本文選題：AMPK + LKB1��； 參考：《南昌大學(xué)》2016年博士論文

【摘要】：背景和目的:全球癌癥死亡人數(shù)正在迅猛上升,癌癥已成為我國(guó)五大致死性疾病之首,嚴(yán)重威脅著我國(guó)人民的健康水平。能量代謝的相對(duì)平衡是抑制腫瘤細(xì)胞過(guò)度生長(zhǎng)的預(yù)防機(jī)制,而腫瘤細(xì)胞依靠代謝重組(如Warburg效應(yīng))獲取能量,克服能量供應(yīng)危機(jī)。因此,腫瘤細(xì)胞的能量代謝途徑已成為腫瘤治療的潛在靶點(diǎn)。細(xì)胞的能量代謝受多條信號(hào)通路的調(diào)控,其中單磷酸腺苷激酶(AMP-activated protein kinase,AMPK)信號(hào)通路是最重要的途徑,該通路參與調(diào)節(jié)Warburg效應(yīng),脂肪酸的合成與谷氨酰胺代謝,并與PI3K-Akt信號(hào)通路、MAPK信號(hào)通路以及多種轉(zhuǎn)錄因子有相互作用,共同調(diào)節(jié)細(xì)胞的增殖、生長(zhǎng)、凋亡等細(xì)胞生物學(xué)行為,發(fā)揮抑癌作用。腫瘤發(fā)生早期,AMPKα1基因的缺失對(duì)癌基因誘發(fā)的腫瘤有促進(jìn)作用,有臨床病理資料顯示腫瘤組織中AMPK活性低于正常組織,AMPK活性不足被認(rèn)為是惡性腫瘤發(fā)生發(fā)展的原因之一。鑒于多種惡性腫瘤組織中AMPK的失活,眾多學(xué)者探索如何利用化學(xué)或生物分子激活A(yù)MPK以抑制腫瘤生長(zhǎng),并取得良好的效果。例如,二甲雙胍、AICAR、A769662、阿斯匹林、水楊酸,黃蓮素等化學(xué)藥物,其中關(guān)于降血糖藥物二甲雙胍的研究最為突出,現(xiàn)已作為抗癌輔助用藥進(jìn)入臨床試驗(yàn)。除了化學(xué)藥物,還發(fā)現(xiàn)一些細(xì)胞因子和應(yīng)激壓力也能激活A(yù)MPK。各種因素激活A(yù)MPK方式不盡相同,但AMPK的充分激活都有賴(lài)于AMPK激酶(AMPKK)的參與,目前已證實(shí)的AMPKK包括:LKB1、CaMKKβ、TAK1等。LKB1作為AMPK最重要的上游激酶,在各種腫瘤組織中表達(dá)不一,對(duì)二甲雙胍等AMPK激活劑的抗癌效果有重要影響,但目前對(duì)于LKB1在應(yīng)激壓力激活A(yù)MPK的作用尚不清楚。此外,我們的前期研究還發(fā)現(xiàn)某些應(yīng)激壓力(細(xì)胞因子刺激或化療藥物)激活A(yù)MPK的過(guò)程中,并未涉及以上AMPKK。例如,可誘導(dǎo)滲透壓改變的山梨醇。這些應(yīng)激壓力不僅誘導(dǎo)腫瘤細(xì)胞的凋亡,還可以引起AMPK活化,但其具體機(jī)制尚不清楚。由此可見(jiàn),為了提高腫瘤細(xì)胞中AMPK的活性,不僅有必要對(duì)LKB1-AMPK途徑進(jìn)行深入研究,還要探索其他的AMPK激活途徑及其在抗腫瘤治療的作用。因此,我們進(jìn)行了以下研究:⑴胃癌及肺癌組織中的p-ampk和lkb1的表達(dá)情況;⑵lkb1過(guò)表達(dá)對(duì)胃癌細(xì)胞sgc-7901生物學(xué)行為的影響;⑶atm和lkb1在抗癌藥物依托泊苷激活ampk中的作用;⑷ampk活化是否增加腫瘤細(xì)胞對(duì)依托泊苷的藥物敏感性;⑸lkb1和mlk3對(duì)ampk的調(diào)節(jié)作用。材料與方法:1.本課題的臨床標(biāo)本來(lái)自南昌大學(xué)第一附屬醫(yī)院手術(shù)切除標(biāo)本(倫理批準(zhǔn)號(hào)2014〔025〕),收集60例胃癌及其對(duì)應(yīng)癌旁組織,采用免疫組化染色分析檢測(cè)胃癌及癌旁組織p-ampk(thr172)及l(fā)kb1的表達(dá)水平;另外,收集了65例不同階段的肺腺癌標(biāo)本,采用免疫組化染色分析不同階段的肺腺癌組織p-ampk(thr172)表達(dá)水平;2.在缺乏lkb1蛋白的sgc-7901細(xì)胞株中轉(zhuǎn)入功能性野生型lkb1基因,構(gòu)建穩(wěn)定表達(dá)lkb1的細(xì)胞株。以mtt法檢測(cè)細(xì)胞生長(zhǎng)和存活,以細(xì)胞劃痕實(shí)驗(yàn)檢測(cè)細(xì)胞遷移,以流式細(xì)胞技術(shù)檢測(cè)細(xì)胞周期、cd44陽(yáng)性表達(dá)細(xì)胞比例、細(xì)胞凋亡比率;3.依托泊苷以濃度依賴(lài)或時(shí)間依賴(lài)的方式作用于前列腺癌細(xì)胞c4-2后,收集蛋白,以免疫印跡法檢測(cè)ampk、atm磷酸化水平;另外,sirna干擾c4-2細(xì)胞中的atm或lkb1,再給依托泊苷刺激后,檢測(cè)ampk、atm的磷酸化水平。4.轉(zhuǎn)入ampkdn突變體到c4-2細(xì)胞抑制ampk活化(c4-2dn),以空載體為對(duì)照(c4-2e),給依托泊苷刺激24小時(shí),以流式細(xì)胞儀檢測(cè)細(xì)胞凋亡情況;選擇ampk活性不同(c4-2dn及c4-2e)細(xì)胞和lkb1表達(dá)不同(a549e及a549-lkb1)的細(xì)胞,給予依托泊苷刺激不同時(shí)間后,以免疫印跡法檢測(cè)凋亡蛋白表達(dá)(cleavedcaspase3\cleavedparp)。5.選擇lkb1表達(dá)不同(a549及a549-lkb1)細(xì)胞,給予各種應(yīng)激因子刺激后,以免疫印跡技術(shù)檢測(cè)p-ampk與p-jnk,篩選出同時(shí)激活ampk與jnk的因子;在hek-293t細(xì)胞中轉(zhuǎn)入mlk3質(zhì)粒,后檢測(cè)ampk的磷酸化水平;以gsh純化技術(shù),獲得基因重組蛋白mlk3和ampk,以體外酶學(xué)實(shí)驗(yàn)檢測(cè)mlk3對(duì)ampk的磷酸化;在hek-293t細(xì)胞中,轉(zhuǎn)入mlk3質(zhì)粒后,用gshbeads將gst-mlk3蛋白捕獲,以免疫印跡檢測(cè)沉淀中是否捕獲內(nèi)源性ampkα1或ampkα2蛋白;將myc-ampk?1或?2和gst-mlk3質(zhì)粒共轉(zhuǎn)染hek-293t細(xì)胞,收集細(xì)胞裂解液,與gshbeads孵育,離心沉淀,收集gshbeads-mlk3和捕獲的蛋白,以anti-gst和anti-myc抗體進(jìn)行免疫印跡分析。結(jié)果1.胃癌及肺癌組織中ampk和lkb1情況。⑴免疫組化結(jié)果顯示胃癌組織p-ampk(thr172)、lkb1的表達(dá)低于癌旁組織(p0.001)。p-ampk(thr172)表達(dá):胃癌中(-)20例,(+)33例,(++)6例,(+++)1例,而癌旁組織中:(-)5例,(+)11例,(++)28例,(+++)16例。lkb1表達(dá):胃癌中(-)23例,(+)25例,(++)12例,(+++)0例,癌旁組織中l(wèi)kb1表達(dá):(-)9例,(+)13例,(++)27,(+++)11例。⑵不同分期肺癌組織中的ampk活化情況,隨著肺腺癌分期的惡性程度增高,p-ampk(thr172)表達(dá)漸漸下降,Ⅰ期Ⅱ期Ⅲ期(spearman相關(guān)分析=-0.397,p0.05)。Ⅰ期(-)4例,Ⅰ期(+)5例,Ⅰ期(++)10例,Ⅰ期(+++)3例;Ⅱ期(-)5例,Ⅱ期(+)10例,Ⅱ期(++)9例,Ⅱ期(+++)3例;Ⅲ期(-)9例,Ⅲ期(+)8例,Ⅲ期(++)1例,Ⅲ期(+++)0例。2.提高lkb1蛋白表達(dá)對(duì)胃癌細(xì)胞sgc-7901生物學(xué)行為的影響在缺乏lkb1的腫瘤細(xì)胞sgc-7901中轉(zhuǎn)入lkb1基因,增加lkb1的表達(dá)可以抑制細(xì)胞的增殖與遷移,減少cd44陽(yáng)性細(xì)胞的比例,提高腫瘤細(xì)胞對(duì)奧沙利鉑、5-氟尿嘧啶和伊立替康三種抗癌藥物敏感性。3.atm和lkb1在依托泊苷激活ampk中的作用⑴westernblot結(jié)果顯示:腫瘤細(xì)胞a549受依托泊苷刺激后,p-atm和p-ampk(thr172)逐漸上升,并呈劑量和時(shí)間依賴(lài)性;⑵sirna干擾c4-2細(xì)胞的atm表達(dá)后,atm表達(dá)下調(diào),給予依托泊苷刺激細(xì)胞,westernblot結(jié)果顯示:p-atm和p-ampk(thr172)無(wú)明顯上升;而對(duì)照組atm表達(dá)正常,提示依托泊苷刺激可以同時(shí)激活atm和ampk,依托泊苷激活ampk過(guò)程中需要atm的參與。⑶sirna干擾c4-2細(xì)胞的lkb1表達(dá),lkb1表達(dá)下降,給予依托泊苷刺激細(xì)胞,westernblot結(jié)果顯示:p-atm表達(dá)逐漸上升,而無(wú)法激活ampk;而對(duì)照組lkb1表達(dá)正常,依托泊苷刺激可以同時(shí)激活atm和ampk。提示在缺乏lkb1的條件下,依托泊苷可以激活atm,但無(wú)法激活ampk。⑷在缺乏lkb1的a549細(xì)胞中,轉(zhuǎn)入lkb1基因,提高lkb1蛋白表達(dá)后,給予依托泊苷刺激細(xì)胞,westernblot結(jié)果顯示:可以同時(shí)激活atm和ampk;而對(duì)照組lkb1表達(dá)無(wú)變化,依托泊苷刺激p-atm表達(dá)逐漸上升,而無(wú)法激活ampk。以上結(jié)果提示:依托泊苷激活ampk過(guò)程中需要atm和lkb1共同參與。4.ampk活化對(duì)腫瘤細(xì)胞藥物敏感性的影響⑴流式細(xì)胞分析顯示,在依托泊苷刺激下c4-2e細(xì)胞(ampk有活性)凋亡細(xì)胞的比例明顯高于c4-2dn(ampk無(wú)活性);⑵依托泊苷刺激后,c4-2e細(xì)胞中的p-ampk(thr172)表達(dá)、cleavedcaspase3表達(dá)和cleavedparp表達(dá)明顯高于c4-2dn細(xì)胞;⑶依托泊苷刺激下,a549-lkb1細(xì)胞的p-ampk(thr172)表達(dá)、cleavedcaspase3表達(dá)和cleavedparp表達(dá)明顯高于對(duì)照組(a549e)。結(jié)果提示,ampk活化或提高lkb1表達(dá)有助于增強(qiáng)腫瘤細(xì)胞的藥物敏感性。5.mlk3對(duì)ampk的調(diào)節(jié)作用⑴a549-lkb1和a549-wt細(xì)胞受山梨醇、aicar等7種因子刺激,僅在山梨醇所刺激a549-lkb1和a549-wt兩種細(xì)胞后,p-ampk和p-jnk都升高;提示山梨醇可以同時(shí)激活ampk和jnk,并不依賴(lài)于lkb1。⑵a549-lkb1和a549-wt細(xì)胞受山梨醇刺激,兩細(xì)胞的p-ampk同時(shí)升高,提示山梨醇可能以不依賴(lài)lkb1的方式激活ampk;⑶在hek293t細(xì)胞中轉(zhuǎn)入mlk3質(zhì)粒,過(guò)表達(dá)mlk3蛋白,然后以免疫印跡法檢測(cè)p-ampk(thr172)水平,結(jié)果顯示mlk3的異常過(guò)表達(dá)可提高p-ampk(thr172)水平。⑷體外激活ampk酶學(xué)實(shí)驗(yàn)顯示,單獨(dú)加入amp或單獨(dú)加入lkb1蛋白對(duì)ampk的磷酸化無(wú)明顯影響;同時(shí)加入amp和lkb1可以引起ampk的磷酸化;單獨(dú)加入mlk3可以引起明顯的ampk磷酸化;同時(shí)加入amp和mlk3引起的ampk磷酸化最強(qiáng)。該結(jié)果提示mlk3可在缺乏amp的情況下磷酸化ampk,說(shuō)明ampk是mlk3的催化底物。⑸MLK3與AMPK間的相互結(jié)合;通過(guò)Pulldown技術(shù)發(fā)現(xiàn),轉(zhuǎn)入GST-MLK3質(zhì)粒的細(xì)胞提取物中發(fā)現(xiàn)AMPKα1的表達(dá),提示轉(zhuǎn)入表達(dá)的MLK3蛋白與細(xì)胞內(nèi)源性AMPKα1亞單位緊密結(jié)合;共轉(zhuǎn)染Myc-AMPK?1或?2和GST-MLK3入HEK-293T細(xì)胞,然后通過(guò)IP技術(shù)證明,外源性表達(dá)GST-MLK3蛋白能與外源性Myc-AMPK?1蛋白發(fā)生特異性結(jié)合。結(jié)論:1.癌組織中AMPK失活可能是惡性腫瘤發(fā)生發(fā)展的原因之一,LKB1表達(dá)下調(diào)可使AMPK失活。在缺乏LKB1表達(dá)的腫瘤細(xì)胞中,提高LKB1蛋白表達(dá)可以抑制細(xì)胞的生長(zhǎng)、轉(zhuǎn)發(fā)移,增強(qiáng)腫瘤細(xì)胞的藥物敏感性。2.依托泊苷激活A(yù)MPK的過(guò)程需要ATM和LKB1共同參與;AMPK活化使得腫瘤細(xì)胞對(duì)依托泊苷誘導(dǎo)凋亡作用更為敏感。3.MLK3能與AMPK?1結(jié)合,并以非LKB1方式直接磷酸化AMPK,MLK3有可能是AMPK新的上游激酶。
[Abstract]:Background and purpose: the number of global cancer deaths is rising rapidly. Cancer has become the first of the five fatal diseases in our country. It is a serious threat to the health of our people. The relative balance of energy metabolism is a preventive mechanism to inhibit the overgrowth of tumor cells, and the tumor cells acquire energy depending on the metabolic recombination (such as the Warburg effect). Energy supply crisis. Therefore, the energy metabolism pathway of tumor cells has become a potential target for cancer treatment. The energy metabolism of cells is regulated by multiple signal pathways, in which the AMP-activated protein kinase (AMPK) signal pathway is the most important pathway. The pathway participates in the regulation of the Warburg effect and the synthesis of fatty acids. Metabolism of glutamine, and the interaction with PI3K-Akt signaling pathway, MAPK signaling pathway and various transcription factors to regulate cell biological behavior, such as proliferation, growth, apoptosis and other cell biological behavior, and play the role of inhibiting cancer. Early tumor occurrence, the deletion of AMPK alpha 1 gene can promote cancer induced tumor, and there are clinicopathological data. The activity of AMPK in tumor tissue is lower than that of normal tissue, and the insufficiency of AMPK activity is considered to be one of the reasons for the development of malignant tumor. In view of the inactivation of AMPK in many malignant tumor tissues, many scholars have explored how to activate AMPK by using chemical or biological molecules to inhibit the growth of the tumor. For example, metformin, AICAR, A76 9662, aspirin, salicylic acid, Huang Liansu and other chemical drugs, among them, the study of metformin, the hypoglycemic drug, is the most prominent. It has now entered the clinical trial as an adjuvant antitumor drug. Besides chemical drugs, some cytokines and stress stress can also activate AMPK. various factors to activate AMPK methods, but AMPK is sufficient. Activation depends on the participation of AMPK kinase (AMPKK). The current confirmed AMPKK, including LKB1, CaMKK beta, and TAK1 as the most important upstream kinase of AMPK, is expressed differently in various tumor tissues and has an important effect on the anticancer effect of the AMPK activator such as metformin, but the effect of LKB1 on stress activation AMPK is not yet clear. In addition, our previous study also found that some stress stress (cytokine stimulation or chemotherapeutic drugs) activation of AMPK did not involve the above AMPKK., which could induce osmotic pressure change of sorbitol. These stress stresses not only induce apoptosis of tumor cells, but also cause AMPK activation, but the specific mechanisms are not yet clear. Therefore, in order to improve the activity of AMPK in tumor cells, it is not only necessary to study the LKB1-AMPK pathway in depth, but also to explore other AMPK activation pathways and their role in antitumor therapy. Therefore, we have carried out the following studies: (1) the expression of p-ampk and LKB1 in gastric and lung cancer tissues; (2) LKB1 over expression of sGC in gastric cancer cells SGC The effects of -7901 biological behavior; (3) the role of ATM and LKB1 in the activation of etoposide in AMPK; (4) whether AMPK activation increases the drug sensitivity of tumor cells to etoposide; the regulatory effect of LKB1 and mlk3 on AMPK. Materials and methods: 1. clinical specimens from the First Affiliated Hospital of Nanchang University ( Ethical approval number 2014 [025]), 60 cases of gastric cancer and their corresponding para cancerous tissues were collected and immunohistochemical staining analysis was used to detect the expression of p-ampk (thr172) and LKB1 in gastric cancer and para cancer tissues. In addition, 65 specimens of lung adenocarcinoma at different stages were collected, and the expression of p-ampk (thr172) expression in lung adenocarcinoma tissues at different stages by immunohistochemical staining was used. Level; 2. in the SGC-7901 cell line with the lack of LKB1 protein into the functional wild type LKB1 gene to construct a stable LKB1 cell line. The cell growth and survival were detected by MTT assay, cell migration was detected by the cell scratch test, the cell cycle, the proportion of CD44 positive cells, the ratio of apoptosis, and the rate of apoptosis were detected by flow cytometry; 3. etoposide was used. Glucoside acts on the prostate cancer cell C4-2 in a concentration dependent or time dependent manner, collecting protein and detecting AMPK, ATM phosphorylation level by immunoblotting; in addition, siRNA interferes with ATM or LKB1 in C4-2 cells, and after stimulation of etoposide, AMPK, ATM phosphorylation level.4. is transferred to ampkdn mutant to C4-2 cells to inhibit activation. -2dn), using the empty carrier as the control (c4-2e), the apoptosis of etoposide was detected by flow cytometry for 24 hours. The cells with different AMPK activity (c4-2dn and c4-2e) and LKB1 were selected to express different (a549e and a549-lkb1) cells. The expression of apoptotic protein was detected by immunoblotting after the stimulation of etoposide at different time (cleavedcaspase3c). Leavedparp).5. chose LKB1 to express different (A549 and a549-lkb1) cells, and after the stimulation of various stress factors, p-ampk and p-JNK were detected by immunoblotting, and the factors that simultaneously activated AMPK and JNK were screened. The mlk3 plasmids were transferred into hek-293t cells and the phosphorylation level of AMPK was detected. PK was used to detect the phosphorylation of mlk3 to AMPK in vitro. In hek-293t cells, after transferring to mlk3 plasmid, gst-mlk3 protein was captured by gshbeads, and the endogenous AMPK a 1 or AMPK alpha 2 protein was captured by immunoblotting, and myc-ampk? 1 or 2 and gst-mlk3 mass were co transfected to the hek-293t cells, and the cell lysate was collected. Incubation, centrifugation, collection of gshbeads-mlk3 and captured proteins, and immunoblotting with anti-GST and anti-myc antibodies. Results 1. the AMPK and LKB1 in gastric cancer and lung cancer. (1) immunohistochemical results showed p-ampk (thr172) in gastric cancer tissue, and the expression of LKB1 was lower than that in the paracancerous group (p0.001).P-ampk (thr172) expression: 20 cases in gastric cancer (-), (+) 33 (+ + +) 6 cases, (+ + +) 1 cases, and (+) 5 cases, (+) 11 cases, (+ +) 28 cases, (+ + +) 16 cases of.Lkb1 expression: gastric cancer (+ +) in 23 cases, (+ +) 12 cases (+ + +) in 0 cases, (+ + + +) LKB1 expression: (+ + +) 27, (+ + +), AMPK activation in different stages of lung cancer, with lung adenocarcinoma stage increased malignancy, p-ampk (p-ampk ( Thr172) gradually decline, stage I stage II stage III (Spearman correlation analysis =-0.397, P0.05). 4 cases (-), I (+) 5 cases, I phase (+ +) 10 cases, phase I (+ + +) 3 cases, II (-) 5 cases, II (+) 10 cases, II (+ +) 9 cases, II (+ + +) 3 cases, III (+) 9 cases, III (+) 8, + + 1 cases, III phase (+ + +).2. improve the expression of LKB1 protein expression of gastric cancer cell s The effect of gc-7901's biological behavior on the LKB1 gene in the tumor cell SGC-7901 lacking LKB1, increasing the expression of LKB1 can inhibit cell proliferation and migration, reduce the proportion of CD44 positive cells, and increase the three kinds of anti-cancer drug sensitivity.3.atm and LKB1 in the tumor cells activated by oxaliplatin, 5- fluorouracil and erinotecan in etoposide activation The effect of AMPK in Westernblot showed that p-atm and p-ampk (thr172) increased gradually after A549 was stimulated by etoposide, and was dosed and time dependent; and after siRNA interfered with ATM expression of C4-2 cells, ATM expression was down, and etoposide stimulated cells. The expression of ATM in the control group was normal, suggesting that etoposide stimulated the activation of ATM and AMPK simultaneously. ATM was needed during the activation of AMPK by etoposide. (3) siRNA interfered with LKB1 expression in C4-2 cells, LKB1 expression decreased, and etoposide stimulated cells. The Westernblot results showed that p-atm expression increased gradually, but no AMPK was activated; while the control group was not activated. The expression of B1 is normal, and etoposide stimulation can activate both ATM and ampk. at the same time. Etoposide can activate ATM under the condition of lack of LKB1. But it can not activate ampk. (4) in A549 cells lacking LKB1, and turn into the LKB1 gene and increase the expression of LKB1 protein to stimulate the cells by etoposide. The Westernblot result shows that ATM and can be activated at the same time. PK, while the expression of LKB1 in the control group was not changed, and the expression of p-atm increased gradually with etoposide stimulation, but the result of the failure to activate ampk. suggested that the effect of etoposide activation on the AMPK process needed both ATM and LKB1 to participate in the effect of.4.ampk activation on the drug sensitivity of the tumor cells (1) flow cytometric analysis showed that the c4-2e cell (AMPK has the presence of etoposide (AMPK) The proportion of apoptotic cells was significantly higher than that of c4-2dn (AMPK inactive); (2) after stimulation of etoposide, the expression of p-ampk (thr172) in c4-2e cells, cleavedcaspase3 expression and cleavedparp expression were significantly higher than that of c4-2dn cells; (3) the expression of p-ampk (thr172) in a549-lkb1 cells under the stimulation of etoposide, cleavedcaspase3 expression and expression clearly expressed. Significantly higher than the control group (a549e), the results suggest that AMPK activation or enhancement of LKB1 expression helps to enhance the drug sensitivity of tumor cells to the regulatory effect of.5.mlk3 on AMPK (1) a549-lkb1 and a549-wt cells are stimulated by 7 factors, such as sorbitol, AICAR and so on. Only after sorbitol stimulates the two cells of a549-lkb1 and a549-wt, p-ampk and p-JNK are elevated. Sorbitol can simultaneously activate AMPK and JNK, which does not depend on lkb1. (lkb1.) a549-lkb1 and a549-wt cells stimulated by sorbitol, and the p-ampk increases at the same time, suggesting that sorbitol may activate AMPK in the manner that does not depend on LKB1; (3) the mlk3 plasmid is transferred into the HEK293T cell, the mlk3 protein is overexpressed, and then the Western blot method is used to detect the p-ampk. The results showed that the abnormal overexpression of mlk3 could increase the level of p-ampk (thr172). 4. In vitro activation of AMPK enzymology experiments showed that adding amp alone or adding LKB1 protein alone had no obvious effect on the phosphorylation of AMPK; meanwhile, AMP and LKB1 could cause phosphorylation of AMPK. The results suggested that the phosphorylation of AMPK caused by mlk3 is the strongest. The results suggest that mlk3 can phosphorylate AMPK under the absence of AMP, indicating that AMPK is a catalytic substrate for mlk3, the binding between MLK3 and AMPK; by Pulldown technology, the expression of AMPK alpha 1 is found in the cell extracts of the GST-MLK3 plasmid, indicating the transfer of the expressed protein to the cell source. Sexual AMPK alpha 1 subunits were tightly combined; CO transfected Myc-AMPK? 1 or 2 and GST-MLK3 into HEK-293T cells, and then IP technique proved that exogenous GST-MLK3 protein could be associated with exogenous Myc-AMPK? 1 protein specific binding. Conclusion: AMPK deactivation in 1. cancer tissues may be one of the causes of malignant tumor development, LKB1 expression downregulation can make AMP K inactivation. In the tumor cells lacking LKB1 expression, increasing the expression of LKB1 protein can inhibit cell growth, transfer and enhance the drug sensitivity of tumor cells..2. etoposide activates AMPK, which requires the joint participation of ATM and LKB1; AMPK activation makes the tumor cells more sensitive to the apoptosis effect of etoposide and AMPK? 1 In combination with direct phosphorylation of AMPK in non LKB1 mode, MLK3 may be a new upstream kinase of AMPK.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R730.5
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本文編號(hào)：1970747