PSMA7 对A549 细胞中RB 途径的影响

目的:探究高表达和干扰PSMA7 对A549 细胞周期以及RB 途径中Cyclin D1、CDK4、P16、Rb 蛋白水平的影响。方法:将pcDNA3.1-PSMA7 高表达和pGPU6/ Hygro-PSMA7-265 干扰载体分别瞬时转染A549 细胞,然后用流式细胞仪检测PSMA7 对细胞周期的影响,再通过Western blot 实验检测Cyclin D1、CDK4、P16、Rb 的蛋白水平。结果:和对照组相比,PSMA7 高表达时周期时相分布无明显差异,但Cyclin D1、CDK4 的蛋白表达水平明显降低,P16、Rb 的表达明显增高;和对照组相比,干扰PSMA7 后细胞的G0/ G1、G2/ M 期比例均降低,而S 期的值增高,均具有差异,而Cyclin D1、CDK4 的蛋白表达水平明显增加,P16、Rb 的表达明显降低,以上结果与对照组相比差异均有统计学意义。结论:PSMA7 在A549 肺腺癌细胞中能够影响RB 途径中Cyclin D1、CDK4、P16、Rb 的蛋白水平的表达,干扰PSMA7 使A549 细胞较早进入S 期。     

TNF-α通过调控KLF4抑制口腔鳞癌细胞CAL27增殖

目的：探讨肿瘤坏死因子-α（TNF-α）通过上调口腔鳞癌细胞CAL27中Krüppel样因子4（KLF4）表达对细胞增殖的影响。方法：用不同浓度的TNF-α（0、25、5、10、20 ng/ml）处理CAL27细胞24 h,免疫印迹试验（Western blot）检测KLF4表达变化;用20 ng/ml TNF-α处理细胞,分别于24 h、48 h、72 h用噻唑蓝（MTT）法检测细胞增殖变化;构建过表达KLF4的CAL27细胞,MTT检测其对细胞增殖的影响;MTT检测沉默KLF4（siKLF4）及siKLF4联合TNF-α 处理对细胞增殖能力的影响。结果：MTT检测发现TNF-α可抑制细胞增殖;TNF-α可呈剂量依赖性地诱导CAL27细胞中KLF4表达;Western blot和qPCR检测显示成功构建过表达KLF4的CAL27细胞;过表达KLF4可抑制细胞增殖;沉默KLF4可部分恢复TNF-α对CAL27细胞增殖的抑制作用。结论：TNF-α可诱导口腔鳞癌细胞CAL27中KLF4表达增加,KLF4可能参与了TNF-α对CAL27细胞增殖的抑制作用。     

敲减microRNA-205靶向PTEN抑制食管癌细胞TE1的增殖并促进细胞凋亡

目的:探讨miRNA-205对食管癌细胞株TE1增殖和凋亡能力的影响及其作用机制.方法:实时定量聚合酶链式反应(qRT-PCR)和Western印迹检测正常食管黏膜细胞Het-1A和不同食管癌细胞株(KYSE70,TE12,TE1)中miRNA-205的mRNA及蛋白表达水平.转染miRNA-205抑制剂下调TE1细胞中miRNA-205的表达,采用CCK-8法和流式细胞术检测细胞增殖能力、细胞周期和细胞凋亡情况;Western印迹检测细胞增殖和细胞凋亡相关CDK2,cyclin D1,P21,Bcl-2,cleavedcaspase-3,caspase-3,p-Rb,Rb,p-Akt和Akt的蛋白表达水平.双荧光素酶报告基因分析法预测及验证其可能的靶基因.结果:MiRNA-205在各型食管癌细胞中高表达.沉默miRNA-205后,TE1细胞的增殖能力降低并表现为细胞周期阻滞,而细胞凋亡率显著升高(p＜0.05).同时细胞中cyclin D1,CDK2,bcl-2,p-Rb及p-Akt的蛋白表达水平均显著降低,p21及cleaved-caspase-3的蛋白表达水平显著升高.双荧光素酶报告基因分析显示PTEN是miRNA-205的可能作用靶点,TE1中共转染miRNA-205抑制剂和PTEN siRNA可部分逆转miRNA-205介导细胞增殖抑制及凋亡诱导作用.结论:沉默miRNA-205可靶向PTEN抑制食管癌细胞株TE1的增殖,并促进其凋亡,提示miRNA-205可作为食管癌诊疗的一个潜在作用靶点.     

Krüppel样因子4在猪脂肪间充质干细胞成脂分化过程中的表达模式

目的探讨猪脂肪间充质干细胞(AMSCs)成脂分化过程中Krüppel样因子4(KLF4)的表达模式。方法采用F3代以上猪AMSCs进行成脂诱导分化,用油红O染色提取法检测成脂分化程度,用RT-PCR和Realtime PCR检测KLF4的表达模式。结果猪AMSCs成脂诱导分化,诱导3 d开始出现脂肪滴,诱导10 d时成脂分化率达60%;RT-PCR检测,在诱导分化2、4、8、12和16 d时都能检测出KLF4的表达;经Real-time PCR检测,上述各诱导分化时间点的表达量分别为对照组的1.6657±0.2269、2.6790±0.0524、2.6293±0.0280、2.3633±0.1568和2.6146±0.1575倍。这些结果表明,KLF4的表达在猪AMSCs成脂诱导分化的第2天就有显著上调,表达高峰出现在成脂分化的第4天,此后持续高表达。结论在猪AMSCs成脂分化过程中,从早期到晚期,KLF4发挥持续性的调控作用。     

miR-211 对上皮性卵巢癌细胞HO8910 增殖及细胞周期相关蛋白的影响

目的:探讨miR-211 与上皮性卵巢癌发生的关系,及其对卵巢癌细胞增殖的影响。方法:对30 例上皮性卵巢癌组织标本及卵巢癌细胞系HO8910 中miR-211、Cyclin D1 和CDK6 表达情况进行研究,同时选取30 例非卵巢癌患者组织标本及正常卵巢上皮细胞株IOSE80 作为对照,并分析上皮性卵巢癌组织及卵巢癌细胞系中miR-211、Cyclin D1、CDK6 表达情况及表达相关性;miR-211 对卵巢癌细胞增殖,以及对Cyclin D1 和CDK6 表达的影响。结果:卵巢癌组织miR-211 相对表达水平显著低于正常组织(P<0.05),卵巢癌细胞中miR-211 相对表达水平显著低于正常卵巢上皮细胞(P<0.05);在上皮性卵巢癌细胞系HO8910 中,第3 天和第4 天miR-211 组细胞数显著低于miR-Ctrl (P<0.05);上皮性卵巢癌组织中Cyclin D1、CDK6相对表达水平显著高于正常卵巢上皮组织(P<0.05);上皮性卵巢癌细胞系中miR-211 显著抑制Cyclin D1 和CDK6 的表达;在卵巢癌组织中,Spearman 相关性分析结果显示miR-211 和Cyclin D1 和CDK6 相对表达水平呈负相关(r =-0.583,P =0.010)。结论:miR-211 可抑制卵巢癌细胞增殖,并抑制周期相关蛋白Cyclin D1 和CDK6 的表达,miR-211 与周期相关蛋白Cyclin D1 和CDK6 在卵巢癌发生中可能存在调控关系。     

Oral squamous cell carcinoma suppressed antitumor immunity through induction of PD-L1 expression on tumor-associated macrophages

Oral squamous cell carcinoma (OSCC) is the most common solid tumor in the oral cavity. Development and progression of OSCC is associated with the elevated presence of inhibitory M2 type tumor-associated macrophages (TAMs). However, the underlying mechanism leading to the enrichment of M2 TAMs and the pathway through which TAMs foster tumor progression are still unclear. In this study, we harvested TAMs and tumor cells from primary OSCC resections of stage II and stage III patients. We showed that compared to peritumoral macrophages, TAMs presented upregulated expression of PD-L1 and elevated capacity in inducing T cell apoptosis. The level of PD-L1 expression directly correlated with the level of T cell apoptosis. Interestingly, peripheral blood monocytes with low initial PD-L1 level had upregulated PD-L1 expression and acquired the ability to induce T cell apoptosis, after incubation with primary tumor cells from OSCC patients. The PD-L1 expression by monocytes depended on interleukin 10 (IL-10), since blockade of IL-10 in the tumor-monocyte coculture abrogated PD-L1 upregulation. IL-10 mRNA expression in tumor cells and monocytes also preceded PD-L1 mRNA expression in monocytes. Furthermore, the IL-10 concentration in the tumor microenvironment directly correlated with the PD-L1 level on TAMs. Together, these results suggest that OSCC could directly suppress antitumor T cell immunity through conditioning TAMs.     

Oncogenic circDHTKD1 promotes tumor growth and metastasis of oral squamous cell carcinoma in vitro and in vivo via upregulating miR-326-mediated GAB1

Circular RNAs (circRNAs) have been extensively elucidated with regard to their significant implications in oral squamous cell carcinoma (OSCC). This study performed the functional investigation of circRNA dehydrogenase E1 and transketolase domain containing 1 (circDHTKD1) in OSCC. RNA expression levels of different molecules were measured via quantitative real-time polymerase chain reaction (qRT-PCR). Cellular behaviors were detected by 3-(4, 5-dimethylthiazol-2-y1)-2,5-diphenyl tetrazolium bromide (MTT) for cell viability, colony formation assay for clonal capacity, flow cytometry for cell apoptosis, wound healing assay for migration, and transwell assay for migration/invasion. Western blot was used for analyzing protein expression. RNA pull-down and dual-luciferase reporter assays were applied to assess the binding between targets. A xenograft tumor model was established in nude mice for in vivo experiments. Our expression analysis revealed that circDHTKD1 was upregulated in OSCC tissues and cells. circDHTKD1 knockdown was shown to impede OSCC cell growth and metastasis but motivate apoptosis. Additionally, circDHTKD1 served as a microRNA-326 (miR-326) sponge and the function of circDHTKD1 was achieved by sponging miR-326 in OSCC cells. Also, miR-326 inhibited OSCC development via targeting GRB2-associated-binding protein 1 (GAB1). circDHTKD1 could sponge miR-326 to alter GAB1 expression. Furthermore, circDHTKD1 contributed to OSCC progression in vivo via the miR-326/GAB1 axis. These data disclosed a specific circDHTKD1/miR-326/GAB1 signal axis in governing the malignant progression of OSCC, showing the considerable possibility of circDHTKD1 as a predictive and therapeutic target for clinical diagnosis and treatment of OSCC.     

Augmented miR-10b expression associated with depressed expression of its target gene KLF4 involved in gastric carcinoma

Previous studies have revealed several targets of miR-10b, such as syndecan-1, HOXD10, TBX5, and E-cadherin. In this study, we aimed to assess whether Krüppel-like factor 4 (KLF4) is a target gene of miR-10b in gastric cancer (GC). Targeting of KLF4 by miR-10b was confirmed by dual-luciferase reporter assays. The expression levels of miR-10b and KLF4 mRNA in 5 different gastric cancer cell lines and 65 pairs of gastric cancer tissues were detected by Real-time PCR. In addition, KLF4 protein in gastric cancer cell lines and 30 GC tissues was measured by western blotting and immunochemistry, respectively. KLF4 is a direct target gene of miR-10b in GC, and its expression is reduced by miR-10b at both mRNA and protein levels. In addition, the expression level of miR-10b was tendentiously upregulated in GC tissues while the expression levels of KLF4 mRNA and protein were decreased in gastric cancer tissues compared with normal adjacent tissue. There was a dramatically inverse correlation between the expression levels of miR-10b and KLF4 mRNA in GC (r = -0.339, P = 0.006). These findings indicate that miR-10b was upregulated in GC and may have a key role in GC pathogenesis and development through the downregulation of its target gene KLF4.     

miR-24-3p靶向KLF6基因调控IL-6/STAT3信号通路影响食管癌细胞的活力和凋亡

目的:探讨微小RNA-24-3p(miR-24-3p)对食管癌细胞活力和凋亡的影响及机制。方法:以人正常食管上皮细胞HEEC为对照,采用RT-qPCR检测食管癌细胞TE11、Eca109和EC9706中miR-24-3p和KLF6 mRNA的表达,Western blot检测KLF6蛋白的表达。用anti-miR-24-3p和KLF6 siRNA转染EC9706细胞,MTT检测细胞活力,流式细胞术检测细胞凋亡率,Western blot检测检测细胞中与增殖、凋亡相关的蛋白以及IL-6/STAT3信号通路相关蛋白的表达,ELISA法检测IL-6的表达。双萤光素酶报告基因实验验证miR-24-3p与KLF6靶向调控的关系。结果:食管癌癌细胞TE11、Eca109和EC9706中miR-24-3p表达上调(P<0.05),KLF6的mRNA和蛋白表达下调(P<0.05)。敲减EC9706细胞miR-24-3p表达可抑制其细胞活力,诱导其凋亡,并抑制细胞CDK4、cyclin D1、CDC25A、p-STAT3、IL-6及Bcl-2的表达,促进caspase-3和Bax的表达。结论:miR-24-3p可靶向KLF6基因调控IL-6/STAT3信号通路影响食管癌细胞的生长和凋亡。     

KLF4对结直肠癌细胞生长抑制及化疗敏感性的影响

目的:探讨Krüppel样因子4(KLF4)对结直肠癌细胞活力、凋亡及顺铂化疗敏感性的影响。方法:Western blot法检测KLF4在结直肠癌Caco2、SW480和HCT116细胞中的表达。将SW480细胞分为pc DNA3. 1组(转染pc DNA3. 1空质粒)、pc DNA3. 1-KLF4组(转染构建的pc DNA3. 1-KLF4过表达质粒)和pc DNA3. 1-KLF4+顺铂组(转染pc DNA3. 1-KLF4 48 h后用1 mg/L顺铂处理细胞48 h),Western blot检测KLF4、p-IκBα、细胞周期素D1(cyclin D1)和生存素(survivin)的蛋白水平; CCK-8法检测各组细胞活力;流式细胞术检测细胞凋亡率; DCFH-DA探针检测活性氧簇(ROS)含量。结果:KLF4在结直肠癌细胞中的表达均显著低于在人结肠黏膜上皮NCM460细胞的表达(P 0. 05)。与pc DNA3. 1组相比,pc DNA3. 1-KLF4组的KLF4蛋白表达显著增高(P 0. 05),细胞活力及cyclin D1和survivin的蛋白表达均显著降低,细胞凋亡率、ROS含量及p-IκBα的蛋白表达均显著增高;而pc DNA3. 1-KLF4+顺铂组细胞活力及cyclin D1和survivin的蛋白表达均显著低于pc DNA3. 1-KLF4组,细胞凋亡率、ROS含量及p-IκBα的蛋白表达均显著高于pc DNA3. 1-KLF4组(P 0. 05)。结论:上调结直肠癌细胞KLF4基因表达可降低肿瘤细胞活力,诱导细胞凋亡,增强顺铂化疗敏感性,其机制可能与提高细胞内ROS含量及下调NF-κB信号关键分子IκBα的磷酸化水平有关。     

Co-expression of XIAP and cyclin D1 complex correlates with a poor prognosis in patients with hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. Despite improved diagnosis and treatment, the prognosis for HCC patients remains poor. The goal of this study was to identify key regulatory proteins and signaling pathways important for cell apoptosis and proliferation as biomarkers for prognostication and targeted therapy. Protein Pathway Array was applied to screen 38 signaling proteins and phosphoproteins in 12 paired HCC tumors and surrounding benign tissues and found that 20 of them, including XIAP, CDK4, CDK6, and Cyclin D1, were overexpressed in HCC tissues. Immunostaining results of XIAP, CDK4, and Cyclin D1 in an additional 59 HCC tissues showed that the expression of XIAP correlated with the expression of CDK4/Cyclin D1, and that the increased expression of these proteins correlated with poor overall survival in these patients. Further studies using the HCC Huh7 cell line transfected with XIAP siRNA or expression vector demonstrated that XIAP regulated the expression of CDK4, CDK6, and Cyclin D1 via NF-êB and PTEN pathways. Finally, inhibition of XIAP using embelin, a XIAP-specific small molecule, leads to an increased apoptosis and decreased cell proliferation via arrest at G1 phase. Taken together, XIAP is a central modulator regulating cell apoptosis and cell cycle progression. Therefore, XIAP together with cell cycle regulatory proteins can be used as prognostic markers and therapeutic targets.     

Zebrafish KLF4 is essential for anterior mesendoderm/pre-polster differentiation and hatching.

Gene knockout studies of Krüppel-like factors (KLFs) in mice have shown essential roles in organogenesis. A screen for KLF family members in zebrafish identified many KLFs. One of these, zebrafish KLF4 (zKLF4) is the homologue of neptune, a Xenopus laevis KLF. zKLF4 is expressed from approximately 80% epiboly a patch of dorsal/anterior mesendodermal cells called the pre-polster and, subsequently, in the polster and hatching gland. Here we investigate the function of zKLF4 using morpholino-based antisense oligonucleotides. Knockdown of zKLF4 resulted in complete absence of hatching gland formation and subsequent hatching in zebrafish. In addition, there was early knockdown of expression of the pre-polster/anterior mesendoderm markers CatL, cap1, and BMP4. These results indicate zKLF4 is expressed within the pre-polster, an early mesendodermal site, and that it plays a critical role in the differentiation of these cells into hatching gland cells.     

Cyclin D3/CDK11p58 Complex Involved in Schwann Cells Proliferation Repression Caused by Lipopolysaccharide

Schwann cells proliferation is the main characterize of kinds PNS inflammation diseases. It has been well documented that cyclin D3 /CDK11^p58 complex inhibits cell function through multiple mechanisms, but the mechanism of cyclin D3/CDK11^p58 complex exerts its repressive role in the Schwann cells proliferation remains to be identified. In the present investigation, we demonstrated that the expression of CDK11^p58 were upregulated in the inflammation caused by LPS, a main part of bactria. Cyclin D3 and the 58-kDa isoform of cyclin-dependent kinase 11 (CDK11^p58) interacted with each other mainly in nuclear region, repressed Schwann cells proliferation and induced cell apoptosis. Overexpression of CDK11^p58 expression might enhance this process, while silence of cyclin D3 reverting it. This work demonstrates for the first time the role of cyclin D3/CDK11^p58 complex in repressing the Schwann cells proliferation and inducing its apoptosis.     

骨形成蛋白4对人胶质瘤干细胞增殖和凋亡的影响

目的 研究骨形成蛋白4(bone morphogenetic protein 4,BMP4)对人胶质瘤干细胞增殖和凋亡的影响.方法 在体外以重组人BMP4蛋白干预人胶质瘤U87细胞系来源的胶质瘤干细胞,免疫荧光鉴定胶质瘤干细胞,通过软琼脂克隆实验、流式细胞仪检测,观察BMP4对胶质瘤干细胞增殖和凋亡的影响.结果 BMP4能显著抑制胶质瘤干细胞的增殖能力,并诱导其凋亡.BMP4组增殖相关蛋白Cyclin D1 表达降低,抗凋亡蛋白Bcl-2表达明显降低,促凋亡蛋白Bax表达显著增多.结论 BMP4可显著抑制胶质瘤U87细胞系来源的胶质瘤干细胞的增殖能力,并诱导其凋亡.