Hedgehog/Gli1信号通路在胶质瘤化疗耐受及血管新生过程中作用的试验研究
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摘要
第一部分
Hedgehog/Gli1信号通路在胶质瘤化疗耐受中作用的实验研究
目的：研究Hedgehog/Gli1信号通路在胶质瘤复发过程中的作用,并就其影响胶质瘤化疗效果的机制进行初步的探讨。
方法：
(1)收集来自30例胶质瘤病人第一次手术及复发后第一次手术切除的肿瘤组织标本60份,采用免疫组化法分析SHH及Glil蛋白的表达。
(2)通过Western Blot,分析U87、SHG4、U251及A172四个胶质瘤细胞系中Hedgehog/Gli1信号通路的活化程度。
(3)以胞外配体SHH活化Hedgehog/Gli1信号通路,采用克隆生存试验,检测这一信号通路活化程度提高后,VCR、VP16、CDDP及ACNU对A172及U251细胞杀伤作用的变化。
(4)以胞外抑制剂Cyclopamine下调Hedgehog/Gli1信号通路的活化程度,观察这一信号通路的活化程度下调后,VCR、VP16、CDDP,及ACNU对U87、SHG44、U251及A172细胞生存率及早期凋亡的影响。
(5)构建以Glil基因为靶向的LV-shGlil,同时构建LV-control作为阴性对照,LV-shGlil及LV-control分别转染U87、SHG44及U251细胞,采用FACS及Western Blot分析Hedgehog/Gli1信号通路活化程度的变化。
(6)使用Cyclopamine及Glil水平的RNAi,下调U87、SHG44及U251细胞Hedgehog/Gli1信号通路的活化程度后,检测MDR1、MRP1、LRP、MGMT, Bcl-2、Survivin等耐药相关基因mRNA表达的变化。
(7)统计学分析：使用SPSS16.0对实验数据进行统计学分析,两组间均数的比较采用t检验,多组间均数的比较采用方差分析,样本率间的差别采用卡方检验,以P<0.05表示差异有统计学意义。
结果：
(1)30例胶质瘤病人中,23例病人的组织样本具有Glil的表达,7例病人的组织样本无Glil的表达。具有Glil表达的这23例胶质瘤病人中,复发的组织样本(n=23)均具有Glil的表达,而相应的原发组织样本中3份无Glil的表达,其余的20份样本具有Glil的表达,复发组(n=23)Gli1的阳性细胞百分比为48.9±20.3%,相应原发组(n=23)的Glil阳性细胞百分比为34.4%±19.8%,二者相比具有显著的统计学意义(P<0.01)。以SHH染色对上述60份组织样本进行分组,SHH阳性组含42份组织样本,38/42(90.5%)的标本伴有Glil的阳性；而SHH阴性组含18份组织样本,仅有5/18(27.8%)的标本中有Glil的表达,统计学分析两组间Glil阳性的样本数也有显著的统计学意义(P<0.01)。
(2)Western Blot结果显示,U87、SHG44、U251及A172四个胶质瘤细胞系均具有Hedgehog/Glil信号通路的活化,在U87及SHG44中这一信号通路活化的程度较高,U251中这一信号通路的活化程度中等,而A172中这一信号通路的活化程度最低。
(3)以胞外配体SHH活化U251及A 172细胞中Hedgehog/Gli1信号通路,随着该信号通路活化程度的提高,SHH组肿瘤细胞较对照组肿瘤细胞,对VCR、VP16、CDDP及ACNU的耐受性明显增强,SHH组肿瘤细胞的克隆存活率显著增加(P<0.01)。
(4)Cyclopamine以浓度依赖性的方式,下调U87、SHG44及U251细胞中Hedgehog/Gli1信号通路的活化程度,而且随着这一信号通路活化程度的降低,Cyclopamine组,VCR、VP16、CDDP及ACNU引起的肿瘤细胞生长抑制及早期凋亡,较对照组明显增加(P<0.01或P<0.05),但在A172细胞中却没有上述现象发现。
(5)U87、SHG44、U251细胞中,Smo水平Cyclopamine的抑制及Glil水平的RNAi均可引起Hedgehog/Gli1信号通路的活化程度显著地降低,而且随着这一信号通路活化程度的下降,MDR1、MRP1、LRP、MGMT、Bcl-2、Survivin等耐药相关基因表达的也明显的下调。
结论：
(1)在部分胶质瘤中存在着异常活化的]Hedgehog/Gli1信号通路,这一信号通路的过度活化与胶质瘤的复发密切相关,SHH在这一信号通路的活化过程中发挥了重要作用。
(2)Hedgehog/Gli1信号通路活化程度增加,促进了胶质瘤细胞对化疗药物的耐受,而该信号通路活化程度的抑制则导致肿瘤细胞对化疗药物敏感性的增加。
(3)胶质瘤中Hedgehog/Gli1信号通路与MDR1、MRP1、LRP、MGMT、Bcl-2、Survivin等一系列耐药相关基因的表达存在一定的相关性。抑制Hedgehog/Gli1信号通路的活性,可相应的下调MDR1、MRP1、LRP、MGMT、Bcl-2、Survivin等耐药相关基因的表达。
(4)干预胶质瘤中Hedgehog/Gli1信号通路的活性,可能代表了一种化疗增敏、提高疗效的治疗措施。
Hedgehog/Gli1信号通路调节胶质瘤血管新生相关基因表达的实验研究
目的：探讨胶质瘤中异常活化的Hedgehog/Gli1信号通路与血管新生的相关性,以及这一信号通路对VEGF、MMP2及MMP9表达的调控作用。
方法：
(1)采用第一部分的组织样本60份,通过免疫组化分析CD34的表达,探讨Glil的表达与MVD计数的相关性。
(2)采用SHH活化Hedgehog/Gli1信号通路,观察该信号通路活化后对VEGF、MMP2及MMP9表达的影响。
(3)采用Cyclopamine抑制Hedgehog/Gli1信号通路,观察这一信号通路的活化程度的下调后,是否伴有VEGF、MMP2及MMP9表达的降低。
(4)在Glil的水平进行RNAi,观察Hedgehog/Gli1信号通路被阻断后,VEGF、MMP2及MMP9表达的变化。
结果：
(1)Glil阳性组(n=43)MVD为27.4±4.6,显著高于Glil阴性组(n=17)的20.9±3.5,组间的MVD差异具有显著的统计学意义(P<0.01)。
(2)Glil低表达组的MVD为23.7±3.5,中度表达组的MVD为27.3±3.4,高度表达组的MVD为33.2±2.9,各组间MVD具有显著的差异(P<0.01),而且Glil的阳性程度与MVD呈正相关(r=0.756,P<0.01)。
(3)Hedgehog/Gli1信号通路活化程度上调后,活化组VEGF、MMP2及MMP9mRNA及蛋白的表达较对照组明显增高。
(4)Cyclopamine抑制Hedgehog/Gli1信号通路后,抑制组VEGF、MMP2及MMP9mRNA及蛋白的表达较对照组明显降低,同时Cyclopamine的抑制作用还具有浓度依赖的特性。
(5)Glil水平的RNAi证实,阻断Hedgehog/Gli1信号通路,对VEGF、MMP2及MMP 9 mRNA及蛋白的表达具有明显的下调作用。
结论：
(1)Hedgehog/Gli1信号通路与胶质瘤的血管新生存在着一定的相关性。
(2)Hedgehog/Gli1信号通路活化程度的提高,可上调VEGF、MMP2及MMP9表达；反之,则导致VEGF、MMP2及MMP9表达的降低。
(3)抑制Hedgehog/Gli1信号通路的活化程度,进而下调VEGF、MMP2、MMP9的表达,可能代表了一种全新的抑制胶质瘤血管新生的治疗策略。
Chapter One
A study of the Hedgehog/Gli1 pathway in chemotherapy resistance of gliomas
Objectives:
This study aimed at investigating roles of the Hedgehog/Gli1 pathway in human primitive and recurrent gliomas and preliminarily exploring mechanisms which affected chemotherapy.
Methods:
(1)Received 60 primary and recurrent glioma samples from the 30 patients, analyzed expression of SHH and Gli1 protein using immunohistochemistry and explored relationship between the Hedgehog/Gli1 pathway and glioma recurrence.
(2)Received the four glioma cell lines including U87, U251, SHG44, A172 and analyzed the active level of their Hedgehog/Gli1 pathway.
(3)Using extracellular active ligand SHH to activate the Hedgehog/Gli1 pathway in U251 and A172 cells, explored whether overactivation of this pathway could promote resistance of tumor cells to chemotherapeutic agents.
(4) Using extracellular inhibiting ligand Cyclopamine to repress the Hedgehog/Gli1 pathway in U87, SHG44, U251, and A172 cells, explored whether this pathway repressed could promote sensitivity of tumor cells to chemotherapeutic agents.
(5)Explored roles of the Hedgehog/Gli1 pathway regulating MDR-related genes including MDR1 and MRP1 etc.
(6)To confirm that effects of Cyclopamine was achieved through Hedgehog/Glil pathway regulating MDR-related genes, we performed LV-shGlil conduction in Glil level in U87, SHG44, and U251.
Results:
(1)In the primary glioma group,20 samples had Glil expression, and 23 samples had Glil expression in the in recurrent glioma group. These 43 samples belonged to 23 patients, every patient had Glil expression in the recurrent glioma group, and 3 didn't have Glil expression in the primary glioma group. Glil-positive rate in the recurrent group (n=23) was 48.9±20.3%, and also Glil-positive rate in the primary group (n=23) was 34.4±19.8%. There was significant difference between these two groups (P<0.01). Moreover, there was significant difference of Glil expression between the SHH-positive and the SHH-negative group (P<0.01).
(2)Among the four lines, Glil was more highly expressed in U87 and SHG44 cells. In U251 lines, Glil expression was modest. Glil expression in A172 cells was quite low. Baseline levels of Glil expression in A172 and U251 were relatively low.
(3) Activation of the Hedgehog/Gli1 pathway supported resistance of U251 and A172 cells to chemotherapeutic agents.
(4)Repression of Glil expression promoted the response of U87, SHG44, and U251 cells to chemotherapeutic agents and contributed to a further increase in cell apoptosis.
(5)The effects of LV-shGli1 and Cyclopamine on the expression of downstream MDR-related genes in glioma cells with the Hedgehog/Glil pathway activity
Conclusions:
(1)There is the existence of Hedgehog/Glil pathway activated in a subset of gliomas, and overactivation of the Hedgehog/Gli1 pathway correlates with glioma recurrence.
(2)Elevated active levels of the Hedgehog/Gli1 pathway promote resistance of glioma cells to chemotherapeutic agents. Conversely, inhibiting this pathway may enhance the effects of chemotherapeutic agents on glioma cell death.
(3)There is certain correlation between activation of the Hedgehog/Gli1 pathway and expression of MDR-related genes including MDR1、MRP1、LRP、MGMT、Bcl-2、Survivin in gliomas. Blockade of the Hedgehog/Glil pathway can downregulate the expressions of MDR1, MRP1, LRP, MGMT, Bcl-2 and Survivin genes.
(4)Interfering the Hedgehog/Gli1 pathway in combination with current chemotherapy regimens may provide a promising approach to improving chemotherapeutic response.
Chapter Two
A study of the Hedgehog/Glil pathway regulating expression of the angiogenesis-related genes in gliomas
Objectives:
This study aimed at investigating relationship between the aberrant active Hedgehog/Glil pathway in glioma recurrence and angiogensis, and roles of this pathway regulating VEGF, MMP2 and MMP9.
Methods:
(1) Received 60 primary and recurrent glioma samples from the 30 patients, analyzed expression of CD34 and Glil protein using immunohistochemistry and explored relationship between the Hedgehog/Gli 1 pathway and MVD.
(2)Using extracellular active ligand SHH to activate the Hedgehog/Gli1 pathway in U251 and A172 cells, explored whether overactivation of this pathway could upregulate expression of VEGF, MMP2 and MMP9.
(3) Using extracellular inhibiting ligand Cyclopamine to repress the Hedgehog/Glil pathway in U87 and SHG44 cells, explored whether this pathway repressed could downregulate expression of VEGF, MMP2 and MMP9.
(5)At the Gli1 level, using RNAi further confirmed that expression of VEGF, MMP2 and MMP9 downregulated was achieved through Hedgehog/Gli1 pathway inhibited.
Results:
(1)MVD in gliomas with Hedgehog/Glil activaton (n=43) was 27.4±4.6, and MVD in gliomas without Hedgehog/Gli1 activaton (n=17) was 20.9±3.5, there was significant difference between these two groups (P<0.01).
(2)MVD in gliomas with high level expression of Gli1 was 23.7±3.5, MVD in gliomas with moderate level expression of Glil was 27.3±3.4, and MVD in gliomas with low level expression of Gli1 was 33.2±2.9. There was significant difference in these three groups. What’more, Glil expression was positively related with MVD (r=0.756, P<0.01).
(3)Overactivation of the Hedgehog/Glil pathway effectively upregulated VEGF, MMP 2 and MMP 9 expression.
(4)Repression of the Hedgehog/Glil pathway activity promoted downregulating expression of VEGF, MMP 2 and MMP 9.
(5)In order to make our results more reliable, we performed RNAi at direct GLi1 level in U87 and SHG44. The data showed that downregulation of Gli1 can indeed inhibit mRNA and protein expression of VEGF, MMP2 and MMP9.
Conclusions:
(1)We show for the first time that status of the Hedgehog/Glil pathway activated is correlated with angiogenesis in gliomas.
(2)The Hedgehog/Glil pathway can effectively regulate expression of VEGF, MMP2 and MMP9.
(3)Inhibiting Hedgehog/Glil pathway, and further downregulating expression of VEGF, MMP2, MMP9 may be a valid therapeutic strategy which represses angiogenesis of gliomas.
Hedgehog/Gli1信号通路在胶质瘤化疗耐受中作用的实验研究
目的：研究Hedgehog/Gli1信号通路在胶质瘤复发过程中的作用,并就其影响胶质瘤化疗效果的机制进行初步的探讨。
方法：
(1)收集来自30例胶质瘤病人第一次手术及复发后第一次手术切除的肿瘤组织标本60份,采用免疫组化法分析SHH及Glil蛋白的表达。
(2)通过Western Blot,分析U87、SHG4、U251及A172四个胶质瘤细胞系中Hedgehog/Gli1信号通路的活化程度。
(3)以胞外配体SHH活化Hedgehog/Gli1信号通路,采用克隆生存试验,检测这一信号通路活化程度提高后,VCR、VP16、CDDP及ACNU对A172及U251细胞杀伤作用的变化。
(4)以胞外抑制剂Cyclopamine下调Hedgehog/Gli1信号通路的活化程度,观察这一信号通路的活化程度下调后,VCR、VP16、CDDP,及ACNU对U87、SHG44、U251及A172细胞生存率及早期凋亡的影响。
(5)构建以Glil基因为靶向的LV-shGlil,同时构建LV-control作为阴性对照,LV-shGlil及LV-control分别转染U87、SHG44及U251细胞,采用FACS及Western Blot分析Hedgehog/Gli1信号通路活化程度的变化。
(6)使用Cyclopamine及Glil水平的RNAi,下调U87、SHG44及U251细胞Hedgehog/Gli1信号通路的活化程度后,检测MDR1、MRP1、LRP、MGMT, Bcl-2、Survivin等耐药相关基因mRNA表达的变化。
(7)统计学分析：使用SPSS16.0对实验数据进行统计学分析,两组间均数的比较采用t检验,多组间均数的比较采用方差分析,样本率间的差别采用卡方检验,以P<0.05表示差异有统计学意义。
结果：
(1)30例胶质瘤病人中,23例病人的组织样本具有Glil的表达,7例病人的组织样本无Glil的表达。具有Glil表达的这23例胶质瘤病人中,复发的组织样本(n=23)均具有Glil的表达,而相应的原发组织样本中3份无Glil的表达,其余的20份样本具有Glil的表达,复发组(n=23)Gli1的阳性细胞百分比为48.9±20.3%,相应原发组(n=23)的Glil阳性细胞百分比为34.4%±19.8%,二者相比具有显著的统计学意义(P<0.01)。以SHH染色对上述60份组织样本进行分组,SHH阳性组含42份组织样本,38/42(90.5%)的标本伴有Glil的阳性；而SHH阴性组含18份组织样本,仅有5/18(27.8%)的标本中有Glil的表达,统计学分析两组间Glil阳性的样本数也有显著的统计学意义(P<0.01)。
(2)Western Blot结果显示,U87、SHG44、U251及A172四个胶质瘤细胞系均具有Hedgehog/Glil信号通路的活化,在U87及SHG44中这一信号通路活化的程度较高,U251中这一信号通路的活化程度中等,而A172中这一信号通路的活化程度最低。
(3)以胞外配体SHH活化U251及A 172细胞中Hedgehog/Gli1信号通路,随着该信号通路活化程度的提高,SHH组肿瘤细胞较对照组肿瘤细胞,对VCR、VP16、CDDP及ACNU的耐受性明显增强,SHH组肿瘤细胞的克隆存活率显著增加(P<0.01)。
(4)Cyclopamine以浓度依赖性的方式,下调U87、SHG44及U251细胞中Hedgehog/Gli1信号通路的活化程度,而且随着这一信号通路活化程度的降低,Cyclopamine组,VCR、VP16、CDDP及ACNU引起的肿瘤细胞生长抑制及早期凋亡,较对照组明显增加(P<0.01或P<0.05),但在A172细胞中却没有上述现象发现。
(5)U87、SHG44、U251细胞中,Smo水平Cyclopamine的抑制及Glil水平的RNAi均可引起Hedgehog/Gli1信号通路的活化程度显著地降低,而且随着这一信号通路活化程度的下降,MDR1、MRP1、LRP、MGMT、Bcl-2、Survivin等耐药相关基因表达的也明显的下调。
结论：
(1)在部分胶质瘤中存在着异常活化的]Hedgehog/Gli1信号通路,这一信号通路的过度活化与胶质瘤的复发密切相关,SHH在这一信号通路的活化过程中发挥了重要作用。
(2)Hedgehog/Gli1信号通路活化程度增加,促进了胶质瘤细胞对化疗药物的耐受,而该信号通路活化程度的抑制则导致肿瘤细胞对化疗药物敏感性的增加。
(3)胶质瘤中Hedgehog/Gli1信号通路与MDR1、MRP1、LRP、MGMT、Bcl-2、Survivin等一系列耐药相关基因的表达存在一定的相关性。抑制Hedgehog/Gli1信号通路的活性,可相应的下调MDR1、MRP1、LRP、MGMT、Bcl-2、Survivin等耐药相关基因的表达。
(4)干预胶质瘤中Hedgehog/Gli1信号通路的活性,可能代表了一种化疗增敏、提高疗效的治疗措施。
Hedgehog/Gli1信号通路调节胶质瘤血管新生相关基因表达的实验研究
目的：探讨胶质瘤中异常活化的Hedgehog/Gli1信号通路与血管新生的相关性,以及这一信号通路对VEGF、MMP2及MMP9表达的调控作用。
方法：
(1)采用第一部分的组织样本60份,通过免疫组化分析CD34的表达,探讨Glil的表达与MVD计数的相关性。
(2)采用SHH活化Hedgehog/Gli1信号通路,观察该信号通路活化后对VEGF、MMP2及MMP9表达的影响。
(3)采用Cyclopamine抑制Hedgehog/Gli1信号通路,观察这一信号通路的活化程度的下调后,是否伴有VEGF、MMP2及MMP9表达的降低。
(4)在Glil的水平进行RNAi,观察Hedgehog/Gli1信号通路被阻断后,VEGF、MMP2及MMP9表达的变化。
结果：
(1)Glil阳性组(n=43)MVD为27.4±4.6,显著高于Glil阴性组(n=17)的20.9±3.5,组间的MVD差异具有显著的统计学意义(P<0.01)。
(2)Glil低表达组的MVD为23.7±3.5,中度表达组的MVD为27.3±3.4,高度表达组的MVD为33.2±2.9,各组间MVD具有显著的差异(P<0.01),而且Glil的阳性程度与MVD呈正相关(r=0.756,P<0.01)。
(3)Hedgehog/Gli1信号通路活化程度上调后,活化组VEGF、MMP2及MMP9mRNA及蛋白的表达较对照组明显增高。
(4)Cyclopamine抑制Hedgehog/Gli1信号通路后,抑制组VEGF、MMP2及MMP9mRNA及蛋白的表达较对照组明显降低,同时Cyclopamine的抑制作用还具有浓度依赖的特性。
(5)Glil水平的RNAi证实,阻断Hedgehog/Gli1信号通路,对VEGF、MMP2及MMP 9 mRNA及蛋白的表达具有明显的下调作用。
结论：
(1)Hedgehog/Gli1信号通路与胶质瘤的血管新生存在着一定的相关性。
(2)Hedgehog/Gli1信号通路活化程度的提高,可上调VEGF、MMP2及MMP9表达；反之,则导致VEGF、MMP2及MMP9表达的降低。
(3)抑制Hedgehog/Gli1信号通路的活化程度,进而下调VEGF、MMP2、MMP9的表达,可能代表了一种全新的抑制胶质瘤血管新生的治疗策略。
Chapter One
A study of the Hedgehog/Gli1 pathway in chemotherapy resistance of gliomas
Objectives:
This study aimed at investigating roles of the Hedgehog/Gli1 pathway in human primitive and recurrent gliomas and preliminarily exploring mechanisms which affected chemotherapy.
Methods:
(1)Received 60 primary and recurrent glioma samples from the 30 patients, analyzed expression of SHH and Gli1 protein using immunohistochemistry and explored relationship between the Hedgehog/Gli1 pathway and glioma recurrence.
(2)Received the four glioma cell lines including U87, U251, SHG44, A172 and analyzed the active level of their Hedgehog/Gli1 pathway.
(3)Using extracellular active ligand SHH to activate the Hedgehog/Gli1 pathway in U251 and A172 cells, explored whether overactivation of this pathway could promote resistance of tumor cells to chemotherapeutic agents.
(4) Using extracellular inhibiting ligand Cyclopamine to repress the Hedgehog/Gli1 pathway in U87, SHG44, U251, and A172 cells, explored whether this pathway repressed could promote sensitivity of tumor cells to chemotherapeutic agents.
(5)Explored roles of the Hedgehog/Gli1 pathway regulating MDR-related genes including MDR1 and MRP1 etc.
(6)To confirm that effects of Cyclopamine was achieved through Hedgehog/Glil pathway regulating MDR-related genes, we performed LV-shGlil conduction in Glil level in U87, SHG44, and U251.
Results:
(1)In the primary glioma group,20 samples had Glil expression, and 23 samples had Glil expression in the in recurrent glioma group. These 43 samples belonged to 23 patients, every patient had Glil expression in the recurrent glioma group, and 3 didn't have Glil expression in the primary glioma group. Glil-positive rate in the recurrent group (n=23) was 48.9±20.3%, and also Glil-positive rate in the primary group (n=23) was 34.4±19.8%. There was significant difference between these two groups (P<0.01). Moreover, there was significant difference of Glil expression between the SHH-positive and the SHH-negative group (P<0.01).
(2)Among the four lines, Glil was more highly expressed in U87 and SHG44 cells. In U251 lines, Glil expression was modest. Glil expression in A172 cells was quite low. Baseline levels of Glil expression in A172 and U251 were relatively low.
(3) Activation of the Hedgehog/Gli1 pathway supported resistance of U251 and A172 cells to chemotherapeutic agents.
(4)Repression of Glil expression promoted the response of U87, SHG44, and U251 cells to chemotherapeutic agents and contributed to a further increase in cell apoptosis.
(5)The effects of LV-shGli1 and Cyclopamine on the expression of downstream MDR-related genes in glioma cells with the Hedgehog/Glil pathway activity
Conclusions:
(1)There is the existence of Hedgehog/Glil pathway activated in a subset of gliomas, and overactivation of the Hedgehog/Gli1 pathway correlates with glioma recurrence.
(2)Elevated active levels of the Hedgehog/Gli1 pathway promote resistance of glioma cells to chemotherapeutic agents. Conversely, inhibiting this pathway may enhance the effects of chemotherapeutic agents on glioma cell death.
(3)There is certain correlation between activation of the Hedgehog/Gli1 pathway and expression of MDR-related genes including MDR1、MRP1、LRP、MGMT、Bcl-2、Survivin in gliomas. Blockade of the Hedgehog/Glil pathway can downregulate the expressions of MDR1, MRP1, LRP, MGMT, Bcl-2 and Survivin genes.
(4)Interfering the Hedgehog/Gli1 pathway in combination with current chemotherapy regimens may provide a promising approach to improving chemotherapeutic response.
Chapter Two
A study of the Hedgehog/Glil pathway regulating expression of the angiogenesis-related genes in gliomas
Objectives:
This study aimed at investigating relationship between the aberrant active Hedgehog/Glil pathway in glioma recurrence and angiogensis, and roles of this pathway regulating VEGF, MMP2 and MMP9.
Methods:
(1) Received 60 primary and recurrent glioma samples from the 30 patients, analyzed expression of CD34 and Glil protein using immunohistochemistry and explored relationship between the Hedgehog/Gli 1 pathway and MVD.
(2)Using extracellular active ligand SHH to activate the Hedgehog/Gli1 pathway in U251 and A172 cells, explored whether overactivation of this pathway could upregulate expression of VEGF, MMP2 and MMP9.
(3) Using extracellular inhibiting ligand Cyclopamine to repress the Hedgehog/Glil pathway in U87 and SHG44 cells, explored whether this pathway repressed could downregulate expression of VEGF, MMP2 and MMP9.
(5)At the Gli1 level, using RNAi further confirmed that expression of VEGF, MMP2 and MMP9 downregulated was achieved through Hedgehog/Gli1 pathway inhibited.
Results:
(1)MVD in gliomas with Hedgehog/Glil activaton (n=43) was 27.4±4.6, and MVD in gliomas without Hedgehog/Gli1 activaton (n=17) was 20.9±3.5, there was significant difference between these two groups (P<0.01).
(2)MVD in gliomas with high level expression of Gli1 was 23.7±3.5, MVD in gliomas with moderate level expression of Glil was 27.3±3.4, and MVD in gliomas with low level expression of Gli1 was 33.2±2.9. There was significant difference in these three groups. What’more, Glil expression was positively related with MVD (r=0.756, P<0.01).
(3)Overactivation of the Hedgehog/Glil pathway effectively upregulated VEGF, MMP 2 and MMP 9 expression.
(4)Repression of the Hedgehog/Glil pathway activity promoted downregulating expression of VEGF, MMP 2 and MMP 9.
(5)In order to make our results more reliable, we performed RNAi at direct GLi1 level in U87 and SHG44. The data showed that downregulation of Gli1 can indeed inhibit mRNA and protein expression of VEGF, MMP2 and MMP9.
Conclusions:
(1)We show for the first time that status of the Hedgehog/Glil pathway activated is correlated with angiogenesis in gliomas.
(2)The Hedgehog/Glil pathway can effectively regulate expression of VEGF, MMP2 and MMP9.
(3)Inhibiting Hedgehog/Glil pathway, and further downregulating expression of VEGF, MMP2, MMP9 may be a valid therapeutic strategy which represses angiogenesis of gliomas.
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