糖原合成激酶3β对骨肉瘤细胞迁移和侵袭的影响

目的 研究糖原合成激酶3β(GSK3β)在骨肉瘤侵袭、转移中的作用。方法 通过Western blot法检测骨肉瘤患者肿瘤组织及骨肉瘤细胞中GSK3β的表达和磷酸化水平;使用两种GSK3β小分子抑制剂处理骨肉瘤细胞,通过细胞迁移及侵袭实验检测GSK3β活性受抑制对骨肉瘤细胞迁移、侵袭的影响。结果 GSK3在骨肉瘤细胞中过表达且存在活性调节异常;在转移及非转移骨肉瘤患者肿瘤组织中均可检测到GSK3表达及活性异常,尤其在转移的骨肉瘤患者肿瘤组织中;抑制内源性GSK3活性,明显降低肿瘤细胞的迁移、侵袭能力。结论 GSK3β促进骨肉瘤细胞的侵袭、迁移,可能为骨肉瘤的临床治疗开辟潜在的靶点奠定理论基础。     

p53抑制干细胞的自我更新能力而增敏奥沙利铂诱导肺癌细胞凋亡的作用及机制

目的:研究p53通过增加肿瘤干细胞不等向分裂的比例而增敏奥沙利铂(Oxaliplatin,L-OHP)诱导肺癌细胞凋亡的作用及机制,探索影响肺癌化疗效果的因素.方法:用Nutlin-3(N-3)调控肺癌细胞野生型p53的表达,研究p53对L-OHP诱导细胞凋亡及对肺癌干细胞不同分裂模式的影响;使用Akt激活剂及shRNA-GSK3β调控表达的肺癌细胞系,研究p53通过Akt调控奥沙利铂诱导的肺癌细胞凋亡,并抑制肺癌干细胞自我更新能力.在肺癌标本中检测GSK3β与磷酸化Akt1的表达及与预后的关系.结果:Nutlin-3上调了A549及H460细胞中野生型p53的表达,有效抑制了细胞增殖,增强L-OHP诱导细胞凋亡效果.Nutlin-3抑制磷酸化Akt1,促进肺癌干细胞不等向分裂,进而抑制肿瘤干细胞的比例,增敏L-OHP诱导肺癌细胞凋亡.Akt1磷酸化活性在p53介导的下游GSK3β表达增加的调控中起关键作用.50nmol/L的L-OHP通过抑制Akt1活性促进凋亡,10μmol/L的Nutlin-3对L-OHP诱导的肺癌细胞凋亡有协同作用,是通过共同调控Akt1/GSK3β通路的活性实现,Akt1的激活与GSK3β的抑制均显著地抵消了p53对L-OHP诱导的肺癌干细胞比例降低.结论:p53通过抑制磷酸化Akt1的水平调控GSK3β表达,促肺癌干细胞不均等分裂,增敏L-OHP诱导凋亡.肺癌中GSK3β的高表达与磷酸化Akt1的低表达预示较好的临床治疗效果.     

miR⁃203a⁃3p通过靶向LASP1介导Akt/GSK⁃3β/Snail信号通路调控肝癌细胞生物学行为

目的 研究miR?203a?3p对肝细胞癌（hepatocellular carcinoma,HCC）细胞生物学行为的影响及其相关分子机制。方法 将miR?203a?3p 模拟物（miR?203a?3p mimics）及阴性对照（miR?NC mimics）、LIM和SH3蛋白1（LIM and SH3 protein 1,LASP1）过表达质粒（pcDNA?LASP1）及阴性对照质粒（pcDNA?NC）分别转染至HepG2细胞。以qRT?PCR法检测细胞miR?203a?3p、LASP1 mRNA表达情况,CCK?8法和异体移植瘤实验分析细胞增殖能力,划痕实验检测细胞迁移能力,Transwell小室实验检测细胞侵袭能力,Annexin V?FITC/PI法检测细胞凋亡,双荧光素酶报告基因检测miR?203a?3p与LASP1的靶向关系,Western blot法检测细胞中LASP1、蛋白激酶B（protein kinase B,Akt）、磷酸化Akt（phosphorylated Akt,p?Akt）、糖原合成酶激酶3β（glycogen synthase kinase?3β,GSK?3β）、磷酸化GSK?3β（phosphorylated GSK?3β,p?GSK?3β）、Snail表达情况。结果 与miR?NC组相比,miR?203a?3p组HepG2细胞增殖活性、迁移率、侵袭数目、LASP1 mRNA和蛋白表达量、p?Akt/Akt和p?GSK?3β/GSK?3β比值、Snail蛋白表达量均显著降低,小鼠移植瘤体积和质量显著减少,细胞凋亡率显著升高（均P<0.01）。Targetscan软件预测显示,miR?203a?3p与LASP1 存在靶向关系;与LASP1?Wt+miR?NC组比较,LASP1?Wt+miR?203a?3p组相对荧光素酶活性显著下降（P<0.001）。与miR?203a?3p+pcDNA?NC组比较,miR?203a?3p+LASP1组HepG2细胞增殖活性、迁移率、侵袭数目、p?Akt/Akt和p?GSK?3β/GSK?3β比值、Snail蛋白表达量均显著升高,小鼠移植瘤体积和质量显著增加,细胞凋亡率显著降低（均P<0.01）。结论 miR?203a?3p可能通过靶向抑制LASP1表达调控Akt/GSK?3β/Snail信号通路活性,从而调控HCC细胞生物学行为。     

糖原合酶激酶-3β促进卵巢癌细胞增殖的实验研究

目的探讨糖原合酶激酶-3β（GSK-3β）对卵巢癌细胞增殖的影响及其意义。方法以蛋白印迹法检测GSK-3β和磷酸化GSK-3β（pGSK-3β）在卵巢癌细胞株SKOV3和ES-2中的表达水平;采用细胞计数法描记细胞生长曲线,以检测GSK-3β抑制剂LiCl和SB216763对SKOV3和ES-2细胞生长的影响;将SKOV3细胞分成4组,将增加GSK-3β活性的质粒GSK-3βS9A及其空载体对照质粒pCS2、降低GSK-3β活性的质粒GID5-6及其对照质粒GID5-6LP,分别用电转法与绿色荧光蛋白（GFP）质粒共同转染入细胞中,采用5-溴脱氧尿嘧啶（BrdU）掺入实验检测GSK-3β活性对卵巢癌细胞增殖的影响;利用G418筛选瞬时转染的SKOV3细胞得到稳定转染株后,采用克隆形成实验,观察改变GSK-3β的活性对卵巢癌细胞增殖的长期影响。结果SKOV3和ES-2细胞均表达GSK-3β和pGSK-3β,SKOV3细胞中pGSK-3β的表达水平比ES-2细胞低,而两者GSK-3β的表达水平相近。GSK-3β抑制剂LiCl和SB216763可抑制SKOV3和ES-2细胞的生长。与对照pCS2相比,转染GSK-3βS9A质粒可提高GSK-3β的活性,增加SKOV3细胞BrdU的掺入率;与对照GID5-6LP相比,转染GID5-6质粒可降低GSK-3β的活性,减少SKOV3细胞BrdU的掺入率。分别与各自的对照质粒相比,稳定转染GSK-3βS9A可形成较多的细胞克隆,而稳定转染GID5-6则形成的细胞克隆较少。结论GSK-3β具有促进卵巢癌细胞增殖的作用,抑制GSK-3β的活性可望成为卵巢癌治疗的新靶点。     

鹰嘴豆芽素A通过抑制Wnt/β-catenin信号通路抑制结肠癌细胞增殖迁移的可能机制

目的 观察鹰嘴豆芽素A(BCA)对结肠癌细胞增殖迁移的影响,并探讨可能机制。方法 取对数期人结肠癌HCT-116细胞,随机分为对照组、BCA组、SB216763[Wnt/β-连环蛋白(β-catenin)通路激活剂]组、联合组。对照组不做处理,BCA组加入BCA(40μmol/L),SB216763组中加入SB216763(20μmol/L),联合组加入BCA(40μmol/L)+SB216763(20μmol/L)。MTT法检测各组细胞增殖能力;划痕实验检测各组细胞迁移能力;血管拟态形成实验检测各组细胞血管新生能力;免疫印迹法检测各组细胞兔抗人糖原合成酶激酶3β(GSK-3β)、β-catenin蛋白表达量。结果 与对照组比较,BCA组24、48、72 h MTT实验吸光度值、迁移率、β-catenin蛋白表达量降低,血管数量/视野减少,GSK-3β蛋白表达量升高(P<0.05),SB216763组24、48、72 h MTT实验吸光度值、迁移率、β-catenin蛋白表达量升高,血管数量/视野增加,GSK-3β蛋白表达量降低(P<0.05);与BCA组比较,联合组24、...     

乙型肝炎病毒X蛋白对肝细胞L02增殖及GSK3β表达的影响

目的:通过研究乙型肝炎病毒X蛋白(hepatitis B virus X protein,HBx)对人肝细胞株L02细胞增殖、细胞周期以及细胞中糖原合成酶激酶3β(glycogen synthase kinase 3β,GSK3β)表达的影响,探讨乙型肝炎病毒(hepatitis B virus,HBV)相关性原发性肝细胞癌(hepatoccellular carcinoma,HCC)的发生机制。方法:用HBx腺病毒(Ad-HBx)感染人肝细胞株L02细胞后,RT-PCR法检测L02细胞中HBx和GSK3β mRNA表达情况;MTT法检测L02细胞的增殖率变化;FCM法检测细胞周期中各时相所占比例;蛋白质印迹法检测HBx、总GSK3β(total-GSK3β,t-GSK3β)、磷酸化GSK3β(phospho-GSK3β,p-GSK3β)、β-连环素(β-catenin)以及细胞用期蛋白(cyclinD1)等蛋白的表达水平。结果:Ad-HBx感染L02细胞后,HBx的mRNA和蛋白均出现表达,细胞增殖率随着时间的延长而增加;G1期细胞所占比例较对照组减少,S期和G2期细胞比例较对照组增加(P<0.05)。感染Ad-HBx后,t-GSK3β在mRNA和蛋白水平上均无明显变化,而p-GSK3β、β-catenin以及cyclinD1蛋白的表达量增加(P<0.05)。结论:HBx可能通过促进人肝细胞株L02细胞中GSK3β的磷酸化,激活Wnt/β-catenin下游信号通路,从而促进细胞的增殖。     

miRNA-199a-5p对骨肉瘤细胞增殖及自噬的影响北大核心CSCD

目的探讨mi RNA-199a-5p对人骨肉瘤细胞株U2OS增殖及自噬的影响及其可能的作用机制。方法实时定量PCR法检测32例骨肉瘤患者的病理组织标本及配对癌旁正常组织中mi RNA-199a-5p的表达水平。将mi RNA-199a-5p mimics转染入U2OS细胞,使其过表达mi RNA-199a-5p,采用CCK-8法和流式细胞仪检测细胞增殖及周期的变化;Western blot检测周期相关蛋白(cyclin D1、P27、p Rb)及自噬相关蛋白(Beclin 1、LC3Ⅱ/Ⅰ)表达水平。结果 mi RNA-199a-5p在骨肉瘤组织及癌细胞U2OS中低表达,且表达水平与肿块大小、远距转移、Enneking临床分期及病理分级密切相关。过表达mi RNA-199a-5p后,U2OS细胞的增殖能力显著降低(P<0.05),同时cyclin D1、p Rb表达水平显著下降,而P27、LC3Ⅱ/Ⅰ、Beclin 1的表达水平显著增加(P<0.05)。结论 mi RNA-199a-5p能够抑制人骨肉瘤细胞株U2OS的增殖并激活细胞自噬;其作用机制可能与下调cyclin D1、上调P27、抑制p Rb蛋白的磷酸化、造成细胞周期G1/S阻滞有关。提示mi RNA-199a-5p可以作为骨肉瘤临床治疗的潜在作用位点。     

shRNA靶向沉默ZEB2表达通过Wnt/β-catenin信号通路降低肺癌细胞增殖活性的实验研究

目的:研究shRNA靶向沉默E盒结合锌指蛋白2（zinc finger E-box binding homeobox,ZEB2）表达对肺癌细胞增殖活性的影响。方法:用shRNA-ZEB2慢病毒和shRNA阴性对照慢病毒感染肺癌细胞,Real time PCR和Western blot检测沉默效果。MTT检测细胞增殖活性,流式细胞术检测细胞凋亡情况,Western blot检测细胞中激活型Caspase-3（Cleaved Caspase-3）、β-连环蛋白（β-catenin）、C-myc蛋白水平。用Wnt/β-catenin信号通路抑制剂处理沉默ZEB2的肺癌细胞,MTT检测细胞增殖,流式细胞术检测细胞凋亡,Western blot检测细胞中激活型Caspase-3、β-catenin、C-myc蛋白水平。结果:shRNA-ZEB2慢病毒可以明显沉默肺癌细胞中ZEB2的表达和转录,shRNA阴性对照慢病毒对肺癌细胞中ZEB2的表达没有影响。沉默ZEB2后的肺癌细胞增殖活性降低,细胞凋亡率升高,细胞中激活型Caspase-3蛋白水平升高,β-catenin、C-myc蛋白水平降低。Wnt/β-catenin信号通路抑制剂下调沉默ZEB2的肺癌细胞株中Wnt/β-catenin信号通路的激活水平,同时可以降低细胞存活率,诱导细胞凋亡,促进细胞中激活型Caspase-3的表达。结论:shRNA靶向沉默ZEB2表达通过Wnt/β-catenin信号通路降低肺癌细胞增殖活性。     

苦参碱通过bcr-abl介导的MEK-ERK通路抑制人慢性粒细胞白血病K562细胞增殖

目的：探讨MEK-ERK通路在苦参碱抑制人慢性粒细胞白血病K562细胞增殖中的分子机制。方法采用Western blot法检测K562细胞内MEK-ERK通路关键分子MEK1、ERK1／2及其上游接头分子Shc、SHP2的总蛋白和磷酸化蛋白的表达;采用反转录聚合酶链反应（RT-PCR）和Western blot检测bcr-abl及有丝分裂原激活的蛋白激酶（MAPK）下游靶蛋白bcl-xL、Cyclin D1、c-myc及p27在转录及蛋白水平的表达。结果苦参碱可明显抑制K562细胞内MEK1、ERK1／2及Shc、SHP2的磷酸化表达,并可在转录及蛋白水平抑制bcr-abl分子表达。同时,RT-PCR和Western blot实验证实,苦参碱处理后,K562细胞内bcl-xL、Cyclin D1、c-myc表达均明显抑制,细胞周期负调控蛋白p27的表达增加。结论苦参碱对K562细胞的抑制效应与bcr-abl介导的MEK-ERK信号通路活性抑制有关,对信号通路中磷酸化蛋白或激酶分子活性的调控可能是其调控MEK-ERK通路活性的重要分子机制。     

槲皮素通过调控p21/cip1和p27/kip1蛋白的稳定性抑制口腔鳞状细胞癌细胞增殖

目的:探究槲皮素通过调控p21/cip1和p27/kip1蛋白的稳定性对口腔鳞状细胞癌细胞增殖的影响。方法:CCK-8试剂盒检测口腔鳞状细胞癌细胞(SCC-15)增殖情况;流式细胞术检测SCC-15细胞周期分布;Western blot检测CyclinD1/CDK复合体、p21/cip1、p27/kip1、p-GSK3β、GSK3β、p53和c-Myc蛋白表达;qRT-PCR检测SCC-15细胞p21/cip1和p27/kip1 mRNA表达;激光共聚焦显微镜检测SCC-15细胞中p21/cip1和p27/kip1蛋白亚细胞定位;放线菌酮蛋白合成抑制实验检测SCC-15细胞中p21/cip1和p27/kip1蛋白半衰期。结果:与对照组相比,槲皮素呈剂量依赖性(6.25μmol/L、12.5μmol/L、25μmol/L、50μmol/L、100μmol/L和200μmol/L)抑制SCC-15细胞增殖(P<0.05),槲皮素在48 h对SCC-15细胞的IC 50为80.07μmol/L。与对照组相比,槲皮素(40μmol/L和80μmol/L)可显著升高SCC-15细胞中G 1/G 0期细胞比例,显著降低S期和G 2/M期细胞比例(P<0.05),呈剂量依赖性抑制CyclinD1/CDK复合体、p-GSK3β和c-Myc蛋白表达(P<0.05),促进p21/cip1和p27/kip1的mRNA和蛋白表达,促进GSK3β和p53的蛋白表达(P<0.05)。与对照组相比,槲皮素(80μmol/L)促进SCC-15细胞中p21/cip1蛋白由细胞质转移至细胞核(P<0.05),显著提高p21/cip1和p27/kip1蛋白生物半衰期(P<0.05),对p27/kip1蛋白的亚细胞定位无显著影响(P>0.05)。结论:槲皮素通过调控p21/cip1和p27/kip1蛋白的稳定性抑制口腔鳞状癌细胞增殖。     

Glycogen synthase kinase 3β as a potential therapeutic target in synovial sarcoma and fibrosarcoma

Soft tissue sarcomas (STSs) are a rare cancer type. Almost half are unresponsive to multi‐pronged treatment and might therefore benefit from biologically targeted therapy. An emerging target is glycogen synthase kinase (GSK)3β, which is implicated in various diseases including cancer. Here, we investigated the expression, activity and putative pathological role of GSK3β in synovial sarcoma and fibrosarcoma, comprising the majority of STS that are encountered in orthopedics. Expression of the active form of GSK3β (tyrosine 216‐phosphorylated) was higher in synovial sarcoma (SYO‐1, HS‐SY‐II, SW982) and in fibrosarcoma (HT1080) tumor cell lines than in untransformed fibroblast (NHDF) cells that are assumed to be the normal mesenchymal counterpart cells. Inhibition of GSK3β activity by pharmacological agents (AR‐A014418, SB‐216763) or of its expression by RNA interference suppressed the proliferation of sarcoma cells and their invasion of collagen gel, as well as inducing their apoptosis. These effects were associated with G0/G1‐phase cell cycle arrest and decreased expression of cyclin D1, cyclin‐dependent kinase (CDK)4 and matrix metalloproteinase 2. Intraperitoneal injection of the GSK3β inhibitors attenuated the growth of SYO‐1 and HT1080 xenografts in athymic mice without obvious detrimental effects. It also mitigated cell proliferation and induced apoptosis in the tumors of mice. This study indicates that increased activity of GSK3β in synovial sarcoma and fibrosarcoma sustains tumor proliferation and invasion through the cyclin D1/CDK4‐mediated pathway and enhanced extracellular matrix degradation. Our results provide a biological basis for GSK3β as a new and promising therapeutic target for these STS types.     

Detection of active fraction of glycogen synthase kinase 3beta in cancer cells by nonradioisotopic in vitro kinase assay

Glycogen synthase kinase 3beta (GSK3beta) is a well-known marker and potential therapeutic target in non-insulin-dependent diabetes mellitus and Alzheimer's disease. Our recent demonstration that GSK3beta has a previously unrecognized role in colorectal cancer facilitates the development of a nonradioisotopic in vitro kinase assay (NRIKA) for detecting GSK3beta activity in gastrointestinal cancer cells. The NRIKA uses a sequential combination of immunoprecipitations to isolate GSK3beta in sample cells' lysates, and an in vitro kinase reaction that uses recombinant beta-catenin protein (substrate) and nonradioisotopic ATP, followed by immunoblotting to detect beta-catenin phosphorylated in serine 33, 37 and/or threonine 41 residues. The NRIKA detected higher expression of active GSK3beta in stomach, colon, pancreas and liver cancer cell lines than in human embryonic kidney cells (HEK293) considered nonneoplastic. Inhibition of cancer cell-derived GSK3beta activity by GSK3beta inhibitors (SB-216763, AR-A014418) was detected by the NRIKA. GSK3beta inhibition attenuated survival and proliferation and induced apoptosis in all types of cancer cells but not in HEK293. These findings supported the idea that the pathologic roles of GSK3beta are definite and common in various types of cancer. The NRIKA provides a basis for evolving a high-throughput tool for testing substances for GSK3beta inhibition, and for screening and identifying novel GSK3beta inhibitors with a view to discovering drugs for treatment of cancer as well as non-insulin-dependent diabetes mellitus and Alzheimer's disease.     

Specific glycogen synthase kinase-3 inhibition reduces neuroendocrine markers and suppresses neuroblastoma cell growth

Objective: Neuroblastoma is a common neuroendocrine (NE) tumor that presents in early childhood, with a high incidence of malignancy and recurrence. The glycogen synthase kinase-3 (GSK-3) pathway is a potential therapeutic target, as this pathway has been shown to be crucial in the management of other NE tumors. However, it is not known which isoform is necessary for growth inhibition. In this study, we investigated the effect of the GSK-3 inhibitor AR-A014418 on the different GSK-3 isoforms in neuroblastoma. Methods: NGP and SH-5Y-SY cells were treated with 0–20 μM of AR-A014418 and cell viability was measured by MTT assay. Expression levels of NE markers CgA and ASCL1, GSK-3 isoforms, and apoptotic markers were analyzed by western blot. Results: Neuroblastoma cells treated with AR-A014418 had a significant reduction in growth at all doses and time points (P &lt; 0.001). A reduction in growth was noted in cell lines on day 6, with 10 μM (NGP-53% vs. 0% and SH-5Y-SY-38% vs. 0%, P &lt; 0.001) treatment compared to control, corresponding with a noticeable reduction in tumor marker ASCL1 and CgA expression. Conclusion: Treatment of neuroblastoma cell lines with AR-A014418 reduced the level of GSK-3α phosphorylation at Tyr279 compared to GSK-3β phosphorylation at Tyr216, and attenuated growth via the maintenance of apoptosis. This study supports further investigation to elucidate the mechanism(s) by which GSK-3α inhibition downregulates the expression of NE tumor markers and growth of neuroblastoma.     

MiR-129-5p靶向HMGB1抑制骨肉瘤细胞增殖和迁移

目的 探讨miR-129-5p对骨肉瘤(OS)细胞增殖和迁移的影响以及对HMGB1的调控作用.方法 RT-PCR和Western blot法分别检测骨肉瘤细胞株MG-63、Saos-2和成骨细胞hFOB1.19中miR-129-5p和HMGB1的表达.生物信息学预测miR-129-5p与HMGB1基因是否存在结合位点,...     

NADPH oxidase 5 (NOX5)—induced reactive oxygen signaling modulates normoxic HIF‐1α and p27Kip1 expression in malignant melanoma and other human tumors

NADPH oxidase 5 (NOX5) generated reactive oxygen species (ROS) have been implicated in signaling cascades that regulate cancer cell proliferation. To evaluate and validate NOX5 expression in human tumors, we screened a broad range of tissue microarrays (TMAs), and report substantial overexpression of NOX5 in malignant melanoma and cancers of the prostate, breast, and ovary. In human UACC‐257 melanoma cells that possesses high levels of functional endogenous NOX5, overexpression of NOX5 resulted in enhanced cell growth, increased numbers of BrdU positive cells, and increased γ‐H2AX levels. Additionally, NOX5‐overexpressing (stable and inducible) UACC‐257 cells demonstrated increased normoxic HIF‐1α expression and decreased p27^Kip1 expression. Similarly, increased normoxic HIF‐1α expression and decreased p27^Kip1 expression were observed in stable NOX5‐overexpressing clones of KARPAS 299 human lymphoma cells and in the human prostate cancer cell line, PC‐3. Conversely, knockdown of endogenous NOX5 in UACC‐257 cells resulted in decreased cell growth, decreased HIF‐1α expression, and increased p27^Kip1 expression. Likewise, in an additional human melanoma cell line, WM852, and in PC‐3 cells, transient knockdown of endogenous NOX5 resulted in increased p27^Kip1 and decreased HIF‐1α expression. Knockdown of endogenous NOX5 in UACC‐257 cells resulted in decreased Akt and GSK3β phosphorylation, signaling pathways known to modulate p27^Kip1 levels. In summary, our findings suggest that NOX5 expression in human UACC‐257 melanoma cells could contribute to cell proliferation due, in part, to the generation of high local concentrations of extracellular ROS that modulate multiple pathways that regulate HIF‐1α and networks that signal through Akt/GSK3β/p27^Kip1.     

HPV16 E6、E7通过糖原合成酶激酶-3β通路影响宫颈鳞癌细胞生物学行为

目的:探讨人乳头瘤病毒16(HPV16)的致癌基因E6、E7对宫颈鳞癌细胞C33A生物学行为的影响及其机制。方法:构建HPV16 E6、E7及E6/E7重组质粒,流式细胞术检测转染前后细胞周期的变化;划痕实验和Transwell实验检测细胞迁移及侵袭能力;克隆形成实验检测细胞的增殖能力;qPCR和Western blot检测相关基因和蛋白表达水平。结果:HPV16 E7重组质粒可引起宫颈鳞癌细胞S期比例显著增加（P＜0.01）,HPV16 E6、E7和E6/E7重组质粒能显著增强宫颈鳞癌细胞C33A的迁移能力（P＜0.05）,同时细胞的克隆形成能力也显著增强（P＜0.05）。HPV16 E6重组质粒可显著上调细胞糖原合成酶激酶-3β(GSK3β) mRNA水平（P＜0.05）;HPV16 E6、E7重组质粒可在24 h内引起GSK3β磷酸化水平升高,同时调控下游靶基因CyclinD1和β-catenin在不同时间点的表达发生变化。结论:HPV16 E6、E7在较短时间（24 h内）可通过GSK3β蛋白磷酸化激活,发挥对GSK3β下游靶基因CyclinD1、β-catenin基因的调控作用,从而促进宫颈鳞癌细胞的增殖、迁移及侵袭。     

miR-1246通过靶向GSK3β促进食管癌转移的机制

目的:探讨miRNA-1246(miR-1246)促进食管癌细胞转移的作用及其机制.方法:Realtime-PCR法检测miR-1246在癌旁组织、食管癌组织、正常食管上皮和食管癌细胞系中的表达差异.Transwell侵袭实验观察转染miR-1246 mimics或inhibitors对食管癌细胞EC109转移能力的影响;裸鼠尾静脉转移实验观察稳定表达miR-1246对食管癌转移能力的影响;生物信息学分析miR-1246的候选靶基因为GSK3β,荧光素酶报告基因实验检测过表达miR-1246后EC109细胞中野生型和突变型GSK3β的荧光素酶活性.Westernblot检测miR-1246对GSK3β蛋白表达的影响.结果:食管癌组织中的miR-1246表达显著高于癌旁组织（P<0.05);多个食管癌细胞中miR-1246的表达比正常食管上皮细胞Het-1A明显增高（P<0.05).与阴性对照相比,miR-1246 mimics显著促进EC109细胞的侵袭能力（P<0.05).反之,miR-1246 inhibitors明显抑制EC109细胞的侵袭能力（P<0.05);体内实验发现稳定过表达miR-1246明显升高EC109细胞的肺转移能力.荧光素酶报告基因结果证实miR-1246能够抑制GSK3β的3'-UTR区荧光素酶活性;转染miR-1246后,EC109细胞中的GSK3β蛋白水平明显低于对照组;miR-1246过表达显著抑制GSK3β-wt,而不能抑制GSK3β-mut的蛋白表达.结论:miR-1246能够通过靶向GSK3β促进食管癌转移,抑制miR-1246的表达或增加GSK3β的表达可能是抑制食管癌转移的有效手段.