【作者】 戴元清；

【导师】 齐琳；

【作者基本信息】 中南大学 ， 泌尿外科， 2011， 博士

【摘要】 研究背景前列腺癌是男性生殖系统最常见肿瘤,在美国肿瘤死亡率位居第二。近年来,我国前列腺癌的发病率呈明显上升趋势。当前,在经典的内分泌治疗广泛的应用的同时,晚期雄激素难治性前列腺癌(HRPC)的治疗仍就是一大难题,目前前列腺癌晚期的化疗方案以多西紫杉醇、米托葸醌等为代表,虽取得一定疗效,但肿瘤缓解率低,副作用较多等使之不能成为最理想的治疗方法。因此,探索和研究新的治疗策略,具有十分重要的临床意义。研究表明,Ackl(activated Cdc42associated kinase1)对恶性肿瘤的生长非常关键,并且作为一种关键转导蛋白,在许多肿瘤相关信号通路,诸如整联蛋白、EGF、PDGF信号通路等中发挥重要作用。它通过酪氨酸磷酸化作用调解着许多蛋白分子,而这些分子对于肿瘤细胞的生长、增殖以及迁移都至关重要。P130CAS适配器蛋白通过介导蛋白复合物的形成在细胞信号通路中发挥重要作用。小RNA干扰技术(RNAi)已经成为近年来发展起来的高效特异的阻断基因表达的新手段,具有广泛的应用前景。尽管已有很多研究涉及Ackl及P130CAS,但其在前列腺癌中的准确机制远未阐明,有待进一步研究。有鉴于此,我们拟通过实验研究Ackl以及P130CAS在前列腺癌细胞株表达情况及可能的相互关系；再构建Ackl基因敲减干扰细胞株的稳定模型,对沉默Ack后的PC-3细胞株进行相关的细胞生物学实验研究及药物干预实验研究,研究AckI蛋白基因敲减后PC-3细胞的生物学行为的改变,以及对药物干预的影响,借以充分挖掘AckI以及P130CAS在前列腺癌的发生发展以及药物治疗方面的作用。研究方法1.采用Western blot及RT-PCR方法检测AckI及P130CAS mRNA和蛋白在激素依赖性前列腺癌细胞株LNCaP及激素非依赖性前列腺癌细胞株PC-3中的表达水平。2.利用慢病毒介导的RNA干扰技术试行构建逆转录shRNA干扰AckI质粒,转染激素非依赖性前列腺癌细胞株PC-3细胞,干扰PC-3细胞AckI蛋白的表达,构建低表达蛋白的模型,并通过荧光显微镜观察、PCR双酶切和免疫印迹对其验证。3.体外采用MTT实验、平板集落形成实验、划痕愈合实验、Transwell实验等方法以及研究沉默后相关小G蛋白家族以及P130CAS蛋白的表达情况,从体外层面进行AckI基因表达调节作用机制研究。4.观察shRNA沉默AckI基因对PC-3细胞多西紫杉醇化疗敏感性的影响,实验分为PC-3+DTX组、PC-3+NC-shRNA+DTX组和PC-3+AckI-shRNA+DTX三组,每组另外设一个未化疗的平行对照组。转染后24小时起给予终浓度为0.25μM多烯紫杉醇,MTT法测各组细胞生长增殖变化；流式细胞术检测细胞的凋亡率和细胞的周期分布。研究结果1. Western blot及RT-PCR结果显示AckI与P130CAS mRNA和蛋白两种侵袭潜能不同的前列腺癌细胞系中均有表达,AckI蛋白相对表达水平为(1.51±0.10VS0.22±0.09),P130CAS为(1.09±0.11VS0.51±0.10),差异有显著差异(P<0.05)；AckI mRNA相对表达水平为(1.22±0.15VS0.12±0.11),P130CAS为(1.19±0.21VS0.32±0.13),差异有显著差异(P<0.05)。结果提示AckI与P130CAS的高表达与前列腺癌细胞的侵袭转移潜能可能相关。2.结果显示AckI的低表达真核细胞载体构建成功,完成了AckI特异性重组慢病毒载体质粒的包装并完成了病毒体的包装与浓缩。通过倒置荧光显微镜观察,转染效率约为75%；通过WB实验观察,AckI基因的表达量明显下调。3.体外细胞研究结果显示敲减AckI的表达可以抑制PC-3细胞的侵袭迁移。划痕实验比较了PC-3细胞AckI基因敲减前后的迁移能力,结果显示敲减后较未干预的细胞迁移能力明显下降,24小时划痕愈合率分别为34%VS89%,差异具有显著性意义(P<0.05)。采用Transwell侵袭小室测定PC-3细胞AckI基因敲减后侵袭能力的不同,结果显示PC-3细胞在AckI基因敲减后侵袭能力明显下降,24小时培养后穿过Transwell的每低倍视野细胞数分别为21.31±4.15VS12.15±3.13,差异具有显著性意义(P<0.05)。而且,AckI表达沉默的PC-3细胞后蛋白的表达为：下调AckI的表达的同时P130cas蛋白的表达下降,上调了Rac1的表达,而cdc42、RhoA、RhoC则无明显改变。P130cas蛋白表达下调(0.94±0.13VS0.32±0.11),Rac1蛋白表达明显上调(0.29±0.08VS0.67±0.10),差异具有统计学意义(P<0.05),cdc42、RhoA、 RhoC蛋白表达无变化(cdc42,0.61±0.14VS0.58±0.19；RhoA,0.67±0.15vs0.58±0.15;RhoC,0.55±0.15vs0.54±0.06).差异具无统计学意义(p>0.05)。表明Ackl可能通过P130cas、Rac1与其下游蛋白进一步作用。AckI可能通过反应性下调Rac1蛋白,上调P130cas蛋白表达在激素非依赖性前列腺癌细胞凋亡中起重要的作用机制。4.化疗48小时后PC-3+AckI-shRNA+DTX组细胞存活数明显低于PC-3+DTX组(?)(?)PC-3+NC-shRNA+DTX组(P<0.05),两化疗对照组间无显著性差异。化疗后48小时PC-3+DTX组、NC-shRNA+DTX组、PC-3+AckI-shRNA+DTX凋亡率分别为23.79%,14.96%以及15.10%,PC-3+AckI-shRNA+DTX组凋亡率高于对照组(P<0.05)。流式细胞仪测定化疗后48小时各组PC-3细胞的周期分布,结果显示未化疗的AckI-shRNA转染组PC-3细胞发生了G0/G1期阻滞解除。结论AckI和P130CAS在Pca细胞中存在高表达,其之间发生相互作用,可能形成复合物在Pca的形成的相关信号通路中担当重要角色,与Pca侵袭性相关。成功通过构建shRNA干扰质粒沉默AckI基因,并发现AckI的表达下调后Pca细胞的增殖、迁移和侵袭能力下降。AckI可能通过调控P130CAS以及Racl的表达从而实现促进Pca细胞生长与演进。AckI-shRNA转染能增加PC-3细胞对多西紫杉醇化疗的敏感性。沉默AckI基因对PC-3细胞多烯紫杉醇的化疗增敏作用与凋亡率升高及G0/G1期阻滞解除有关。更多还原

【Abstract】 BackgroundProstate cancer is the most common malignancy generated from the male reproductive system. In the United States, it is the second cause of cancer mortality which is only less than lung cancer. During recent years, detection rate of prostate cancer has been increasing in China. Currently, endocrine therapy is a very effective method for ADPC, however, HRPC is still a therapeutic challenge not to overcome. Docetaxel and Mitoxantrone are the representative drugs for the advanced prostate cancer. Although these drugs, to some extent, can postpone the progression of the disease, it is still not an ideal, effective therapy for HRPC. Therefore, to explore and study novel therapeutic strategies for AIPC is of clinical significance.AckI(activated Cdc42associated kinase1) has been shown to be critical for malignant tumors growth, and to be a key transducer in several cancer-related signaling pathway such as integrins, EGF, PDGF pathways and so on. It regulates activity of a number of proteins by tyrosine phosphorylation especially proteins critical for cell migration, cell growth, and proliferation. P130CAS adaptor proteins play an important role during cellular signaling by mediating the formation of protein complexes. A new very efficient and special tool for gene knockdown, RNAi(RNA interference) technology, has developed rapidly and has a wide application prospect during recent years. Indeed, the detailed mechanism of AckI and p130cas in prostate cancer remain far from clear and is needed to be investigated, though many studies have shown some mechanism about it.. In view of this, we carried out the present study to investigate the expression of AckI and p130cas in Pca as well as the role of it in the growth and metastasis of Pca and the possible underlying molecular mechanism. By using plasmid transfection and RNA interference(shRNA), as well as a series of in vitro assays, we got results as follows.Methods and ResultsFirst of all, Western blot and RT-PCR methods were used to examine the expression levels of AckI and P130CAS in androgen-dependent prostate cancer(ADPC) cells LNCaP and andandrogen-independent prostate cancer(AIPC) PC-3cells. As a result, the expression levels of AckI and P130CAS was lower in LNCaP than PC-3with significant difference(P<0.05), indicating that AckI and P130CAS may play an important role in the development of prostate cancer.Second, by the aid of the Technology of RNA interference via lentivirus which Can transcribe a short hairpin RNA against AckI, we have successfully generated AckI knockdown PC-3cells lines. The interfection result was confirmed by western bolt and RT-PCR and fluorescence microscopy was used to determine the fluorescence.Third, Cell proliferation was quantitated by the3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl-tetrazolium Bromide (MTT) assay and Colony formation assay, Cell migration was measured by in vitro wound healing assay and transwell healing assay. As a result, Inhibition of the expression of Ackl lead to decrease of the invasion ablitiy of PC-3cells migration in vitro. The results of scratch healing showed that migration ability of PC-3after AckI knockdown was significantly impaired,24-hour scratch healing rates of PC-3cells before and after AckI knockdown were34%VS89%, the difference was significan(P<0.05). Different invasive ability were found in pc-3cells before and after AckI knockdown by Transwell assay. After24hours cell culture, cell count of permeate through Transwell(10w power field)were21.31±4.15VS12.15±3.13, difference was significant(P<0.05). These results indicate that in vitro inhibition of AckI expression in PC-3cells can inhibit the invasion and metastasis of prostate cancer.We further detected the expression of cdc42, RhoA, RhoC, Racl, P130CAS protein levels in PC-3cells respectively after AckI gene knockdown. Down-regulating the expression of AckI-silenced in PC-3cells lead to down-regulated expression of p130cas protein expression (0.94±0.13VS0.32±0.11), up-regulated expression of Rac1(0.29±0.08VS0.67±0.10),with statistically significant difference(p<0.05).No statistically significant difference was found in cdc42, RhoA and RhoC protein expression(cdc42,0.61±0.14VS0.58±0.19; RhoA,0.67±0.15vs0.58±0.15; RhoC,0.55±0.15vs0.54±0.06).In the end, we evaluated the influence of knocking down of AckI gene with shRNA on the chemosensitivity of Docetaxel in PC-3cell line. PC-3cell was divided into three groups as previous study, Docetaxel was given as final concentration of0.25μM at24h after transfection. MTT test was used to measure the proliferation of PC3cell. Flow cytometer was used to measure cell apoptosis, cycle distribution. Inhibition ratio of PC3+AckI-shRNA+DTX group is higher than PC3+DTX group and PC3+NC-shRNA+DTX group (P<0.05, at48h), while there was no difference between two control groups(P>0.05). Cell apoptosis rate of PC3+AckI-shRNA+DTX group, PC3+DTX group and PC3+NC-shRNA+DTX group was23.79%,14.96%and15.10%respectively at48h after docetaxe1given. The apoptosis rate of PC3+AckI-shRNA+DTX group was the highest (P<0.05). Flow cytometry analysis showed there was less G0/G1phase arrest in PC3+AckI-shRNA group than two control groups without chemotherapy.Conclusions Our experiments have demonstrated AckI was overexpressed in AIPC cell and played an important role in the invasion and metastasis of Pca. we eidentified that P130cas was involved in AckI and AckI may promote invasion and metastasis of human prostate cancer via regulating P130cas and Racl. What’s more, AckI-shRNA can enhance chemosensitivity of docetaxel in PC-3cell in vitro by knocking down of AckI gene expression. The possible mechanisms involved in chemosensitization of AckI-shRNA include increasing apoptosis rate and releasing cell cycle arrest of G0/G1. Our findings suggest AckI as a novel prognostic marker and a potential therapeutic target for treatment of Pca.更多还原