【作者】 岳丹；

【导师】 任常山；

【作者基本信息】 中国医科大学 ， 肿瘤学， 2010， 博士

【摘要】 前言前列腺癌(prostate cancer, PC)在欧美等国家发病率极高,死亡率在男性疾病中位居第二位,仅次于肺癌。近年来,PC的发病率在我国明显上升,且发病年龄正趋于年轻化。激素抑制治疗是目前最有效的治疗方法,PC早期无症状,多数病人就诊已属晚期,除了骨和淋巴转移,PC最主要的死因是激素依赖性前列腺癌(androgen dependent prostate cancer, ADPC)演变成激素非依赖性前列腺癌(androgen independent prostate cancer, AIPC),然而,AIPC对去势治疗和化疗无效,细胞继续生长,肿瘤停止退化。因此在PC的诊断和治疗上也有待于寻找新的作用靶点。肝细胞生长因子(hepatocyte growth factor, HGF)是一种基质细胞源性生长因子,HGF通过作用于细胞膜表面的特异性跨膜受体c-Met发挥生物学作用,如诱导肿瘤细胞的离散、增殖、转移、侵袭及血管形成等。近年来许多报道证实c-Met、HGF在前PC组织过度表达,与PC的转移、侵袭相关,PC病人血浆中的HGF水平明显升高,因此提出HGF与PSA联合做为PC诊断的新的重要指标。NK4是1997年Date等发现的一种新型的HGF拮抗剂,已经在体外实验中证实,NK4在浓度从10ng／ml到1000ng／ml范围内完全不产生任何生物学效应,但具有完全的HGF拮抗的作用。NK4与c-Met结合,可以竞争性地抑制HGF和c-Met的相互作用,影响HGF/c-Met系统的信号转导,从而抑制HGF所诱导细胞的增殖、运动和形态改变,但其本身不能诱导c-Met的酪氨酸磷酸化。NK4蛋白或NK4表达基因的注入,在多种不同类型肿瘤实验模型均能显示其具有抑制肿瘤侵袭、生长、转移和血管生成等作用。有研究证明HGF可以增强前列腺癌的侵袭能力。此外,HGF从旁分泌转变成自分泌可能是前列腺癌表型转换的重要原因。HGF及其拮抗剂NK4在前列腺癌发生发展的作用及其机制还有待于进一步研究。HGF与细胞膜上的受体c-Met结合,受体激活后,通过与之偶联的磷脂酶C(phospholipase C, PLC)水解磷酸肌醇二磷酸(phosphatidylinositol diphosphate,PIP2),生成三磷酸肌醇(inositol triphosphate,IP3)和二酰基甘油(diacylglycerol,DAG)。IP3作用于内质网或肌浆网导致Ca2+释放,钙池耗竭引起外Ca2+内流。DAG也可以直接激活受体操纵性钙通道(receptor operated calcium channels, ROCC),引起细胞外Ca2+内流。细胞内游离Ca2+升高,激活一些蛋白磷酸酶,使底物蛋白磷酸化,将外界信号级联放大,进入核内,影响DNA复制,导致细胞恶变及肿瘤细胞的增殖分化。细胞内Ca2+直接参与调控肿瘤的生长,侵袭,转移和分化。而发生于细胞膜上的促使Ca2+进入细胞内的机制尚不清楚。瞬时受体电位(transient receptor potential, TRP)通道家族对细胞起着稳定和调控作用,其表达升高促进恶性肿瘤的生长。TRPC通道是TRP的一个亚族,包括TRPC(1-7), TRPC6通道作为胞内钙信号产生的一个重要途径,调节着第二信使Ca2+的浓度及各种蛋白酶的活性,从而直接或间接影响细胞的各种生物学行为。有研究证实：TRPC6是HGF诱导的钙内流的调停者,TRPC6介导HGF诱导肾小管细胞增殖；HGF和表皮生长因子诱导TRPC6的过表达从而促进肝癌细胞的增殖等。因此,研究和开发高选择性和高特异TRP通道药物,可为肿瘤的治疗提供新的思路。RNA干扰技术(RNA interference)能有效沉默特异基因的表达,本实验应用RNAi沉默TRPC6基因,研究TRPC6阳离子通道在HGF诱导的PC增殖中的作用。根据以上研究背景,为了明确HGF对PC生长的作用机制并探寻治疗PC的途径,本课题从以下方面进行研究：①将构建的pBudCE4.1-EGFP/NK4真核表达载体稳定转染前列腺癌DU145细胞中,观察其对前列腺癌细胞增殖、迁移和侵袭的影响,以及如何调节HGF受体c-Met及其下游的ERK1和Akt1／2信号通路,进一步明确HGF拮抗剂NK4基因的抗肿瘤作用机制。②检测TRPC6在BPH、PC及前列腺癌细胞系中的表达,探讨TRPC6的表达与PC进展的关系。利用siRNA技术对前列腺癌DU145细胞进行TRPC6干扰,检测胞浆游离Ca2+浓度([Ca2+]i)、细胞增殖和细胞周期等指标,进一步探讨TRPC6对PC增殖作用的影响及与HGF的相关性,为防治PC提供新思路和新靶点。材料与方法一、实验材料1、细胞株前列腺癌细胞株Du145、PC3和22Rvl购于中国医学科学院基础医学研究所。2、组织标本收集中国医科大学盛京医院2004年至2008年手术或穿刺活检的PC和BPH组织标本,142个PC患者得到153张切片(131个患者各一张切片,11个患者各2张切片),20个BPH患者各一张切片。二、主要研究方法1、建立稳定表达NK4蛋白的细胞系pBudCE4.1-EGFP/NK4质粒的转化、扩增、提纯,酶切鉴定。Zeocin筛选转染最佳浓度,Lipofectamine 2000转染DU145细胞株,Zeocin筛选稳定表达NK4细胞株,应用Western blot进行分泌NK4蛋白的检测。2、MTT法测定细胞增殖能力噻唑蓝[3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT]染色法检测转染NK4基因以及TRPC6干扰后DU145细胞增殖的变化。3、Transwell法测定细胞迁移能力细胞密度为1×105／ml的细胞悬液0.2ml加入上室,下室加入含有10ng／ml HGF的10%胎牛血清(fetal bovine serum, FBS)培养基0.5 ml,37℃,5%CO2培养箱孵育6h,擦净小室上面的细胞,用甲醇／冰醋酸固定30min,Giemsa染色,光镜下计数细胞。4、Matrigel小室测定细胞侵袭能力Transwell小室涂布Matrigel基质胶,细胞密度为1×105／ml的细胞悬液0.2ml加入上室,下室加入含有10ng/ml HGF的10%FBS培养基0.5 ml,37℃,5%CO2培养箱孵育24h,擦净小室上面的细胞,用甲醇／冰醋酸固定30min, Giemsa染色,光镜下计数细胞。5、免疫组化检测TRPC6在PC、BPH组织中的表达采用链霉素抗生物素蛋白氧化物酶免疫组化法(streptavidin-peroxidase conjugated method, SP法),标本石蜡切片,TRPC6一抗体孵育,4℃过夜,磷酸盐缓冲液(phosphate buffer solution, PBS)缓冲液代替一抗为阴性对照。以棕黄色颗粒着色,膜着色清楚、连续且明显高于背景为阳性。结果判定：每张切片随机选取5个视野,按阳性细胞所占细胞数量百分比分为5级：阳性细胞数1%-10%为0,1%～5%为1级,5%～10%为2级,10%～0%为3级,20%～50%为4级,>50%为5级。6、RT-PCR检测TRPC6 mRNA的表达收集Du145、22Rvl、PC3细胞,采用Trizol试剂提取总RNA,检测纯度及含量,反转录成cDNA,以TRPC6基因引物进行PCR扩增,以β-actin为内参照,条带进行灰度值分析。7、Western blot检测NK4、c-Met、p-c-Met、Akt1/2、p-Akt1/2、ERK1、p-ERK1、β-actin、TRPC6蛋白的表达收集细胞提取总蛋白,BCA法检测蛋白质含量。进行SDS-PAGE电泳,转印至PVDF膜。封闭,NK4、c-Met、p-c-Met、Akt1/2、p-Akt1/2、ERK1、p-ERK1、TRPC6、β-actin一抗孵育过夜,相应的二抗孵育,增强型化学发光(enhance chemiluminescence, ECL),拍照,以β-actin为内参照,条带进行灰度值分析。8、TRPC6 siRNA及干扰效率检测TRPC6 siRNA的设计,合成3对siRNA,使用荧光标记质粒FAM-siRNA检测转染效率,根据筛选出的最佳条件进行RNA干扰,Western blot筛选干扰效率。9、钙成像细胞数约为1×105／ml播种在共聚焦皿,5μM Fura 2-AM37℃孵育,再用HEPESbuffer saline孵育1h,在检测过程中用OAG刺激,用荧光倒置显微镜检测并记录[Ca2+]i变化。10、流式细胞仪检测细胞周期制成1×107／ml细胞悬液,95%乙醇固定30min,PBS冲洗后用RNase处理15min,用碘化丙啶(propidium iodide, PI)染液作用细胞30min,流式细胞仪检测细胞周期分布。11、统计学分析采用SPSS11.5统计软件包对数据进行统计分析,数据结果以x±s表示,以P<0.05为判断差异显著性标准。结果1、对质粒酶切鉴定,琼脂糖凝胶电泳可见约1326bp的片段,表明NK4片段插入pBudCE4.1-EGFP载体上,最终得到pBudCE4.1-EGFP/NK4双基因表达载体。Western blot检测发现,稳定转染NK4基因的DU145细胞能够自分泌分子量约50KD的NK4蛋白。2、MTT检测显示,HGF对前列腺癌细胞具有增殖作用,单纯的NK4基因转染对细胞增殖没有影响,而在HGF存在的情况下,自分泌的NK4可以抑制HGF对DU145细胞的增殖作用。Transwell小室检测发现自分泌NK4减弱HGF诱导的DU145细胞的运动。Matrigel侵袭小室检测发现转染NK4基因抑制HGF诱导的DU145细胞的侵袭。3、Western blot检测发现,c-Met在前列腺癌DU145细胞中表达,HGF诱导磷酸化的p-c-Met、p-ERK1和p-Akt1／2增多,而自分泌NK4可以有效抑制HGF诱导的c-Met、ERK1和Akt1／2蛋白的磷酸化。4、免疫组织化学检测发现TRPC6在BPH中弱表达,在PC中的表达明显高于BPH。TRPC6的表达与PC的组织分级、Gleason评分及前列腺外转移有关。RT-PCR检测Du145、PC3和22Rvl前列腺癌细胞中有TRPC6 mRNA的表达。5、转染TRPC6 siRNA质粒后TPRC6表达减少,siRNA1和siRNA2两组在干扰后72h干扰率达到60%～70%。OAG诱导[Ca2+]i增加,而在细胞外Ca2+缺乏的情况下OAG诱导不能引起[Ca2+]i增加；siRNA TRPC6使[Ca2+]i降低,同时OAG诱导Ca2+内流明显下降。另一方面,HGF刺激使DU145细胞TRPC6蛋白表达增多,HGF使OAG诱导[Ca2+]i增加；siRNA TRPC6使细胞阻滞在G2／M期。结论1、转染NK4基因的DU145细胞可以自分泌NK4蛋白。NK4可以抑制HGF诱导的c-Met及其下游的ERK和Akt1／2蛋白的活化,从而调节肿瘤细胞的增殖、迁移和侵袭。NK4作用于HGF/c-Met可以作为治疗PC的一个有效靶点。2、TRPC6在BPH中弱表达,在PC中的表达明显高于BPH。TRPC6的表达与PC的组织分级、Gleason评分及前列腺外转移有关。TRPC6在22Rvl、PC3和DU145细胞系中均表达。3、TRPC6通道能够调节DU145细胞的Ca2+内流。阻断TRPC6通道可以抑制DU145细胞的增殖,使细胞周期阻滞在G2／M期。HGF可以增加TRPC6蛋白的表达水平,使OAG诱导的Ca2+内流增加。因此,TRPC6通道介导的Ca2+内流在HGF诱导的细胞增殖中起重要作用。更多还原

【Abstract】 IntroductionProstate cancer (PC) is one of the leading threats to men’s health in western societies. Following metastasis to bone and lymph nodes, the primary cause of prostate cancer mortality is the progression from androgen dependent (AD) to andro gen-independent (AI) growth. In the early stages of the disease, androgen-ablation therapy can cause tumor regression, and is currently the most successful treatment option. However, once the tumor achieves androgen independence, there is no effective therapy. Therefore, new prognostic markers and therapeutic strategies may be of great benefit to prostate cancer patients.Hepatocyte growth factor (HGF) is a heterodimers molecule with a number of biological activities, including regulation of migration, invasion and angiogenesis in cancer. HGF is composed of aα-chain, containing the N-terminal hairpin domain and four kringle domains, and a serine protease-likeβ-chain. Over expression of HGF and its receptor, c-Met, in prostate cancer has been reported. Moreover, a higher plasma level of HGF in prostate cancer patients is associated with an advanced stage of malignancy and a poor prognosis.Recently, the HGF antagonist, NK4, was developed. It is a variant form of HGF, comprising the N-terminal and the subsequent four kringle domains of HGF. Since NK4 retains the capacity to bind to the HGF receptor, c-Met, it competes with HGF and inhibits the biological activity of HGF, such as cell proliferation, migration and morphologicchanges. But NK4 itself can’t induce c-Met phosphorylation. Competitive inhibitory effects of NK4 on HGF/c-Met have been demonstrated in some types of human cancer cells. In addition, HGF is intimately involved in growth of human prostate cancer and that progression from the androgen-dependent to the androgen-independent state. Using these characteristics of prostate cancer, we have found a new approach for investigating prostate cancer.Cytosolic Ca2+ is an important transducer of HGF signal. HGF combines with c-Met to active phospholipase C (PLC), and then phosphatidylinositol diphosphate (PIP2) is hydrolyzed, generating inositol triphosphate (IP3) and diacylglycerol (DAG). DAG can activate receptor operated calcium channels (ROCC) and caused Ca2+ influx. The increasing of intracellular free Ca2+ active some protein phosphatase, thereby, affected cell DNA replication, leading to cell malignant transformation and tumor cell proliferation and differentiation. Intracellular Ca2+ is related to tumor proliferation, which is involved in the regulation of tumor growth, invasion, metastasis and differentiation. However, the mechanism of Ca2+ entry is unclear.Transient receptor potential (TRP) channels are a large family of nonselective cation channels. TRPC channels, a subfamily of TRP channels in mammalian cells, include 7 members (TRPC1～7), and are mostly permeable to Ca2+. TRPC6, an important way of intracellular calcium signal generation, could regulate intracellular calcium concentration and enzyme activity, which directly or indirectly affect cell biological behaviour. Recently, it has been shown that in Huh-7 cells, the expression of EGF and HGF receptors in hepatocarcinoma probably induces TRPC6 expression, increasing the rate of cell proliferation. Additionally, TRPC6 is critical for HGF-induced growth, migration and morphogenesis in human renal tubular cell line HK2. RNA interference could effectively knock down the expression of target genes. We used TRPC6siRNA to explore the role of TRPC6 in HGF-induced prostate cancer cell proliferation.The above results suggest HGF has a critical role in the growth of PC. To clarify the role of HGF on the growth of PC, We designed the following experiment:①We used the pBudCE4.1-EGFP/NK4 expressing NK4 gene to investigate the anti-tumor effect and the possible mechanism of NK4 on human prostate cancer DU145 cells. This study may supply an experimental basis for the clinical application of NK4 in prostate cancer treatment.②We studied TRPC6 protein expression in benign and malignant human prostate tissues, and correlated TRPC6 protein immunostaining with the stage, grade and androgen responsiveness of the tumors. Furthermore, study was designed to investigate the possible relationship between TRPC6 and HGF and the function of TRPC6 channels in cell proliferation of prostate cancer by using physiological and molecular techniques.Material and methodsⅠ. Material1. Cell linesThe 22Rv1 and DU145 prostate cancer cell lines were obtained from Institute of Basic Medical Sciences Chinese Academy of Medical Sciences.2. SpecimenClinical specimens were collected from the patients registered at Shengjing Hospital of China Medical University (Shenyang, China) between 2004 and 2008.153 prostate cancer samples derived from 142 patients (1 sample in 131 patients,2 samples in 11 patients) and 20 benign prostate tissues derived from BPH patients for TRPC6 protein staining.Ⅱ. Methods1. Plasmid constructionPlasmid pBudCE4.1-EGFP/NK4 was constructed, transformed, amplified and purified. Plasmids were transfected into DU145 cells by Lipofectamine 2000. The cells were selected in the presence of Zeocin, and resistant clones were obtained. Western blot was carried out to detect the expression of NK4 protein.2.MTTAfter transfection, the proliferation of cell was detected by MTT. 3. In vitro migration assayTranswell chamber was used to determine the motility of prostate cancer cells.0.2 ml cells(1×105/ml)was added to the upper well. The chambers were placed in lower wells containing 0.5 ml of RPMI 1640 supplemented with 10% fetal calf serum and 10 ng/ml of HGF for 12 h in 5% CO2 at 37℃. At the end of the incubation period, cells in the upper chamber were removed, and the migrating cells were fixed with cold methanol/acetic acid and then stained with Giemsa. The number of migrating cells was evaluated by random observation of five fields on every section. The average number of cells per field was defined as migration index.4. In vitro invasion assayMatrigel-coated invasion chambers were used to determine the motility of prostate cancer cells.0.2 ml cells (1×105/ml) was added to the upper well. The chambers were placed in lower wells containing 0.5 ml of RPMI 1640 supplemented with 10% fetal calf serum and 10 ng/ml of HGF for 12 h in 5% CO2 at 37℃. At the end of the incubation period, cells in the upper chamber were removed from the top of the filter and the migrating cells were fixed with cold methanol/acetic acid for 30 min, and then stained with Giemsa. The number of migrating cells was evaluated by random observation.5. ImmunohistochemistrySections from paraffin-embedded specimens were deparaffinized and rehydrated, then were incubated at room temperature overnight in an anti-TRPC6 antibody. In control experiments, the primary antibody was replaced with PBS. The TRPC6 in immunohistochemistry score was evaluated by a grading system ranging from 0 to 5:0, 0%～1% of tumor cells are positive; 1,1%～5% of tumor cells positive; 2,5%～10% of tumor cells positive; 3,10%～20% of tumor cells positive; 4,20%～50% of tumor cells positive; 5,> 50% of tumor cells positive.6. RT-PCR analysis Total RNA was isolated from cells using Trizol Reagent. Total RNA was then reverse-transcribed into cDNA. For the PCR reaction, specific sense and antisense primers were selected. The bands were quantified densitometrically.7. Western blotTotal proteins from prostate cancer cells were harvested. Protein samples were separated by electrophoresis on SDS-PAGE and transferred to PVDF membranes. Immunoblotting was performed with anti-c-Met, anti-phosphorylation- c-Met, anti-Aktl/2, anti-phosphorylation-Akt1/2, anti-ERK1, anti-phosphorylation- ERK1, anti-HGF-α, anti-TRPC6 and anti-β-actin antibody, then developed with the enhanced chemiluminescence system using specific peroxidase-conjugated anti-IgG secondary antibodies. The bands were quantified densitometrically.8. Silencing of TRPC6Transfection was accomplished using 3 target sequences for TRPC6. FAM-siRNA plasmids and Western blot were used to detect transfection and silence efficiency respectively.9. Intracellular Ca2+ measurementsCells grown on glass bottom cell culture dishes were placed in external solution and loaded with 5μM Fura 2-acetoxy methyl ester (Fura 2-AM) at 37℃. Cells were washed, and dye was allowed to deesterify for 1 h. Fluorescence intensity was recorded over the entire surface of each single cell and intracellular Ca2+ was evaluated from the ratio of the fluorescence emission intensities excited at the two wavelengths.10. Cell cycle analysisCell suspensions were transferred to 95% ethanol while mixing thoroughly and stored at room temperature for 30 min. Cells were washed three times with PBS and then treated with ribonuclease for 15 min. Propidium iodide was added and cells were allowed to incubate for an additional 30 min. Using flow cytometry, cell cycle was measured. 11. Statistical analysisDate analyses were performed with the Statistical Package for Social Sciences software (SPSS11.5). P< 0.05 was regarded as statistically significant.Results1. Construction and identification of eukaryotic expression plasmid. Enzyme analysis and agarose gel electrophoresis showed that there were 1326bp band. The two eukaryotic expression vectors containing pBudCE4.1-EGFP/NK4 were successfully constructed. DU145 cells of transferred NK4 gene expressed NK4 protein.2. MTT showed that HGF-induced cell proliferation was suppressed by autocrine NK4, NK4 alone had no effect on cell proliferation. Cell migration index increased after HGF treatment. Although again, this HGF-induced motility was significantly inhibited by autocrine NK4. Autocrine NK4 suppressed HGF-induced tumor cell invasion.3. Western blot showed that c-Met was detected in DU145 cells. HGF induced phosphorylation of c-Met, ERK1 and Aktl/2. And that was inhibited by autocrine NK4.4. Immunohistochemistry showed that TRPC6 expression in PC was higher than BPH. The increasing staining recorded was associated with the histological grade, Gleason score and extraprostatic extension of prostate cancer. TRPC6 was expressed in the DU145, PC3 and 22Rvl prostate cancer cell lines.5. Western blot revealed a 70%～80% decrease of TRPC6 protein expression in the TRPC6 siRNA1 (siRNA1) and TRPC6 siRNA2 (siRNA2) groups. HGF up-regulated TRPC6 expression. OAG increased [Ca2+]i in the presence of extracellular Ca2+, but not in the absence of extracellular Ca2+. A further experiment showed OAG-induced Ca2+ entry significantly decreased after TRPC6 siRNA. After treatment with HGF, OAG-induced Ca2+ entry increased in DU145 cells. TRPC6 knockdown arrested DU145 cells at G2/M phase. Conclusion1. Genetic modification of DU145 cells with NK4 cDNA yields a significant effect on their proliferation, migration and invasion through inhibiting the HGF-induced phosphorylation of c-Met, ERK1 and Akt1/2. Molecular targeting of HGF/c-Met by NK4 could be applied as a novel therapeutic approach to prostate cancer.2. TRPC6 was expressed in BPH, prostate cancer and, in the DU145, PC3 and 22Rv1 prostate cancer cell lines, and was associated with the cancer’s histological grade, Gleason score and extraprostatic extension status.3. TRPC6 could regulate calcium influx in DU145 cells. Knockdown of TRPC6 channels in DU145 cells suppressed the cell proliferation and arrested cell cycle at G2/M phase. HGF increases the expression of TRPC6, which results in an increase of OAG-induced calcium entry. So the Ca2+ inflow regulated by TRPC6 channels is essential for HGF-induced growth proliferation of prostate cancer.更多还原