缺氧诱导因子1α诱导前列腺癌LNCaP细胞上皮间质转化的体内外研究

目的:探讨在体内外环境下缺氧诱导因子1α(HIF-1α)过表达对前列腺癌细胞发生上皮间质转化(EMT)并导致肿瘤侵袭能力升高的影响。方法:对已构建的稳定表达HIF-1α的人前列腺癌LNCaP细胞(LN-CaP/HIF-1α)复苏后培养,对HIF-1α过表达进行鉴定。MTT法测定细胞增殖;检测转染前后培养液上清PSA水平;软琼脂成瘤实验比较两种细胞体外成瘤能力;将转染前后的LNCaP细胞注射免疫裸鼠皮下建立皮下肿瘤模型,观察肿瘤生长情况;收集肿瘤标本进一步行免疫组化处理。结果:免疫荧光和Western印迹证实LNCaP/HIF-1α细胞中HIF-1α过表达。同LNCaP细胞相比,转染HIF-1α的人前列腺癌LNCaP细胞培养液中PSA水平明显降低;MTT法显示其更具有增殖活性,体外成瘤能力更强。体内实验显示皮下肿瘤成瘤率提高,成瘤时间提前。肿瘤标本免疫组化提示转染组E钙粘蛋白表达下调,波形蛋白表达上调。结论:HIF-1α过表达能够封闭E钙粘蛋白,上调波形蛋白表达,提示HIF-1α过表达可能通过诱导EMT增强LNCaP细胞侵袭能力。     

缺氧诱导因子1α诱导前列腺癌LNCaP细胞上皮间质转化的体内外研究

目的:探讨在体内外环境下缺氧诱导因子1α(HIF-1α)过表达对前列腺癌细胞发生上皮间质转化(EMT)并导致肿瘤侵袭能力升高的影响.方法:对已构建的稳定表达HIF-1α的人前列腺癌LNCaP细胞(LNCaP/HIF-1α)复苏后培养,对HIF-1α过表达进行鉴定.MTT法测定细胞增殖;检测转染前后培养液上清PSA水平;软琼脂成瘤实验比较两种细胞体外成瘤能力;将转染前后的LNCaP细胞注射免疫裸鼠皮下建立皮下肿瘤模型,观察肿瘤生长情况;收集肿瘤标本进一步行免疫组化处理.结果:免疫荧光和Western印迹证实LNCaP/HIF-1α细胞中HIF-1α过表达.同LNCaP细胞相比,转染HIF-1α的人前列腺癌LNCaP细胞培养液中PSA水平明显降低;MTT法显示其更具有增殖活性,体外成瘤能力更强.体内实验显示皮下肿瘤成瘤率提高,成瘤时间提前.肿瘤标本免疫组化提示转染组E钙粘蛋白表达下调,波形蛋白表达上调.结论:HIF-1α过表达能够封闭E钙粘蛋白,上调波形蛋白表达,提示HIF-1α过表达可能通过诱导EMT增强LNCaP细胞侵袭能力.     

Snail和LEF-1转录因子的相互作用是TGF-β1诱导的上皮细胞间质转化（EMT）的必须因素

转化生长因子beta-1（TGF-β1）已被证实在胚胎起源各个阶段、进展性疾病的各个时期都有诱导上皮细胞间质转化（EMT）的作用。细胞极性变化及粘连蛋白（如E-钙粘蛋白）丢失的典型表现就是细胞形态的改变。此项研究揭示了TGF—β1作用于MDCK细胞72h内，EMT与claudin-1、claudin-2、occludin以及E-钙粘蛋白表达丢失的关系。已有证据表明，这种粘连蛋白表达丢失是通过非依赖Smad机制包括MEK和PI3通路产生的，     

缺氧诱导因子1α诱导人前列腺癌细胞上皮间质化改变的研究

目的:研究人前列腺癌细胞在缺氧诱导因子1α(HIF-1α)诱导下能否发生上皮细胞间质转化态(EMT)改变,进而致侵袭能力增强,并初步分析其分子机制。方法:应用RT-PCR技术检测LNCaP细胞及其亚细胞系C4、C4-2、C4-2B这4种EMT阴性的人前列腺癌细胞中波形蛋白(vimentin)mRNA表达情况,并凭此筛选出适合于进一步作转染诱导试验的细胞。用脂质体Lipofectamine2000包装重组真核表达载体pCDNA3.1(-)/HIF-1α和pCD-NA3.1(-)空质粒后,分别转染上步试验所挑选出的人前列腺癌细胞LNCaP,600μg/mLG418筛选抗性克隆。免疫荧光及Western印迹法鉴定HIF-1α表达,Western印迹法检测EMT标志蛋白——上皮型钙粘素(E-cadherin)和vimentin的表达,Transwell验证转染后LNCaP细胞侵袭能力的改变。结果:RT-PCR证实4种EMT阴性的细胞中,仅LNCaP表达有vimentin编码基因,适合作转染诱导试验。免疫荧光也观察到HIF-1α转染细胞胞质中荧光亮度较空质粒转染细胞和未转染细胞明显增强。Western印迹法证实HIF-1α转染细胞发生了EMT转化,其E-cad-herin表达缺失,而vimentin表达增加。同时,Transwell体外侵袭试验也发现,LNCaP/HIF1α细胞的体外侵袭能力显著高于LNCaP细胞和LNCaP/pCDNA3.1(-)细胞。结论:HIF-1α过表达可以通过调节两种EMT相关蛋白诱导人前列腺癌细胞LNCaP发生EMT改变并致其侵袭能力增强。     

多种转移潜能不同的人前列腺癌细胞“上皮细胞间质转化态”特性鉴定及其意义

目的:对多种转移潜能不同的人前列腺癌细胞“上皮细胞间质转化态”(EMT)特性进行鉴定,并从粘附因素和细胞骨架蛋白角度分析其骨转移潜能获得的分子机制。方法:用W estern印迹法鉴定LNCaP及其亚细胞系C4、C4-2和ArCaP亚细胞系IF11、IA8,以及PC-3、Du145等细胞中上皮型钙粘素(E-cadherin)、神经型钙粘素(N-cadherin)和波形纤维蛋白(V im entin)的表达差异情况,并分析其在前列腺癌转移过程中的作用。结果:E-cad-herin在PC-3、LNCaP、C4、C4-2中表达较高,但在Du145、IF11、IA8中表达极低;而V im entin的表达情况恰恰与E-cadherin相反;N-cadherin在IF11、IA8细胞中呈现显著的高表达状态。结论:转移潜能不同的人前列腺癌细胞株之间存在EMT表型的表达差异,其中PC-3、LNCaP、C4、C4-2是未发生EMT改变的细胞,Du145、IF11、IA8却是EMT化的细胞。EMT表型差异蛋白在解释前列腺癌转移机制方面占据着重要地位。     

蛋白激酶A、信号转导分子和转录激动子3调节转化生长因子-β1诱导的细胞凋亡和上皮细胞间质转化

细胞凋亡和上皮细胞间质转化（EMT）对于正常发育和机体自稳非常重要。凋亡和上皮细胞间质转化这些事件的改变常与一些病理过程相关，比如肿瘤形成和转移、肝脏与肾脏的纤维化疾病、胚胎发育异常等。TGF-β1诱导凋亡和EMT同时发生的机制尚未完全明了。作研究了TGF—G1诱导细胞凋亡和EMT的潜在机制。TGF—β1诱导细胞凋亡和EMT与蛋白激酶A、信号转导分子、转录激活子3（STAT3）的活化相关。     

E-cadherin相关性前列腺癌上皮-间质转化调控机制的研究进展

前列腺癌(PCa)是老年男性中常见的恶性肿瘤。在欧美地区男性中,其发病率居恶性肿瘤的第一位,死亡率位居第二[1]。在我国,随着人口老龄化,前列腺癌患者数量呈逐渐上升趋势。前列腺癌的特点是发病隐匿,晚期易转移、易耐药、预后较差。其中,转移性前列腺癌的5年生存率仅为29.3%[2]。因此,深入了解前列腺癌侵袭和转移的潜在机制对于前列腺癌,尤其是转移性前列腺癌的诊断与治疗显得尤为迫切。     

不同前列腺癌细胞系及其皮下肿瘤上皮间质转化特性鉴定

目的:通过比较不同人前列腺癌细胞系在SC ID鼠皮下成瘤特性,鉴定前列腺癌细胞系及其皮下肿瘤上皮、间质标志蛋白的表达情况,探讨前列腺癌细胞系成瘤过程与上皮-间质转化(EMT)之间关系。方法:将DU145、Tsu、PC3和LNCaP 4种人前列腺癌细胞系分别接种于SC ID鼠皮下,比较其成瘤特性;用W estern印迹法鉴定人前列腺癌细胞系及其相应皮下肿瘤钙粘蛋白、波形纤维蛋白的表达,并比较其成瘤前后的区别,探讨上皮间质转化与成瘤的关系。结果:EMT阳性细胞(DU145和Tsu)成瘤率、成瘤速度高于EMT阴性细胞(PC3和LNCaP),4种细胞系成瘤的上皮性的标志蛋白钙粘蛋白表达出现不同变化:DU145表达下调,PC3、LNCaP表达缺失,Tsu表达上调,共同特点是间质性的标志蛋白波形纤维蛋白表达消失。结论:EMT阳性细胞成瘤能力高于EMT阴性细胞,皮下肿瘤的形成过程中的确存在间质-上皮转化(MET)现象。     

转录因子12对前列腺癌进展及预后的影响

目的:探讨转录因子12(TCF12)对前列腺癌进展及预后的影响。方法:利用在线生信分析网站UALCAN、基因表达谱动态分析(GEPIA)、基因、蛋白质相互作用关系检索工具(STRING)及METASCAPE分析TCF12在前列腺组织中表达(高表达组和低表达组)及其与预后的关系,预测TCF12相关基因发挥作用的可能信号通...     

罗格列酮对肾小管上皮细胞间质转化的影响

目的探讨罗格列酮对转化生长因子 β（TGF β）诱导的肾小管上皮细胞间质转化（EMT）过程的影响。方法体外培养人肾小管上皮细胞HK 2,并给予不同浓度TGF β及罗格列酮处理,观察HK 2形态学变化,利用免疫印迹检测过氧化物酶体增殖激活物受体γ（PPARγ）、SMAD家族成员2/3、钙粘附蛋白 E（E cadherin）及波形蛋白（Vimentin）水平变化,通过实时荧光定量聚合酶链反应（PCR）术检测PPARγ、E cadherin、Vimentin、锌指转录因子Snail及Slug的mRNA水平变化,并利用双荧光素酶报告基因检测TGF β及罗格列酮对E cadherin启动子活性的影响。结果TGF β可诱导HK 2细胞发生伪足增多变长、细胞间隙变大等EMT样形态学变化,进而激活TGF β下游的SMAD2/3信号通路,导致上皮细胞标志E cadherin表达明显减少,伴有间质细胞标志的Vimentin表达增高,转录因子Snail及Slug的mRNA水平分别升高6倍以上,伴随E cadherin启动子活性下降70%。罗格列酮可以显著抑制上述TGF β诱导的EMT过程,表现为HK 2细胞足减少变短,细胞间隙变小等,同时伴有E cadherin表达增加,Vimentin表达降低,Snail及Slug的mRNA水平明显降低,E cadherin启动子活性恢复到对照组水平。结论罗格列酮可通过激活PPARγ促进E cadherin转录活性及蛋白表达,拮抗TGF β诱导的EMT过程。     

Clinical significance of runt‐related transcription factor 1 polymorphism in prostate cancer

What’s known on the subject? and What does the study add? Although most clinically localized prostate cancer patients who underwent radical prostatectomy have a favorable outcome, molecular markers capable of providing prognostic information are still urgently needed to identify high‐risk patients who might benefit from aggressive treatment. In this study, we found that RUNX1 rs2253319 polymorphism was significantly associated with higher risks of advanced pathological stage, lymph node metastasis, and time to disease recurrence. Our results suggest that a simple and pretreatment analysis of genetic variants might add prognostic value to the currently used indicators for outcome prediction in patients receiving radical prostatectomy.

OBJECTIVE

To investigate the association of RUNX1 rs2253319 with clinicopathological characteristics of prostate cancer (PCa) and disease recurrence after radical prostatectomy (RP).

PATIENTS AND METHODS

Taking advantage of the systematic stage and grade for each tumor in a cohort of 314 patients with localized PCa receiving RP, we evaluated the associations of RUNX1 rs2253319 with age at diagnosis, preoperative prostate‐specific antigen (PSA) level, Gleason score, surgical margin, pathologic stage, status of lymph node metastasis, and PSA recurrence after RP.

RESULTS

The minor allele, T, and the minor homozygote TT genotype of RUNX1 rs2253319 were significantly associated with a 1.49‐ to 2.76‐fold higher risk for advanced pathologic stage and a 3.35‐ to 9.52‐fold higher risk for lymph node metastasis. RUNX1 rs2253319 TT genotype was also associated with poorer PSA‐free survival compared with the major homozygote CC genotype in Kaplan–Meier analysis (log‐rank test, P= 0.038) and multivariate Cox proportional hazards model adjusting for age and PSA concentration (P= 0.045).

CONCLUSION

RUNX1 rs2253319 is associated with adverse clinicopathological features and might be a prognostic factor for the recurrence of PSA in patients with PCa receiving RP.     

Comparison of two in vivo models for prostate cancer: Orthotopic and intratesticular inoculation of LNCaP or PC-3 cells

BACKGROUND: The critical events in the clinical course of prostate cancer are the occurrence of metastasis and the induction of the hormone-refractory status of the disease. In order to investigate the factors responsible for these events, we need appropriate in vivo models. MATERIALS AND METHODS: Orthotopic and intratesticular models were created by the injection of LNCaP cells or PC-3 cells into the prostate or testis of severe combined immunodeficient mice. RESULTS: LNCaP cells in the intratesticular model showed a higher incidence of tumor formation and lymph node metastasis when compared with those in the orthotopic model, while PC-3 cells were highly tumorigenic and metastastic in both models. A high concentration of androgens might play a role in tumor aggressiveness of LNCaP cells, given that enhanced mRNA expressions of integrin alphaV and vascular endothelial growth factor was induced by dehydrotestosterone administration in vitro. The high expression of metastasis-related genes, including the urokinase plasminogen activator system, metalloproteinases and vascular endothelial growth factor-C, might be attributed to the high metastatic potential in both models. Interestingly, testicular xenografts of LNCaP cells were able to survive on the subcutis back of castrated male mice as well female mice. CONCLUSIONS: Intratesticular models of prostate cancer appear to be suitable for studying the mechanisms of metastasis and for evaluating various treatment strategies.     

Frequency and clinical significance of transition zone cancer in prostate cancer screening

Approximately 20% of prostate cancers originate in the transition zone (TZ). Although transrectal ultrasound (TRUS) and systematic biopsies have improved peripheral zone (PZ) cancer diagnosis, additional biopsies directed into the TZ may further improve cancer detection. To evaluate the frequency and clinical significance of TZ cancers, we added two TZ biopsies to the routinely performed sextant biopsies. Three hundred forty patients (aged 45–75) from our prostate-specific antigen (PSA) screening study (21,078 volunteers) with negative rectal examination findings underwent systematic and TZ biopsies with three-dimensional ultrasound equipment. All patients had elevated PSA levels according to age-specific reference ranges. Ninety-eight of 340 men (28.5%) had biopsies positive for cancer. Of these 98 cancers, 28 (28%) originated in the TZ only and 5 (5%) were located in the TZ as well as the PZ. Eight men showed TZ abnormalities on ultrasound images, of whom four had biopsies positive for TZ cancer. The TZ cancers detected were pathologically significant in 96% (27 of 28). Seventy-one percent (20 of 28) of pathologically staged cancers were found to be organ confined and all combined TZ and PZ cancers were advanced tumors. We conclude that TZ biopsies enhance the cancer detection rate in prostate cancer screening and should therefore be added to the routinely done sextant biopsies in men with PSA elevation and normal digital rectal examination findings. Prostate 30:130–135, 1997. © 1997 Wiley-Liss, Inc.     

环氧化酶2在不同前列腺癌细胞系中的表达

目的:研究环氧化酶2(COX-2)在不同前列腺癌细胞系中的表达,探讨COX-2在前列腺癌侵袭进展及转移潜能获得机制中的可能作用。方法:应用Western印迹及RT-PCR鉴定LNCaP及其亚细胞系C4-2和AR-CaP亚细胞系IF11、IA8,以及PC-3细胞中COX-2的表达情况,并初步分析其在不同特性前列腺癌细胞系转移侵袭过程中的作用。结果:Western印迹结果显示:COX-2蛋白在PC-3细胞中表达相对较高,在IF11、IA8、LNCaP和C4-2细胞中表达缺失,差异具有统计学意义(P<0.05)。COX-2mRNA表达结果同蛋白一致。结论:不同来源、不同转移潜能的前列腺癌细胞株中COX-2表达存在差异。高表达COX-2可能在PC-3细胞高侵袭转移潜能获得方面起着一定作用,而与其他细胞系转移作用无关。     

骨桥蛋白基因在前列腺癌组织中的表达及临床意义简

目的 研究骨桥蛋白（OPN）基因在前列腺癌中的表达及临床意义。 方法 收集我院2008年1月至2011年5月行前列腺癌手术后的石蜡包埋标本80例、良性前列腺增生组织石蜡包埋标本35例。应用免疫组化MaxVision法,检测前列腺癌和前列腺增生组织中OPN的表达情况,分析 OPN的表达与临床病理参数之间的关系。结果 OPN蛋白在前列腺癌和前列腺增生组织中的阳性表达率分别为72.50%（58/80）和8.57%（3/35）, 差异具有统计学意义（P＜0.05）。OPN蛋白阳性表达率与前列腺癌的T、 M分期正相关,差异有统计学意义（P＜0.05）;OPN蛋白阳性表达率与前列腺癌的年龄、N分期和Gleason评分无相关性,差异没有统计学意义（P＞0.05）。结论 OPN蛋白在前列腺癌组织中高表达,提示OPN基因可能与前列腺癌的发生和转移过程有关。     

前列腺癌中神经营养因子及其受体的表达

目的:研究神经营养因子(NTFs)及其受体在前列腺癌中的表达。方法:采用免疫印迹法检测35例前列腺癌患者前列腺组织中神经生长因子(NGF)、脑源性神经营养因子(BDNF)以及其受体TrkA、TrkB、p75的表达,并以10例意外死亡年轻人正常前列腺组织中表达水平作对照。结果:与对照组相比,癌组织中NGF表达下降,而BDNF表达上调,差异均具有显著性(P<0.01);另外,癌组织中p75表达下调,但TrkA、TrkB表达均上调,且差异都具有显著性(P<0.05)。结论:NGF、BDNF及其受体表达变化与前列腺癌发生发展有关,可作为诊断前列腺癌的参考指标。     

Investigations of prostate epithelial stem cells and prostate cancer stem cells

Although both prostate epithelial stem cells and prostate cancer stem cells are implicated in the differentiation of the normal prostate gland and carcinogenesis of prostate cancer, there has, until recently, been little information regarding their biology. This review summarizes the recent advancements in cell biological research including various in vitro culture systems that have offered the characterization and isolation of prostate epithelial stem cells and prostate cancer stem cells. In addition, the stromal niche or microenvironment of stem cells plays an essential role in proliferation and differentiation of normal stem cells. Stroma surrounding cancer cells, which also provide another unique niche, may involve the initiation and development of cancer stem cells. Investigation of stem cells and their microenvironments in the prostate should lead to the elucidation of biological features and the development of novel treatments for prostate cancer.