Hedgehog信号通路通过基质金属蛋白酶-7调节卵巢癌的侵袭与转移
发布时间：2018-08-20 20:06
【摘要】：研究背景与目的： 卵巢癌是妇科肿瘤中死亡率非常高的恶性肿瘤，在近三十年中死亡率并没有呈下降趋势。由于其发病隐匿，早期诊断相当困难，一旦发现已有远处转移。Hedgehog(Hh)信号通路参与胚胎的发育及成人体内稳态的维持，在机体发育中扮演重要的角色。近年来研究显示多种恶性肿瘤的发生、发展与Hh信号通路的异常激活有关，包括卵巢癌。但Hh信号通路怎么参与调控卵巢癌的侵袭与转移的分子机制仍不清楚。本实验从细胞分子水平及动物模型水平来初步探讨Hh信号通路参与卵巢癌侵袭与转移的分子机制及寻找卵巢癌的早期诊断治疗靶点，为卵巢癌新药研发提供理论根据。 研究方法： (1)根据NCBI查询到的MMP7cDNA序列在Invitrogen网站上设计3个靶向MMP7的干扰序列，连接到pcDNA6.2-GW/EmGFP-miR载体中构建重组质粒，分别命名为miRMMP7-158、314、688。用脂质体2000将MMP7干扰质粒转染人卵巢癌SKO-V3细胞，并用倒置荧光显微镜观察荧光强度以确定转染效率。蛋白免疫印迹（Western blot）筛选有效的干扰片段。 (2)以优化了的转染条件【plasmid（ug）：Lipofectamine2000(ul)=1:3】转染人卵巢癌细胞SKO-V3，并通过划痕实验和Transwell细胞侵袭实验检测细胞迁移与侵袭能力。 (3)分别用GANT61（Hh信号通路的抑制剂）与SHH信号通路配体条件培养液（Hh信号通路激活剂）处理人卵巢癌细胞SKO-V3，然后通过real-TimePCR和Western blot实验检测MMP7mRNA水平和蛋白水平的表达。 (4)用GANT61裸鼠皮下注射后检测人卵巢癌裸鼠皮下移植瘤组织中MMP7蛋白表达水平的改变。 结果： (1)成功构建干扰质粒（miRMMP7-158、314、688），该质粒转染SKO-V3细胞的荧光率大于80%。经Western blot检测MMP7蛋白表达，，其中MMP7-158为最佳干扰质粒（p＜0.05）。 (2)转染阴性对照质粒组其侵袭与转移能力均明显强于转染干扰质粒组（p＜0.05）；转染阴性对照质粒的SKO-V3细胞，加入SHH配体条件培养液组其侵袭与迁移能力明显强于加入对照培养液组（p＜0.05）；转染miRNAMMP7-158质粒的SKO-V3细胞，加入SHH配体条件培养液组其侵袭与迁移能力与加入对照培养液组无差异（p＞0.05）。 (3)经SHH配体条件培养液刺激后，SKO-V3细胞中MMP7的mRNA及蛋白水平的表达随处理时间延长逐渐增高（p＜0.05）；而经GANT61（20μM）处理后的SKO-V3细胞中MMP7的mRNA及蛋白表达水平随处理时间延长逐渐降低（p＜0.05）。 (4)免疫组化结果显示，经GANT61皮下裸鼠注射的人卵巢癌裸鼠皮下移植瘤组织中，MMP7蛋白表达明显低于对照组（p＜0.05）。 结论： 1. SHH配体条件培养液激活Hh信号通路，卵巢癌细胞的侵袭与迁移能力明显增强，干扰MMP7表达后，能有效抑制卵巢癌细胞的侵袭与迁移能力，在干扰掉MMP7的SKO-V3细胞中无论是否激活Hh信号通路，卵巢癌细胞的侵袭与迁移能力皆减弱，以上结果提示Hh信号通路很可能是通过MMP7的表达来调控卵巢癌的侵袭与转移。 2.采用Gli特异性小分子抑制剂GANT61处理SKO-V3细胞后， MMP7的mRNA及蛋白表达水平下降；用SHH配体条件培养液激活Hh信号通路后，卵巢癌细胞SKO-V3中MMP7的mRNA及蛋白表达水平增强；GANT61可能有效抑制人卵巢癌裸鼠皮下移植瘤模型中肿瘤的体积及重量，且实验组中MMP7的蛋白表达水平下降；提示MMP7可能是Hh信号通路的下游靶基因，Hh信号通路有望成为卵巢癌诊疗策略的新靶点，为抗癌新药研发提供理论依据。
[Abstract]:Background and purpose:
Ovarian cancer is a very high mortality malignancy in gynecological tumors. The mortality rate has not shown a downward trend in the past 30 years. Owing to its latent onset, early diagnosis is very difficult. Once distant metastasis is found, the Hedgehog (Hh) signaling pathway plays an important role in embryonic development and maintenance of homeostasis in adults. Recent studies have shown that the occurrence and development of many malignant tumors are related to the abnormal activation of Hh signaling pathways, including ovarian cancer. However, the molecular mechanism of how Hh signaling pathways participate in the regulation of ovarian cancer invasion and metastasis remains unclear. The molecular mechanism of invasion and metastasis of nest cancer and the target of early diagnosis and treatment of ovarian cancer will provide theoretical basis for the development of new drugs for ovarian cancer.
Research methods:
(1) According to the sequence of MMP7 cDNA inquired by NCBI, three interference sequences targeting MMP7 were designed on Invitrogen website and linked to pcDNA6.2-GW/EmGFP-miR vector to construct recombinant plasmids, which were named microMMP7-158,314,688 respectively. MMP7 interference plasmids were transfected into human ovarian cancer SKO-V3 cells by liposome 2000, and the fluorescence intensity was observed by inverted fluorescence microscope. To determine the transfection efficiency, Western blot was used to screen effective interference fragments.
(2) Human ovarian cancer cell SKO-V3 was transfected with optimized transfection conditions [plasmid (ug): Lipofectamine 2000 (ul) = 1:3], and the ability of migration and invasion was tested by scratch test and Transwell cell invasion test.
(3) Human ovarian cancer cells SKO-V3 were treated with GANT61 (inhibitor of Hh signaling pathway) and SHH signaling ligand conditioned medium (Hh signaling pathway activator) respectively. The expression of MMP7 mRNA and protein was detected by real-time PCR and Western blot.
(4) After subcutaneous injection of GANT61 into nude mice, the expression of MMP7 protein in human ovarian cancer xenografts was detected.
Result:
(1) The interfering plasmid (MiMMP7-158,314,688) was successfully constructed, and the fluorescence rate of the plasmid transfected SKO-V3 cells was more than 80%. The expression of MMP7 protein was detected by Western blot, and MMP7-158 was the best interfering plasmid (p < 0.05).
(2) The invasive and metastatic abilities of SKO-V3 cells transfected with negative control plasmids were significantly stronger than those of SKO-V3 cells transfected with interfering plasmids (p < 0.05); the invasive and metastatic abilities of SKO-V3 cells transfected with negative control plasmids and cultured with SHH ligand conditioned medium were significantly stronger than those of SKO-V3 cells transfected with MiNAMMP7-158 plasmids (p < 0.05). The invasion and migration ability of the ligand conditioned medium group was not different from that of the control medium (P > 0.05).
(3) After SHH ligand conditioned medium stimulation, the expression of MMP7 mRNA and protein in SKO-V3 cells increased gradually with the prolongation of treatment time (p < 0.05), while the expression of MMP7 mRNA and protein in SKO-V3 cells treated with GANT61 (20 mu M) decreased gradually with the prolongation of treatment time (p < 0.05).
(4) Immunohistochemical staining showed that the expression of MMP7 protein in subcutaneous transplanted human ovarian cancer tissues of nude mice injected with GANT61 was significantly lower than that of control group (p < 0.05).
Conclusion:
1. SHH ligand conditioned medium activates the Hh signaling pathway, and the invasion and migration of ovarian cancer cells are significantly enhanced. Interference with MMP7 expression can effectively inhibit the invasion and migration of ovarian cancer cells. In SKO-V3 cells interfering with MMP7, the invasion and migration of ovarian cancer cells are weakened whether or not the Hh signaling pathway is activated. The results suggest that Hh signaling pathway may regulate the invasion and metastasis of ovarian cancer through the expression of MMP7.
2. After the SKO-V3 cells were treated with Gli-specific small molecule inhibitor GANT61, the expression of MMP7 mRNA and protein decreased; after the Hh signaling pathway was activated by SHH ligand conditioned medium, the expression of MMP7 mRNA and protein in ovarian cancer SKO-V3 cells increased; GANT61 may effectively inhibit the tumor in human ovarian cancer xenograft model in nude mice. The results showed that MMP7 may be the downstream target gene of Hh signaling pathway, and Hh signaling pathway may become a new target of ovarian cancer diagnosis and treatment strategy, providing theoretical basis for the development of new anticancer drugs.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31
本文编号：2194842
[Abstract]:Background and purpose:
Ovarian cancer is a very high mortality malignancy in gynecological tumors. The mortality rate has not shown a downward trend in the past 30 years. Owing to its latent onset, early diagnosis is very difficult. Once distant metastasis is found, the Hedgehog (Hh) signaling pathway plays an important role in embryonic development and maintenance of homeostasis in adults. Recent studies have shown that the occurrence and development of many malignant tumors are related to the abnormal activation of Hh signaling pathways, including ovarian cancer. However, the molecular mechanism of how Hh signaling pathways participate in the regulation of ovarian cancer invasion and metastasis remains unclear. The molecular mechanism of invasion and metastasis of nest cancer and the target of early diagnosis and treatment of ovarian cancer will provide theoretical basis for the development of new drugs for ovarian cancer.
Research methods:
(1) According to the sequence of MMP7 cDNA inquired by NCBI, three interference sequences targeting MMP7 were designed on Invitrogen website and linked to pcDNA6.2-GW/EmGFP-miR vector to construct recombinant plasmids, which were named microMMP7-158,314,688 respectively. MMP7 interference plasmids were transfected into human ovarian cancer SKO-V3 cells by liposome 2000, and the fluorescence intensity was observed by inverted fluorescence microscope. To determine the transfection efficiency, Western blot was used to screen effective interference fragments.
(2) Human ovarian cancer cell SKO-V3 was transfected with optimized transfection conditions [plasmid (ug): Lipofectamine 2000 (ul) = 1:3], and the ability of migration and invasion was tested by scratch test and Transwell cell invasion test.
(3) Human ovarian cancer cells SKO-V3 were treated with GANT61 (inhibitor of Hh signaling pathway) and SHH signaling ligand conditioned medium (Hh signaling pathway activator) respectively. The expression of MMP7 mRNA and protein was detected by real-time PCR and Western blot.
(4) After subcutaneous injection of GANT61 into nude mice, the expression of MMP7 protein in human ovarian cancer xenografts was detected.
Result:
(1) The interfering plasmid (MiMMP7-158,314,688) was successfully constructed, and the fluorescence rate of the plasmid transfected SKO-V3 cells was more than 80%. The expression of MMP7 protein was detected by Western blot, and MMP7-158 was the best interfering plasmid (p < 0.05).
(2) The invasive and metastatic abilities of SKO-V3 cells transfected with negative control plasmids were significantly stronger than those of SKO-V3 cells transfected with interfering plasmids (p < 0.05); the invasive and metastatic abilities of SKO-V3 cells transfected with negative control plasmids and cultured with SHH ligand conditioned medium were significantly stronger than those of SKO-V3 cells transfected with MiNAMMP7-158 plasmids (p < 0.05). The invasion and migration ability of the ligand conditioned medium group was not different from that of the control medium (P > 0.05).
(3) After SHH ligand conditioned medium stimulation, the expression of MMP7 mRNA and protein in SKO-V3 cells increased gradually with the prolongation of treatment time (p < 0.05), while the expression of MMP7 mRNA and protein in SKO-V3 cells treated with GANT61 (20 mu M) decreased gradually with the prolongation of treatment time (p < 0.05).
(4) Immunohistochemical staining showed that the expression of MMP7 protein in subcutaneous transplanted human ovarian cancer tissues of nude mice injected with GANT61 was significantly lower than that of control group (p < 0.05).
Conclusion:
1. SHH ligand conditioned medium activates the Hh signaling pathway, and the invasion and migration of ovarian cancer cells are significantly enhanced. Interference with MMP7 expression can effectively inhibit the invasion and migration of ovarian cancer cells. In SKO-V3 cells interfering with MMP7, the invasion and migration of ovarian cancer cells are weakened whether or not the Hh signaling pathway is activated. The results suggest that Hh signaling pathway may regulate the invasion and metastasis of ovarian cancer through the expression of MMP7.
2. After the SKO-V3 cells were treated with Gli-specific small molecule inhibitor GANT61, the expression of MMP7 mRNA and protein decreased; after the Hh signaling pathway was activated by SHH ligand conditioned medium, the expression of MMP7 mRNA and protein in ovarian cancer SKO-V3 cells increased; GANT61 may effectively inhibit the tumor in human ovarian cancer xenograft model in nude mice. The results showed that MMP7 may be the downstream target gene of Hh signaling pathway, and Hh signaling pathway may become a new target of ovarian cancer diagnosis and treatment strategy, providing theoretical basis for the development of new anticancer drugs.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31
【参考文献】
相关期刊论文 前7条
1 Qi Chen;Guolan Gao;Shiwen Luo;;Hedgehog signaling pathway and ovarian cancer[J];Chinese Journal of Cancer Research;2013年03期
2 郝亚琴;欧阳小波;代剑波;王立;;Hedgehog信号通路在胃癌侵袭转移中的作用及分子机制[J];中国生物制品学杂志;2012年02期
3 潘登;李艳;;Hedgehog-Gli信号通路与肿瘤[J];现代肿瘤医学;2011年03期
4 黄修燕;黄自丽;郑起;;Hedgehog-Gli信号通路失调控对肝细胞癌生长和转移潜能的影响及其靶向治疗[J];中华肿瘤防治杂志;2010年06期
5 玄延花;李贵铃;金铁峰;林贞花;;子宫颈癌中Gli蛋白过表达的临床病理学意义[J];临床与实验病理学杂志;2009年02期
6 顾燕萍;李兆申;高军;龚燕芳;吴红玉;金晶;郭杰芳;满晓华;;Gli基因在胰腺癌中的表达变化及其临床意义[J];胃肠病学;2007年10期
7 郭杰芳;李兆申;金震东;高军;龚燕芳;金晶;满晓华;;GLI1基因在胰腺癌组织中的表达及其临床意义[J];中华医学杂志;2007年12期
本文编号：2194842
本文链接：https://www.wllwen.com/yixuelunwen/fuchankeerkelunwen/2194842.html
最近更新
教材专著
