鞘糖脂微结构域1相关磷酸化蛋白基因的筛选及其对转移性前列腺癌细胞生物学行为的影响

目的 筛选肿瘤转移相关新基因,探讨鞘精脂微结构域1相关磷酸化蛋白基因(PAG1)转染对人前列腺癌细胞系PC-3M的高转移亚系PC-3M-1E8体外生物学行为的影响.方法 利用PC-3M高转移亚系PC-3M-1E8、低转移亚系PC-3M-284,人肺巨细胞痛细胞系(PG)高转移亚系PG-BE1和低转移亚系PG-LH7 cDNA制作4张基因芯片,筛选出PC-3M和PG高、低转移亚系共同差异表达基因.对在两个转移亚系共同表达下调的PAG1基因做进一步研究,采用即时定量PCR及Western blot验证PAG1在PC-3M细胞系中的表达.构建pcDNA3.0-PAG1真核表达载体,稳定转染PC-3M-1E8细胞.MTT比色实验及软琼脂集落形成实验检测肿瘤细胞体外增殖能力;流式细胞术检测肿瘤细胞周期及凋亡;Matrigel穿膜实验检测肿瘤细胞体外侵袭能力.结果 基因芯片初步筛选出PC-3M高、低转移亚系差异表达基因共327个,上调基因123个,下调基因204个.PG高、低转移亚系差异表达基因共281个,上调基因167个,下调基因114个.PC-3M与PG高转移亚系共同表达下调基因9个、上调基因8个.即时定量PCR及Western blot证实PAG1在PC-3M高转移亚系中表达低于低转移亚系.MTT比色及软琼脂集落形成实验显示转染pcDNA3.0-PAG1组细胞增殖速度明显低于转染空载体组和未转染组(均P<0.05).细胞周期检测转染pcDNA3.0-PAG1组比转染空载体组和未转染组处于G_0～G_1期的细胞百分数明显增加(P<0.05).转染pcDNA3.0-PAG1组与转染空载体组和未转染组相比凋亡细胞百分率无显著差异(P>0.05).体外穿膜侵袭实验结果表明转染pcDNA3.0-PAG1组比转染空载体组和未转染组穿膜细胞数目明显减少,分别为(35.1±4.9)、(127.6±6.6)和(135.0±5.0)个(P<0.05).结论 利用同一母系来源的高、低转移亚系制作基因芯片可以摒除转移无关基因的干扰,筛选出差异表达的肿瘤转移相关基因.PAG1基因稳定转染能抑制人前列腺癌高转移亚系PC-3M-1E8细胞的体外增殖能力和侵袭能力,PAG1基因可能是一个潜在的肿瘤增殖、侵袭和转移的抑制基因.     

肿瘤转移抑制基因LASS2/TMSG1全长及其截短体对人前列腺癌细胞生物功能的影响

目的构建肿瘤转移抑制基因LASS2/TMSG1全长及其截短体质粒,探讨其对人前列腺癌细胞生长、迁移、侵袭等生物学功能的影响。方法构建LASS2/TMSG1基因全长以及3个截短体的慢病毒质粒(pCDH-CMV-MCS-EF1-copGFP-T2A-Puro),并将其稳定转染到人前列腺癌细胞系PC-3M-1E8中,采用Western blot法鉴定稳定转染的效果;然后采用软琼脂集落形成实验、细胞划痕修复试验、Transwell体外侵袭实验等分析LASS2/TMSG1基因全长以及3个截短体对人前列腺癌细胞系PC-3M-1E8生物学功能的影响。结果 LASS2/TMSG1蛋白和缺失Homeobox(Hox)结构域的截短蛋白,可以有效抑制PC-3M-1E8细胞的增殖、迁移和侵袭能力(P均0.001);缺失TLC结构域的LASS2/TMSG1蛋白则丧失抑制前列腺癌细胞生长的功能。结论 LASS2/TMSG1蛋白的TLC结构域是抑制前列腺癌细胞生长的关键功能域。     

液泡型ATP酶C亚基ATP6V0C在人前列腺癌细胞系中的表达及其意义

目的探讨液泡型ATP酶C亚基ATP6V0C在不同转移潜能人前列腺癌细胞系中的表达及其意义。方法采用半定量RT-PCR、荧光实时定量RT-PCR和Western blot法检测ATP6V0C在人不同转移潜能前列腺癌细胞系PC-3M-1E8、PC-3M(高转移潜能)和PC-3M-2B4、PC-3(低转移潜能)中的表达。结果 ATP6V0C在PC-3M-1E8、PC-3M细胞系中的mRNA及蛋白表达量均明显高于在PC-3M-2B4、PC-3细胞系中的表达,其中,ATP6V0C在PC-3M-1E8中的表达最高,差异具有统计学意义(P<0.05)。结论 ATP6V0C在高转移潜能人前列腺癌细胞系中的表达明显高于低转移潜能人前列腺癌细胞系,证明其和肿瘤的转移密切相关,有可能成为判断人前列腺癌侵袭和转移的重要指标及治疗前列腺癌的新靶点。     

前列腺癌细胞系中TMSG-1的表达及其意义

目的 探讨一种新的肿瘤转移抑制基因TMSG-1(tumor metastasis suppressor gene 1,TMSG-1)在不同转移潜能前列腺癌细胞系中的表达及其意义.方法 采用半定量RT-PCR、Western blotting、细胞爬片免疫组化PV-9000法来检测TMSG-1在人不同转移潜能前列腺癌细胞系PC-3M-2B4(低转移潜能)和PC-3M-IE8(高转移潜能)中的表达情况.结果 TMSG-1在PC-3M-2B4(低转移潜能)细胞系中的mRNA及蛋白表达量均明显高于在PC-3M-IE8(高转移潜能)细胞系中的表达,具有统计学意义(P<0.05).结论 TMSG-1在低转移潜能前列腺癌细胞系中的表达明显高于在高转移潜能前列腺癌细胞系中的表达,证明它是一种肿瘤转移抑制基因,有可能成为判断前列腺癌细胞浸润及转移的重要预后指标.     

增殖抑制基因抑制乳腺癌细胞生长和侵袭及其相关机制研究

目的 研究增殖抑制基因(HSG)对乳腺癌MDA-MB-231细胞(简称231细胞)生物学行为的影响并对其作用机制进行探讨.方法 构建HSG全长真核表达载体,通过脂质体瞬时转染法使其在231细胞中高表达,通过MTT比色实验检查肿瘤细胞的体外增殖能力、Matrigel穿膜实验检测肿瘤细胞体外侵袭能力和应用流式细胞术检测肿瘤细胞的细胞周期及凋亡情况;并应用GST-pulldown法检测HSG高表达对乳腺癌细胞Ras蛋白活性的影响.结果 将构建好的重组人HSG真核表达质粒pcDNA3-MYC-HSG转染至231细胞中,MTT比色实验结果显示HSG的过表达可以抑制肿瘤细胞的增殖;Matrigel侵袭实验显示转染HSG的肿瘤细胞的穿膜细胞数(HSG/231组,78.5个±5.8个)与转染空载体组(vector/231组,131.1个±14.5个)相比明显减少.流式细胞分析结果显示转染HSG/231组细胞出现了G_0/G_1期阻滞(56.3%±2.3%),且凋亡细胞的百分比与对照组(vector/231组为50.4%±1.9%)相比有所增加.Ras蛋白活性检测发现转染HSG/231组的乳腺癌细胞Ras蛋白活性明显下降.结论 HSG表达上调可抑制高侵袭性231细胞的体外增殖和侵袭能力,使其出现G_0/G_1期细胞周期阻滞,并可促进细胞凋亡.HSG对乳腺癌细胞的抑制作用可能与其抑制细胞Ras活性有关.     

干扰素诱导蛋白p204对大鼠血管平滑肌细胞增殖的影响及其机制

目的: 研究干扰素诱导蛋白p204对大鼠血管平滑肌细胞(VSMCs)增殖的影响并探讨其可能的机制。方法: 应用干扰素α(IFN-α)和p204基因(Ifi204)的小干扰RNA(siRNA)瞬时干预体外培养的VSMCs,用MTT法测定细胞活力反映细胞增殖,流式细胞术分析细胞周期,用实时 qRT-PCR法和Western blotting 分别检测mRNA和蛋白表达。结果: IFN-α可诱导大鼠VSMCs p204 mRNA和蛋白表达上调,抑制VSMCs细胞活力和细胞周期G₁/S转换,伴Ras蛋白表达减少,Raf和ERK磷酸化水平下降。转染Ifi204 siRNA可抑制p204表达,提高VSMCs细胞活力和促进细胞周期G₁/S转换,伴Ras蛋白表达增多,Raf和ERK磷酸化水平升高。结论: p204表达可抑制鼠VSMCs的增殖,该效应可能与p204抑制Ras/Raf/MEK/ERK信号通路的激活有关。     

过表达miR-151a-3p抑制PC-3前列腺癌细胞增殖和迁移

目的研究上调微小RNA-151a-3p(miR-151a-3p)对前列腺癌细胞增殖和迁移的影响和机制。方法采用实时荧光定量PCR检测PC-3M、C4-2B、22RV1、DU-145、PC-3、LNCaP人前列腺癌细胞和RWPE-1人正常前列腺上皮细胞中miR-151a-3p的表达量。以miR-151a-3p表达量最低的PC-3细胞为研究对象,生物信息学预测并采用双荧光素酶报告基因实验检验miR-151a-3p的潜在靶基因。转染miR-151a-3p模拟物或阴性对照微小RNA(miR-NC)至PC-3细胞。实时荧光定量PCR检测miR-151a-3p和潜在靶基因mRNA的表达量,Western blot法检测靶基因及下游信号通路蛋白表达量。采用MTT法检测PC-3细胞的增殖,Transwell~(TM)实验检测PC-3细胞的迁移能力。结果 miR-151a-3p在前列腺癌细胞的表达量明显低于RWPE-1人正常前列腺上皮细胞,其中PC-3细胞中的表达量最低。生物信息学预测及双荧光素酶报告基因实验表明NIMA相关激酶2(NEK2)是miR-151a-3p的潜在靶基因。转染miR-151a-3p模拟物后,PC-3细胞中miR-151a-3p的表达量明显上升,NEK2基因的表达量明显降低,NEK2基因下游磷脂酰肌醇3激酶-蛋白激酶B-哺乳动物雷帕霉素靶蛋白(PI3K-AKT-mTOR)信号通路蛋白水平降低。上调miR-151a-3p明显抑制前列腺癌PC-3细胞的增殖和迁移。结论miR-151a-3p在前列腺癌细胞中表达降低,上调miR-151a-3p的表达可通过降低NEK2基因及下游PI3K-AKT-mTOR信号通路蛋白的表达抑制前列腺癌PC-3细胞的增殖和迁移。     

p16和p27表达对前列腺癌细胞系的作用

目的 探讨p16和p27Kip1 基因蛋白对抑制前列腺癌细胞增殖和调控其凋亡的作用.方法 构建携带人P16和p27基因的腺病毒载体,转染体外培养的前列腺癌细胞系PC-3,采用RT-PCR、Western blot检测目的 基因的表达.通过细胞生长试验、流式细胞仪检测PC-3转染前后细胞增殖和凋亡的变化.结果 病毒滴度Ad-p16为115×10^8 pfuPml 、Ad-p27为112×10^9 pfuPml ,RT-PCR 检测可见p16-mRNA（520bp）和p27Kip12mRNA （320bp）表达,Western blot检测有p16 蛋白（65KD）和P27蛋白（27KD） 特异表达,并可明显抑制PC-3细胞的增殖,诱导其凋亡,联合基因治疗组与单基因组相比差异显著（P＜0.01）.结论 p16和p27可明显抑制前列腺癌细胞株PC-3的增殖,增加细胞的凋亡,转染携带p16和p27的重组腺病毒载体有望成为治疗前列腺癌的有效方法.     

PSMA对LNCaP细胞生长、迁移及ERK蛋白活性的影响

目的: 利用我们已经成功沉默前列腺特异性膜抗原(PSMA)的LNCaP前列腺癌细胞株,探讨PSMA对LNCaP细胞磷酸化胞外信号调节激酶(ERK)及细胞生长、迁移的影响,为进一步研究PSMA在前列腺癌发展中的作用提供理论基础。方法: 实验对象包括携带可稳定抑制PSMA表达siRNA慢病毒的LNCaP细胞组(实验组),携带对任何基因无干扰作用siRNA慢病毒的LNCaP细胞组(空转组),同时建立未进行处理的普通LNCaP细胞组(对照组),分别对在一般培养基及添加ERK蛋白上游抑制剂的3组细胞,使用Western blotting和细胞免疫化学的方法检测MAPK/ERK蛋白活性,并用MTT描绘细胞生长曲线、Transwell观察细胞迁移情况。结果: 在一般培养基的3组细胞中,Western blotting提示实验组磷酸化ERK蛋白表达明显低于对照组和空转组;免疫细胞化学结果显示实验组染色明显比对照组和空转组弱,阳性细胞数较少;MTT绘制生长曲线,得到实验组细胞的增殖生长能力较对照组、空转组降低;Transwell结果提示实验组较对照组和空转组细胞的增殖迁移能力降低。在ERK磷酸化被抑制的情况下,3组细胞磷酸化ERK蛋白均低表达,MTT及Transwell检测显示其生长迁移能力都处于低水平,且与在一般培养基中实验组的效应类似。结论: 初步发现PSMA可能通过上调前列腺癌LNCaP细胞ERK蛋白的活性,从而在其生长、迁移中起正向调节作用。     

不同分型腺性膀胱炎中CK20、Ki67、Ras P21蛋白表达及 临床意义研究

目的 探讨CK20、Ki67、Ras P21蛋白三种肿瘤标志物在正常膀胱组织、不同类型腺性膀胱炎及膀胱腺癌组织中表达的特点。方法 将腺性膀胱炎经典型（泌尿上皮型）20例、肠上皮型20例、前列腺上皮型20例,正常膀胱组织20例、膀胱腺癌组织20例,采用免疫组织化学法,检查CK20、Ki67、Ras P21在各分型腺性膀胱炎中的表达,并与正常膀胱组织、膀胱腺癌组织组进行比较。结果 肠上皮型和前列腺上皮型腺性膀胱炎 CK20、Ki67、Ras P21阳性表达与正常膀胱黏膜组织比较,差异具有统计学意义（P＜0.05）。而经典型（泌尿上皮型）CK20、Ki67、Ras P21的表达与正常膀胱黏膜组织比较,差异无统计学意义（P＞0.05）,肠上皮型和前列腺上皮型腺性膀胱炎及膀胱腺癌中CK20、Ki67、Ras P21高表达。肠上皮型和前列腺上皮型腺性膀胱炎可能为癌前病变。结论 对于肠上皮型及前列腺型腺性膀胱炎,应进行积极的手术治疗及抗癌药物膀胱灌注,并严密术后随访。     

应用RNA干扰技术研究激素非依赖性高转移潜能前列腺癌细胞中survivin的表达及意义

目的研究 survivin 在激素非依赖性高转移潜能前列腺癌中的表达及其与前列腺癌的生物学行为及侵袭和转移潜能的相关性。方法应用 RNA 干扰技术构建 survivin 真核表达载体,转染人激素非依赖性前列腺癌高转移亚系 PC-3M-1E8细胞系。通过细胞生长曲线、肿瘤细胞裸鼠异种接种、软琼脂集落形成实验检测体内、体外细胞生长能力;流式细胞术检测 survivin RNA 干扰质粒对细胞周期及细胞凋亡的影响,Western blot 检测 caspase3活性片段,观察 survivin 抑制细胞凋亡的情况;Matrigel 穿膜实验检测肿瘤细胞体外侵袭能力。结果稳定转染 survivin RNA 干扰质粒的 PC-3M-1E8细胞中 survivin 的 mRNA 及蛋白水平明显降低,蛋白水平与阴性对照组相比,约下降78%～80%,差异有统计学意义(P<0.01);体外培养细胞生长速度及裸鼠体内肿瘤生长速度均明显减慢,锚着不依赖性生长的能力(软琼脂克隆形成数:14.33±3.51)与阴性对照组(52.33±6.81)及空白对照组(54.00±6.00)相比明显降低(P<0.01);凋亡细胞比例明显增加,空白对照组、阴性对照组及干扰阳性组的凋亡比例分别为5.88±0.99、6.97±1.60、16.40±1.95,干扰阳性组的凋亡比例显著高于对照组(P<0.01),并伴有 caspase3活性片段的表达增加;细胞阻滞在 G_0/G_1期(干扰阳性组、阴性对照组及空白对照组的细胞 G_0/G_1期的比例分别为52.71±1.10、43.59±1.83及43.65±3.44,P<0.05),并在细胞形态学上出现多核巨细胞现象;细胞体外侵袭能力明显降低,干扰阳性组的细胞(38.67±6.59)与阴性对照组(46.07±9.97)及空白对照组(47.87±9.58)相比穿膜细胞数明显减少(P<0.05)。结论 survivin 在激素非依赖性高转移潜能前列腺癌中高表达,并与细胞凋亡、细胞生长及肿瘤侵袭有关。抑制 survivin 的表达可能成为临床治疗激素非依赖性前列腺癌的方法之一。     

Overexpression of Pim-1 during progression of prostatic adenocarcinoma

AIMS: Pim-1 is a serine/threonine kinase that has been shown to play an integral role in the development of a number of human cancers, such as haematolymphoid malignancies. Recently, evidence has shown Pim-1 to be important in prostatic carcinogenesis. In order to further our understanding of its role in prostate cancer, we investigated Pim-1 expression in normal, premalignant, and malignant prostate tissue. METHODS: Using immunohistochemistry, Pim-1 expression was analysed in prostate tissue from 120 radical prostatectomy specimens. In each case, Pim-1 staining was evaluated in benign prostatic epithelium, high grade prostatic intraepithelial neoplasia (PIN), and prostatic adenocarcinoma. The number of positively staining cells was estimated, and the intensity of staining was scored on a scale of 0 to 3+. RESULTS: Pim-1 immunoreactivity was identified in 120 cases (100%) of adenocarcinoma, 120 cases (100%) of high grade PIN, and 62 cases (52%) of benign glands. The number of cells staining in benign epithelium (mean 34%) was much lower than that in high grade PIN (mean 80%; p<0.0001) or adenocarcinoma (mean, 84%; p<0.0001). There was no significant difference between high grade PIN and adenocarcinoma in the percentage of cells staining positively for Pim-1 (p = 0.34). The staining intensity for Pim-1 was significantly lower in benign prostatic epithelium than in PIN and adenocarcinoma (p<0.001). There was no statistically significant correlation between the level of Pim-1 expression and Gleason score, patient age, tumour stage, lymph node metastasis, perineural invasion, vascular invasion, surgical margin status, extraprostatic extension, or seminal vesicle invasion. CONCLUSIONS: Pim-1 expression is elevated in PIN and prostatic adenocarcinoma compared with benign prostatic epithelium. This finding suggests that upregulation of Pim-1 may play a role in prostatic neoplasia.     

CD44 expression is a feature of prostatic small cell carcinoma and distinguishes it from its mimickers

Small cell neuroendocrine carcinoma of the prostate is a rare variant of prostatic cancer that shares morphologic similarity with prostatic adenocarcinoma of Gleason 5 pattern. It has also been considered morphologically and immunohistochemically indistinguishable from small cell neuroendocrine carcinomas of other origins. CD44 is a cell-surface molecule proposed to identify cancer stem/progenitor cells in prostate cancer. We performed immunohistochemical study for CD44 expression in 11 cases of prostatic small cell neuroendocrine carcinoma and compared its patterns of expression with 73 cases of prostatic adenocarcinoma and 47 cases of small cell neuroendocrine carcinomas of other organs. Strong and diffuse membrane staining for CD44 was observed in 100% of the prostatic small cell neuroendocrine carcinomas. In conventional adenocarcinomas of the prostate, positive staining was only seen in rare, scattered tumor cells; and CD44 staining was negative in most of the small cell neuroendocrine carcinomas of nonprostate origin. The difference in CD44 expression between small cell neuroendocrine carcinomas of the prostate and those of other organs are statistically significant (P < .001). Our study demonstrates the utility of immunohistochemical staining for CD44 in distinguishing prostatic small cell neuroendocrine carcinoma from its mimickers including prostatic adenocarcinoma of Gleason 5 pattern and small cell neuroendocrine carcinomas of other organs. CD44 is the first marker that shows a high degree of tissue/organ specificity for small cell neuroendocrine carcinomas. Because CD44 is a putative marker of prostate cancer stem cells, the strong and diffuse expression of CD44 and the lack of expression of prostate luminal differentiation markers androgen receptor and prostatic specific antigen in prostatic small cell neuroendocrine carcinomas suggest that the tumor cells may retain cancer stem cell features.     

Expression of HLA-like structures on a permanent human tumor line PC-93

A permanent human tumor-line PC-93 was HLA allotyped with the use of a complement-dependent cytotoxicity test supplemented with absorption experiments. In comparison with the peripheral blood lymphocytes of the patient, whose adenocarcinoma of the prostate was the origin of PC-93, remarkable differences were observed. The tumor line lacked the expression of the genetically appropriate HLA-A1, A2 and B15 antigens. Instead of these antigens, cross-reactive HLA antigens seemed to be expressed on PC-93, i.e. All, A28 and B17. Absorption experiments showed that the antibodies reactive with the neo-HLA antigens were not the same as those reactive with the specific HLA antigens. This alien expression of HLA-like structures was specific for the patients' prostatic tumor line, as EB-virus transformed B-lymphocytes of the patient expressed the genetically appropriate HLA antigens.     

Low frequency of BK virus in prostatic adenocarcinomas

BK virus (BKV) exhibits many oncogenic properties and has been associated with a variety of tumors in humans. BKV has not been well studied in the context of prostate neoplasia; however, an association of BKV with prostatic adenocarcinoma has been suggested based on the detection of viral DNA sequences and expression of viral proteins in clinical samples. To further investigate the reported association of BKV with prostatic adenocarcinoma and the potential role of the virus in prostate tumorigenesis, 30 cases of adenocarcinoma of the prostate were analyzed for evidence of BKV infection by in situ hybridization and immunohistochemistry. In situ hybridization analysis detected BKV DNA in 2 of 30 (7%) prostatic adenocarcinomas, with positive signals focally identified in less than 1% of the neoplastic cells in both cases. However, none of the tumors evaluated demonstrated evidence of BKV large tumor antigen expression by immunohistochemistry. Among prostatic adenocarcinomas that showed no evidence of BKV infection, BKV DNA was focally observed in the adjacent non-neoplastic prostate tissue in four cases by in situ hybridization in the absence of BKV large tumor antigen immunoreactivity. The findings of the present study indicate rare cases of prostatic adenocarcinoma may be associated with BKV infection. However, lack of localization of BKV to a large population of the neoplastic cells and absence of BKV large tumor antigen expression suggest that the virus does not play a role in the pathogenesis of prostate cancer.     

基质金属蛋白酶及其组织抑制因子在人前列腺组织及各种类型前列腺细胞中的表达

目的：研究基质金属蛋白酶（MMPs）及其组织抑制因子（TIMPs）在人前列腺组织及各种类型细胞中的表达。方法： 用半定量RT－PCR的方法，对癌变和非癌变部分的前列腺组织、原代培养的平滑肌细胞、成纤维细胞、上皮细胞以及4种前列腺上皮细胞系（BPH－1、LNCaP、DU－145和PC－3）中MMP2、MMP7和MMP9、膜型基质金属蛋白酶1和3（MT1－MMP和MT3－MMP）及其组织抑制因子1和2（TIMP－1和TIMP－2）的mRNA 水平进行了测定。结果：MMP-2主要在前列腺基质细胞中表达；MMP-7和MMP-9则在前列腺上皮细胞中有较高的表达；MT1-MMP、MT3-MMP、TIMP-1和TIMP-2在前列腺基质细胞和上皮细胞中均有表达，但MT1-MMP和MT3-MMP在成纤维细胞中的表达量较高；另外，各种基质金属蛋白酶及其组织抑制因子在各种前列腺细胞系中也存在差异表达。结论： MMPs和TIMPs在前列腺组织及其各种类型细胞中的差异表达提示：它们可能在前列腺癌的转移中起着不同的作用。