RNA干扰抑制Skp2表达对人喉癌细胞系Hep-2影响的实验研究
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摘要
研究背景与目的
喉癌是危害人体健康和生命的恶性肿瘤,约占耳鼻咽喉恶性肿瘤的10-35%,其中以喉鳞状细胞癌(LSCC)最为多见,可达90%。喉癌在我国东北地区是高发区,而且逐年增长。目前,喉切除术是治疗喉癌的主要手段,但术后对患者的生活质量会产生较大影响,且对于晚期及全身状态不佳的患者无法有效实施,虽可辅以放疗及化疗,效果仍不理想。因此,深入探讨喉癌的发生机制,寻找利用基因干预的手段进行治疗是目前喉癌研究的热点。
Skp2基因是最近发现的癌基因,与肿瘤的增殖、分化有关。在对喉癌的研究中发现,Skp2表达与临床分期及淋巴结转移正相关,提示该基因表达上调可能与LSCC的侵袭转移能力密切相关；同时,其表达与抑癌基因p27表达强度呈明显负相关,表明在LSCC的进展过程中,Skp2表达的上调可能通过泛素—蛋白酶体途径导致p27表达减少,使其不能有效发挥细胞周期负调控因子的抑癌作用,促使癌细胞获得更高的侵袭转移能力。因此,我们推测Skp2可能是喉部恶性肿瘤基因治疗的靶点。
RNA干扰(RNA interference, RNAi)是新近发展起来的一种封闭基因表达的有效方法,它比反义寡核苷酸技术能更高效地抑制目的基因的表达。已有研究证实化学合成siRNA (small interfering RNA)能高效地介导RNAi作用,但作用持时间短,而采用siRNA表达载体尤其是慢病毒载体的方法能有效延长作用时间。
本研究试图通过细胞体外及动物体内试验,利用慢病毒介导的RNA干扰技术抑制癌基因Skp2的表达,观察其对人喉癌细胞系Hep-2细胞的增殖、调亡以及对抑癌基因p27表达的影响,为今后应用该技术研究和治疗喉癌提供实验依据。
方法
1、针对人Skp2基因的RNAi载体构建
利用计算机辅助设计软件,设计合成四条针对Skp2基因cds区的siRNA序列,同时设计一条对于任何基因无干扰效果序列Negative用于对照组实验,并将其定向克隆入真核表达载体pSIHl-Hl-copGFP shRNA Vector,获得五个重组质粒：pSIHl-negative、pSIHl-siRNA1、pSIH1-siRNA2、pSIHl-siRNA3、pSIH1-siRNA4。
2、抑制Skp2表达对人喉癌细胞系Hep-2影响的体外实验
(1)在Hep-2细胞中针对Skp2基因筛选有效的干扰序列：利用脂质体将重组质粒转染Hep-2细胞,荧光实时定量PCR实验检测转染前后细胞中的Skp2 mRNA的变化,根据结果选择一个含有最强抑制效果的干扰序列的质粒用于后续试验；
(2)用慢病毒系统建立干扰Skp2基因的稳定细胞株：以三载体包装的含有筛选出的pSIH-siRNA3和pSIH-negative质粒的慢病毒原液感染Hep-2细胞,观察转染效果,获得Hep2-siRNA, Hep2-neg两组稳定转染细胞株；
(3)荧光实时定量PCR检测稳定转染细胞株中Skp2和p27 mRNA的表达；
(4) Western blot法检测Hep2, Hep2-siRNA, Hep2-neg三组细胞中的Skp2和p27蛋白表达量的变化；
(5)MTT法检测Hep2, Hep2-siRNA, Hep2-neg三组细胞的增殖情况；
(6)流式细胞仪检测三组细胞凋亡情况。
3、抑制Skp2表达对人喉癌细胞系Hep-2影响的体内实验
(1)裸鼠随机分成三组,将三组细胞按照1.0×107个/ml的浓度,每只鼠0.2ml背部皮下接种；
(2)观察裸鼠的成瘤情况,30天后处死裸鼠,经皮下剥离肿瘤组织,比较各组瘤体大小和瘤重；
(3)将瘤组织切片进行常规病理检查,并采用免疫组化方法对三组裸鼠瘤组织中Skp2和p27蛋白的表达进行检测。
1、利用PCR分析,DNA测序证实靶向Skp2特异性siRNA表达载体pSIHl-negative、pSIHl-siRNA1、pSIHl-siRNA2、pSIH1-siRNA3、pSIH1-siRNA4重组质粒已成功构建。
2、五组重组质粒转染Hep-2细胞后对Skp2 mRNA抑制率分别为12%,42%,14%,46%和29%,但细胞转染效率不高,仅为50%左右；重组质粒pSIH1-siRNA3对Hep-2细胞Skp2基因的抑制作用最强。
3、应用慢病毒包装系统包装pSIH1-negative和pSIHl-siRNA3质粒后以病毒原液感染Hep-2细胞,转染效率可提高至90%左右,且转染细胞株可稳定传代培养。
4、应用荧光实时定量PCR检测Hep2, Hep2-siRNA, Hep2-neg三组细胞中的Skp2和p27 mRNA表达量的变化,显示Hep2-siRNA细胞中Skp2 mRNA表达量与Hep2组细胞中的表达量相比下调了74%,而p27 mRNA表达量则上调了38%,Hep2-neg组无明显变化。
5、Western blot法检测Hep2, Hep2-siRNA, Hep2-neg三组细胞中的Skp2和p27蛋白表达量的变化,显示Hep2-siRNA细胞中Skp2蛋白表达量与Hep2组细胞中的表达量相比下调了约76%,p27蛋白表达量则上调了约89%,Hep2-neg组无明显变化。
6、MTT法检测细胞生长增殖情况结果显示Hep2-siRNA组细胞增殖明显减慢,而Hep2-neg组细胞与Hep2组相比,细胞增殖无明显变化。
7、流式细胞仪检测结果显示Hep2, Hep2-neg和Hep2-siRNA三组细胞凋亡率分别为(2.86±0.21)%,(3.58±0.16)%和(16.64±0.17)%, Hep2-siRNA组与其他两组比较细胞凋亡率明显增加,Hep2-neg组和Hep2组之间无显著差异。
8、裸鼠成瘤实验显示与两对照组相比,转染阳性质粒组细胞裸鼠致瘤活性明显降低,生长减慢,瘤体体积明显较小；而转染阴性质粒组细胞裸鼠致瘤性及生长活性与正常培养组相比无明显差异。接种后30天转染阳性质粒组瘤体重量较对照组明显减轻,抑瘤率为79.55%；而转染阴性质粒组细胞裸鼠致瘤体重量与正常培养组比较无明显差异。
9、荷瘤裸鼠瘤体HE染色切片结果显示正常培养组及转染阴性质粒组肿瘤组织中央可见坏死灶,癌细胞增生活跃,癌巢形成明显,瘤组织以实质为主,间质少见；转染阳性质粒组瘤体中央坏死减少,肿瘤组织间质较多见。正常培养组及转染阴性质粒组比较,瘤体病理学特征无明显差异。
10、荷瘤裸鼠瘤体免疫组化结果显示正常培养组及转染阴性质粒组肿瘤组织中Skp2蛋白高表达,p27蛋白表达显著下调；转染阳性质粒组裸鼠瘤体中Skp2蛋白表达明显减弱,p27蛋白表达显著上调。
结论
1、针对Skp2基因设计的慢病毒介导的siRNA表达载体能从mRNA和蛋白水平特异、高效地抑制人喉癌细胞系Hep-2中癌基因Skp2的表达,并上调抑癌基因p27的表达；同时,抑制肿瘤细胞的恶性增殖,增加其凋亡率。
2、在喉癌的发生发展中,Skp2与p27的表达呈明显负相关,靶向Skp2基因的siRNA对Hep-2细胞的抑制作用可能是通过上调p27基因水平来实现的。
3、利用RNA干扰技术抑制Skp2的表达有望成为喉癌基因治疗的合理策略之一。
Research Background and Objective
Laryngeal carcinoma is a kind of malignant tumor which does great harm to human health and life. And it accounts for about 10-35% in all ENT malignant tumors. Among them, laryngeal squamous cell carcinoma (LSCC) is the most frequent one, and it accounts for 90%. The morbidity of LSCC in northeast is highest in China, and increases year by year. Laryngectomy is the main method for LSCC treatment. Nevertheless, satisfactory treatment results have not been achieved even though by laryngectomy accompanied with radiotherapy and/or chemotherapy, because of poorer quality of life after treatments and their limitation to deal with some patients in advanced stage and/or worse general body state. Therefore, to research the pathological mechanism of LSCC, and to further investigate gene therapy method are focal points in present LSCC research.
As a recently discovered gene, Skp2 is related to proliferation and differentiation. It has been discovered in previous laryngeal carcinoma researches that Skp2 expression was positive correlated to clinical stage and lymph node metastasis. And it indicated that increased expression of Skp2 gene was probably related to invasion and metastasis of LSCC. Moreover, significant negative correlation is discovered between the expression of Skp2 gene and p27 antioncogene. And it indicated that increased expression of Skp2 maybe lead to decreased expression of p27 which as a negative regulatory factor in cell cycle through ubiquitin-proteasomes pathway in LSCC development. Therefore, the anticancer ability of p27 gene was inhibited, and cancer cells got better invasion and metastasis ability. To sum up, it is suspected that Skp2 probably is the target for the gene therapy of laryngeal malignant tumor.
RNA interference is a recently developed method to block gene expression, which provides better expression inhibition of target gene than antisense oligonucleotides ribozyme technique. It is confirmed that chemosynthesized small interfering RNA (siRNA) was able to efficiently mediate RNA interference effect, but its active duration was short. Method applied siRNA expression vector especial lentivirus vector could prolong active duration effectively.
We aimed to observe the influence of inhibition of Skp2 expression with RNA interference technique by lentivirus on proliferation and apoptosis of human laryngeal carcinoma cell line Hep-2 and p27 expression in cell experiments in vivo and animal experiments. And we suppose to provide experimental foundation for further research and treatment of laryngeal cancer.
Methods
1. RNAi vector construction for human Skp2 gene
Four siRNA sequences of Skp2 gene cds region and a negative sequence were designed with computer-aided software. The sequences were directed cloned in eukaryotic expressive pSIHl-Hl-copGFP shRNA Vector. And five recombinant plasmids such as pSIHl-negative, pSIHl-siRNAl, pSIHl-siRNA2, pSIHl-siRNA3, pSIH1-siRNA4 were got.
2. Inhabitation of Skp2 expression of human laryngeal carcinoma cell line Hep-2 in vivo
(1) To select effective interference consequence targeted to Skp2 gene in Hep-2 cell:Recombinant plasmids were transfected into Hep-2 cell with liposome. Skp2 mRNA before and after transfection was detected with fluorescent realtime quantitated PCR, and the best inhibited interference consequence was selected for following experiments.
(2) To construct the stable cell line interfered Skp2 gene with lentivirus system: Hep-2 cells were infected with lentivirus stock solution of tri-vehicle package contained selected pSIH-siRNA3 and pSIH-negative plasmids. The transfected effects were observed, and two stable Hep2-siRNA, Hep2-neg transfected cell lines were got.
(3) To detect mRNA expression of Skp2 and p27 in stable transfected cell lines with fluorescent realtime quantitated PCR.
(4) To detect protein expression of Skp2 and p27 in Hep2, Hep2-siRNA, Hep2-neg cells with Western blot.
(5) To detect the proliferation of Hep2, Hep2-siRNA, Hep2-neg cells with MTT.
(6) To detect the apoptosis of Hep2, Hep2-siRNA, Hep2-neg cells with flow cytometer.
3. The effect of Skp2 expressive inhibition on human laryngeal carcinoma cell line Hep-2 in vitro
(1) Athymic mice were divided into three groups. And cells of three groups were subcutaneous inoculated into each mouse back in dosage of 1.0×107个/ml.
(2) Carcinoma development was observed. Athymic mice were executed after 30 days, and carcinoma tissues were separated subcutaneously. The size and weight of carcinoma were compared.
(3) Regular pathologic examinations were performed with histological section of carcinoma. The protein expression of Skp2 and p27 were detected in athymic mice carcinoma of three groups with immunohistochemistry.
Results
1. pSIHl-negative, pSIHl-siRNAl, pSIHl-siRNA2, pSIHl-siRNA3 and pSIHl-siRNA4 recombinant plasmids targeted to Skp2 specific siRNA were successfully constructed with PCR and DNA sequencing.
2. Inhibition ratios of Skp2 mRNA in Hep-2 cells after transfected by five recombinant plasmids are 12%,42%,14%,46% and 29%. And the transfective efficiency is merely 50%. Among them, the inhibitory effect of pSIHl-siRNA3 group on Skp2 gene was biggest in Hep-2 cells.
3. The transfective efficiency increased to about 90%, and transfected cell lines were able to passage stably after Hep-2 cells were infected by virus stock solution contained packaged pSIHl-negative and pSIH1-siRNA3 plasmids with lentivirus system.
4. The Skp2 and p27 mRNA expression in Hep2, Hep2-siRNA and Hep2-neg groups were detected with fluorescent realtime quantitated PCR. The Skp2 mRNA expression in Hep2-siRNA cells decreased about 74%, and p27 mRNA expression increased about 38% contrasted to Hep2 cells. But apparent change of gene expression has not been found in Hep2-neg group.
5. Skp2 and p27 protein expressions in Hep2, Hep2-siRNA and Hep2-neg groups were detected with Western blot. Skp2 protein expression in Hep2-siRNA cells decreased about 76%, and p27 protein expression increased about 89% contrasted to Hep2 cells. Apparent change of protein expression has not been found in Hep2-neg group either.
6. Cell proliferation in Hep2-siRNA group apparently slowed down, and apparent change of cell proliferation had not been found between Hep2-neg and Hep2 group with MTT.
7. The apoptosis ratios were (2.86±0.21)%, (3.58±0.16)% and (16.64±0.17)% in Hep2, Hep2-neg and Hep2-siRNA group with flow cytometer. The apoptosis ratio in Hep2-siRNA group was apparently bigger than Hep2 and Hep2-neg group. But significant difference of cell apoptosis had not been found between Hep2-neg and Hep2 group.
8. Tumorigenesis activity apparently decreased, tumor growth slowed down and tumor volume obviously appeared less in athymic mice carcinoma cell transfected with positive plasmid. But no significant difference was found in oncogenicity and growth activity between negative plasmid group and controls. Weight of tumors was apparently lightened, and oncoinhibition ratio was 79.55% in positive plasmid group on the 30th day after tansfection. No significant difference was found in tumor weight between negative plasmid group and controls.
9. Necrosis, active cellular proliferation and obvious cancer nest were observed in tumor center of bearing cancer mice in negative plasmid group and controls by HE dyeing. Most of tumor was composed of parenchyma, and mesenchymal was rare. Less necrosis and more mesenchymal were observed in tumor center in positive plasmid group. There was no significant difference on pathological character between negative plasmid group and controls.
10. Increased expression of Skp2 protein and decreased expression of p27 protein were observed in tumor of bearing cancer mice in negative plasmid group and controls by immunohistochemistry. But decreased expression of Skp2 protein and increased expression of p27 protein were found in tumor of bearing cancer mice in positive plasmid group.
Conclusions
1. The mRNA and protein expressions of oncogene Skp2 in human laryngeal carcinoma cell line Hep-2 were specifically and efficiently inhibited by lentivirus-mediated siRNA expression vectors which were designed focused on Skp2 gene, but the expression of anti-oncogene p27 was increased. Meanwhile, the proliferation of carcinoma cell was inhibited, and apoptosis rate increased.
2. The expression of Skp2 and p27 was obviously negative correlated in laryngeal carcinoma generation and development. The inhabitation effects of siRNA targeted to Skp2 on Hep-2 cell were probably derived from the increased expression of p27 gene.
3. It is supposed that inhabitation of Skp2 expression with RNA interference technique maybe worked in gene therapy for laryngeal carcinoma.
喉癌是危害人体健康和生命的恶性肿瘤,约占耳鼻咽喉恶性肿瘤的10-35%,其中以喉鳞状细胞癌(LSCC)最为多见,可达90%。喉癌在我国东北地区是高发区,而且逐年增长。目前,喉切除术是治疗喉癌的主要手段,但术后对患者的生活质量会产生较大影响,且对于晚期及全身状态不佳的患者无法有效实施,虽可辅以放疗及化疗,效果仍不理想。因此,深入探讨喉癌的发生机制,寻找利用基因干预的手段进行治疗是目前喉癌研究的热点。
Skp2基因是最近发现的癌基因,与肿瘤的增殖、分化有关。在对喉癌的研究中发现,Skp2表达与临床分期及淋巴结转移正相关,提示该基因表达上调可能与LSCC的侵袭转移能力密切相关；同时,其表达与抑癌基因p27表达强度呈明显负相关,表明在LSCC的进展过程中,Skp2表达的上调可能通过泛素—蛋白酶体途径导致p27表达减少,使其不能有效发挥细胞周期负调控因子的抑癌作用,促使癌细胞获得更高的侵袭转移能力。因此,我们推测Skp2可能是喉部恶性肿瘤基因治疗的靶点。
RNA干扰(RNA interference, RNAi)是新近发展起来的一种封闭基因表达的有效方法,它比反义寡核苷酸技术能更高效地抑制目的基因的表达。已有研究证实化学合成siRNA (small interfering RNA)能高效地介导RNAi作用,但作用持时间短,而采用siRNA表达载体尤其是慢病毒载体的方法能有效延长作用时间。
本研究试图通过细胞体外及动物体内试验,利用慢病毒介导的RNA干扰技术抑制癌基因Skp2的表达,观察其对人喉癌细胞系Hep-2细胞的增殖、调亡以及对抑癌基因p27表达的影响,为今后应用该技术研究和治疗喉癌提供实验依据。
方法
1、针对人Skp2基因的RNAi载体构建
利用计算机辅助设计软件,设计合成四条针对Skp2基因cds区的siRNA序列,同时设计一条对于任何基因无干扰效果序列Negative用于对照组实验,并将其定向克隆入真核表达载体pSIHl-Hl-copGFP shRNA Vector,获得五个重组质粒：pSIHl-negative、pSIHl-siRNA1、pSIH1-siRNA2、pSIHl-siRNA3、pSIH1-siRNA4。
2、抑制Skp2表达对人喉癌细胞系Hep-2影响的体外实验
(1)在Hep-2细胞中针对Skp2基因筛选有效的干扰序列：利用脂质体将重组质粒转染Hep-2细胞,荧光实时定量PCR实验检测转染前后细胞中的Skp2 mRNA的变化,根据结果选择一个含有最强抑制效果的干扰序列的质粒用于后续试验；
(2)用慢病毒系统建立干扰Skp2基因的稳定细胞株：以三载体包装的含有筛选出的pSIH-siRNA3和pSIH-negative质粒的慢病毒原液感染Hep-2细胞,观察转染效果,获得Hep2-siRNA, Hep2-neg两组稳定转染细胞株；
(3)荧光实时定量PCR检测稳定转染细胞株中Skp2和p27 mRNA的表达；
(4) Western blot法检测Hep2, Hep2-siRNA, Hep2-neg三组细胞中的Skp2和p27蛋白表达量的变化；
(5)MTT法检测Hep2, Hep2-siRNA, Hep2-neg三组细胞的增殖情况；
(6)流式细胞仪检测三组细胞凋亡情况。
3、抑制Skp2表达对人喉癌细胞系Hep-2影响的体内实验
(1)裸鼠随机分成三组,将三组细胞按照1.0×107个/ml的浓度,每只鼠0.2ml背部皮下接种；
(2)观察裸鼠的成瘤情况,30天后处死裸鼠,经皮下剥离肿瘤组织,比较各组瘤体大小和瘤重；
(3)将瘤组织切片进行常规病理检查,并采用免疫组化方法对三组裸鼠瘤组织中Skp2和p27蛋白的表达进行检测。
1、利用PCR分析,DNA测序证实靶向Skp2特异性siRNA表达载体pSIHl-negative、pSIHl-siRNA1、pSIHl-siRNA2、pSIH1-siRNA3、pSIH1-siRNA4重组质粒已成功构建。
2、五组重组质粒转染Hep-2细胞后对Skp2 mRNA抑制率分别为12%,42%,14%,46%和29%,但细胞转染效率不高,仅为50%左右；重组质粒pSIH1-siRNA3对Hep-2细胞Skp2基因的抑制作用最强。
3、应用慢病毒包装系统包装pSIH1-negative和pSIHl-siRNA3质粒后以病毒原液感染Hep-2细胞,转染效率可提高至90%左右,且转染细胞株可稳定传代培养。
4、应用荧光实时定量PCR检测Hep2, Hep2-siRNA, Hep2-neg三组细胞中的Skp2和p27 mRNA表达量的变化,显示Hep2-siRNA细胞中Skp2 mRNA表达量与Hep2组细胞中的表达量相比下调了74%,而p27 mRNA表达量则上调了38%,Hep2-neg组无明显变化。
5、Western blot法检测Hep2, Hep2-siRNA, Hep2-neg三组细胞中的Skp2和p27蛋白表达量的变化,显示Hep2-siRNA细胞中Skp2蛋白表达量与Hep2组细胞中的表达量相比下调了约76%,p27蛋白表达量则上调了约89%,Hep2-neg组无明显变化。
6、MTT法检测细胞生长增殖情况结果显示Hep2-siRNA组细胞增殖明显减慢,而Hep2-neg组细胞与Hep2组相比,细胞增殖无明显变化。
7、流式细胞仪检测结果显示Hep2, Hep2-neg和Hep2-siRNA三组细胞凋亡率分别为(2.86±0.21)%,(3.58±0.16)%和(16.64±0.17)%, Hep2-siRNA组与其他两组比较细胞凋亡率明显增加,Hep2-neg组和Hep2组之间无显著差异。
8、裸鼠成瘤实验显示与两对照组相比,转染阳性质粒组细胞裸鼠致瘤活性明显降低,生长减慢,瘤体体积明显较小；而转染阴性质粒组细胞裸鼠致瘤性及生长活性与正常培养组相比无明显差异。接种后30天转染阳性质粒组瘤体重量较对照组明显减轻,抑瘤率为79.55%；而转染阴性质粒组细胞裸鼠致瘤体重量与正常培养组比较无明显差异。
9、荷瘤裸鼠瘤体HE染色切片结果显示正常培养组及转染阴性质粒组肿瘤组织中央可见坏死灶,癌细胞增生活跃,癌巢形成明显,瘤组织以实质为主,间质少见；转染阳性质粒组瘤体中央坏死减少,肿瘤组织间质较多见。正常培养组及转染阴性质粒组比较,瘤体病理学特征无明显差异。
10、荷瘤裸鼠瘤体免疫组化结果显示正常培养组及转染阴性质粒组肿瘤组织中Skp2蛋白高表达,p27蛋白表达显著下调；转染阳性质粒组裸鼠瘤体中Skp2蛋白表达明显减弱,p27蛋白表达显著上调。
结论
1、针对Skp2基因设计的慢病毒介导的siRNA表达载体能从mRNA和蛋白水平特异、高效地抑制人喉癌细胞系Hep-2中癌基因Skp2的表达,并上调抑癌基因p27的表达；同时,抑制肿瘤细胞的恶性增殖,增加其凋亡率。
2、在喉癌的发生发展中,Skp2与p27的表达呈明显负相关,靶向Skp2基因的siRNA对Hep-2细胞的抑制作用可能是通过上调p27基因水平来实现的。
3、利用RNA干扰技术抑制Skp2的表达有望成为喉癌基因治疗的合理策略之一。
Research Background and Objective
Laryngeal carcinoma is a kind of malignant tumor which does great harm to human health and life. And it accounts for about 10-35% in all ENT malignant tumors. Among them, laryngeal squamous cell carcinoma (LSCC) is the most frequent one, and it accounts for 90%. The morbidity of LSCC in northeast is highest in China, and increases year by year. Laryngectomy is the main method for LSCC treatment. Nevertheless, satisfactory treatment results have not been achieved even though by laryngectomy accompanied with radiotherapy and/or chemotherapy, because of poorer quality of life after treatments and their limitation to deal with some patients in advanced stage and/or worse general body state. Therefore, to research the pathological mechanism of LSCC, and to further investigate gene therapy method are focal points in present LSCC research.
As a recently discovered gene, Skp2 is related to proliferation and differentiation. It has been discovered in previous laryngeal carcinoma researches that Skp2 expression was positive correlated to clinical stage and lymph node metastasis. And it indicated that increased expression of Skp2 gene was probably related to invasion and metastasis of LSCC. Moreover, significant negative correlation is discovered between the expression of Skp2 gene and p27 antioncogene. And it indicated that increased expression of Skp2 maybe lead to decreased expression of p27 which as a negative regulatory factor in cell cycle through ubiquitin-proteasomes pathway in LSCC development. Therefore, the anticancer ability of p27 gene was inhibited, and cancer cells got better invasion and metastasis ability. To sum up, it is suspected that Skp2 probably is the target for the gene therapy of laryngeal malignant tumor.
RNA interference is a recently developed method to block gene expression, which provides better expression inhibition of target gene than antisense oligonucleotides ribozyme technique. It is confirmed that chemosynthesized small interfering RNA (siRNA) was able to efficiently mediate RNA interference effect, but its active duration was short. Method applied siRNA expression vector especial lentivirus vector could prolong active duration effectively.
We aimed to observe the influence of inhibition of Skp2 expression with RNA interference technique by lentivirus on proliferation and apoptosis of human laryngeal carcinoma cell line Hep-2 and p27 expression in cell experiments in vivo and animal experiments. And we suppose to provide experimental foundation for further research and treatment of laryngeal cancer.
Methods
1. RNAi vector construction for human Skp2 gene
Four siRNA sequences of Skp2 gene cds region and a negative sequence were designed with computer-aided software. The sequences were directed cloned in eukaryotic expressive pSIHl-Hl-copGFP shRNA Vector. And five recombinant plasmids such as pSIHl-negative, pSIHl-siRNAl, pSIHl-siRNA2, pSIHl-siRNA3, pSIH1-siRNA4 were got.
2. Inhabitation of Skp2 expression of human laryngeal carcinoma cell line Hep-2 in vivo
(1) To select effective interference consequence targeted to Skp2 gene in Hep-2 cell:Recombinant plasmids were transfected into Hep-2 cell with liposome. Skp2 mRNA before and after transfection was detected with fluorescent realtime quantitated PCR, and the best inhibited interference consequence was selected for following experiments.
(2) To construct the stable cell line interfered Skp2 gene with lentivirus system: Hep-2 cells were infected with lentivirus stock solution of tri-vehicle package contained selected pSIH-siRNA3 and pSIH-negative plasmids. The transfected effects were observed, and two stable Hep2-siRNA, Hep2-neg transfected cell lines were got.
(3) To detect mRNA expression of Skp2 and p27 in stable transfected cell lines with fluorescent realtime quantitated PCR.
(4) To detect protein expression of Skp2 and p27 in Hep2, Hep2-siRNA, Hep2-neg cells with Western blot.
(5) To detect the proliferation of Hep2, Hep2-siRNA, Hep2-neg cells with MTT.
(6) To detect the apoptosis of Hep2, Hep2-siRNA, Hep2-neg cells with flow cytometer.
3. The effect of Skp2 expressive inhibition on human laryngeal carcinoma cell line Hep-2 in vitro
(1) Athymic mice were divided into three groups. And cells of three groups were subcutaneous inoculated into each mouse back in dosage of 1.0×107个/ml.
(2) Carcinoma development was observed. Athymic mice were executed after 30 days, and carcinoma tissues were separated subcutaneously. The size and weight of carcinoma were compared.
(3) Regular pathologic examinations were performed with histological section of carcinoma. The protein expression of Skp2 and p27 were detected in athymic mice carcinoma of three groups with immunohistochemistry.
Results
1. pSIHl-negative, pSIHl-siRNAl, pSIHl-siRNA2, pSIHl-siRNA3 and pSIHl-siRNA4 recombinant plasmids targeted to Skp2 specific siRNA were successfully constructed with PCR and DNA sequencing.
2. Inhibition ratios of Skp2 mRNA in Hep-2 cells after transfected by five recombinant plasmids are 12%,42%,14%,46% and 29%. And the transfective efficiency is merely 50%. Among them, the inhibitory effect of pSIHl-siRNA3 group on Skp2 gene was biggest in Hep-2 cells.
3. The transfective efficiency increased to about 90%, and transfected cell lines were able to passage stably after Hep-2 cells were infected by virus stock solution contained packaged pSIHl-negative and pSIH1-siRNA3 plasmids with lentivirus system.
4. The Skp2 and p27 mRNA expression in Hep2, Hep2-siRNA and Hep2-neg groups were detected with fluorescent realtime quantitated PCR. The Skp2 mRNA expression in Hep2-siRNA cells decreased about 74%, and p27 mRNA expression increased about 38% contrasted to Hep2 cells. But apparent change of gene expression has not been found in Hep2-neg group.
5. Skp2 and p27 protein expressions in Hep2, Hep2-siRNA and Hep2-neg groups were detected with Western blot. Skp2 protein expression in Hep2-siRNA cells decreased about 76%, and p27 protein expression increased about 89% contrasted to Hep2 cells. Apparent change of protein expression has not been found in Hep2-neg group either.
6. Cell proliferation in Hep2-siRNA group apparently slowed down, and apparent change of cell proliferation had not been found between Hep2-neg and Hep2 group with MTT.
7. The apoptosis ratios were (2.86±0.21)%, (3.58±0.16)% and (16.64±0.17)% in Hep2, Hep2-neg and Hep2-siRNA group with flow cytometer. The apoptosis ratio in Hep2-siRNA group was apparently bigger than Hep2 and Hep2-neg group. But significant difference of cell apoptosis had not been found between Hep2-neg and Hep2 group.
8. Tumorigenesis activity apparently decreased, tumor growth slowed down and tumor volume obviously appeared less in athymic mice carcinoma cell transfected with positive plasmid. But no significant difference was found in oncogenicity and growth activity between negative plasmid group and controls. Weight of tumors was apparently lightened, and oncoinhibition ratio was 79.55% in positive plasmid group on the 30th day after tansfection. No significant difference was found in tumor weight between negative plasmid group and controls.
9. Necrosis, active cellular proliferation and obvious cancer nest were observed in tumor center of bearing cancer mice in negative plasmid group and controls by HE dyeing. Most of tumor was composed of parenchyma, and mesenchymal was rare. Less necrosis and more mesenchymal were observed in tumor center in positive plasmid group. There was no significant difference on pathological character between negative plasmid group and controls.
10. Increased expression of Skp2 protein and decreased expression of p27 protein were observed in tumor of bearing cancer mice in negative plasmid group and controls by immunohistochemistry. But decreased expression of Skp2 protein and increased expression of p27 protein were found in tumor of bearing cancer mice in positive plasmid group.
Conclusions
1. The mRNA and protein expressions of oncogene Skp2 in human laryngeal carcinoma cell line Hep-2 were specifically and efficiently inhibited by lentivirus-mediated siRNA expression vectors which were designed focused on Skp2 gene, but the expression of anti-oncogene p27 was increased. Meanwhile, the proliferation of carcinoma cell was inhibited, and apoptosis rate increased.
2. The expression of Skp2 and p27 was obviously negative correlated in laryngeal carcinoma generation and development. The inhabitation effects of siRNA targeted to Skp2 on Hep-2 cell were probably derived from the increased expression of p27 gene.
3. It is supposed that inhabitation of Skp2 expression with RNA interference technique maybe worked in gene therapy for laryngeal carcinoma.
引文
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