Non-small cell lung cancer (NSCLC) is a disease related to inflammation. Proinflammatory cytokines such as interleukin 17 (IL-17) can induce cancer cell proliferation, metastasis and immune escape. Although NSCLC immune escape is partly due to the interaction between PD-1 and PD-L1 and PD-L1 expression can be upregulated in cancer cells upon stimulation with IL-17, the underlying mechanism of IL-17-triggered PD-L1 gene transcription in NSCLC cells remains elusive. RT‒PCR, real-time PCR, and IB were used to assess the levels of PD-L1, MEF2C, and TRIM31 in NSCLC tissues as well as in IL-17–stimulated H1299 or PC9 cells. Bioinformatics analysis, luciferase assays, and ChIP were utilized to investigate the transcriptional mechanism of the PD-L1 gene. Co-IP/IB was used to examine the interaction between MEF2C and PD-L1, including MEF2C ubiquitination. IHC staining was carried out to analyse the expression of IL-17RA, MEF2C, TRIM31, and PD-L1 in NSCLC tissue arrays. The corresponding plasmids were constructed and identified. An isograft model was used to verify the findings in vitro. PD-L1, MEF2C and TRIM31 expression levels were increased in NSCLC tissues and NSCLC cells exposed to IL-17. Mechanistically, MEF2C could bind to the − 778 to -475 nt and − 336 to -97 nt regions of the PD-L1 promoter. TRIM31 could mediate MEF2C K63-linked polyubiquitination at Lys 25, increasing MEF2C recruitment to the PD-L1 promoter and PD-L1 gene transcription. MEF2C, TRIM31 or PD-L1 gene silencing effectively suppressed MEF2C K63-linked polyubiquitination, PD-L1 induction and NSCLC growth in mice inoculated with Lewis lung cancer (LLC) cells transfected with the corresponding shRNA and treated with IL-17. IL-17 induces PD-L1 gene transcription in NSCLC cells through TRIM31-dependent MEF2C K63-linked polyubiquitination.

The cancer cell metastasis is a major death reason for patients with non-small cell lung cancer (NSCLC).Although researchers have disclosed that interleukin 17 (IL-17) can increase matrix metalloproteinases (MMPs)induction causing NSCLC cell metastasis, the underlying mechanism remains unclear. In the study, we found thatIL-17 receptor A (IL-17RA), p300, p-STAT3, Ack-STAT3, and MMP19 were up-regulated both in NSCLC tissues andNSCLC cells stimulated with IL-17. p300, STAT3 and MMP19 overexpression or knockdown could raise or reduceIL-17-induced p-STAT3, Ack-STAT3 and MMP19 level as well as the cell migration and invasion. Mechanisminvestigation revealed that STAT3 and p300 bound to the same region (-544 to-389 nt) of MMP19 promoter, andp300 could acetylate STAT3-K631 elevating STAT3 transcriptional activity, p-STAT3 or MMP19 expression and thecell mobility exposed to IL-17. Meanwhile, p300-mediated STAT3-K631 acetylation and its Y705-phosphorylationcould interact, synergistically facilitating MMP19 gene transcription and enhancing cell migration and invasion.Besides, the animal experiments exhibited that the nude mice inoculated with NSCLC cells by silencing p300, STAT3or MMP19 gene plus IL-17 treatment, the nodule number, and MMP19, Ack-STAT3, or p-STAT3 production in thelung metastatic nodules were all alleviated. Collectively, these outcomes uncover that IL-17-triggered NSCLCmetastasis involves up-regulating MMP19 expression via the interaction of STAT3-K631 acetylation by p300 and itsY705-phosphorylation, which provides a new mechanistic insight and potential strategy for NSCLC metastasis and therapy

The cancer cell metastasis is a major death reason for patients with non-small cell lung cancer (NSCLC). Although researchers have disclosed that interleukin 17 (IL-17) can increase matrix metalloproteinases (MMPs) induction causing NSCLC cell metastasis, the underlying mechanism remains unclear. In the study, we found that IL-17 receptor A (IL-17RA), p300, p-STAT3, Ack-STAT3, and MMP19 were up-regulated both in NSCLC tissues and NSCLC cells stimulated with IL-17. p300, STAT3 and MMP19 overexpression or knockdown could raise or reduce IL-17-induced p-STAT3, Ack-STAT3 and MMP19 level as well as the cell migration and invasion. Mechanism investigation revealed that STAT3 and p300 bound to the same region (−544 to −389 nt) of MMP19 promoter, and p300 could acetylate STAT3-K631 elevating STAT3 transcriptional activity, p-STAT3 or MMP19 expression and the cell mobility exposed to IL-17. Meanwhile, p300-mediated STAT3-K631 acetylation and its Y705-phosphorylation could interact, synergistically facilitating MMP19 gene transcription and enhancing cell migration and invasion. Besides, the animal experiments exhibited that the nude mice inoculated with NSCLC cells by silencing p300, STAT3 or MMP19 gene plus IL-17 treatment, the nodule number, and MMP19, Ack-STAT3, or p-STAT3 production in the lung metastatic nodules were all alleviated. Collectively, these outcomes uncover that IL-17-triggered NSCLC metastasis involves up-regulating MMP19 expression via the interaction of STAT3-K631 acetylation by p300 and its Y705-phosphorylation, which provides a new mechanistic insight and potential strategy for NSCLC metastasis and therapy.

目的:研究人胚肾293T(HEK 293T,简称293T)细胞中外源性肿瘤坏死因子受体相关因子(tumor necrosis factor receptor-associated factor 6,TRAF6)与 Krüppel样因子5(Krüppel-like factor 5,KLF5)的结合及TRAF6多聚泛素化修饰KLF5 的方式和修饰的位点.方法:将构建的Flag-TRAF6、HA-KLF5、泛素过表达质粒、shTRAF6小干扰质粒和TRAF6 C70A位点突变质粒行不同组合转染293T细胞48 h.用免疫沉淀(immunoprecipitation,IP)和免疫印迹(immunoblotting,IB)实验检查TRAF6与KLF5的结合以及KLF5 K63或K48多聚泛素化水平.此外,构建KLF5全部赖氨酸突变的质粒,分别与TRAF6质粒共转染293T细胞.用前述IP/IB检测KLF5 K63连接的多聚泛素化修饰,并确定KLF5 K63泛素化修饰的位点.结果:293T细胞中TRAF6能与KLF5结合;TRAF6过表达和基因沉默或TRA6酶活性缺失能相应上调或下调KLF5 K63的多聚泛素化;KLF5被TRAF6 K63多聚泛素化修饰的位点是其第99位和第100位的赖氨酸.结论:TRAF6能与KLF5相互作用,并对KLF5-K99和K100进行K63多聚泛素化修饰.

Rat Thy-1 nephritis (Thy-1N) is an experimental model for studying human mesangioproliferative glomerulonephritis (MsPGN), and its pathological features are glomerular mesangial cell (GMC) proliferation and extracellular matrix (ECM) accumulation. Although we have confirmed that renal lesions of Thy-1N rats are sublytic C5b-9-dependent, and ECM accumulation is related to tissue inhibitor of matrix metalloproteinase (TIMP) inhibiting matrix metalloproteinase (MMP) activity, whether sublytic C5b-9 can induce TIMP production by GMC in Thy-1N rat and the underlying mechanism remains unclear. In the study, we proved that the expressions of TIMP3, krϋppel-like transcription factor 5 (KLF5) and tumor necrosis factor receptor-associated factor 6 (TRAF6) were simultaneously up-regulated both in the renal tissues of Thy-1N rats (in vivo) and in the GMC exposed to sublytic C5b-9 (in vitro). Further mechanism exploration discovered that KLF5 and TRAF6 as two upstream molecules could induce TIMP3 gene transcription through binding to the same region i.e., -1801nt to -1554nt (GGGGAGGGGC) and -228nt to -46nt (GCCCCGCCCC) of TIMP3 promoter. In the process, TRAF6 mediated KLF5 K63-linked ubiquitination at K99 and K100 enhancing KLF5 nuclear localization and binding to TIMP3 promoter, augmenting its gene activation. Furthermore, the experiments in vivo exhibited that silencing KLF5, TRAF6 or TIMP3 gene could markedly lessen renal KLF5 K63-linked ubiquitination or TIMP3 induction, ECM accumulation and other pathological changes of Thy-1N rats. Besides, the positive expressions of above-mentioned these proteins and ECM accumulation and their correlation in the renal tissues of MsPGN patients were also demonstrated. Overall, our findings implicate that KLF5 and TRAF6 play a promoting role in sublytic C5b-9-triggered TIMP3 gene transcription and expression, which might provide a novel mechanistic insight into rat Thy-1N and human MsPGN.

Interleukin-17 (IL-17), a potent proinflammatory cytokine, can trigger the metastasis of non-small cell lung cancer (NSCLC). However, the underlying mechanism involved in IL-17-induced NSCLC cell metastasis remains unclear. In this study, we found that not only the expression of IL-17, IL-17RA, and/or general control nonrepressed protein 5 (GCN5), SRY-related HMG-BOX gene 4 (SOX4), and matrix metalloproteinase 9 (MMP9) was increased in the NSCLC tissues and in the IL-17-stimulated NSCLC cells, but also IL-17 treatment could enhance NSCLC cell migration and invasion. Further mechanism exploration revealed that IL-17-upregulated GCN5 and SOX4 could bind to the same region (-915 to -712 nt) of downstream MMP9 gene promoter driving its gene transcription. In the process, GCN5 could mediate SOX4 acetylation at lysine 118 (K118, a newly identified site) boosting MMP9 gene expression as well as cell migration and invasion. Moreover, the SOX4 acetylation or MMP9 induction and metastatic nodule number in the lung tissues of the BALB/c nude mice inoculated with the NSCLC cells stably infected by corresponding LV-shGCN5 or LV-shSOX4, LV-shMMP9 plus IL-17 incubation were markedly reduced. Overall, our findings implicate that NSCLC metastasis is closely associated with IL-17-GCN5-SOX4-MMP9 axis.

目的:检查人胶质母细胞瘤(glioblastoma,GBM)组织和细胞系中ZBTB3的表达,并探讨ZBTB3对GBM细胞增殖和克隆形成的影响及其调控机制.方法:通过GEPIA2数据库分析GBM患者肿瘤组织中ZBTB3的表达情况.RT-PCR、qPCR和Western blot检测GBM细胞系(U251、U373、U87)中ZBTB3的mRNA和蛋白表达水平,筛选出ZBTB3表达最高的U87细胞.CCK-8和克隆形成实验检测沉默ZBTB3基因对U87细胞增殖和克隆形成的影响.用p38MAPK、AMPK、Akt1抑制剂处理U87细胞后,Western blot 检测 p38MAPK、AMPK、Akt1 的磷酸化水平,RT-PCR、qPCR 和 Western blot 测定 ZBTB3 的 mRNA 和蛋白表达水平,CCK-8和克隆形成实验检测细胞增殖和克隆形成.结果:GBM患者肿瘤组织中ZBTB3的表达显著高于正常组织.U251、U373和U87细胞中均可见ZBTB3的表达,其中U87细胞表达最高.沉默ZBTB3基因能明显抑制U87细胞的增殖和克隆形成.抑制AMPK既能显著降低U87细胞ZBTB3的表达水平,又可明显减弱U87细胞增殖和克隆形成.结论:GBM组织和细胞系中ZBTB3的表达显著上调,GBM细胞中AMPK活化并上调ZBTB3基因的表达,促进GBM细胞的增殖和克隆形成.

目的:探讨长链非编码RNA01518(long intergenic non-protein coding RNA 01518,LINC01518)基因过表达或沉默对白介素(interleukin,IL)-17诱导非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞增殖的影响.方法:Western blot检测NSCLC 细胞系(PC9、H1299 和 H1975)IL-17 受体 A(IL-17 receptor A,IL-17RA)的表达后,用 IL-17 刺激 H1299 和 PC9 细胞不同时间,利用CCK-8实验测定细胞的增殖水平.LncRNA芯片筛查IL-17刺激H1299和PC9细胞3h后LncRNA上调的结果,从中选取某些上调的LncRNA进行RT-PCR和Real-time PCR验证.此外,将构建的pcDNA3.1/LINC01518或shLINC01518质粒转染H1299细胞,用CCK-8、EdU和克隆形成实验检查细胞的增殖情况.结果:3种NSCLC细胞系均有IL-17RA的表达.实验发现,IL-17刺激H1299和PC9细胞后能提高其增殖水平,同时受刺激的细胞内LINC01518的上调最为显著,且过表达LINC01518可提高H1299细胞的增殖水平,而沉默LINC01518基因则能抑制IL-17诱导的细胞增殖.结论:LINC01518能促进IL-17诱导的H1299细胞增殖.

目的:探讨IL-17刺激非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞上调腺病毒E1A相关300 kDa蛋白(ad-enoviral E1A binding protein of 300 kDa,p300)调控其细胞迁移、侵袭及生成基质金属蛋白酶2(matrix metalloproteinase 2,MMP2)的作用.方法:用IL-17刺激NSCLC细胞(H1299)后行划痕和Transwell实验观察细胞迁移和侵袭的变化.然后用Western blot检查p300和MMP2蛋白的表达.同时,将p300过表达质粒(pcDNA3.1/p300)或shp300干扰质粒分别转染H1299细胞(后者再行IL-17刺激),用前述方法测定细胞迁移、侵袭和MMP2的表达.结果:用IL-17刺激H1299细胞后能显著诱导其迁移和侵袭,并上调p300和MMP2的表达.过表达p300可增强细胞的迁移与侵袭,并提高MMP2的生成,但沉默p300基因后由IL-17诱导的细胞迁移和侵袭能力及MMP2的表达均显著降低.结论:IL-17上调的p300能促进H1299细胞的迁移与侵袭以及MMP2基因的表达.