热休克蛋白27在头颈部鳞状细胞癌细胞迁移和侵袭中的作用研究
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摘要
头颈部鳞状细胞癌是威胁人类健康的主要肿瘤之一,已成为全球第六大好发性肿瘤,每年约有逾50万病例发生,且恶性程度高,已成为第五大致死肿瘤。每年的死亡数约为14,000例,约占全部肿瘤诊断病例的6%。根据American Cancer Society数据显示,尽管随着外科技术水平的提高和放疗化疗方案的成熟,在1996-2003年间,其死亡率已显著降低,但是,其复发和转移病例的术后存活时间仅为6个月。而这其中,导致低存活率的主要因素就是区域性淋巴结转移或者远处脏器转移。因此针对肿瘤侵袭转移的相关基因研究成为肿瘤研究的热点之一
热休克蛋白27(heat shock protein,Hsp27)是小分子量热休克蛋白家族中最具代表性的成员,其重要的生物学功能是保护细胞免受环境中自由基、热、缺血和毒性物质等各种应激因素导致的损伤,促进蛋白质的正确折叠、组装和纠正蛋白质的错误结构。此外,Hsp27也参与微丝的稳定,细胞增殖、分化、及细胞凋亡的信号转导调节等。
有报道显示Hsp27与多种肿瘤的发生和转移有关,其作为一种应激蛋白,在肿瘤中的表达程度与肿瘤分期及预后有一定的相关性。但有研究发现Hsp27在某些肿瘤中过表达,而同时在某些肿瘤中低表达或无表达。如在前列腺癌,人肝细胞肝癌,肾细胞癌,口腔舌鳞癌及乳腺癌,胃癌中,Hsp27均呈高表达状态,并被认为是预后不良的标志性因子。同时有诸多研究的结果与以上研究截然相反,显示Hsp27是预后良好的标志性因子,在诸多患有恶性纤维素瘤,成神经细胞瘤,子宫内膜腺瘤和食道癌的患者中,都有报道认为Hsp27高水平表达预示预后良好。也有更多的研究支持,即使对同一病理分型的肿瘤进行有关Hsp27的研究,其与肿瘤恶性性及转移和侵袭的关系也不完全一致；此外,不同组织来源的肿瘤,不同个体间,也存在同样现象。因此,目前,对于Hsp27的研究,尤其Hsp27与头颈部鳞状细胞癌的关系尚无定论。
本课题中使用美国密歇根大学肿瘤中心Thomas E.Carey实验室建立并保存的来自同一病人原发肿瘤和同期存在的淋巴结转移灶的两种细胞系UM-SCC-22A/UM-SCC-22B,首先分别运用MTS,细胞划痕试验,Matrigel细胞侵袭实验及裸鼠活体内生物发光技术,观察此两种细胞系生物学行为的异同,发现相对于低转移潜能头颈鳞癌细胞系UM-SCC-22A,高转移潜能头颈鳞癌细胞系UM-SCC-22B表现出极强的迁移和侵袭特性。即,来源于同一病患同期原位癌和淋巴结转移灶的两种细胞系,在体内外转移生物学行为上存在着显著差异,可被用于进行头颈鳞癌转移行为的研究模型。而且,在此模型中我们发现其两者的Hsp27mRNA和蛋白水平表达存在显著性差异,即,UM-SCC-22A低表达Hsp27,同时,UM-SCC-22B高表达Hsp27,因此,我们将此实验模型用于进行Hsp27在头颈鳞癌迁移和侵袭中的作用研究,以期能够揭示两者之间的关系。
作为一种新的基因治疗方法,RNA干扰(RNA interference,RNAi)以其特异性、高效性成为研究的热点。慢病毒载体具有可感染分裂细胞及非分裂细胞、转移基因片段容量较大、目的基因表达时间长、不易诱发宿主免疫反应等优点,已成为当前基因治疗中的理想载体。活体内荧光示踪系统的应用也拓展了肿瘤转移动物实验模型的建立思路,尤其通过裸鼠左心房内注射肿瘤细胞所建立的肿瘤转移动物实验模型,相对于鼠尾注射,皮下注射、原位注射等操作,具有成瘤率高、成瘤速度快、动物存活率高并且可以更好的模拟肿瘤细胞定植、侵袭、转移过程等优点,已逐渐发展成为成熟且被广泛应用的肿瘤转移动物实验模型。
在本课题中,我们利用RNAi干扰技术,设计并合成针对头颈部鳞状细胞癌细胞Hsp27基因的小干扰RNA片段,通过阳离子脂质体瞬时转染Hsp27过表达肿瘤细胞,同时,购买已建立的载有特异性抑制Hsp27基因表达的小发夹RNA的慢病毒质粒,包装生产慢病毒颗粒,长效转染Hsp27过表达肿瘤细胞；此外,设计构建生产过表达Hsp27基因的慢病毒颗粒,长效转染Hsp27低表达肿瘤细胞。运用MTS,细胞划痕,Matrigel细胞侵袭实验等观察体外实验中肿瘤细胞相应的迁移和侵袭能力改变,并同时通过建立裸鼠活体内肿瘤转移生物发光模型,观察Hsp27基因沉默/过表达后,肿瘤细胞体内转移能力的改变。以期进一步研究头颈部鳞状细胞癌细胞转移机制,及其与Hsp27表达之间的关系,探讨抑制头颈部鳞状细胞癌转移的策略,同时探索头颈部鳞状细胞癌基因治疗的靶点。
本课题包括四部分
第一部分：不同转移潜能头颈部鳞状细胞癌细胞株(UM-SCC-22A/UM-SCC-22B)生物学行为异同及Hsp27mRNA和蛋白的表达
1、不同转移潜能头颈部鳞状细胞癌细胞株生物学行为异同
对2种已知转移潜能的头颈部鳞状细胞癌细胞系UM-SCC-22A/UM-SCC-22B进行常规培养,采用MTS细胞增殖实验,细胞划痕实验及transwell侵袭实验,建立裸鼠活体内肿瘤转移生物发光模型,观察不同转移潜能细胞系的转移能力。结果显示：常规接种细胞培养24、48小时后,此两种细胞系增殖能力无明显差异(P>0.05)。细胞划痕试验表明,划痕产生48小时后,UM-SCC-22B细胞系划痕愈合率达66.50%,UM-SC-22A仅为18.69%,即UM-SCC-22B细胞体外迁移能力为UM-SCC-22A的3.56倍。Matrigel侵袭实验显示,接种105细胞于上室40小时后,UM-SCC-22B细胞系有53.56个细胞/观察视野穿透Matrigel,粘附于上室底膜下表面,UM-SCC-22A细胞系有25.45个细胞/观察视野,即UM-SCC-22B细胞体外侵袭能力为UM-SCC-22A的2.11倍。生物发光显示,UM-SCC-22B组细胞裸鼠左心房接种后,成瘤率和转移率明显高于UM-SCC-22A组。
2、不同转移潜能头颈部鳞状细胞癌细胞株中Hsp27mRNA和蛋白的表达
对通过体内外实验证实,具有不同转移潜能的头颈部鳞状细胞癌细胞系UM-SCC-22A/UM-SCC-22B进行常规培养,采用实时荧光定量PCR和Western blotting方法对Hsp27基因在mRNA和蛋白质水平的表达进行检测,分析其表达丰度与头颈部鳞状细胞癌细胞转移潜能的关系。实验发现,2种头颈部鳞状细胞癌细胞系均有Hsp27基因表达,高转移潜能细胞系UM-SCC-22B中Hsp27表达水平明显高于UM-SCC-22A,差异显著(P<0.01)。UM-SCC-22B细胞系Hsp27 mRNA水平是UM-SCC-22A的22.38倍,western blotting结果显示UM-SCC-22B细胞中,位于27kb位置条带明显强于UM-SCC-22A。头颈部鳞状细胞癌细胞系Hsp27表达丰度与其转移能力相关,随着转移能力的上升,Hsp27表达水平升高。
第二部分：瞬时转染siRNA干扰Hsp27基因效果鉴定及其对UM-SCC-22B迁移和侵袭的影响
1、siRNA干扰Hsp27基因及其干扰效果鉴定
根据siRNA设计原则,设计合成靶向Hsp27基因的siRNA,采用阳离子脂质体试剂瞬时转染高转移潜能头颈部鳞状细胞癌细胞系UM-SCC-22B,应用实时荧光定量PCR和Western Blotting检测RNA干扰后Hsp27基因的沉默效果。结果显示,siRNA Hsp27瞬时转染24小时后,Hsp27 mRNA水平明显下调,其干扰效率为93%,与空白对照组及阴性对照组比较,差异具有显著性(P<0.01)。siRNA Hsp27瞬时转染48小时后,Hsp27蛋白水平明显下调,western blotting结果显示,与空白对照组及阴性对照组比较,位于27kb位置条带明显强度明显下降,与实时荧光定量PCR结果一致。
2、siRNA干扰Hsp27基因对头颈部鳞状细胞癌细胞转移和侵袭的影响
于转染siRNA后48小时,应用细胞划痕试验检测Hsp27基因表达下调后,高转移潜能头颈部鳞状细胞癌细胞UM-SCC-22B迁移能力变化；应用Matrigel侵袭实验检测Hsp27基因表达下调后,UM-SCC-22B侵袭能力的改变。转染siRNA下调UM-SCC-22B细胞Hsp27基因表达后,细胞划痕后24小时,空白对照组和阴性对照组细胞划痕开始愈合,而siRNA转染组几乎没有愈合；72小时后空白对照组和阴性对照组愈合率达71.66%,50.57%,而siRNA转染组仅有少量愈合,愈合率为18.30%,与其它两组比较差异显著(P<0.01)。Matrigel侵袭实验结果显示,接种上室40小时后,siRNA转染组穿膜细胞数明显减少,每个观察视野仅有26.91个细胞,是空白对照组穿膜细胞数的50.24%,此结果与空白对照组和阴性对照组相比,具有显著性差异(P<0.01)
第三部分：Hsp27基因shRNA慢病毒靶向沉默Hsp27的效果鉴定及对UM-SCC-22B生物学行为的影响
1、Hsp27基因shRNA慢病毒载体的鉴定及其靶向沉默热休克蛋白27的效果鉴定
将购自Open Biosystem,可表达shRNA Hsp27的慢病毒载体质粒pLKO.1-shRNA-Hsp27质粒,转染入UM-SCC-22B细胞,运用实时荧光定量PCR检测Hsp27表达,初步鉴定质粒正确性和有效性。结果显示,转染24,48小时后,Hsp27 mRNA表达量分别降至42%,24%,干扰效果明显。密歇根大学载体中心包装后,得到高滴度慢病毒颗粒pLenti-shRNA-Hsp27,长效转染入UM-SCC-22B后,运用实时荧光定量PCR和western blotting检测Hsp27表达。结果显示,shRNA Hsp27长效转染后,Hsp27 mRNA水平明显下调,其干扰效率为94%,与空白对照组及阴性对照组比较,差异具有显著性(P<0.01)。Hsp27蛋白水平明显下调,western blotting结果显示,与空白对照组及阴性对照组比较,位于27kb位置条带明显强度明显下降,与实时荧光定量PCR结果一致。
2.Hsp27基因shRNA慢病毒转染后对头颈部鳞状细胞癌细胞生物学行为的影响
慢病毒颗粒pLenti-shRNA-Hsp27长效转染UM-SCC-22B,并高效干扰Hsp27表达后,采用MTS细胞增殖实验,细胞划痕实验及Matrigel侵袭实验,建立裸鼠活体内肿瘤转移生物发光模型,观察Hsp27表达抑制后,UM-SCC-22B转移能力变化。结果显示：常规接种细胞培养24、48小时后,与空白组和阴性对照组相比,shRNA Hsp27组增殖能力无明显差异(P>0.05)。细胞划痕试验表明,划痕产生72小时后,空白对照组细胞划痕愈合率达71.4%,shRNA Hsp27实验组仅为16.3%,即shRNA Hsp27干扰后,空白组细胞迁移能力为实验组4.38倍。Matrigel侵袭实验显示,接种105细胞于上室40小时后,shRNA Hsp27实验组有29.6个细胞／观察视野穿透Matrigel,粘附于上室底膜下表面,空白对照组细胞有60个细胞／观察视野,即空白组细胞体外侵袭能力为shRNA Hsp27组的2.03倍。荧光示踪显示,各组细胞裸鼠左心房接种后,空白组动物模型成瘤率和转移率明显高于实验组。
第四部分：重组Hsp27表达慢病毒载体的构建、鉴定及其对头颈部鳞状细胞癌细胞生物学行为的影响
1、重组Hsp27表达慢病毒载体的构建和鉴定
设计引入BamHI, Xbal两个酶切位点的人Hsp27基因引物,利用pOTB7-Hsp27全长cDNA质粒,PCR扩增Hsp27基因,平端酶切,回收纯化。利用BamHI, Xbal酶切位点,T4连接酶连接Hsp27基因片段和pLentiLox RSV慢病毒载体。双酶切电泳验证目的Hsp27基因片段连接正确,转化感受态DH5-alpha大肠杆菌,小提后进行重组质粒基因测序。测序正确,成功构建过表达Hsp27基因慢病毒载体pLenti-RSV-Hsp27。大提后送密歇根大学载体中心包装生产过表达Hsp27慢病毒颗粒pLenti-RSV-Hsp27。长效转染入低转移潜能头颈部鳞状细胞癌细胞系UM-SCC-22A后,实时荧光定量PCR和western blotting检测Hsp27表达。结果显示,转染后,Hsp27基因mRNA和蛋白水平均明显升高。过表达组Hsp27 mRNA水平是对照组的15.07倍,western blotting结果显示,过表达组27kb蛋白条带强度显著增高。
2、过表达Hsp27对头颈部鳞状细胞癌细胞生物学行为的影响
慢病毒颗粒plenti-RSV-Hsp27长效转染UM-SCC-22A,并高效表达Hsp27表达后,采用MTS细胞增殖实验,细胞划痕实验及Matrigel侵袭实验,建立裸鼠活体内荧光示踪肿瘤转移动物模型,观察Hsp27表达增加后,UM-SCC-22A转移能力变化。结果显示：常规接种细胞培养24、48小时后,与空白对照组相比,过表达Hsp27组增殖能力无明显差异(P>0.05)。细胞划痕试验表明,划痕产生48小时后,空白对照组细胞划痕愈合率为19.84%,过表达Hsp27实验组为51.33%,即Hsp27过表达后,细胞迁移能力为空白对照组2.56倍。Matrigel侵袭实验显示,接种105细胞于上室40小时后,Hsp27过表达组有59.13个细胞/观察视野穿透Matrigel,粘附于上室底膜下表面,空白对照组细胞有29.35个细胞/观察视野,即过表达组细胞体外侵袭能力为空白对照组的2.01倍。荧光示踪显示,各组细胞裸鼠左心房接种后,过表达组动物模型成瘤率和转移率明显高于对照组。
结论：
1.成功建立头颈部鳞状细胞癌转移细胞实验模型,建立头颈部鳞状细胞癌裸鼠活体内肿瘤转移生物发光模型。
2.头颈部鳞状细胞癌Hsp27的表达与其转移潜能密切相关,随其转移潜能的升高,其表达丰度升高。头颈部鳞状细胞癌低转移潜能细胞系UM-SCC-22A/高转移潜能细胞系UM-SCC-22B,可作为良好的实验模型,深入研究Hsp27表达调控与头颈部鳞状细胞癌转移间的关系。
3.体外合成,瞬时转染siRNA干扰技术可高效下调高转移潜能头颈部鳞状细胞癌细胞系UM-SCC-22B Hsp27基因的表达。
4.瞬时转染siRNAHsp27,高转移潜能头颈部鳞状细胞癌细胞系Hsp27基因下调后,显著抑制其细胞迁移和侵袭能力。Hsp27基因可作为有效头颈部鳞状细胞癌转移的治疗靶点。
5.长效转染慢病毒颗粒pLenti-shRNA-Hsp27能够高效、特异的沉默高转移潜能头颈部鳞状细胞癌细胞系UM-SCC-22B的Hsp27基因表达。
6.长效转染慢病毒颗粒pLenti-shRNA-Hsp27可显著抑制高转移潜能头颈部鳞状细胞癌细胞系UM-SCC-22B的体内外转移能力。
7.成功构建过表达Hsp27基因慢病毒载体pLenti-RSV-Hsp27。长效转染慢病毒颗粒pLenti-RSV-Hsp27可高效、特异性增加低转移潜能头颈部鳞状细胞癌细胞系UM-SCC-22A的Hsp27基因表达。
8.长效转染慢病毒颗粒pLenti-RSV-Hsp27可显著提高低转移潜能头颈部鳞状细胞癌细胞系UM-SCC-22A的体内外转移能力。
Head and neck squamous cell carcinoma (HNSCC) is the 6th most prevalent cancer types worldwide with an incidence of more than 500,000 cases annually and a high mortality rate, making it the fifth leading cause of cancer related death. In the United States, it accounts for 6% of all cancer diagnoses and results in an estimated 14,000 deaths annually. Despite the advanced therapeutic regimens used in treating HNSCC, oral cavity cancer survival during 1996-2003 was less than 50% on average according to the data from American Cancer Society. Furthermore, patients with recurrent or metastatic HNSCC have median survival of approximately 6 months. The major contributing factors for low survival include local-regional relapse, lymph node or distant metastatic spread of the primary tumor. The small Heat Shock Protein 27(Hsp27) belongs to heat shock protein family, and acts as a molecular chaperone. Expression of Hsp27 can be induced by environmental stress, such as heat shock, heavy metals, oxidants, infection, inflammation, ischemia. Hsp27 has been shown to have various cellular functions to promote differentiation, proliferation, cell growth, and motility. In addition, it has been reported that Hsp27 is associated with various carcinomas. RNA interference(RNAi) is a potential approach for gene therapy, thanks to its high efficience in silencing target sequence-specific gene, it has been used as a useful method in molecular biology study. And Lentivirus vector has become a common vector to expressing specific gene, as it could be transfected into separated/unseparated cells with larger gene fragment without the host immune response induced. To establish a head and neck squamous cell cancer model with stable expression of luciferase for in vivo imaging is a rising method to detect the tumor progression,metastasis and drug sensitivity. Especially, the left intracardic injection has been used as a better way to set up the in vivo imaging mice model, compared with tail vein injection,in site injection and other methods, it induces tumor quicker, easier to practice and has a higher survival rate for mice.
To figure out how Hsp27 expression regulate head and neck squamous cell carcinoma metastasis behavior and find out a noval target for HNSCC treatment, in the present study, we silenced Hsp27 gene expression in head and neck squamous cell cancer cells UM-SC-22B which shows a high potential of metastasis by using temporary transfection of Hsp27 siRNA and stable transfection of Lentivirus with shRNA Hsp27. At the same time, we overexpressed Hsp27 expression in UM-SC-22A with lower metastasis potentcy by re-constructuring a Lentivirus vector expressing Hsp27 gene. And we used MTS assay to detect the proliferation changes of cells, wound-healing motility assay and Matrigel invasion assay to detect the metastasis ability changes before and after silencing or overexpressing Hsp27 gene in vitro. Otherwise, we set up a metastasis in vivo imaging mice model to detect the tumor cells metastasis ability in vivo.
The whole dissertation consists of four parts:
Part 1 Biology behaviors and Hsp27 expression in HNSCC cell lines with different metastasis potential
1. Biology behaviors in HNSCC cell lines with different metastasis potential
Proliferation of the primary and metastatic HNSCC cell lines was evaluated using the MTS proliferation assay. Metastatic behavior was assessed using migration and invasion assays in vitro. Set up imaging mice model to detect the metastasis behavior in vivo. MTS assays showed that the primary (UM-SCC-22A) and metastatic (UM-SCC-22B) HNSCC have similar proliferation rates after cultured for 24/48h(p>0.05). However, UM-SCC-22B derived from the metastasis showed 2.3 to 3.6-fold higher migration ability and 2-fold higher invasion ability than UM-SCC-22A. And UM-SCC-22B shows high rate of metastasis of kidney and thing-bone in vivo.
2. Hsp27 expression in HNSCC cell lines with different metastasis potential
The expression of Hsp27 in primary and metastatic cell lines derived from the primary HNSCC and a synchronous lymph node metastasis in the same patient was determined using real-time PCR and western blotting. Real-time PCR demonstrated that Hsp27 mRNA is 22.4-fold higher in metastatic UM-SCC-22B than primary UM-SCC-22A. Similarly, Western blotting showed that Hsp27 is rarely detectable in UM-SCC-22A whereas UM-SCC-22B expresses a higher level of Hsp27 protein.
These data indicate higher expression of Hsp27 shows in UM-SCC-22B with higher metastasis potential, and Hsp27 may regulate metastatic potential of HNSCC cancer cells.
Part 2 Hsp27 expression and biology behaviors changes in vitro after silencing Hsp27 by transfected with siRNA Hsp27 in UM-SCC-22B
1. Hsp27 expression changes in vitro after silencing Hsp27 by transfected with siRNA Hsp27 in UM-SCC-22B
Designed a specific siRNA sequence to knocking down Hsp27 in the highly migratory metastatic HNSCC cell line UM-SCC-22B. The expression of Hsp27 in UM-SCC-22B before and after silencing Hsp27 was determined using real-time PCR and western blotting. Compared to blank control group, after transfected with siRNA Hsp27 by lipofectamine 2000, the mRNA level of Hsp27 was reduced by 93%.And at 48h after transfection, the protein level of Hsp27 was dramatically decreased in consistent.
2. biology behaviors changes in vitro after silencing Hsp27 by transfected with siRNA Hsp27 in UM-SCC-22B
Furthermore, proliferation of UM-SCC-22B before and after silencing Hsp27 was evaluated using the MTS proliferation assay. Metastatic behavior was assessed using migration and invasion assays in vitro. MTS assays showed that the UM-SCC-22B before and after silencing Hsp27 have similar proliferation rates after cultured for 24/48h (p>0.05). However, compared to blank control group,siRNA knockdown of Hsp27 decreased metastatic behaviors of UM-SCC-22B by 3 to 4-fold in migration and 2-fold in cell invasion reducing cell invasion and migration.
These data indicate temporarily silencing Hsp27 expression by siRNA may decrease metastasis in head and neck squamous cell cancer cells.
Part 3 Bilology behaviors and Hsp27 expression changes after knocking down Hsp27 by tranfected with Lentivirus-shRNA-Hsp27 in UM-SCC-22B
1. Hsp27 expression changes after knocking down Hsp27 by tranfected with Lentivirus-shRNA- Hsp27 in UM-SCC-22B
Phurcase the Lentivirus vector plasmid with shRNA Hsp27 from Open Biosystem. After temperarily transfected into UM-SCC-22B, using real-time PCR to detect the mRNA of Hsp27 expression. The result shows that compared with blank control group, after 24/48h, the mRNA level of Hsp27 reduced to 42%/24%. Then produce lentivirus particals with shRNA Hsp27,stably transfected into UM-SCC-22B,using real-time PCR and western blotting assay to detect the mRNA and protein level changes of Hsp27 expression. The data shows mRNA level of Hsp27 was decreased by 94%,comparing to blank control group, meanwhile, the protein level was also significantly reduced in consistent.
2. Bilology behaviors changes after knocking down Hsp27 by tranfected with Lentivirus-shRNA- Hsp27 in UM-SCC-22B
Using MTS assay to detect the proliferation changes of UM-SCC-22B before and after stable transfected with lentivirus particals pLenti-shRNA-Hsp27 with shRNA Hsp27. Metastatic behavior was assessed using migration and invasion assays in vitro. MTS assays showed that the UM-SCC-22B before and after knocking down Hsp27 have similar proliferation rates after cultured for 24/48h (p>0.05). However, compared to blank control group,shRNA knockdown of Hsp27 decreased metastatic behaviors of UM-SCC-22B by 4.38-fold in migration and 2.03-fold in cell invasion reducing cell invasion and migration. Besides, UM-SCC-22B with shRNA Hsp27 shows lower rate of metastasis of kidney and thing-bone in vivo.
These data indicate stably knocking down Hsp27 expression by shRNA may decrease metastasis in head and neck squamous cell cancer cells.
Part 4 Bilology behaviors and Hsp27 expression changes after over-expressing Hsp27 by tranfected with Lentivirus- Hsp27 in UM-SCC-22A
1.Re-constructured Lentivirus vector expressiong Hsp27 and Hsp27 expression changes after over-expressing Hsp27 by tranfected with Lentivirus- Hsp27 in UM-SCC-22A
The full length cDNA pOTB7-Hsp27 plasmid was got from Invitrogen, design specific primers with BamHI, Xbal restriction enzyme sites,PCR was used to get the fragment of Hsp27 sequence with BamHI, Xbal. The fragment of Hsp27 and the pLentiLox-RSV lentivirus vector were ligated by T4 DNA ligase.After transferring them into the DH5-alpha competent cells, restracted the recombined plamides DNA.The recombinant clones were identified by PCR with 'the primers and digested it by restriction enzyme sites BamHI, Xbal for further identification. Then the positice recombinant pLenti-RSV-Hsp27 plasmid was sequenced. Packaging and producing pLenti-RSV-Hsp27 were processed.Stably transfected UM-SCC-22A with lentivirus particals pLenti-RSV-Hsp27,using real-time PCR and western blotting assay to detect the Hsp27 expression changes. The data shows mRNA level of Hsp27 was increased by 15.07-fold,and protein level of Hsp27 was also induced dramatically.
2. Bilology behaviors changes after over-expressing Hsp27 by tranfected with Lentivirus-Hsp27 in UM-SCC-22A
Using MTS assay to detect the proliferation changes of UM-SCC-22A before and after stable transfected with lentivirus particals pLenti-RSV-Hsp27 with expressing Hsp27. Metastatic behavior was assessed using migration and invasion assays in vitro. MTS assays showed that the UM-SCC-22A before and after overexpressing Hsp27 have similar proliferation rates after cultured for 24/48h (p>0.05). However, compared to blank control group, overexprssing Hsp27 increased metastatic behaviors of UM-SCC-22A by 2.56-fold in migration and 2.01-fold in cell invasion reducing cell invasion and migration. Besides, UM-SCC-22A with overexpressing Hsp27 shows higher rate of metastasis of kidney and thing-bone in vivo.
These data indicate overexpressing Hsp27 expression by stably transfected lentivirus particals pLenti-RSV-Hsp27 may increase metastasis in head and neck squamous cell cancer cells.
Conclusion:
1.Successfully set up a head and neck squamous cell cancer metastasis cell model in vitro and imaging in vivo mice model.
2.Hsp27 could regulate the metastasis behavior of HNSCC, and it show higher expression in HNSCC cells with higher metastasis ability.
3.Temperarily transfected with siRNA Hsp27 could efficiently decrease Hsp27 expression on mRNA an protein levels in UM-SCC-22B.
4.silencing Hsp27 expression by siRNA could reduce the metastasis ability of UM-SCC-22B.
5.Stably transfected with lentivirus -shRNA-Hsp27 could efficiently decrease Hsp27 expression on mRNA an protein levels in UM-SCC-22B.
6. Stably transfected with lentivirus -shRNA-Hsp27 could reduce the metastasis ability of UM-SCC-22B.
7. Successfully re-constructured Lentivirus vector expressiong Hsp27. Stably transfected with lentivirus -RSV-Hsp27 could efficiently increase Hsp27 expression on mRNA an protein levels in UM-SCC-22A.
8. Stably transfected with lentivirus -RSV-Hsp27 could induce the metastasis ability of UM-SCC-22A.
热休克蛋白27(heat shock protein,Hsp27)是小分子量热休克蛋白家族中最具代表性的成员,其重要的生物学功能是保护细胞免受环境中自由基、热、缺血和毒性物质等各种应激因素导致的损伤,促进蛋白质的正确折叠、组装和纠正蛋白质的错误结构。此外,Hsp27也参与微丝的稳定,细胞增殖、分化、及细胞凋亡的信号转导调节等。
有报道显示Hsp27与多种肿瘤的发生和转移有关,其作为一种应激蛋白,在肿瘤中的表达程度与肿瘤分期及预后有一定的相关性。但有研究发现Hsp27在某些肿瘤中过表达,而同时在某些肿瘤中低表达或无表达。如在前列腺癌,人肝细胞肝癌,肾细胞癌,口腔舌鳞癌及乳腺癌,胃癌中,Hsp27均呈高表达状态,并被认为是预后不良的标志性因子。同时有诸多研究的结果与以上研究截然相反,显示Hsp27是预后良好的标志性因子,在诸多患有恶性纤维素瘤,成神经细胞瘤,子宫内膜腺瘤和食道癌的患者中,都有报道认为Hsp27高水平表达预示预后良好。也有更多的研究支持,即使对同一病理分型的肿瘤进行有关Hsp27的研究,其与肿瘤恶性性及转移和侵袭的关系也不完全一致；此外,不同组织来源的肿瘤,不同个体间,也存在同样现象。因此,目前,对于Hsp27的研究,尤其Hsp27与头颈部鳞状细胞癌的关系尚无定论。
本课题中使用美国密歇根大学肿瘤中心Thomas E.Carey实验室建立并保存的来自同一病人原发肿瘤和同期存在的淋巴结转移灶的两种细胞系UM-SCC-22A/UM-SCC-22B,首先分别运用MTS,细胞划痕试验,Matrigel细胞侵袭实验及裸鼠活体内生物发光技术,观察此两种细胞系生物学行为的异同,发现相对于低转移潜能头颈鳞癌细胞系UM-SCC-22A,高转移潜能头颈鳞癌细胞系UM-SCC-22B表现出极强的迁移和侵袭特性。即,来源于同一病患同期原位癌和淋巴结转移灶的两种细胞系,在体内外转移生物学行为上存在着显著差异,可被用于进行头颈鳞癌转移行为的研究模型。而且,在此模型中我们发现其两者的Hsp27mRNA和蛋白水平表达存在显著性差异,即,UM-SCC-22A低表达Hsp27,同时,UM-SCC-22B高表达Hsp27,因此,我们将此实验模型用于进行Hsp27在头颈鳞癌迁移和侵袭中的作用研究,以期能够揭示两者之间的关系。
作为一种新的基因治疗方法,RNA干扰(RNA interference,RNAi)以其特异性、高效性成为研究的热点。慢病毒载体具有可感染分裂细胞及非分裂细胞、转移基因片段容量较大、目的基因表达时间长、不易诱发宿主免疫反应等优点,已成为当前基因治疗中的理想载体。活体内荧光示踪系统的应用也拓展了肿瘤转移动物实验模型的建立思路,尤其通过裸鼠左心房内注射肿瘤细胞所建立的肿瘤转移动物实验模型,相对于鼠尾注射,皮下注射、原位注射等操作,具有成瘤率高、成瘤速度快、动物存活率高并且可以更好的模拟肿瘤细胞定植、侵袭、转移过程等优点,已逐渐发展成为成熟且被广泛应用的肿瘤转移动物实验模型。
在本课题中,我们利用RNAi干扰技术,设计并合成针对头颈部鳞状细胞癌细胞Hsp27基因的小干扰RNA片段,通过阳离子脂质体瞬时转染Hsp27过表达肿瘤细胞,同时,购买已建立的载有特异性抑制Hsp27基因表达的小发夹RNA的慢病毒质粒,包装生产慢病毒颗粒,长效转染Hsp27过表达肿瘤细胞；此外,设计构建生产过表达Hsp27基因的慢病毒颗粒,长效转染Hsp27低表达肿瘤细胞。运用MTS,细胞划痕,Matrigel细胞侵袭实验等观察体外实验中肿瘤细胞相应的迁移和侵袭能力改变,并同时通过建立裸鼠活体内肿瘤转移生物发光模型,观察Hsp27基因沉默/过表达后,肿瘤细胞体内转移能力的改变。以期进一步研究头颈部鳞状细胞癌细胞转移机制,及其与Hsp27表达之间的关系,探讨抑制头颈部鳞状细胞癌转移的策略,同时探索头颈部鳞状细胞癌基因治疗的靶点。
本课题包括四部分
第一部分：不同转移潜能头颈部鳞状细胞癌细胞株(UM-SCC-22A/UM-SCC-22B)生物学行为异同及Hsp27mRNA和蛋白的表达
1、不同转移潜能头颈部鳞状细胞癌细胞株生物学行为异同
对2种已知转移潜能的头颈部鳞状细胞癌细胞系UM-SCC-22A/UM-SCC-22B进行常规培养,采用MTS细胞增殖实验,细胞划痕实验及transwell侵袭实验,建立裸鼠活体内肿瘤转移生物发光模型,观察不同转移潜能细胞系的转移能力。结果显示：常规接种细胞培养24、48小时后,此两种细胞系增殖能力无明显差异(P>0.05)。细胞划痕试验表明,划痕产生48小时后,UM-SCC-22B细胞系划痕愈合率达66.50%,UM-SC-22A仅为18.69%,即UM-SCC-22B细胞体外迁移能力为UM-SCC-22A的3.56倍。Matrigel侵袭实验显示,接种105细胞于上室40小时后,UM-SCC-22B细胞系有53.56个细胞/观察视野穿透Matrigel,粘附于上室底膜下表面,UM-SCC-22A细胞系有25.45个细胞/观察视野,即UM-SCC-22B细胞体外侵袭能力为UM-SCC-22A的2.11倍。生物发光显示,UM-SCC-22B组细胞裸鼠左心房接种后,成瘤率和转移率明显高于UM-SCC-22A组。
2、不同转移潜能头颈部鳞状细胞癌细胞株中Hsp27mRNA和蛋白的表达
对通过体内外实验证实,具有不同转移潜能的头颈部鳞状细胞癌细胞系UM-SCC-22A/UM-SCC-22B进行常规培养,采用实时荧光定量PCR和Western blotting方法对Hsp27基因在mRNA和蛋白质水平的表达进行检测,分析其表达丰度与头颈部鳞状细胞癌细胞转移潜能的关系。实验发现,2种头颈部鳞状细胞癌细胞系均有Hsp27基因表达,高转移潜能细胞系UM-SCC-22B中Hsp27表达水平明显高于UM-SCC-22A,差异显著(P<0.01)。UM-SCC-22B细胞系Hsp27 mRNA水平是UM-SCC-22A的22.38倍,western blotting结果显示UM-SCC-22B细胞中,位于27kb位置条带明显强于UM-SCC-22A。头颈部鳞状细胞癌细胞系Hsp27表达丰度与其转移能力相关,随着转移能力的上升,Hsp27表达水平升高。
第二部分：瞬时转染siRNA干扰Hsp27基因效果鉴定及其对UM-SCC-22B迁移和侵袭的影响
1、siRNA干扰Hsp27基因及其干扰效果鉴定
根据siRNA设计原则,设计合成靶向Hsp27基因的siRNA,采用阳离子脂质体试剂瞬时转染高转移潜能头颈部鳞状细胞癌细胞系UM-SCC-22B,应用实时荧光定量PCR和Western Blotting检测RNA干扰后Hsp27基因的沉默效果。结果显示,siRNA Hsp27瞬时转染24小时后,Hsp27 mRNA水平明显下调,其干扰效率为93%,与空白对照组及阴性对照组比较,差异具有显著性(P<0.01)。siRNA Hsp27瞬时转染48小时后,Hsp27蛋白水平明显下调,western blotting结果显示,与空白对照组及阴性对照组比较,位于27kb位置条带明显强度明显下降,与实时荧光定量PCR结果一致。
2、siRNA干扰Hsp27基因对头颈部鳞状细胞癌细胞转移和侵袭的影响
于转染siRNA后48小时,应用细胞划痕试验检测Hsp27基因表达下调后,高转移潜能头颈部鳞状细胞癌细胞UM-SCC-22B迁移能力变化；应用Matrigel侵袭实验检测Hsp27基因表达下调后,UM-SCC-22B侵袭能力的改变。转染siRNA下调UM-SCC-22B细胞Hsp27基因表达后,细胞划痕后24小时,空白对照组和阴性对照组细胞划痕开始愈合,而siRNA转染组几乎没有愈合；72小时后空白对照组和阴性对照组愈合率达71.66%,50.57%,而siRNA转染组仅有少量愈合,愈合率为18.30%,与其它两组比较差异显著(P<0.01)。Matrigel侵袭实验结果显示,接种上室40小时后,siRNA转染组穿膜细胞数明显减少,每个观察视野仅有26.91个细胞,是空白对照组穿膜细胞数的50.24%,此结果与空白对照组和阴性对照组相比,具有显著性差异(P<0.01)
第三部分：Hsp27基因shRNA慢病毒靶向沉默Hsp27的效果鉴定及对UM-SCC-22B生物学行为的影响
1、Hsp27基因shRNA慢病毒载体的鉴定及其靶向沉默热休克蛋白27的效果鉴定
将购自Open Biosystem,可表达shRNA Hsp27的慢病毒载体质粒pLKO.1-shRNA-Hsp27质粒,转染入UM-SCC-22B细胞,运用实时荧光定量PCR检测Hsp27表达,初步鉴定质粒正确性和有效性。结果显示,转染24,48小时后,Hsp27 mRNA表达量分别降至42%,24%,干扰效果明显。密歇根大学载体中心包装后,得到高滴度慢病毒颗粒pLenti-shRNA-Hsp27,长效转染入UM-SCC-22B后,运用实时荧光定量PCR和western blotting检测Hsp27表达。结果显示,shRNA Hsp27长效转染后,Hsp27 mRNA水平明显下调,其干扰效率为94%,与空白对照组及阴性对照组比较,差异具有显著性(P<0.01)。Hsp27蛋白水平明显下调,western blotting结果显示,与空白对照组及阴性对照组比较,位于27kb位置条带明显强度明显下降,与实时荧光定量PCR结果一致。
2.Hsp27基因shRNA慢病毒转染后对头颈部鳞状细胞癌细胞生物学行为的影响
慢病毒颗粒pLenti-shRNA-Hsp27长效转染UM-SCC-22B,并高效干扰Hsp27表达后,采用MTS细胞增殖实验,细胞划痕实验及Matrigel侵袭实验,建立裸鼠活体内肿瘤转移生物发光模型,观察Hsp27表达抑制后,UM-SCC-22B转移能力变化。结果显示：常规接种细胞培养24、48小时后,与空白组和阴性对照组相比,shRNA Hsp27组增殖能力无明显差异(P>0.05)。细胞划痕试验表明,划痕产生72小时后,空白对照组细胞划痕愈合率达71.4%,shRNA Hsp27实验组仅为16.3%,即shRNA Hsp27干扰后,空白组细胞迁移能力为实验组4.38倍。Matrigel侵袭实验显示,接种105细胞于上室40小时后,shRNA Hsp27实验组有29.6个细胞／观察视野穿透Matrigel,粘附于上室底膜下表面,空白对照组细胞有60个细胞／观察视野,即空白组细胞体外侵袭能力为shRNA Hsp27组的2.03倍。荧光示踪显示,各组细胞裸鼠左心房接种后,空白组动物模型成瘤率和转移率明显高于实验组。
第四部分：重组Hsp27表达慢病毒载体的构建、鉴定及其对头颈部鳞状细胞癌细胞生物学行为的影响
1、重组Hsp27表达慢病毒载体的构建和鉴定
设计引入BamHI, Xbal两个酶切位点的人Hsp27基因引物,利用pOTB7-Hsp27全长cDNA质粒,PCR扩增Hsp27基因,平端酶切,回收纯化。利用BamHI, Xbal酶切位点,T4连接酶连接Hsp27基因片段和pLentiLox RSV慢病毒载体。双酶切电泳验证目的Hsp27基因片段连接正确,转化感受态DH5-alpha大肠杆菌,小提后进行重组质粒基因测序。测序正确,成功构建过表达Hsp27基因慢病毒载体pLenti-RSV-Hsp27。大提后送密歇根大学载体中心包装生产过表达Hsp27慢病毒颗粒pLenti-RSV-Hsp27。长效转染入低转移潜能头颈部鳞状细胞癌细胞系UM-SCC-22A后,实时荧光定量PCR和western blotting检测Hsp27表达。结果显示,转染后,Hsp27基因mRNA和蛋白水平均明显升高。过表达组Hsp27 mRNA水平是对照组的15.07倍,western blotting结果显示,过表达组27kb蛋白条带强度显著增高。
2、过表达Hsp27对头颈部鳞状细胞癌细胞生物学行为的影响
慢病毒颗粒plenti-RSV-Hsp27长效转染UM-SCC-22A,并高效表达Hsp27表达后,采用MTS细胞增殖实验,细胞划痕实验及Matrigel侵袭实验,建立裸鼠活体内荧光示踪肿瘤转移动物模型,观察Hsp27表达增加后,UM-SCC-22A转移能力变化。结果显示：常规接种细胞培养24、48小时后,与空白对照组相比,过表达Hsp27组增殖能力无明显差异(P>0.05)。细胞划痕试验表明,划痕产生48小时后,空白对照组细胞划痕愈合率为19.84%,过表达Hsp27实验组为51.33%,即Hsp27过表达后,细胞迁移能力为空白对照组2.56倍。Matrigel侵袭实验显示,接种105细胞于上室40小时后,Hsp27过表达组有59.13个细胞/观察视野穿透Matrigel,粘附于上室底膜下表面,空白对照组细胞有29.35个细胞/观察视野,即过表达组细胞体外侵袭能力为空白对照组的2.01倍。荧光示踪显示,各组细胞裸鼠左心房接种后,过表达组动物模型成瘤率和转移率明显高于对照组。
结论：
1.成功建立头颈部鳞状细胞癌转移细胞实验模型,建立头颈部鳞状细胞癌裸鼠活体内肿瘤转移生物发光模型。
2.头颈部鳞状细胞癌Hsp27的表达与其转移潜能密切相关,随其转移潜能的升高,其表达丰度升高。头颈部鳞状细胞癌低转移潜能细胞系UM-SCC-22A/高转移潜能细胞系UM-SCC-22B,可作为良好的实验模型,深入研究Hsp27表达调控与头颈部鳞状细胞癌转移间的关系。
3.体外合成,瞬时转染siRNA干扰技术可高效下调高转移潜能头颈部鳞状细胞癌细胞系UM-SCC-22B Hsp27基因的表达。
4.瞬时转染siRNAHsp27,高转移潜能头颈部鳞状细胞癌细胞系Hsp27基因下调后,显著抑制其细胞迁移和侵袭能力。Hsp27基因可作为有效头颈部鳞状细胞癌转移的治疗靶点。
5.长效转染慢病毒颗粒pLenti-shRNA-Hsp27能够高效、特异的沉默高转移潜能头颈部鳞状细胞癌细胞系UM-SCC-22B的Hsp27基因表达。
6.长效转染慢病毒颗粒pLenti-shRNA-Hsp27可显著抑制高转移潜能头颈部鳞状细胞癌细胞系UM-SCC-22B的体内外转移能力。
7.成功构建过表达Hsp27基因慢病毒载体pLenti-RSV-Hsp27。长效转染慢病毒颗粒pLenti-RSV-Hsp27可高效、特异性增加低转移潜能头颈部鳞状细胞癌细胞系UM-SCC-22A的Hsp27基因表达。
8.长效转染慢病毒颗粒pLenti-RSV-Hsp27可显著提高低转移潜能头颈部鳞状细胞癌细胞系UM-SCC-22A的体内外转移能力。
Head and neck squamous cell carcinoma (HNSCC) is the 6th most prevalent cancer types worldwide with an incidence of more than 500,000 cases annually and a high mortality rate, making it the fifth leading cause of cancer related death. In the United States, it accounts for 6% of all cancer diagnoses and results in an estimated 14,000 deaths annually. Despite the advanced therapeutic regimens used in treating HNSCC, oral cavity cancer survival during 1996-2003 was less than 50% on average according to the data from American Cancer Society. Furthermore, patients with recurrent or metastatic HNSCC have median survival of approximately 6 months. The major contributing factors for low survival include local-regional relapse, lymph node or distant metastatic spread of the primary tumor. The small Heat Shock Protein 27(Hsp27) belongs to heat shock protein family, and acts as a molecular chaperone. Expression of Hsp27 can be induced by environmental stress, such as heat shock, heavy metals, oxidants, infection, inflammation, ischemia. Hsp27 has been shown to have various cellular functions to promote differentiation, proliferation, cell growth, and motility. In addition, it has been reported that Hsp27 is associated with various carcinomas. RNA interference(RNAi) is a potential approach for gene therapy, thanks to its high efficience in silencing target sequence-specific gene, it has been used as a useful method in molecular biology study. And Lentivirus vector has become a common vector to expressing specific gene, as it could be transfected into separated/unseparated cells with larger gene fragment without the host immune response induced. To establish a head and neck squamous cell cancer model with stable expression of luciferase for in vivo imaging is a rising method to detect the tumor progression,metastasis and drug sensitivity. Especially, the left intracardic injection has been used as a better way to set up the in vivo imaging mice model, compared with tail vein injection,in site injection and other methods, it induces tumor quicker, easier to practice and has a higher survival rate for mice.
To figure out how Hsp27 expression regulate head and neck squamous cell carcinoma metastasis behavior and find out a noval target for HNSCC treatment, in the present study, we silenced Hsp27 gene expression in head and neck squamous cell cancer cells UM-SC-22B which shows a high potential of metastasis by using temporary transfection of Hsp27 siRNA and stable transfection of Lentivirus with shRNA Hsp27. At the same time, we overexpressed Hsp27 expression in UM-SC-22A with lower metastasis potentcy by re-constructuring a Lentivirus vector expressing Hsp27 gene. And we used MTS assay to detect the proliferation changes of cells, wound-healing motility assay and Matrigel invasion assay to detect the metastasis ability changes before and after silencing or overexpressing Hsp27 gene in vitro. Otherwise, we set up a metastasis in vivo imaging mice model to detect the tumor cells metastasis ability in vivo.
The whole dissertation consists of four parts:
Part 1 Biology behaviors and Hsp27 expression in HNSCC cell lines with different metastasis potential
1. Biology behaviors in HNSCC cell lines with different metastasis potential
Proliferation of the primary and metastatic HNSCC cell lines was evaluated using the MTS proliferation assay. Metastatic behavior was assessed using migration and invasion assays in vitro. Set up imaging mice model to detect the metastasis behavior in vivo. MTS assays showed that the primary (UM-SCC-22A) and metastatic (UM-SCC-22B) HNSCC have similar proliferation rates after cultured for 24/48h(p>0.05). However, UM-SCC-22B derived from the metastasis showed 2.3 to 3.6-fold higher migration ability and 2-fold higher invasion ability than UM-SCC-22A. And UM-SCC-22B shows high rate of metastasis of kidney and thing-bone in vivo.
2. Hsp27 expression in HNSCC cell lines with different metastasis potential
The expression of Hsp27 in primary and metastatic cell lines derived from the primary HNSCC and a synchronous lymph node metastasis in the same patient was determined using real-time PCR and western blotting. Real-time PCR demonstrated that Hsp27 mRNA is 22.4-fold higher in metastatic UM-SCC-22B than primary UM-SCC-22A. Similarly, Western blotting showed that Hsp27 is rarely detectable in UM-SCC-22A whereas UM-SCC-22B expresses a higher level of Hsp27 protein.
These data indicate higher expression of Hsp27 shows in UM-SCC-22B with higher metastasis potential, and Hsp27 may regulate metastatic potential of HNSCC cancer cells.
Part 2 Hsp27 expression and biology behaviors changes in vitro after silencing Hsp27 by transfected with siRNA Hsp27 in UM-SCC-22B
1. Hsp27 expression changes in vitro after silencing Hsp27 by transfected with siRNA Hsp27 in UM-SCC-22B
Designed a specific siRNA sequence to knocking down Hsp27 in the highly migratory metastatic HNSCC cell line UM-SCC-22B. The expression of Hsp27 in UM-SCC-22B before and after silencing Hsp27 was determined using real-time PCR and western blotting. Compared to blank control group, after transfected with siRNA Hsp27 by lipofectamine 2000, the mRNA level of Hsp27 was reduced by 93%.And at 48h after transfection, the protein level of Hsp27 was dramatically decreased in consistent.
2. biology behaviors changes in vitro after silencing Hsp27 by transfected with siRNA Hsp27 in UM-SCC-22B
Furthermore, proliferation of UM-SCC-22B before and after silencing Hsp27 was evaluated using the MTS proliferation assay. Metastatic behavior was assessed using migration and invasion assays in vitro. MTS assays showed that the UM-SCC-22B before and after silencing Hsp27 have similar proliferation rates after cultured for 24/48h (p>0.05). However, compared to blank control group,siRNA knockdown of Hsp27 decreased metastatic behaviors of UM-SCC-22B by 3 to 4-fold in migration and 2-fold in cell invasion reducing cell invasion and migration.
These data indicate temporarily silencing Hsp27 expression by siRNA may decrease metastasis in head and neck squamous cell cancer cells.
Part 3 Bilology behaviors and Hsp27 expression changes after knocking down Hsp27 by tranfected with Lentivirus-shRNA-Hsp27 in UM-SCC-22B
1. Hsp27 expression changes after knocking down Hsp27 by tranfected with Lentivirus-shRNA- Hsp27 in UM-SCC-22B
Phurcase the Lentivirus vector plasmid with shRNA Hsp27 from Open Biosystem. After temperarily transfected into UM-SCC-22B, using real-time PCR to detect the mRNA of Hsp27 expression. The result shows that compared with blank control group, after 24/48h, the mRNA level of Hsp27 reduced to 42%/24%. Then produce lentivirus particals with shRNA Hsp27,stably transfected into UM-SCC-22B,using real-time PCR and western blotting assay to detect the mRNA and protein level changes of Hsp27 expression. The data shows mRNA level of Hsp27 was decreased by 94%,comparing to blank control group, meanwhile, the protein level was also significantly reduced in consistent.
2. Bilology behaviors changes after knocking down Hsp27 by tranfected with Lentivirus-shRNA- Hsp27 in UM-SCC-22B
Using MTS assay to detect the proliferation changes of UM-SCC-22B before and after stable transfected with lentivirus particals pLenti-shRNA-Hsp27 with shRNA Hsp27. Metastatic behavior was assessed using migration and invasion assays in vitro. MTS assays showed that the UM-SCC-22B before and after knocking down Hsp27 have similar proliferation rates after cultured for 24/48h (p>0.05). However, compared to blank control group,shRNA knockdown of Hsp27 decreased metastatic behaviors of UM-SCC-22B by 4.38-fold in migration and 2.03-fold in cell invasion reducing cell invasion and migration. Besides, UM-SCC-22B with shRNA Hsp27 shows lower rate of metastasis of kidney and thing-bone in vivo.
These data indicate stably knocking down Hsp27 expression by shRNA may decrease metastasis in head and neck squamous cell cancer cells.
Part 4 Bilology behaviors and Hsp27 expression changes after over-expressing Hsp27 by tranfected with Lentivirus- Hsp27 in UM-SCC-22A
1.Re-constructured Lentivirus vector expressiong Hsp27 and Hsp27 expression changes after over-expressing Hsp27 by tranfected with Lentivirus- Hsp27 in UM-SCC-22A
The full length cDNA pOTB7-Hsp27 plasmid was got from Invitrogen, design specific primers with BamHI, Xbal restriction enzyme sites,PCR was used to get the fragment of Hsp27 sequence with BamHI, Xbal. The fragment of Hsp27 and the pLentiLox-RSV lentivirus vector were ligated by T4 DNA ligase.After transferring them into the DH5-alpha competent cells, restracted the recombined plamides DNA.The recombinant clones were identified by PCR with 'the primers and digested it by restriction enzyme sites BamHI, Xbal for further identification. Then the positice recombinant pLenti-RSV-Hsp27 plasmid was sequenced. Packaging and producing pLenti-RSV-Hsp27 were processed.Stably transfected UM-SCC-22A with lentivirus particals pLenti-RSV-Hsp27,using real-time PCR and western blotting assay to detect the Hsp27 expression changes. The data shows mRNA level of Hsp27 was increased by 15.07-fold,and protein level of Hsp27 was also induced dramatically.
2. Bilology behaviors changes after over-expressing Hsp27 by tranfected with Lentivirus-Hsp27 in UM-SCC-22A
Using MTS assay to detect the proliferation changes of UM-SCC-22A before and after stable transfected with lentivirus particals pLenti-RSV-Hsp27 with expressing Hsp27. Metastatic behavior was assessed using migration and invasion assays in vitro. MTS assays showed that the UM-SCC-22A before and after overexpressing Hsp27 have similar proliferation rates after cultured for 24/48h (p>0.05). However, compared to blank control group, overexprssing Hsp27 increased metastatic behaviors of UM-SCC-22A by 2.56-fold in migration and 2.01-fold in cell invasion reducing cell invasion and migration. Besides, UM-SCC-22A with overexpressing Hsp27 shows higher rate of metastasis of kidney and thing-bone in vivo.
These data indicate overexpressing Hsp27 expression by stably transfected lentivirus particals pLenti-RSV-Hsp27 may increase metastasis in head and neck squamous cell cancer cells.
Conclusion:
1.Successfully set up a head and neck squamous cell cancer metastasis cell model in vitro and imaging in vivo mice model.
2.Hsp27 could regulate the metastasis behavior of HNSCC, and it show higher expression in HNSCC cells with higher metastasis ability.
3.Temperarily transfected with siRNA Hsp27 could efficiently decrease Hsp27 expression on mRNA an protein levels in UM-SCC-22B.
4.silencing Hsp27 expression by siRNA could reduce the metastasis ability of UM-SCC-22B.
5.Stably transfected with lentivirus -shRNA-Hsp27 could efficiently decrease Hsp27 expression on mRNA an protein levels in UM-SCC-22B.
6. Stably transfected with lentivirus -shRNA-Hsp27 could reduce the metastasis ability of UM-SCC-22B.
7. Successfully re-constructured Lentivirus vector expressiong Hsp27. Stably transfected with lentivirus -RSV-Hsp27 could efficiently increase Hsp27 expression on mRNA an protein levels in UM-SCC-22A.
8. Stably transfected with lentivirus -RSV-Hsp27 could induce the metastasis ability of UM-SCC-22A.
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