HPIP在腸癌中的功能以及機(jī)制研究
發(fā)布時(shí)間：2019-06-21 10:26
【摘要】：結(jié)直腸癌(colorectal cancer CRC)是世界上最常見(jiàn)的惡性腫瘤之一,在西方國(guó)家,在腫瘤死亡率里排名第三,大約10%的癌癥發(fā)病率和致死率。因此,闡明結(jié)直腸腫瘤發(fā)生發(fā)展的分子機(jī)制對(duì)于結(jié)直腸癌個(gè)體治療是至關(guān)重要的。雖然人們普遍認(rèn)為結(jié)直腸癌是一種異質(zhì)性疾病,與受體酪氨酸激酶(RTK)及其在胞內(nèi)下游多條信號(hào)途徑中多種癌基因、抑癌基因的突變、失活、激活有關(guān),但是總體說(shuō)來(lái),我們對(duì)結(jié)直腸癌發(fā)生、發(fā)展背后潛在的分子生物學(xué)改變的認(rèn)識(shí)仍然有限。人造血相關(guān)的PBX相互作用蛋白質(zhì)(hematopoietic PBX—interacting protein, HPIP),最早是在2000年由Abramovich等以前B淋巴細(xì)胞白血病轉(zhuǎn)錄因子(pre-B-cell leukemia transcription factor 1,PBX 1)為誘餌,通過(guò)酵母雙雜交技術(shù),從胎兒肝臟cDNA文庫(kù)中篩選得到而被命名的。HPIP蛋白共由731個(gè)氨基酸組成,且與已知蛋白無(wú)明顯的同源性。HPIP除了能和PBX1結(jié)合之外,還可以與PBX2和PBX3相互作用,并且和PBX1一樣,能在原始的造血細(xì)胞CD34(+)亞群中表達(dá)。Lu Q等發(fā)現(xiàn)Pbxl可以與它的原癌基因衍生物E2A-Pbx1共同結(jié)合于DNA的模序ATCAATCAA序列,而HPIP可通過(guò)與PB X的相互作用從而抑制E2A-PBX1的轉(zhuǎn)錄激活,因此,HPIP具有能調(diào)節(jié)造血干細(xì)胞正常生長(zhǎng)分化過(guò)程,抑制白血病發(fā)生的作用。HPIP作為個(gè)較新的基因,它的生物學(xué)功能研究尚在起步階段,我們的前期研究發(fā)現(xiàn)乳腺癌細(xì)胞外源轉(zhuǎn)入HPIP后,其生長(zhǎng)速度與空載體相比明顯加快,同時(shí)又檢測(cè)到宮頸癌、肺癌、肝癌等多種細(xì)胞系中HPIP廣泛表達(dá),提示HPIP可能參與細(xì)胞正向生長(zhǎng)增殖的調(diào)控,具有一定的癌基因特性,但是HPIP基因在胃腸道腫瘤的功能至今無(wú)研究,抑制HPIP是否可以作為腸癌診斷以及治療的新策略尚不知曉。眾所周知,目前,早期大腸癌治愈率高達(dá)90%-100%,而晚期CRC的5年生存率只有10%左右。而傳統(tǒng)腫瘤標(biāo)志物,如糖抗原199(CA199)、癌胚抗原(CEA)等在早期大腸癌診斷方面,敏感度僅為10%-40%,且大部分特異性不強(qiáng)。因此,我們以HPIP為研究對(duì)象,研究其在腸癌組織和細(xì)胞中的表達(dá)、功能及可能的機(jī)制,以期待發(fā)現(xiàn)新的腫瘤標(biāo)志物。我們的研究首次揭示了HPIP在腸癌中的關(guān)鍵作用以及部分機(jī)制。首先,我們?cè)陔S機(jī)選擇的64例腸癌患者石蠟標(biāo)本中,運(yùn)用免疫組化檢測(cè)發(fā)現(xiàn)癌組織HPIP表達(dá)明顯高于相應(yīng)癌旁組織；并且發(fā)現(xiàn)HPIP的表達(dá)與腸癌預(yù)后呈負(fù)相關(guān),HPIP表達(dá)越高,患者無(wú)病生存期和總生存期越短。其次我們從7株腸癌細(xì)胞中選擇HCT-8,HCT-116,SW480三株細(xì)胞,瞬時(shí)轉(zhuǎn)染HPIP過(guò)表達(dá)質(zhì)粒(pcDNA3-Flag-HPIP)的HCT-116較裸細(xì)胞和對(duì)照細(xì)胞的生長(zhǎng)速度明顯增快,轉(zhuǎn)染慢病毒包裝質(zhì)粒pcdh-HPIP構(gòu)建的穩(wěn)定過(guò)表達(dá)HPIP的SW480細(xì)胞上也得出此結(jié)論。而轉(zhuǎn)染慢病毒包裝質(zhì)粒(PSIH-HPIP-RNAi)構(gòu)建的敲低HPIP的穩(wěn)定克隆細(xì)胞HCT-8和SW480細(xì)胞的生長(zhǎng)速度較相應(yīng)裸細(xì)胞和對(duì)照細(xì)胞明顯減慢。說(shuō)明HPIP促進(jìn)腸癌細(xì)胞的增殖,其次,通過(guò)流式細(xì)胞術(shù)的細(xì)胞周期檢測(cè)以及Annexin V-FITC PI雙染色方法的凋亡檢測(cè)證實(shí)HPIP促進(jìn)了大腸癌細(xì)胞G1期向S期,G2期向M期的轉(zhuǎn)化,同時(shí)抑制了腸癌細(xì)胞凋亡,并意外發(fā)現(xiàn)其降低了腸癌對(duì)奧沙利鉑的化療敏感性。再次,我們通過(guò)劃痕實(shí)驗(yàn)、Transwell實(shí)驗(yàn)發(fā)現(xiàn)HPIP增強(qiáng)了腸癌細(xì)胞遷移和侵襲能力,并促進(jìn)了上皮細(xì)胞向間質(zhì)轉(zhuǎn)化(EMT)。更重要的是在機(jī)制研究中,我們通過(guò)添加AKT和ERK的抑制劑發(fā)現(xiàn)過(guò)表達(dá)HPIP的HCT-116細(xì)胞、SW480細(xì)胞與對(duì)照細(xì)胞相比促進(jìn)生長(zhǎng)的幅度均明顯下降,促進(jìn)劃痕愈合的能力也明顯下降甚至消失。驗(yàn)證了HPIP促進(jìn)腸癌細(xì)胞增殖、遷移是通過(guò)激活A(yù)KT、MAPK/ERK通路,一旦AKT、ERK被抑制,HPIP將不能起作用,最后,通過(guò)裸鼠成瘤實(shí)驗(yàn),再次證實(shí)敲低HPIP抑制腸癌細(xì)胞在裸鼠體內(nèi)的生長(zhǎng)速度。我們的研究結(jié)果暗示HPIP在腸癌的發(fā)生發(fā)展中起重要作用,如果把HPIP作為抗結(jié)直腸癌治療的藥物新靶標(biāo),構(gòu)建效果良好的HPIP小干擾RNA或拮抗HPIP的化學(xué)小分子,將HPIP有效敲低或抑制其功能,從而阻礙腫瘤生長(zhǎng)、轉(zhuǎn)移,將有可能為腸癌的靶向治療的開(kāi)創(chuàng)出新的分子藥物,也可能會(huì)為解決部分化療藥物耐藥問(wèn)題提供新的啟示。
[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.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R735.3
本文編號(hào)：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.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R735.3
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