【作者】 张晓芳；

【导师】 周庚寅；

【作者基本信息】 山东大学 ， 病理学与病理生理学， 2009， 博士

【摘要】 【研究目的】乳腺癌是女性最常见的恶性肿瘤之一。除手术治疗外,化疗和内分泌治疗是乳腺癌治疗的重要手段。对化疗药物产生多药耐药是导致乳腺癌化疗失败的重要因素。所谓多药耐药是指肿瘤对一种药物产生耐药的同时,对其他结构、作用机制不同的药物也产生抗药性。乳腺癌多药耐药的机制较复杂,而过表达ATP膜转运家族蛋白是重要的因素之一。由多药耐药基因1(mdr1)编码的P-糖蛋白就是该家族的一员。与其它膜转运蛋白相同,P-糖蛋白通过水解ATP产生的能量,将进入细胞内的药物泵出细胞,从而降低药物对细胞的杀伤作用,使细胞产生耐药。目前报道显示许多抗肿瘤药物包括长春新碱、多柔米星、紫杉醇等均为P-糖蛋白的作用底物。50%的乳腺癌耐药与P-糖蛋白的表达相关。因此逆转由P-糖蛋白介导的多药耐药将对乳腺癌的治疗起重要作用。为了逆转P-糖蛋白介导的肿瘤多药耐药,人们采用了许多方法,包括化学药物治疗、天然药物治疗、免疫学治疗、基因治疗等。化学合成的抑制剂可以增加肿瘤细胞对化疗药物的敏感性。但是化学药物在达到治疗浓度时往往具有较大的毒性。选择性阻断耐药蛋白mRNA的表达是一种有效的逆转耐药的方法,本课题组曾经分别应用反义寡核苷酸以及核酶等方法逆转P-gp介导的MDR的研究,各种方法均能在不同程度上抑制P-gp的作用,但未能达到预期的结果;这可能与核酶的抑制效率较低以及反义寡核苷酸和核酶的降解效率受靶基因mRNA二级结构的影响较大,不容易筛选靶序列有关,此外耐药肿瘤细胞中存在其他的机制也可能是逆转效果不理想的原因。近期研究发现,葡萄糖神经酰胺合成酶(GCS)也是引起肿瘤多药耐药的因素之一。GCS催化UDP-glucose上的糖基与神经酰胺相结合,使细胞逃避神经酰胺介导的促凋亡作用。神经酰胺,是由细胞膜神经鞘磷脂水解产生的一种脂质分子,是介导细胞凋亡的第二信使,参与了肿瘤细胞对化疗和放疗的反应。研究发现,GCS糖基化产物葡萄糖神经酰胺(Glucosylceramide,GlcCer)在耐药肿瘤细胞中的表达水平明显升高,抑制神经酰胺的糖基化可以逆转对化疗药物的耐药性。许多化疗药物可以抑制GCS的活性并增加细胞内神经酰胺的合成。如PPPP(1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-proranol)、PDMP(1-phenyl-2-dsdecanoylamino-3-morpholino-1-propanol)和cyclosorin A。但在临床实验中这些药物特异性不高,且具有较强副作用,限制了其应用。RNA干扰技术是新近研究中有效的分析基因功能的工具。其高效的基因沉默功能使其一经发现就表现出强大的生命力。该技术通过导入细胞双链RNA(dsRNA)而导致序列特异性的基因沉默。dsRNA在体内可以被一种具有RNaseⅢ样活性的dsRNA特异性核酸内切酶(Dicer)作用切割成21-23bp长的小干扰RNA(siRNA)。siRNA接着与RNA诱导的沉默复合物(RNA-induced silencing complex,RISC)结合,特异性识别和切割目的mRNA。由于RNAi具有特异性和高效性,并且和反义寡核苷酸相比,siRNA介导的基因沉默更有效,持续时间更长,因此,我们拟应用携带RNA干扰序列的重组质粒载体同时沉默乳腺癌中mdr1基因和GCS基因的表达,从而提高乳腺癌多药耐药的逆转效率。【研究方法】1.分别设计并合成针对mdr1基因和GCS基因的双链干扰RNA序列,将其定向插入到质粒载体pSUPER中,经酶切及测序鉴定。然后选用合适的酶切位点酶切,获得含有RNA干扰序列及其上游的H1启动子的片段,并将其插入到含有绿色荧光蛋白(EGFP)和稳定筛选标记的质粒载体pEGFP-C1中,构建稳定表达载体pEGFP-MDR1和pEGFP-GCS。酶切及测序鉴定正确后,大量扩增。2.应用脂质体将上述干扰质粒载体同时转染到高表达P-糖蛋白和GCS的乳腺癌细胞系MCF-7/ADM中,并设空白对照组及仅转染一种干扰质粒组。培养48h后,通过RT-PCR检测各组细胞中mdr1 mRNA和GCS mRNA的表达情况,应用western blot检测P-糖蛋白和GCS蛋白表达情况,应用罗丹明外排实验检测P-糖蛋白外排功能,应用MTT法检测各组细胞对阿霉素、长春新碱的半数细胞抑制浓度(IC50)的改变。3.各组细胞转染不同载体后,继续培养48h,加用G418对其进行筛选。约2周后得到稳定表达干扰RNA序列的细胞系。继续培养3周后,通过RT-PCR检测各组细胞中mdr1 mRNA和GCS mRNA的表达情况,应用western blot检测P-糖蛋白和GCS蛋白表达情况,应用罗丹明外排实验检测P-糖蛋白外排功能,应用MTT法检测细胞耐药指数的改变。从而对RNA干扰的长期逆转效率进行评价。【实验结果】1.转染48h后,应用半定量RT-PCR检测各组细胞中mdr1 mRNA和GCS mRNA的表达情况,结果显示:与MCF-7/ADM相比(我们假定MCF-7/ADM组的相对mRNA水平为100%),M0组(转染质粒pEGFP-MDR1)、G0组(转染质粒pEGFP-GCS)、GM0组(共转染质粒pEGFP-MDR1和pEGFP-GCS)的mdr1 mRNA的表达水平分别降为1.105±0.99%、38.75±9.7%和0.4491±0.22%,而GCS mRNA的表达水平分别降为7.464±3.0%、38.34±16%and 3.142±0.64%,显著低于MCF-7/ADM组及对照组(P＜0.01)。进一步分析发现,GM0细胞的MDR1 mRNA和GCS mRNA表达水平也低于单独干扰组。Western blot检测结果表明GM0组细胞两种蛋白水平明显减低,且显著低于M0和G0组。罗丹明外排实验显示:耐药细胞的荧光强度为2.727%±0.4834,G0、M0细胞荧光强度分别为33.71%±2.267和17.25%±1.024,而GM0组细胞荧光强度为50.12±6.349。RNA干扰组细胞与MCF-7/ADM组相比,差异具有统计学意义(P＜0.01)。而GM0组与G0、M0组相比,P-gp功能显著降低。MTT法检测细胞对化疗药物的耐受性,发现G0、M0、GM0组细胞对阿霉素和长春新碱耐药性明显降低,GM0组明显低于G0和M0组。此外分析结果可以发现,抑制mdr1基因的表达会引起GCS基因表达的降低,而下调GCS基因也会导致mdr1基因表达下调。2.筛选出稳定表达细胞系后,培养3周,应用半定量RT-PCR检测各组细胞中mdr1 mRNA和GCS mRNA的表达情况,结果显示:M1组(转染质粒pEGFP-MDR1)、G1组(转染质粒pEGFP-GCS)、GM1组(共转染质粒pEGFP-MDR1和pEGFP-GCS)mdr1 mRNA的表达水平分别降为MCF-7/ADM组的46.04±3.8%、13.39±0.69%和0.6715±0.71%,而GCS mRNA的表达水平分别降为64.11±2.7%、25.28±5.9%and 4.321±2.7%,显著低于MCF-7/ADM组(P＜0.01)。且GM1细胞的MDR1和GCS mRNA表达水平也低于单独干扰组。而与瞬时转染组相比,mRNA水平无明显差异。Western blot检测结果表明GM1细胞两种蛋白水平明显减低,且显著低于M1和G1组。罗丹明外排实验显示:G1、M1、GM1细胞荧光强度均较MCF-7/ADM细胞内荧光强度显著增强,而GM1组荧光强度明显高于G1和M1组。三组细胞与瞬时转染组相比无明显差异。MTT法检测细胞对化疗药物的耐受性,发现G1、M1、GM1组细胞对阿霉素和长春新碱耐药性明显降低,与瞬时转染组相比差异不大。【结论】1.RNA干扰可以通过抑制靶基因mRNA的表达,从而显著降低蛋白的产生。2.瞬时转染结果表明抑制mdr1基因或GCS基因可以有效逆转乳腺癌多药耐药,而同时抑制mdr1基因和GCS基因比单独抑制更有效。3.稳定转染后检测结果显示携带RNA干扰片度的质粒可以较长时间抑制靶基因的表达(至少3周)。4.实验结果表明,mdr1基因表达下调可以引起GCS基因的表达部分抑制,反之亦然,说明乳腺癌中mdr1基因与GCS基因可以相互影响,发挥协调作用。更多还原

【Abstract】 【Background】Breast cancer is one of the most common malignant tumor of women.In addition to surgical treatment,chemotherapy and endocrine therapy are important tools in breast cancer treatment.However,multidrug resistance(MDR) to chemotherapeutic agents is a major obstacle to successful chemotherapy in patients with breast cancer. The so-called multidrug resistance refers to that when a kind of tumor become resistent to one drug,it will also become resistent to other drugs which have different structure and mechanisms.The mechanism of MDR in breast cancer is complex,while overexpression of the members of the adenosine triphosphate(ATP)-binding cassette(ABC) membrane transporter family are one of the most important factors.P-glycoprotein(P-gp),coded by multidrug resistance gene-1(mdrl),as other members of ABC membrane transporter family,acts as a drug efflux pump,lowers intracellular drug levels to sublethal concentrations and helps cells to escape from death.It has been reported that notably natural product anticancer agents,including vinca alkaloids,anthracyclines (daunorubicin,adriamycin),and taxanes are the substrates of P-gp and P-gp covers 50%of MDR in breast cancer.So reversing MDR mediated by P-gp will play an important role in the therapy of breast cancer.In order to reverse MDR mediated by P-gp,a number of methods were adopted, including chemical treatment,natural drug therapy,immunology treatment and gene therapy.Chemical synthesis of inhibitors can increase tumor cell sensitivity to chemotherapeutic drugs.However,chemical drugs at therapeutic concentrations often have greater toxicity.At present,it is thought that inhibiting resistance protein mRNA expression selectively may be an effective stragedy to reverse drug resistance.Our group once applicated antisense oligonucleotide as well as ribozyme to reverse P-gp-mediated MDR and the methods were able to inhibit P-gp at different levels,but failed to achieve the desired results.This may be related to the following factors:First, ribozyme has less efficient inhibition of target gene;Second,the degradation of ribozyme and antisense oligonucleotides were limited by the secondary structure of target gene mRNA,so it is difficult to select efficient sequence;Third,drug-resistant tumor cells exist other mechanisms.Recent study reveals that glucosylceramide synthase(GCS) may be one of the factors which could cause tumor multidrug resistance.Glucosylceramide synthase (GCS) allows cells to escape from ceramide-induced cellular apoptosis by mediating ceramide glycosylation.Some researches revealed that accumulation of glucosylceramide(GC),the product of the glycosylation event,is a characteristic of some multidrug-resistant cancer cells and tumors derived from patients who are less responsive to chemotherapy.Many chemotherapy drugs can inhibit the activity of GCS and to increase intracellular ceramide synthesisRNA interference is a new and effective research tool for gene function analysis by introducing double-stranded RNA into cells and leading to the sequence-specific destruction.RNAi can be induced by double stranded RNA(dsRNA),which is cleaved into 21-23bp RNA fragment,knowm as small interference RNA(siRNA),by an RnaseⅢ-like enzyme,Dicer.Then,siRNA is incorporated into a protein complex called RNA-induced silencing complex(RISC) which recognizes and cleaves mRNA in a sequence-specific manner.Because RNAi has the specificity and efficiency,and compared to antisense oligonucleotide,siRNA-mediated gene silencing is more effective and longer duration,we planned to co-suppress the mdrl and GCS gene by plasmids with RNA interference sequences to improve the efficiency of MDR reverse in breast cancer.【Methods】1.The siRNA sequence targeted to mdrl or GCS were designed and inserted into plasmid vector pSUPER,then confirmed by enzyme digestion and DNA sequencing. After that,RNA interference sequence and H1 promoter sequence were obtained by digesting the plasmids by appropriate restriction enzymes and then inserted into pEGFP-C1 with Enhanced Green Fluorescence Protein(EGFP) and stable selective marker.After comfirmed by enzyme digestion and DNA sequencing,the plasmids were amplified.2.Co-transfection of the plasmids with RNA interference sequences into the cell lines MCF-7/ADM which over-expression P-gp and GCS,meanwhile,the blank control group and only a kind of interference plasmid transfection group were set up, respecively.After another 48h culture,RT-PCR was adopted to detected the expression of mdrl mRNA and GCS mRNA,western blot was used to analysis the P-gp and GCS protein.The function of P-gp was evaluated by Rhodamine 123 (Rh123) effiux assay.MTT method was performed to evaluate the 50%inhibition of concertration of adriamycin and vincristine.3.After transfection with different vectors,G418 was used to select stable expression cell lines.And after 2 weeks,cell lines which stably express RNA interference sequences were established.After cultured for another 3 weeks,RT-PCR was adopted to detected the expression of mdrl mRNA and GCS mRNA,western blot was used to analysis the P-gp and GCS protein.The function of P-gp was evaluated by Rhodamine 123(Rh123) efflux assay.MTT method was performed to evaluate the 50%inhibition of concertration of adrimaycin and vincristine.Through the above experiments,the efficiency of the long-term reversal by RNA interference was evaluated.【Results】1.After transfecion for 48h,RT-PCR was adopted to detected the expression of mdrl mRNA and GCS mRNA,and the result demonstrated that compared with MCF-7/ADM cells(we considered the relative mRNA levels in MCF-7/ADM cells as 100%),the relative expression of mdrl mRNA in G0,M0 and GM0 decreased to 1.105±0.99%,38.75±9.7%and 0.4491±0.22%,respectively,and that of GCS mRNA decreased to 7.464±3.0%,38.34±16%and 3.142±0.64%,respectively,significantly lower than that of the controls(P<0.01).From further analysis,we found that the mRNA levels in the cells GM0 were much lower than that of G0 or M0(P<0.05).The results of western blot showed that the levels of the two proteins were significantly decreased in the cells GM0 and that the levels of the two proteins in GM0 cells were signifiantly lower than that in G0 or M0 cells.Rh123 effiux test indicated that the fluoresence intensity of the resistence cell lines were 2.727%±0.4834,and that of G0 or M0 were 33.71%±2.267 and 17.25%±1.024,while that in GM0 were 50.12±6.349. Compared to that in the resistence cell lines,the function of P-gp in GM0 down-regulated significantly.MTT method showed that the drug resistence index of G0,M0 and GM0 to adriamycin and vincristine declined significantly,while that of GM0 was also much lower than that of G0 and M0.Furthermore,down-regulated of mdrl could induce decrease of GCS while inhibition of GCS also induced suppression of mdrl.2.After stable cell lines were seleced,RT-PCR was used to detect the expression of mdrl mRNA and GCS mRNA,and the result demonstrated that the relative expression of mdrl mRNA in G1,M1 and GM1 decreased to 46.04±3.8%、13.39±0.69%and 0.6715±0.71%,respectively,and that of GCS mRNA decreased to 64.11±2.7%、25.28±5.9%and 4.321±2.7%respectively,significantly lower than that in MCF-7/ADM(P<0.01).From further analysis,we found that the mRNA levels in the cells GM1 were much lower than that of G1 or M1(P<0.05),while that had no significant differences with the corresponding transient transfection groups.The results of western blot showed that the levels of the two proteins were significantly decreased in the cells GM1 and that the levels of the two proteins in GM1 cells were signifiantly lower than that in G1 or M1 cells.Rh123 efflux test indicated that the fluorescence intensity of G1,M1 and GM1 cell lines all decreased compared to that of MCF-7/ADM.Compared to that in the resistence cell lines,the function of P-gp in RNAi group down-regulated significantly.MTT method displayed that the drug resistence of G1,M1 and GM1 to adriamycin and vincristine declined significantly and there were no significant differences between the stable transfection groups and the corresponding transient -transfection cells.【Conclusions】1.RNA interference could significantly decrease the production of protein by inhibition the expression of mRNA.2.The result of transient transfection demonstrated that suppression of mdrl or GCS gene could efficiently reverse MDR in breast cancer,while co-suprression of both mdrl and GCS was more efficient than inhibiting alone.3.The result of stable transfection showed that the plasmids with RNA interference sequences could inhibit the expression of target gene for a long period(at least for 3 weeks).4.The result of the experiment indicated that down-regulated of mdrl could induce decrease of GCS while inhibition of GCS also induced suppression of mdrl which revealed that mdrl and GCS could affect each other in breast cancer.更多还原