RNAi技术干扰骨肉瘤细胞Dll4表达抑制侵袭转移和血管生成的体内外实验研究
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摘要
目的:探讨Dll4在骨肉瘤组织的表达及意义;靶向沉默Dll4的表达对骨肉瘤侵袭转移和血管生成的作用机制。
方法:
(1)采用免疫组化的方法检测62例骨肉瘤组织中Dll4蛋白的表达,结合临床病理资料分析,研究Dll4的表达与临床病理特征的关系。
(2)构建全长的逆转录病毒pSUPER-Dll4-siRNA质粒,通过鉴定后,将其稳定转染到PT67细胞培养,获得分泌Dll4蛋白病毒血清,干预沉默人骨肉瘤细胞株SOSP-9607E10中Dll4的表达;干预沉默人微血管内皮细胞系(HMEC-1),体外采用划痕实验、Transwell侵袭实验、血管成管实验、MTT和流式细胞仪检测等方法,研究Dll4调控骨肉瘤细胞分化增殖、骨肉瘤侵袭运动和骨肉瘤血管成管的分子机制验证Dll4调控骨肉瘤血管成管的分子机制导致肿瘤生物学行为的改变。
结果:
(1)Notch1、Dll4在骨肉瘤和骨软骨瘤中的表达
Notch1蛋白的阳性表达主要在肿瘤细胞的细胞质或细胞膜,呈棕黄色,散在分布。62例骨肉瘤中有53例呈阳性表达,阳性率为85.5%(53/62),对照组为21.2%(9/43),两组差异具有显著性(P＜0.05)。Dll4蛋白的阳性表达主要在肿瘤细胞的细胞质或细胞膜,部分表达在血管内皮细胞。62例骨肉瘤中有55例呈阳性表达,阳性率为88.7%(54/62),对照组为23.6%(10/43),两者之间差别具有显著性(P＜0.05),Notch1和Dll4在骨肉瘤中的表达高于在骨软骨瘤中的表达,提示Notch1和Dll4可能参与了骨肉瘤的发生、发展。
(2).Notch1、Dll4表达及其与骨肉瘤病理临床指标的关系
Notch1与Dll4在骨肉瘤中表达存在正相关关系,相关系数rs为0.842,P=0.027。Notch1及Dll4蛋白表达与骨肉瘤组织分化程度、Ennecking分期和有无转移密切相关(P＜0.05),但与肿瘤直径、性别、年龄、ALP(碱性磷酸酶)无关(P＞0.05)。
(3)骨肉瘤中Notch1和Dll4表达与MVD表达的关系
Notch1高表达组MVD为35.2±9.3,显著高于低表达组28.3±3.4,此外,Dll4高表达组MVD为36.2±7.2,显著高于低表达组29.5±2.2,差别具有显著性(P＜0.05)。骨肉瘤中Notch1和Dll4表达与MVD表达的具有相关性(P＜0.05),Notch1及Dll4蛋白高表达可能在骨肉瘤血管生成进程中起重要作用。
(4)Dll4的表达与骨肉瘤的侵袭转移潜能密切相关
Dll4 mRNA和蛋白在SOSP-9607H9及SOSP-9607E10两株细胞中的表达。结果显示:在SOSP-9607H9及SOSP-9607E10两个骨肉瘤细胞株细胞中,Dll4 mRNA和蛋白表达水平分别0.43±0.11 vs0.86±0.09、0.47±0.05vs 0.82±0.08,具有显著性差异(P＜0.05)。
(5)逆转录病毒介导的小RNA干扰特异性沉默SOSP-9607E10癌细胞系中Dll4的表达
SOSP-9607E10~(Dll4RNAi+)中Dll4的表达被明显抑制,较对照组而言,其抑制效率超过70%(P＜0.05),而作为对照的SOSP-9607E10~(Dll4RNAi-)中Dll4的表达较SOSP-9607E10细胞系无明显变化。以上结果提示本研究中采用逆转录病毒介导的小RNA干扰能够有效特异的沉默SOSP-9607E10骨肉瘤细胞中Dll4的表达。
(6)体外实验中抑制Dll4的表达可以抑制骨肉瘤细胞的侵袭迁移而不影响其增殖和凋亡
SOSP-9607E10~(Dll4RNAi+)较SOSP-9607E10~(Dll4RNAi-)细胞迁移能力明显下降,24小时划痕愈合率分别为62%vs 77%,差异具有显著性意义(P＜0.05)。采用Transwell侵袭小室测定法比较SOSP-9607E10~(Dll4RNAi+)和SOSP-9607E10~(Dll4RNAi-)的侵袭能力,结果显示SOSP-9607E10~(Dll4RNAi+)和SOSP-9607E10~(Dll4RNAi-)细胞侵袭能力明显下降,24小时培养后穿过Transwell的每低倍视野细胞数分别为63±11 vs129±13,差异具有显著性意义(P＜0.05)。用MTT法比较SOSP-9607E10~(Dll4RNAi+)和SOSP-9607E10~(Dll4RNAi-)的增殖能力,差异不具有显著性意义(P＞0.05)。检测SOSP-9607E10~(Dll4RNAi+)和SOSP-9607E10~(Dll4RNAi-)的凋亡情况,两组细胞差异不具有显著性意义(P＞0.05)。
(7)体内抑制Dll4的表达可以抑制骨肉瘤细胞的成瘤能力
比较Mice~(Dll4RNAi+)和Mice~(Dll4RNAi-)两组裸鼠模型中SOSP-9607E10原发瘤的大小,结果显示皮下种植45天后SOSP-9607E10原发瘤的大小(cm~3)分别为3.01±0.22和3.45±0.23,差异具有显著性意义(P＜0.05),比较Mice~(Dll4RNAi+)和Mice~(Dll4RNAi-)两组裸鼠模型SOSP-9607E10原发瘤中的MVD,结果显示Mice~(Dll4RNAi+)和Mice~(Dll4RNAi-)两组裸鼠模型SOSP-9607E10原发瘤中每高倍镜视野平均MVD值分别为29±2和37±3,差异具有显著性意义(P＜0.05)。
(8)体外实验中抑制Dll4的表达可以抑制HMEC-1人微血管内皮细胞的侵袭迁移而不影响其增殖和凋亡
实验组HMEC-1~(Dll4RNAi+)较对照组HMEC-1~(Dll4RNAi-)细胞迁移能力明显下降,24小时划痕愈合率分别为69%vs 92%,差异具有显著性意义(P＜0.05)。细胞侵袭能力也明显下降,24小时培养后穿过Transwell的每低倍视野细胞数分别为49±6 vs 70±3,差异具有显著性意义(P＜0.05)。MTT法比较HMEC-1~(Dll4RNAi+)较对照组HMEC-1~(Dll4RNAi-)的增殖能力,差异不具有显著性意义(P＞0.05)。两组细胞的凋亡差异不具有显著性意义(P＞0.05)。
(9)体外实验中抑制Dll4的表达可以抑制HMEC-1人微血管内皮细胞的血管生成
HMEC-1~(Dll4RNAi+)组不能成管;对照组成管指数为2397±726,实验组成管指数为489±215,两组差异具有显著性意义(P＜0.05)。三维血管新生模型对照组和实验组中每10个微载体成管数分别为45±9和9±7,差异具有显著性意义(P＜0.05),这些结果说明体外抑制Dll4的表达可以抑制HMEC-1人微血管内皮细胞的血管生成。
(10)体内实验中抑制Dll4的表达可以抑制VEGF诱导的血管生成
实验组Matrigel中的新生血管少于对照组,实验组和对照组Matrigel中新生血管总长度分别为23634±6421μm和32145±4314μm,差异具有显著性意义(P＜0.05)这说明体内实验中抑制的Dll4表达可以抑制VEGF诱导的新生血管生成。
结论:Dll4在骨肉瘤中的高表达可能参与了骨肉瘤的发生发展,Dll4可能作为预测骨肉瘤不良预后的肿瘤标志物;Dll4可能通过调控血管成管分子机制参与了骨肉瘤的侵袭转移;Dll4可能成为抗骨肉瘤侵袭转移和血管生成的基因治疗新靶点。
Objective:To investigate the significance of Dll4 expressed in osteosarcoma tissues;construct targeted to silence the expression of Dll4 osteosarcoma invasion and metastasis and angiogenesis in rats.
Methods:(1)The expression of Dll4 protein in 62 cases of osteosarcoma were detected by immunohistochemical method, combined with the clinical pathological data to analysis Dll4 expression relation to clinicopath olo gical characteristics.(2)By constructing full-length retroviral pSUPER-Dll4-siRNA plasmid,through the identification of post-its stable transfection PT67 cell culture to obtain virus serum protein secretion Dll4,intervention silence human osteosarcoma cell line SOSP-9607E10 Medium Dll4 expression, intervention silence the Department of Human microvascular endothelial cells(HMEC-1),in vitro experiments using scratches,Transwell invasion assay,blood vessels into a tube experiment,MTT and flow cytometry methods to study the regulation and control Dll4 osteosarcoma cell differentiation and proliferation,Osteosarcoma osteosarcoma invasion sports and vascular tube into the molecular mechanism of regulation and control to verify Dll4 osteosarcoma vascular tube into the molecular mechanisms leading to tumor biological behavior changes.
Results:
(1) The expression of Notch1 and Dll4 in osteosarcoma or osteochondroma.The positive expression of Notch1 protein in tumor cells mainly in the cytoplasm or cell membrane,showing brown, scattered.62 cases of osteosarcoma in 53 cases of positive expression, the positive rate was 85.5%,21.2%for the control group,the difference between the two was significant(P＜0.05).Dll4 protein expression mainly in the cytoplasm of tumor cells or cell membranes,some expression in vascular endothelial cells.62 cases of osteosarcoma in 55 cases of positive expression,the positive rate was 88.7%,23.6%for the control group,the difference between the two was significant(P＜0.05). Dll4 in osteosarcoma than in osteochondroma expression,suggesting that Notch1 and Dll4 may be involved in the occurrence of osteosarcoma, development.
(2) The expression of Notch1 and Dll4 in osteosarcoma and its relationship with clinical indicators of the relationship between pathology.Notch1 and Dll4 expression in osteosarcoma exist a positive correlation between(P＜0.05),correlation coefficient rs for the 0.842,P =0.027.Notch1 and Dll4 protein expression and differentiation of osteosarcoma,Ennecking staging and metastasis are closely related(P＜0.05),but the diameter of tumor,gender,age,ALP(alkaline phosphatase) unrelated(P＞0.05).
(3) The expression of Notch1 and Dll4 in osteosarcoma relationship to MVD.Notch1 high expression group MVD for 35.2±9.3, significantly higher than the low expression group 28.3±3.4,In addition,Dll4 high expression group MVD for 36.2±7.2,significantly higher than the low expression group 29.5±2.2,the difference was significant(P＜0.05).The results suggest that Notch1 and Dll4 protein expression and MVD between the closely related(P＜0.05),Notch1 and Dll4 protein expression in osteosarcoma may angiogenesis plays an important role in the process.
(4) Dll4 at different metastatic potential osteosarcoma invasion of differentially expressed.Dll4mRNA and protein at SOSP-9607H9 and SOSP-9607E10 2 cells.The results showed that:At SOSP-9607H9 and SOSP-9607E 10 two osteosarcoma cell line cells,Dll4mRNA and protein expression level of significant difference,respectively,0.43±0.11 vs 0.86±0.09,0.47±0.05vs 0.82±0.08(P＜0.05).The results suggest: Dll4 expression in osteosarcoma is closely related to invasion and metastasis.
(5) retrovirus-mediated small interference RNA silencing of specific cancer cell line SOSP-9607E10 Medium Dll4 expression.SOSP-9607E10~(Dll4RNAi+) Medium Dll4 expression was significantly inhibited than the control group,its inhibition efficiencies in excess of 70%(P＜0.05),and as a contrast to SOSP-9607E10~(Dll4RNAi-)in Dll4 expression than the cell line SOSP- 9607E10-free change significantly.These results suggest that the present study using retroviral-mediated small RNA interference can effectively silence specific SOSP-9607E10 osteosarcoma cells in Dll4 expression.
(6) in vitro inhibition of Dll4 expression can inhibit the invasion of osteosarcoma cell migration without affecting their proliferation and apoptosis.SOSP-9607E10~(Dll4RNAi+) and SOSP-9607E10~(Dll4RNAi-)cell migration capacity decreased 24-hour scratch healing rates were 62%vs 77%,difference statistically significant(P＜0.05).Using Transwell invasion chamber assay compared SOSP-9607E10~(Dll4RNAi+) and SOSP-9607E10 ~(Dll4RNAi-) invasion capacity,results show that SOSP-9607E10~(Dll4RNAi+) and SOSP-9607E10~(Dll4RNAi-)cell invasion decreased 24 hours after the train crossed the Transwell cell per low magnification field of vision respectively 63±11 vs 129±13,difference was significant(P＜0.05).Using MTT method SOSP-9607E10~(Dll4RNAi+) and SOSP-9607E10 ~(Dll4RNAi-)proliferation ability,cell proliferation results showed discrepancies in the ability of the two groups is not significant (P＞0.05).Thus the use of Annexin V and PI double staining method and flow cytometry detection of SOSP-9607E10~(Dll4RNAi+) and SOSP-9607 E10 ~(Dll4RNAi-)in apoptosis,the results showed differences in two groups of cells in apoptosis is not significant(P＞0.05).These results suggest that in vitro inhibit the expression of Dll4 inhibits the invasion of osteosarcoma cell migration without affecting their proliferation and apoptosis.
(7) in vivo inhibited the expression of Dll4 inhibits osteosarcoma tumor cells the ability.Comparison Mice~(Dll4RNAi+) and Mice~(Dll4RNAi-)two nude mice model of SOSP-9607E10 primary tumor size,results showed that 45 days after subcutaneous plant SOSP-9607E10 primary tumor size (cm3) were 3.01±0.22 and 3.45±0.23,difference was significant(P＜0.05),compared MiceDll4RNAi + and Mice Dll4RNAi-two nude mice model of SOSP-9607E10 primary tumors of the MVD,showed Mice~(Dll4RNAi+) and Mice~(Dll4RNAi-)two nude mice model of SOSP-9607E10 original hair tumors in each high-power microscope field of vision MVD values were an average of 29±2 and 37±3,difference significant(P＜0.05),which suggests the expression of Dll4 inhibition in vivo can inhibit the growth of osteosarcoma may be associated with inhibition of tumor angiogenesis relevant.
(8) in vitro inhibition of Dll4 expression can inhibit HMEC-1 Human microvascular endothelial cell invasion without affecting the migration of its proliferation and apoptosis.Experimental group, HMEC-1~(Dll4RNAi+) than the control group,HMEC-1~(Dll4RNAi-)cell migration capacity decreased 24-hour scratch healing rates were 69%vs 92%, difference statistically significant(P＜0.05).Cell invasion ability was significantly decreased 24 hours after culture through Transwell times each low vision cell count were 49±6 vs 70±3,difference significant (P＜0.05).MTT method HMEC-1~(Dll4RNAi+) than the control group, HMEC-1~(Dll4RNAi-)proliferation ability,cell proliferation results showed discrepancies in the ability of the two groups is not significant(P＞0.05). Differences between two groups of apoptosis has no significant(P＞0.05).These results suggest that inhibition in vitro can inhibit the expression of Dll4 Human microvascular endothelial cells HMEC-1 invasion of migration without affecting their apoptosis.
(9) in vitro in inhibiting the expression of Dll4 inhibits HMEC-1 Human microvascular endothelial cell angiogenesis HMEC-1~(Dll4RNAi+) endothelial cells in Matrigel formed should not mesh structure;control the composition of control index for 2397±726,the experimental index for the composition of tube 489±215,with differences between the two groups was significant(P＜0.05).Three-dimensional angiogenesis model control group and experimental group in each of 10 Microcarrier into possession of a few were 45±9 and 9±7,with difference significant(P＜0.05),These results indicate that in vitro inhibit the expression of Dll4 inhibits HMEC-1 Human microvascular endothelial cell angiogenesis.
(10) in vivo in inhibiting the expression of Dll4 inhibits VEGF-induced angiogenesis.Experimental group in Matrigel neovascularization than the control group,experimental group and control group in Matrigel neovascularization separately for the total length of 23634±6421μm and 32145±4314μm,difference statistically significant(P＜0.05) This shows that in vivo inhibition Dll4 expression can inhibit VEGF-induced angiogenesis.
Conclusion:Dll4 in osteosarcoma high expression may be involved in the occurrence and development of osteosarcoma,Dll4 may be used as prediction of poor prognosis in osteosarcoma tumor markers;Dll4 may control blood vessels into the molecular mechanisms involved in control of osteosarcoma invasion and metastasis;Dll4 likely to become anti-osteosarcoma invasion and metastasis and angiogenesis in a new target for gene therapy.
方法:
(1)采用免疫组化的方法检测62例骨肉瘤组织中Dll4蛋白的表达,结合临床病理资料分析,研究Dll4的表达与临床病理特征的关系。
(2)构建全长的逆转录病毒pSUPER-Dll4-siRNA质粒,通过鉴定后,将其稳定转染到PT67细胞培养,获得分泌Dll4蛋白病毒血清,干预沉默人骨肉瘤细胞株SOSP-9607E10中Dll4的表达;干预沉默人微血管内皮细胞系(HMEC-1),体外采用划痕实验、Transwell侵袭实验、血管成管实验、MTT和流式细胞仪检测等方法,研究Dll4调控骨肉瘤细胞分化增殖、骨肉瘤侵袭运动和骨肉瘤血管成管的分子机制验证Dll4调控骨肉瘤血管成管的分子机制导致肿瘤生物学行为的改变。
结果:
(1)Notch1、Dll4在骨肉瘤和骨软骨瘤中的表达
Notch1蛋白的阳性表达主要在肿瘤细胞的细胞质或细胞膜,呈棕黄色,散在分布。62例骨肉瘤中有53例呈阳性表达,阳性率为85.5%(53/62),对照组为21.2%(9/43),两组差异具有显著性(P＜0.05)。Dll4蛋白的阳性表达主要在肿瘤细胞的细胞质或细胞膜,部分表达在血管内皮细胞。62例骨肉瘤中有55例呈阳性表达,阳性率为88.7%(54/62),对照组为23.6%(10/43),两者之间差别具有显著性(P＜0.05),Notch1和Dll4在骨肉瘤中的表达高于在骨软骨瘤中的表达,提示Notch1和Dll4可能参与了骨肉瘤的发生、发展。
(2).Notch1、Dll4表达及其与骨肉瘤病理临床指标的关系
Notch1与Dll4在骨肉瘤中表达存在正相关关系,相关系数rs为0.842,P=0.027。Notch1及Dll4蛋白表达与骨肉瘤组织分化程度、Ennecking分期和有无转移密切相关(P＜0.05),但与肿瘤直径、性别、年龄、ALP(碱性磷酸酶)无关(P＞0.05)。
(3)骨肉瘤中Notch1和Dll4表达与MVD表达的关系
Notch1高表达组MVD为35.2±9.3,显著高于低表达组28.3±3.4,此外,Dll4高表达组MVD为36.2±7.2,显著高于低表达组29.5±2.2,差别具有显著性(P＜0.05)。骨肉瘤中Notch1和Dll4表达与MVD表达的具有相关性(P＜0.05),Notch1及Dll4蛋白高表达可能在骨肉瘤血管生成进程中起重要作用。
(4)Dll4的表达与骨肉瘤的侵袭转移潜能密切相关
Dll4 mRNA和蛋白在SOSP-9607H9及SOSP-9607E10两株细胞中的表达。结果显示:在SOSP-9607H9及SOSP-9607E10两个骨肉瘤细胞株细胞中,Dll4 mRNA和蛋白表达水平分别0.43±0.11 vs0.86±0.09、0.47±0.05vs 0.82±0.08,具有显著性差异(P＜0.05)。
(5)逆转录病毒介导的小RNA干扰特异性沉默SOSP-9607E10癌细胞系中Dll4的表达
SOSP-9607E10~(Dll4RNAi+)中Dll4的表达被明显抑制,较对照组而言,其抑制效率超过70%(P＜0.05),而作为对照的SOSP-9607E10~(Dll4RNAi-)中Dll4的表达较SOSP-9607E10细胞系无明显变化。以上结果提示本研究中采用逆转录病毒介导的小RNA干扰能够有效特异的沉默SOSP-9607E10骨肉瘤细胞中Dll4的表达。
(6)体外实验中抑制Dll4的表达可以抑制骨肉瘤细胞的侵袭迁移而不影响其增殖和凋亡
SOSP-9607E10~(Dll4RNAi+)较SOSP-9607E10~(Dll4RNAi-)细胞迁移能力明显下降,24小时划痕愈合率分别为62%vs 77%,差异具有显著性意义(P＜0.05)。采用Transwell侵袭小室测定法比较SOSP-9607E10~(Dll4RNAi+)和SOSP-9607E10~(Dll4RNAi-)的侵袭能力,结果显示SOSP-9607E10~(Dll4RNAi+)和SOSP-9607E10~(Dll4RNAi-)细胞侵袭能力明显下降,24小时培养后穿过Transwell的每低倍视野细胞数分别为63±11 vs129±13,差异具有显著性意义(P＜0.05)。用MTT法比较SOSP-9607E10~(Dll4RNAi+)和SOSP-9607E10~(Dll4RNAi-)的增殖能力,差异不具有显著性意义(P＞0.05)。检测SOSP-9607E10~(Dll4RNAi+)和SOSP-9607E10~(Dll4RNAi-)的凋亡情况,两组细胞差异不具有显著性意义(P＞0.05)。
(7)体内抑制Dll4的表达可以抑制骨肉瘤细胞的成瘤能力
比较Mice~(Dll4RNAi+)和Mice~(Dll4RNAi-)两组裸鼠模型中SOSP-9607E10原发瘤的大小,结果显示皮下种植45天后SOSP-9607E10原发瘤的大小(cm~3)分别为3.01±0.22和3.45±0.23,差异具有显著性意义(P＜0.05),比较Mice~(Dll4RNAi+)和Mice~(Dll4RNAi-)两组裸鼠模型SOSP-9607E10原发瘤中的MVD,结果显示Mice~(Dll4RNAi+)和Mice~(Dll4RNAi-)两组裸鼠模型SOSP-9607E10原发瘤中每高倍镜视野平均MVD值分别为29±2和37±3,差异具有显著性意义(P＜0.05)。
(8)体外实验中抑制Dll4的表达可以抑制HMEC-1人微血管内皮细胞的侵袭迁移而不影响其增殖和凋亡
实验组HMEC-1~(Dll4RNAi+)较对照组HMEC-1~(Dll4RNAi-)细胞迁移能力明显下降,24小时划痕愈合率分别为69%vs 92%,差异具有显著性意义(P＜0.05)。细胞侵袭能力也明显下降,24小时培养后穿过Transwell的每低倍视野细胞数分别为49±6 vs 70±3,差异具有显著性意义(P＜0.05)。MTT法比较HMEC-1~(Dll4RNAi+)较对照组HMEC-1~(Dll4RNAi-)的增殖能力,差异不具有显著性意义(P＞0.05)。两组细胞的凋亡差异不具有显著性意义(P＞0.05)。
(9)体外实验中抑制Dll4的表达可以抑制HMEC-1人微血管内皮细胞的血管生成
HMEC-1~(Dll4RNAi+)组不能成管;对照组成管指数为2397±726,实验组成管指数为489±215,两组差异具有显著性意义(P＜0.05)。三维血管新生模型对照组和实验组中每10个微载体成管数分别为45±9和9±7,差异具有显著性意义(P＜0.05),这些结果说明体外抑制Dll4的表达可以抑制HMEC-1人微血管内皮细胞的血管生成。
(10)体内实验中抑制Dll4的表达可以抑制VEGF诱导的血管生成
实验组Matrigel中的新生血管少于对照组,实验组和对照组Matrigel中新生血管总长度分别为23634±6421μm和32145±4314μm,差异具有显著性意义(P＜0.05)这说明体内实验中抑制的Dll4表达可以抑制VEGF诱导的新生血管生成。
结论:Dll4在骨肉瘤中的高表达可能参与了骨肉瘤的发生发展,Dll4可能作为预测骨肉瘤不良预后的肿瘤标志物;Dll4可能通过调控血管成管分子机制参与了骨肉瘤的侵袭转移;Dll4可能成为抗骨肉瘤侵袭转移和血管生成的基因治疗新靶点。
Objective:To investigate the significance of Dll4 expressed in osteosarcoma tissues;construct targeted to silence the expression of Dll4 osteosarcoma invasion and metastasis and angiogenesis in rats.
Methods:(1)The expression of Dll4 protein in 62 cases of osteosarcoma were detected by immunohistochemical method, combined with the clinical pathological data to analysis Dll4 expression relation to clinicopath olo gical characteristics.(2)By constructing full-length retroviral pSUPER-Dll4-siRNA plasmid,through the identification of post-its stable transfection PT67 cell culture to obtain virus serum protein secretion Dll4,intervention silence human osteosarcoma cell line SOSP-9607E10 Medium Dll4 expression, intervention silence the Department of Human microvascular endothelial cells(HMEC-1),in vitro experiments using scratches,Transwell invasion assay,blood vessels into a tube experiment,MTT and flow cytometry methods to study the regulation and control Dll4 osteosarcoma cell differentiation and proliferation,Osteosarcoma osteosarcoma invasion sports and vascular tube into the molecular mechanism of regulation and control to verify Dll4 osteosarcoma vascular tube into the molecular mechanisms leading to tumor biological behavior changes.
Results:
(1) The expression of Notch1 and Dll4 in osteosarcoma or osteochondroma.The positive expression of Notch1 protein in tumor cells mainly in the cytoplasm or cell membrane,showing brown, scattered.62 cases of osteosarcoma in 53 cases of positive expression, the positive rate was 85.5%,21.2%for the control group,the difference between the two was significant(P＜0.05).Dll4 protein expression mainly in the cytoplasm of tumor cells or cell membranes,some expression in vascular endothelial cells.62 cases of osteosarcoma in 55 cases of positive expression,the positive rate was 88.7%,23.6%for the control group,the difference between the two was significant(P＜0.05). Dll4 in osteosarcoma than in osteochondroma expression,suggesting that Notch1 and Dll4 may be involved in the occurrence of osteosarcoma, development.
(2) The expression of Notch1 and Dll4 in osteosarcoma and its relationship with clinical indicators of the relationship between pathology.Notch1 and Dll4 expression in osteosarcoma exist a positive correlation between(P＜0.05),correlation coefficient rs for the 0.842,P =0.027.Notch1 and Dll4 protein expression and differentiation of osteosarcoma,Ennecking staging and metastasis are closely related(P＜0.05),but the diameter of tumor,gender,age,ALP(alkaline phosphatase) unrelated(P＞0.05).
(3) The expression of Notch1 and Dll4 in osteosarcoma relationship to MVD.Notch1 high expression group MVD for 35.2±9.3, significantly higher than the low expression group 28.3±3.4,In addition,Dll4 high expression group MVD for 36.2±7.2,significantly higher than the low expression group 29.5±2.2,the difference was significant(P＜0.05).The results suggest that Notch1 and Dll4 protein expression and MVD between the closely related(P＜0.05),Notch1 and Dll4 protein expression in osteosarcoma may angiogenesis plays an important role in the process.
(4) Dll4 at different metastatic potential osteosarcoma invasion of differentially expressed.Dll4mRNA and protein at SOSP-9607H9 and SOSP-9607E10 2 cells.The results showed that:At SOSP-9607H9 and SOSP-9607E 10 two osteosarcoma cell line cells,Dll4mRNA and protein expression level of significant difference,respectively,0.43±0.11 vs 0.86±0.09,0.47±0.05vs 0.82±0.08(P＜0.05).The results suggest: Dll4 expression in osteosarcoma is closely related to invasion and metastasis.
(5) retrovirus-mediated small interference RNA silencing of specific cancer cell line SOSP-9607E10 Medium Dll4 expression.SOSP-9607E10~(Dll4RNAi+) Medium Dll4 expression was significantly inhibited than the control group,its inhibition efficiencies in excess of 70%(P＜0.05),and as a contrast to SOSP-9607E10~(Dll4RNAi-)in Dll4 expression than the cell line SOSP- 9607E10-free change significantly.These results suggest that the present study using retroviral-mediated small RNA interference can effectively silence specific SOSP-9607E10 osteosarcoma cells in Dll4 expression.
(6) in vitro inhibition of Dll4 expression can inhibit the invasion of osteosarcoma cell migration without affecting their proliferation and apoptosis.SOSP-9607E10~(Dll4RNAi+) and SOSP-9607E10~(Dll4RNAi-)cell migration capacity decreased 24-hour scratch healing rates were 62%vs 77%,difference statistically significant(P＜0.05).Using Transwell invasion chamber assay compared SOSP-9607E10~(Dll4RNAi+) and SOSP-9607E10 ~(Dll4RNAi-) invasion capacity,results show that SOSP-9607E10~(Dll4RNAi+) and SOSP-9607E10~(Dll4RNAi-)cell invasion decreased 24 hours after the train crossed the Transwell cell per low magnification field of vision respectively 63±11 vs 129±13,difference was significant(P＜0.05).Using MTT method SOSP-9607E10~(Dll4RNAi+) and SOSP-9607E10 ~(Dll4RNAi-)proliferation ability,cell proliferation results showed discrepancies in the ability of the two groups is not significant (P＞0.05).Thus the use of Annexin V and PI double staining method and flow cytometry detection of SOSP-9607E10~(Dll4RNAi+) and SOSP-9607 E10 ~(Dll4RNAi-)in apoptosis,the results showed differences in two groups of cells in apoptosis is not significant(P＞0.05).These results suggest that in vitro inhibit the expression of Dll4 inhibits the invasion of osteosarcoma cell migration without affecting their proliferation and apoptosis.
(7) in vivo inhibited the expression of Dll4 inhibits osteosarcoma tumor cells the ability.Comparison Mice~(Dll4RNAi+) and Mice~(Dll4RNAi-)two nude mice model of SOSP-9607E10 primary tumor size,results showed that 45 days after subcutaneous plant SOSP-9607E10 primary tumor size (cm3) were 3.01±0.22 and 3.45±0.23,difference was significant(P＜0.05),compared MiceDll4RNAi + and Mice Dll4RNAi-two nude mice model of SOSP-9607E10 primary tumors of the MVD,showed Mice~(Dll4RNAi+) and Mice~(Dll4RNAi-)two nude mice model of SOSP-9607E10 original hair tumors in each high-power microscope field of vision MVD values were an average of 29±2 and 37±3,difference significant(P＜0.05),which suggests the expression of Dll4 inhibition in vivo can inhibit the growth of osteosarcoma may be associated with inhibition of tumor angiogenesis relevant.
(8) in vitro inhibition of Dll4 expression can inhibit HMEC-1 Human microvascular endothelial cell invasion without affecting the migration of its proliferation and apoptosis.Experimental group, HMEC-1~(Dll4RNAi+) than the control group,HMEC-1~(Dll4RNAi-)cell migration capacity decreased 24-hour scratch healing rates were 69%vs 92%, difference statistically significant(P＜0.05).Cell invasion ability was significantly decreased 24 hours after culture through Transwell times each low vision cell count were 49±6 vs 70±3,difference significant (P＜0.05).MTT method HMEC-1~(Dll4RNAi+) than the control group, HMEC-1~(Dll4RNAi-)proliferation ability,cell proliferation results showed discrepancies in the ability of the two groups is not significant(P＞0.05). Differences between two groups of apoptosis has no significant(P＞0.05).These results suggest that inhibition in vitro can inhibit the expression of Dll4 Human microvascular endothelial cells HMEC-1 invasion of migration without affecting their apoptosis.
(9) in vitro in inhibiting the expression of Dll4 inhibits HMEC-1 Human microvascular endothelial cell angiogenesis HMEC-1~(Dll4RNAi+) endothelial cells in Matrigel formed should not mesh structure;control the composition of control index for 2397±726,the experimental index for the composition of tube 489±215,with differences between the two groups was significant(P＜0.05).Three-dimensional angiogenesis model control group and experimental group in each of 10 Microcarrier into possession of a few were 45±9 and 9±7,with difference significant(P＜0.05),These results indicate that in vitro inhibit the expression of Dll4 inhibits HMEC-1 Human microvascular endothelial cell angiogenesis.
(10) in vivo in inhibiting the expression of Dll4 inhibits VEGF-induced angiogenesis.Experimental group in Matrigel neovascularization than the control group,experimental group and control group in Matrigel neovascularization separately for the total length of 23634±6421μm and 32145±4314μm,difference statistically significant(P＜0.05) This shows that in vivo inhibition Dll4 expression can inhibit VEGF-induced angiogenesis.
Conclusion:Dll4 in osteosarcoma high expression may be involved in the occurrence and development of osteosarcoma,Dll4 may be used as prediction of poor prognosis in osteosarcoma tumor markers;Dll4 may control blood vessels into the molecular mechanisms involved in control of osteosarcoma invasion and metastasis;Dll4 likely to become anti-osteosarcoma invasion and metastasis and angiogenesis in a new target for gene therapy.
引文
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[22] Hainaud P, Confreres JO, Villemain A, et al. The Role of the Vascular Endothelial Growth Factor-Delta-like 4 Ligand/Notch4-Ephrin B2 Cascade in Tumor Vessel Remodeling and Endothelial Cell Functions. Cancer Research, 2006(66):8501-8510.
[23] Bergers G, Benjamin LE. Tumorigenesis and the angiogenic switch. Nat Rev Cancer, 2003, 3(6): 401-410.
[24] Jain RK, Duda DG, Clark JW, et al. Lessons from phase III clinical trials on anti-VEGF therapy for cancer. Nat Clin Pract Oncol, 2006, 3(1): 24-40.
[25] FerraraN, KerbelRS. Angiogenesis as a therapeutic target. Nature, 2005,438(7070): 967-974.
[26] Ridgway J, Zhang Gu, Wu Yan, et al. Inhibition of D114 signalling inhibits tumour growth by deregulating angiogenesis. Nature, 2006, 444(7122): 1083-1087
[27] Nognera-Troise I, Daly C, Papadopoulos NJ, et al. Blockade of D114 inhibits tumour growth by promoting non-productive angiogenesis. Nature, 2006,444 (7122): 1032-1037.
[28] Scehnet JS, Jiang W, Ram Kumar S, et al. Inhibition of D114-mediated signaling induces proliferation of immature vessels and results in poor tissue perfusion. Blood, 109(11): 4753-4760.
[1] Kaya M, Wada T, Akatsuka T, et al. Vascular endothelial growth factor expression in untreated osteosarcoma is predictive of pulmonary metastasis and poor prognosis. Clin Cancer Res, 2000, 6:572-577.
[2] Lee YH, Tokunaga T, Oshika Y, et al. Cell-retained isoforms of vascular endothelial growth factor (VEGF) are correlated with poor prognosis in osteosarcoma. Eur J Cancer, 1999, 35:1089-1093.
[3] Starzec A, Vassy R, Martin A, et al. Antiangiogenic and antitumor activities of peptide inhibiting the vascular endothelial growth factor binding to neuropilin-1.Life Sciences, 2006,79: 2370-2381.
[4] He Z, Tessier-Lavigne M. Neuropilin is a receptor for the axonal chemorepellent Semaphorin Ⅲ. Cell, 1997, 90:739-751.
[5] Guttmann-Raviv N, Kessler O, Shraga-Heled N, et al. The neuropilins and their role in tumorigenesis and tumor progression. Cancer Lett, 2006,231:1-11.
[6] Handa A, Tokunaga T, Tsuchida T, et al. Neuropilin-2 expression affects the increased vascularization and is a prognostic factor in osteosarcoma. Int J Oncol, 2000,17:291-295.
[7] Sulzbacher I, Birner P, Trieb K, et al. Expression of platelet-derived growth factor-AA is associated with tumor progression in osteosarcoma. Mod Pathol, 2003,16:66-71.
[8] Tang Y, Nakada MT, Kesavan P, et al. Extracellular matrix metalloproteinase inducer stimulates tumor angiogenesis by elevating vascular endothelial cell growth factor and matrix metalloproteinases. Cancer Res, 2005, 65:3193-3199.
[9] Uchibori M, Nishida Y, Nagasaka T, et al. Increased expression of membranetype matrix metalloproteinase-1 is correlated with poor prognosis in patients with osteosarcoma. Int J Oncol, 2006,28: 33-42.
[10] Foukas AF, Deshmukh NS, Grimer RJ, et al. Stage-ⅡB osteosarcomas around the knee. A study of MMP-9 in surviving tumour cells. J Bone Joint Surg Br,2002,84:706-711.
[11] Holzer G, Obermair A, Koschat M, et al. Concentration of vascular endothelial growth factor (VEGF) in the serum of patients with malignant bone tumors.Med Pediatr Oncol, 2001, 36:601-604.
[12] Rutkowski P, Kaminska J, Kowalska M, et al. Cytokine and cytokine receptor serum levels in adult bone sarcoma patients: correlations with local tumor extent and prognosis. J Surg Oncol, 2003, 84:151-159.
[13] Kaya M, Wada T, Kawaguchi S, et al. Increased pre-therapeutic serum vascular endothelial growth factor in patients with early clinical relapse of osteosarcoma. Br J Cancer, 2002, 86:864-869.
[14] Holash J, Maisonpierre PC, Compton D, et al. Vessel cooption, regression,and growth in tumors mediated by angiopoietins and VEGF. Science, 1999, 284: 1994-1998.
[15] Patan S. Vasculogenesis and angiogenesis. Cancer Treat Res, 2004,117:3-32.3 Balasubramanian L, Evens AM. Targeting angiogenesis for the treatment of sarcoma. Curr Opin Oncol, 2006,18:354-359.
[16] Heymach JV. Angiogenesis and antiangiogenic approaches to sarcomas.Curr Opin Oncol, 2001,13:261-269.
[17] Poon RT, Fan ST, Wong J. Clinical implications of circulating angiogenic factors in cancer patients. J Clin Oncol, 2001,19:1207-1225.
[18] Poncelet C, Fauvet R, Feldmann G, et al. Prognostic value of von Willebrand factor, CD34, CD31, and vascular endothelial growth factor expression in women with uterine leiomyosarcomas. J Surg Oncol, 2004,86:84-90.
[19] Kreuter M, Bieker R, Bielack SS, et al. Prognostic relevance of increased angiogenesis in osteosarcoma. Clin Cancer Res, 2004,10:8531-8537.
[20] Mikulic D, Ilic I, Cepulic M, et al. Tumor angiogenesis and outcome in osteosarcoma. Pediatr Hematol Oncol, 2004, 21:611-619.
[21] Mantadakis E, Kim G, Reisch J, et al. Lack of prognostic significance of intratumoral angiogenesis in nonmetastatic osteosarcoma. J Pediatr Hematol Oncol,2001,23:286-289.
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[1] Shutter JR, Scully S, Fan Wei, et al. D114, a novel Notch ligand expressed in arterial endothelium. Genes Dev, 2000, 14(11): 1313-1318.
[2] Gale NW, Dominguez MG, Noguera I, et al. Haploinsufficiency of delta-like 4 ligand results in embryonic lethality due to major defects in arterial and vascular development. Proc Natl Acad Sci U S A, 2004, 101(45): 15949-15954.
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[10] Kleer CG, van Golen KL, Zhang Y, et al. Characterization of RhoC expression in benign and malignant breast disease: a potential new marker for small breast carcinomas with metastatic ability. Am J Pathol 2002; 160: 579-584.
[11] Gale NW, DominguezMG, Noguera I, et al. Haploin sufficiency of delta-like 4 ligand results in embryonic lethality due to major defects in arterial and vascular development. Proc Natl Acad Sci USA, 2004, 101 (45): 15949 -15954.
[12] Hellstrom M, Phng LK, Hofmann JJ, et al. DU4 signalling through Notchl regulates form ation of tip cells during angiogenesis. Nature, 2007, 445(7129): 776-780.
[13] Gerhardt H, Golding M, Fruttiger M, et al. VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J Cell Biol, 2003, 161(6):1163-1177.
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[15] Williams CK, Li JL, Murga M, et al. Up-regulation of the Notch ligand Delta- like 4 inhibits VEGF-induced endothelial cell function. Blood. 2006; 107: 931-939.
[16] Suchting, S, Freitas, C, Noble, F, et al. The Notch ligand Delta-like 4 negatively regulates endothelial tip cell formation and vessel branching. Proc. Natl.Acad. Sci. 2007, 104: 3225-3230
[17] Leslie JD, Ariza—McNaughton L, Bermange AL, et al. Endothelial signalling by the Notch ligand Delta-like 4 restricts an6ogenesis. Development, 2007,134(5): 839-844.
[18] Lobov IB, Renard RA, Papadopoulos N, et al. Delta-like ligand 4(D114)is induced by VEGF as a negative regulator of angiogenie sprouting. Proe Natl Acad Sci USA, 2007, 104(9): 3219-3224.
[19] MailhosC, ModlichU, Lewis J, etal. Delta4, an endothelial specific notch ligand expressed at sites of physiological and tumor angiogen esis. Differentia tion,2001, 69(2-3): 135-144.
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[21] Patel NS, Li JL, Generali D,et al. Up-regulation of Delta-like 4 Ligand in Human Tumor Vasculature and the Role of Basal Expression in Endothelial Cell Function. Cancer Research, 2005 65(19), 8690-8697.
[22] Hainaud P, Confreres JO, Villemain A, et al. The Role of the Vascular Endothelial Growth Factor-Delta-like 4 Ligand/Notch4-Ephrin B2 Cascade in Tumor Vessel Remodeling and Endothelial Cell Functions. Cancer Research, 2006(66):8501-8510.
[23] Bergers G, Benjamin LE. Tumorigenesis and the angiogenic switch. Nat Rev Cancer, 2003, 3(6): 401-410.
[24] Jain RK, Duda DG, Clark JW, et al. Lessons from phase III clinical trials on anti-VEGF therapy for cancer. Nat Clin Pract Oncol, 2006, 3(1): 24-40.
[25] FerraraN, KerbelRS. Angiogenesis as a therapeutic target. Nature, 2005,438(7070): 967-974.
[26] Ridgway J, Zhang Gu, Wu Yan, et al. Inhibition of D114 signalling inhibits tumour growth by deregulating angiogenesis. Nature, 2006, 444(7122): 1083-1087
[27] Nognera-Troise I, Daly C, Papadopoulos NJ, et al. Blockade of D114 inhibits tumour growth by promoting non-productive angiogenesis. Nature, 2006,444 (7122): 1032-1037.
[28] Scehnet JS, Jiang W, Ram Kumar S, et al. Inhibition of D114-mediated signaling induces proliferation of immature vessels and results in poor tissue perfusion. Blood, 109(11): 4753-4760.
[1] Kaya M, Wada T, Akatsuka T, et al. Vascular endothelial growth factor expression in untreated osteosarcoma is predictive of pulmonary metastasis and poor prognosis. Clin Cancer Res, 2000, 6:572-577.
[2] Lee YH, Tokunaga T, Oshika Y, et al. Cell-retained isoforms of vascular endothelial growth factor (VEGF) are correlated with poor prognosis in osteosarcoma. Eur J Cancer, 1999, 35:1089-1093.
[3] Starzec A, Vassy R, Martin A, et al. Antiangiogenic and antitumor activities of peptide inhibiting the vascular endothelial growth factor binding to neuropilin-1.Life Sciences, 2006,79: 2370-2381.
[4] He Z, Tessier-Lavigne M. Neuropilin is a receptor for the axonal chemorepellent Semaphorin Ⅲ. Cell, 1997, 90:739-751.
[5] Guttmann-Raviv N, Kessler O, Shraga-Heled N, et al. The neuropilins and their role in tumorigenesis and tumor progression. Cancer Lett, 2006,231:1-11.
[6] Handa A, Tokunaga T, Tsuchida T, et al. Neuropilin-2 expression affects the increased vascularization and is a prognostic factor in osteosarcoma. Int J Oncol, 2000,17:291-295.
[7] Sulzbacher I, Birner P, Trieb K, et al. Expression of platelet-derived growth factor-AA is associated with tumor progression in osteosarcoma. Mod Pathol, 2003,16:66-71.
[8] Tang Y, Nakada MT, Kesavan P, et al. Extracellular matrix metalloproteinase inducer stimulates tumor angiogenesis by elevating vascular endothelial cell growth factor and matrix metalloproteinases. Cancer Res, 2005, 65:3193-3199.
[9] Uchibori M, Nishida Y, Nagasaka T, et al. Increased expression of membranetype matrix metalloproteinase-1 is correlated with poor prognosis in patients with osteosarcoma. Int J Oncol, 2006,28: 33-42.
[10] Foukas AF, Deshmukh NS, Grimer RJ, et al. Stage-ⅡB osteosarcomas around the knee. A study of MMP-9 in surviving tumour cells. J Bone Joint Surg Br,2002,84:706-711.
[11] Holzer G, Obermair A, Koschat M, et al. Concentration of vascular endothelial growth factor (VEGF) in the serum of patients with malignant bone tumors.Med Pediatr Oncol, 2001, 36:601-604.
[12] Rutkowski P, Kaminska J, Kowalska M, et al. Cytokine and cytokine receptor serum levels in adult bone sarcoma patients: correlations with local tumor extent and prognosis. J Surg Oncol, 2003, 84:151-159.
[13] Kaya M, Wada T, Kawaguchi S, et al. Increased pre-therapeutic serum vascular endothelial growth factor in patients with early clinical relapse of osteosarcoma. Br J Cancer, 2002, 86:864-869.
[14] Holash J, Maisonpierre PC, Compton D, et al. Vessel cooption, regression,and growth in tumors mediated by angiopoietins and VEGF. Science, 1999, 284: 1994-1998.
[15] Patan S. Vasculogenesis and angiogenesis. Cancer Treat Res, 2004,117:3-32.3 Balasubramanian L, Evens AM. Targeting angiogenesis for the treatment of sarcoma. Curr Opin Oncol, 2006,18:354-359.
[16] Heymach JV. Angiogenesis and antiangiogenic approaches to sarcomas.Curr Opin Oncol, 2001,13:261-269.
[17] Poon RT, Fan ST, Wong J. Clinical implications of circulating angiogenic factors in cancer patients. J Clin Oncol, 2001,19:1207-1225.
[18] Poncelet C, Fauvet R, Feldmann G, et al. Prognostic value of von Willebrand factor, CD34, CD31, and vascular endothelial growth factor expression in women with uterine leiomyosarcomas. J Surg Oncol, 2004,86:84-90.
[19] Kreuter M, Bieker R, Bielack SS, et al. Prognostic relevance of increased angiogenesis in osteosarcoma. Clin Cancer Res, 2004,10:8531-8537.
[20] Mikulic D, Ilic I, Cepulic M, et al. Tumor angiogenesis and outcome in osteosarcoma. Pediatr Hematol Oncol, 2004, 21:611-619.
[21] Mantadakis E, Kim G, Reisch J, et al. Lack of prognostic significance of intratumoral angiogenesis in nonmetastatic osteosarcoma. J Pediatr Hematol Oncol,2001,23:286-289.