新型多胺代谢酶抑制剂SI-4650对结肠癌CT-26细胞增殖的影响及其机制

目的:探讨本实验室新发现的新型多胺代谢酶小分子抑制剂SI-4650对结肠癌CT-26细胞增殖、自噬和凋亡的影响。方法:体外培养CT-26细胞,以0μmol·L^-1SI-4650处理48 h细胞为正常对照组,单独2.5 mmol·L^-13-MA处理细胞为自噬抑制对照组,40、80μmol·L^-1SI-4650处理48 h细胞以及3-MA联合40、80μmol·L^-1SI-4650处理48 h细胞为4个实验组,化学发光法检测CT-26细胞中多胺代谢酶SMO和APAO酶活性的变化,HPLC法检测细胞中多胺含量的变化,CCK8法检测CT-26细胞增殖能力变化; PI单染结合流式细胞术分析细胞周期; Western blot法分析细胞自噬; PI/FITC-Annexin V双染、JC-1荧光探针和Fluo-3 AM钙离子荧光探针分别结合流式细胞术以及Western blot法分析细胞凋亡。结果:与正常对照组比较,40、80μmol·L^-1SI-4650实验组细胞生长抑制率分别为36.9...     

SPOP对卵巢癌细胞生物学行为的影响及其分子机制的研究

探讨人卵巢癌组织中斑点状蛋白(SPOP)的表达及对其卵巢癌细胞生物行为学的影响及相关机制。采用免疫组化技术分别检测正常卵巢组织10例、卵巢癌组织90例的组织芯片中SPOP的表达;体外培养永生化人卵巢癌细胞株OVCAR-3、SKOV3及永生化的人卵巢表皮上皮细胞株HOSE,RT-PCR、WB检测3种细胞株SPOP的表达。用慢病毒稳定转染OVCAR-3、HOSE细胞株,构建过表达、敲低SPOP的细胞株。流式细胞仪检测转染后细胞株的凋亡,CCK8检测转染后细胞株的增殖情况,Transwell小室检测转染后细胞株的迁移侵袭情况。WB检测转染后细胞株PI3K-Akt通路相关蛋白的表达情况。组织学免疫组化评分显示卵巢癌组织染色明显高于正常卵巢组织。细胞学实验显示,成功构建过表达、敲低SPOP的OVCAR-3细胞,过表达组、过表达空载组、敲低组、敲低空载组细胞凋亡及增殖能力无明显差异;各组细胞迁移及侵袭结果与空载组有明显差异,且差异有统计学意义(p0.05)。过表达组细胞P-GSK3β(Ser9)表达水平明显低于敲低组(p0.05),MMP-9表达水平明显高于敲低组(p0.05)。SPOP在卵巢癌组织中表达上升,且具有促进卵巢癌细胞迁移侵袭的作用,其调节机制可能与p-GSK3β(Ser9)通路相关。     

纳米ZnO对人肺上皮细胞BEAS-2B细胞增殖和凋亡的影响

目的:探讨纳米ZnO对人肺上皮细胞BEAS-2B细胞增殖、凋亡的影响及分子机制。方法:用终浓度为3、6、12μg/ml的纳米ZnO处理BEAS-2B细胞12 h和24 h,对照组未加入纳米ZnO,各设3复孔,CCK-8法检测细胞活力,分析半致死浓度。筛选3、6μg/ml纳米ZnO处理BEAS-2B细胞24 h,各设3复孔,倒置显微镜观察细胞形态,Hochest33342染色观察细胞核,AO染色及扫描电镜观察细胞凋亡形态,流式细胞术检测活性氧水平、细胞周期进程、细胞凋亡;Western blot检测Bcl-2、Bax蛋白表达水平。结果:与对照组相比,纳米ZnO处理组细胞活力显著下降(P<0.01),处理24 h时IC₅₀为6.13μg/ml;纳米ZnO处理细胞24 h后,3μg/ml和6μg/ml组的活性氧水平显著升高(P<0.05,P<0.01)。6μg/ml处理组细胞周期阻滞于G2/M期、染色质固缩凝集、出现凋亡小体、细胞凋亡率显著增加(P<0.01)、Bcl-2蛋白表达显著降低(P<0.05)、Bax蛋白表达显著升高(P<0...     

前列腺癌干细胞拮抗剂盐霉素对人前列腺癌PC3细胞增殖和凋亡的影响

为了探讨前列腺癌干细胞拮抗剂盐霉素对人前列腺癌PC3细胞增值、凋亡和迁移的影响,本研究用前列腺癌干细胞拮抗剂盐霉素处理人前列腺癌PC3细胞,用CCK-8检测细胞的增殖能力,用流式细胞术检测细胞的凋亡状况,蛋白免疫印迹技术检测凋亡蛋白Bax和Bcl-2,划痕实验检测细胞迁移。用前列腺癌干细胞拮抗剂盐霉素处理人前列腺PC3细胞后,其细胞增殖能力显著下降,凋亡细胞显著增加,促凋亡蛋白Bax的表达明显增加,抗凋亡蛋白Bcl-2的表达显著降低,且细胞划痕修复率显著降低。前列腺癌干细胞拮抗剂盐霉素可诱导人前列腺癌PC3细胞活力降低、凋亡增多和迁移抑制。     

蟾毒灵对人卵巢癌SKOV-3细胞增殖的影响

目的：探讨蟾毒灵对人卵巢癌SKOV-3细胞增殖抑制和凋亡的影响,为卵巢癌临床治疗提供依据和分子基础。方法：不同浓度蟾毒灵处理卵巢癌SKOV-3细胞后,MTT法检测细胞增殖抑制作用,细胞免疫化学染色法检测细胞的凋亡,Western Blot法检测Bax,Bcl-2,Caspase-3蛋白以及计算Bax／Bcl-2的比值。结果：蟾毒灵能够抑制SKOV-3细胞的增殖,且成时间和剂量依赖性,免疫荧光显示蟾毒灵对SKOV-3细胞具有凋亡作用,Western Blot检测发现蟾毒灵能够促进Caspase-3蛋白的活化,提高Bax／Bcl-2的比值。结论：蟾毒灵在体外能够抑制卵巢癌SKOV-3细胞的增殖和促进卵巢癌SKOV-3细胞的凋亡。     

蟾毒灵对人卵巢癌SKOV-3 细胞增殖的影响

目的：探讨蟾毒灵对人卵巢癌SKOV-3细胞增殖抑制和凋亡的影响,为卵巢癌临床治疗提供依据和分子基础。方法：不同浓 度蟾毒灵处理卵巢癌SKOV-3 细胞后,MTT 法检测细胞增殖抑制作用,细胞免疫化学染色法检测细胞的凋亡,Western Blot 法检 测Bax,Bcl-2,Caspase-3 蛋白以及计算Bax/Bcl-2 的比值。结果：蟾毒灵能够抑制SKOV-3 细胞的增殖,且成时间和剂量依赖性, 免疫荧光显示蟾毒灵对SKOV-3 细胞具有凋亡作用,Western Blot 检测发现蟾毒灵能够促进Caspase-3 蛋白的活化,提高 Bax/Bcl-2 的比值。结论：蟾毒灵在体外能够抑制卵巢癌SKOV-3 细胞的增殖和促进卵巢癌SKOV-3 细胞的凋亡。     

小鼠胚胎对人卵巢癌细胞生物学行为影响的体外研究

探讨体外共培养环境中小鼠胚胎对人卵巢癌细胞HO8910PM的影响.通过小鼠胚胎与肿瘤细胞体外共培养模型观察小鼠胚胎对肿瘤细胞的形态及生长行为的影响,Annexin V-EGFP/PI原位检测与小鼠胚胎共培养后肿瘤细胞的凋亡情况,MTT粘附实验、transwell迁移及侵袭实验研究与小鼠胚胎共培养后的人卵巢癌细胞的粘附性、迁移性及侵袭性的变化.共培养时小鼠胚胎能够侵入人卵巢癌细胞并推开肿瘤细胞形成自己的生长空间,激光共聚焦显微镜下见胚胎周围的肿瘤细胞凋亡增加,与对照组比较共培养后的HO8910PM肿瘤细胞的粘附性、迁移性及侵袭性均显著降低(P0.05、P0.01).结果表明体外共培养体系中小鼠胚胎能够侵袭肿瘤细胞,促进人卵巢癌细胞的凋亡,并使其粘附性、迁移及侵袭相关恶性行为降低.     

Neuronatin对人视网膜色素上皮细胞增殖迁移的影响

目的:研究胚胎时期表达部位广泛、丰度高,而成年后分化表达的印记基因Neuronatin(Nnat)的两种剪接形式Nnatα和Nnatβ对人视网膜色素上皮细胞(RPE)增殖、迁移的影响。方法:构建Nnatα、β两种剪接形式的表达质粒,转染RPE获得表达该基因的稳定表达细胞株;CCK-8实验检测稳定表达细胞株的增殖能力,流式细胞仪分析细胞周期,细胞划痕实验检测其迁移能力。结果:成功构建了Nnatα和Nnatβ表达质粒,并获得了Nnatα和Nnatβ基因稳定表达PRE细胞株。CCK-8实验结果显示cNNATα组与对照组相比较,增值率为23.33%(P0.05),cNNATβ组相较于对照组无显著性差异,细胞周期分析cNNATα组和cNNATβ组细胞在G2-S期的百分率分别为18.60%、11.11%,对照组细胞的为9.94%;相较于对照组,cNNATα组的细胞迁移能力显著增强,cNNATβ组的细胞迁移能力微弱增强。结论:Nnatα对RPE有一定的增殖作用,其影响主要在S期;同时,Nnatα显著促进RPE细胞的迁移能力。     

OCT4 介导卵泡刺激素对永生化人卵巢上皮细胞株增殖、 凋亡和侵袭能力的影响

目的:探讨OCT4基因对卵泡刺激素作用下的永生化人卵巢上皮细胞株(Moody细胞)增殖、凋亡和侵袭能力影响。方法:将不同浓度的FSH(0、25、50、100mIU/ml)作用于Moody细胞48小时,应用Western-blot技术检测OCT4表达情况。采用慢病毒介导将重组质粒OCT4稳定转染至人卵巢上皮细胞株中,应用Western-blot法鉴定OCT4蛋白表达情况。FSH以50 mIU/ml作为工作浓度,实验对象分为4组:①siCon组,转染空载体的阴性对照组;②OCT4组:稳定转染OCT4基因的Moody细胞组;③FSH+siCon组:以FSH处理的siCon组;④FSH+OCT4组:以FSH处理的OCT4组。采用MTT比色法检测各组细胞的增殖情况,流式细胞仪检测各组细胞凋亡情况,Transwell侵袭实验检测各组细胞侵袭能力的变化。结果:(1)随着FSH浓度的增加,Moody细胞中OCT4蛋白表达逐渐增高,在FSH浓度为50 mIU/ml时达最高;(2)OCT4基因成功转染至Moody细胞中,经Western-blot检测该基因在细胞中进行蛋白高表达;(3)FSH+OCT4组细胞增殖活性明显增高,同时凋亡率降低,与另外三组相比差异具有统计学意义(P<0.05);(4)在FSH作用下,转染OCT4后明显增强了细胞的侵袭能力,与另外三组相比差异具有统计学意义(P<0.05)。结论:OCT4介导了FSH对人卵巢上皮细胞增殖、凋亡、侵袭活性的调控。     

根皮素对HepG2肝癌细胞增殖和凋亡影响的实验研究

目的：探讨根皮素对人肝癌HepG2细胞增殖和凋亡的影响。方法：MTT法检测不同浓度（30、40、50 ug/mL）根皮素对肝癌 HepG2 细胞增殖的抑制作用,流式细胞技术检测根皮素对HepG2细胞凋亡的影响,ELISA 法检测不同浓度根皮素干预后细胞中 Bcl-2 和Bax表达的变化,Western blot法检测30 ug/mL根皮素在8,16,24 小时后检测p-AKT 蛋白表达情况。结果：30、40 和50 ug/mL 的根皮素对肝癌HepG2 细胞的增殖均有抑制作用（P＜0.01）,同时,30、40 和50 ug/mL 的根皮素在24 小时后可诱导 HepG2 细胞发生早期凋亡,凋亡率分别为0.1321± 0.0224, 0.2607± 0.0457, 0.3712± 0.0884（P＜0.01）;另外,30、40 和50 ug/mL的 根皮素作用24 小时后细胞内Bcl-2 表达降低,Bax 表达增高（P＜0.01）;最后,30 ug/mL根皮素可以明显减少p-AKT 表达量,这 种作用呈现时间依赖性。结论：根皮素能够抑制肝癌HepG2 细胞的增殖和促进细胞凋亡。     

Vinculin presents unfavorable prediction in ovarian cancer and prevents proliferation and migration of ovarian cancer cells

The influences of Vinculin on many cancers were blurry, including ovarian cancer. Thus, we concentrated on the efficient role of Vinculin in ovarian cancer and explored the potential mechanism(s). Expression of Vinculin in ovarian cancer tissues and cell lines was investigated by real‐time polymerase chain reaction, immunohistochemistry, and Western blot. The Kaplan–Meier manner with the logrank was performed to assess overall survival. We further evaluated the relations between Vinculin expression and clinicopathological features of ovarian cancer. Moreover, Vinculin was overexpressed or silenced by respectively transfection with pcDNA‐Vinculin or small interfering (si‐Vinculin) into human ovarian cancer cell line Caov3 or human ovarian epithelial cell line (HOEpiC). Thereafter, cell viability, cell apoptosis, and migration were checked by 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide, flow cytometer, and scratch assay, respectively. Likewise, the apoptosis‐ and migration‐related proteins were distinguished by Western blot. Compared to the nontumor tissues or HOEpiC cells, Vinculin was significantly lower expressed in the ovarian cancer tissues and cells. Furthermore, we found out that Vinculin was primarily distributed at the cell membrane and cytoplasm. Moreover, Vinculin was negatively associated with International Federation of Gynecology and Obstetrics stage, grade, and distant metastasis. Overexpression of Vinculin dramatically weakened cell viability and migration and stimulated apoptosis. Conversely, suppression of Vinculin showed opposite results. Vinculin presents unfavorable prediction in ovarian cancer and inhibits ovarian cancer proliferation and migration.     

The G-protein-coupled estrogen receptor agonist G-1 suppresses proliferation of ovarian cancer cells by blocking tubulin polymerization

The G-protein-coupled estrogen receptor 1 (GPER) has recently been reported to mediate the non-genomic action of estrogen in different types of cells and tissues. G-1 (1-[4-(6-bromobenzo[1,3] dioxol-5yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl]-ethanone) was developed as a potent and selective agonist for GPER. G-1 has been shown to induce the expression of genes and activate pathways that facilitate cancer cell proliferation by activating GPER. Here we demonstrate that G-1 has an anticancer potential with a mechanism similar to vinca alkaloids, the commonly used chemotherapy drugs. We found that G-1 blocks tubulin polymerization and thereby interrupts microtubule assembly in ovarian cancer cells leading to the arrest of cell cycle in the prophase of mitosis and the suppression of ovarian cancer cell proliferation. G-1 treatment also induces apoptosis of ovarian cancer cells. The ability of G-1 to target microtubules to suppress ovarian cancer cell proliferation makes it a promising candidate drug for treatment of ovarian cancer.     

miR-670-5p对肺癌细胞增殖、迁移、侵袭的影响

目的: 探讨miR-670-5p对肺癌细胞增殖、迁移和侵袭的影响,分析其调控WW结构域氧化还原酶基因(WWOX)的机制。方法: 收集2016年1月至2017年10月收治的28例肺癌组织和对应癌旁组织,实时荧光定量PCR(RT-qPCR)检测肺癌组织、癌旁组织中miR-670-5p的表达水平。将肺癌细胞A549分为anti-miR-NC组(转染anti-miR-NC)、anti-miR-670-5p组(转染anti-miR-670-5p)、anti-miR-670-5p+si-NC组(转染anti-miR-670-5p与si-NC)、anti-miR-670-5p+si-WWOX组(转染anti-miR-670-5p与si-WWOX)。转染48 h后,RT-qPCR或蛋白质印记(Western blot)检测转染效果。细胞计数试剂盒(CCK-8)检测细胞活力;Transwell实验检测细胞迁移和侵袭能力;Western blot检测P21、上皮细胞钙粘蛋白(E-cadherin)和基质金属蛋白酶2(MMP-2)蛋白的表达水平。双荧光素酶报告基因实验和Western blot验证miR-670-5p和WWOX的靶向关系。结果: 肺癌组织中miR-670-5p的表达水平较癌旁组织显著升高(P＜0.05)。抑制miR-670-5p可抑制MMP-2蛋白表达(P＜0.05),促进P21和E-cadherin表达(P＜0.05),抑制A549细胞增殖、迁移和侵袭(P＜0.05)。WWOX是miR-670-5p的靶基因,miR-670-5p负调控WWOX表达。抑制WWOX可部分逆转anti-miR-670-5p对A549细胞增殖、迁移和侵袭的影响(P＜0.05)。结论: miR-670-5p通过靶向WWOX能够促进肺癌细胞增殖、迁移、侵袭。     

Polysaccharides derived from Balanophora polyandra significantly suppressed the proliferation of ovarian cancer cells through P53‐mediated pathway

Ovarian cancer (OC) is ranked the first among the cancers threatening women's health. It attracts tremendous attention of cancer researchers because of its extremely high mortality rate. Recent studies have indicated that traditional herbal medicines (THMs) can play a pivotal role in cancer prevention and treatment. THMs are gaining popularity as a source of anti‐cancer agents. The plant of Balanophora polyandra, which has been used as a traditional herbal medicine, has been known for exhibiting potential haemostatic, analgesic, anti‐inflammatory and anti‐cancer properties. However, few studies on inhibitory effect of B. polyandra on OC have been performed. In the present study, we found that B. polyandra polysaccharides (BPP) induced cell cycle arrest at S phase, triggered apoptosis and inhibited migration and invasion of OC cells. Furthermore, we also found that there was a potential and close relationship between BPP and P53‐mediated pathway. Overall, these findings suggest that BPP can be a potential therapeutic agent for the treatment of OC.     

miR-449a对人乳腺癌细胞MCF-7增殖及迁移能力的影响

目的：通过敲低微小RNA （microRNA,miRNA）-449a的方法研究miR-449a对人乳腺癌细胞MCF-7的增殖和迁移能力的影响。方法：采用miRNA芯片在乳腺癌细胞MCF-7和人正常乳腺细胞MCF-10A筛选具有表达差异的miRNA;化学合成法制备miR-449a的抑制剂（inhibitor）,转染后经real-time PCR验证表达的变化;细胞增殖CCK-8实验对转染后细胞增殖能力进行检测;划痕实验检测细胞转移能力,transwell小室实验检测细胞侵袭的改变;蛋白免疫印迹法（Western blot）实验对MCF-7细胞增殖和迁移相关的β-catenin和E-cadherin蛋白进行检测;通过生物信息学软件预测miR-449a潜在靶基因为Notch 1,荧光素酶实验检测Notch 1是miR-449a的靶基因。结果：分别收集MCF-7和MCF-10A细胞,芯片结果显示miR-449a在MCF-7细胞的表达水平显著高于MCF-10A;本研究将细胞分为未处理组（Mock组）,阴性对照组（negative control组,NC组）和处理组,通过收集不同组MCF-7细胞进行试验,CCK-8结果显示miR-449a下调后MCF-7细胞增殖能力显著降低;划痕实验结果显示miR-449a表达降低导致MCF-7细胞转移能力降低;transwell实验结果显示MCF-7细胞侵袭受到抑制;Western blot结果发现miR-449a敲低后β-catenin表达降低,E-cadherin表达增加;荧光素酶试验结果显示,miR-449a能够显著降低Notch 1-3'-UTR质粒的荧光素活性（P<0.01）。结论：在乳腺癌细胞MCF-7中敲低miR-449a能够显著抑制癌细胞增殖和迁移,而这一变化可能通过降低Notch 1蛋白表达实现的。     

Effect of a synthetic cannabinoid agonist on the proliferation and invasion of gastric cancer cells

Although cannabinoids are associated with antineoplastic activity in a number of cancer cell types, the effect in gastric cancer cells has not been clarified. In the present study, we investigated the effects of a cannabinoid agonist on gastric cancer cell proliferation and invasion. The cannabinoid agonist WIN 55,212‐2 inhibited the proliferation of human gastric cancer cells in a dose‐dependent manner and that this effect was mediated partially by the CB₁ receptor. We also found that WIN 55,212‐2 induced apoptosis and down‐regulation of the phospho‐AKT expression in human gastric cancer cells. Furthermore, WIN 55,212‐2 treatment inhibited the invasion of gastric cancer cells, and down‐regulated the expression of MMP‐2 and VEGF‐A through the cannabinoid receptors. Our results open the possibilities in using cannabinoids as a new gastric cancer therapy. J. Cell. Biochem. 110: 321–332, 2010. © 2010 Wiley‐Liss, Inc.     

lncRNA MEG3 modified epithelial-mesenchymal transition of ovarian cancer cells by sponging miR-219a-5p and regulating EGFR

This study was aimed to verify whether there existed any associations between long noncoding RNA MEG3/miR-219a-5p/EGFR axis and the development of ovarian cancer (OC). As a whole, we gathered 317 pairs of OC tissues and surgical marginal normal tissues and simultaneously acquired four OC cell lines (ie, A2780, Caov-3, OVCAR-3, and SKOV-3) and human normal ovarian surface epithelial cell line. Moreover, pcDNA3.1-MEG3, si-MEG3, miR-219a-5p mimic, miR-219a-5p inhibitor, pcDNA3.1-EGFR, and si-EGFR were, respectively, transfected into the OC cells, and their impacts on viability, proliferation, apoptosis, invasion, and migration of OC cells were assessed via conduction of MTT assay, colony formation assay, flow cytometry assay, transwell assay, and scratch assay. Ultimately, dual-luciferase reporter gene assay was performed to testify the targeted relationships among maternally expressed gene 3 (MEG3), miR-219a-5p, and estimated glomerular filtration rate (EGFR). It was indicated that underexpressed MEG3 and miR-219a-5p were significantly associated with unfavorable prognosis of patients with OC when compared with overexpressed MEG3 and miR-219a-5p (P < .05). In addition, the OC cells transfected with si-MEG3 or miR-219a-5p inhibitor exhibited stronger viability, proliferation, invasion, and migration than untreated cells (P < .05). Correspondingly, the apoptotic percentage of OC cells was reduced observably under treatments of si-MEG3 and miR-219a-5p inhibitor (P < .05). Moreover, MEG3 exerted modulatory effects on the expression of miR-219a-5p (P < .05), and there was a sponging relationship between them (P < .05). Finally, EGFR expression was modified by both MEG3 and miR-219a-5p significantly (P < .05), and raising EGFR expression could changeover the impacts of MEG3 and miR-219a-5p on the above-mentioned activity of OC cells (P < .05). Conclusively, MEG3 could serve as a promising biomarker for diagnosis and treatment of OC, considering its involvement with OC etiology via regulation of miR-219a-5p/EGFR axis.