GWAS study in Germany (N = 8956) served as exposure, whilst the UK biobank GWAS study (N = 456 348) served as an outcome. The case-control study was conducted at the First Affiliated Hospital of Wenzhou Medical University from December 2018 to October 2020 and included 184 HCC patients, 63 ICC patients and 40 healthy controls. RESULTS: A total of 57 features were available for MR analysis, and protective causal associations were identified for Family_Ruminococcaceae (OR = 0.46 [95% CI, 0.26-0.82]; p = .009) and Genus_Porphyromonadaceae (OR = 0.59 [95% CI, 0.42-0.83]; p = .003) with HCC, and for Family_Porphyromonadaceae (OR = 0.36 [95% CI, 0.14-0.94]; p = .036) and Genus_Bacteroidetes (OR = 0.55 [95% CI, 0.34-0.90]; p = .017) with ICC respectively. The case-control study results showed that the healthy controls had a higher relative abundance of Family_Ruminococcaceae (p = .00033), Family_Porphyromonadaceae (p = .0055) and Genus_Bacteroidetes (p = .021) than the liver cancer patients. CONCLUSIONS: This study demonstrates that Ruminococcaceae, Porphyromonadaceae and Bacteroidetes are related to a reduced risk of liver cancer (HCC or ICC), suggesting potential significance for the prevention and control of liver cancer.

Diminished Rbfox1 increases vascular constriction by dynamically regulating alternative splicing of CaV1.2 calcium channel in hypertension.

Calcium influx from depolarized CaV1.2 calcium channels triggers the contraction of vascular smooth muscle cells (VSMCs), which is important for maintaining vascular myogenic tone and blood pressure. The function of CaV1.2 channel can be subtly modulated by alternative splicing (AS), and its aberrant splicing involves in the pathogenesis of multiple cardiovascular diseases. The RNA-binding protein Rbfox1 is reported to regulate the AS events of CaV1.2 channel in the neuronal development, but its potential roles in vascular CaV1.2 channels and vasoconstriction remain undefined. Here, we detect Rbfox1 is expressed in rat vascular smooth muscles. Moreover, the protein level of Rbfox1 is dramatically decreased in the hypertensive small arteries from spontaneously hypertensive rats in comparison with normotensive ones from Wistar-Kyoto rats. In VSMCs, Rbfox1 could dynamically regulate the AS of CaV1.2 exons 9* and 33. By whole-cell patch clamp, we identify knockdown of Rbfox1 induces the hyperpolarization of CaV1.2 current-voltage relationship curve in VSMCs. Furthermore, siRNA-mediated knockdown of Rbfox1 increases the K+-induced constriction of rat mesenteric arteries. In summary, our results indicate Rbfox1 modulates vascular constriction by dynamically regulating CaV1.2 alternative exons 9* and 33. Therefore, our work elucidates the underlying mechanisms for CaV1.2 channels regulation and provides a potential therapeutic target for hypertension.

A novel mutation in KCNH2 yields loss-of-function of hERG potassium channel in long QT syndrome 2.

Mutations in hERG (human ether-à-go-go-related gene) potassium channel are closely associated with long QT syndromes. By direct Sanger sequencing, we identified a novel KCNH2 mutation W410R in the patient with long QT syndrome 2 (LQT2). However, the electrophysiological functions of this mutation remain unknown. In comparison to hERGWT channels, hERGW410R channels have markedly decreased total and surface expressions. W410R mutation dramatically reduces hERG channel currents (IKr) and shifts its steady-state activation curve to depolarization. Moreover, hERGW410R channels make dominant-negative effects on hERGWT channels. Significantly, we find hERG channel blocker E-4031 could partially rescue the function of hERGW410R channels by increasing the membrane expression. By using in silico model, we reveal that hERGW410R channels obviously elongate the repolarization of human ventricular myocyte action potentials. Collectively, W410R mutation decreases the currents of hERG channel, because of diminished membrane expression of mutant channels, that subsequently leads to elongated repolarization of cardiomyocyte, which might induce the pathogenesis of LQT2. Furthermore, E-4031 could partially rescue the decreased activity of hERGW410R channels. Thus, our work identifies a novel loss-of-function mutation in KCNH2 gene, which might provide a rational basis for the management of LQT2.

Inhibition of miR-135a-5p attenuates vascular smooth muscle cell proliferation and vascular remodeling in hypertensive rats.

Proliferation of vascular smooth muscle cells (VSMCs) greatly contributes to vascular remodeling in hypertension. This study is to determine the roles and mechanisms of miR-135a-5p intervention in attenuating VSMC proliferation and vascular remodeling in spontaneously hypertensive rats (SHRs). MiR-135a-5p level was raised, while fibronectin type III domain-containing 5 (FNDC5) mRNA and protein expressions were reduced in VSMCs of SHRs compared with those of Wistar-Kyoto rats (WKYs). Enhanced VSMC proliferation in SHRs was inhibited by miR-135a-5p knockdown or miR-135a-5p inhibitor, but exacerbated by miR-135a-5p mimic. VSMCs of SHRs showed reduced myofilaments, increased or even damaged mitochondria, increased and dilated endoplasmic reticulum, which were attenuated by miR-135a-5p inhibitor. Dual-luciferase reporter assay shows that FNDC5 was a target gene of miR-135a-5p. Knockdown or inhibition of miR-135a-5p prevented the FNDC5 downregulation in VSMCs of SHRs, while miR-135a-5p mimic inhibited FNDC5 expressions in VSMCs of both WKYs and SHRs. FNDC5 knockdown had no significant effects on VSMC proliferation of WKYs, but aggravated VSMC proliferation of SHRs. Exogenous FNDC5 or FNDC5 overexpression attenuated VSMC proliferation of SHRs, and prevented miR-135a-5p mimic-induced enhancement of VSMC proliferation of SHR. MiR-135a-5p knockdown in SHRs attenuated hypertension, normalized FNDC5 expressions and inhibited vascular smooth muscle proliferation, and alleviated vascular remodeling. These results indicate that miR-135a-5p promotes while FNDC5 inhibits VSMC proliferation in SHRs. Silencing of miR-135a-5p attenuates VSMC proliferation and vascular remodeling in SHRs via disinhibition of FNDC5 transcription. Either inhibition of miR-135a-5p or upregulation of FNDC5 may be a therapeutically strategy in attenuating vascular remodeling and hypertension.

Exclusion of alternative exon 33 of CaV1.2 calcium channels in heart is proarrhythmogenic.

Alternative splicing changes the CaV1.2 calcium channel electrophysiological property, but the in vivo significance of such altered channel function is lacking. Structure-function studies of heterologously expressed CaV1.2 channels could not recapitulate channel function in the native milieu of the cardiomyocyte. To address this gap in knowledge, we investigated the role of alternative exon 33 of the CaV1.2 calcium channel in heart function. Exclusion of exon 33 in CaV1.2 channels has been reported to shift the activation potential -10.4 mV to the hyperpolarized direction, and increased expression of CaV1.2Δ33 channels was observed in rat myocardial infarcted hearts. However, how a change in CaV1.2 channel electrophysiological property, due to alternative splicing, might affect cardiac function in vivo is unknown. To address these questions, we generated mCacna1c exon 33-/--null mice. These mice contained CaV1.2Δ33 channels with a gain-of-function that included conduction of larger currents that reflects a shift in voltage dependence and a modest increase in single-channel open probability. This altered channel property underscored the development of ventricular arrhythmia, which is reflected in significantly more deaths of exon 33-/- mice from ß-adrenergic stimulation. In vivo telemetric recordings also confirmed increased frequencies in premature ventricular contractions, tachycardia, and lengthened QT interval. Taken together, the significant decrease or absence of exon 33-containing CaV1.2 channels is potentially proarrhythmic in the heart. Of clinical relevance, human ischemic and dilated cardiomyopathy hearts showed increased inclusion of exon 33. However, the possible role that inclusion of exon 33 in CaV1.2 channels may play in the pathogenesis of human heart failure remains unclear.

Swietenine extracted from Swietenia relieves myocardial hypertrophy induced by isoprenaline in mice.

As a traditional plant medicine in tropical areas, Swietenia macrophylla seeds are usually applied for some chronic diseases, including hypertension, diabetes, and so on. Few studies have been carried out to identify the effective elements in seed extract and their indications. In this study, we first investigated the functions of the swietenine, an extract from S. macrophylla seeds, using a model of myocardial hypertrophy induced by isoprenaline (ISO). At cellular level, H9c2 cell hypertrophy was also established through the treatment with ISO. The cardiac pathological remodeling was evaluated by echocardiography and histological analysis. Western blot and RT-qPCR were used to detect the expression of possible hypertrophy-promoting genes. Here, our results indicated that swietenine remarkably attenuated ISO-induced myocardial hypertrophy in vivo and in vitro. Moreover, Akt phosphorylation, ANP and BNP mRNA expression were efficiently decreased. Based on these findings, we concluded that swietenine might be a promising anti-hypertrophic agent against cardiac hypertrophy.

[A preliminary study on the construction of the triad medical experimental system].

The traditional medical experiment based on animal studies fails to reflect competency-oriented goal, and is not closely combined with clinical and scientific research, which does not meet the need for early clinical and scientific training. In order to cultivate the first-class medical talents, medical experimental teaching should conform to the trend of modern medical education, innovate teaching ideas and models, and update the hardware and software in time. Therefore, our teaching center adopts the triad medical experimental system which consists of "animal experiments, human functional experiments, and electronic standardized patient (ESP)-based virtual simulation experiments", and uses one system to integrate basic and clinical medicine, practice and virtual learning, teaching and scientific training. The system retains the core content of traditional animal experiments, and includes the most mature and widely used human physiological experiments to increase students' learning experience. With medical simulation experiment, it explains the specific physiological and pathophysiological processes of human body to improve students' cognitive and thinking ability. Here, we provide a systematic description on our triad medical experimental system, and discuss the experience to establish this novel system.

[Building virtual simulation teaching platform based on electronic standardized patient].

Informatization is an effective way to promote the reform and innovation of higher education and improve its quality. Virtual simulation teaching is indispensable in the educational informatization. Here, we describe the development and current situation of virtual simulation teaching, and introduce electronic standardized patient (ESP) based-virtual human body system powered by the real-time human physiological parameters. We also discuss how to build an ESP-based community in the teaching of human physiology, preclinical integrated case learning and other teaching projects. These ESP-based virtual simulation projects display the advantages of interdisciplinary fusion and the combination of basic and clinical knowledge, and open up the third type of functional experiments. Therefore, ESP-based virtual simulation teaching platform presumably becomes a considerable option for the first-class course construction in physiology.

Novel compound heterozygous CLCNKB gene mutations (c.1755A>G/c.848_850delTCT) cause classic Bartter syndrome.

Inactivated variants in CLCNKB gene encoding the basolateral chloride channel ClC-Kb cause classic Bartter syndrome characterized by hypokalemic metabolic alkalosis and hyperreninemic hyperaldosteronism. Here, we identified two cBS siblings presenting hypokalemia in a Chinese family due to novel compound heterozygous CLCNKB mutations (c.848_850delTCT/c.1755A>G). Compound heterozygosity was confirmed by amplifying and sequencing the patient's genomic DNA. The synonymous mutation c.1755A>G (Thr585Thr) was located at +2 bp from the 5' splice donor site in exon 15. Further transcript analysis demonstrated that this single nucleotide mutation causes exclusion of exon 15 in the cDNA from the proband and his mother. Furthermore, we investigated the expression and protein trafficking change of c.848_850delTCT (ΔTCT) and exon 15 deletion (ΔE15) mutation in vitro. The ΔE15 mutation markedly decreased the expression of ClC-Kb and resulted in a low-molecular-weight band (~55 kDa) trapping in the endoplasmic reticulum, while the ΔTCT mutant only decreased the total and plasma membrane ClC-Kb protein expression but did not affect the subcellular localization. Finally, we studied the physiological functions of mutations by using whole cell patch-clamp and found that the ΔE15 or ΔTCT mutation decreased the current of the ClC-Kb/barttin channel. These results suggested that the compound defective mutations of the CLCNKB gene are the molecular mechanism of the two cBS siblings.

Long Non-Coding RNA MEG3 Functions as a Competing Endogenous RNA to Regulate HOXA11 Expression by Sponging miR-181a in Multiple Myeloma.

BACKGROUND/AIMS: Long non-coding RNA maternally expressed gene 3 (MEG3) has been reported to play an essential role in cancer progression and metastasis. However, the overall biological role and regulatory mechanism of MEG3 in multiple myeloma (MM) development and progression remains largely ill-defined. METHODS: MEG3 and miR-181a expression of MM patients were analyzed by publicly available MM data sets. Cell counting kit-8 and flow cytometry analysis were used to identify the function of MEG3 on MM in vitro. Additionally, we conducted tumor formation experiments in mice models to explain the role of MEG3 on MM in vivo. Then, several mechanism experiments, including dual-luciferase reporter assay and RNA immunoprecipitation were performed to evaluate the emulative relationship between MEG3 and miR-181a. RESULTS: In this research, we found that MEG3 was downregulated in MM patients, which was linked with tumor progression. In addition, we demonstrated that miR-181a was overexpressed in MM patients in consistent with its cancer-promoting function. Importantly, several mechanism experiments revealed that MEG3, acting as an endogenous competitive RNA, could contend with miR-181a to inhibit tumor progression. Furthermore, as the target mRNA of miR-181a, homeobox gene A11(HOXA11) could be positively regulated by MEG3 through sponging miR-181a competitively in vitro. CONCLUSION: Our present work supplies the first discovery of a MEG3/miR-181a/HOXA11 regulatory network in MM and highlights that MEG3 may serve as a promising target for MM therapy in the future.

TRPV2-induced Ca2+-calcineurin-NFAT signaling regulates differentiation of osteoclast in multiple myeloma.

BACKGROUND: Myeloma bone disease (MBD) can cause bone destruction and increase the level of Ca2+ concentration in the bone marrow microenvironment by stimulating osteoclastic differentiation. Nevertheless, the relationships between MBD and highly efficient stimuli of Ca2+ in multiple myeloma (MM) progression, and possible regulatory mechanisms are poorly defined. Here, we reported that the nonselective cation channel transient receptor potential vanilloid 2 (TRPV2) plays a functional role in Ca2+ oscillations and osteoclastogenesis. METHODS: To investigate the expression of TRPV2 in MM, we analyzed publicly available MM data sets and performed immunohistochemistry in MM patients. The correlations between TRPV2 expression levels and osteoclast-related cytokines were analyzed. Fluo-4 staining and ELISA assays were used to assess the regulated function of TRPV2 in intracellular Ca2+ and cytokines. Western blotting and Chromatin immunoprecipitation (ChIP) assays were performed to explore the signaling pathway of TRPV2-induced osteoclastic differentiation. Real-time PCR, Western blotting, ELISA and tartrate-resistant acid phosphatase (TRAP) staining were performed to detect the biological effects of TRPV2 inhibitor on osteoclastogenesis. RESULTS: The functional expression of TRPV2, involved in the osteolysis through gating the calcium influx, was changed in the MM cells cultured in a high Ca2+ environment. Mechanistically, TRPV2 modulates nuclear factor-κB ligand (RANKL)-dependent osteoclastic differentiation through the Ca2+-calcineurin-NFAT signaling pathway. Of clinical relevance, systemic administration with SKF96365 could attenuate the MM-induced osteoclast formation in vitro. CONCLUSIONS: Our study uncovers the possible roles of TRPV2, which enhances MBD, suggesting that targeting osteocyte-MM cells interactions through blockade of TRPV2 channel may provide a promising treatment strategy in MM.

FNDC5 overexpression and irisin ameliorate glucose/lipid metabolic derangements and enhance lipolysis in obesity.

Irisin is a cleaved and secreted fragment of fibronectin type III domain containing 5 (FNDC5), and contributes to the beneficial effects of exercise on metabolism. Here we report the therapeutical effects of FNDC5/irisin on metabolic derangements and insulin resistance in obesity, and show the lipolysis effect of irisin and its signal molecular mechanism. In obese mice, lentivirus mediated-FNDC5 overexpression enhanced energy expenditure, lipolysis and insulin sensitivity, and reduced hyperlipidemia, hyperglycemia, hyperinsulinism, blood pressure and norepinephrine levels; it increased hormone-sensitive lipase (HSL) expression and phosphorylation, and reduced perilipin level and adipocyte diameter in adipose tissues. Subcutaneous perfusion of irisin reduced hyperlipidemia and hyperglycemia, and improved insulin resistance. Either FNDC5 overexpression or irisin perfusion only induced a tendency toward a slight decrease in body weight in obese mice. In 3T3-L1 adipocytes, irisin enhanced basal lipolysis rather than isoproterenol-induced lipolysis, which were prevented by inhibition of adenylate cyclase or PKA; irisin increased the HSL and perilipin phosphorylation; it increased PKA activity, and cAMP and HSL mRNA levels, but reduced perilipin expression. These results indicate that FNDC5/irisin ameliorates glucose/lipid metabolic derangements and insulin resistance in obese mice, and enhances lipolysis via cAMP-PKA-HSL/perilipin pathway. FNDC5 or irisin can be taken as an effective therapeutic strategy for metabolic disorders.

Salusin-ß contributes to vascular remodeling associated with hypertension via promoting vascular smooth muscle cell proliferation and vascular fibrosis.

Vascular smooth muscle cell (VSMC) proliferation and vascular fibrosis are closely linked with hypertension and atherosclerosis. Salusin-ß is a bioactive peptide involved in the pathogenesis of atherosclerosis. However, it is still largely undefined whether salusin-ß is a potential candidate in the VSMC proliferation and vascular fibrosis. Experiments were carried out in human vascular smooth muscle cells (VSMCs) and in rats with intravenous injection of lentivirus expressing salusin-ß. In vitro, salusin-ß promoted VSMCs proliferation, which was attenuated by adenylate cyclase inhibitor SQ22536, PKA inhibitor Rp-cAMP, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478, ERK inhibitor U0126 or cAMP response element binding protein (CREB) inhibitor KG501. It promoted the phosphorylation of ERK1/2, CREB and EGFR, which were abolished by SQ22536 or Rp-cAMP. Furthermore, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478 diminished the salusin-ß-evoked ERK1/2 and CREB phosphorylation. On the other hand, salusin-ß increased collagen-I, collagen-III, fibronectin and connective tissue growth factor (CTGF) mRNA and phosphorylation of Smad2/3, which were prevented by ALK5 inhibitor A83-01. In vivo, salusin-ß overexpression increased the media thickness, media/lumen ratio coupled with ERK1/2, CREB, EGFR and Smad2/3 phosphorylation, as well as the mRNA of collagen-I, collagen-III, fibronectin, transforming growth factor-ß1 (TGF-ß1) and CTGF in arteries. Moreover, salusin-ß overexpression in rats caused severe hypertension. Intravenous injection of salusin-ß dose-relatedly increased blood pressure, but excessive salusin-ß decreased blood pressure and heart rate. These results indicate that salusin-ß promotes VSMC proliferation via cAMP-PKA-EGFR-CREB/ERK pathway and vascular fibrosis via TGF-ß1-Smad pathway. Increased salusin-ß contributes to vascular remodeling and hypertension.

Up-Regulation of MiR-452 Inhibits Metastasis of Non-Small Cell Lung Cancer by Regulating BMI1.

BACKGROUND/AIMS: MicroRNAs (miRNAs) have been regarded as a new class of regulators in cellular processes in non-small cell lung cancer (NSCLC). However, the relationship between miR-452 and the development of NSCLC remains unclear. METHODS: qRT-PCR was used to detect the expression of miR-452 and its target gene in NSCLC samples (n=60). The transwell assay was used to test the cell invasion capability. The regulation mechanism was confirmed by luciferase reporter assay and western blot assay. RESULTS: In the current study, a relatively lower miR-452 and higher BMI1 expression levels were confirmed to be associated with advanced tumor stage and more extent of lymph nodes metastasis. In vitro, down-regulated miR-452 could enhance cell invasion capability. Furthermore, miR-452 modulated BMI1 expression by binding to its 3'-UTR. The enhancement of cell invasion capability induced by down-regulated miR-452 was eliminated by repression of BMI1. CONCLUSIONS: Our results suggest that miR-452 plays a vital role in development of NSCLC, and this miR-452-BMI1 pathway might generate a novel insight into the treatment of NSCLC.

Irisin inhibits hepatic gluconeogenesis and increases glycogen synthesis via the PI3K/Akt pathway in type 2 diabetic mice and hepatocytes.

Increased glucose production and reduced hepatic glycogen storage contribute to metabolic abnormalities in diabetes. Irisin, a newly identified myokine, induces the browning of white adipose tissue, but its effects on gluconeogenesis and glycogenesis are unknown. In the present study, we investigated the effects and underlying mechanisms of irisin on gluconeogenesis and glycogenesis in hepatocytes with insulin resistance, and its therapeutic role in type 2 diabetic mice. Insulin resistance was induced by glucosamine (GlcN) or palmitate in human hepatocellular carcinoma (HepG2) cells and mouse primary hepatocytes. Type 2 diabetes was induced by streptozotocin/high-fat diet (STZ/HFD) in mice. In HepG2 cells, irisin ameliorated the GlcN-induced increases in glucose production, phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) expression, and glycogen synthase (GS) phosphorylation; it prevented GlcN-induced decreases in glycogen content and the phosphoinositide 3-kinase (PI3K) p110α subunit level, and the phosphorylation of Akt/protein kinase B, forkhead box transcription factor O1 (FOXO1) and glycogen synthase kinase-3 (GSK3). These effects of irisin were abolished by the inhibition of PI3K or Akt. The effects of irisin were confirmed in mouse primary hepatocytes with GlcN-induced insulin resistance and in human HepG2 cells with palmitate-induced insulin resistance. In diabetic mice, persistent subcutaneous perfusion of irisin improved the insulin sensitivity, reduced fasting blood glucose, increased GSK3 and Akt phosphorylation, glycogen content and irisin level, and suppressed GS phosphorylation and PEPCK and G6Pase expression in the liver. Irisin improves glucose homoeostasis by reducing gluconeogenesis via PI3K/Akt/FOXO1-mediated PEPCK and G6Pase down-regulation and increasing glycogenesis via PI3K/Akt/GSK3-mediated GS activation. Irisin may be regarded as a novel therapeutic strategy for insulin resistance and type 2 diabetes.

The Mediation/Moderation Effects of Gut Microbiota on Sleep Quality and Primary Liver Cancer: A Mendelian Randomization and Case-Control Study.

Background: Primary liver cancer (PLC) is a fatal malignancy, sleep quality and gut microbiota were shown to be associated with PLC. However, the mechanism of how sleep quality affects PLC is unclear. This study aims to investigate the mediation/moderation effects of gut microbiota on sleep quality and the occurrence of PLC. Methods: The causality of sleep quality and the occurrence of PLC was detected through the Mendelian randomization (MR) analysis based on the data including 305,359 individuals (Finland Database) and 456,348 participants (UK Biobank). The primary method used for MR analysis was inverse-variance weighted analysis. Gut microbiota' mediation/moderation effects were uncovered in the case-control study including 254 patients with PLC and 193 people with benign liver diseases through the mediation/moderation effect analyses. People's sleep quality was evaluated through the Pittsburgh sleep quality index (PSQI). Results: Poor sleep quality could lead to PLC through the MR analysis (P = 0.026). The case-control study uncovered that Actinobacteria had mediation effects on the relationship between PSQI score, self-sleep quality, and the occurrence of PLC (P = 0.048, P = 0.046). Actinobacteria and Bifidobacterium could inhibit the development of PLC caused by short night sleep duration (P = 0.021, P = 0.022). Erysipelotrichales could weaken the influence of daytime dysfunction on PLC (P = 0.033). Roseburia modulated the contribution of nocturnal insomnia and poor sleep quality to PLC (P = 0.009, P = 0.017). Conclusion: Poor sleep quality was associated with PLC. Gut microbiota' mediation/moderation effects on poor sleep quality and the occurrence of PLC prompted an insightful idea for the prevention of PLC.

Enhanced isradipine sensitivity in vascular smooth muscle cells due to hypoxia-induced Cav1.2 splicing and RbFox1/Fox2 downregulation.

Calcium influx via the L-type voltage-gated Cav1.2 calcium channel in smooth muscle cells regulates vascular contraction. Calcium channel blockers (CCBs) are widely used to treat hypertension by inhibiting Cav1.2 channels. Using the vascular smooth muscle cell line, A7r5 and primary culture of cerebral vascular smooth muscle cells, we found that the expression and function of Cav1.2 channels are downregulated during hypoxia. Furthermore, hypoxia induces structural changes in Cav1.2 channels via alternative splicing. The expression of exon 9* is upregulated, whereas exon 33 is downregulated. Such structural alterations of Cav1.2 channels are caused by the decreased expression of RNA-binding proteins RNA-binding protein fox-1 homolog 1 and 2 (RbFox1 and RbFox2). Overexpression of RbFox1 and RbFox2 prevents hypoxia-induced exon 9* inclusion and exon 33 exclusion. Importantly, such structural alterations of the Cav1.2 channel partly contribute to the enhanced sensitivity of Cav1.2 to isradipine (a CCB) under hypoxia. Overexpression of RbFox1 and RbFox2 successfully reduces isradipine sensitivity in hypoxic smooth muscle cells. Our results suggest a new strategy to manage ischemic diseases such as stroke and myocardial infarction.

Association of glucose-lowering drug target and risk of gastrointestinal cancer: a mendelian randomization study.

BACKGROUND & AIMS: Glucose-lowering drug is associated with various cancers, but the causality with gastrointestinal cancer risk is rarely reported. We aimed to explore the causality between them in this Mendelian randomization (MR) study. METHODS: Two-sample MR, summary-data-based (SMR), mediation MR, and colocalization analyses was employed. Ten glucose-lowering drug targets (PPARG, DPP4, GLP1R, INSR, SLC5A2, ABCC8, KCNJ11, ETFDH, GPD2, PRKAB1) and seven types of gastrointestinal cancer (anal carcinoma, cardia cancer, gastric cancer, hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC), pancreatic cancer, rectum cancer) were included. Patients with gastrointestinal cancers from six different large GWAS databases, including the UK Biobank and Finnish cohorts were incorporated, for discovery and external validation. Meta-analysis was employed to integrate the results from both discovery and validation cohorts, thereby ensuring the reliability of findings. RESULTS: ABCC8/KCNJ11 were associated with pancreatic cancer risk in both two-sample MR (odds ratio (OR): 15.058, per standard deviation unit (SD) change of glucose-lowering durg target perturbation equivalent to 1 SD unit of HbA1c lowering; 95% confidence interval (95% CI): 3.824-59.295; P-value = 0.0001) and SMR (OR: 1.142; 95% CI: 1.013-1.287; P-value = 0.030) analyses. The mediation effect of body mass index (OR: 0.938; 95% CI: 0.884-0.995; proportion of mediation effect: 3.001%; P-value = 0.033) on ABCC8/KCNJ11 and pancreatic cancer was uncovered. Strong connections of DPP4 with anal carcinoma (OR: 0.123; 95% CI: 0.020-0.745; P-value = 0.023) and ICC (OR: 7.733; 95% CI: 1.743-34.310; P-value = 0.007) were detected. PPARG was associated with anal carcinoma (OR: 12.909; 95% CI: 3.217-51.795; P-value = 0.0003), HCC (OR: 36.507; 95% CI: 8.929-149.259; P-value < 0.0001), and pancreatic cancer (OR: 0.110; 95% CI: 0.071-0.172; P-value < 0.0001). SLC5A2 was connected with pancreatic cancer (OR: 8.096; 95% CI: 3.476-18.857; P-value < 0.0001). Weak evidence indicated the connections of GLP1R, GPD2, and PRKAB1 with anal carcinoma, cardia cancer, ICC, and rectum cancer. In addition, the corresponding results were consistently validated in both the validation cohorts and the integrated outcomes. CONCLUSIONS: Some glucose-lowering drugs were associated with gastrointestinal cancer risk, which might provide new ideas for gastrointestinal cancer treatment.