Claudin-15 overexpression inhibits proliferation and promotes apoptosis of Schwann cells in vitro

Our previous experiments have discovered that Claudin-15 was up-regulated in Schwann cells of the distal nerve stumps of rat models of sciatic nerve injury.However,how Claudin-15 affects Schwann cell function is still unknown.This study aimed to identify the effects of Claudin-15 on proliferation and apoptosis of Schwann cells cultured in vitro and explore the underlying mechanisms.Primary Schwann cells were obtained from rats.Claudin-15 in Schwann cells was knocked down using siRNA(siRNA-1 group)compared with the negative control siRNA transfection group(negative control group).Claudin-15 in Schwann cells was overexpressed using pGV230-Claudin-15 plasmid(pGV230-Claudin-15 group).The pGV230 transfection group(pGV230 group)acted as the control of the pGV230-Claudin-15 group.Cell proliferation was analyzed with EdU assay.Cell apoptosis was analyzed with flow cytometric analysis.Cell migration was analyzed with Transwell inserts.The mRNA and protein expressions were analyzed with quantitative polymerase chain reaction assay and western blot assay.The results showed that compared with the negative control group,cell proliferation rate was up-regulated;p-AKT/AKT ratio,apoptotic rate,p-c-Jun/c-Jun ratio,mRNA expression of protein kinase C alpha,Bcl-2 and Bax were down-regulated;and mRNA expression of neurotrophins basic fibroblast growth factor and neurotrophin-3 were increased in the siRNA-1 group.No significant difference was found in cell migration between the negative control and siRNA-1 groups.Compared with the pGV230 group,the cell proliferation rate was down-regulated;apoptotic rate,p-c-Jun/c-Jun ratio and c-Fos protein expression increased;mRNA expression of protein kinase C alpha and Bax decreased;and mRNA expressions of neurotrophins basic fibroblast growth factor and neurotrophin-3 were up-regulated in the pGV230-Claudin-15 group.The above results demonstrated that overexpression of Claudin-15 inhibited Schwann cell proliferation and promoted Schwann cell apoptosis in vitro.Silencing of Claudin-15 had the reverse effect and provided neuroprotective effect.This study was approved by the Experimental Animal Ethics Committee of Jilin University of China(approval No.2016-nsfc001)on March 5,2016.

结直肠癌伴肝转移组织中PEA15和ZFP57的表达及与预后的关系

目的:研究伴肝转移结直肠癌(CRC)癌组织中增殖和凋亡衔接子蛋白15(PEA15)、锌指蛋白57(ZFP57)的表达及与预后的关系。方法:选取2014年2月—2017年2月收治的100例CRC患者,其中40例伴肝转移,60例无肝转移。检测CRC癌组织和癌旁组织中PEA15、ZFP57的表达。单因素及多因素Logistic回归分析CRC肝转移的影响因素。分析不同PEA15、ZFP57表达的伴肝转移CRC患者生存预后差异。结果:相比于癌旁组织,无肝转移组和肝转移组CRC癌组织中PEA15、ZFP57相对表达量均明显较高(P<0.05);相比于无肝转移组CRC癌组织,肝转移组CRC癌组织中PEA15、ZFP57相对表达量明显较高(P<0.05)。单因素分析显示,肿瘤浸润深度、淋巴结转移、PEA15表达、ZFP57表达均与CRC肝转移有关(P<0.05)。多因素Logistic回归分析结果显示,肿瘤浸润深度T_(3)~T_(4)、有淋巴结转移、PEA15高表达、ZFP57高表达均是CRC肝转移的独立危险因素(P<0.05)。PEA15、ZFP57高表达的伴肝转移CRC患者的3年总体生存率及平均生存时间均显著低于PEA15、ZFP57低表达的伴肝转移CRC患者(P<0.05)。结论:伴肝转移CRC癌组织中PEA15、ZFP57表达异常升高,PEA15高表达、ZFP57高表达是影响CRC肝转移的危险因素,可能作为判断伴肝转移CRC患者生存预后的指标。

SH2B1沉默对乳腺癌细胞增殖和凋亡及PI3K/Akt通路的影响

目的:探讨RNA干扰技术(RNAi)沉默Src同源性2B衔接蛋白1(SH2B1)对乳腺癌细胞增殖、凋亡及磷脂酰肌醇-3激酶/丝氨酸-苏氨酸蛋白激酶(PI3K/Akt)信号通路的影响。方法:体外培养人正常乳腺上皮细胞MCF-10a与乳腺癌细胞MCF-7,采用实时荧光定量PCR(qRT-PCR)与蛋白免疫印迹(WB)法检测细胞中SH2B1mRNA及蛋白表达水平。采用RNAi技术将SH2B1阴性对照质粒、si-SH2B1分别转染至MCF-7细胞,分别命名为si-NC组、si-SH2B1组,未经处理的MCF-7细胞命名为空白对照组。采用RT-PCR法检测各组MCF-7细胞中SH2B1、B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关蛋白X(Bax)、天冬氨酸蛋白水解酶3(Caspase-3)mRNA表达水平;CCK8法检测细胞增殖情况;流式细胞仪检测细胞凋亡及细胞周期情况;Westernblot法检测Bcl-2、Bax、Caspase-3、PI3K及其磷酸化蛋白(p-PI3K)、Akt及其磷酸化蛋白(p-Akt)等蛋白表达情况。结果:与MCF-10a细胞相比,MCF-7细胞中SH2B1mRNA及蛋白表达均显著升高(P<0.05);与空白对照组、si-NC组相比,si-SH2B1组SH2B1mRNA及蛋白表达、S期与G2/M期DNA量、Bcl-2mRNA及蛋白表达均显著降低(P<0.05);MCF-7细胞增殖抑制率、细胞凋亡率、G0/G1期DNA量、Bax、Caspase-3mRNA及蛋白表达均显著升高(P<0.05);WB法显示,si-SH2B1组MCF-7细胞中p-PI3K、p-Akt蛋白表达均显著低于空白对照组、si-NC组(P<0.05)。结论:SH2B1沉默可抑制乳腺癌细胞增殖,诱导细胞周期阻滞及细胞凋亡,其作用机制可能与下调PI3K/Akt信号通路有关。

CARMA3在胆管癌细胞中的表达及功能

目的:探讨接头蛋白CARMA3在胆管癌细胞中的表达及功能。方法:用qRT-PCR技术检测胆管癌HUCCT1细胞与RBE细胞以及正常胆管细胞HIBEC中CARMA3的mRNA表达。用siRNA技术沉默HUCCT1细胞与RBE细胞中CARMA3的表达后,分别通过CCK-8法、流式细胞术、Transwell实验检测细胞增殖、凋亡、细胞周期以及转移和侵袭能力的变化。结果:两种胆管癌细胞中CARMA3的mRNA表达水平均明显高于正常胆管细胞HIBEC(HUCCT1 vs.HIBEC:t=5.321,P=0.011;RBE vs.HIBEC:t=5.932,P=0.008)。沉默CARMA3的表达后,两种胆管癌细胞的增殖、转移和细胞侵袭能力被明显抑制,G_1期阻滞明显增加,细胞凋亡率明显升高(均P<0.05)。结论:CARMA3在胆管癌细胞中表达上调,其作用可能与促进细胞增殖、侵袭、转移并抑制细胞凋亡。

XB130对胶质瘤细胞株LN229增殖和凋亡的影响

目的 探讨下调胶质瘤细胞株LN229中XB130的表达对细胞增殖和凋亡的影响.方法 选取LN229细胞作为研究对象,分为对照组、无义序列siRNA转染组和XB130 siRNA转染组进行细胞培养,细胞转染siRNA后,采用Western blot和免疫荧光检测XB130的表达情况;用MTT检测细胞的增殖活性;流式细胞仪检测细胞凋亡及周期.结果 转染siRNA后,对照组、无义序列siRNA转染组、XB130 siRNA转染组XB130蛋白相对表达量分别为1.00 ±0.10、1.00±0.13、0.52±0.06,XB130平均免疫荧光强度分别为0.085±0.012、0.072±0.009、0.013±0.002,表明XB130siRNA转染组XB130蛋白表达显著降低,差异有统计学意义（P＜0.05）;MTr结果表明XB130 siRNA转染组细胞增殖受到明显抑制,差异有统计学意义（P＜0.05）;流式细胞仪检测发现对照组、无义序列siRNA转染组、XB130 siRNA转染组细胞凋亡率分别为（12.45±0.96）％、（13.64±0.85）％、（20.26±1.42）％,G0/G1期细胞比例分别为（62.85±1.85）％、（63.04±1.74）％、（72.67±1.93）％,表明XB130 siRNA转染组细胞凋亡率显著高于其他两组,差异有统计学意义（P＜0.05）,G0/G1期细胞比例也显著高于其他两组,差异有统计学意义（P＜0.05）.结论 沉默胶质瘤细胞中XB130的表达能显著抑制细胞的增殖并促进其凋亡,表明XB130蛋白可促进胶质瘤细胞的增殖。