【作者】 孙延波；

【导师】 杨贵卢；

【作者基本信息】 吉林大学 ， 免疫学， 2002， 博士

【摘要】 为探讨Heregulin β1作用乳腺癌细胞后差异性基因表达的情况,采用抑制性差减杂交技术选择性地扩增差异表达的基因片段,经逆向斑点杂交和Northern blot证实在一乳腺癌细胞系（T47D细胞）中线粒体ATP合成酶6和线粒体细胞色素C氧化酶亚单位Ⅱ基因为差异性表达基因。为进一步观察erbB2受体水平对上述差异性表达的基因的影响,经Northern blot分析,发现在erbB2受体过表达的人乳腺上皮细胞中(HB2^erbB2细胞),线粒体细胞色素C氧化酶亚单位Ⅱ基因的表达呈下降状态。说明erbB2受体的过表达可抑制细胞色素C氧化酶亚单位Ⅱ基因的表达。通过应用不同信号传导途径抑制剂,发现细胞色素C氧化酶亚单位Ⅱ基因的表达可通过ERKs和P13K途径进行调节。更多还原

【Abstract】 Members of the epidermal growth receptor family of tyrosine kinase, including Epidermal growth factor receptor ( EGFR), erbB2, erbB3 and erbB4 receptor. All members of this family are single-chain membrane-spanning proteins that have significant sequence homology to one another. It is known that an intracellular region of these receptors comprises a tyrosine kinase domain and a carboxyl tail that contains critical tyrosine phosphorylation sites. These receptors are activated by members of the EGF family of ligands. Based upon their specificities there are two classes of ligands. The first group of ligands binds EGFR and includes: EGF, transforming growth factor a , amphiregulin and betacellulin. Heregulins which bind erbB3 and erbB4 form the second class of EGF-related peptides. Unlike its other family members, no ligand has yet been identified for erbB2.Although no known ligand has been identified, erbB2 plays a pivotal role both in the erbB receptor signaling and cellular phenotypes. Clinical data show that amplification of the erbB2 is found in 30% of breast cancer patients and is associated with poor prognosis. On the other hand, erbB2 is the preferred heterodimerazation partner for all erbB family members. For example, heregulin induces the formation of heterodimers between erbB2 and erbB3 or between erbB2 and erbB4, thereby transactivating erbB receptors. Heregulin, a secretory glycoprotein, is produced by both stroma cells and tumor cells, and regulates growth, differentiation, rearrangement of cytoskeleton and formation of filopodiarelated to migration in breast cancer cells. Evidence suggests that heregulin induces expression of vascular endothelial growth factor and up-regulation of transcription factors. The activation of signalings ( such as MAPK or PI3K ) is required for mitogenic effect of heregulin on the cells. Therefore, suppression subtractive hybridization was performed to identify differentially expressed genes in breast cancer cells.ATP synthase 6 and Cytochrome C oxidase subunit II (COX II) are Heregulin- responsiveT47D cells were exposed to recombinant human heregulin 3 1 for specified periods. Total RNA and mRNA were isolated and then were subjected to subtractive hybridization analysis. Clones derived from subtractive hybridization were analyzed by reverse dot blot to confirm differential expression. With reverse dot blot hybridization, 2 differentially expressed cDNA fragments were verified. These heregulin-induced fragments were sequenced and compared for homology to any entry in existing nuclear acid database available through the National Center for Biotechnology Information (GenBank). Both fragments exhibited 100% homology with human ATP synthase 6 and human cytochrome C oxidase subunit II, respectively. The expression of ATP synthase 6 and COX II were further examined by Noethern blot hybridization using specific cDNA probes for ATP synthase 6 and COX II respectively. Time sequence analysis of ATP synthase 6 expression showed that heregulin treatment produced a significant up-regulation of ATP synthase 6 at 1 h in T47D cells. COX II is highly expressed in T47D cells at 6 h after stimulation with heregulin.Heregulin-regulated COX II expression is dependent on erbB2 levelsThe relative levels of erbB2 expression determine signal transduction and biological activities elicited by herergulin. Over-expression of erbB2 is believed to be associatedwith tumor aggressiveness and a poor prognosis. T47D cells and HB2 cells expressed similar amount of erbB2, while HBErbB2 cells possessed much higher level of erbB2. Northern blot analysis showed that like in T47D cells,heregulin also induced significant up-regulation of COX II in HB2 cells. However, heregulin had an opposite effect on HBElbB2 cells. It suggests that heregulin-regulated COX II mRNA expression is dependenton the levels of erbB2 expression.Heregulin-regulated COX II expression requires activation of ERK and PI3KTo determine which pathway is involved in heregulin regulation of COX II, we used specific chemical inhibitors to block either MAP kinase or PI3 kinase pathway. The results showed that both PD98059 and LY294004 blocked heregulin-regulated COX II expression, while SB203580 had little effect on COX II expression. Western blot demonstrated that heregulin induced quick phosphorylation of ERK1 and ERK2 both inT47D and HB2 cells. On the other hand, heregulin-mediated phosphorylations of p38MAP kinase and Akt in HB2ErbB cells were much intensive and sustained than both T47D and HB2 cells.In conclusion, heregulin elicits diverse biological activities, and the mitochondially encoded genes may be influenced by heregulin. The levels of erbB2 expression are decisive for the complexity of signal transduction and the diverse biological activities of heregulin. These findings suggest that there is a cross-talk between ERK and PI3K in HRG-mediated COXII expression.更多还原