Lentivirus vectors construction of SiRNA targeting interference GPC3 gene and its biological effects on liver cancer cell lines Huh-7

Objective:To build GPC3 gene short hairpin interference RNA(shRNA)slow virus veclor.observe expression of Huh-7 GPC3 gene in human liver cell line proliferation apoptosis and the effect of GPC3 gene influencing on liver cancer cell growth,and provide theoretical basis for genc therapy of liver cancer.Methods:Hepatocellular carcinoma cell line Huh-7 wsa transfected by a RNA interference technique.GPC3 gene expression in a variety of liver cancer cell lines was detected by fluorescence quantitative PCR.Targeted GPC3 gene seqnences of small interfering RNA(siRNA)PGC-shRNA-GPC3 were restructured.Stable expression cell linse of siRNA were screened and established with the heplp of liposomes(lipofectamine^(TM2000))as carrier transfcetion of human liver cell lines.In order to validate siRNA interference efficiency.GPC3 siRNA mRNA expression was detected after transfection by using RT-PCR and Western blot.The absorbance value of the cells of blank group,untransfection group and transfection group,the cell cycle and cell apoptosis were calculated,and effects of GPC3 gene nn Huh-7 cell proliferation and apoptosis were observed.Results:In the liver cancer cell lines Huh-7 GPC3 gene showed high expression.PGC-shRNA-GPC3 recombinant plasmid was constructde successfully via sequencing validation.Stable recombinant plasmid transfected into liver cancer cell linse Huh-7can obviously inhibit GPC3 mRNA expression level.Conclusions:The targeted GPC3 siRNA can effectively inhibit the expression of GPC3.

Potential roles of EZH2, Bmi-1 and mi R-203 in cell proliferation and invasion in hepatocellular carcinoma cell line Hep3B

AIM: To investigate the potential roles of enhancer of zeste homolog2(EZH2), Bmi-1 and mi R-203 in cell proliferation and invasion in hepatocellular carcinoma(HCC) cell line Hep3 B.METHODS: A total of 73 patients who underwent surgical resection at Fuzong Clinical Medical College of Fujian Medical University were enrolled in this study. Hep3 B cells were cultivated in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37?℃. Vectors that containing c DNA of the EZH2 gene or mi R-203 targeted sh RNA plasmid were constructed, and then transfected into Hep3 B cells. The m RNA expression of mi R-203, EZH2, and Bmi-1 was analyzed using quantitative real-time polymerase chain reaction analysis, and the protein levels of EZH2 and Bmi-1 were detected by Western blot analysis. Effect of EZH2 or mi R-203 on cell proliferation was observed by methyl thiazolyl tetrazolium assay, and cell apoptosis was assessed using flow cytometry. Besides, effect of EZH2 or mi R-203 on tumor cell invasion was detected using Transwell assay.RESULTS: The m RNA levels of EZH2 and Bmi-1 in HCC tissues and in Hep3 B cells were significantly higher compared with those in normal samples(P < 0.01), while mi R-203 level was significantly lower in HCC tissues(P < 0.01). Hep3 B cells transfected with EZH2-sh RNA or mi R-203-sh RNA showed lower expression levels of EZH2 and Bmi-1(P < 0.05). Compared with controls, Hep3 B cells transfected with EZH2-sh RNA had relative slow cell proliferation, indicating that low expression of EZH2 and Bmi-1 and overexpression of mi R-203 could inhibit Hep3 B cell proliferation(P < 0.05). The average apoptosis rate of Hep3 B cells transfected with EZH2-sh RNA vector was about 18.631%, while that of Hep3 B cells transfected with sh RNA vector was about 5.33%, suggesting that EZH2 was down-regulated by transfecting with EZH2-sh RNA, and the down-regulated EZH2 contributed to the cell apoptosis. Low expression of EZH2 and Bmi-1 and overexpression of mi R-203 could reduce Hep3 B cell invasion(P < 0.05).CONCLUSION: Our study suggests that EZH2 and Bmi-1 are up-regulated while mi R-203 is downregulated in Hep3 B cells. Mi R-203 may contribute to the metastasis and enhance apoptosis of HCC cells by regulating EZH2 and Bmi-1. Our study may provide a theoretical basis for metastasis of HCC and targeted therapy of HCC.

白藜芦醇通过介导GAL-3表达促进卵巢癌细胞凋亡并增强其化疗感性

目的 探讨白藜芦醇(RES)对卵巢癌细胞凋亡和化疗敏感性的影响及其作用机制。方法研究样本为卵巢癌SKOV3细胞,分为3组,每组11株,均进行常规培养,对照组不给予干预,顺铂组给予10μmol/L顺铂干预,RES+顺铂组给予50μmol/L RES+10μmol/L顺铂干预。采用CCK-8实验检测细胞活力,BrdU实验检测细胞增殖,Annexin V-FITC/PI双染细胞凋亡试验检测细胞凋亡,比色测定试验检测caspase-3活性,Transwell细胞迁移实验检测细胞迁移能力,侵袭试验检测细胞侵袭能力,蛋白质印迹分析caspase-3、GAL-3、p-Akt、AKT、Iκβα、P65、Bcl-2、 p-IKKα/β蛋白相对表达。结果 RES+顺铂组、顺铂组、对照组细胞活力分别为(62±14)%、(74±16)%、(98±16)%,细胞增殖率分别为(59±16)%、(76±17)%、(97±17)%,RES+顺铂组、顺铂组细胞活力、细胞增殖率显著低于对照组,RES+顺铂组显著低于顺铂组(P<0.05);RES+顺铂组、顺铂组、对照组细胞凋亡率分别为(64.29±13.14)%、(47.48±9.52)%、(14.72±3.03)%,RES+顺铂组、顺铂组细胞凋亡率显著高于对照组,RES+顺铂组显著高于顺铂组(P<0.05);RES+顺铂组caspase-3活性、蛋白相对表达显著高于顺铂组、对照组(P<0.05);RES+顺铂组、顺铂组细胞迁移量、细胞侵袭量显著低于对照组,RES+顺铂组显著低于顺铂组(P<0.05);RES+顺铂组GAL-3、p-Akt蛋白相对表达显著低于顺铂组、对照组(P<0.05),各组AKT蛋白表达差异无统计学意义(P<0.05);RES+顺铂组、顺铂组p-IKKα/β、P65、Bcl-2、蛋白相对表达显著低于对照组,RES+顺铂组显著低于顺铂组(P<0.05);RES+顺铂组、顺铂组Iκβα蛋白相对表达显著高于对照组,RES+顺铂组显著高于顺铂组(P<0.05)。结论 RES诱导癌细胞凋亡可能与GAL-3水平降低有关。其作用机制可能是通过调节卵巢癌细胞凋亡、转移相关蛋白表达抑制其增殖、侵袭并增强其对顺铂的敏感性。

MiR-25-3p attenuates the proliferation of tongue squamous cell carcinoma cell line Tca8113

Objective:To investigate the effects of miR-25-3p on the occurrence,development and proliferation of tongue squamous cell carcinoma cells.Methods:To establish tongue squamous cell carcinoma cell line Tca8113 that stably and highly express miR-25-3p using recombinant reiroviral vector-mediated gene transfer method.The proliferation of transfected Tca8113 was detected by thiazolyl blue tetrazolium bromide(MTT)and cell colony formation assays.eyclnD1,p21^(cipt)and p27^(kipt)mRNA expressions in the transfected Tca-8113 were detected by quantitative PCR.cyclinD1,p21^(cipt),p27^(kipt),AKT,p-AKT,FOXOt and p-FOX01 expressions in the transfected Tca8113 were detected by western blot analysis.In addition,miR-25-3p expression in the tongue squamous cell carcinoma cell line and tissue specimen was also detected by quantitative PCR.Results:Quantitative PCR showed that mitt-25-3p expression in the tongue squamous cell carcinoma cell lines and tissue specimen was significantly lower than that in the adjacent tissue.MTT and cell colony formation assays showed that after miR-25-3p overexpression,the proliferation of transfected Tca8113 was obviously attenuated.Western blot analysis and quantitative PCR showed that after miR-25-3p overexpression.p21^(cipt)and p27^(kipt)expressions were upregulated,while cyclinD1,AKT,FOXO1 expressions were downregulated,and AKT and FOXO1 phosphorylation was inactivated in the transfected Tca8113 cells.Conclusions:MiR-25-3p inhibited the proliferation of tongue squamous cell carcinoma cells and regulated cell cycle-related protein expression,playing an important role in the occurrence and development of squamous cell carcinoma of the tongue.

Contragestazol (DL111-IT) inhibits proliferation of human androgen-independent prostate cancer cell line PC3 in vitro and in vivo

Aim： To evaluate the antiproliferative activity of contragestazol （DL111-IT） on the human prostate cancer cell line PC3 in vitro and in vivo and to elucidate its potential molecular mechanisms. Methods： The cell killing ability of DL111-IT was measured by the 3-（4,5-dimethylthia-zol,2-yl）-2,5-diphenyltetrazolium bromide （MTT） reagent assay method and the tumor xenograft model. The cell cycle was analyzed by flow cytometry and protein expression, including retinoblastoma （pRb）, cyclin-dependent kinase 4 （CDK4） and cyclin D 1, was detected by Western blotting. Results： DL111-IT exhibited high efficiency on cell growth inhibition of the human androgen-independent prostate cancer cell line PC3. The drug concentration that yielded 50 % cell inhibition （IC50 value） was 9.9 mg/mL. In the PC3 tumor xenograft study, DL111-IT （1.25 mg/kg-20.0 mg/kg） given once a day for 10 days significantly inhibited tumor growth, with the inhibition rate ranging from 21% to 50 %. Flow cytometric analysis indicated that DL111-IT could cause GI arrest in the PC3 cell line, but not apoptosis. DL111-IT enhanced pRb expression and down-regulated CDK4 and cyclin D 1 expression, suggesting that cell cycle regulation might contribute to the anticancer property of DL 111- IT. Conclusion： DL111-1T inhibits the proliferation of human androgen-independent prostate cancer cell line PC3 in vitro and in vivo by a cell cycle regulation pathway.

The U937 cell line induced to express CD14 protein by 1,25-dihydroxyvitamin D3 and be sensitive to endotoxin stimulation

BACKGROUND: CD14 was first described as a differentia- tion antigen on the surface of myeloid lineage cells. It acts as a glycosylphosphatidylinositol ( GPI)-anchored receptor for the complex of lipopolysaccharide (LPS) and plays a key role in the activation of LPS-induced monocytes. The purpose of this study was to observe the expression of CD14 protein and its gene in the human U937 promonocytic cell line when these cells were exposed to 1,25-dihydroxyvita- min D3 ( VitD3 ) and investigate their sensitivity to endo- toxin stimulation. METHODS: U937 cells were exposed to (0.1 μmol) VitD3 for 24 hours and were induced to express the CD14 mRNA gene and CD14 protein, then their responses were observed when they were stimulated with different concentrations of LPS for different time. RESULTS: The U937 cells induced by VitD3 were found to stably express CD14 mRNA and CD14 protein. And CD14 protein enhanced the sensitivity of U937/CD14 cells to li- popolysaccharide ( LPS ) stimulation. NF-ΚB in U937/ CD14 cells can be activated with low concentration of LPS (1 ng/ml-10 ng/ml), the TNF-α mRNA gene was in- duced , and then TNF-α was produced and released into the supernatant of culture. CONCLUSION: VitD3 can induce U937 cell to express the CD14 gene and CD14 protein and enhance the response of this type of cells to LPS stimulation.

Inhibitory effect of a new gossypol derivative apogossypolone (ApoG2) on xenograft of human prostate cancer cell line PC-3

Objective: To investigate the inhibitory effect of apogossypolone (ApoG2) on prostate cancer cell line PC-3 in vivo, and explore its mechanism. Methods: The models of transplantation tumors in Balb/c nu/nu mice were established via subcutaneous injection of PC-3 cells and the tumor-transplanted mice were divided into 4 groups: control group and three ApoG2 treatment groups, with 10 mice in each group. Volumes of the tumor were estimated every 2 d and the morphology of tumor tissues was observed. Immunohistochemistry was employed to observe the expression of Bcl-2, PCNA, CD31, caspase-3 and caspase-8 in tumor tissues. Results: ApoG2 (2.5 mg/kg-10 mg/kg) given intraperitoneally once a day can obviously inhibit the growth of subcutaneous prostatic carcinoma implant. The tumor volume decreased obviously when the treatment dosage was bigger than 5.0 mg/kg (P<0.01). Meanwhile, ApoG2 decreased the expression of PCNA and CD31, and enhanced the expression of caspases-3, caspase-8 in tumor tissues. Conclusion: ApoG2 exert an inhibitory effect on prostatic carcinoma possibly by inducing apoptosis and inhibiting tumor angiogenesis.

THE DIFFERENTIATION OF HUMAN GASTRIC ADENOCAR-CINOMA CELL LINE MGc80-3 INDUCED BY DIBUTYRYL cAMP IN VITRO

For providing some experimental basis in establishing malignant phenotypic reversed indexes of gastric carcinoma cells, human gastric adenocar-cinoma cell line MGc80-3 was induced by dBcAMP in vitro to appraise the effect of gastric carcinoma cell differentiation by chemical inducers.Under light microscope, MGc80-3 cells, after treated with 1 mM dBcAMP, tended to be flat and disperse, and their volume gradually enlarged, with their uncleus relatively smaller and their shape rather regular. Morphological changes, like norma differentiated epithelial cells, were observed. The cells attached firmly, grew slowly, their growth curve showed inhibitory rate amounted to 52.87%, and cellular division exponent displayed their peak value 1.5 times less than that of MGc80-3 cells. It was clear that dBcAMP could effectively inhibit the multiplication activity of MGc80-3 cells. After dBcAMP treatment, remarkable changes of cell surface charges was indicated by cell electrophoresis, the ratio dropped to 3.043 from 3.988, and their re-tardant ratio reached up to 31.2%. cAMP content in cells after this treatment, detected by cAMP and cGMP radioimmunoassay, was enhanced by 2.42 times, and cAMP/cGMP ratio, by 1.73 times. Thus, cAMP level within MGc80-3 cells was raised obviously by dBcAMP. Heterotransplantation experiments showed that tuntorigenic rate of MGc80-5 cells (transplanted subcutaneously to BALB/c mice) amounted to 100%, and that of the cells after this treatment was only 5.6%. Their tumorigenic ability was extremely reduced.These results confirmed that dBcAMP was able to change malignant phenotypic characteristics of MGc80-3 cells and produce a reversed alteration: Thus, it has a remarkable inductive effect in differentiating gastric carcinoma cells. All these characteristics were also considered as the reference indexes in appraising reversed effect for the homologous cancer cells.

Effects of 4-(3-Chloro-Benzyl)-6,7-Dimethoxy-Quinazoline on Kinetics of P120-Catenin and Periplakin in Human Buccal Mucosa Squamous Carcinoma Cell Line

In order to detect molecular markers for the epidermal growth factor inhibitor 4-(3-chloro-benzyl)- 6,7-dimethoxy-quinazoline (tyrphostin), we investigated the kinetics of p120-catenin and periplakin in the human buccal mucosa squamous cancer cell line BICR 10 treated with 3 nM tyrphostin. Growth of BICR 10 cells was inhibited by treatment with tyrphostin. Although changes were not observed in the expression of EGFR and p120-catenin, expression of Akt, Src and periplakin in BICR 10 treated with 3 nM tyrphostin tended to decrease. In addition, phosphorylation of EGFR, Akt and Src was inhibited by treatment with tyrphostin. On immunocytochemical staining, immunoreactions with phosphorylated EGFR, phosphorylated Akt and phosphorylated p120-catenin were weak in BICR 10 treated with tyrphostin. There was a slight immunocy to chemical reaction to periplakin in BICR 10 cells induced by tyrphostin. In conclusion, the decrease in phosphorylation in EGFR and p120-catenin by tyrphostin, following the decrease in Src or Akt phosphorylation, may inhibit expression of several growth factors associated with the proliferation and migration of cancer cells.

3-甲基腺嘌呤对转化生长因子-β诱导大鼠肝脏星形细胞株HSC-T6活化及自噬的影响观察

目的观察3-甲基腺嘌呤(3-MA)对转化生长因子-β(TGF-β)诱导的大鼠肝星形细胞株HSC-T6活化及自噬的影响。方法取对数生长期的HSC-T6细胞分为空白组、TGF-β+PBS组、TGF-β+3-MA组。TGF-β+3-MA组细胞加入浓度为2 ng/mL TGF-β培养72 h后加入3-MA(0.5 mg/mL)处理24 h,TGF-β+PBS组细胞加入浓度为2 ng/mL TGF-β培养72 h后加入等量PBS处理24 h,空白组加入等量PBS处理,采用实时荧光定量PCR法检测各组细胞活化标志物α-SMA、TGF-βmRNA和自噬标志物LC3、Beclin-1、Atg5 mRNA,采用Western blotting法检测各组细胞活化标志物α-SMA、TGF-β蛋白和自噬标志物LC3、Beclin-1、Atg5蛋白。结果TGF-β+PBS组、TGF-β+3-MA组细胞活化标志物α-SMA、TGF-βmRNA和蛋白均高于空白组,且TGF-β+3-MA组均低于TGF-β+PBS组(P均<0.05)。TGF-β+PBS组、TGF-β+3-MA组细胞自噬标志物LC3、Beclin-1、Atg5 mRNA和蛋白均高于空白组,且TGF-β+3-MA组均低于TGF-β+PBS组(P均<0.05)。结论3-MA可抑制TGF-β诱导的大鼠肝星形细胞株HSC-T6活化及自噬。

过表达膜定位IL-3的293T细胞外泌体的纯化及体外功能验证

目的体外验证过表达膜定位IL-3的293T细胞外泌体的功能,为在阿尔茨海默病模型动物的体内功能验证奠定基础。方法利用本课题组的专利结构,构建能定位于外泌体膜上的重组IL-3慢病毒载体,包装病毒感染293T细胞,筛选稳定表达细胞株。用流式细胞测量术和免疫荧光技术对IL-3的膜定位进行验证;超滤离心纯化IL-3外泌体,透射电镜观察外泌体形态;纳米流式测量术检测外泌体粒径分布及浓度;Western blot检测IL-3及外泌体相关标志蛋白质表达;免疫荧光技术检测其对小胶质细胞系BV-2吞噬Aβ淀粉样蛋白能力的影响。结果经过载体构建、病毒感染、嘌呤霉素筛选和验证,得到稳定过表达膜定位IL-3的293T细胞株;收集纯化外泌体,在透射电镜下可见直径50~100 nm的双层膜囊泡结构;免疫印迹结果显示CD63、ALIX、TSG101等多种外泌体标志蛋白质检测阳性,且与对照相比富含IL-3,提示IL-3外泌体纯化成功;免疫荧光技术检测结果显示IL-3外泌体能在体外促进BV-2细胞对Aβ淀粉样蛋白的吞噬作用。结论过表达膜定位IL-3的基因修饰293T细胞外泌体在体外兼具IL-3和外泌体的作用,能够促进小胶质细胞的吞噬作用,为阿尔茨海默病的临床治疗提供新的思路。

Comparative Analysis of Protein Expression Concomitant with DNA Methyltransferase 3A Depletion in a Melanoma Cell Line

DNA methyltransferase 3A (Dnmt3a), a de novo methyltransferase, has attracted a great deal of attention for its important role played in tumorigenesis. We have previously demonstrated that melanoma is unable to grow in-vivo in conditions of Dnmt3a depletion in a mouse model. In this study, we cultured the Dnmt3a depletion B16 melanoma (Dnmt3a-D) cell line to conduct a comparative analysis of protein expression con-comitant with Dnmt3a depletion in a melanoma cell line. After two-dimensional separation, by gel electro-phoresis and liquid chromatography, combined with mass spectrometry analysis (1DE-LC-MS/MS), the re-sults demonstrated that 467 proteins were up-regulated and 535 proteins were down-regulated in the Dnmt3a-D cell line compared to the negative control (NC) cell line. The Genome Ontology (GO) and KEGG pathway were used to further analyze the altered proteins. KEGG pathway analysis indicated that the MAPK signaling pathway exhibited a greater alteration in proteins, an interesting finding due to the close relation-ship with tumorigenesis. The results strongly suggested that Dnmt3a potentially controls the process of tu-morigenesis through the regulation of the proteins (JNK1, p38α, ERK1, ERK2, and BRAF) involved in tu-mor-related pathways, such as the MAPK signaling pathway and melanoma pathway.

The establishment of a stable PC-3 cell line overexpressing micro RNA-145

Objective To establish stable prostate cancer bone metastasis cell line overexpressing microRAN-145 (miR-145)for the study of the mechanism of miR-145 in bone metastasis.Methods pMSCV-miR-145 plasmids and retroviruses of pMSCV-vector

RNA干扰C-erbB-2对肺腺癌细胞calu-3增殖的影响

背景与目的:C-erbB-2基因在人乳腺癌、卵巢癌、肺癌中扩增及过量表达,与肿瘤恶性程度增高、转移能力增强、化疗抵抗密切相关,且与预后不良有关。RNA干扰(RNA interfering,RNAi)是一种新的基因治疗技术,能有效、特异性地抑制细胞内基因表达。本实验研究靶向C-erbB-2基因的siRNA(small interferingRNA)对C-erbB-2过表达的肺腺癌细胞calu-3增殖的影响。方法:化学合成的靶向C-erbB-2基因siRNA在脂质体的介导下转染calu-3细胞,观察转染前后细胞表型的改变;通过逆转录聚合酶链反应(reversetranscription-polymerasechainreaction,RT-PCR)和流式细胞术检测转染前后细胞中C-erbB-2mRNA和蛋白水平的变化,四甲基偶氮唑蓝(MTT)法评价对calu-3细胞增殖的抑制作用,流式细胞术检测细胞周期的变化和细胞凋亡情况。结果:靶向C-erbB-2基因siRNA降低了calu-3细胞中C-erbB-2mRNA和蛋白的表达,转染C-erbB-2siRNA48h后calu-3细胞出现C-erbB-2蛋白表达下调。C-erbB-2蛋白阳性率为(25.04±1.56)%,与未转染对照组、空载体组、非特异性siRNA组比较[(98.24±2.23)%、(95.67±1.98)%和(94.79±0.87)%]有显著性差异(P<0.01)。同时C-erbB-2siRNA对calu细胞增殖有抑制作用,使细胞周期阻滞于G0/G1期[C-erbB-2siRNA组(56.6±3.6)%,未转染对照组(45.5±3.2)%,P<0.01],并能诱导细胞凋亡。结论:化学合成的靶向C-erbB-2基因siRNA能有效抑制C-erbB-2基因在calu-3细胞中的表达,并对calu-3细胞增殖有抑制作用。

复方苦参注射液对PC-3细胞凋亡及cyclinE蛋白表达的影响

目的:研究复方苦参注射液对人前列腺癌PC-3细胞凋亡及cyclinE蛋白表达的影响。方法:用复方苦参注射液对体外培养的人前列腺癌PC-3细胞进行药物处理,采用MTT比色分析法测定复方苦参注射液不同剂量组对PC-3细胞生长的调控作用;采用流式细胞仪分析复方苦参注射液对PC-3细胞周期分布的影响,免疫细胞化学方法观察复方苦参注射液对PC-3细胞周期调控蛋白cyclinE的影响。结果:复方苦参注射液可抑制PC-3细胞的增殖,诱导其凋亡,改变细胞周期分布,使G0/G1期PC-3细胞比例增高,同时还可使cyclinE蛋白表达下降。上述作用具有时间和剂量的依赖性。结论:复方苦参注射液可诱导人前列腺癌PC-3细胞凋亡,改变细胞周期分布,影响细胞周期调控蛋白cyclinE的表达,从而抑制细胞增殖。

奥曲肽对人类前列腺癌PC-3细胞凋亡及其对cyclinE蛋白表达的影响

目的:研究生长抑素类似物奥曲肽对人类前列腺癌PC-3细胞凋亡及其对cyclinE蛋白表达的影响。方法:用奥曲肽对体外培养的人类前列腺癌PC-3细胞进行药物处理,采用MTT比色分析法测定奥曲肽不同剂量组对前列腺癌PC-3细胞生长的调控作用;采用流式细胞术分析奥曲肽对PC-3细胞的周期分布的影响,免疫细胞化学方法观察奥曲肽对前列腺癌PC-3细胞周期调控蛋白cyclinE的影响。结果:奥曲肽可抑制前列腺癌PC-3细胞的增殖,诱导其凋亡,改变细胞周期分布,使G0/G1期细胞比例增高,S期比例降低,同时还可使cyclinE蛋白表达下降。上述作用具有剂量和时间依赖性。结论:奥曲肽可诱导前列腺癌PC-3细胞凋亡,改变细胞周期分布,影响细胞周期调控蛋白表达,从而抑制细胞增殖。

miRNA-155 Modulates the Malignant Biological Characteristics of NK/T-Cell Lymphoma Cells by Targeting FOXO3a Gene

This study investigated the effects of miRNA-155 on malignant biological characteristics of NK/T-cell lymphoma cell lines and the possible mechanism. The expression of miRNA-155 was detected in lymphoma cell lines from different sources （SNK-6, YTS, Jurkat and DOHH2） by real-time PCR. Lentiviral vectors （pLL3.7） that could overexpress or downexpress miRNA-155 were constructed. Recombinant lentiviral particles were prepared and purified, and their titers determined. The expression of miRNA-155 in the infected SNK-6 cells and the cell proliferation were detected by PCR and CCK-8, respectively. Flow cytometry was used to determine the apoptosis of infected SNK-6 cells. The target of miRNA155 was predicted from Targetscan website. The effect of miRNA155 on FOXO3a expression was examined by Western blotting. The results showed that among the human NK/T-cell lymphoma cell lines SNK-6, YTS, Jurkat and DOHH2, the expression of miRNA-155 was highest in SNK-6. The infection efficiency of the recombinant lentivirns in SNK-6 was more than 70% at multiplicity of infection （MOI） of 100. The expression of miRNA-155 was significantly increased in SNK-6 cells infected by lentivirus vectors with high expression of miRNA-155 （4 times higher than the control group）, and profoundly decreased in those infected with lentivirnses with low expression of miRNA-155. The proliferation of letivirns-infected SNK-6 cells was decreased as the expression of miRNA-155 reduced. The apoptosis rate was increased with the reduction in the expression of miRNA-155. FOXO3a was found to be a possible target of miRNA155, as suggested by Targetscan website. Western blotting showed that the expression of FOXO3a was significantly elevated in SNK-6 cells with miRNA-155 inhibition. It was concluded that reduction in miRNA-155 expression can inhibit the proliferation of SNK-6 lymphoma cells and promote their apoptosis, which may be associated with regulation of FOXO3a gene.

Over-expression of LRIG3 Suppresses Growth and Invasion of Bladder Cancer Cells

The purpose of this study was to investigate the impact of leucine-rich repeats and immu- noglobulin-like domains 3 （LRIG3） on the biological features of bladder cancer cell lines. The plasmids of over-expressed LRIG3 and the blank plasmid serving as control were transfected into the bladder cancer cell lines, T24, EJ and BIU-87, and the expression levels of LRIG3 mRNA and protein were de- tected by using real-time PCR and Western blotting. The changes in the cell cycle and apoptosis were examined by using flow cytometry. The invasive ability was measured by Transwell assay, and CCK-8 assays were used to measure the proliferation of cells. As compared with the control group, the LRIG3 mRNA and protein expression levels in LRIG3 cDNA-transfected group were raised significantly （P〈0.05）. The average number of cells with up-regulated LRIG3 passing through the inserted filter was decreased significantly as compared with the control group （P〈0.05）. Up-regulation of LRIG3 also could inhibit proliferation and induce apoptosis of T24, EJ and BIU-87 cells. Except BIU-87, the T24 and EJ cells transfected with LIRG3 eDNA were arrested in G0/G1 phase compared to the control group （P〈0.05）. In conclusion, the over-expression of LRIG3 could influence the cell cycle and invasion, in- hibit proliferation and induce apoptosis in the three bladder cancer cell lines.

Transport and uptake of clausenamide enantiomers in CYP3A4-transfected Caco-2 cells： an insight into the efflux-metabolism alliance

Aim The present study developed a CYP3A4-expressed Caco-2 monolayer model at which effects of the efflux-metabolism alliance on the transport and uptake of clausenamide（CLA） enantiomers as CYP3A4 substrates were investigated. The apparent permeability coefficients （Papp） of （ - ） and （ ＋ ）CLA were higher in the ab- sorptive direction than those in the secretory direction with efflux ratios（ER） of 0. 709 ± 0.411 and 0. 867± 0. 250 （ Х10^-6 -1 cm · s ）, respectively. Their bidirectional transports were significantly reduced by （75.6 ± 87.5）% af- ter treatment with verapamil （ a P-glycoprotein inhibitor） that increased the rate of metabolism by CYP3 A4, whereas the CYP3A4 inhibitor ketoconazole treatment markedly enhanced the basolateral to apical flux of （ - ） and （ ＋ ） CLA with ERs being 2. 934 ± 1. 432 and 1. 877 ± 0. 148 （ Х 10^-6 cm/s） respectively. These changes could be blocked by the duel CYP3A4/P-glycoprotein inhibitor cyclosporine A, consequently, Papp values for CLA enanti- omers in both directions were significantly greater than those obtained by using verapamil or ketoconazole, and their ERs were similar to those following （ - ） or （ ＋ ）-isomer treatment alone. Furthermore, the uptake of （ - ）CLA was more than that of （ ＋ ）CLA in the transfected cells. Incubation with ketoeonazole decreased the intracellular concentrations of the two enantiomers. This effect disappeared in the presence of a CYP3A4 inducer dexametha- sone. These results indicated that CYP3A4 could influence P-gp efflux, transport and uptake of CLA enantiomers as CYP3A4 substrates and that a duel inhibition to CYP3A4/ P-glycoprotein could enhance their absorption and bioavailability, which provides new insight into the efflux-metabolism alliance and will benefit the clinical pharma- cology of （？） CLA as a candidate drug for treatment of Alzheimer＇ s disease.

Apoptotic effect and mechanisms of AHPN on human skin malignant melanoma cell A375

Objective： To study apoptotic effects of synthetic retinoic acid 6-[3-（1-adamantyl）-4-hydroxyphenyl]-2-naphthalene carboxylic acid（AHPN） on human skin malignant melanoma A375 cells in comparison with the natural ligand all-trans-retinoic acid（ATRA） in vitro and the mechanisms related to the actions of AHPN. Methods：MTT assay was used to determine the anti-proliferative effects of AHPN and ATRA on A375 cells. Flow cytometry was performed to investigate the influence of AHPN and ATRA on cell cycle and cell apoptosis. In addition, transfection and luciferase activity assays were employed to explore the mechanisms of how AHPN executes its proapoptotic function. Results：Firstly, AHPN promoted apoptosis and GI arrest in A375 cells compared with ATRA. Secondly, the activity of NF-K B in A375 cells treated with AHPN increased 2-3 times compared with solvent DMSO treatment. Conclusion：AHPN, in comparison with ATRA, is a more effective alternative for therapy of malignant melanoma. The potentially proapoptotic function of AHPN requires activation of NF-K B.