花姜酮对胰腺癌细胞迁移和侵袭的影响及机制的研究

目的:探讨花姜酮(zerumbone)对人胰腺癌细胞株BxPC-3和Panc-1迁移和侵袭的影响及其相关基因的表达。方法:以BxPC-3和Panc-1为研究对象,通过四甲基偶氮唑盐(MTT)实验选择毒性较小的花姜酮浓度,用细胞划痕实验、Transwell小室迁移和侵袭实验检测药物对细胞迁移和侵袭能力的影响,蛋白质印迹(Western blot)检测各细胞组CXC族趋化因子受体4(CXCR4)、促分裂原活化蛋白酶激酶1/2(MEK1/2)、磷酸化MEK 1/2(p-MEK1/2)、细胞外调节激酶1/2(ERK1/2)、磷酸化ERK1/2(p-ERK1/2)蛋白表达变化。结果:花姜酮对BxPC-3和Panc-1细胞的生长均有抑制作用,且随着药物浓度的增高和作用时间的延长而增强(P<0.05)。低浓度药物作用后BxPC-3和Panc-1的划痕迁移率都低于对照组(P<0.05)。与对照组相比,Transwell迁移和侵袭实验中BxPC-3和Panc-1用药组的平均穿膜细胞数均减少(P     

蛋白激酶C对大肠癌细胞转移特性调控的实验研究

目的:探讨蛋白激酶C(protein kinase C.PKC)对大肠癌细胞转移特性的调控作用。方法:采用羊膜侵袭培养系统和明胶酶谱分析的方法,研究了PKC激活剂PMA对人结肠癌细胞株HT-29体外侵袭作用的影响及PKC抑制剂Staurosporine(sP)对PMA的拮抗作用,以及研究了这种体外的侵袭作用与细胞分泌Ⅳ型胶原酶,包括72kDⅣ型胶原酶(MMP-2)和92kDⅣ型胶原酶(MMP-9)的关系。结果:PMA可显著增强HT-29细胞的侵袭性,与对照组相比,具有显著性差异(P<0.01).而SP则可拮抗PMA的这种诱导作用。PMA还可增加HT-29细胞分泌MMP-2和MMP-9,而SP则可拮抗PMA的这种诱导作用,抑制MMP-2和MMP-9的分泌。结论:蛋白激酶C可能是调控肿瘤细胞侵袭、转移的重要因素。PKC的激活可诱导肿瘤细胞侵袭性增强和分泌MMP-2和MMP-9增加,SP可能通过抑制PMA对PKC的激活,进而抑制了肿瘤细胞的侵袭和分泌MMP-2和MMP-9。MMP-2和MMP-9的分沁与肿瘤细胞侵袭性有密切关系。     

普鲁卡因通过miR-15a-5p/PI3K-AKT3途径调控乳腺癌细胞增殖、迁移和侵袭的机制

目的探讨普鲁卡因对乳腺癌细胞MCF-7增殖、迁移和侵袭的影响及其作用机制。方法以不同浓度(0.5、1.0、2.5、5.0、10.0 mmol/L)普鲁卡因处理MCF-7细胞,噻唑蓝(MTT)法检测细胞增殖;5.0 mmol/L普鲁卡因处理MCF-7细胞48 h后,Transwell法检测迁移和侵袭,实时荧光定量-聚合酶链反应(qRT-PCR)检测miR-15a-5p的表达。转染miR-15a-5p mimic至MCF-7细胞,检测细胞增殖、迁移和侵袭。靶基因预测软件预测miR-15a-5p和AKT3靶向结合位点,双荧光素酶报告基因实验检测二者靶向关系。转染miR-15a-5p inhibitor至MCF-7细胞,并用5.0 mmol/L普鲁卡因处理48 h,检测细胞增殖、迁移和侵袭,Western印迹检测蛋白激酶B(AKT)3的表达。结果与Con组相比,普鲁卡因能明显抑制MCF-7细胞增殖、迁移和侵袭(P<0.001),促进细胞中miR-15a-5p的表达(P<0.01)。过表达miR-15a-5p可抑制MCF-7细胞增殖、迁移和侵袭(P<0.001,P<0.01,P<0.05)。双荧光素酶报告基因实验证实miR-15a-5p和AKT3的靶向结合有关。抑制miR-15a-5p表达减弱了普鲁卡因对MCF-7细胞增殖、迁移、侵袭及AKT3表达的抑制作用(P<0.05)。结论普鲁卡因能够抑制乳腺癌细胞MCF-7的增殖、迁移和侵袭能力,其机制可能与调控miR-15a-5p/磷脂酰肌激-3-激酶(PI3K)-AKT3途径有关。     

丹参酚酸B靶向SOX2OT/miRNA-34a/SOX2轴抑制大肠癌细胞侵袭转移

目的:验证SOX2OT/miRNA(miR)-34a/SOX2信号轴对人大肠癌LOVO细胞侵袭转移的影响以及丹参酚酸B(SalB)通过该信号轴浓度依赖性抑制LOVO细胞侵袭转移.方法:双荧光素酶报告基因检测SOX2OT与miR-34a、miR-34a与SOX2的靶向结合;瞬时转染SOX2OT质粒和miR-34a mim...     

黄连素调节非小细胞肺癌细胞糖酵解水平并抑制A549细胞EMT的发生

目的本研究旨在探讨黄连素(berberine,BBR)在抑制肺癌侵袭转移中的应用价值并探索相关机制。方法 CCK8细胞活性检测法检测不同浓度的BBR对A549细胞增殖活性的影响;迁移和侵袭实验检测BBR对A549细胞侵袭和迁移能力的影响;Western blot检测BBR对A549细胞间质转化(epithelial-mesenchymal transition, EMT)相关蛋白表达的影响;代谢通量测定检查BBR对A549细胞糖酵解水平的调节作用;PCR检测BBR对糖酵解相关酶HK1、HK2、LDHA基因表达的影响;使用重组HK2,研究激活糖酵解进程对BBR抑制EMT进程的影响。结果低剂量BBR(8μm/L)轻微上调细胞增殖活性,高浓度BBR(64 um/L)下调细胞增殖活性;高剂量BBR能够抑制A549细胞发生EMT,减弱癌细胞的侵袭和迁移能力;BBR通过下调HK2表达,抑制A549糖酵解水平;BBR抑制A549细胞侵袭和迁移能力是通过调节糖酵解水平实现的。结论高浓度BBR下调A549细胞内HK2的表达进而调节肺癌细胞糖酵解;BBR抑制A549细胞侵袭和迁移能力可能与抑制糖酵解活性有关。     

胰腺星状细胞对胰腺癌AsPC-1细胞侵袭转移的影响

目的 探讨胰腺星状细胞(PSCs)对胰腺癌细胞侵袭转移的影响及基质细胞因子-I(SDF-1)在此过程中的作用.方法 常规分离、培养PSCs,收集及浓缩PSCs条件培养液(PSC-CM),应用不同浓度的PSC-CM、抗SDF-1抗体(anti-SDF-1)及两者联合应用处理AsPC-1细胞,采用MTT法检测AsPC-1细胞的增殖,Transwell小室检测细胞迁移,体外侵袭实验观察细胞侵袭能力.结果 对照组及0.25、0.5、1 μg/μl PSC-CM组AsPC-1细胞增殖的吸光度值(A490)分别为0.437 ±0.041、0.472±0.048、0.553±0.057、0.690±0.051,PSC-CM呈剂量依赖性促进细胞增殖,其中0.5、1μg/μl PSC-CM组与对照组间以及0.5 μg/μl与1μg/μl PSC-CM组间的差异具有统计学意义(P值均＜0.05).对照组、anti-SDF-1组、PSC-CM组及PSC-CM± anti-SDF-1组细胞增殖的A490值分别为0.407±0.028、0.416±0.030、0.629±0.048、0.481±0.049;穿膜细胞数分别为(35.3±7.1)、(34.8±5.6)、(140.9±12.7)、(56.5±5.9)个;侵袭细胞数分别为(27.1±2.9)、(29.1±4.2)、(81.5±8.2)、(46.4±4.4)个.anti-SDF-1组与对照组间的差异无统计学意义,PSC-CM组细胞的增殖、迁移、侵袭能力较对照组显著增强(P＜0.05或P＜0.01),PSC-CM± anti-SDF-1组细胞的增殖、迁移、侵袭能力较PSC-CM组显著降低,但仍显著高于对照组(P＜0.05或P＜0.01).结论 PSC-CM可促进AsPC-1的增殖、迁移及侵袭,其机制为部分通过SDF-1/CXCR4受体配体系统.     

肝癌患者外周血Th17/Treg细胞和相关细胞因子的表达及IL-17对肝癌细胞侵袭的影响

目的探讨肝癌患者体内辅助性T细胞(Th)17/调节性T细胞(Treg)比例和相关细胞因子的表达水平及白细胞介素(IL)-17影响肝癌细胞侵袭的机制。方法选取在该院就诊的40例肝癌患者为观察组,同期在医院进行体检的40例健康人为对照组。采用流式细胞术检测两组人群外周血Th17和Treg细胞比例,酶联免疫吸附(ELISA)法检测两组外周血中IL-17、IL-10、IL-6和转化生长因子(TGF)-β的表达水平。在人肝癌细胞系SMMC7721中瞬时转染IL-17 siRNA及阴性对照,并采用实时荧光定量PCR(qRT-PCR)、ELISA及免疫印迹(Western blot)法检测沉默后IL-17 mRNA及蛋白水平的表达变化,然后采用Transwell法检测侵袭变化,qRT-PCR和Western印迹检测MMP-2、MMP-9表达水平。结果与对照组比较,观察组外周血中Th17细胞和Treg细胞上升,Th17/Treg比例下降(P<0.05);观察组外周血中IL-17、IL-10、IL-6和TGF-β表达水平均高于对照组(P<0.05)。与阴性对照比较,SMMC7721转染IL-17 siRNA后,IL-17表达水平显著下降(P<0.05),干扰IL-17后SMMC7721细胞侵袭能力显著下降(P<0.05),MMP-2和MMP-9表达水平明显降低(P<0.05)。结论 Th17/Treg在肝癌患者外周血中存在失衡现象,IL-17、IL-10、IL-6和TGF-β等细胞因子在肝癌患者外周血中高表达,沉默IL-17可抑制肝癌细胞侵袭并抑制MMP-2和MMP-9表达,IL-17可能通过调控MMP-2和MMP-9参与调节肝癌细胞的侵袭。     

麝黄消瘤方对小鼠肝癌血管生成的抑制作用

目的:观察麝黄消瘤方(SHXLF)对肝癌H22模型小鼠癌细胞侵袭和转移的影响,探讨其作用机制。方法:以小鼠H22肝癌淋巴道转移模型为对象,观察其淋巴结指数、脾指数、胸腺指数,光镜下观察肝癌H22细胞局部侵袭和淋巴结转移程度。免疫组化SP法观察局部瘤组织血管内皮细胞生长因子(VEGF)表达水平和微血管密度(MVD)。结果:麝黄消瘤方可使局部瘤组织的侵袭和腘窝淋巴结转移程度及淋巴结指数有降低趋势;大中剂量组可使胸腺指数明显升高(P<0.05)。中药各治疗组可明显降低肝癌组织VEGF表达水平和MVD(P<0.05)。结论:SHXLF可抑制H22肝癌小鼠癌组织在局部及淋巴结的侵袭转移程度。其作用机制可能是通过调整免疫功能,杀伤癌细胞,抑制肿瘤血管生成(降低VEGF和MVD)而实现的。     

Cdc42基因在表皮生长因子促进HepG2细胞丝状伪足形成中的作用

目的:探讨表皮生长因子(EGF)对HepG2细胞丝状伪足形成和细胞体外侵袭能力的影响以及细胞分裂周期蛋白42(Cdc42)在其中的作用.方法:将Cdc42 siRNA转染HepG2细胞株,RT-PCR和Western blot检测EGF组、EGF+siRNA干扰组、siRNA干扰组及空白对照组细胞Cdc42 mRNA转录以及蛋白的表达水平,微丝绿色荧光探针(actin-tracker green)染色检测HepG2细胞丝状伪足形成,侵袭小室检测细胞体外侵袭能力.结果:经EGF处理后HepG2细胞可见明显丝状伪足形成,EGF组细胞Cdc42 mRNA及总蛋白(Total-Cdc42)表达量无显著变化,但活性Cdc42(Active-Cdc42)显著增高(0.713±0.021vs0.423±0.015,P<0.05),siRNA干扰细Cdc42表达及活性受到明显抑制(0.118±0.017 vs 1.128±0.024;0.351±0.021 vs 0.936±0.024,均P<0.05),并抑制EGF诱导的细胞丝状伪足形成,EGF组细胞体外侵袭能力显著高于其余各组(98.43+3.11vs61,09±3.58,50.53±2.34,62.73±2.64,均P<0.05).结论:EGF通过激活Cdc42诱导HepG2细胞丝状伪足形成并增强其体外侵袭能力.     

吴茱萸碱对肝癌HepG2细胞侵袭和迁徙的影响

目的:研究吴茱萸碱对人肝癌细胞Hep G2细胞外调节蛋白激酶(extracellular-regulated protein kinases 1/2,ERK1/2)表达的影响.方法:Transwell小室检测吴茱萸碱对Hep G2细胞侵袭及迁徙的影响.集落形成实验检测吴茱萸碱对Hep G2细胞集落形成能力的影响.Western blot检测Hep G2细胞ERK1/2、phospho-ERK1/2(P-ERK1/2)蛋白.结果:吴茱萸碱5、25、50μmol/L作用后,H e p G 2细胞侵袭细胞数分别为3 2.3个±1.3个、22.3个±2.5个、10.6个±3.7个,对照组为97.6个±6.3个,差异有统计学意义(P<0.05);迁徙细胞数为57.7个±3.2个、26.8个±1.6个、15.4个±3.9个,对照组为106.3个±3.2个,差异有统计学意义(P<0.05).5、25、50μmol/L吴茱萸碱作用Hep G2细胞集落形成数分别为147.0个±12.1个、94.3个±4.0个、0个,对照组为231.3个±15.4个,差异有统计学意义(P<0.05).Western blot分析5、25、50μmol/L吴茱萸碱处理Hep G2细胞24 h及30μmol/L吴茱萸碱处理Hep G2细胞24、48 h后,ERK1/2蛋白的表达无明显变化,但其活化形式P-ERK1/2蛋白的表达量减少.结论:吴茱萸碱可显著抑制人肝癌Hep G2细胞侵袭和迁徙及克隆增殖能力,其机制可能与吴茱萸碱抑制ERK1/2蛋白活化有关.     

微小RNA-190低表达调控肺癌A549细胞增殖、侵袭和凋亡的实验研究

目的 探讨微小RNA(miR)-190对肺癌A549细胞增殖、侵袭和凋亡的影响及其机制.方法 将体外培养的A549细胞分为对照(Ctrl)组、抑制剂阴性对照(anti-miR-NC)组和miR-190抑制剂(anti-miR-190)组,转染miR-190抑制剂及其阴性对照后,采用实时荧光定量PCR检测检测A549细胞...     

miR-539通过靶向调控DCLK1抑制非小细胞肺癌的侵袭能力

目的探讨非小细胞肺癌组织中miR-539的表达水平及其抑制肺癌细胞侵袭的作用机制。方法通过基因表达数据库dbDEMC分析miR-539在非小细胞肺癌组织及正常肺组织中的表达量差异。根据235例miR-539高表达的非小细胞肺癌患者和102例miR-539低表达的非小细胞肺癌患者随访资料,分析miR-539与非小细胞肺癌患者预后的相关性。利用生物信息预测结合双荧光素酶报告基因实验研究miR-539的潜在靶基因。在肺癌A549细胞中分别过表达miR-539、转染miR-539抑制剂,利用Transwell法检测肺癌A549细胞的侵袭能力。结果dbDEMC数据库资料显示miR-539在非小细胞肺癌组织中的表达量显著低于其在正常肺组织中的表达量(P=3.10×10-4)。生存分析发现miR-539的表达量与肺癌患者预后正相关(P=0.033)。通过在线生物信息学预测结合双荧光素报告实验发现miR-539在转录水平减少DCLK1的表达。Transwell侵袭实验发现,过表达miR-539可以抑制肺癌A549细胞的侵袭能力,而沉默miR-539可以增加A549细胞的侵袭能力。结论miR-539是肺癌发生中的抑癌基因,可能是通过靶向沉默DCLK1的表达,而抑制肺癌细胞A549的侵袭能力。     

miRNA-126靶向抑制A549细胞迁移和侵袭能力的研究

目的研究microRNA-126（miR-126）对非小细胞肺癌（NSCLC）细胞迁移和侵袭能力的影响。方法利用脂质体介导法将十勾建的miR126过表达质粒转染至NSCI．C细胞株A549细胞（A549／miR-126组）,并设空质粒转染组（A549／MOCK组）和空白对照组（A549组）。利用RT-PCR技术检测3组细胞中EGFI。7（miR126的靶标）的表达水平,采川细胞划痕实验观察细胞迁移能力差异,采用Transwell小室法分析3组细胞侵袭能力的差异。结果RT—PCR检测A549／miR-126组、A549／MOCK组和A549组细胞中EGFI。7mRNA分别为2．32±0．088、1．43±0．026和1．00±0．000,差异有统计学意义（P〈0．01）;细胞划痕实验显示A549／miR—126组、A549jM（）CK组和A549组细胞平均迁移距离分别为3．0μm、2．65μm和0．5μm,平均抑制率分别为0、11．25％和83．75％,差异有统计学意义（P〈O．05）;Transwell小室实验显示,A549／miR126组24hr侵袭细胞数为（28．6±2．322）个,36h侵袭细胞数为（29．2±3．7683）个,A549／M（）CK组24h为（49．8±3．7014）个,差异有统计学意义（P〈0．05）。结论miR-126可上调EGFL7mRNA的表达,并可能通过表达产物EGFI。7蛋白抑制A549细胞的迁移和侵袭能力。     

Depletion of circ_0088046 suppressed cell growth and motility of hepatocellular carcinoma via circ_0088046-miR-1299-RTKN2 ceRNA pathway

Circular RNAs (circRNAs) have been verified to be important modulators and therapeutic targets of human hepatocellular carcinoma (HCC). This study aims to explore the role and mechanism of circ_0088046 in HCC progression. Quantitative real-time polymerase chain reaction (qRT-PCR), western blot and immunohistochemistry assays were used to detect the mRNA and protein expression of circ_0088046, miR-1299, Rhotekin 2 (RTKN2), Bax, Bcl-2, E-cadherin and Ki-67. Cell proliferation was investigated by 5-Ethynyl-2′-deoxyuridine (EdU) assay and cell colony formation assay. Cell apoptosis rate was measured by flow cytometry. Transwell migration and invasion assays were adopted to assess cell migration and invasion. The molecular target relationship between miR-1299 and circ_0088046 or RTKN2 were analysed by dual-luciferase reporter assay and RNA immunoprecipitation assay. An animal experiment was conducted to demonstrate the effect of circ_0088046 on tumour formation in vivo. High levels of circ_0088046 and RTKN2, and low levels of miR-1299 were displayed in HCC tissues and cells. Circ_0088046 absence repressed cell proliferation, migration and invasion, but boosted apoptosis of HCC cells. MiR-1299 was a target of circ_0088046 and miR-1299 inhibitor restored circ_0088046 silencing-mediated inhibitory impacts on HCC cell malignancy. MiR-1299 could directly target RTKN2, and overexpressed RTKN2 rescued the suppressive effects caused by miR-1299 mimic. In addition, circ_0088046 silencing constrained tumour formation in vivo. Circ_0088046 contributed to HCC cell malignancy via modulating the miR-1299/RTKN2 axis.     

Down-Regulation of PTEN Expression Modulated by Dysregulated miR-21 Contributes to the Progression of Esophageal Cancer

Background and Aim

miR-21, a putative tumor oncomiR, is a frequently overexpressed miRNA in a variety of tumors. Because it targets tumor-suppressor genes it has been linked to tumor progression. In this study we investigated the role of miR-21 in esophageal squamous cell carcinoma (ESCC), and its possible mechanism.

Methods

Expression of miR-21 was detected by stem–loop RT-PCR in tissue from 76 invasive ESCC at stage I–IV and in their corresponding para-cancerous histological normal tissues (PCHNT). Thirty endoscopic esophageal mucosal biopsy specimens from non-tumor patients were used as controls. Expression of PTEN in 76 paired ESCC and PCHNT was investigated by real-time RT-PCR and an immunohistochemical method, respectively. Paired tumor and PCHNT specimens of 20 ESCC cases were randomly selected for western blot analysis. The effect of miR-21 on PTEN expression was assessed in the ESCC cell line with an miR-21 inhibitor to reduce miR-21 expression. Furthermore, the roles of miR-21 in cell biology were analyzed by use of miR-21 inhibitor-transfected cells.

Results

Stem–loop RT-PCR revealed miR-21 was significantly overexpressed in ESCC tissues and cell lines. Overexpression of miR-21 correlated with tumor status, lymph node metastasis, and clinical stage. We demonstrated that knockdown of miR-21 significantly increased expression of PTEN protein. Consequent PTEN expression reduced cell proliferation, invasion, and migration.

Conclusions

Our findings suggest that miR-21 could be a potential oncomiR, probably by regulation of PTEN, and a novel prognostic factor for ESCC patients.     

长链非编码RNA GIHCG对非小细胞肺癌恶性表型的影响

目的探讨长链非编码RNA GIHCG(lncRNA GIHCG)对非小细胞肺癌(NSCLC)恶性表型的影响。 方法使用qRT-PCR法分别检测50例NSCLC的肿瘤组织及其癌旁组织中lncRNA GIHCG及正常人支气管上皮细胞系E16HB,NSCLC细胞系A549、H1299、H1650、H2087中lncRNA GIHCG的表达。分别使用小干扰RNA(siRNA-1、siRNA-2)及对照转染A549和H1299细胞,实验设siRNA-1转染组、siRNA-2转染组、阴性对照组和空白对照组。各组转染48 h后采用CCK-8实验检测细胞增殖活性,Transwell实验检测细胞侵袭能力,流式细胞术检测各组细胞周期变化。 结果qRT-PCR结果显示,lncRNA GIHCG在肺癌组织中的表达水平明显高于癌旁组织,同时非小细胞肺癌细胞系A549、H1299、H1650、H2087中lncRNA GIHCG的表达均高于16HBE细胞,差异有统计学意义(P<0.05)。转染48 h后,与空白对照组和阴性对照相比,siRNA-1组和siRNA-2组细胞存活率明显下降,穿膜细胞数明显减少,G0/G1期细胞比例明显增加,S期细胞减少,差异均有统计学意义,而阴性对照组和空白对照组上述指标差异均无统计学意义。 结论lncRNA GIHCG高表达于NSCLC组织及细胞系中,沉默lncRNA GIHCG表达可明显抑制NSCLC恶性生物学表型。     

Trichosanthin inhibits cell growth and metastasis by promoting pyroptosis in non-small cell lung cancer

BackgroundA number of studies have demonstrated that trichosanthin (TCS) can induce apoptosis in numerous types of tumor cell lines. However, whether TCS can induce pyroptosis has not yet been reported. This study aimed to investigate the role of TCS and its inhibitory effect on tumor growth by modulating pyroptosis in non-small cell lung cancer (NSCLC).MethodsEffects of different concentrations of TCS on the cell viability, proliferation, migration and invasion of NSCLC were detected by Cell Counting Kit-8 (CCK-8), colony formation, migration, and invasion assays. Immunofluorescence was used to detect the effect of TCS on the expression of pyroptosis marker protein gasdermin-D (GSDMD)-N in A549 cells. A tumor xenograft animal model was established by injecting A549 cells into nude mice.ResultsIn the present study, we found that TCS significantly inhibited the proliferation, migration, and invasion of A549 cells in a concentration-dependent manner. In addition, TCS at a high concentration (40 µg/mL) significantly promoted the expression of pyroptosis-related proteins [GSDMD-N, NLR family pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, and GSDMD], which showed an inhibitory effect on the pyroptosis of A549 cells. Additionally, we found that necrosulfonamide (NSA) significantly reversed the inhibitory effect of high concentrations of TCS on the pyroptosis of A549 cells. The in vivo experiments showed that TCS effectively reduced the tumor volume and inhibited the expression of Ki-67, whereas it increased the expression of GSDMD-N.ConclusionsTaken together, these results indicated that TCS could inhibit the progression of NSCLC by promoting pyroptosis. These findings provide further information on the possible underlying mechanism of TCS in the treatment of NSCLC.