MicroRNA-760 acts as a tumor suppressor in gastric cancer development via inhibiting G-protein-coupled receptor kinase interacting protein-1 transcription

BACKGROUND Gastric cancer(GC)has become a serious threat to people's health.Accumulative evidence reveals that dysregulation of numerous microRNAs(miRNAs)has been found during malignant formation.So far,the role of microRNA-760(miR-760)in the development of GC is largely unknown.AIM To measure the expression level of miR-760 in GC and investigate its role in gastric tumorigenesis.METHODS Real-time quantitative polymerase chain reaction and Western blot analysis were used to measure the expression of miR-760 and G-protein-coupled receptor kinase interacting protein-1(GIT1).Cell growth was detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT)and cell colony formation assays.Apoptosis was assessed by flow cytometric analysis.The relationship between miR-760 and GIT1 was verified by luciferase reporter assay.RESULTS The results showed that the expression of miR-760 was decreased in GC and associated with poor clinical outcomes in GC patients.Furthermore,miR-760 restrained cell proliferation and cell colony formation and induced apoptosis in GC cells.In addition,miR-760 directly targeted GIT1 and negatively regulated its expression in GC.GIT1 was upregulated in GC and predicted a worse prognosis in GC patients.We also found that upregulation of GIT1 weakened the inhibitory CONCLUSION In conclusion,miR-760 targets GIT1 to inhibit cell growth and promote apoptosis in GC cells.Our data demonstrate that miR-760 may be a potential target for the treatment of GC.

Evidence, hypotheses and significance of MAP kinase TNNI3K interacting with its partners

TNNI3K is a cardiac-specific and cardiac troponin I(cT n I)-interacting MAP kinase, known to play important roles in promoting cardiac differentiation, maintenance of beating rhythm and contractual force. The molecular structure of TNNI3 K contains three kinds of domain: a seven or ten NH2-terminal ankyrin repeat domain followed by a protein kinase domain and a COOH-terminal serine-rich domain. There are many binding sites in the structure of TNNI3 K for binding to ATP, magnesium, nucleotide, protein kinase C, antioxidant protein 1(AOP-1) and cT n I, indicating TNNI3 K has many interacting partners. This review summarizes the evidence, hypothesis and significance of TNNI3 K interacting with TNNI3 and its other putative interaction partners. From the literature, the interaction partners of TNNI3 K are divided into 2 types following their phenotypic pattern of functions, positive interaction(to increase the cardiac performance) or negative interaction(to suppress the cardiac performance). Following their binding sites, it also can be divided into other 2 types: binding to C-terminal domain(e.g., cT n I) or binding to both ankyrin repeat domain and C-terminal domains(AOP-1).To date, a well understood partner of TNNI3 K is cT nI, from the molecular structure, physiological function, mechanisms and its significance in some physiological and pathophysiological conditions. There are many reasons to believe that, with more understanding on the TNNI3 K interacting with its partners, we can understand more roles of TNNI3 K in some cardiac diseases.

Perspective of future drugs targeting sterile 20/SPS1-related proline/alanine-rich kinase for blood pressure control

According to a genome-wide association study,intronic SNPs within the human sterile 20/SPS1-related proline/alanine-rich kinase(SPAK) gene was linked to 20% of the general population and may be associated with elevated blood pressure. As cell volume changes,mammalian SPAK kinases respond to phosphorylate and regulate cation-coupled chloride co-transporter activity. To our knowledge,phosphorylation of upstream with-no-lysine(K)(WNK) kinases would activate SPAK kinases. The activation of WNK-OSR1/SPAK cascade on the kidneys and aortic tissue is related to the development of hypertension. Several regulators of the WNK pathway such as the Kelch kinase protein 3-Cullin 3 E3 ligase,hyperinsulinemia,and low potassium intake to mediate hypertension have been identified. In addition,the SPAK kinases may affect the action of renin-angiotensin-aldosterone system on blood pressure as well. In 2010,two SPAK knock-in and knock-out mouse models have clarified the pathogenesis of lowering blood pressure by influencing the receptors on the kidneys and aortic smooth muscle. More recently,two novel SPAK inhibitors for mice,Stock 1S-14279 and Closantel were discovered in 2014. Targeting of SPAK seems to be promising for future antihypertensive therapy. Therefore we raised some viewpoints for the issue for the antihypertensive therapy on the SPAK(gene or kinase).

IL-1β activates p44/42 and p38 mitogen-activated protein kinases via different pathways in cat esophageal smooth muscle cells

AIM： To examine the pathway related to the IL-1β induced activation of mitogen-activated protein （MAP） kinases in cat esophageal smooth muscle cells. METHODS： Culture of the esophageal smooth muscle cells from cat was prepared. Specific inhibitors were treated before applying the IL-β3. Western blot analysis was performed to detect the expressions of COX, iNOS and MAP kinases. RESULTS： In the primary cultured cells, although IL-β3 failed to upregulate the COX and iNOS levels, the levels of the phosphorylated forms of 1344142 HAP kinase and p38 MAP kinase increased in both concentration- and time-dependent manner, of which the level of activation reached a maximum within 3 and 18 h, respectively. The pertussis toxin reduced the level of p44/42 MAP kinase phosphorylation. Tyrphostin 51 and genistein also inhibited this activation. Neomycin decreased the density of the p44/42 HAP kinase band to the basal level. Phosphokinase C （PKC） was found to play a mediating role in the IL-1β-induced p44/42 MAP kinase activity. In contrast, the activation of p38 MAP kinase was inhibited only by a pretreatment with forskolin, and was unaffected by the other compounds. CONCLUSION： Based on these results, IL-1β-induced p44/42 MAP kinase activation is mediated by the Gi protein, tyrosine kinase, phospholipase C （PLC） and PKC. The pathway for p38 MAP kinase phosphorylation is different from that of p44/42 MAP kinase, suggesting that it plays a different role in the cellular response to IL- 1β.

Physiological roles of mitogen-activated-protein-kinase-activated p38-regulated/activated protein kinase

Mitogen-activated protein kinases(MAPKs)are a family of proteins that constitute signaling pathways involved in processes that control gene expression,cell division, cell survival,apoptosis,metabolism,differentiation and motility.The MAPK pathways can be divided into conventional and atypical MAPK pathways.The first group converts a signal into a cellular response through a relay of three consecutive phosphorylation events exerted by MAPK kinase kinases,MAPK kinase,and MAPK.Atypical MAPK pathways are not organized into this three-tiered cascade.MAPK that belongs to both conventional and atypical MAPK pathways can phosphorylate both non-protein kinase substrates and other protein kinases.The latter are referred to as MAPK-activated protein kinases.This review focuses on one such MAPK-activated protein kinase,MAPK-activated protein kinase 5(MK5)or p38-regulated/activated protein kinase(PRAK).This protein is highly conserved throughout the animal kingdom and seems to be the target of both conventional and atypical MAPK pathways.Recent findings on the regulation of the activity and subcellular localization,bona fide interaction partners and physiological roles of MK5/PRAK are discussed.

Effects of tyrosine kinase inhibitor E7080 and eNOS inhibitor L-NIO on colorectal cancer alone and in combination

Objective：To investigate the effects of E7080 and N5-（1-iminoethyl）-L-ornithine dihydrochloride （L-NIO）on colorectal cancer alone and in combination.Methods：HT29 colorectal cancer cell line from Sap Institute was used.Real-time cell analysis （xCELLigence system） was performed to determine the effects of E7080 and L-NIO on colorectal cell proliferation.While apoptosis was determined with Annexin V staining,and the effect of agents on angiogenesis was determined with chorioallantoic membrane （CAM） model.Results：We found that E7080 has a strong antiproliferative effect with an half maximum inhibition of concentration （IC50） value of 5.60×10-8 mol/L.Also it has been observed that E7080 showed antiangiogenic and apoptotic effects on HT29 colorectal cancer cells.Antiangiogenic scores of E7080 were 1.2,t.0 and 0.6 for 100,10 and 1 nmol/L E7080 concentrations,respectively.Furthermore,apoptosis has been detected in 71％ of HT29 colorectal cancer cells after administration of 100 nmol/L E7080 which may indicate strong apoptotic effect.Meanwhile administration of L-NIO alone did not show any effect,but the combination of E7080 with L-NIO increased the antiproliferative,antiangiogenic and apoptotic effects of E7080.Conclusions：Results of this study indicate that E7080 may be a good choice in treatment of colorectal tumors.Furthermore the increased effects of E7080 when combined with L-NIO raise the possibility to use a lower dose of E7080 and therefore avoid/minimize the side effects observed with E7080.

Correlation between receptor-interacting protein 140 expression and directed differentiation of human embryonic stem cells into neural stem cells

Overexpression of receptor-interacting protein 140（RIP140） promotes neuronal differentiation of N2 a cells via extracellular regulated kinase 1/2（ERK1/2） signaling.However,involvement of RIP140 in human neural differentiation remains unclear.We found both RIP140 and ERK1/2 expression increased during neural differentiation of H1 human embryonic stem cells.Moreover,RIP140 negatively correlated with stem cell markers Oct4 and Sox2 during early stages of neural differentiation,and positively correlated with the neural stem cell marker Nestin during later stages.Thus,ERK1/2 signaling may provide the molecular mechanism by which RIP140 takes part in neural differentiation to eventually affect the number of neurons produced.

Involvement of ERK1/2 and p38 MAPK in up-regulation of 14-3-3 protein induced by hydrogen peroxide preconditioning in PC12 cells

Objective To investigate the protective effects of hydrogen peroxide preconditioning （HPP） on the pheochromocytoma （PC12） cells treated with 1-methyl-4-phenylpyridinium （MPP^＋） and to explore the potential mechanisms. Methods The viability and apoptosis of PC 12 cells were determinded by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay and 4′,6′-diamidino-2-phenylindole （DAPI） staining, respectively. The expressions of 14-3-3 protein and phospholylated p38 mitogen-activated protein kinase （MAPK） were determined by Western blot. Enzyme-linked immunosorbent assay （ELISA） was used to measure the activity of extracellular signal-regulated protein kinase 1/2 （ERK1/2）. Results The cell viability decreased and the number of apoptotic cells increased dramatically in MPP^＋ group compared with that in Control group. HPP induced a significant increase in cell viability and a marked decrease in population of apoptotic cells of the MPP^＋- treated PC 12 cells, accompanied with up-regulation of 14-3-3 protein and increase of ERK 1/2 and p38 MAPK activities. The 14-3-3 protein expression was positively correlated with the phosphorylation of ERK1/2. Furthermore, inhibition of the ERK1/2 with PD98059 abolished the 14-3-3 protein up-regulation in PC 12 cells induced by HPP. Conclusion HPP protects PC 12 cells against MPP＋ toxicity by up-regulating 14-3-3 protein expression through the ERK1/2 and p38 MAPK signaling pathways.

The involvement of p38 MAPK in transforming growth factor β1-induced apoptosis in murine hepatocytes

We reported in this manuscript that TGF-beta1 induces apoptosis in AML12 murine hepatocytes, which is associated with the activation of p38 MAPK signaling pathway. SB202190, a specific inhibitor of p38 MAPK, strongly inhibited the TGF-beta1-induced apoptosis and PAI-1 promoter activity. Treatment of cells with TGF-beta1 activates p38. Furthermore, over-expression of dominant negative mutant p38 also reduced the TGF-beta1-induced apoptosis. The data indicate that the activation of p38 is involved in TGF-beta1-mediated gene expression and apoptosis.

MiR-451 inhibits proliferation of esophageal carcinoma cellline EC9706 by targeting CDKN2D and MAP3K1

AIM To investigate the underlying molecularmechanisms of miR-451 to inhibit proliferation ofesophageal carcinoma cell line EC9706.METHODS： Assays for cell growth, apoptosis andinvasion were used to evaluate the effects of miR-451expression on EC cells. Luciferase reporter and Westernblot assays were used to test whether cyclin-dependentkinase inhibitor 2D （CDKN2D） and MAP3K1 act as majortargets of miR-451.RESULTS： The results showed that CDKN2D andMAP3K1 are direct targets of miR-451. CDKN2D andMAP3K1 overexpression reversed the effect of miR-451.MiR-451 inhibited the proliferation of EC9706 bytargeting CDKN2D and MAP3K1.CONCLUSION： These findings suggest that miR-451might be a novel prognostic biomarker and a potentialtarget for the treatment of esophageal squamous cellcarcinoma in the future.

Casein kinase 2 interacting protein 1 positively regulates caudal-related homeobox 1 in intestinal-type gastric cancer

Background:Gastric cancer(GC)is one of the most common malignancies,and intestinal-type GC is the main histopathologic type of GC in China.We previously reported that casein kinase 2 interacting protein 1(CKIP-1)acts as a candidate tumor suppressor in intestinal-type GC.CKIP-1 participates in the regulation of multiple signaling pathways,including the Wnt/b-catenin pathway,of which caudal-related homeobox 1(CDX1)may be a downstream target gene.The purpose of this study was to investigate the relationship between CKIP-1 and CDX1 in intestinal-type GC.Methods:Sixty-seven gastroscopy biopsy specimens and surgically resected gastric specimens were divided into four groups:gastric mucosa group,intestinal metaplasia(IM)group,dysplasia group,and intestinal-type GC group.The expression levels of CKIP-1 and CDX1 were detected in these groups and GC cell lines,and the correlations between these expression levels were analyzed.SGC7901 and BGC823 cells were divided into CKIP-1 shRNA groups and CKIP-1 over-expression groups,and CDX1 expression was detected.b-Catenin expression was detected in intestinal-type GC tissue samples and CKIP-1 shRNA and CKIP-1 overexpression SGC7901 cells,and its correlation with CKIP-1 expression in intestinal-type GC tissue was analyzed.The Wnt/b-catenin pathway inhibitor DKK-1 and activator LiCl were incubated with SGC7901 cells,BGC823 cells,and CKIP-1 shRNA and CKIP-1 over-expression SGC7901 and BGC823 cells,following which CDX1 and Ki-67 expression were detected.Results:The expression levels of CKIP-1 and CDX1 were lower in patients with intestinal-type GC than in patients with IM and dysplasia(both P<0.05).CKIP-1 and CDX1 expression levels were positively correlated in IM,dysplasia,and intestinal-type GC tissue and cell lines(r=0.771,P<0.01;r=0.597,P<0.01;r=0.654,P<0.01;r=0.811,P<0.01,respectively).CDX1 expression was decreased in the CKIP-1 shRNA groups and increased in the CKIP-1 over-expression groups of SGC7901 and BGC823 cells compared to that in the corresponding control groups(both P<0.05).CKIP-1 expression was negatively correlated with b-catenin expression in intestinal-typeGCpatients(r=0.458,P<0.01).Compared to the control group,b-catenin expression was increased in the CKIP-1 shRNA SGC7901 cell group and decreased in the CKIP-1 over-expression SGC7901 cell group(P<0.05).CDX1 expression was increased inSGC7901 andBGC823 cells treatedwithDKK-1,DKK-1 increasedCDX1 expression and decreased Ki-67 expression in the CKIP-1 shRNA group;the opposite result was observed in SGC7901 and BGC823 cells treated with LiCl,and LiCl decreased CDX1 expression and increased Ki-67 expression in the CKIP-1 over-expression group(both P<0.05).Conclusions:Through the Wnt/b-catenin signaling pathway,CKIP-1 may positively regulate CDX1 in intestinal-type GC.

p38α MAP kinase phosphorylates RCAN1 and regulates its interaction with calcineurin

RCAN1, also known as DSCR1, is an endogenous regulator of calcineurin, a serine/threonine protein phosphatase that plays a critical role in many physiological processes. In this report, we demonstrate that p38a MAP kinase can phosphorylate RCAN1 at multiple sites in vitro and show that phospho-RCAN1 is a good protein substrate for calcineurin. In addition, we found that unphosphorylated RCANI noncompetitively inhibits calcineurin protein phosphatase activity and that the phosphorylation of RCAN1 by p38a MAP kinase decreases the binding affinity of RCAN1 for calcineurin. These findings reveal the molecular mechanism by which p38a MAP kinase regulates the function of RCAN1/calcineurin through phosphorylation.

榄香烯阻碍大鼠胶质瘤C6细胞ERK信号通路中Hsp90/Raf-1分子复合体的形成

目的探讨莪术提取物榄香烯抑制胶质瘤细胞体外增殖的机制。方法采用细胞计数、免疫共沉淀、Western印迹等方法,分别检测不同浓度、不同作用时间榄香烯对C6胶质瘤细胞的增殖影响、对C6细胞中与Raf-1结合形成分子复合体的Hsp90水平的影响、对C6细胞Hsp90、Raf-1、ERK蛋白质表达的影响。结果榄香烯对大鼠C6胶质瘤细胞具有明显的抑制增殖作用,该增殖抑制效应呈剂量、时间依赖性。榄香烯可明显下调C6细胞中与Raf-1结合的Hsp90水平及C6细胞的磷酸化Raf-1、ERK表达,但不影响总Hsp90的表达水平。结论榄香烯能有效抑制胶质瘤细胞的体外增殖,破坏Hsp90的分子伴侣功能,阻碍Hsp90/Raf-1复合体的形成,使Raf-1不能被活化,最终下调胶质瘤细胞赖以生存的Raf/MEK/ERK通路,这可能是榄香烯抑制C6细胞增殖的机制。

RIP1/RIP3-MLKL-信号通路与慢性心力衰竭的发生、发展及预后相关

目的通过检测正常人与慢性心力衰竭(HF)患者血浆中受体相互作用的蛋白激酶RIP1、RIP3和混合谱系激酶结构域样蛋白MLKL的表达水平,探讨程序性坏死信号通路RIP1/RIP3-MLKL在HF过程中的表达规律及临床意义。方法入选2020年2月~2020年10月在我院接受治疗的慢性HF患者(NYHAⅡ~Ⅳ级)。正常对照组(n=21),Ⅱ级(n=20),Ⅲ级(n=33),Ⅳ级(n=43)。采集所有患者的空腹静脉血,ELISA法和Western blotting分别检测RIP1/RIP3-MLKL表达水平和通路蛋白表达情况,同时对心功能Ⅳ级患者进行为期5个月的临床随访,评估临床预后指标。结果与正常对照组相比,心功能Ⅱ、Ⅲ、Ⅳ级组RIP1、RIP3、MLKL表达量均增加(P<0.01);与II级组相比,心功能Ⅲ、Ⅳ级组RIP1、RIP3、MLKL表达量均增加(P<0.01);与Ⅲ级组相比,心功能Ⅳ级组RIP1、MLKL表达量增加(P<0.05)。WB检测RIP1/RIP3-MLKL通路中各蛋白表达量,均存在上调趋势。经pearson相关性分析,HF患者RIP1/RIP3-MLKL表达量与Scr、AST、LVEDD、NT-ProBNP呈正相关(r=0.375、0.231、0.238、0.339、0.299、0.373、0.228、0.256、0.253、0.184、0.189、0.187,P<0.05),与LVEF呈负相关(r=-0.268、-0.234、-0.322,P<0.05)。此外对临床随访结果运用独立样本t检验分析,发现在心功能Ⅳ级患者中RIP1/RIP3-MLKL表达量更高组临床预后指标更差。结论RIP1-RIP3-MLKL信号通路在HF患者中表达明显增加,与HF严重程度呈正相关;慢性HF的发生、发展及预后可能与RIP1/RIP3-MLKL信号通路的激活与表达有关。

EB病毒LMP-1在鼻咽癌细胞中通过JNK介导AP-1活化

EB病毒潜伏膜蛋白 1 (latentmembraneprotein 1 ,LMP 1 )活化激活蛋白 1 (activatorprotein 1 ,AP 1 )信号传导途径与其致瘤作用密切相关 .为了探讨LMP 1活化AP 1信号传导的分子机制 ,在可诱导调控LMP 1表达的鼻咽癌细胞系L7中 ,首先通过荧光酶双报道系统确定了LMP 1表达能激活AP 1 ;在此基础上 ,用c JunPathDetect系统确定LMP 1表达活化AP 1是通过c Jun的磷酸化 (活化 )介导 .虽然LMP 1不能上调c Jun上游主要调节激酶c JunN端激酶 (c JunN terminalkinase ,JNK)的蛋白表达 ,但能显著促进JNK的磷酸化 (活化 ) ;在L7细胞中导入JNK相互作用蛋白 (JNK interactingprotein ,JIP)基因 ,抑制JNK的核移位能显著抑制LMP 1诱导的AP 1活化 ,同时对NFкВ活化也有部分抑制作用 .结果表明 ,EB病毒LMP 1在鼻咽癌细胞中通过JNK介导AP

p38MAPK在周期性机械牵张诱导肺泡巨噬细胞表达HMGB1中的作用

目的:研究p38 MAPK在周期性机械牵张诱导肺泡巨噬细胞(AM)表达高迁移率族蛋白B1(HMGB1)中的作用。方法:大鼠AM随机分为A、B、C3组,A组为对照组;B组细胞施加20%牵张应变,牵张时间为4h;C组细胞的牵张模式与B组相同,在牵张前用p38 MAPK特异性抑制剂SB203580(40μmol/L)预处理2h。利用RT-PCR法检测HMGB1 mRNA的表达,Western blotting检测HMGB1蛋白表达和p38 MAPK的活性。结果:与对照组相比,AM施加20%牵张应变可诱导HMGB1蛋白和mRNA表达明显增加、p38 MAPK活性明显增高(均P<0.05),SB203580可显著抑制牵张应变的这种诱导作用(均P<0.05)。结论:周期性机械牵张可能通过p38 MAPK信号通路,调节肺泡巨噬细胞HMGB1 mRNA和蛋白的表达。

HB-EGF对HSC-T6细胞Ras/ERK通路的激活及对MMP-2、TIMP-1表达的影响

目的观察肝素结合性表皮生长因子样生长因子(HB-EGF)激活肝星状细胞(HSC)过程中Ras/ERK信号通路的变化和对金属基质蛋白酶(MMP)及其抑制物的影响。方法传代培养HSC-T6细胞,分空白对照组、HB-EGF处理组(20,40,80ng/mL),采用QRT-PCR法检测MMP-2、金属蛋白酶组织抑制剂-1(TIMP-1)及表皮生长因子受体(EGFR)mRNA的表达,并确定HB-EGF刺激HSC-T6Ras/ERK通路(ERK1、ERK2、P38MAPK)活化的最佳浓度及最佳时间,检测该通路活化情况。结果 QRT-PCR检测提示,MMP-2的表达随HB-EGF干预时间的延长增加,TIMP-1的表达随干预时间的延长递减;40ng/mL HB-EGF培养HSC-T6 48h后,EGFR mRNA的表达较空白对照组明显上调(P<0.05);选择40ng/mL HB-EGF培养48h为最佳刺激时间与浓度,该处理组ERK1mRNA的表达较空白对照组明显上调(P<0.05),但ERK2、P38MAPK的变化无统计学意义;Western-blot检测提示该处理组ERK1蛋白的表达较空白对照组上调(P<0.05)。结论HB-EGF可影响HSC-T6的MMP-2、TIMP-1的表达;HB-EGF通过促进HSC-T6表达EGFR、ERK1,有效激活细胞内Ras/ERK信号通路。

GIT1通过BMP-2/p-Smad1/5/8信号通路调节骨折愈合

目的 :探讨G蛋白偶联受体激酶结合蛋白1(G-protein coupled receptor kinase-interacting protein 1,GIT1)影响骨折愈合的具体机制。方法:分别取GIT1基因敲除小鼠和同窝野生型小鼠制作股骨骨折模型,每组30只。造模成功后2周,免疫组化检测骨痂区软骨细胞矿化及p-Smad1/5/8表达量。分别取2组小鼠的骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs),予以10 ng/m L的骨形态发生蛋白2(bone morphogenetic protein 2,BMP2)刺激后检测Smad1/5/8磷酸化水平和核转入能力;取C3H10T1/2细胞,分别用1μg p GL3重组质粒和1μg GIT1-si RNA共转染细胞,荧光报告素酶技术检测细胞内GIT1蛋白对BMP2转录水平的影响。结果:与同窝野生型小鼠相比,GIT1基因敲除小鼠软骨内骨化减弱,骨痂区软骨细胞堆积,骨折愈合延迟,p-Samd1/5/8表达水平明显减少;与GIT1基因敲除小鼠相比,BMP2可明显提高同窝野生型小鼠BMCs中p-Samd1/5/8核转入能力;GIT1蛋白表达的缺失可明显降低BMP2的转录水平。结论 :GIT1可通过调节Samd1/5/8的磷酸化及BMP2信号转导影响骨折部位的软骨内骨化。

受体相互作用蛋白激酶1(RIPK1)调控人结肠癌HT-29细胞程序性坏死的分子机制

目的研究受体相互作用蛋白激酶1(receptor interacting protein kinase 1,RIPK1)在人结肠HT-29细胞程序性坏死过程中的作用,并探讨E3泛素连接酶三重结构域包含蛋白16(tripartite domain containing protein 16,Trim16)对其作用的潜在调控机制。方法用肿瘤坏死因子α(tumor necrosis factorα,TNFα)建立HT-29细胞的程序性坏死模型,采用Annexin V-FITC/PI双染色法检测其对凋亡、坏死细胞数目的影响。分别用Western blot和qRT-PCR检测RIPK1在细胞程序性坏死中的表达。构建稳定表达Flag标记的RIPK1的HT-29细胞株,采用Flag标记的pulldown试验结合质谱检测发现与RIPK1有相互作用的新蛋白。应用NiNTA pulldown试验检测筛选出的E3泛素连接酶对RIPK1泛素化的调控。结果 TNFα能够成功诱导HT-29细胞程序性坏死。HT-29细胞在TNFα和半胱天蛋白酶抑制剂z-VAD处理后,表现出RIPK1、RIPK3、混合系列蛋白激酶样结构域(mixed lineage kinase domain-like protein,MLKL)表达水平显著增加,并伴随着炎性因子白介素1α(IL1α)和白介素6(IL6)水平明显升高。Flag标记的pulldown试验结合质谱检测发现一个与RIPK1有相互作用的E3泛素连接酶Trim16,体外实验表明Trim16可以增强RIPK1的泛素化程度,其可能调节RIPK1在程序性坏死过程中的作用。结论 RIPK1在TNFα和z-VAD诱导人结肠癌HT-29细胞的程序性坏死过程中起重要作用,E3泛素连接酶Trim16对此过程可能有重要调控作用。

磷酸化的MAPK作用激酶1表达与鼻咽癌发生发展及预后的关系

目的探讨磷酸化的MAPK作用激酶1(pMnK1)表达与鼻咽癌病人发生发展及预后的关系。方法选取2015年1月至2017年7月南阳市中心医院收集的90例鼻咽癌病理学标本、90例经病理学检查证实鼻部慢性炎症的鼻黏膜组织(对照组),应用免疫组化染色分析检测pMnk1蛋白表达,并按照年龄、性别、组织学类型、临床分期、淋巴结转移情况进行分组分析,分析pMnk1蛋白阳性与阴性表达的鼻咽癌病人2年生存率及预后。结果鼻咽癌组的pMnK1蛋白阳性率74.44%高于对照组的26.67%,差异有统计学意义(P<0.05);不同TNM分期、淋巴结转移的鼻咽癌组的pMnK1蛋白阳性率比较,差异有统计学意义(P<0.05);经2年随访,pMnK1蛋白阳性的鼻咽癌病人失访4例、阴性病人失访1例,pMnK1蛋白阳性的鼻咽癌病人2年生存率61.90%与阴性病人的77.27%,差异无统计学意义(P>0.05);pMnK1蛋白阳性的鼻咽癌病人的生存时间20.0个月短于阴性组23.0个月,差异有统计学意义[Log-rank(Mantel-Cox)=3.285,P<0.05]。结论鼻咽癌组织中pMnK1蛋白呈高表达,并且与肿瘤的发生发展及不良预后关系密切。