HPIP在肠癌中的功能以及机制研究
发布时间：2019-06-21 10:26
【摘要】：结直肠癌(colorectal cancer CRC)是世界上最常见的恶性肿瘤之一,在西方国家,在肿瘤死亡率里排名第三,大约10%的癌症发病率和致死率。因此,阐明结直肠肿瘤发生发展的分子机制对于结直肠癌个体治疗是至关重要的。虽然人们普遍认为结直肠癌是一种异质性疾病,与受体酪氨酸激酶(RTK)及其在胞内下游多条信号途径中多种癌基因、抑癌基因的突变、失活、激活有关,但是总体说来,我们对结直肠癌发生、发展背后潜在的分子生物学改变的认识仍然有限。人造血相关的PBX相互作用蛋白质(hematopoietic PBX—interacting protein, HPIP),最早是在2000年由Abramovich等以前B淋巴细胞白血病转录因子(pre-B-cell leukemia transcription factor 1,PBX 1)为诱饵,通过酵母双杂交技术,从胎儿肝脏cDNA文库中筛选得到而被命名的。HPIP蛋白共由731个氨基酸组成,且与已知蛋白无明显的同源性。HPIP除了能和PBX1结合之外,还可以与PBX2和PBX3相互作用,并且和PBX1一样,能在原始的造血细胞CD34(+)亚群中表达。Lu Q等发现Pbxl可以与它的原癌基因衍生物E2A-Pbx1共同结合于DNA的模序ATCAATCAA序列,而HPIP可通过与PB X的相互作用从而抑制E2A-PBX1的转录激活,因此,HPIP具有能调节造血干细胞正常生长分化过程,抑制白血病发生的作用。HPIP作为个较新的基因,它的生物学功能研究尚在起步阶段,我们的前期研究发现乳腺癌细胞外源转入HPIP后,其生长速度与空载体相比明显加快,同时又检测到宫颈癌、肺癌、肝癌等多种细胞系中HPIP广泛表达,提示HPIP可能参与细胞正向生长增殖的调控,具有一定的癌基因特性,但是HPIP基因在胃肠道肿瘤的功能至今无研究,抑制HPIP是否可以作为肠癌诊断以及治疗的新策略尚不知晓。众所周知,目前,早期大肠癌治愈率高达90%-100%,而晚期CRC的5年生存率只有10%左右。而传统肿瘤标志物,如糖抗原199(CA199)、癌胚抗原(CEA)等在早期大肠癌诊断方面,敏感度仅为10%-40%,且大部分特异性不强。因此,我们以HPIP为研究对象,研究其在肠癌组织和细胞中的表达、功能及可能的机制,以期待发现新的肿瘤标志物。我们的研究首次揭示了HPIP在肠癌中的关键作用以及部分机制。首先,我们在随机选择的64例肠癌患者石蜡标本中,运用免疫组化检测发现癌组织HPIP表达明显高于相应癌旁组织；并且发现HPIP的表达与肠癌预后呈负相关,HPIP表达越高,患者无病生存期和总生存期越短。其次我们从7株肠癌细胞中选择HCT-8,HCT-116,SW480三株细胞,瞬时转染HPIP过表达质粒(pcDNA3-Flag-HPIP)的HCT-116较裸细胞和对照细胞的生长速度明显增快,转染慢病毒包装质粒pcdh-HPIP构建的稳定过表达HPIP的SW480细胞上也得出此结论。而转染慢病毒包装质粒(PSIH-HPIP-RNAi)构建的敲低HPIP的稳定克隆细胞HCT-8和SW480细胞的生长速度较相应裸细胞和对照细胞明显减慢。说明HPIP促进肠癌细胞的增殖,其次,通过流式细胞术的细胞周期检测以及Annexin V-FITC PI双染色方法的凋亡检测证实HPIP促进了大肠癌细胞G1期向S期,G2期向M期的转化,同时抑制了肠癌细胞凋亡,并意外发现其降低了肠癌对奥沙利铂的化疗敏感性。再次,我们通过划痕实验、Transwell实验发现HPIP增强了肠癌细胞迁移和侵袭能力,并促进了上皮细胞向间质转化(EMT)。更重要的是在机制研究中,我们通过添加AKT和ERK的抑制剂发现过表达HPIP的HCT-116细胞、SW480细胞与对照细胞相比促进生长的幅度均明显下降,促进划痕愈合的能力也明显下降甚至消失。验证了HPIP促进肠癌细胞增殖、迁移是通过激活AKT、MAPK/ERK通路,一旦AKT、ERK被抑制,HPIP将不能起作用,最后,通过裸鼠成瘤实验,再次证实敲低HPIP抑制肠癌细胞在裸鼠体内的生长速度。我们的研究结果暗示HPIP在肠癌的发生发展中起重要作用,如果把HPIP作为抗结直肠癌治疗的药物新靶标,构建效果良好的HPIP小干扰RNA或拮抗HPIP的化学小分子,将HPIP有效敲低或抑制其功能,从而阻碍肿瘤生长、转移,将有可能为肠癌的靶向治疗的开创出新的分子药物,也可能会为解决部分化疗药物耐药问题提供新的启示。
[Abstract]:Colorectal cancer (CRC) is one of the world's most common malignant tumors, and in the western countries, the third, about 10% of the cancer morbidity and mortality in the tumor mortality rate. Therefore, it is of vital importance to elucidate the molecular mechanism of colorectal cancer development for the treatment of colorectal cancer. Although it is generally believed that colorectal cancer is a heterogeneous disease, it is associated with a receptor tyrosine kinase (RTK) and a variety of oncogenes in a plurality of signal pathways downstream of the cell, the mutation, inactivation and activation of the tumor suppressor gene, but in general, we are responsible for colorectal cancer, The knowledge of potential molecular biology changes behind development is still limited. The human hematopoiesis-related PBX interaction protein (HPIP) is the first to be named after screening from the fetal liver cDNA library by yeast two-hybrid technique by using a yeast two-hybrid technique, such as the pre-B-cell leukebia trans-expression factor 1 (PBX 1). The HPIP protein is composed of 731 amino acids and has no obvious homology with known proteins. HPIP, in addition to being able to bind to PBX1, can also interact with PBX2 and PBX3 and, like PBX1, can be expressed in the original hematopoietic cell CD34 (+) subpopulation. Lu Q et al found that Pbxl can be combined with its proto-oncogene derivative E2A-Pbx1 in the sequence ATCAATCAA of the DNA, and the HPIP can inhibit the transcriptional activation of E2A-PBX1 by the interaction with the PB X, so that the HPIP has the function of regulating the normal growth and differentiation of the hematopoietic stem cells and inhibiting the occurrence of the leukemia. As a new gene, HPIP is a new gene, its biological function research is still in the initial stage, our earlier study found that the growth rate of breast cancer cells was significantly faster than that of empty vector, and cervical cancer and lung cancer were detected at the same time. HPIP is widely expressed in various cell lines, such as liver cancer, and it is suggested that the HPIP may be involved in the regulation of the proliferation of the positive growth of the cell, and has certain oncogene characteristics, but the function of the HPIP gene in the gastrointestinal tumor has not been studied yet. It is unknown whether or not the HPIP can be a new strategy for the diagnosis and treatment of intestinal cancer. It is well known that the cure rate of early colorectal cancer is as high as 90% -100%, while the 5-year survival rate of late CRC is about 10%. The sensitivity of the traditional tumor markers, such as the sugar antigen 199 (CA199), the carcinoembryonic antigen (CEA), and the like in the early diagnosis of large intestine cancer, is only 10% to 40%, and most of the specificity is not strong. Therefore, we study the expression, function and possible mechanism of its expression, function and possible mechanism in intestinal cancer tissues and cells with HPIP, in order to find new tumor markers. Our study first revealed the key role of HPIP in intestinal cancer and some of the mechanisms. First, we used the immunohistochemical method to detect the expression of HPIP in the paraffin specimen of 64 patients with intestinal cancer randomly selected, and found that the expression of HPIP was negatively correlated with the prognosis of intestinal cancer. The higher the expression of HPIP, the shorter the patient's disease-free survival and overall survival. Second, we selected HCT-8, HCT-116 and SW480 three cells from 7 intestinal cancer cells, and the growth rate of HCT-116, which was transiently transfected with HPIP over-expression plasmid (pcDNA3-Flag-HPIP), was significantly higher than that of naked cells and control cells. The growth rate of HCT-8 and SW480 cells, which was constructed by the transfection of the lentiviral packaging plasmid (PSH-HPIP-RNAi), was slower than that of the corresponding naked and control cells. The results showed that HPIP promoted the proliferation of intestinal cancer cells, and then the cell cycle detection by flow cytometry and the apoptosis test of Annexin V-FITC PI double staining method confirmed that HPIP promoted the transformation of the G1 phase and the G2 phase to the M phase of the colorectal cancer cells, and at the same time inhibited the apoptosis of the intestinal cancer cells, And unexpectedly found that it reduced the chemosensitivity of the intestinal cancer to oxaliplatin. Again, through the scratch test, the Transwell experiment found that HPIP enhanced the migration and invasion of intestinal cancer cells and promoted epithelial cell-to-mesenchymal transition (EMT). More importantly, in the mechanism study, we found that HCT-116 cells expressing HPIP were found by the addition of inhibitors of AKT and ERK. The effect of HPIP on the proliferation and migration of intestinal cancer cells was verified by the activation of AKT, MAPK/ ERK pathway. Once the AKT and ERK were inhibited, the HPIP could not function, and finally, the growth rate of the low-HPIP-inhibited intestinal cancer cells in the nude mice was confirmed by tumor-forming experiments in nude mice. Our findings suggest that the HPIP plays an important role in the development of intestinal cancer, and if the HPIP is used as a new target for the treatment of colorectal cancer, the HPIP is constructed with a good effect of HPIP small interfering RNA or a chemical small molecule which antagonizes the HPIP, the HPIP is effectively knocked down or its function is suppressed, and the growth of the tumor is inhibited, The transfer will be a new molecular medicine for the targeted treatment of intestinal cancer, and it may also provide a new inspiration for solving the drug-resistance problem of some of the chemotherapy drugs.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.3
本文编号：2504006
[Abstract]:Colorectal cancer (CRC) is one of the world's most common malignant tumors, and in the western countries, the third, about 10% of the cancer morbidity and mortality in the tumor mortality rate. Therefore, it is of vital importance to elucidate the molecular mechanism of colorectal cancer development for the treatment of colorectal cancer. Although it is generally believed that colorectal cancer is a heterogeneous disease, it is associated with a receptor tyrosine kinase (RTK) and a variety of oncogenes in a plurality of signal pathways downstream of the cell, the mutation, inactivation and activation of the tumor suppressor gene, but in general, we are responsible for colorectal cancer, The knowledge of potential molecular biology changes behind development is still limited. The human hematopoiesis-related PBX interaction protein (HPIP) is the first to be named after screening from the fetal liver cDNA library by yeast two-hybrid technique by using a yeast two-hybrid technique, such as the pre-B-cell leukebia trans-expression factor 1 (PBX 1). The HPIP protein is composed of 731 amino acids and has no obvious homology with known proteins. HPIP, in addition to being able to bind to PBX1, can also interact with PBX2 and PBX3 and, like PBX1, can be expressed in the original hematopoietic cell CD34 (+) subpopulation. Lu Q et al found that Pbxl can be combined with its proto-oncogene derivative E2A-Pbx1 in the sequence ATCAATCAA of the DNA, and the HPIP can inhibit the transcriptional activation of E2A-PBX1 by the interaction with the PB X, so that the HPIP has the function of regulating the normal growth and differentiation of the hematopoietic stem cells and inhibiting the occurrence of the leukemia. As a new gene, HPIP is a new gene, its biological function research is still in the initial stage, our earlier study found that the growth rate of breast cancer cells was significantly faster than that of empty vector, and cervical cancer and lung cancer were detected at the same time. HPIP is widely expressed in various cell lines, such as liver cancer, and it is suggested that the HPIP may be involved in the regulation of the proliferation of the positive growth of the cell, and has certain oncogene characteristics, but the function of the HPIP gene in the gastrointestinal tumor has not been studied yet. It is unknown whether or not the HPIP can be a new strategy for the diagnosis and treatment of intestinal cancer. It is well known that the cure rate of early colorectal cancer is as high as 90% -100%, while the 5-year survival rate of late CRC is about 10%. The sensitivity of the traditional tumor markers, such as the sugar antigen 199 (CA199), the carcinoembryonic antigen (CEA), and the like in the early diagnosis of large intestine cancer, is only 10% to 40%, and most of the specificity is not strong. Therefore, we study the expression, function and possible mechanism of its expression, function and possible mechanism in intestinal cancer tissues and cells with HPIP, in order to find new tumor markers. Our study first revealed the key role of HPIP in intestinal cancer and some of the mechanisms. First, we used the immunohistochemical method to detect the expression of HPIP in the paraffin specimen of 64 patients with intestinal cancer randomly selected, and found that the expression of HPIP was negatively correlated with the prognosis of intestinal cancer. The higher the expression of HPIP, the shorter the patient's disease-free survival and overall survival. Second, we selected HCT-8, HCT-116 and SW480 three cells from 7 intestinal cancer cells, and the growth rate of HCT-116, which was transiently transfected with HPIP over-expression plasmid (pcDNA3-Flag-HPIP), was significantly higher than that of naked cells and control cells. The growth rate of HCT-8 and SW480 cells, which was constructed by the transfection of the lentiviral packaging plasmid (PSH-HPIP-RNAi), was slower than that of the corresponding naked and control cells. The results showed that HPIP promoted the proliferation of intestinal cancer cells, and then the cell cycle detection by flow cytometry and the apoptosis test of Annexin V-FITC PI double staining method confirmed that HPIP promoted the transformation of the G1 phase and the G2 phase to the M phase of the colorectal cancer cells, and at the same time inhibited the apoptosis of the intestinal cancer cells, And unexpectedly found that it reduced the chemosensitivity of the intestinal cancer to oxaliplatin. Again, through the scratch test, the Transwell experiment found that HPIP enhanced the migration and invasion of intestinal cancer cells and promoted epithelial cell-to-mesenchymal transition (EMT). More importantly, in the mechanism study, we found that HCT-116 cells expressing HPIP were found by the addition of inhibitors of AKT and ERK. The effect of HPIP on the proliferation and migration of intestinal cancer cells was verified by the activation of AKT, MAPK/ ERK pathway. Once the AKT and ERK were inhibited, the HPIP could not function, and finally, the growth rate of the low-HPIP-inhibited intestinal cancer cells in the nude mice was confirmed by tumor-forming experiments in nude mice. Our findings suggest that the HPIP plays an important role in the development of intestinal cancer, and if the HPIP is used as a new target for the treatment of colorectal cancer, the HPIP is constructed with a good effect of HPIP small interfering RNA or a chemical small molecule which antagonizes the HPIP, the HPIP is effectively knocked down or its function is suppressed, and the growth of the tumor is inhibited, The transfer will be a new molecular medicine for the targeted treatment of intestinal cancer, and it may also provide a new inspiration for solving the drug-resistance problem of some of the chemotherapy drugs.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.3
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，本文编号：2504006
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