长非编码RNA SNAI3-AS1调控人软骨细胞C28/I2增殖能力及退变的机制研究

摘要 目的：为了探究长非编码RNA SNAI3-AS1（LncRNA SNAI3-AS1,即SNAI3-AS1）在骨性关节炎（osteoarthritis,OA）进展中的作用与机制。方法：通过全转录组测序筛选出在OA中差异表达的lncRNA SNAI3-AS1,并通过实时荧光定量PCR（qRT-PCR）检测SNAI3-AS1在软骨细胞退变模型中的表达情况。在软骨细胞C28/I2中分别转染SNAI3-AS1特异性siRNA或真核过表达质粒,分别敲低或过表达SNAI3-AS1,通过MTT、平板克隆形成和EdU掺入实验检测细胞增殖活力,Western Blot检测炎症和细胞外基质蛋白的表达情况。通过生物信息学网站预测SNAI3-AS1相互作用的miRNA和下游靶基因,并通过双荧光素酶报告基因和RIP实验进行验证。结果：相较于正常软骨细胞, SNAI3-AS1的表达水平在OA中显著下调。敲低正常软骨细胞中SNAI3-AS1的表达后,软骨细胞的增殖能力减弱并促进了软骨细胞的退变,而在OA模型的软骨细胞中过表达SNAI3-AS1后,软骨细胞的增殖活力加强并抑制了软骨细胞的退变。在机制上,SNAI3-AS1可充当竞争性内源性RNA（ceRNA）,经海绵吸附miR-2278间接上调PRELP,发挥促进软骨细胞增殖和抑制其退变的作用。结论：LncRNA SNAI3-AS1通过LncRNA SNAI3-AS1/ miR-2278/PRELP轴参与骨性关节炎的发生发展过程。     

MiR-127-5p通过靶向调节adipoR1促进骨关节炎软骨细胞的增殖

脂联素（adiponection）与骨关节炎（osteoarthritis, OA）的发病密切相关,且主要通过其受体adipoR1发挥作用。而骨关节炎中脂联素的表达是否受miRNA表达的影响却未见报道。本文旨在研究miR-127-5p对骨关节炎软骨细胞中脂联素及细胞增殖的影响。分离培养人原代OA软骨细胞及对应正常细胞,甲苯胺蓝染色和II型胶原免疫细胞化学染色进行鉴定。 Real-time PCR结果表明,OA软骨细胞中miR-127-5p的表达与正常软骨细胞中的相比较显著下降。MiR-127-5p转染可显著降低荧光素酶报告基因的荧光强度（P<0.05）,表明adipoR1为miR-127-5p的靶向基因。MiR-127-5p mimic转染软骨细胞后,MTT法研究结果表明,miR-127-5p mimic 可显著促进软骨细胞增殖,Western 印迹结果表明,脂联素及其受体（adipoR1）表达显著上升,p65的表达以及p38、ERK1/2以及IkBα的磷酸化水平显著下降。ELISA结果表明,MMP-1、MMP-3、MMP-13的含量显著下降。实验结果提示,miR-127-5p通过靶向下调adipoR1及脂联素的表达,促进软骨细胞增殖,并且抑制NF-κB信号通路,进而抑制炎性反应。     

多效生长因子（Pleiotrophin）基因沉默后的基因表达谱初步分析

多效生长因子（pleiotrophin,PTN指蛋白,Ptn指基因）是一种可同肝素结合的分泌性的生长/分化因子,具有刺激细胞粘附、迁移、存活、生长和分化等功能.我们前期研究发现,Ptn稳定沉默可以显著降低细胞的生长及成瘤能力.为进一步了解Ptn表达沉默后小鼠基因转录谱的变化,用小鼠表达谱芯片比较了对照及Ptn沉默细胞的基因表达差异.在检测的24 000个基因中,Ptn沉默后上调2倍以上的基因有240个,下调2倍以上的基因有129个.值得引起注意的是,在Ptn沉默的MEFs细胞中,同DDK综合症相关的基因家族,schlafen（Slfn）家族的Slfn 2、Slfn 3、Slfn 4以及基质金属蛋白酶（matrix metalloproteinase,MMP）家族的Mmp 3、Mmp 10、Mmp 13表达均显著上调;而可促进内皮细胞运动,参与血管发生的基因angiomotin(Amot)表达显著下调.通过研究,获得了一系列Ptn沉默后表达变化的基因信息.     

RIP3蛋白的研究进展

受体相互作用蛋白-3是丝/苏氨酸蛋白激酶家族成员（RIPs）之一,该蛋白家族作为细胞重要应激传感分子,在调控细胞存活、细胞凋亡和细胞坏死通路中发挥重要作用.近年研究发现,RIP3参与肿瘤坏死因子TNF-α诱导的细胞程序性坏死生物学过程,是TNF-α诱导的细胞凋亡与坏死不同死亡途径转换的关键开关分子.本文就RIP3分子的发现、结构特点、细胞亚定位、生理功能及其分子机制进行综述,并对RIP3分子的研究进行了展望.     

受体相互作用蛋白3——细胞凋亡与坏死的调节阀

综述了受体相互作用蛋白(RIPs)蛋白结构和RIP3调控细胞凋亡与坏死机制的研究进展．受体相互作用蛋白3(receptor-interacting protein 3, RIP3)是丝/苏氨酸蛋白激酶家族成员之一,该蛋白质家族包含一类高度保守的丝/苏氨酸激酶结构域．RIP家族激酶作为细胞应激传感分子,在调控细胞凋亡、细胞坏死和存活通路中发挥重要作用．近年发现,RIP3参与肿瘤坏死因子TNFα诱导的细胞程序化坏死的生物学过程．认识RIP3调控TNFα诱导的细胞凋亡与坏死不同死亡途径转换的分子机制,有助于发现肿瘤治疗的新策略．     

葡萄糖诱导绿色杜氏藻转录组及相关通路差异分析

绿色杜氏藻是研究耐盐机理的模式绿藻．葡萄糖不仅是营养物质,而且还是信号物质．目前,对绿色杜氏藻转录组、糖处理后差异表达基因和β-胡萝卜素生物合成途径关键基因表达还不清楚．本研究通过Illumina HiSeqTM 2000高通量测序,获得葡萄糖处理和未处理绿色杜氏藻转录组信息．利用P value值和差异倍数对样本进行差异表达分析,共111条转录本存在差异表达,3条为上调转录本,108条为下调转录本．利用RT-qPCR检验差异表达分析的准确性. 结果表明,转录本表达结果与转录组分析结果一致．GO功能富集结果表明,71条下调转录本与代谢相关,占所有下调转录本的65.74%．KEGG富集分析结果表明,21条KEGG通路含89条下调转录本,14条通路与代谢相关．代谢中通路最多的为能量代谢（6条）,含63条下调转录本．能量代谢中与光合作用相关的下调转录本最多,为29条．通过分析找到2条与β-胡萝卜素生物合成相关通路（MVA/MEP途径及β-胡萝卜素合成途径）,并发现通路的关键基因hmgs、dxs、dxr、psy、pds、chyb,对其进行差异表达分析,均不存在差异表达．研究表明,葡萄糖抑制了绿色杜氏藻光合作用,代谢受阻,但未影响β-胡萝卜素生物合成相关通路及关键基因．     

重组荞麦胰蛋白酶抑制剂通过下调己糖激酶Ⅱ抑制HepG2细胞生长

糖酵解过度活跃是肿瘤细胞能量代谢的显著特征。抑制过度糖酵解已经成为一种新的癌症疗法。重组荞麦胰蛋白酶抑制剂 (recombinant buckwheat trypsin inhibitor, rBTI)可以通过上调磷酸酶及张力蛋白同源基因 (PTEN) 进而抑制HepG2细胞增殖。有关rBTI对肿瘤细胞能量代谢的影响仍未见报道。本研究中的MTT和ATP检测分析表明,rBTI以剂量依赖性方式抑制细胞活力及胞内ATP含量。qRT-PCR和Western印迹分析表明,rBTI处理HepG2细胞后,己糖激酶Ⅱ转录显著下调,但是糖酵解过程中的其他酶及葡萄糖转运蛋白基因在转录水平未发生显著变化,同时己糖激酶Ⅱ蛋白水平的表达也显著下调。酶活性分析也表明,rBTI能显著降低己糖激酶的活性。进一步分析表明, rBTI使细胞内PTEN转录及表达水平明显上调,己糖激酶Ⅱ转录和p-AKT,p-mTOR、己糖激酶Ⅱ的表达下调。当PTEN抑制剂phen存在时,可阻断rBTI诱导的己糖激酶 Ⅱ表达下降,表明rBTI能通过上调PTEN进而影响己糖激酶Ⅱ的表达。免疫荧光及Western印迹分析显示,rBTI作用后减弱了己糖激酶 Ⅱ在线粒体的定位,导致己糖激酶Ⅱ与线粒体电压依赖性阴离子通道蛋白 (voltage-dependent anion channel, VDAC) 分离,促使己糖激酶Ⅱ从线粒体转位到细胞质,降低糖酵解的效率。上述结果证明,rBTI对肿瘤细胞能量代谢的调控作用主要通过抑制PI3K/AKT信号通路,下调己糖激酶Ⅱ的表达并影响空间定位,进而抑制肿瘤细胞糖酵解过程,导致癌细胞生长受到抑制。     

IL-6通过MAPK/ERK信号通路调节BMSCs的软骨定向分化

白细胞介素-6（IL-6）是参与骨髓间充质干细胞（BMSCs）软骨定向分化的重要调节因子. MAPK/ERK信号通路可介导骨关节炎软骨损伤. 然而,IL-6调节BMSCs定向分化为软骨细胞的分子机制尚不清楚. IL-6通过激活MAPK/ERK信号途径,抑制BMSCs的成软骨分化. 本文发现,BMSCs在体外向软骨细胞分化时, Il-6基因表达水平显著下调,同时分泌到培养基中的IL-6蛋白水平亦明显降低. 重组IL-6可抑制BMSCs向软骨细胞分化,软骨分化标志蛋白Runx2和Sox9的诱导表达亦相应下调. IL-6可诱导MAPK/ERK信号通路活化,加入ERK特异性阻断剂后,Runx2和Sox9的诱导表达恢复正常.结果提示,IL-6通过激活MAPK/ERK信号通路抑制BMSCs的软骨细胞分化.炎症因子IL-6对软骨细胞的再生具有不利的影响,该研究为软骨组织工程研究和骨关节炎等软骨疾病的治疗提供有价值的参考.     

苦参碱对IL-1β诱导的小鼠体外骨关节炎的作用研究

目的探讨苦参碱对IL-1β诱导小鼠软骨细胞退行性变修复作用。方法提取3～5 dC 57乳鼠软骨细胞,分为空白对照组,骨关节炎(OA)组,骨关节炎加苦参碱(OA+Mat)组,分别处理24 h后,行HE染色,观察各组小鼠的细胞形态及数量,并提取各组小鼠的总RNA,RT-qPCR反应检测炎症及软骨标志基因的表达。结果与OA组相比,OA+Mat组细胞形态与空白对照组一样良好,细胞数量与空白对照组相近,且比OA组多,炎症及软骨标志基因的表达均比OA组高(P0.05)。结论苦参碱能降低白介素1诱导的小鼠体外骨关节炎症标志基因的表达,对小鼠关节炎有治疗作用,且能上调关节炎细胞中软骨标志基因COL2a1的表达。     

p53调控骨关节炎软骨细胞凋亡

肿瘤抑制蛋白p53是一种可以有效调节哺乳动物细胞生长的核磷酸化蛋白质。p53表达增加能够激活一系列细胞基因,通过抑制多个细胞周期蛋白依赖性激酶导致细胞周期停滞并凋亡。有研究表明,骨关节炎(osteoarthritis,OA)软骨细胞中,p53的表达高于正常软骨细胞,通过下调p53表达能够减少软骨细胞凋亡,进而预防和缓解骨关节炎病变,这可能与线粒体凋亡途径密切相关,但是具体机制尚不明确。本文通过综述近年来p53调控骨关节炎软骨细胞凋亡的文献资料,为骨关节炎机制和治疗有关研究提供理论基础。     

The pro‐inflammatory effect of NR4A3 in osteoarthritis

NR4A3 is a member of nuclear receptor subfamily 4, which is an important regulator of cellular function and inflammation. In this study, high expression of NR4A3 in human osteoarthritis (OA) cartilage was firstly observed. To explore the relationship between NR4A3 and OA, we used a lentivirus overexpression system to simulate its high expression and study its role in OA. Additionally, siRNA‐mediated knockdown of NR4A3 was used to confirm the findings of overexpression experiments. The results showed the stimulatory effect of IL‐1β on cartilage matrix‐degrading enzyme expression such as MMP‐3, 9, INOS and COX‐2 was enhanced in NR4A3‐overexpressed chondrocytes and decreased in NR4A3‐knockdown chondrocytes at both mRNA and protein levels, while IL‐1β‐induced chondrocyte‐specific gene (collagen 2 and SOX‐9) degradation was only regulated by NR4A3 at protein level. Furthermore, overexpression of NR4A3 would also enhance EBSS‐induced chondrocytes apoptosis, while knockdown of NR4A3 decreased apoptotic level after EBSS treatment. A pathway study indicated that IL‐1β‐induced NF‐κB activation was enhanced by NR4A3 overexpression and reduced by NR4A3 knockdown. We suggest that NR4A3 plays a pro‐inflammatory role in the development of OA, and we also speculate that NR4A3 mainly regulates cartilage matrix‐degrading gene expression under inflammatory conditions via the NF‐κB pathway.     

MiR‐155 promotes interleukin‐1β‐induced chondrocyte apoptosis and catabolic activity by targeting PIK3R1‐mediated PI3K/Akt pathway

Osteoarthritis (OA) is a common joint disease characterized by progressive cartilage degradation, in which elevated chondrocyte apoptosis and catabolic activity play an important role. MicroRNA‐155 (miR‐155) has recently been shown to regulate apoptosis and catabolic activity in some pathological circumstances, yet, whether and how miR‐155 is associated with OA pathology remain unexplored. We report here that miR‐155 level is significantly up‐regulated in human OA cartilage biopsies and also in primary chondrocytes stimulated by interleukin‐1β (IL‐1β), a pivotal pro‐catabolic factor promoting cartilage degradation. Moreover, miR‐155 inhibition attenuates and its overexpression promotes IL‐1β‐induced apoptosis and catabolic activity in chondrocytes in vitro. We also demonstrate that the PIK3R1 (p85α regulatory subunit of phosphoinositide 3‐kinase (PI3K)) is a target of miR‐155 in chondrocytes, and more importantly, PIK3R1 restoration abrogates miR‐155 effects on chondrocyte apoptosis and catabolic activity. Mechanistically, PIK3R1 positively regulates the transduction of PI3K/Akt pathway, and a specific Akt inhibitor reverses miR‐155 effects on promoting chondrocyte apoptosis and catabolic activity, phenocopying the results obtained via PIK3R1 knockdown, hence establishing that miR‐155 promotes chondrocyte apoptosis and catabolic activity through targeting PIK3R1‐mediated PI3K/Akt pathway activation. Altogether, our study discovers novel roles and mechanisms of miR‐155 in regulating chondrocyte apoptosis and catabolic activity, providing an implication for therapeutically intervening cartilage degradation and OA progression.     

Effect of microRNA-145 on IL-1β-induced cartilage degradation in human chondrocytes

MicroRNA-145 has been shown to regulate chondrocyte homeostasis. It seems that miR-145 is implicated in cartilage dysfunction in Osteoarthritis (OA). However, the functional role of miR-145 in interleukin-1 beta (IL-1β)-induced extracellular matrix (ECM) degradation of OA cartilage has never been clarified. Here, we show that miR-145 expression increased in OA chondrocytes and in response to IL-1β stimulation. We confirm that mothers against decapentaplegic homolog 3 (Smad3), a key factor in maintaining chondrocyte homeostasis, is directly regulated by miR-145. Modulation of miR-145 affects the expression of Smad3 causing a change of its downstream target gene expression as well as IL-1β-induced ECM degradation in OA chondrocytes. This indicates that miR-145 contributes to impaired ECM in OA cartilage probably in part via targeting Smad3.     

miR-29b inhibits TGF-β1-induced cell proliferation in articular chondrocytes

Transforming growth factor β1 (TGF-β1) is a known regulator of chondrocyte proliferation and promotes cartilage repair in osteoarthritis (OA). microRNA-29b-3p (miR-29b-3p) is downregulated by TGF-β1 and overexpressed in OA cartilage. However, the ability of miR-29b-3p to mediate the chondrocyte pro-proliferative effects of TGF-β1 is not yet understood. This current study aimed to investigate the effect of miR-29b-3p on TGF-β1-induced cell proliferation in murine articular chondrocytes. The stimulation of chondrocytes by TGF-β1 for 24 h resulted in the downregulation of miR-29b-3p expression. The ratio of G0/G1 phase cells decreased in response to TGF-β1 whereas the ratio of S phase cells was increased. Consistent with this observation, miR-29b-3p overexpression inhibited TGF-β1’s ability to promote the ratio of S phase cells and downregulate the ratio of G0/G1 phase cells. These findings suggest that the downregulation of miR-29b-3p is a likely requirement for TGF-β1-mediated proliferation of murine articular chondrocytes. Furthermore, implying that miR-29b-3p expression may be involved in reduced chondrocyte proliferation in OA.     

Shear-induced Interleukin-6 Synthesis in Chondrocytes: ROLES OF E PROSTANOID (EP) 2 AND EP3 IN cAMP/PROTEIN KINASE A- AND PI3-K/Akt-DEPENDENT NF-κB ACTIVATION*

Mechanical overloading of cartilage producing hydrostatic stress, tensile strain, and fluid flow can adversely affect chondrocyte function and precipitate osteoarthritis (OA). Application of high fluid shear stress to chondrocytes recapitulates the earmarks of OA, as evidenced by the release of pro-inflammatory mediators, matrix degradation, and chondrocyte apoptosis. Elevated levels of cyclooxygenase-2 (COX-2), prostaglandin (PG) E₂, and interleukin (IL)-6 have been reported in OA cartilage in vivo, and in shear-activated chondrocytes in vitro. Although PGE₂ positively regulates IL-6 synthesis in chondrocytes, the underlying signaling pathway of shear-induced IL-6 expression remains unknown. Using the human T/C-28a2 chondrocyte cell line as a model system, we demonstrate that COX-2-derived PGE₂ signals via up-regulation of E prostanoid (EP) 2 and down-regulation of EP3 receptors to raise intracellular cAMP, and activate protein kinase A (PKA) and phosphatidylinositol 3-kinase (PI3-K)/Akt pathways. PKA and PI3-K/Akt transactivate the NF-κB p65 subunit via phosphorylation at Ser-276 and Ser-536, respectively. Binding of p65 to the IL-6 promoter elicits IL-6 synthesis in sheared chondrocytes. Selective knockdown of EP2 or ectopic expression of EP3 blocks PKA- and PI3-K/Akt-dependent p65 activation and markedly diminishes shear-induced IL-6 expression. Similar inhibitory effects on IL-6 synthesis were observed by inhibiting PKA, PI3-K, or NF-κB using pharmacological and/or genetic interventions. Reconstructing the signaling network regulating shear-induced IL-6 expression in chondrocytes may provide insights for developing therapeutic strategies for arthritic disorders and for culturing artificial cartilage in bioreactors.     

microRNA-206 is required for osteoarthritis development through its effect on apoptosis and autophagy of articular chondrocytes via modulating the phosphoinositide 3-kinase/protein kinase B-mTOR pathway by targeting insulin-like growth factor-1

microRNA (miR) has been shown to be involved in the treatment of diseases such as osteoarthritis (OA). This study aims to investigate the role of miR-206 in regulating insulin-like growth factor-1 (IGF-1) in chondrocyte autophagy and apoptosis in an OA rat model via the phosphoinositide 3-kinase (P13K)/protein kinase B (AKT)-mechanistic target of rapamycin (mTOR) signaling pathway. Wistar rats were used to establish the OA rat model, followed by the observation of histopathological changes, Mankin score, and the detection of IGF-1-positive expression and tissue apoptosis. The underlying regulatory mechanisms of miR-206 were analyzed in concert with treatment by an miR-206 mimic, an miR-206 inhibitor, or small interfering RNA against IGF-1 in chondrocytes isolated from OA rats. Then, the expression of miR-206, IGF-1, and related factors in the signaling pathway, cell cycle, and apoptosis, as well as inflammatory factors, were determined. Subsequently, chondrocyte proliferation, cell cycle distribution, apoptosis, autophagy, and autolysosome were measured. OA articular cartilage tissue exhibited a higher Mankin score, promoted cell apoptotic rate, increased expression of IGF-1, Beclin1, light chain 3 (LC3), Unc-51-like autophagy activating kinase 1 (ULK1), autophagy-related 5 (Atg5), caspase-3, and Bax, yet exhibited decreased expression of miR-206, P13K, AKT, mTOR, and Bcl-2. Besides, miR-206 downregulated the expression of IGF-1 and activated the P13K/AKT signaling pathway. Moreover, miR-206 overexpression and IGF-1 silencing inhibited the interleukins levels (IL-6, IL-17, and IL-18), cell apoptotic rate, the formation of autolysosome, and cell autophagy while promoting the expression of IL-1β and cell proliferation. The findings from our study provide a basis for the efficient treatment of OA by investigating the inhibitory effects of miR-206 on autophagy and apoptosis of articular cartilage in OA via activating the IGF-1-mediated PI3K/AKT-mTOR signaling pathway.