MiR-146a-5p targeting SMAD4 and TRAF6 inhibits adipogenensis through TGF-β and AKT/mTORC1 signal pathways in porcine intramuscular preadipocytes

Background: Intramuscular fat(IMF) content is a vital parameter for assessing pork quality. Increasing evidence has shown that microRNAs(miRNAs) play an important role in regulating porcine IMF deposition. Here, a novel miRNA implicated in porcine IMF adipogenesis was found, and its effect and regulatory mechanism were further explored with respect to intramuscular preadipocyte proliferation and differentiation.Results: By porcine adipose tissue miRNA sequencing analysis, we found that miR-146a-5p is a potential regulator of porcine IMF adipogenesis. Further studies showed that miR-146a-5p mimics inhibited porcine intramuscular preadipocyte proliferation and differentiation, while the miR-146a-5p inhibitor promoted cell proliferation and adipogenic differentiation. Mechanistically, miR-146a-5p suppressed cell proliferation by directly targeting SMAD family member 4(SMAD4) to attenuate TGF-β signaling. Moreover, miR-146a-5p inhibited the differentiation of intramuscular preadipocytes by targeting TNF receptor-associated factor 6(TRAF6) to weaken the AKT/mTORC1 signaling downstream of the TRAF6 pathway.Conclusions: MiR-146a-5p targets SMAD4 and TRAF6 to inhibit porcine intramuscular adipogenesis by attenuating TGF-β and AKT/mTORC1 signaling, respectively. These findings provide a novel miRNA biomarker for regulating intramuscular adipogenesis to promote pork quality.

Hepatitis C virus core protein-induced miR-93-5p upregulation inhibits interferon signaling pathway by targeting IFNAR1

AIM To investigate the mechanism by which hepatitis C virus(HCV) core protein-induced mi R-93-5 p up-regulation regulates the interferon(IFN) signaling pathway.METHODS HCV-1 b core protein was exogenously expressed in Huh7 cells using pc DNA3.1(+) vector. The expression of mi R-93-5 p and interferon receptor 1(IFNAR1) was measured using quantitative reverse transcriptionpolymerase chain reaction and Western blot. The protein expression and phosphorylation level of STAT1 were evaluated by Western blot. The overexpression and silencing of mi R-93-5 p and IFNAR1 were performed using mi R-93-5 p agomir and antagomir, and pc DNA3.1-IFNAR1 and IFNAR1 si RNA, respectively. Luciferase assay was used to identify whether IFNAR1 is a target of mi R-93-5 p. Cellular experiments were also conducted.RESULTS Serum mi R-93-5 p level was increased in patients with HCV-1 b infection and decreased to normal level after HCV-1 b clearance, but persistently increased in those with pegylated interferon-α resistance, compared with healthy subjects. Serum mi R-93-5 p expression had an AUC value of 0.8359 in distinguishing patients with pegylated interferon-α resistance from those with pegylated interferon-α sensitivity. HCV-1 b core protein increased mi R-93-5 p expression and induced inactivation of the IFN signaling pathway in Huh7 cells. Furthermore, IFNAR1 was identified as a direct target of mi R-93-5 p, and IFNAR1 restore could rescue mi R-93-5 p-reduced STAT1 phosphorylation, suggesting that the mi R-93-5 p-IFNAR1 axis regulates the IFN signaling pathway.CONCLUSION HCV-1 b core protein-induced mi R-93-5 p up-regulation inhibits the IFN signaling pathway by directly targeting IFNAR1, and the mi R-93-5 p-IFNAR1 axis regulates STAT1 phosphorylation. This axis may be a potential therapeutic target for HCV-1 b infection.

Chaihu Longgu Muli Decoction relieving temporal lobe epilepsy in rats by inhibiting TLR4 signaling pathway through miR-146a-3p and miR-146a-5p

Objective To explore the effect and mechanism of Chaihu Longgu Muli Decoction(柴胡龙骨牡蛎汤,CHLGMLD)in rats with temporal lobe epilepsy(TLE).Methods A total of 80 Sprague-Dawley(SD)male rats were randomized into control(CON),model(MOD),carbamazepine(CBZ,0.1 g/kg),CHLGMLD low dose(CHLGMLD-L,12.5 g/kg),and high dose(CHLGMLD-H,25 g/kg)groups,with 16 rats in each group.TLE rat models were established in the four groups with the use of lithium-pilocarpine except for the CON group.After the successful establishment of TLE models,all drugs were administered through gavage,and distilled water was given to rats in the CON and MOD groups for four weeks.The frequency and duration of seizures before and after treatment were recorded for the evaluation of the alleviation degree.Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the expression levels of miR-146a-3p and miR-146a-5p.The expression levels of toll-like receptor 4(TLR4),interleukin-1 receptor-associated kinase 1(IRAK1),tumor necrosis factor(TNF)receptor-associated factor 6(TRAF6),TAK1-binding protein(TAB),nuclear factor-kappa B(NF-κB),and interleukin-1 beta(IL-1β)in hippocampus were tested by immunofluorescence assay.Correlation analysis between the above factors and expressions of miR-146a-3p and miR-146a-5p were performed separately.Results CHLGMLD decreased the frequency(P<0.05)and duration(P<0.01)of seizures in rats.CHLGMLD down-regulated the expression levels of miR-146a-5p and miR-146a-3p(P<0.05),and inhibited the expression levels of TLR4,IRAK1,TRAF6,TAB,NF-κB,and IL-1β(P<0.01).The correlation analysis revealed that the expression levels of TLR4,IRAK1,TRAF6,TAB,NF-κB,and IL-1β were positively correlated with the expression levels of miR-146a-3p and miR-146a-5p detected by qRT-PCR,respectively(P<0.01).Conclusion CHLGMLD can inhibite the TLR4 signaling pathway by lowering the expression levels of miR-146a-3p and miR-146a-5p to alleviate hippocampal dentate gyrus inflammation in TLE rats,thus relieving seizures.

miR-146a-5p affects inflammation response of trophoblast by inhibiting TRAF6/NF-кB signaling pathway

Objective:To investigate the association of Micro-rna(miR)-146a-5p expression with preeclampsia,and further explore the potential mechanism involved.Methods:Compared with the blank control group,the expressions of miR-146a-5p and TRAF6 were detected in lipopolysaccharide(LPS)-induced JEG-3 cells.Chorionic carcinoma cell JEG-3 in vitro culture are divided into control,miR-146a-5p mimic+lipopolysaccharide(lps),miR-146a-5p mimic and miR-146a-5p inhibitor groups.qRT-PCR analysis were used to detect the mRNA of miR-146a-5p,IL-1β,IL-6,IL-8 and TNF-α.Western blot assays were carried out to determine the protein expression of TRAF6/NF-кB pathway related proteins.Results:1.miR-146a expression in miR-146a mimic group were significantly higher than the other three groups(P<0.05).2.Compared with the control group,the expression level of miR-146a-5p in JEG-3 cells induced by LPS was significantly increased,and the expression level of TRAF6 was significantly reduced(P<0.05).3.Compared with the control group,the mRNA expression levels of IL-1β,IL-6,IL-8,and TNF-αdecreased significantly after using miR-146a mimic(P<0.05).After adding miR-146a inhibitor,the mRNA expression levels of IL-1β,IL-6,IL-8,and TNF-αwere significantly increased(P<0.05).However,compared with the mimic+LPS group,the difference was not statistically significant(all P>0.05).The results of Western Blot showed that the expression of TRAF6 and NF-κB protein in JEG-3 cells decreased significantly after adding miR-146a mimic and increased after adding miR-146a inhibitor.Conclusion:MiR-146-5p can affect the inflammation response of Maternal-fetal interface by inhibiting TRAF6/NF-кB signaling pathway in preeclampsia.

经皮穴位电刺激对早发性卵巢功能不全大鼠卵巢颗粒细胞凋亡的作用机制

目的 探讨经皮穴位电刺激(TEAS)对早发性卵巢功能不全(POI)大鼠卵巢颗粒细胞凋亡的作用机制。方法 将40只SPF级SD雌性大鼠按照随机数字表法分成4组,对照组、模型组、TEAS组和激素组。除对照组外,其余各组均使用雷公藤多苷片连续灌胃14 d建立POI大鼠模型。造模成功后,TEAS组给予电子针疗仪刺激20 min,1次/d,共干预2周;激素组给予戊酸雌二醇溶液1 mg/(kg·d)灌胃,对照组和模型组仅给予相同时间的抓取和固定,不予任何治疗。采用酶联免疫吸附试验检测大鼠血清卵泡刺激素(FSH)、雌二醇(E_(2))、抗米勒管激素(AMH)水平,TUNEL检测卵巢颗粒细胞凋亡情况,Western blot法检测卵巢组织微小RNA-23a(miR-23a)、SMAD同源物5(SMAD5)、半胱氨酸天冬氨酸蛋白酶3(Caspase-3)蛋白的表达。结果 与对照组比较,模型组大鼠血清E_(2)、AMH水平降低,FSH水平升高(P<0.05);颗粒细胞凋亡率均升高(P<0.05);与模型组比较,TEAS组及激素组大鼠血清E_(2)、AMH水平均升高,FSH水平均降低,颗粒细胞凋亡率均下降(P<0.05)。与对照组比较,模型组大鼠卵巢SMAD5蛋白表达减少,而mi R-23a、Caspase-3蛋白表达增加(P<0.05);与模型组比较,TEAS组及激素组大鼠卵巢SMAD5蛋白表达增加,mi R-23a、Caspase-3蛋白表达减少(P<0.05)。结论 TEAS能够有效调节POI模型大鼠血清激素水平,可能通过mi R-23a/SMAD5信号通路,下调凋亡调控蛋白Caspase-3的表达,从而减少颗粒细胞的过度凋亡,改善卵巢功能。

Melatonin influences the biological characteristics of keloid fibroblasts through the Erk and Smad signalling pathways

Background:Keloids are abnormal fibrous hyperplasias that are difficult to treat.Melatonin can be used to inhibit the development of certain fibrotic diseases but has never been used to treat keloids.We aimed to discover the effects and mechanisms of melatonin in keloid fibroblasts(KFs).Methods:Flow cytometry,CCK-8 assays,western blotting,wound-healing assays,transwell assays,collagen gel contraction assays and immunofluorescence assays were applied to demonstrate the effects and mechanisms of melatonin in fibroblasts derived from normal skin,hypertrophic scars and keloids.The therapeutic potential of the combination of melatonin and 5-fluorouracil(5-FU)was investigated in KFs.Results:Melatonin significantly promoted cell apoptosis and inhibited cell proliferation,migration and invasion,contractile capability and collagen production in KFs.Further mechanistic studies demonstrated that melatonin could inhibit the cAMP/PKA/Erk and Smad pathways through the membrane receptor MT2 to alter the biological characteristics of KFs.Moreover,the combination of melatonin and 5-FU remarkably promoted cell apoptosis and inhibited cell migration and invasion,contractile capability and collagen production in KFs.Furthermore,5-FU suppressed the phosphorylation of Akt,mTOR,Smad3 and Erk,and melatonin in combination with 5-FU markedly suppressed the activation of the Akt,Erk and Smad pathways.Conclusions:Collectively,melatonin may inhibit the Erk and Smad pathways through the mem-brane receptor MT2 to alter the cell functions of KFs,while combination with 5-FU could exert even more inhibitory effects in KFs through simultaneous suppression of multiple signalling pathways.