抑癌基因PDCD4对肿瘤和巨噬细胞自噬的抑制效应及其分子机制
摘要
研究目的
程序性凋亡因子4(programmed cell death protein4, PDCD4)是近年发现一种重要的抑癌基因,通过抑制蛋白转录和翻译抑制肿瘤的发生、发展从而发挥抑癌基因的功能。PDCD4转基因小鼠抵抗表皮乳头状瘤向皮肤癌的恶性转化；而PDCD4基因敲除小鼠较野生型小鼠更容易罹患淋巴瘤。另外PDCD4在人多数肿瘤组织(如：肺癌、肝癌、神经胶质瘤、卵巢癌、结直肠癌等)中低表达甚至缺失表达,预示着肿瘤恶性程度高和不良预后。体外实验发现PDCD4抑制肿瘤细胞的恶性表型,并增强肿瘤的化疗敏感性。总之,越来越多的证据表明PDCD4是一种重要的抑癌基因。然而,PDCD4抑制肿瘤的机制目前尚不清楚,在不同细胞系中的作用也不尽相同。我们和其他学者的前期研究证明在肝细胞肝癌、卵巢癌、神经胶质瘤、乳腺癌以及胃癌中PDCD4具有促进肿瘤细胞凋亡的作用；但有学者提出不同的观点,如,PDCD4抑制宫颈癌HeLa细胞的凋亡,而对结肠癌的凋亡无影响。同样,PDCD4对细胞周期的影响在不同肿瘤来源细胞系中也不尽相同,具有明显的细胞特异性。因此,目前有关PDCD4抑癌机制均不足以解释PDCD4在不同肿瘤中普遍存在的抑制作用。
(巨)自噬是细胞的一种生理和病理学过程,为细胞自身代谢提供能量并对某些细胞器进行更新,从而维持细胞的自身稳态。自噬在肿瘤细胞中的作用具有双重性,一方面,自噬介导的细胞凋亡或死亡抑制肿瘤的发展,而另一方面,自噬作为一种代偿反应,能为细胞提供生存所必须的能量,使肿瘤细胞免于死亡,从而促进肿瘤生长。另外,自噬在免疫细胞(如巨噬细胞)的功能调节中也发挥重要作用,如巨噬细胞通过自噬调节其在脂质代谢等方面的作用。目前,已知多种基因参入自噬过程中,统称为自噬相关基因(Autophagy Related Genes, ATG),如ATG5、ATG6(又名BECN1/Beclinl)、ATG8(又名LC3, microtubule-associated protein1light chain3)和ATG12。其中,ATG5参与了自噬小体形成的早期过程,它与ATG12结合形成ATG5-ATG12复合体,对自噬小体的延长、闭环起到至关重要的作用,并且能够调控LC3家族蛋白脂化并锚定到自噬小体双分子膜上而BECN1作用在ATG5的上游通路,它能调控上游的P13K通路并且募集其他ATG相关蛋白进而达到调节自噬的目的。
为了明确PDCD4在自噬中的作用,探索其作用机制,以及对肿瘤和巨噬细胞功能的影响。本论文从以下几个方面进行了研究：一、PDCD4对肿瘤细胞自噬及其相关功能的影响发现PDCD4具有抑制肿瘤细胞自噬的作用,且其作用具有普遍性；发现PDCD4对肿瘤细胞生长的抑制依赖于其对自噬的抑制；二、Pdcd4对巨噬细胞自噬及其相关功能的影响证实Pdcd4能够抑制多种刺激诱导的巨噬细胞自噬；发现Pdcd4通过抑制巨噬细胞自噬促进巨噬细胞泡沫化形成,并且在动脉粥样硬化发生发展过程中也发挥至关重要作用；三、PDCD4抑制自噬的分子机制发现PDCD4对自噬的调节不依赖于BECN1通路,而是通过PDCD4-EIF4A-ATG5轴抑制ATG5的蛋白翻译实现的。
研究方法
一、PDCD4对肿瘤细胞自噬及其相关功能的影响
1.在PDCD4过表达和干扰体系中,应用western blot的方法检测自噬相关标志蛋白LC3B-II及SQSTM1的表达,用免疫荧光的方法检测自噬小体形成。
2.应用Pepstatin A和E64d联合用药进行自噬流分析,排除自噬流损伤的影响。
3.建立裸鼠成瘤模型,在成瘤后分别瘤内注射生理盐水、空载体及PDCD4载体,进行western blot检测LC3B-II及SQSTM1的表达,应用组织免疫荧光方法检测自噬小体形成。
4.用3-MA、ATG5小干扰阻断自噬或用z-VAD阻断凋亡,应用CCK8方法检测PDCD4对细胞活性的影响。
二、Pdcd4对巨噬细胞自噬及其相关功能的影响
1.在pdcd4-/-小鼠和野生型小鼠腹腔原代巨噬细胞和骨髓来源巨噬细胞(BMDM)及小鼠白血病来源巨噬细胞系RAW264.7中检测利用western blot和免疫荧光方法检测多种刺激下(EBSS、LPS、雷帕霉素、ox-LDL)自噬水平变化。
2.油红O染色方法观察野生型和pdcd4-/-小鼠腹腔原代巨噬细胞泡沫化发生
3.通过荧光染色的方法观察胆固醇、自噬小体、溶酶体在巨噬细胞中的共定位情况。用MDC染色方法观察pdcd4-/-小鼠和野生型小鼠腹腔原代巨噬细胞溶酶体活化情况。
4.用荧光定量PCR的方法,鉴定Pdcd4缺失对Lxr-a, Fas, Ppar-y, Leptin, C/EBPa-R, Abcal和Abcgl表达的影响。
5.建立apoE-/-和pdcd4-/-apoE-/-动脉粥样硬化模型,用western blot方法检测小鼠血管蛋白Lc3及Sqstm1的表达。
6.通过免疫荧光双染的方法观察apoE-/-和pdcd4-/-apoE-/-小鼠主动脉根斑块局部自噬及中性脂质沉积情况；分别观察自噬小体数量、Sqstm1表达情况以及与巨噬细胞的共定位关系。用骨髓细胞移植实验,重复上述实验
7.通过免疫组化染色方法检测人颈动脉内膜切除手术后的斑块组织标本PDCD4的表达情况,通过免疫荧光染色的方法观察自噬小体形成。
三. PDCD4抑制自噬的分子机制
1.在过表达、沉默表达体系中,通过western blot方法检测BECN1、ATG5、 ATG12、LC3蛋白表达。
2.用免疫组化方法分别检测肝癌、卵巢癌、神经胶质瘤、裸鼠成瘤组织中PDCD4、ATG5的表达,分析PDCD4和ATG5蛋白表达关系。
3.在人胚肾细胞系HEK293中,过表达PDCD4,应用实时定量PCR方法检测PDCD4、ATG5mRNA表达水平,用western blot方法检测PDCD4、BECN1、 ATG5、LC3的蛋白水平。应用RIP方法检测PDCD4蛋白与ATG5、ATG12mRNA结合情况。
4.构建PDCD4突变载体(mutPDCD4),应用RNA免疫共沉淀方法检测其与ATG5mRNA结合能力,应用western blot方法检测BECN1、ATG5、ATG12、LC3蛋白表达,应用细胞免疫荧光方法检测自噬小体的形成；应用CCK8方法检测细胞活性。
5.在HEK293细胞系中,共转染ATG5和PDCD4质粒,应用western blot方法检测ATG5和LC3蛋白表达。
结果
一. PDCD4对肿瘤细胞自噬及其相关功能的影响
(一)体外实验发现PDCD4具有抑制肿瘤细胞自噬的作用
1.过表达PDCD4能抑制自噬发生及自噬小体的形成结果发现PDCD4过表达能下调自噬标志蛋白LC3表达,上调SQSTM1的表达,且不依赖自噬流,说明PDCD4能够负调控自噬的形成。同时,我们应用细胞免疫荧光技术,观察自噬小体形成,结果表明PDCD4过表达能明显抑制自噬小体的形成。
2.沉默表达PDCD4能促进自噬发生及自噬小体的形成结果表明PDCD4沉默后,自噬标志蛋白LC3表达上调,SQSTM1表达下调,而这种变化不依赖自噬流。细胞免疫荧光表明干扰PDCD4后自噬小体形成明显增多。
(二)裸鼠移植瘤实验证明PDCD4具有抑制自噬的作用
为了获得体内实验PDCD4抑制自噬的证据,我们建立了裸鼠成瘤模型。Western blot结果表明注射PDCD4载体组中LC3II-B表达受到抑制而SQSTM1表达上调；免疫组织荧光检测显示PDCD4载体注射组自噬小体形成受到明显抑制。LC3和PDCD4共定位实验表明,在PDCD4高表达的区域LC3B表达降低,而PDCD4低表达则能引起LC3B表达增强。
(三)PDCD4对肿瘤细胞生长的抑制依赖于其对自噬的抑制
1.3-MA阻断实验证实PDCD4对细胞生长的影响依赖于自噬结果显示PDCD4过表达能明显抑制细胞生长,3-MA阻断自噬使细胞生长都受到明显抑制,且PDCD4过表达引起的生长差异亦被消除。而敲除PDCD4后,细胞活性明显上调,而一旦自噬被3-MA阻断后,上调的细胞活性又受到明显抑制。
2.ATG5小干扰实验证实PDCD4对细胞生长的影响依赖于自噬我们沉默ATG5基因,并进行相应的过表达和干扰PDCD4, western blot检测LC3表达,结果同样证实了PDCD4对细胞活性的影响确依赖于其对自噬的抑制作用。
3.凋亡抑制实验证实PDCD4对细胞生长的影响不依赖于凋亡结果显示抑制凋亡后细胞形态良好,PDCD4依然能抑制细胞活性；而抑制自噬后细胞则出现细胞破碎、核固缩等细胞死亡的现象,说明PDCD4抑制细胞活性不依赖于其对凋亡的促进作用。
二、Pdcd4对巨噬细胞自噬及其相关功能的影响
(一)Pdcd4能抑制不同刺激诱导的巨噬细胞自噬
1. Pdcd4能够抑制EBSS、LPS、雷帕霉素诱导的巨噬细胞、淋巴细胞自噬EBSS、LPS (1μug/ml)、雷帕霉素(100nM)刺激细胞不同时间点,western blot结果显示,pdcd4-/-小鼠巨噬细胞中的LC3-II蛋白表达水平明显高于野生型；免疫荧光的结果显示,pdcd4-/-小鼠巨噬细胞、脾脏淋巴细胞中的自噬小体形成明显多于野生型。
2. Pdcd4能够抑制ox-LDL诱导的巨噬细胞自噬ox-LDL50μg/ml刺激24小时后,pdcd4-/-小鼠巨噬细胞中的自噬小体形成明显多于野生型小鼠。而Sqstml荧光染色的结果显示,pdcd4-/-小鼠和野生型小鼠中巨噬细胞中Sqstm1荧光强度都明显升高,二者无明显差异。
(二)Pdcd4介导的自噬对巨噬细胞泡沫化的影响
1. Pdcd4缺失能减轻巨噬细胞泡沫化形成Pdcd4缺失能导致巨噬细胞脂质沉积和泡沫化程度明显降低。EBSS或雷帕霉素诱导自噬均能导致pdcd4-/-小鼠和野生型巨噬细胞泡沫化程度降低,而pdcd4-/-小鼠巨噬细胞更低。渥曼青霉素(WM)、3-MA阻断自噬,或Pepstatin+E64d阻断自噬流野生型和pdcd4-/-小鼠巨噬细胞泡沫化程度均明显升高,但两组间的差异消失。
2. Pdcd4缺失促进自噬依赖的胆固醇流出Pdcd4缺失及其介导自噬不影响巨噬细胞吞噬功能。荧光双染结果显示,Pdcd4缺失导致巨噬细胞内的胆固醇沉积明显减少,而自噬小体明显增多,二者负相关且共定位,溶酶体的活性明显高于野生型。另外,细胞内溶酶体与胆固醇,及自噬小体和溶酶体存在共定位关系。Pdcd4缺失能够导致Abcal和Abcgl及其上游调控基因Lxra表达上调。综上所述,PDCD4缺失促进自噬依赖的胆固醇流出。
(三)Pdcd4介导的巨噬细胞自噬在动脉粥样硬化中的作用
1. Pdcd4缺失促进动脉粥样硬化斑块局部巨噬细胞自噬Pdcd4-/-apoE-/-小鼠血管蛋白中Lc3及Sqstm1表达明显低于apoE-/-, pdcd4-/-apoE-/-小鼠主动脉根斑块局部的脂质沉积明显减少,而自噬小体却明显增多,多位于巨噬细胞中。
2.骨髓移植实验证明Pdcd4调节巨噬细胞自噬为了排除内皮细胞及平滑肌细胞影响,我们进行了骨髓细胞移植实验。结果显示,与移植野生型小鼠骨髓组相比,Pdcd4-/-小鼠骨髓移植组,自噬明显要升高,且多发生在巨噬细胞中。
3.人局部血管斑块中PDCD4表达和自噬成负相关对人颈动脉内膜切除术后的病理标本染色显示PDCD4表达与自噬水平存在一定的负相关关系。
一、PDCD4抑制自噬的分子机制
(一)PDCD4抑制ATG5蛋白表达
1.体外实验证实PDCD4抑制ATG5蛋白表达Western blot结果显示,在过表达、沉默表达体系中,在肿瘤细胞和巨噬细胞中,PDCD4对BECN1的表达无影响；而却能抑制ATG5的表达,进而抑制了ATG12-ATG5复合体形成。
2.临床标本及裸鼠成瘤组织中PDCD4抑制ATG5蛋白表达在肝癌、卵巢癌、神经胶质瘤及裸鼠成瘤组织中,PDCD4和ATG5表达存在负性相关,PDCD4高表达组ATG5表达明显受到抑制。
PDCD4抑制ATG5表达依赖其ma3功能域
1. PDCD4蛋白与ATG5mRNA结合PDCD4抑制ATG5蛋白表达,但对ATG5mRNA表达无影响,表明PDCD4对ATG5的调控发生在翻译水平而非转录水平。RIP实验结果表明PDCD4与ATG5mRNA结合。
2. PDCD4抑制ATG5介导的自噬依赖于它的ma3域PDCD4突变分析表明,PDCD4ma3突变的PDCD4失去与ATG5mRNA结合的能力,并且失去了抑制自噬、ATG5蛋白表达以及细胞活性的能力,表明PDCD4抑制ATG5介导的自噬依赖于它的ma3域。
结论
1. PDCD4在多种肿瘤细胞、免疫细胞中抑制自噬发生,且PDCD4抑制细胞生长依赖于其对自噬的抑制作用。
2. Pdcd4能通过抑制自噬减低胆固醇流出,促进巨噬细胞泡沫化形成,并在动脉粥样硬化进程中发挥重要作用。
3.从机制上,PDCD4通过其ma3抑制ATG5的翻译进而抑制了ATG12-ATG5复合体的形成,抑制了自噬的发生,进而抑制肿瘤细胞生存能力。
创新性和研究的意义
1.首先提出并证明了“抑癌基因PDCD4抑制自噬”的观点,而这种抑制作用普遍存在于多种肿瘤细胞系和免疫细胞,具有普遍性。同时我们研究发现PDCD4抑制细胞生长依赖于其对自噬的抑制,这就为解释PDCD4抑癌作用提供了新的理论基础。
2.首次发现Pdcd4抑制巨噬细胞自噬进而抑制了自噬介导的胆固醇流出,促进了泡沫化的形成。同时首次论证了Pdcd4对血源性巨噬细胞自噬抑制作用。这是首次发现抑癌基因Pdcd4在脂质代谢中的作用,为下一步研究其在代谢相关疾病(动脉粥样硬化和糖尿病)的功能鉴定了基础。
3.首次证明PDCD4抑制ATG5蛋白表达,由于ATG5是自噬发生发展过程中非常重要必不可少的一个基因,因此为PDCD4功能研究发开辟了新的领域。
Objective:
PDCD4(programmed cell death4), an important tumor suppressor, inhibits carcinogenesis, progression and invasion via suppressing gene transcription and translation. It has been reported that Pdcd4transgenic mice show significant resistance to tumor induction whereas pdcd4-/-mice develop spontaneously lymphomas. In addition, loss or reduction of PDCD4expression has been observed in multiple types of human tumors, such as glioma, lung, ovarian, liver tumor, implicating development and progression of these human tumors. Further, PDCD4suppresses the malignant phenotype and enhances the chemosensitivity of tumors. Overall, these are indications that PDCD4is a tumor suppressor. However, the mechanisms by which PDCD4inhibits tumors remain unclear and conflict in different tumors. Some researchers have demonstrated that PDCD4promotes tumor cell apoptosis in glioma, hepatocellular, ovarian, breast and gastric tumor. However, others have reported that PDCD4plays an antiapoptotic role in HeLa cells or has no impact on apoptosis in colon carcinoma. Furthermore, PDCD4plays inhibitory functions or no effect on cell cycles. These suggest that roles of PDCD4in apoptosis and cell cycle may be limited to certain cell types, which cannot explain the suppression of PDCD4on multiple tumors.
(Macro)autophagy, a conserved catabolic process whereby cellular proteins and organelles are engulfed by autophagosomes, digested in lysosomes, and recycled to sustain cellular homeostasis, has dual roles in tumor. Some data support the idea that autophagy is classified as an antioncogenic mechanism; However, accumulating evidences strongly support that autophagy enhances tumorigenesis and protects tumor cells from death. In addition, autophagy also plays an important role in regulation of macrophage functions such as lipid metabolism, which is involved in the initiation and development of atherosclerosis. The process of autophagosome formation is regulated by several autophagy-related genes(ATG), such as ATG5, ATG6(known as BECN1), ATG8(microtubule-associated protein1light chain3, LC3) and ATG12. ATG5, a protein involved at the early stage of autophagosome formation, contributes to elongation and closure of the autophagosomes. BECN1, acting upstream of autophagosome formation, governs the autophagy process by the subsequent recruitment of additional ATG proteins for initiating autophagosome formation.
In our current study, we will investigate these questions from the following several aspects:(1) Effect of PDCD4on tumor autophagy and its related functions. PDCD4suppresses autophagy in multiple cell types and PDCD4-attenuated autophagy is required for its inhibition of tumor cell growth.(2) Effect of Pdcd4on macrophage autophagy and its related functions. Pdcd4suppresses macrophage autophagy and then cholesterol efflux, resulting in formation of foam cell, contributing to the initiation and development of atherosclerosis.(3) Molecular mechanisms by which PDCD4suppresses autophagy. PDCD4suppresses autophagy dependent on inhibition of PDCD4on ATG5protein via PDCD4-EIF4A-ATG5axis but neither BECN1nor ATG12.
Methods
1. Effect of PDCD4on tumor autophagy and its related functions
(1) After plasmid transfection or siRNA interference, autophagy related markers LC3B-Ⅱ, SQSTMl/p62were detected by western blot. IF was carried out to demonstrate the formation of autophagosome.
(2) Pepstatin A plus E64d were applied to block autophagic flux.
(3) Xenograft animal model was established, treated with N.S, Mock and PDCD4plasmid. IHC and IF were applied to indicate PDCD4expression and autophagosome formation; western blot analysis to show LC3B-Ⅱ and SQSTM1expression.
(4) After plasmid or siRNA transfection, cells were treated with3-MA or siATG5to inhibit autophagy or z-VAD to inhibit apoptosis, cell viability was detected by CCK8.
2. Effect of Pdcd4on macrophage autophagy and its related functions
(1) Western blot and IF were used to show autophagy level in wide type and pdcd4-/-mice primary peritoneal macrophages, BMDM, RAW264.7cells and lymphocytes after treated by EBSS, LPS, rapamycin and ox-LDL.
(2) Oil Red "O" staining was used to show foam cell formation.
(3) IF was used to demonstrate co-location of cholesterol, lysosome and autophagosome. MDC staining to show activated lysosome in macrophage
(4) Real time PCR was used to determine the expression of Lxr-a, Fas, Ppar-y, Leptin, C/EBPa-R,Abcal and Abcgl.
(5) Western blot was used to show the expression of Lc3b and Sqstml in vessel protein of apoE-/-and pdcd4-/-apoE-/-AS model mice.
(6) IF was used to show co-location of cholesterol, autophagosome and macrophage, Sqstml expression in apoE-/-and pdcd4-/-apoE-/-AS model mice aortic root local plaque. After bone marrow transplantation assay, above experiments were repeated.
(7) IHC was applied to demonstrate PDCD4expression and IF to show autophagosome in human plaque.
3. Molecular mechanisms by which PDCD4suppresses autophagy
(1) After plasmid transfection or siRNA interference, cells were subjected to EBSS, and PDCD4, BECN1, ATG5, ATG12and LC3B were analyzed by western blot.
(2) Samples from human liver, ovarian cancers, glioma and xenograft tumors were stained with an anti-PDCD4and ATG5antibody for IHC analysis.
(3) After plasmid transfection, HEK293cells were subjected to western blot to show PDCD4, ATG5expression; real time PCR to show mRNA levels of PDCD4, ATG5; RNA-IP to show binding between PDCD4protein and ATG5or ATG12mRNA.
(4) After transfection with a mutant plasmid, cells was subjected to western blot to demonstrate BECN1, ATG5, ATG12, LC3; IF to show the formation of autophagosome; CCK8to demonstrate cell viability.
(5) HEK293cells were cotransfected with PDCD4and ATG5plasmid. PDCD4, ATG5, and LC3B-I/LC3B-II were analyzed by western blot analysis.
Results
Ⅰ. Effect of PDCD4on tumor autophagy and its related functions
1. PDCD4suppresses autophagy in multiple cell types in vitro
(1) Forced PDCD4suppresses autophagy Overexpressed PDCD4in Skov3and U87cells led to decreases in the amount of LC3B-Ⅱ and increases in the amount of SQSTM1in absence or presence of Pep A and E64d detected by western blot, and a decrease in the number of autophagosomes detected by IF.
(2) Silencing of PDCD4expression promotes autophagy Silencing of PDCD4 expression in HepG2and HeLa cells led to increases in the amount of LC3B-Ⅱ and decreases in the amount of SQSTM1in absence or presence of Pep A and E64d detected by western blot, and activated autophagosomes formation detected by IF.
2. PDCD4suppresses autophagy in murine xenograft tumors
Murine xenograft tumors were established and treated with Mock and PDCD4plasmid. Western blot revealed LC3B-Ⅱ expression was decreased and SQSTM1was increased in the PDCD4group, as confirmed by IF:a decrease in the number of autophagosomes in the PDCD4group.
3. PDCD4-attenuated autophagy required for its inhibition of proliferation
(1)3-MA blockage assay indicates PDCD4-attenuated autophagy is required for its inhibition to tumor growth Skov3-Mock cells grew significantly faster than Skov3-PDCD4cells, retarded by3-MA mediated blockage of autophagy. Knockdown of PDCD4expression caused an increase of cell viability in HepG2, which was inhibited by3-MA-mediated blockage of autophagy.
(2) Si-ATG5assay demonstrates that PDCD4-attenuated autophagy is required for tumor inhibition Si-ATG5caused depletion of autophagy both in Skov3and HepG2cells, which was enhanced by overexpression of PDCD4in Skov3cells and retarded by silencing of PDCD4in HepG2cells, and the cell viability after treatment with siATG5was similar to that of cells treated with3-MA.
(3) Blockage assay of apoptosis by Z-VAD confirms that PDCD4inhibits cell viability independent on apoptosis Blockage of apoptosis by z-VAD caused cells with a good status, cell proliferation was still suppressed by PDCD4, whereas blockage of autophagy by3-MA led to cell fragmentation, nuclear condensation, suggesting PDCD4inhibits cell viability independent on apoptosis.
Ⅱ. Effect of Pdcd4on macrophage autophagy and its related functions
1. Pdcd4inhibits macrophage autophagy induced by various stimuli
(1) Pdcd4deficiency upregulates immunocytes autophagy induced by various stimuli Western blot and IF revealed Pdcd4deficiency caused upregulated autophagy level in primary peritoneal macrophages, BMDM, mouse RAW264.7and even in spleen lymphocytes after treated by EBSS, LPS, rapamycin.
(2) Pdcd4deficiency upregulates autophagy induced by ox-LDL IF showed that Pdcd4deficiency led to an increase in the number of autophagosomes, but no obvious difference in fluorescence intensity of Sqstm1induced by ox-LDL.
2. Effects of Pdcd4-attenuated autophagy on macrophage-derived foam cell formation
(1) Pdcd4deficiency attenuates macrophage-derived foam cell formation dependent on autophagy Oil Red "0" staining was used in wide type and pdcd4-/-mouse primary macrophage, demonstrating that Pdcd4deficiency caused a decrease in lipid accumulation and attenuation of foam cell formation, enhanced by EBSS and rapamycin, and restored by WM,3-MA and Pep A+E64d.
(2) Pdcd4controls autophagy dependent cholesterol efflux Uptake tests demonstrated no differences between wide type and pdcd4-/-primary macrophage. A decrease in cholesterol accumulation and an increase in number of autophagosomes, and activated lysosome were observed in pdcd4-/-mouse primary macrophage. Lysosome was colocalized with autophagosome and cholesterol. Real time PCR showed Pdcd4deficiency led to an increase in amount of Abcal, Abcgl and Lxra.
3. Effects of Pdcd4-attenuated macrophage autophagy on atherosclerosis
(1) Pdcd4deficiency promotes macrophage autophagy and lipid accumulation in atherosclerotic plaque in apoE-/-mice Western blot and IF showed Pdcd4deficiency led to an increase in amount of Lc3and Sqstm1, and a decrease in cholesterol accumulation and an increase in number of autophagosomes, localizing in macrophage in atherosclerotic plaque.
(2) Pdcd4-attenuated macrophage autophagy may play an important role in AS via bone marrow transplantation Pdcd4deficiency led to a decrease in cholesterol accumulation and an increase in number of autophagosome, colocalizing with macrophage in local plaque after bone marrow transplantation.
(3) Relationship between PDCD4expression and autophagy in human atherosclerotic plaque PDCD4expression were detected by IHC and autophagosomes were analyzed by IF. The correlationship analysis demonstrated a negative relation between PDCD4expression and autophagy level in human plaque.
Ⅲ.Molecular mechanisms by which PDCD4suppresses autophagy
1. PDCD4inhibits autophagy-related gene ATG5protein expression
(1) PDCD4inhibits ATG5protein expression in vitro Western blot demonstrated in both tumor cells and macrophages BECN1was not affected by PDCD4under starvation, LPS or rapamycin. Higher PDCD4expression remarkably inhibited ATG5expression and formation of the ATG12-ATG5complex.
(2) PDCD4inhibits ATG5protein expression in vivo ATG5expression in human liver, ovarian cancers, gliomas and murine xenograft with negative or low PDCD4expression was significantly higher than that with higher PDCD4expression.
2. PDCD4inhibits ATG5expression and autophagy via its ma3domains
(1) PDCD4protein binds ATG5mRNA ATG5protein was suppressed significantly, while ATG5mRNA was unaffected by PDCD4expression, suggesting the inhibition of PDCD4on ATG5occurs translationally. A RIP assay was applied to demonstrate an association between ATG5mRNA and PDCD4protein.
(2) PDCD4inhibits ATG5-mediated autophagy via its ma3domains PDCD4mutation analysis demonstrated only wild-type PDCD4but not mutant PDCD4led to ATG5downregulation, and a decrease in the number of autophagosomes, suppressed cell viability and an association with ATG5mRNA, suggesting PDCD4inhibited ATG5by binding of PDCD4-ma3with EIF4A-ATG5mRNA.
Conclusions
1. PDCD4inhibits activation of autophagy and formation of autophagosome in tumor cells and immunocytes. PDCD4-attenuated autophagy is required for inhibition of tumor cell growth.
2. Pdcd4promotes macrophage-derived foam cell formation via suppressing autophagy and cholesterol efflux, at last promotes the initiation and development of atherosclerosis.
3. Mechanically, PDCD4inhibits ATG5protein expression and further inhibits ATG12-ATG5complex formation in vitro and in vivo. The inhibition of PDCD4on ATG5occurs translationally, and RIP assay demonstrates ATG5but not ATG12mRNA, binds with PDCD4protein. Once PDCD4fails to binding with ATG5, it loses the ability to suppress ATG5expression, autophagy and cell viability.
Originality and Significance
1. For the first time, we demonstrate PDCD4inhibits autophagy and formation of autophagosome in multiple cells including tumor and immune cells, an universal phenomenon. We report here that inhibition of PDCD4on autophagy contributes to its tumor suppressor activity and effect of Pdcd4on lipid metabolism and atherosclerosis.
2. Importantly, PDCD4inhibits the expression of an essential autophagy related gene, ATG5and the formation of ATG12-ATG5complex, and its ma3domains are required for PDCD4-mediated inhibition of autophagy. Our findings indicate that PDCD4negatively regulates autophagy by targeting ATG5, which provides a novel mechanism of tumor suppression.
程序性凋亡因子4(programmed cell death protein4, PDCD4)是近年发现一种重要的抑癌基因,通过抑制蛋白转录和翻译抑制肿瘤的发生、发展从而发挥抑癌基因的功能。PDCD4转基因小鼠抵抗表皮乳头状瘤向皮肤癌的恶性转化；而PDCD4基因敲除小鼠较野生型小鼠更容易罹患淋巴瘤。另外PDCD4在人多数肿瘤组织(如：肺癌、肝癌、神经胶质瘤、卵巢癌、结直肠癌等)中低表达甚至缺失表达,预示着肿瘤恶性程度高和不良预后。体外实验发现PDCD4抑制肿瘤细胞的恶性表型,并增强肿瘤的化疗敏感性。总之,越来越多的证据表明PDCD4是一种重要的抑癌基因。然而,PDCD4抑制肿瘤的机制目前尚不清楚,在不同细胞系中的作用也不尽相同。我们和其他学者的前期研究证明在肝细胞肝癌、卵巢癌、神经胶质瘤、乳腺癌以及胃癌中PDCD4具有促进肿瘤细胞凋亡的作用；但有学者提出不同的观点,如,PDCD4抑制宫颈癌HeLa细胞的凋亡,而对结肠癌的凋亡无影响。同样,PDCD4对细胞周期的影响在不同肿瘤来源细胞系中也不尽相同,具有明显的细胞特异性。因此,目前有关PDCD4抑癌机制均不足以解释PDCD4在不同肿瘤中普遍存在的抑制作用。
(巨)自噬是细胞的一种生理和病理学过程,为细胞自身代谢提供能量并对某些细胞器进行更新,从而维持细胞的自身稳态。自噬在肿瘤细胞中的作用具有双重性,一方面,自噬介导的细胞凋亡或死亡抑制肿瘤的发展,而另一方面,自噬作为一种代偿反应,能为细胞提供生存所必须的能量,使肿瘤细胞免于死亡,从而促进肿瘤生长。另外,自噬在免疫细胞(如巨噬细胞)的功能调节中也发挥重要作用,如巨噬细胞通过自噬调节其在脂质代谢等方面的作用。目前,已知多种基因参入自噬过程中,统称为自噬相关基因(Autophagy Related Genes, ATG),如ATG5、ATG6(又名BECN1/Beclinl)、ATG8(又名LC3, microtubule-associated protein1light chain3)和ATG12。其中,ATG5参与了自噬小体形成的早期过程,它与ATG12结合形成ATG5-ATG12复合体,对自噬小体的延长、闭环起到至关重要的作用,并且能够调控LC3家族蛋白脂化并锚定到自噬小体双分子膜上而BECN1作用在ATG5的上游通路,它能调控上游的P13K通路并且募集其他ATG相关蛋白进而达到调节自噬的目的。
为了明确PDCD4在自噬中的作用,探索其作用机制,以及对肿瘤和巨噬细胞功能的影响。本论文从以下几个方面进行了研究：一、PDCD4对肿瘤细胞自噬及其相关功能的影响发现PDCD4具有抑制肿瘤细胞自噬的作用,且其作用具有普遍性；发现PDCD4对肿瘤细胞生长的抑制依赖于其对自噬的抑制；二、Pdcd4对巨噬细胞自噬及其相关功能的影响证实Pdcd4能够抑制多种刺激诱导的巨噬细胞自噬；发现Pdcd4通过抑制巨噬细胞自噬促进巨噬细胞泡沫化形成,并且在动脉粥样硬化发生发展过程中也发挥至关重要作用；三、PDCD4抑制自噬的分子机制发现PDCD4对自噬的调节不依赖于BECN1通路,而是通过PDCD4-EIF4A-ATG5轴抑制ATG5的蛋白翻译实现的。
研究方法
一、PDCD4对肿瘤细胞自噬及其相关功能的影响
1.在PDCD4过表达和干扰体系中,应用western blot的方法检测自噬相关标志蛋白LC3B-II及SQSTM1的表达,用免疫荧光的方法检测自噬小体形成。
2.应用Pepstatin A和E64d联合用药进行自噬流分析,排除自噬流损伤的影响。
3.建立裸鼠成瘤模型,在成瘤后分别瘤内注射生理盐水、空载体及PDCD4载体,进行western blot检测LC3B-II及SQSTM1的表达,应用组织免疫荧光方法检测自噬小体形成。
4.用3-MA、ATG5小干扰阻断自噬或用z-VAD阻断凋亡,应用CCK8方法检测PDCD4对细胞活性的影响。
二、Pdcd4对巨噬细胞自噬及其相关功能的影响
1.在pdcd4-/-小鼠和野生型小鼠腹腔原代巨噬细胞和骨髓来源巨噬细胞(BMDM)及小鼠白血病来源巨噬细胞系RAW264.7中检测利用western blot和免疫荧光方法检测多种刺激下(EBSS、LPS、雷帕霉素、ox-LDL)自噬水平变化。
2.油红O染色方法观察野生型和pdcd4-/-小鼠腹腔原代巨噬细胞泡沫化发生
3.通过荧光染色的方法观察胆固醇、自噬小体、溶酶体在巨噬细胞中的共定位情况。用MDC染色方法观察pdcd4-/-小鼠和野生型小鼠腹腔原代巨噬细胞溶酶体活化情况。
4.用荧光定量PCR的方法,鉴定Pdcd4缺失对Lxr-a, Fas, Ppar-y, Leptin, C/EBPa-R, Abcal和Abcgl表达的影响。
5.建立apoE-/-和pdcd4-/-apoE-/-动脉粥样硬化模型,用western blot方法检测小鼠血管蛋白Lc3及Sqstm1的表达。
6.通过免疫荧光双染的方法观察apoE-/-和pdcd4-/-apoE-/-小鼠主动脉根斑块局部自噬及中性脂质沉积情况；分别观察自噬小体数量、Sqstm1表达情况以及与巨噬细胞的共定位关系。用骨髓细胞移植实验,重复上述实验
7.通过免疫组化染色方法检测人颈动脉内膜切除手术后的斑块组织标本PDCD4的表达情况,通过免疫荧光染色的方法观察自噬小体形成。
三. PDCD4抑制自噬的分子机制
1.在过表达、沉默表达体系中,通过western blot方法检测BECN1、ATG5、 ATG12、LC3蛋白表达。
2.用免疫组化方法分别检测肝癌、卵巢癌、神经胶质瘤、裸鼠成瘤组织中PDCD4、ATG5的表达,分析PDCD4和ATG5蛋白表达关系。
3.在人胚肾细胞系HEK293中,过表达PDCD4,应用实时定量PCR方法检测PDCD4、ATG5mRNA表达水平,用western blot方法检测PDCD4、BECN1、 ATG5、LC3的蛋白水平。应用RIP方法检测PDCD4蛋白与ATG5、ATG12mRNA结合情况。
4.构建PDCD4突变载体(mutPDCD4),应用RNA免疫共沉淀方法检测其与ATG5mRNA结合能力,应用western blot方法检测BECN1、ATG5、ATG12、LC3蛋白表达,应用细胞免疫荧光方法检测自噬小体的形成；应用CCK8方法检测细胞活性。
5.在HEK293细胞系中,共转染ATG5和PDCD4质粒,应用western blot方法检测ATG5和LC3蛋白表达。
结果
一. PDCD4对肿瘤细胞自噬及其相关功能的影响
(一)体外实验发现PDCD4具有抑制肿瘤细胞自噬的作用
1.过表达PDCD4能抑制自噬发生及自噬小体的形成结果发现PDCD4过表达能下调自噬标志蛋白LC3表达,上调SQSTM1的表达,且不依赖自噬流,说明PDCD4能够负调控自噬的形成。同时,我们应用细胞免疫荧光技术,观察自噬小体形成,结果表明PDCD4过表达能明显抑制自噬小体的形成。
2.沉默表达PDCD4能促进自噬发生及自噬小体的形成结果表明PDCD4沉默后,自噬标志蛋白LC3表达上调,SQSTM1表达下调,而这种变化不依赖自噬流。细胞免疫荧光表明干扰PDCD4后自噬小体形成明显增多。
(二)裸鼠移植瘤实验证明PDCD4具有抑制自噬的作用
为了获得体内实验PDCD4抑制自噬的证据,我们建立了裸鼠成瘤模型。Western blot结果表明注射PDCD4载体组中LC3II-B表达受到抑制而SQSTM1表达上调；免疫组织荧光检测显示PDCD4载体注射组自噬小体形成受到明显抑制。LC3和PDCD4共定位实验表明,在PDCD4高表达的区域LC3B表达降低,而PDCD4低表达则能引起LC3B表达增强。
(三)PDCD4对肿瘤细胞生长的抑制依赖于其对自噬的抑制
1.3-MA阻断实验证实PDCD4对细胞生长的影响依赖于自噬结果显示PDCD4过表达能明显抑制细胞生长,3-MA阻断自噬使细胞生长都受到明显抑制,且PDCD4过表达引起的生长差异亦被消除。而敲除PDCD4后,细胞活性明显上调,而一旦自噬被3-MA阻断后,上调的细胞活性又受到明显抑制。
2.ATG5小干扰实验证实PDCD4对细胞生长的影响依赖于自噬我们沉默ATG5基因,并进行相应的过表达和干扰PDCD4, western blot检测LC3表达,结果同样证实了PDCD4对细胞活性的影响确依赖于其对自噬的抑制作用。
3.凋亡抑制实验证实PDCD4对细胞生长的影响不依赖于凋亡结果显示抑制凋亡后细胞形态良好,PDCD4依然能抑制细胞活性；而抑制自噬后细胞则出现细胞破碎、核固缩等细胞死亡的现象,说明PDCD4抑制细胞活性不依赖于其对凋亡的促进作用。
二、Pdcd4对巨噬细胞自噬及其相关功能的影响
(一)Pdcd4能抑制不同刺激诱导的巨噬细胞自噬
1. Pdcd4能够抑制EBSS、LPS、雷帕霉素诱导的巨噬细胞、淋巴细胞自噬EBSS、LPS (1μug/ml)、雷帕霉素(100nM)刺激细胞不同时间点,western blot结果显示,pdcd4-/-小鼠巨噬细胞中的LC3-II蛋白表达水平明显高于野生型；免疫荧光的结果显示,pdcd4-/-小鼠巨噬细胞、脾脏淋巴细胞中的自噬小体形成明显多于野生型。
2. Pdcd4能够抑制ox-LDL诱导的巨噬细胞自噬ox-LDL50μg/ml刺激24小时后,pdcd4-/-小鼠巨噬细胞中的自噬小体形成明显多于野生型小鼠。而Sqstml荧光染色的结果显示,pdcd4-/-小鼠和野生型小鼠中巨噬细胞中Sqstm1荧光强度都明显升高,二者无明显差异。
(二)Pdcd4介导的自噬对巨噬细胞泡沫化的影响
1. Pdcd4缺失能减轻巨噬细胞泡沫化形成Pdcd4缺失能导致巨噬细胞脂质沉积和泡沫化程度明显降低。EBSS或雷帕霉素诱导自噬均能导致pdcd4-/-小鼠和野生型巨噬细胞泡沫化程度降低,而pdcd4-/-小鼠巨噬细胞更低。渥曼青霉素(WM)、3-MA阻断自噬,或Pepstatin+E64d阻断自噬流野生型和pdcd4-/-小鼠巨噬细胞泡沫化程度均明显升高,但两组间的差异消失。
2. Pdcd4缺失促进自噬依赖的胆固醇流出Pdcd4缺失及其介导自噬不影响巨噬细胞吞噬功能。荧光双染结果显示,Pdcd4缺失导致巨噬细胞内的胆固醇沉积明显减少,而自噬小体明显增多,二者负相关且共定位,溶酶体的活性明显高于野生型。另外,细胞内溶酶体与胆固醇,及自噬小体和溶酶体存在共定位关系。Pdcd4缺失能够导致Abcal和Abcgl及其上游调控基因Lxra表达上调。综上所述,PDCD4缺失促进自噬依赖的胆固醇流出。
(三)Pdcd4介导的巨噬细胞自噬在动脉粥样硬化中的作用
1. Pdcd4缺失促进动脉粥样硬化斑块局部巨噬细胞自噬Pdcd4-/-apoE-/-小鼠血管蛋白中Lc3及Sqstm1表达明显低于apoE-/-, pdcd4-/-apoE-/-小鼠主动脉根斑块局部的脂质沉积明显减少,而自噬小体却明显增多,多位于巨噬细胞中。
2.骨髓移植实验证明Pdcd4调节巨噬细胞自噬为了排除内皮细胞及平滑肌细胞影响,我们进行了骨髓细胞移植实验。结果显示,与移植野生型小鼠骨髓组相比,Pdcd4-/-小鼠骨髓移植组,自噬明显要升高,且多发生在巨噬细胞中。
3.人局部血管斑块中PDCD4表达和自噬成负相关对人颈动脉内膜切除术后的病理标本染色显示PDCD4表达与自噬水平存在一定的负相关关系。
一、PDCD4抑制自噬的分子机制
(一)PDCD4抑制ATG5蛋白表达
1.体外实验证实PDCD4抑制ATG5蛋白表达Western blot结果显示,在过表达、沉默表达体系中,在肿瘤细胞和巨噬细胞中,PDCD4对BECN1的表达无影响；而却能抑制ATG5的表达,进而抑制了ATG12-ATG5复合体形成。
2.临床标本及裸鼠成瘤组织中PDCD4抑制ATG5蛋白表达在肝癌、卵巢癌、神经胶质瘤及裸鼠成瘤组织中,PDCD4和ATG5表达存在负性相关,PDCD4高表达组ATG5表达明显受到抑制。
PDCD4抑制ATG5表达依赖其ma3功能域
1. PDCD4蛋白与ATG5mRNA结合PDCD4抑制ATG5蛋白表达,但对ATG5mRNA表达无影响,表明PDCD4对ATG5的调控发生在翻译水平而非转录水平。RIP实验结果表明PDCD4与ATG5mRNA结合。
2. PDCD4抑制ATG5介导的自噬依赖于它的ma3域PDCD4突变分析表明,PDCD4ma3突变的PDCD4失去与ATG5mRNA结合的能力,并且失去了抑制自噬、ATG5蛋白表达以及细胞活性的能力,表明PDCD4抑制ATG5介导的自噬依赖于它的ma3域。
结论
1. PDCD4在多种肿瘤细胞、免疫细胞中抑制自噬发生,且PDCD4抑制细胞生长依赖于其对自噬的抑制作用。
2. Pdcd4能通过抑制自噬减低胆固醇流出,促进巨噬细胞泡沫化形成,并在动脉粥样硬化进程中发挥重要作用。
3.从机制上,PDCD4通过其ma3抑制ATG5的翻译进而抑制了ATG12-ATG5复合体的形成,抑制了自噬的发生,进而抑制肿瘤细胞生存能力。
创新性和研究的意义
1.首先提出并证明了“抑癌基因PDCD4抑制自噬”的观点,而这种抑制作用普遍存在于多种肿瘤细胞系和免疫细胞,具有普遍性。同时我们研究发现PDCD4抑制细胞生长依赖于其对自噬的抑制,这就为解释PDCD4抑癌作用提供了新的理论基础。
2.首次发现Pdcd4抑制巨噬细胞自噬进而抑制了自噬介导的胆固醇流出,促进了泡沫化的形成。同时首次论证了Pdcd4对血源性巨噬细胞自噬抑制作用。这是首次发现抑癌基因Pdcd4在脂质代谢中的作用,为下一步研究其在代谢相关疾病(动脉粥样硬化和糖尿病)的功能鉴定了基础。
3.首次证明PDCD4抑制ATG5蛋白表达,由于ATG5是自噬发生发展过程中非常重要必不可少的一个基因,因此为PDCD4功能研究发开辟了新的领域。
Objective:
PDCD4(programmed cell death4), an important tumor suppressor, inhibits carcinogenesis, progression and invasion via suppressing gene transcription and translation. It has been reported that Pdcd4transgenic mice show significant resistance to tumor induction whereas pdcd4-/-mice develop spontaneously lymphomas. In addition, loss or reduction of PDCD4expression has been observed in multiple types of human tumors, such as glioma, lung, ovarian, liver tumor, implicating development and progression of these human tumors. Further, PDCD4suppresses the malignant phenotype and enhances the chemosensitivity of tumors. Overall, these are indications that PDCD4is a tumor suppressor. However, the mechanisms by which PDCD4inhibits tumors remain unclear and conflict in different tumors. Some researchers have demonstrated that PDCD4promotes tumor cell apoptosis in glioma, hepatocellular, ovarian, breast and gastric tumor. However, others have reported that PDCD4plays an antiapoptotic role in HeLa cells or has no impact on apoptosis in colon carcinoma. Furthermore, PDCD4plays inhibitory functions or no effect on cell cycles. These suggest that roles of PDCD4in apoptosis and cell cycle may be limited to certain cell types, which cannot explain the suppression of PDCD4on multiple tumors.
(Macro)autophagy, a conserved catabolic process whereby cellular proteins and organelles are engulfed by autophagosomes, digested in lysosomes, and recycled to sustain cellular homeostasis, has dual roles in tumor. Some data support the idea that autophagy is classified as an antioncogenic mechanism; However, accumulating evidences strongly support that autophagy enhances tumorigenesis and protects tumor cells from death. In addition, autophagy also plays an important role in regulation of macrophage functions such as lipid metabolism, which is involved in the initiation and development of atherosclerosis. The process of autophagosome formation is regulated by several autophagy-related genes(ATG), such as ATG5, ATG6(known as BECN1), ATG8(microtubule-associated protein1light chain3, LC3) and ATG12. ATG5, a protein involved at the early stage of autophagosome formation, contributes to elongation and closure of the autophagosomes. BECN1, acting upstream of autophagosome formation, governs the autophagy process by the subsequent recruitment of additional ATG proteins for initiating autophagosome formation.
In our current study, we will investigate these questions from the following several aspects:(1) Effect of PDCD4on tumor autophagy and its related functions. PDCD4suppresses autophagy in multiple cell types and PDCD4-attenuated autophagy is required for its inhibition of tumor cell growth.(2) Effect of Pdcd4on macrophage autophagy and its related functions. Pdcd4suppresses macrophage autophagy and then cholesterol efflux, resulting in formation of foam cell, contributing to the initiation and development of atherosclerosis.(3) Molecular mechanisms by which PDCD4suppresses autophagy. PDCD4suppresses autophagy dependent on inhibition of PDCD4on ATG5protein via PDCD4-EIF4A-ATG5axis but neither BECN1nor ATG12.
Methods
1. Effect of PDCD4on tumor autophagy and its related functions
(1) After plasmid transfection or siRNA interference, autophagy related markers LC3B-Ⅱ, SQSTMl/p62were detected by western blot. IF was carried out to demonstrate the formation of autophagosome.
(2) Pepstatin A plus E64d were applied to block autophagic flux.
(3) Xenograft animal model was established, treated with N.S, Mock and PDCD4plasmid. IHC and IF were applied to indicate PDCD4expression and autophagosome formation; western blot analysis to show LC3B-Ⅱ and SQSTM1expression.
(4) After plasmid or siRNA transfection, cells were treated with3-MA or siATG5to inhibit autophagy or z-VAD to inhibit apoptosis, cell viability was detected by CCK8.
2. Effect of Pdcd4on macrophage autophagy and its related functions
(1) Western blot and IF were used to show autophagy level in wide type and pdcd4-/-mice primary peritoneal macrophages, BMDM, RAW264.7cells and lymphocytes after treated by EBSS, LPS, rapamycin and ox-LDL.
(2) Oil Red "O" staining was used to show foam cell formation.
(3) IF was used to demonstrate co-location of cholesterol, lysosome and autophagosome. MDC staining to show activated lysosome in macrophage
(4) Real time PCR was used to determine the expression of Lxr-a, Fas, Ppar-y, Leptin, C/EBPa-R,Abcal and Abcgl.
(5) Western blot was used to show the expression of Lc3b and Sqstml in vessel protein of apoE-/-and pdcd4-/-apoE-/-AS model mice.
(6) IF was used to show co-location of cholesterol, autophagosome and macrophage, Sqstml expression in apoE-/-and pdcd4-/-apoE-/-AS model mice aortic root local plaque. After bone marrow transplantation assay, above experiments were repeated.
(7) IHC was applied to demonstrate PDCD4expression and IF to show autophagosome in human plaque.
3. Molecular mechanisms by which PDCD4suppresses autophagy
(1) After plasmid transfection or siRNA interference, cells were subjected to EBSS, and PDCD4, BECN1, ATG5, ATG12and LC3B were analyzed by western blot.
(2) Samples from human liver, ovarian cancers, glioma and xenograft tumors were stained with an anti-PDCD4and ATG5antibody for IHC analysis.
(3) After plasmid transfection, HEK293cells were subjected to western blot to show PDCD4, ATG5expression; real time PCR to show mRNA levels of PDCD4, ATG5; RNA-IP to show binding between PDCD4protein and ATG5or ATG12mRNA.
(4) After transfection with a mutant plasmid, cells was subjected to western blot to demonstrate BECN1, ATG5, ATG12, LC3; IF to show the formation of autophagosome; CCK8to demonstrate cell viability.
(5) HEK293cells were cotransfected with PDCD4and ATG5plasmid. PDCD4, ATG5, and LC3B-I/LC3B-II were analyzed by western blot analysis.
Results
Ⅰ. Effect of PDCD4on tumor autophagy and its related functions
1. PDCD4suppresses autophagy in multiple cell types in vitro
(1) Forced PDCD4suppresses autophagy Overexpressed PDCD4in Skov3and U87cells led to decreases in the amount of LC3B-Ⅱ and increases in the amount of SQSTM1in absence or presence of Pep A and E64d detected by western blot, and a decrease in the number of autophagosomes detected by IF.
(2) Silencing of PDCD4expression promotes autophagy Silencing of PDCD4 expression in HepG2and HeLa cells led to increases in the amount of LC3B-Ⅱ and decreases in the amount of SQSTM1in absence or presence of Pep A and E64d detected by western blot, and activated autophagosomes formation detected by IF.
2. PDCD4suppresses autophagy in murine xenograft tumors
Murine xenograft tumors were established and treated with Mock and PDCD4plasmid. Western blot revealed LC3B-Ⅱ expression was decreased and SQSTM1was increased in the PDCD4group, as confirmed by IF:a decrease in the number of autophagosomes in the PDCD4group.
3. PDCD4-attenuated autophagy required for its inhibition of proliferation
(1)3-MA blockage assay indicates PDCD4-attenuated autophagy is required for its inhibition to tumor growth Skov3-Mock cells grew significantly faster than Skov3-PDCD4cells, retarded by3-MA mediated blockage of autophagy. Knockdown of PDCD4expression caused an increase of cell viability in HepG2, which was inhibited by3-MA-mediated blockage of autophagy.
(2) Si-ATG5assay demonstrates that PDCD4-attenuated autophagy is required for tumor inhibition Si-ATG5caused depletion of autophagy both in Skov3and HepG2cells, which was enhanced by overexpression of PDCD4in Skov3cells and retarded by silencing of PDCD4in HepG2cells, and the cell viability after treatment with siATG5was similar to that of cells treated with3-MA.
(3) Blockage assay of apoptosis by Z-VAD confirms that PDCD4inhibits cell viability independent on apoptosis Blockage of apoptosis by z-VAD caused cells with a good status, cell proliferation was still suppressed by PDCD4, whereas blockage of autophagy by3-MA led to cell fragmentation, nuclear condensation, suggesting PDCD4inhibits cell viability independent on apoptosis.
Ⅱ. Effect of Pdcd4on macrophage autophagy and its related functions
1. Pdcd4inhibits macrophage autophagy induced by various stimuli
(1) Pdcd4deficiency upregulates immunocytes autophagy induced by various stimuli Western blot and IF revealed Pdcd4deficiency caused upregulated autophagy level in primary peritoneal macrophages, BMDM, mouse RAW264.7and even in spleen lymphocytes after treated by EBSS, LPS, rapamycin.
(2) Pdcd4deficiency upregulates autophagy induced by ox-LDL IF showed that Pdcd4deficiency led to an increase in the number of autophagosomes, but no obvious difference in fluorescence intensity of Sqstm1induced by ox-LDL.
2. Effects of Pdcd4-attenuated autophagy on macrophage-derived foam cell formation
(1) Pdcd4deficiency attenuates macrophage-derived foam cell formation dependent on autophagy Oil Red "0" staining was used in wide type and pdcd4-/-mouse primary macrophage, demonstrating that Pdcd4deficiency caused a decrease in lipid accumulation and attenuation of foam cell formation, enhanced by EBSS and rapamycin, and restored by WM,3-MA and Pep A+E64d.
(2) Pdcd4controls autophagy dependent cholesterol efflux Uptake tests demonstrated no differences between wide type and pdcd4-/-primary macrophage. A decrease in cholesterol accumulation and an increase in number of autophagosomes, and activated lysosome were observed in pdcd4-/-mouse primary macrophage. Lysosome was colocalized with autophagosome and cholesterol. Real time PCR showed Pdcd4deficiency led to an increase in amount of Abcal, Abcgl and Lxra.
3. Effects of Pdcd4-attenuated macrophage autophagy on atherosclerosis
(1) Pdcd4deficiency promotes macrophage autophagy and lipid accumulation in atherosclerotic plaque in apoE-/-mice Western blot and IF showed Pdcd4deficiency led to an increase in amount of Lc3and Sqstm1, and a decrease in cholesterol accumulation and an increase in number of autophagosomes, localizing in macrophage in atherosclerotic plaque.
(2) Pdcd4-attenuated macrophage autophagy may play an important role in AS via bone marrow transplantation Pdcd4deficiency led to a decrease in cholesterol accumulation and an increase in number of autophagosome, colocalizing with macrophage in local plaque after bone marrow transplantation.
(3) Relationship between PDCD4expression and autophagy in human atherosclerotic plaque PDCD4expression were detected by IHC and autophagosomes were analyzed by IF. The correlationship analysis demonstrated a negative relation between PDCD4expression and autophagy level in human plaque.
Ⅲ.Molecular mechanisms by which PDCD4suppresses autophagy
1. PDCD4inhibits autophagy-related gene ATG5protein expression
(1) PDCD4inhibits ATG5protein expression in vitro Western blot demonstrated in both tumor cells and macrophages BECN1was not affected by PDCD4under starvation, LPS or rapamycin. Higher PDCD4expression remarkably inhibited ATG5expression and formation of the ATG12-ATG5complex.
(2) PDCD4inhibits ATG5protein expression in vivo ATG5expression in human liver, ovarian cancers, gliomas and murine xenograft with negative or low PDCD4expression was significantly higher than that with higher PDCD4expression.
2. PDCD4inhibits ATG5expression and autophagy via its ma3domains
(1) PDCD4protein binds ATG5mRNA ATG5protein was suppressed significantly, while ATG5mRNA was unaffected by PDCD4expression, suggesting the inhibition of PDCD4on ATG5occurs translationally. A RIP assay was applied to demonstrate an association between ATG5mRNA and PDCD4protein.
(2) PDCD4inhibits ATG5-mediated autophagy via its ma3domains PDCD4mutation analysis demonstrated only wild-type PDCD4but not mutant PDCD4led to ATG5downregulation, and a decrease in the number of autophagosomes, suppressed cell viability and an association with ATG5mRNA, suggesting PDCD4inhibited ATG5by binding of PDCD4-ma3with EIF4A-ATG5mRNA.
Conclusions
1. PDCD4inhibits activation of autophagy and formation of autophagosome in tumor cells and immunocytes. PDCD4-attenuated autophagy is required for inhibition of tumor cell growth.
2. Pdcd4promotes macrophage-derived foam cell formation via suppressing autophagy and cholesterol efflux, at last promotes the initiation and development of atherosclerosis.
3. Mechanically, PDCD4inhibits ATG5protein expression and further inhibits ATG12-ATG5complex formation in vitro and in vivo. The inhibition of PDCD4on ATG5occurs translationally, and RIP assay demonstrates ATG5but not ATG12mRNA, binds with PDCD4protein. Once PDCD4fails to binding with ATG5, it loses the ability to suppress ATG5expression, autophagy and cell viability.
Originality and Significance
1. For the first time, we demonstrate PDCD4inhibits autophagy and formation of autophagosome in multiple cells including tumor and immune cells, an universal phenomenon. We report here that inhibition of PDCD4on autophagy contributes to its tumor suppressor activity and effect of Pdcd4on lipid metabolism and atherosclerosis.
2. Importantly, PDCD4inhibits the expression of an essential autophagy related gene, ATG5and the formation of ATG12-ATG5complex, and its ma3domains are required for PDCD4-mediated inhibition of autophagy. Our findings indicate that PDCD4negatively regulates autophagy by targeting ATG5, which provides a novel mechanism of tumor suppression.
引文
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