HRP-3在肝癌组织中上调表达的生物学意义探讨
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摘要
本文以我室已获得发明专利授权的HRP3(Hepatoma-derived growth factor
related protein 3)基因为研究对象，对其功能进行了初步的研究。HRP3是HDGF(Hepatoma-derived growth factor)家族成员之一，它在正常情况下仅主要表达于人的睾丸，脑和心脏，其他组织中虽然也有表达，但表达很少。然而，我们发现，它在肝癌组织或肝癌细胞株中高丰度表达。为了探讨其在肝癌中高丰度表达的生理或病理学意义，我们用免疫组化方法证实它确在肝癌细胞中上调表达，且在肝癌组织内的血管或血管区的丰度最高。同时，我们用在哺乳动物中瞬时表达以及细菌中原核表达的方法检测了HRP-3是否能像HDGF那样可以分泌到细胞外，结果用HA或GST单克隆抗体在浓缩的培养基上清液中检测到了HA-HRP-3或GST-HRP-3融合蛋白，证明它确实能被分泌或释放到细胞外的培养基中。此外，EGFP-HRP-3的融合蛋白亚细胞定位分析结果又显示HRP-3能定位在核中，这些结果提示HRP-3具有分泌蛋白和核定位蛋白的双重特性。
为了探讨HRP-3作为分泌蛋白的功能，我们用原核表达的HRP-3蛋白作为普通培养基的添加物质，分别培养成纤维细胞NIH3T3，人脐静脉内皮细胞HUVEC和肝癌细胞HepG2，其结果显示HRP-3蛋白在50-1000ng／mL浓度具有促生长因子的活性，尤其对于HUVEC细胞的促增殖作用最强，其次是NIH3T3细胞，对肝癌细胞也有明显的促增殖作用。这一结果提示HRP-3在肝癌组织中表达丰度显著上调，在病理学上具有促进瘤体内血管生长和肝癌细胞增殖的意义。
基于上述发现，我们又进一步探讨了HRP-3促细胞生长的分子机制，结果显示当用50-1000ng／mL浓度的HRP-3重组蛋白作用于NIH3T3细胞，作用5-10min，可以使MAPK通路的MEK和ERK1／2蛋白激酶的磷酸化增强，而这种激活作用又能被MEK通路的特异抑制剂PD98059抑制，但对由P38和JNK激酶介导的通路则不受影响。为检查对MAPK通路的这种激活过程是否具有生理效应，我们又用双荧光素酶报告反应系统分别检查了MAPK通路下游的应答状态，结果显示，在外源重组蛋白HRP-3加入后的1-4h，SRE和E-box(C-Myc)元件下游的荧光素酶蛋白的表达增强，从而在分子水平说明HRP-3基因在肝癌中的上调表达所产生的促肿瘤生长作用，是通过细胞内和组织间隙HRP-3蛋白的增加，以增强血管内皮细胞和肝癌细胞内的MAPK信号来实现的。
同时，我们还发现稳定表达外源HRP-3的细胞株显示出增殖或生长速度的加快，但其分子机制除了增加培养液中的HRP-3蛋白的分泌量之外，HRP-3是否还存在细胞核内发挥促增殖调节作用，还有待于进一步研究。
此外，本文还进行了另一项小的研究工作，即从人脑cDNA文库克隆了一个新的含CRAL-TRIO结构域的成员，细胞内视黄醛结合蛋白类似蛋白(Cellular
retinaldehyde binding protein-like，CRALBPL)基因。它定位在染色体8q12.2，包含1694个碱基，开放阅读框编码354个氨基酸，CRAL-TRIO结构域位于118-279位氨基酸。在人18种组织cDNA的RT-PCR分析显示CRALBPL主要在脑中表达。CRAL-TRIO结构域的比对分析显示CRALBPL与CRALBP有45％的相似性。通过Western分析表明CRALBPL的分子量约为40kD，EGFP-CRALBPL融合蛋白在转染的Hela细胞中定位于细胞质。由于CRALBP是在脊椎动物的视觉过程中作为一个下游的蛋白发挥作用，它作为内源性配体携带11-顺式视黄醇和11-顺式视黄醛，可能是视觉周期的一个功能组分。CRALBPL作为CRALBP的同源基因，其克隆和鉴定为视觉生理研究提供了一种新的候选分子材料。
HRP-3(hepatoma-derived growth factor related protein 3) is a new member of HDGF (hepatoma-derived growth factor) family and we had got an international patent for this novel protein. In current study, we carried the study on the function of HRP-3. In normal physical condition, it expressed mainly in human testis, brain and heart, but less in other tissues. Howerer, its expression level was higher in hepatocellular carcinoma (HCC) and hepatoma cells. To discuss the significance of HRP-3 in physiological and pathological process in HCC, we proved its expression level was up-regulated in hepatoma cells especially in the vessels or vessel region in HCC. Then we applied the transient expression in mammalian cells and bacteria expression to examine whether HRP-3 could be secreted from cells like HDGF protein. The results showed that HA-HRP-3 and GST-HRP-3 fusion protein could be detected in the concentrated culture medium using the anti-HA or anti-GST monoclonal antibody. Moreover, the subcelluar location showed that enhanced green fluorescent protein (EGFP)-tagged HRP-3 fusion protein was in nucleus. These results suggested that HRP-3 had the characteristic as secreted protein and nuclear protein.
To find the effect of HRP-3 as a secreted protein, we recombinanted the HRP-3 protein and used it as a supplement in the foundamental cell culture medium. The results displayed that when the concentration of HRP-3 was within 50-1000ng/mL, it could promote the proliferation of NIH3T3, HUVEC and HepG2 cells. HRP-3 had the most distinct effect on HUVECs, followed by NIH3T3 cells. HRP-3 could also stimulate the growth of HepG2 cells. It implied that the increased HRP-3 expression in HCC may potentially related with the growth of vessels in tumor and hepatoma cells during the pathogenesis of hepatocellular carcinoma.
According to these studies, we investigated the mechanism on cells proliferation induced by HRP-3. Adding HRP-3 protein by the concentration of 50-1000ng/mL to NIH3T3 cells, the phosphorylation of MEK and ERK could be rapidly, transiently and specially enhanced in 5-10min, and this activation could be inhibited by the MEK
inhibitor PD98059. But the P38 and JNK signal pathway were not influenced by HRP-3. To confirm the physiological effect of the kinase activation, we used the dual-luciferase reporter system to check the response of MAPK downstream signal elements, the results showed that HRP-3 protein could enhance E-box and SRE (DNA responsone elements) dependent transcription in l-4h. These finding suggested that HRP-3 up-regulated expression in HCC stimulated the tumor growth by increasing its intracellular and extracellular expression to strengthen the MAPK signal in hepatoma cells and vascular endothelial cells.
In addition, we found the growth rate was higher and the MAPK signal was enhanced in the cell lines stablly expressed the exogenous HRP-3. We deduced it may induced by the increased HRP-3 expression in the culture medium, but whether it had the effect in the nucleus needs more study in future.
Moreover, we had taken another simple study on cloning and characterize of a novel human cellular retinaldehyde binding protein-like (CRALBPL) gene. CRALBPL is a new member of a widespread lipid binding SEC14-like protein family with CRAL-TRIO domain, which was predicted and subsequently isolated from the human adult brain cDNA library. The CRALBPL gene was mapped to human chromosome 8q12.2, consisted of 1694 bp and had a open reading frame in length encoding a putative 354 amino acids with a CRAL-TRIO domain in 118-279 aa. The result of RT-PCR amplification in 18 human tissues indicated that CRALBPL gene was mainly expressed in brain. The alignment of CRAL-TRIO domain showed that CRALBPL had 45% identity with human CRALBP. Subcellular location revealed that CRALBPL protein was located in the cytoplasm of Hela cells. Western blotting indicated that the CRALBPL had a molecular weight of about 40kD. For CRALBP playing a role in the vertebrate visual process as a substrate-routing protein, it carries 11-cis-retinol and 11-cis-retinaldehyde as endogenous ligands and may be a functional component of the visual cycle. The clone of novel CRALBPL gene will develop a new and broad field to understand visual process.
related protein 3)基因为研究对象，对其功能进行了初步的研究。HRP3是HDGF(Hepatoma-derived growth factor)家族成员之一，它在正常情况下仅主要表达于人的睾丸，脑和心脏，其他组织中虽然也有表达，但表达很少。然而，我们发现，它在肝癌组织或肝癌细胞株中高丰度表达。为了探讨其在肝癌中高丰度表达的生理或病理学意义，我们用免疫组化方法证实它确在肝癌细胞中上调表达，且在肝癌组织内的血管或血管区的丰度最高。同时，我们用在哺乳动物中瞬时表达以及细菌中原核表达的方法检测了HRP-3是否能像HDGF那样可以分泌到细胞外，结果用HA或GST单克隆抗体在浓缩的培养基上清液中检测到了HA-HRP-3或GST-HRP-3融合蛋白，证明它确实能被分泌或释放到细胞外的培养基中。此外，EGFP-HRP-3的融合蛋白亚细胞定位分析结果又显示HRP-3能定位在核中，这些结果提示HRP-3具有分泌蛋白和核定位蛋白的双重特性。
为了探讨HRP-3作为分泌蛋白的功能，我们用原核表达的HRP-3蛋白作为普通培养基的添加物质，分别培养成纤维细胞NIH3T3，人脐静脉内皮细胞HUVEC和肝癌细胞HepG2，其结果显示HRP-3蛋白在50-1000ng／mL浓度具有促生长因子的活性，尤其对于HUVEC细胞的促增殖作用最强，其次是NIH3T3细胞，对肝癌细胞也有明显的促增殖作用。这一结果提示HRP-3在肝癌组织中表达丰度显著上调，在病理学上具有促进瘤体内血管生长和肝癌细胞增殖的意义。
基于上述发现，我们又进一步探讨了HRP-3促细胞生长的分子机制，结果显示当用50-1000ng／mL浓度的HRP-3重组蛋白作用于NIH3T3细胞，作用5-10min，可以使MAPK通路的MEK和ERK1／2蛋白激酶的磷酸化增强，而这种激活作用又能被MEK通路的特异抑制剂PD98059抑制，但对由P38和JNK激酶介导的通路则不受影响。为检查对MAPK通路的这种激活过程是否具有生理效应，我们又用双荧光素酶报告反应系统分别检查了MAPK通路下游的应答状态，结果显示，在外源重组蛋白HRP-3加入后的1-4h，SRE和E-box(C-Myc)元件下游的荧光素酶蛋白的表达增强，从而在分子水平说明HRP-3基因在肝癌中的上调表达所产生的促肿瘤生长作用，是通过细胞内和组织间隙HRP-3蛋白的增加，以增强血管内皮细胞和肝癌细胞内的MAPK信号来实现的。
同时，我们还发现稳定表达外源HRP-3的细胞株显示出增殖或生长速度的加快，但其分子机制除了增加培养液中的HRP-3蛋白的分泌量之外，HRP-3是否还存在细胞核内发挥促增殖调节作用，还有待于进一步研究。
此外，本文还进行了另一项小的研究工作，即从人脑cDNA文库克隆了一个新的含CRAL-TRIO结构域的成员，细胞内视黄醛结合蛋白类似蛋白(Cellular
retinaldehyde binding protein-like，CRALBPL)基因。它定位在染色体8q12.2，包含1694个碱基，开放阅读框编码354个氨基酸，CRAL-TRIO结构域位于118-279位氨基酸。在人18种组织cDNA的RT-PCR分析显示CRALBPL主要在脑中表达。CRAL-TRIO结构域的比对分析显示CRALBPL与CRALBP有45％的相似性。通过Western分析表明CRALBPL的分子量约为40kD，EGFP-CRALBPL融合蛋白在转染的Hela细胞中定位于细胞质。由于CRALBP是在脊椎动物的视觉过程中作为一个下游的蛋白发挥作用，它作为内源性配体携带11-顺式视黄醇和11-顺式视黄醛，可能是视觉周期的一个功能组分。CRALBPL作为CRALBP的同源基因，其克隆和鉴定为视觉生理研究提供了一种新的候选分子材料。
HRP-3(hepatoma-derived growth factor related protein 3) is a new member of HDGF (hepatoma-derived growth factor) family and we had got an international patent for this novel protein. In current study, we carried the study on the function of HRP-3. In normal physical condition, it expressed mainly in human testis, brain and heart, but less in other tissues. Howerer, its expression level was higher in hepatocellular carcinoma (HCC) and hepatoma cells. To discuss the significance of HRP-3 in physiological and pathological process in HCC, we proved its expression level was up-regulated in hepatoma cells especially in the vessels or vessel region in HCC. Then we applied the transient expression in mammalian cells and bacteria expression to examine whether HRP-3 could be secreted from cells like HDGF protein. The results showed that HA-HRP-3 and GST-HRP-3 fusion protein could be detected in the concentrated culture medium using the anti-HA or anti-GST monoclonal antibody. Moreover, the subcelluar location showed that enhanced green fluorescent protein (EGFP)-tagged HRP-3 fusion protein was in nucleus. These results suggested that HRP-3 had the characteristic as secreted protein and nuclear protein.
To find the effect of HRP-3 as a secreted protein, we recombinanted the HRP-3 protein and used it as a supplement in the foundamental cell culture medium. The results displayed that when the concentration of HRP-3 was within 50-1000ng/mL, it could promote the proliferation of NIH3T3, HUVEC and HepG2 cells. HRP-3 had the most distinct effect on HUVECs, followed by NIH3T3 cells. HRP-3 could also stimulate the growth of HepG2 cells. It implied that the increased HRP-3 expression in HCC may potentially related with the growth of vessels in tumor and hepatoma cells during the pathogenesis of hepatocellular carcinoma.
According to these studies, we investigated the mechanism on cells proliferation induced by HRP-3. Adding HRP-3 protein by the concentration of 50-1000ng/mL to NIH3T3 cells, the phosphorylation of MEK and ERK could be rapidly, transiently and specially enhanced in 5-10min, and this activation could be inhibited by the MEK
inhibitor PD98059. But the P38 and JNK signal pathway were not influenced by HRP-3. To confirm the physiological effect of the kinase activation, we used the dual-luciferase reporter system to check the response of MAPK downstream signal elements, the results showed that HRP-3 protein could enhance E-box and SRE (DNA responsone elements) dependent transcription in l-4h. These finding suggested that HRP-3 up-regulated expression in HCC stimulated the tumor growth by increasing its intracellular and extracellular expression to strengthen the MAPK signal in hepatoma cells and vascular endothelial cells.
In addition, we found the growth rate was higher and the MAPK signal was enhanced in the cell lines stablly expressed the exogenous HRP-3. We deduced it may induced by the increased HRP-3 expression in the culture medium, but whether it had the effect in the nucleus needs more study in future.
Moreover, we had taken another simple study on cloning and characterize of a novel human cellular retinaldehyde binding protein-like (CRALBPL) gene. CRALBPL is a new member of a widespread lipid binding SEC14-like protein family with CRAL-TRIO domain, which was predicted and subsequently isolated from the human adult brain cDNA library. The CRALBPL gene was mapped to human chromosome 8q12.2, consisted of 1694 bp and had a open reading frame in length encoding a putative 354 amino acids with a CRAL-TRIO domain in 118-279 aa. The result of RT-PCR amplification in 18 human tissues indicated that CRALBPL gene was mainly expressed in brain. The alignment of CRAL-TRIO domain showed that CRALBPL had 45% identity with human CRALBP. Subcellular location revealed that CRALBPL protein was located in the cytoplasm of Hela cells. Western blotting indicated that the CRALBPL had a molecular weight of about 40kD. For CRALBP playing a role in the vertebrate visual process as a substrate-routing protein, it carries 11-cis-retinol and 11-cis-retinaldehyde as endogenous ligands and may be a functional component of the visual cycle. The clone of novel CRALBPL gene will develop a new and broad field to understand visual process.
引文
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