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.

Deamidation enables pathogenic SMAD6 variants to activate the BMP signaling pathway

The BMP signaling pathway plays a crucial role in regulating early embryonic development and tissue homeostasis.SMAD6 encodes a negative regulator of BMP,and rare variants of SMAD6 are recurrently found in individuals with birth defects.However,we observed that a subset of rare pathogenic variants of SMAD6 consistently exhibited positive regulatory effects instead of the initial negative effects on the BMP signaling pathway.We sought to determine whether these SMAD6 variants have common pathogenic mechanisms.Here,we showed that pathogenic SMAD6 variants accompanying this functional reversal exhibit similar increases in deamidation.Mechanistically,increased deamidation of SMAD6 variants promotes the accumulation of the BMP receptor BMPR1A and the formation of new complexes,both of which lead to BMP signaling pathway activation.Specifically,two residues,N262 and N404,in SMAD6 were identified as the crucial sites of deamidation,which was catalyzed primarily by glutamine-fructose-6-phosphate transaminase 2(GFPT2).Additionally,treatment of cells harboring SMAD6 variants with a deamidase inhibitor restored the inhibitory effect of SMAD6 on the BMP signaling pathway.Conversely,when wild-type SMAD6 was manually simulated to mimic the deamidated state,the reversed function of activating BMP signaling was reproduced.Taken together,these findings show that deamidation of SMAD6 plays a crucial role in the functional reversal of BMP signaling activity,which can be induced by a subset of various SMAD6 variants.Our study reveals a common pathogenic mechanism shared by these variants and provides a potential strategy for preventing birth defects through deamidation regulation,which might prevent the off-target effects of gene editing.

The role of Smad6 in immunity of the pearl oyster Pinctada fucata martensii

Inhibitory Smads(I-Smads),which belong to the Smad family and inhibit bone morphogenic protein 2(BMP2)signaling by a variety of mechanisms,can suppress innate immunity responses in vertebrates.However,there are no reports for the role of Smad6 in immunity in mollusks.In this study,we showed that Smad6 of the pearl oyster Pinctada fucata martensii was located in the Smad6 cluster of the phylogenetic tree;mRNA expression of Smad6 and Smad3 was up-regulated after lipopolysaccharide and polyinosinic:polycytidylic challenge;and transcript levels of Smad6 and Smad3 showed opposite patterns during wound healing.Under salinity stress,water inflow and outflow in the gills appear to be regulated by BMP2-Smads signals,and BMP2-Smads signaling may be closely related to the immune response.Our results indicate that Smad6 is involved in immunity,that it plays a positive role in the response to immune challenge and an inhibitory role during wound healing,and that Smad6 and Smad3 may work against each other.

逍遥散及其拆方对Erastin诱导的LO-2肝细胞系铁死亡的影响

目的 探讨逍遥散及其拆方对Erastin诱导肝细胞铁死亡的影响及机制。方法 使用铁死亡诱导剂Erastin诱导肝细胞LO-2铁死亡,用逍遥散及各功效拆方(逍遥散去疏肝药组、逍遥散去健脾药组、逍遥散去养血药组)的含药血清进行干预,阳性对照药使用铁死亡抑制剂Ferrostatin-1。通过CCK-8检测细胞活力,ELISA法检测细胞上清液中谷丙转氨酶(alanine aminotransferase,ALT)、谷草转氨酶(aspartate aminotransferase,AST)水平,比色法检测细胞中丙二醛(malondialdehyde,MDA)、还原型谷胱甘肽(reduced glutathione,GSH)、Fe^(2+)等含量,荧光酶标仪检测脂质过氧化活性氧(reactive oxygen species,ROS),RT-PCR检测细胞中谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)、前列腺素内过氧化物合酶2(prostaglandin-endoperoxide synthase 2,PTGS2)、铁调素(hepcidin)、膜铁转运蛋白(ferroportin,FPN1)mRNA表达,Western blot检测细胞中骨形态发生蛋白6(bone morphogenetic protein6,BMP6)、铁调素调节蛋白(hemojuvelin,HJV)、Smad同源物4(mothers against decapentaplegic homolog 4,SMAD4)蛋白表达。结果 erastin诱导肝细胞铁死亡后,细胞活力较对照组显著下降(P<0.05),细胞外上清液中ALT、AST含量显著上升(P<0.05),细胞中MDA、ROS、Fe^(2+)、PTGS2与hepcidin mRNA表达及BMP6/HJV/Smad4信号通路蛋白显著上升(P<0.05),GSH含量及GPX4、FPN1 mRNA表达显著下降(P<0.05)。除逍遥散去养血药组在Smad4蛋白表达方面与模型组无显著差异(P>0.05),逍遥散及各功效拆方及阳性对照药Ferrostatin-1均能显著逆转上述指标的变化(P<0.05)。与整方组疗效比较,逍遥散去疏肝药组AST、MDA、GSH、ROS、Fe^(2+)、BMP6、hepcidin、FPN1指标具有显著差异(P<0.05);除逍遥散去健脾药组在Smad4蛋白表达方面与整方组无显著差异(P>0.05),逍遥散去健脾药组及逍遥散去养血药组对所检测指标均有显著改善(P<0.05)。结论 逍遥散可抑制Erastin诱导的肝细胞铁死亡,其机制可能与调控BMP6/HJV/SMAD4信号通路及其下游hepcidin-ferroportin轴有关,而逍遥散各功效拆方均在一定程度上参与了逍遥散抑制肝细胞铁死亡的过程。