RTN4B在肝癌发生发展过程中的作用
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摘要
肝癌是我国高发恶性肿瘤之一,特别是东南沿海地区为肝癌高发地带,其发病率居各种恶性肿瘤的第四位,死亡率居第一位。目前临床上有效治疗肝癌的方法只有手术切除,并且病人术后5年生存率低。通过分子生物学技术探讨肝癌发生发展的规律,将有助于寻找有效治疗肝癌药物的靶标。本室1998年克隆的新基因RTN4B,在正常肝组织中不表达但在肝癌组织中却异常高表达,提示该基因可能与肝癌的发生发展存在某种联系。肝脏是一个供血丰富的器官,肝癌细胞克隆化生长过程中常被结缔组织包膜所包裹,处于包膜内外的肝癌细胞和正常肝细胞的供血和供氧条件则然不同。近年有文献报道,RTN4B能够诱导血管内皮细胞的粘附和迁移,并且参与血管损伤后的重塑。依据这一提示,我们推测RTN4B在肝癌中的异常表达很可能与肝癌内的血管生长有关,如果能证实RTN4B可促进肝癌血管生成并参与肝癌的快速生长,那么RTN4B就将成为筛选有效治疗肝癌药物的一个重要靶点。为此,我们选择了RTN4B作为本文的研究对象。
我们发现含有外源RTN4B的L02和QGY—7703的稳定表达细胞株,与稳定转染空载体的同源细胞株在生长速度上并无显著差异,但是当把含外源RTN4B的稳定细胞株接种于裸鼠腋部皮下之后,却令人惊奇地观察到RTN4B的稳定细胞株能够显著促进裸鼠皮下的肝癌瘤体的生长。长出的瘤体经组化检测,显示稳定表达外源RTN4B的瘤体内血管密度明显高于稳定转染空载体的瘤体内的血管密度。提示RTN4B在肝癌组织内特异表达的生物学意义可能是促进肿瘤组织内血管生成。
通过细胞组化和FACS技术,我们发现RTN4B定位于细胞质膜上,还可分泌到胞外基质中。培养上清液的超离心沉淀物镜检结果提示分泌过程很可能是通过外排体(exosome)实现的。经对各种肝癌细胞株的培养上清和细胞裂解物进行免疫印迹分析,我们发现在所有的肝癌细胞株以及它们生长的培养基中均可检测到RTN4B蛋白。
我们还观察到由酵母表达系统产生并经亲和纯化获得的RTN4B重组蛋白,在体外能够诱导人血管内皮细胞的粘附、迁移及伸展。运用血管生成研究中的经典实验:小鼠角膜模型及鸡尿囊膜模型,检测RTN4B对血管生成的作用,发现在动物模型水平上RTN4B具有诱导血管生成的作用。
当用RNAi技术有效消减SMMC-7721细胞的RTN4B mRNA后,裸鼠皮下的肝癌细胞瘤体比野生型SMMC-7721肝癌细胞瘤体显著减小,组化结果显示血管密度也显著疏于野生型SMMC-7721细胞瘤体的血管密度,证明消减RTN4B可抑制肿瘤内血管生成进而抑制肿瘤的生长速度。
我们进一步采用制作大鼠肝动脉结扎模型的方法,观察大鼠肝动脉结扎后,在低氧条件下正常肝内RTN4B的表达变化,结果发现RTN4B的表达能被低氧环境所诱导。
综上所述,本项研究发现和证明在肝癌的发生发展过程中产生的低氧环境可诱导RTN4B大量表达,并分泌到细胞外促进肿瘤内血管的生成,进而加速肝癌瘤体的生长。消减肿瘤细胞内的RTN4B则可以抑制肿瘤内血管的生成进而抑制肿瘤的发展。RTN4B在肝癌内诱导血管生成的这一生物学特性,为我们筛选治疗肝癌的有效药物提供了一个重要的药靶,为设计新的肝癌治疗方法和策略提供了新的实验依据。
Hepatocellular carcinoma(HCC) is one of the most common malignancies with the fourth highest incidence rate in China,especially in the region of southeast coast. At present,surgical resection is the only effective way for clinical treatment of HCC. However,even by resection,5-year survival prognosis is rare.Our lab has been working on disclosing the mechanism of HCC development and progression through cancer molecular biology research,in searching for novel strategies for effective anti-tumor therapies.
Our lab cloned RTN4B gene by virtual cloning in 1998 for the first time.Both immunohistochemistry and RT-PCR experiments showed that RTN4B was significantly up-regulated in HCC tissues.Meanwhile,we were surprised to find that RTN4B was widely expressed in 15 human normal tissues except liver.Therefore, what's the role of RTN4B in HCC? What's the internal relationship between its high expression level and HCC development and progression?
As we know,liver is an organ with sufficient blood supply and the cloning hepatoma cells are usually capsuled in HCC.So there is difference in the blood and oxygen supply between normal hepatic cells and HCC cells outside and inside the capsule separately.
In 2004.4,Lisette Acevedo et al.reported that RTN4B could promote vascular endothelial cells' adhesion and migration in vitro and it was also involved in the regulation of vascular remodeling in vivo after injury.Based on these implications,we hypothesized that over-expressed RTN4B protein in HCC might be involved in tumor angiogenesis.RTN4B probably promotes tumor growth through regulation of tumor vascular development.If this hypothesis was demonstrated,RTN4B was probably a novel and effective target in drug screen for anti-tumor therapies. Moreover,exploring the biological function of RTN4B in tumor angiogenesis will bring insights into the research work on the mechanism and rules of HCC development and progression.So we selected RTN4B as our study object and have carried out extensive research on it.
We first successfully established two RTN4B stably expressing liver cancer cell lines,L02 and QGY.Through comparison,we found no significant difference on cell growth rate between ceils stably transfected with RTN4B and control cells. Conversely,cells expressing RTN4B could evidently promote tumor growth when subcutaneously injected into nude mice.Immunohistochemical staining showed that the vessel density of the tumor formed by cells expressing RTN4B was much higher that that of control cells.These results suggest that the over-expressed RTN4B in HCC might be involved in tumor angiogenesis.
Both immunofluorescent cell staining and FACS experiments showed that RTN4B was localized to cell membrane in cancer cells.Further research work found RTN4B could be secreted into extracellular matrix,probably through exosome.By performing western blotting of cell lyses and supernatants,we also demonstrated that RTN4B was widely expressed in nearly all the liver cancer cell lines and it could also be secreted into corresponding cells' culture medium.
Recombinant RTN4B protein was successfully prepared through yeast expression system and protein affinity purification technique.We carried out Boyden assay in vitro,finding this protein was able to enhance the chemotaxis of human umbilicus vascular endothelial cells(HUVECs).While in vivo models,recombinant RTN4B protein could also promote angiogenesis in the classic angiogenesis experiment,mouse corneal pockets assay and Chick Embryo Model,which provided a further support for the biological activity of RTN4B in the organism level.
Furthermore,we confirmed endogenous RTN4B function with RNA interference (RNAi) technique.SiRNA effectively down-regulating RTN4B was transfected into SMMC-7721 cell line by lentivirus-derived vector and nude mice were subsequently injected.Tumor sizes in the group transfected with RTN4B siRNA were evidently smaller than those transfected with siRNA control.Further immunohistochemistry results showed the vascular density of tumors in the group transfected with RTN4B siRNA were also remarkably lower than those transfected with siRNA control.Taken together,RNAi experiment demonstrated the involvement of RTN4B in tumor angiogenesis in a reverse way:down regulation of RTN4B could inhibit tumor growth through influencing on tumor angiogenesis.
By utilizing rat hepatic artery ligation(HAL) model,we found the artificial hypoxia environment could induce the expression of RTN4B.So the hypoxia microenvironment formed in HCC development may be the reason of the expression of RTN4B in HCC.
Taken together,our results are mainly consistent with our former hypothesis.The hypoxia microenvironment formed in HCC development induces the expression and secretion of RTN4B. Extracellular RTN4B is then activate tumor vascular endothelial ceils and promote tumor angiogenesis,thus realizing its regulatory function in HCC development and progression. Down-regulation of RTN4B could inhibit tumor angiogenesis and thereby prevent tumor growth. The specific pro-angiogenic function of RTN4B in HCC presents us a new therapeutic target in HCC treatment and also provides us new theoretical evidences and implications for improved strategies.
我们发现含有外源RTN4B的L02和QGY—7703的稳定表达细胞株,与稳定转染空载体的同源细胞株在生长速度上并无显著差异,但是当把含外源RTN4B的稳定细胞株接种于裸鼠腋部皮下之后,却令人惊奇地观察到RTN4B的稳定细胞株能够显著促进裸鼠皮下的肝癌瘤体的生长。长出的瘤体经组化检测,显示稳定表达外源RTN4B的瘤体内血管密度明显高于稳定转染空载体的瘤体内的血管密度。提示RTN4B在肝癌组织内特异表达的生物学意义可能是促进肿瘤组织内血管生成。
通过细胞组化和FACS技术,我们发现RTN4B定位于细胞质膜上,还可分泌到胞外基质中。培养上清液的超离心沉淀物镜检结果提示分泌过程很可能是通过外排体(exosome)实现的。经对各种肝癌细胞株的培养上清和细胞裂解物进行免疫印迹分析,我们发现在所有的肝癌细胞株以及它们生长的培养基中均可检测到RTN4B蛋白。
我们还观察到由酵母表达系统产生并经亲和纯化获得的RTN4B重组蛋白,在体外能够诱导人血管内皮细胞的粘附、迁移及伸展。运用血管生成研究中的经典实验:小鼠角膜模型及鸡尿囊膜模型,检测RTN4B对血管生成的作用,发现在动物模型水平上RTN4B具有诱导血管生成的作用。
当用RNAi技术有效消减SMMC-7721细胞的RTN4B mRNA后,裸鼠皮下的肝癌细胞瘤体比野生型SMMC-7721肝癌细胞瘤体显著减小,组化结果显示血管密度也显著疏于野生型SMMC-7721细胞瘤体的血管密度,证明消减RTN4B可抑制肿瘤内血管生成进而抑制肿瘤的生长速度。
我们进一步采用制作大鼠肝动脉结扎模型的方法,观察大鼠肝动脉结扎后,在低氧条件下正常肝内RTN4B的表达变化,结果发现RTN4B的表达能被低氧环境所诱导。
综上所述,本项研究发现和证明在肝癌的发生发展过程中产生的低氧环境可诱导RTN4B大量表达,并分泌到细胞外促进肿瘤内血管的生成,进而加速肝癌瘤体的生长。消减肿瘤细胞内的RTN4B则可以抑制肿瘤内血管的生成进而抑制肿瘤的发展。RTN4B在肝癌内诱导血管生成的这一生物学特性,为我们筛选治疗肝癌的有效药物提供了一个重要的药靶,为设计新的肝癌治疗方法和策略提供了新的实验依据。
Hepatocellular carcinoma(HCC) is one of the most common malignancies with the fourth highest incidence rate in China,especially in the region of southeast coast. At present,surgical resection is the only effective way for clinical treatment of HCC. However,even by resection,5-year survival prognosis is rare.Our lab has been working on disclosing the mechanism of HCC development and progression through cancer molecular biology research,in searching for novel strategies for effective anti-tumor therapies.
Our lab cloned RTN4B gene by virtual cloning in 1998 for the first time.Both immunohistochemistry and RT-PCR experiments showed that RTN4B was significantly up-regulated in HCC tissues.Meanwhile,we were surprised to find that RTN4B was widely expressed in 15 human normal tissues except liver.Therefore, what's the role of RTN4B in HCC? What's the internal relationship between its high expression level and HCC development and progression?
As we know,liver is an organ with sufficient blood supply and the cloning hepatoma cells are usually capsuled in HCC.So there is difference in the blood and oxygen supply between normal hepatic cells and HCC cells outside and inside the capsule separately.
In 2004.4,Lisette Acevedo et al.reported that RTN4B could promote vascular endothelial cells' adhesion and migration in vitro and it was also involved in the regulation of vascular remodeling in vivo after injury.Based on these implications,we hypothesized that over-expressed RTN4B protein in HCC might be involved in tumor angiogenesis.RTN4B probably promotes tumor growth through regulation of tumor vascular development.If this hypothesis was demonstrated,RTN4B was probably a novel and effective target in drug screen for anti-tumor therapies. Moreover,exploring the biological function of RTN4B in tumor angiogenesis will bring insights into the research work on the mechanism and rules of HCC development and progression.So we selected RTN4B as our study object and have carried out extensive research on it.
We first successfully established two RTN4B stably expressing liver cancer cell lines,L02 and QGY.Through comparison,we found no significant difference on cell growth rate between ceils stably transfected with RTN4B and control cells. Conversely,cells expressing RTN4B could evidently promote tumor growth when subcutaneously injected into nude mice.Immunohistochemical staining showed that the vessel density of the tumor formed by cells expressing RTN4B was much higher that that of control cells.These results suggest that the over-expressed RTN4B in HCC might be involved in tumor angiogenesis.
Both immunofluorescent cell staining and FACS experiments showed that RTN4B was localized to cell membrane in cancer cells.Further research work found RTN4B could be secreted into extracellular matrix,probably through exosome.By performing western blotting of cell lyses and supernatants,we also demonstrated that RTN4B was widely expressed in nearly all the liver cancer cell lines and it could also be secreted into corresponding cells' culture medium.
Recombinant RTN4B protein was successfully prepared through yeast expression system and protein affinity purification technique.We carried out Boyden assay in vitro,finding this protein was able to enhance the chemotaxis of human umbilicus vascular endothelial cells(HUVECs).While in vivo models,recombinant RTN4B protein could also promote angiogenesis in the classic angiogenesis experiment,mouse corneal pockets assay and Chick Embryo Model,which provided a further support for the biological activity of RTN4B in the organism level.
Furthermore,we confirmed endogenous RTN4B function with RNA interference (RNAi) technique.SiRNA effectively down-regulating RTN4B was transfected into SMMC-7721 cell line by lentivirus-derived vector and nude mice were subsequently injected.Tumor sizes in the group transfected with RTN4B siRNA were evidently smaller than those transfected with siRNA control.Further immunohistochemistry results showed the vascular density of tumors in the group transfected with RTN4B siRNA were also remarkably lower than those transfected with siRNA control.Taken together,RNAi experiment demonstrated the involvement of RTN4B in tumor angiogenesis in a reverse way:down regulation of RTN4B could inhibit tumor growth through influencing on tumor angiogenesis.
By utilizing rat hepatic artery ligation(HAL) model,we found the artificial hypoxia environment could induce the expression of RTN4B.So the hypoxia microenvironment formed in HCC development may be the reason of the expression of RTN4B in HCC.
Taken together,our results are mainly consistent with our former hypothesis.The hypoxia microenvironment formed in HCC development induces the expression and secretion of RTN4B. Extracellular RTN4B is then activate tumor vascular endothelial ceils and promote tumor angiogenesis,thus realizing its regulatory function in HCC development and progression. Down-regulation of RTN4B could inhibit tumor angiogenesis and thereby prevent tumor growth. The specific pro-angiogenic function of RTN4B in HCC presents us a new therapeutic target in HCC treatment and also provides us new theoretical evidences and implications for improved strategies.
引文
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