miR-205下调ASPP2表达促进食管癌转移及EMT的作用及机制研究
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摘要
研究背景及目的：
食管癌是常见的消化道肿瘤，全世界每年约有30万人死于食管癌。其发病率和死亡率各国差异很大。我国是世界上食管癌高发地区之一，每年平均病死约15万人。肿瘤转移和复发是食管癌预后差的主要原因之一，目前根治性食管癌切除后5年复发率仍高达40%以上。因此，如果可以从遗传学以及表观遗传学的角度来深入研究食管癌进展和食管癌转移的机制，将能有效的为食管癌的诊断和治疗提供新靶点和策略。
ASPP2是凋亡调控蛋白ASPP (apoptosis stimulating proteins of p53)家族的一个重要的肿瘤抑制分子。ASPP2+/-的小鼠能自发形成肿瘤，且ASPP2杂合缺失能增加小鼠γ射线诱发肿瘤的发生率。研究也表明ASPP2在结肠癌、乳腺癌及血液系统肿瘤中都存在表达下调，我们前期也证明ASPP2在肝癌细胞系及临床病例中均存在启动子区高甲基化所致的基因沉默，提示我们ASPP2是一个重要的广谱的抑癌基因。
已知的ASPP2的表达调控方式主要包括：①E2F1的转录激活，②启动子区甲基化导致的转录抑制，③泛素化促进蛋白降解；其中甲基化是多种肿瘤中ASPP2表达水平下降的主要原因。但我们的研究结果表明甲基化不是食管癌中ASPP2表达水平下降的主要原因；检测食管癌细胞系中ASPP2的内源性表达水平，发现ASPP2的mRNA与蛋白表达水平存在很大差异，提示ASPP2存在转录后蛋白水平的调控方式。
miRNA是近年来发现的一系列的内源性单链非编码的小RNAs分子，长度约18-25个核苷酸。其广泛分布于真核生物中，成熟的miRNA在RNA诱导沉默复合物（RISC）的引导下与靶信使RNA的3’非编码区（3’-UTR）完全或不完全配对，降解靶信使RNA或阻遏其转录后翻译，从而参与靶基因的表达调控。研究表明，在哺乳动物中miRNA主要是通过抑制转录后翻译的方式调控靶基因表达，是基因转录后翻译水平调控的重要方式。miRNA的表达具有组织和疾病特异性，使其有望成为新型的肿瘤标记物；此外，miRNA可以分泌且稳定存在于外周循环，为其成为新的无创的诊断性生物标记提供了有利条件。
肿瘤转移是肿瘤细胞、宿主细胞和组织微环境之间相互作用的序贯连续过程。上皮细胞间质化(Epithelial-mesenchymal transition，EMT)在肿瘤转移中的作用越来越引起人们的重视。2011年Lu X等发现ASPP2在中枢神经系统发育中发挥了重要的作用，ASPP2作为Par-3的结合分子，通过调节神经细胞的极性影响细胞的增殖及组织重构。同时，Shigeo等在MDCK细胞中证明ASPP2通过影响PAR复合体的形成调节上皮细胞顶-基地极性的建立和维持。但是，ASPP2是否参与了癌性EMT的调控还尚无报道。
研究方法及结果：
1.免疫组化检测ASPP2在食管癌组织中的表达及其与临床指标的关系
我们通过免疫组化技术，检测了ASPP2和E-cadherin在食管癌中的表达情况。在正常的食管粘膜中ASPP2主要分布于棘细胞层到角质细胞层，而在基底层细胞表达缺失，并且，ASPP2在食管癌原发灶及转移灶中的表达水平均较正常食管上皮细胞有显著下调。通过分析ASPP2与多项临床指标之间的关系，发现ASPP2与食管癌的浸润深度（P=0.004）、淋巴结转移（P=0.026）及远处转移（P=0.043）呈负相关，而与其它临床指标无明显相关性。通过Wilcoxon rank sum检验分析ASPP2表达水平与E-cadherin表达情况的关系，发现在E-cadherin表达正常组ASPP2的表达水平显著高于E-cadherin表达异常组（P<0.001），提示我们ASPP2可能参与了EMT的调控。Log-Rank检验分析ASPP2的表达和患者生存的关系发现，发现ASPP2低表达组患者的总体生存期显著差于ASPP2高表达组（37.67±3.10vs.49.70±3.16，month），结果提示我们食管癌中ASPP2的表达水平较正常组织显著下降，且ASPP2低表达与食管癌转移及预后差呈正相关。
2. miR-205抑制ASPP2转录后翻译下调ASPP2的表达
我们利用QRT-PCR及Westen blots两种方法分别检测了ASPP2在正常食管粘膜细胞Het-1A细胞及11株食管癌细胞系KYSE30、KYSE70、KYSE140、KYSE150、KYSE180、KYSE410、TE3、TE7、TE10、TE11、TE12中的mRNA和蛋白表达水平。发现食管癌细胞系中ASPP2的mRNA水平和蛋白表达水平不一致，提示在食管癌中ASPP2存在转录后水平的调控机制来抑制其表达。通过Microlnspector、miRanda及targetscan三个预测软件预测ASPP2的调控miRNA，并通过luciferase报告基因系统验证，发现miR-205能与ASPP23’URT的23nt-31nt结合。通过转染miR-205的mimics或inhibitor来调节miR-205的表达水平，然后检测ASPP2的蛋白表达水平和mRNA表达水平，发现miR-205能够显著下调ASPP2蛋白水平而对RNA水平无明显影响。结果提示miR-205能够通过与ASPP23’URT区结合进而抑制ASPP2翻译下调其表达。
3. miR-205在组织及血清中的表达及其与临床指标的关系
我们利用Taqman探针法QRT-PCR技术检测了117例食管癌组织（石蜡标本）中miR-205的表达水平，这部分标本有完整的临床资料及预后分析；同时检测了56对食管癌组织及相应血清（均为冰冻标本）中miR-205的表达水平，其中28例有相应的癌旁正常食管粘膜，其miR-205的表达水平也一并做了检测。通过配对T检验比较28对癌和癌旁组织中miR-205的表达水平，发现癌组织中miR-205的表达水平较癌旁组织显著增高。通过Wilcoxon rank sum检验比较，发现淋巴结转移组或远处转移组中miR-205的表达水平显著高于无转移组（P<0.05）。通过Wilcoxon rank sum检验分析miR-205和ASPP2的表达水平的关系，发现ASPP2高表达组中miR-205的表达水平显著低于ASPP2低表达组（P<0.05），进一步验证了miR-205对ASPP2的负调控。Log-Rank检验分析miR-205的表达和患者生存的关系发现，miR-205高表达组患者的总体生存期显著差于miR-205低表达组（40.66±3.28vs.49.70±3.34，month）。通过pearsoncorelation检测，分析组织中miR-205和血清中miR-205的相关性，发现血清中miR-205的表达水平与组织中miR-205的表达水平成正相关。提示我们血清中miR-205能很好的反应原位瘤中miR-205的表达情况，为miR-205成为食管癌预后指标提供更有利的依据。
4.下调ASPP2促进食管癌转移及EMT的作用
选取ASPP2高表达细胞株KYSE30，利用慢病毒载体干扰KYSE30细胞内源性ASPP2的表达水平，和对照组相比，下调内源性ASPP2表达后KYSE30细胞的迁移能力，侵袭能力，裸鼠皮下肿瘤的浸润生长能力均显著增强，体内实验转移模型中肺转移灶及肝转移显著增加。检测EMT相关指标，下调内源性ASPP2表达后KYSE30细胞呈典型间充质表型，伴随E-cadherin mRNA及蛋白表达水平的下降、N-cadherin、Fibronectin表达水平上升。证实下调ASPP2能显著促进食管癌转移及EMT进程。
5.过表达ASPP2阻滞miR-205介导的转移能力增加及EMT进程
利用慢病毒载体上调KYSE30细胞miR-205的表达水平，72小时后分别向实验组和对照组细胞瞬时转染pcASPP2上调ASPP2的表达水平，结果显示上调miR-205表达后KYSE30细胞的迁移能力，侵袭能力显著增强，过表达ASPP2后miR-205的作用被阻滞。检测EMT相关指标，发现上调miR-205表达后KYSE30细胞E-cadherin的表达水平显著下降，过表达ASPP2后E-cadherin的表达水平被恢复。利用慢病毒载体下调KYSE150内源性miR-205的表达水平，和对照组相比，下调内源性miR-205表达后KYSE150细胞的迁移能力，侵袭能力、划痕修复能力及裸鼠皮下肿瘤的浸润生长能力均显著下降，体内转移模型中肺转移灶的数量显著减少。检测EMT相关指标，下调内源性miR-205表达后E-cadherin mRNA及蛋白表达水平显著增加，N-cadherin、Fibronectin表达水平下降。证实miR-205-能通过下调ASPP2促进食管癌转移及EMT进程。
结论：
我们首次检测ASPP2在食管癌中的表达水平，发现ASPP2在食管癌细胞系中，原发瘤及淋巴转移灶中表达水平均显著下降，且ASPP2的表达下调与食管癌转移及预后不良呈正相关，提示它在食管癌的转移中发挥了重要的作用。阐明miR-205通过抑制ASPP2转录后翻译下调ASPP2表达的分子机制，揭示ASPP2的全新调控方式。以食管癌为模型从EMT的角度，阐明ASPP2抑制肿瘤转移的作用和机制；同时分析miR-205与食管癌转移和预后的关系，评价血清miR-205作为食管癌预后指标的价值，为临床诊断提供帮助。
Background and Aims：
ASPP2, a member of the ankyrin-repeat-, SH3-domain-and proline-rich-region-containing protein (ASPP) family is frequently decreased in various cancers.Clinical data reveals that reduced expression of ASPP2is related with poor clinicaloutcomes in diffuse large B-cell lymphoma and tumor metastasis and poorrecurrence-free survival in breast cancer patients. The effects and underlying mechanismof ASPP2on tumor metastasis are remains unknown.
Previous studies have suggested that the expression of ASPP1and ASPP2could beactivated by E2F or inactivated by DNA methylation. Hypermethylation of ASPP1andASPP2promoters is found in several tumor cell lines expressing wildtype p53. wepreviously showed that ASPP1and ASPP2were frequently methylated in HCCs. What’sthe regulation mechanism of ASPP2in Esophageal Squamous Cell Carcinoma (ESCC)remains unknown.
In order to metastasis to distant oranges, tumor cells must acquire motility andinvasiveness properties to disseminate from primary tumors. Recent evidences havesuggested that a subset of epithelial tumor cells acquires such abilities by undergoingepithelial-mesenchymal transition (EMT), characterized by the loss of cell polarity, andgain of mesenchymal differentiation properties. A key step in EMT is down-regulation ofE-cadherin. Recently, ASPP2is found to be involved in the regulation of cell polaritythrough binding with the PAR complex protein Par-3at cell junctions in neuralprogenitor cells or epithelial cells. The importance of cell polarity and adherens junctionin EMT indicates a possible role of ASPP2in the regulation of EMT and tumormetastasis.
Methods and Results：
In current study; we analyzed ASPP2expression in132Esophageal Squamous CellCarcinoma (ESCC) samples and revealed that ASPP2expression was down-regulated inprimary tumor and lymph metastasis compared with the normal esophageal tissue. Further,decreased ASPP2expression correlated with tumor metastasis poor prognosis in ESSCpatients.
MicroRNAs (miRNAs) inhibit translation or induce mRNA degradation in general by binding to the3’ untranslational region (3’UTR) of target mRNAs. Since ASPP2wasregulated at protein level in ESSC, we analyzed potential miRNA target ASPP2throughtargetscan，Microlnspector and targetscan, the results showed that miR-205, miR-221andmiR-222may target ASPP2. Luciferase report systerm demonstrats that miR-205can bindwith ASPP23’UTR. We detect expression of miR-205in ESCC cell lince, the data showsthat expression of miR-205is sharply increased in ESSC cell lines compared with that inthe normal esophageal cell line. Further, The expression of miR-205in ESCC cells weredeceased by miR-205inhibitor or increased by miR-205mimics, then mRNA and proteinlevels of ASPP2are detected, the results show miR-205decreased ASPP2expression byinhibits it’s translation.
The expression of ASPP2and miR-205were deceased by RNA interference orincreased by transected with over expression plasmid, then tumorigenicity and metastasispotentials of ESSC cells were studied in vitro and in nude mice. Wound-healing,migrationg and invasion assays show silence of ASPP2incerase migration and invasionpotentials of ESSC cells; overexpression miR-205may increase migration and invasionpotentials of ESSC cells as well, which can be inhibit by ASPP2. Futher, silence of ASPP2or overexpression of miR-205promot tumor metastasis in vivo. RT-PCR,Western blotand Immunofluorscence were used to analysis EMT. The results show silence of ASPP2induce morphological and molecular changes of EMT, overexpression of miR-205aslomay induce morphological and molecular changes of EMT and that can be inhibit byASPP2.
Colusion：
Our findings present functional and mechanistic insight into the critical role of ASPP2in the progression and metastasis of cancer.
食管癌是常见的消化道肿瘤，全世界每年约有30万人死于食管癌。其发病率和死亡率各国差异很大。我国是世界上食管癌高发地区之一，每年平均病死约15万人。肿瘤转移和复发是食管癌预后差的主要原因之一，目前根治性食管癌切除后5年复发率仍高达40%以上。因此，如果可以从遗传学以及表观遗传学的角度来深入研究食管癌进展和食管癌转移的机制，将能有效的为食管癌的诊断和治疗提供新靶点和策略。
ASPP2是凋亡调控蛋白ASPP (apoptosis stimulating proteins of p53)家族的一个重要的肿瘤抑制分子。ASPP2+/-的小鼠能自发形成肿瘤，且ASPP2杂合缺失能增加小鼠γ射线诱发肿瘤的发生率。研究也表明ASPP2在结肠癌、乳腺癌及血液系统肿瘤中都存在表达下调，我们前期也证明ASPP2在肝癌细胞系及临床病例中均存在启动子区高甲基化所致的基因沉默，提示我们ASPP2是一个重要的广谱的抑癌基因。
已知的ASPP2的表达调控方式主要包括：①E2F1的转录激活，②启动子区甲基化导致的转录抑制，③泛素化促进蛋白降解；其中甲基化是多种肿瘤中ASPP2表达水平下降的主要原因。但我们的研究结果表明甲基化不是食管癌中ASPP2表达水平下降的主要原因；检测食管癌细胞系中ASPP2的内源性表达水平，发现ASPP2的mRNA与蛋白表达水平存在很大差异，提示ASPP2存在转录后蛋白水平的调控方式。
miRNA是近年来发现的一系列的内源性单链非编码的小RNAs分子，长度约18-25个核苷酸。其广泛分布于真核生物中，成熟的miRNA在RNA诱导沉默复合物（RISC）的引导下与靶信使RNA的3’非编码区（3’-UTR）完全或不完全配对，降解靶信使RNA或阻遏其转录后翻译，从而参与靶基因的表达调控。研究表明，在哺乳动物中miRNA主要是通过抑制转录后翻译的方式调控靶基因表达，是基因转录后翻译水平调控的重要方式。miRNA的表达具有组织和疾病特异性，使其有望成为新型的肿瘤标记物；此外，miRNA可以分泌且稳定存在于外周循环，为其成为新的无创的诊断性生物标记提供了有利条件。
肿瘤转移是肿瘤细胞、宿主细胞和组织微环境之间相互作用的序贯连续过程。上皮细胞间质化(Epithelial-mesenchymal transition，EMT)在肿瘤转移中的作用越来越引起人们的重视。2011年Lu X等发现ASPP2在中枢神经系统发育中发挥了重要的作用，ASPP2作为Par-3的结合分子，通过调节神经细胞的极性影响细胞的增殖及组织重构。同时，Shigeo等在MDCK细胞中证明ASPP2通过影响PAR复合体的形成调节上皮细胞顶-基地极性的建立和维持。但是，ASPP2是否参与了癌性EMT的调控还尚无报道。
研究方法及结果：
1.免疫组化检测ASPP2在食管癌组织中的表达及其与临床指标的关系
我们通过免疫组化技术，检测了ASPP2和E-cadherin在食管癌中的表达情况。在正常的食管粘膜中ASPP2主要分布于棘细胞层到角质细胞层，而在基底层细胞表达缺失，并且，ASPP2在食管癌原发灶及转移灶中的表达水平均较正常食管上皮细胞有显著下调。通过分析ASPP2与多项临床指标之间的关系，发现ASPP2与食管癌的浸润深度（P=0.004）、淋巴结转移（P=0.026）及远处转移（P=0.043）呈负相关，而与其它临床指标无明显相关性。通过Wilcoxon rank sum检验分析ASPP2表达水平与E-cadherin表达情况的关系，发现在E-cadherin表达正常组ASPP2的表达水平显著高于E-cadherin表达异常组（P<0.001），提示我们ASPP2可能参与了EMT的调控。Log-Rank检验分析ASPP2的表达和患者生存的关系发现，发现ASPP2低表达组患者的总体生存期显著差于ASPP2高表达组（37.67±3.10vs.49.70±3.16，month），结果提示我们食管癌中ASPP2的表达水平较正常组织显著下降，且ASPP2低表达与食管癌转移及预后差呈正相关。
2. miR-205抑制ASPP2转录后翻译下调ASPP2的表达
我们利用QRT-PCR及Westen blots两种方法分别检测了ASPP2在正常食管粘膜细胞Het-1A细胞及11株食管癌细胞系KYSE30、KYSE70、KYSE140、KYSE150、KYSE180、KYSE410、TE3、TE7、TE10、TE11、TE12中的mRNA和蛋白表达水平。发现食管癌细胞系中ASPP2的mRNA水平和蛋白表达水平不一致，提示在食管癌中ASPP2存在转录后水平的调控机制来抑制其表达。通过Microlnspector、miRanda及targetscan三个预测软件预测ASPP2的调控miRNA，并通过luciferase报告基因系统验证，发现miR-205能与ASPP23’URT的23nt-31nt结合。通过转染miR-205的mimics或inhibitor来调节miR-205的表达水平，然后检测ASPP2的蛋白表达水平和mRNA表达水平，发现miR-205能够显著下调ASPP2蛋白水平而对RNA水平无明显影响。结果提示miR-205能够通过与ASPP23’URT区结合进而抑制ASPP2翻译下调其表达。
3. miR-205在组织及血清中的表达及其与临床指标的关系
我们利用Taqman探针法QRT-PCR技术检测了117例食管癌组织（石蜡标本）中miR-205的表达水平，这部分标本有完整的临床资料及预后分析；同时检测了56对食管癌组织及相应血清（均为冰冻标本）中miR-205的表达水平，其中28例有相应的癌旁正常食管粘膜，其miR-205的表达水平也一并做了检测。通过配对T检验比较28对癌和癌旁组织中miR-205的表达水平，发现癌组织中miR-205的表达水平较癌旁组织显著增高。通过Wilcoxon rank sum检验比较，发现淋巴结转移组或远处转移组中miR-205的表达水平显著高于无转移组（P<0.05）。通过Wilcoxon rank sum检验分析miR-205和ASPP2的表达水平的关系，发现ASPP2高表达组中miR-205的表达水平显著低于ASPP2低表达组（P<0.05），进一步验证了miR-205对ASPP2的负调控。Log-Rank检验分析miR-205的表达和患者生存的关系发现，miR-205高表达组患者的总体生存期显著差于miR-205低表达组（40.66±3.28vs.49.70±3.34，month）。通过pearsoncorelation检测，分析组织中miR-205和血清中miR-205的相关性，发现血清中miR-205的表达水平与组织中miR-205的表达水平成正相关。提示我们血清中miR-205能很好的反应原位瘤中miR-205的表达情况，为miR-205成为食管癌预后指标提供更有利的依据。
4.下调ASPP2促进食管癌转移及EMT的作用
选取ASPP2高表达细胞株KYSE30，利用慢病毒载体干扰KYSE30细胞内源性ASPP2的表达水平，和对照组相比，下调内源性ASPP2表达后KYSE30细胞的迁移能力，侵袭能力，裸鼠皮下肿瘤的浸润生长能力均显著增强，体内实验转移模型中肺转移灶及肝转移显著增加。检测EMT相关指标，下调内源性ASPP2表达后KYSE30细胞呈典型间充质表型，伴随E-cadherin mRNA及蛋白表达水平的下降、N-cadherin、Fibronectin表达水平上升。证实下调ASPP2能显著促进食管癌转移及EMT进程。
5.过表达ASPP2阻滞miR-205介导的转移能力增加及EMT进程
利用慢病毒载体上调KYSE30细胞miR-205的表达水平，72小时后分别向实验组和对照组细胞瞬时转染pcASPP2上调ASPP2的表达水平，结果显示上调miR-205表达后KYSE30细胞的迁移能力，侵袭能力显著增强，过表达ASPP2后miR-205的作用被阻滞。检测EMT相关指标，发现上调miR-205表达后KYSE30细胞E-cadherin的表达水平显著下降，过表达ASPP2后E-cadherin的表达水平被恢复。利用慢病毒载体下调KYSE150内源性miR-205的表达水平，和对照组相比，下调内源性miR-205表达后KYSE150细胞的迁移能力，侵袭能力、划痕修复能力及裸鼠皮下肿瘤的浸润生长能力均显著下降，体内转移模型中肺转移灶的数量显著减少。检测EMT相关指标，下调内源性miR-205表达后E-cadherin mRNA及蛋白表达水平显著增加，N-cadherin、Fibronectin表达水平下降。证实miR-205-能通过下调ASPP2促进食管癌转移及EMT进程。
结论：
我们首次检测ASPP2在食管癌中的表达水平，发现ASPP2在食管癌细胞系中，原发瘤及淋巴转移灶中表达水平均显著下降，且ASPP2的表达下调与食管癌转移及预后不良呈正相关，提示它在食管癌的转移中发挥了重要的作用。阐明miR-205通过抑制ASPP2转录后翻译下调ASPP2表达的分子机制，揭示ASPP2的全新调控方式。以食管癌为模型从EMT的角度，阐明ASPP2抑制肿瘤转移的作用和机制；同时分析miR-205与食管癌转移和预后的关系，评价血清miR-205作为食管癌预后指标的价值，为临床诊断提供帮助。
Background and Aims：
ASPP2, a member of the ankyrin-repeat-, SH3-domain-and proline-rich-region-containing protein (ASPP) family is frequently decreased in various cancers.Clinical data reveals that reduced expression of ASPP2is related with poor clinicaloutcomes in diffuse large B-cell lymphoma and tumor metastasis and poorrecurrence-free survival in breast cancer patients. The effects and underlying mechanismof ASPP2on tumor metastasis are remains unknown.
Previous studies have suggested that the expression of ASPP1and ASPP2could beactivated by E2F or inactivated by DNA methylation. Hypermethylation of ASPP1andASPP2promoters is found in several tumor cell lines expressing wildtype p53. wepreviously showed that ASPP1and ASPP2were frequently methylated in HCCs. What’sthe regulation mechanism of ASPP2in Esophageal Squamous Cell Carcinoma (ESCC)remains unknown.
In order to metastasis to distant oranges, tumor cells must acquire motility andinvasiveness properties to disseminate from primary tumors. Recent evidences havesuggested that a subset of epithelial tumor cells acquires such abilities by undergoingepithelial-mesenchymal transition (EMT), characterized by the loss of cell polarity, andgain of mesenchymal differentiation properties. A key step in EMT is down-regulation ofE-cadherin. Recently, ASPP2is found to be involved in the regulation of cell polaritythrough binding with the PAR complex protein Par-3at cell junctions in neuralprogenitor cells or epithelial cells. The importance of cell polarity and adherens junctionin EMT indicates a possible role of ASPP2in the regulation of EMT and tumormetastasis.
Methods and Results：
In current study; we analyzed ASPP2expression in132Esophageal Squamous CellCarcinoma (ESCC) samples and revealed that ASPP2expression was down-regulated inprimary tumor and lymph metastasis compared with the normal esophageal tissue. Further,decreased ASPP2expression correlated with tumor metastasis poor prognosis in ESSCpatients.
MicroRNAs (miRNAs) inhibit translation or induce mRNA degradation in general by binding to the3’ untranslational region (3’UTR) of target mRNAs. Since ASPP2wasregulated at protein level in ESSC, we analyzed potential miRNA target ASPP2throughtargetscan，Microlnspector and targetscan, the results showed that miR-205, miR-221andmiR-222may target ASPP2. Luciferase report systerm demonstrats that miR-205can bindwith ASPP23’UTR. We detect expression of miR-205in ESCC cell lince, the data showsthat expression of miR-205is sharply increased in ESSC cell lines compared with that inthe normal esophageal cell line. Further, The expression of miR-205in ESCC cells weredeceased by miR-205inhibitor or increased by miR-205mimics, then mRNA and proteinlevels of ASPP2are detected, the results show miR-205decreased ASPP2expression byinhibits it’s translation.
The expression of ASPP2and miR-205were deceased by RNA interference orincreased by transected with over expression plasmid, then tumorigenicity and metastasispotentials of ESSC cells were studied in vitro and in nude mice. Wound-healing,migrationg and invasion assays show silence of ASPP2incerase migration and invasionpotentials of ESSC cells; overexpression miR-205may increase migration and invasionpotentials of ESSC cells as well, which can be inhibit by ASPP2. Futher, silence of ASPP2or overexpression of miR-205promot tumor metastasis in vivo. RT-PCR,Western blotand Immunofluorscence were used to analysis EMT. The results show silence of ASPP2induce morphological and molecular changes of EMT, overexpression of miR-205aslomay induce morphological and molecular changes of EMT and that can be inhibit byASPP2.
Colusion：
Our findings present functional and mechanistic insight into the critical role of ASPP2in the progression and metastasis of cancer.
引文
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