目的 探究免疫球蛋白A(IgA)肾病进程与转录因子EB(TFEB)自噬通路的相关性.方法 选取2021年12月至2022年12月在该院诊断并收治的IgA肾病患者24例作为研究对象,按Katafuchi积分标准将患者分成4组,每组6例.收集患者一般指标(性别和年龄),实验室指标[血肌酐(Scr)、尿素(UREA)、24 h尿蛋白、空腹血清和肾穿活检标本].免疫组织化学染色观察肾穿活检标本中TFEB、微管相关蛋白1轻链3B(MAP1LC3B,简称LC3B)、p62和肾病蛋白(nephrin)表达水平.采用Pearson或Spearman相关性分析和单因素logistic回归分析TFEB自噬通路及其蛋白与IgA肾病分级的关系.结果 各组患者性别与年龄比较差异无统计学意义(P>0.05).与Katafuchi 0级患者比较,KatafuchiⅢ级患者Scr水平,以及肾穿组织TE-FB、LC3B蛋白表达水平升高,p62、nephrin蛋白表达水平降低,差异有统计学意义(P<0.05).相关性分析结果显示,IgA肾病分级与患者性别、年龄、24 h尿蛋白、尿素无相关性(P>0.05),与Scr水平和TEFB、LC3B蛋白表达水平呈正相关(P<0.05),与p62、nephrin蛋白表达水平呈负相关(P<0.05).单因素logistic回归分析结果显示,Scr和TEFB、LC3B、p62、nephrin蛋白为IgA肾病分级的影响因素(P<0.05).结论 IgA肾病分级与TFEB自噬通路存在高度相关性;Scr高水平,TFEB、LC3B高表达,p62和nephrin低表达是IgA肾病患者病情恶化的潜在标志.

在转录延伸过程中,P-TEFb激酶(由激酶CDK9 和Cyclin T1 组成的二聚体)是释放停滞的Pol Ⅱ这一步的关键调节因子.在人类细胞中,它可参与形成三个更大的复合体,即超级延伸复合体(super elongation complex,SEC)、BRD4/P-TEFb 和 7SK snRNP,其活性在前两者中不被抑制,而在后者中被抑制.除 P-TEFb 外,SEC还含有AFF1 或 4、AF9 或ENL、ELL1、2 或 3,EAF1 或 2 等无酶活亚基,这些亚基被认为可通过P-TEFb影响转录延伸.本课题组前期的研究和其他实验室的研究工作均已表明AFF1、AF9 和ENL亚基均可调控转录启始,但尚不清楚 AFF4 是否具有相似的功能.在调控基因表达的选择性方面,近期有研究表明 BRD4/P-TEFb在人类结直肠腺癌细胞DLD-1 中主要负责除热激基因以外的其他基因的停滞释放,而SEC负责热激基因的停滞释放.为了深入理解AFF4 的功能,本研究利用RNA干扰技术在人类红系白血病细胞HEL中敲低了AFF4,发现RNA聚合酶Ⅱ(Pol Ⅱ)的羧基端重复区(C-terminal domain,CTD)内第二个丝氨酸(Serine 2,Ser-2)的磷酸化水平降低.利用RNA-seq和CUT&Tag确定了AFF4 的直接靶基因,利用ChIP-seq和PRO-seq发现AFF4 对转录启始的影响不明确、对Pol Ⅱ的停滞释放的调控不局限于热激基因,利用ChIP-qPCR发现AFF4 缺失降低了P-TEFb的染色质结合.上述研究结果表明,AFF4 对Pol Ⅱ的停滞释放的调控可能具有一定的细胞类型特异性,即在一些细胞内仅调控热激基因的停滞释放,而在另一些细胞中调控的对象却不仅限于热激基因.

Regulation of transcription is fundamental to the control of cellular gene expression and function.Although recent studies have revealed a role for the oncoprotein MYC in amplifying global transcription,little is known as to how the global transcription is suppressed.Here we report that SUMO and MYC mediate opposite effects upon global transcription by controlling the level of CDK9 sumoylation.On one hand,SUMO suppresses global transcription via sumoylation of CDKg,the catalytic subunit of P-TEFb kinase essential for productive transcriptional elongation.On the other hand,MYC amplifies global transcription by antagonizing CDK9 sumoylation.Sumoylation of CDK9 blocks its interaction with Cyclin T1 and thus the formation of active P-TEFb complex.Transcription profiling analyses reveal that SUMO represses global transcription,particularly of moderately to highly expressed genes and by generating a sumoylation-resistant CDK9 mutant,we confirm that sumoylation of CDK9 inhibits global transcription.Together,our data reveal that SUMO and MYC oppositely control global gene expression by regulating the dynamic sumoylation and desumoylation of CDK9.

The persistence of latent HIV-1 reservoirs throughout combination antiretroviral therapy (cART) is a major barrier on the path to achieving a cure for AIDS.It has been shown that bromodomain and extra-terminal (BET) inhibitors could reactivate HIV-1 latency,but restrained from clinical application due to their toxicity and side effects.Thus,identifying a new type of BET inhibitor with high degrees of selectivity and safety is urgently needed.Apabetalone is a small-molecule selective BET inhibitor specific for second bromodomains,and has been evaluated in phase Ⅲ clinical trials that enrolled patients with high-risk cardiovascular disorders,dyslipidemia,and low HDL cholesterol.In the current study,we examined the impact of apabetalone on HIV-1 latency.We showed that apabetalone (10-50 μmol/L) dose-dependently reactivated latent HIV-1 in 4 types of HIV-1 latency cells in vitro and in primary human CD4+ T cells ex vivo.In ACH2 cells,we further demonstrated that apabetalone activated latent HIV-1 through Tatdependent P-TEFB pathway,i.e.,dissociating bromodomain 4 (BDR4) from the HIV-1 promoter and recruiting Tat for stimulating HIV-1 elongation.Furthermore,we showed that apabetalone (10-30 μmol/L) caused dose-dependent cell cycle arrest at the G1/G0 phase in ACH2 cells,and thereby induced the preferential apoptosis of HIV-1 latent cells to promote the death of reactivated reservoir cells.Notably,cardiovascular diseases and low HDL cholesterol are known as the major side effects of cART,which should be prevented by apabetalone.In conclusion,apabetalone should be an ideal bifunctional latency-reversing agent for advancing HIV-1 eradication and reducing the side effects of BET inhibitors.

目的 探究红景天苷对创伤后应激障碍(post-traumatic stress disorder,PTSD)大鼠空间记忆、学习能力、恐惧的影响及可能机制.方法 采用国际上认可的单一延长刺激(single prolonged stress,SPS )方法建立PTSD大鼠模型,应用Morris水迷宫实验观察红景天苷对SPS大鼠空间学习记忆能力的影响,使用旷场实验观察红景天苷对SPS大鼠恐惧的影响,采用免疫印迹法及免疫荧光方法检测海马神经元mTOR、TEFB、LC3、PGC-1 α的蛋白水平和免疫反应性.结果 SPS+红景天苷组大鼠空间记忆能力较SPS组大鼠有明显改善,焦虑水平有所下降.免疫荧光染色显示了SPS大鼠海马神经元TFEB、LC3、mTOR、PGC-1α的改变,Western blot检测显示SPS大鼠海马神经元LC3、PGC-1α水平上升(P＜0.05),SPS+红景天苷组与SPS组相比,PGC-1 α、LC3显著降低(P＜0.05).结论 红景天苷可能通过调节SPS大鼠海马神经元细胞自噬途径影响大鼠的焦虑行为及空间记忆能力.

Cyclin-dependent kinase(CDK)9 associates mainly with cyclin T1 and forms the positive transcription elongation factor b(p-TEFb)complex responsible for transcriptional regulation.It has been shown that CDK9 modulates the expression and activity of oncogenes,such as MYC and murine double minute 4(MDM4),and it also plays an important role in development and/or maintenance of the malignant cell phenotype.Malfunction of CDK9 is frequently observed in numerous cancers.Recent studies have highlighted the function of CDK9 through a variety of mechanisms in cancers,including the formation of new complexes and epigenetic alterations.Due to the importance of CDK9 activation in cancer cells,CDK9 inhibitors have emerged as promising candidates for cancer therapy.Natural product-derived and chemically synthesized CDK9 inhibitors are being examined in preclinical and clinical research.In this review,we summarize the current knowledge on the role of CDK9 in transcriptional regulation,epigenetic regulation,and different cellular factor interactions,focusing on new advances.We show the importance of CDK9 in mediating tumorigenesis and tumor progression.Then,we provide an overview of some CDK9 inhibitors supported by multiple oncologic preclinical and clinical investigations.Finally,we discuss the perspective and challenge of CDK9 modulation in cancer.

目的 探讨miR-214对异丙酚麻醉诱导的神经元损伤的保护作用及生物学机制.方法 7日龄SD雄性大鼠随机分为4组(每组15只):生理盐水组(NS)、异丙酚麻醉开腹探查组(模型)、miR-NC组和miR-214组.除NS组外,其余组别建立异丙酚麻醉开腹探查模型.miR-NC组和miR-214组分别在麻醉前将miR-NC-agomir或miR-214-agomir注射到海马内.采用免疫荧光法检测海马mTORC1的表达,TUNEL染色检测海马组织细胞凋亡,RT-qPCR分析海马组织中miR-214表达.分别将miR-214抑制剂和mTORC1抑制剂转染入HT22海马神经元细胞,然后暴露于异丙酚.采用流式细胞术分析细胞的存活情况和Western blot分析TEFB、C-caspase3蛋白表达.结果 与NS组相比,模型组大鼠海马中miR-214明显下调(P<0.05),并且海马神经细胞凋亡显著增加(P<0.05).与模型组相比,miR-214组海马神经元凋亡减弱(P<0.05).生物信息学预测证明mTORC1和miR-214之间存在特异性结合位点.免疫荧光检测显示,与NS组比较,模型组诱导海马神经组织中mTORC1表达增加(P<0.05),miR-214治疗显著降低了mTORC1表达(P<0.05).此外,与NC对照组相比,si-mTORC1转染导致暴露于异丙酚的HT22海马神经元细胞的凋亡率显著降低,并且TFEB表达显著增加(P<0.01),以及C-caspase3降低(P<0.05).而miR-214抑制剂转染显著逆转了si-mTORC1的保护作用以及诱导的TFEB、C-caspase3蛋白表达的变化(P<0.05).结论 miR-214可能通过mTORC1-TFEB通路减轻异丙酚神经毒性,提高神经元的存活率.