结直肠癌中AKAP12基因甲基化及其肿瘤抑制作用
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摘要
结直肠癌(Colorectal cancer,CRC)是我国最常见的恶性肿瘤之一,居肿瘤发病率第4位,居癌症死亡率第3位[1]。随着饮食结构日渐精细化,高脂肪和低纤维素饮食,致使结直肠癌的发病率呈明显上升趋势,在大中城市较明显,每年新增病例数近百万,同时近50万人死于该病,五年生存率约为64%[2]。其生长方式以局部浸润为主,晚期主要沿淋巴管扩散,外科手术切除为主的综合治疗虽有一定进步,但总体术后5年生存率无明显提高,主要原因在于术后复发率高达40-70%,因而非常有必要加强CRC的发病机制及其预防治疗的相关研究。
A激酶锚定蛋白12(AKAP12或AKAP250或Gravin),位于6q24-q25,最早是从重症肌无力病人血清中分离出的,主要分布细胞质中。是细胞特异性AKAPs,主要表达在β_2肾上腺素受体周围。主要参与PKA和PKC复合物的形成[3],细胞骨架的重塑[4],同时亦是一重要的β_2肾上腺素受体复合物的调节基因[5],参与了蛋白定向、信号转导、G蛋白偶联受体蛋白信号转导途径以及细胞凋亡[6]等。最近的研究显示AKAP12除了上述功能以外,还发现它在多种肿瘤中缺失,是一种潜在的抑癌基因,已经报道的缺失可见于黑素瘤[7]、乳腺癌[8]、前列腺癌[9]、胃癌[10]、血液系统恶性肿瘤[11]等。
基因启动子区域的甲基化是抑癌基因(Tumor suppressor gene,TSGs)失活的重要机制之一[12],并能直接促进肿瘤的恶性行为[13-20]。因此寻找由异常DNA甲基化引起的肿瘤抑制基因失活在肿瘤研究中就显得尤其重要。作为潜在的肿瘤抑制基因,甲基化报道也见于AKAP12的研究中,Choi等[10]在胃癌组织和细胞株中均发现AKAP12表达下降或缺失,并发现了高甲基化(56%)的存在,并与其表达缺失相对应。Christian等[11]则在白血病的研究中,发现白血病中CD~(34+)细胞中其表达降低,并伴有CpG位点高甲基化。
除此之外还陆续发现了AKAP12其他的一些良性功能,Choi等[10]在对胃癌的研究中发现它具有抑制肿瘤生长的功能。Vant等[11]在慢性淋巴细胞性白血病中发现AKAP12基因与肿瘤的预后呈正相关。Dae-Kwan Yoon等[21]的研究中,AKAP12基因还可以通过影响细胞生长周期蛋白CDKI-cyclin D1的方式使肿瘤细胞生长停滞,并且阻碍细胞核分裂,促进细胞自身凋亡机制的启动。在前列腺癌的研究发现,AKAP12能够起到较为明显的抑制癌细胞生长的作用[22]。与AKAP12基因具有同源性的大鼠SSeCKS基因在人类的NIH3T3型纤维母细胞瘤中可以因过度表达而使得肿瘤细胞生长停滞在G1期[23;24]。AKAP12在血脑屏障中具有抑制血管内皮细胞生长因子(VEGF)活性的功能,从而抑制肿瘤的发展[25]。这些研究都慢慢揭示了AKAP12抑癌作用。
(1)AKAP12在结直肠癌组织中的表达及其甲基化
结直肠癌中多个抑癌基因失活的致癌模式逐渐为人们所认识,目前已明确与结直肠癌有关的抑癌基因包括ApC、p53、p16、p15、MCC、hMLH1等。为了探究结直肠癌中AKAP12是否存在表达下降或者缺失,如果存在下调,其AKAP12启动子区域的甲基化是不是失活的重要原因。因此我们检测了45例结直肠组织及其癌旁对照中的表达及其启动子区域甲基化状态,并同时分析其与临床病理资料的相关性。
结果显示,有68.9%的标本中检测到该基因的表达下降或缺失,在不同分级和分化的结直肠癌中并没有统计学意义。经过MSP检测有26份标本(57.8%)检测出AKAP12基因启动子区域的甲基化,其中在表达下调的31份标本中,有20份标本(64.5%)检测出甲基化,且与结直肠癌分期相关(P=0.02),在Dukes'C+D的标本中发现更多甲基化,提示我们甲基化可能与病程进展相关。统计分析发现,AKAP12的表达下降与甲基化发生之间没有相关性,是不是还存在甲基化之外的其他可能原因影响该基因的转录表达?我们将在细胞实验中做初步的探索。亚硫酸盐测序的结果显示不同的位点发生甲基化的频率也不同,不同标本17个位点发生甲基化的频率并不相同,其中9、10、11、12位点高发可能与肿瘤发生发展的关系更为密切。
(2)AKAP12在结直肠癌细胞中的表达和甲基化研究
我们尝试在细胞层次上加以深入,检测了5株结直肠癌细胞株(LoVo,Colo205,SW480,LS174T和Colo320)的mRNA和蛋白表达情况及甲基化状态。通过甲基化酶的抑制剂(5-吖-2-脱氧胞苷酸,5-Aza-dC)和组蛋白乙酰化的抑制剂(Trichostatin A,TSA)作用,观察去甲基化和乙酰化能否恢复其基因和蛋白水平的表达。
结果显示SW480,LoVo和Colo320中,AKAP12在基因和蛋白水平均表达缺失。同时在LoVo和Colo320中检测到完全甲基化,Colo205、SW480和LS174T检测到部分甲基化。亚硫酸盐测序结果也与此基本相符。在三株存在表达缺失的细胞株(Colo320、LoVo和SW480)中,经5-Aza-dC作用后表达上升分别5.4倍,6.3倍和3.6倍,TSA作用后表达分别上升6.0倍,9.3倍和21.7倍,共同作用后表达分别上升35.2倍,13.8倍和120.9倍,佐证甲基化是该基因表达缺失的重要原因,并且存在一些其他原因(如组蛋白的乙酰化)也协同作用导致该基因的表达改变,并可通过去甲基化和乙酰化恢复其表达。
(3)AKAP12抑制结直肠癌体内体外生长和转移
无限增殖和凋亡受阻是肿瘤细胞的重要特征,作为一种潜在的肿瘤抑制基因,其抑制肿瘤细胞增殖和诱导其凋亡的能力至关重要。同时,在肿瘤特别是结直肠癌这种以侵润方式生长的肿瘤中,降低肿瘤细胞的迁徙、侵袭能力,也是抑癌基因发挥作用的重要方面。本研究将构建pCMV6-NEO-AKAP12真核表达载体,转染未表达AKAP12,且具有高转移能力的结直肠癌细胞系LoVo[26],AKAP12恢复表达后,观察体内和体外研究中的影响,研究其在肿瘤细胞中生长、凋亡、迁徙、侵袭、转移等方面的作用,探讨其对肿瘤生物学特性的影响。
结果显示,LoVo细胞转染表达AKAP12后,细胞生长曲线和凋亡检测可以明显看出,表达AKAP12蛋白能够抑制肿瘤细胞生长、迁徙、侵袭、非依赖生长能力,并能诱导其凋亡。体内实验发现接种LoVo-AKAP12转染组的裸鼠肿瘤生长和转移明显受到抑制。
(4)甲基化特异性高分辨熔解曲线(Methylation specific high-resolutionmelting,MS-HRM)快速定量检测结直肠癌患者外周血中AKAP12甲基化
我们立足于组织细胞学实验的基础上,检测体液(主要是外周血)中AKAP12异常甲基化的DNA,如果能实现定量检测,将为结直肠的诊断、筛查和检测复发转移提供另一种思路。本研究旨在前期研究的基础上,通过MS-HRM法初步探索检测AKAP12启动子区域的甲基化情况,探讨外周血中AKAP12高甲基化与肿瘤发生发展之间的潜在关系。
结果发现,80例结直肠癌标本中有38例(47.5%)出现不同程度甲基化,我们发现外周血中AKAP12的甲基化程度与结直肠癌的病理分级和恶性程度相关。同时用MSP方检测,只有＞10%的标本都出现甲基化条带,而采用HRM方法可检测低至1%甲基化水平,比MSP方法更敏感。并具有较好的灵敏度、特异性和重复性,为结直肠癌高危人群的筛选、病程的评估,特别是转移和复发提供更多的信息,也可能发展成为一种新的检测标志物。
综上所述,本研究发现AKAP12在结直肠癌组织中表达下调,并存在启动子区域高甲基化,且与肿瘤的病理分级相关。LoVo、Colo320和SW480中发现AKAP12表达缺失,发现LoVo和Colo320完全甲基化,LoVo、Colo320和SW480经去甲基化和去乙酰化处理能部分恢复其表达。同时,本研究构建了稳定表达的真核载体pCMV6-NEO-AKAP12,经体内体外实验证实,恢复AKAP12表达能抑制结直肠癌细胞的生长、迁徙、侵袭、悬浮生长能力,并能诱导肿瘤细胞凋亡。裸鼠模型中,AKAP12能抑制结直肠癌的生长和转移,显示了其肿瘤抑制作用。最后本研究还首次建立了甲基化特异的高分辨融解曲线法,用于检测外周血中AKAP12甲基化程度,发现直肠癌外周血中AKAP12的高甲基化,且与结直肠癌的病理分级相关。
Colorectal cancer is the third most common form of cancer and the second leading cause of cancer mortality in the world.[2;14;27]To establish more effective diagnostic and therapeutic strategies against this deadly disease,it is essential to understand its molecular pathology.One of the key molecular pathogenic elements underlying human cancer is inactivation of tumor suppressor genes.
A kinase anchor protein 12(AKAP12/AKAP250/Gravin) was first isolated as a protein recognized by serum from myasthenia gravis patients.It is one of the A kinase anchoring proteins(AKAPs),which belongs to a family of scaffolding proteins, and organizes the complex of PKA and PKC.It is also an important regulator of theβ_2-adrenergic receptor complex,which controls cell signaling,cell adhesion, mitogenesis and differentiation.AKAP12 has been mapped to chromosome 6q24-25.2.Accumulating evidence indicates that DNA hypermethylation in the AKAP12 promoter region and concurrent underexpression of the gene was noted in a variety of human cancers,including gastric cancer,esophageal cancer,lung cancer, myeloma cells and myeloid malignancies[10;29-32]Downregulation of AKAP12 expression suggests that the inactivation of AKAP12 expression may be linked to oncogenesis.A recent report using microarray data in silico genetic searches suggests that AKAP12 methylation is associated with the downregulation of AKAP12 in colon caner and suggests that AKAP12 may be a potential tumor suppressor gene candidate.However more information regarding the methylation and expression status of this gene and it's inhibiting progression and metastasis remains to be determined in this solid tumor.
(1) AKAP12 expression and promoter methylation in colorectal cancer tissues
To determine whether AKAP12 is downregulated in colorectal cancer tissues,we examined the expression of this gene.AKAP12 expression levels were determined by real-time RT-PCR in 45 tumor-normal pairs.AKAP12 mRNA was decreased in 31 of 45(68.9%) colorectal cancer tissues.The frequency of AKAP12 downregulation did not correlated with patients' age,gender,Duke's stage and differentiation.To determine whether the decreasing of AKAP12 expression was mediated by promoter hypermethylation,the methylation status of the 5'CpG island of AKAP12 gene was determined by MSP.The CpG islands of AKAP12 were methylated in 26 of 45(57.8%) tumor cases.Hypermethylation of AKAP12 mRNA was significantly associated with more advanced Duke's stage(P=0.02).The frequency of AKAP12 downregulation did not correlated with hypermethylation.To gain more information on the methylation status,particularly for the upstream region of the basic promoter and around the translation start sites,a 228bp fragment of the AKAP12 promoter region,containing 17 CpG dinucleotides,was sequenced.
(2) AKAP12 expression and promoter methylation in colorectal cancer cell lines
By RT-PCR,we first determined the expression of AKAP12 gene in five colorectal cell lines.Complete loss of AKAP12 transcript occurred in SW480,LoVo and Colo320 cells.In contrast,AKAP12 transcript remained in LS174T and Colo205 cells.Consistent with RT-PCR assay,Western blot analysis showed the lack of AKAP12 protein expression in SW480,LoVo and Colo320 and positive expression in Colo205 and LS174T.To determine if methylation was responsible for down regulation of AKAP12 in carcinoma cancer cell lines,we performed MSP analysis. Only methylated DNA was seen in LoVo and Colo320 cells whereas LS174T, Colo205 and SW480 showed partial methylation.To assess whether methylation of AKAP12 promoter was associated with transcriptional silencing,we analyzed AKAP12 mRNA expression in the presence or absence of the demethylating agent 5-Aza-dC and the histone deacetylase inhibitor TSA.Treatment of Colo320,LoVo and SW480 cells harboring extensive methylation with 5-Aza-dC and TSA upregulated the expression of AKAP12 by 5.4-fold,6.3-fold,3.6-fold and 6.0-fold, 9.3-fold,21.7fold,respectively.Interestingly the highest levels(35.2-fold,13.8-fold and 120.9-fold) of gene expression were observed when the cell lines were cultured with both agents,suggesting that aberreant histone deacetylation is also an important mechanism for inactivation of this gene and 5-aza-dC and TSA act synergistically to enhance transcriptional reactivation.
(3) AKAP12 inhabit the progression growth and invasion in vitro and in vivo
Cell proliferation and apoptosis assays were performed by WST and flow-cytometric analysis.AKAP12 inhibited colorectal cells growth,proliferation and apoptosis.We also to detect the local invasion of the cell lines and the results suggested that AKAP12 could decrease the invasive,migratory and transformation capacity of the colorectal cells in vitro.
We have found that AKAP12 have the inhibitory ability of growth and invasion on colorectal cancer in vitro,we considered that if the function of AKAP12 could be maintained in vivo? So we conducted successfully the subcutaneous xenotransplantation of colorectal in nude severe combined immunodeficient mice. Tumor size of the LoVo-AKAP12 animals was significantly smaller than that of animals from control groups(P＜0.01).The results also suggested that AKAP12 could decrease the metastasis capacity by detection the Alu sequence.
(4) Rapid determination of AKAP12 methylation levels in peripheral blood using methylation-sensitive high resolution melting(MS-HRM) analysis
The aim of this study was to apply methylation specific high resolution melting (MS-HRM) technology to detect quantitatively AKAP12 methylation in peripheral blood from 80 colorectal cancer patients and 20 healthy volunteers and in three colorectal cancer cell lines.In this study 38 of the 80 colorectal cancer samples (47.5%) were found to be methylated at the AKAP12 promoter region.AKAP12 methylation was significantly higher in the colorectal cancer samples with differentiation(P=0.03).We also compared the results generated by MS-HRM with a traditional methylation-specific PCR(MSP) assay.We found that intra-assay variability ranged from 6.14-9.90%and inter-assay variability ranged from 14.5-17.2%.The AKAP12 MS-HRM assay was able to reproducibly detect 1% methylated DNA,whereas the MSP method was unable to detect less than 10% methylation in our this study.We demonstrate here for the first time,the utility of quantitative AKAP12 MS-HRM analysis of promoter methylation in peripheral blood samples.
A激酶锚定蛋白12(AKAP12或AKAP250或Gravin),位于6q24-q25,最早是从重症肌无力病人血清中分离出的,主要分布细胞质中。是细胞特异性AKAPs,主要表达在β_2肾上腺素受体周围。主要参与PKA和PKC复合物的形成[3],细胞骨架的重塑[4],同时亦是一重要的β_2肾上腺素受体复合物的调节基因[5],参与了蛋白定向、信号转导、G蛋白偶联受体蛋白信号转导途径以及细胞凋亡[6]等。最近的研究显示AKAP12除了上述功能以外,还发现它在多种肿瘤中缺失,是一种潜在的抑癌基因,已经报道的缺失可见于黑素瘤[7]、乳腺癌[8]、前列腺癌[9]、胃癌[10]、血液系统恶性肿瘤[11]等。
基因启动子区域的甲基化是抑癌基因(Tumor suppressor gene,TSGs)失活的重要机制之一[12],并能直接促进肿瘤的恶性行为[13-20]。因此寻找由异常DNA甲基化引起的肿瘤抑制基因失活在肿瘤研究中就显得尤其重要。作为潜在的肿瘤抑制基因,甲基化报道也见于AKAP12的研究中,Choi等[10]在胃癌组织和细胞株中均发现AKAP12表达下降或缺失,并发现了高甲基化(56%)的存在,并与其表达缺失相对应。Christian等[11]则在白血病的研究中,发现白血病中CD~(34+)细胞中其表达降低,并伴有CpG位点高甲基化。
除此之外还陆续发现了AKAP12其他的一些良性功能,Choi等[10]在对胃癌的研究中发现它具有抑制肿瘤生长的功能。Vant等[11]在慢性淋巴细胞性白血病中发现AKAP12基因与肿瘤的预后呈正相关。Dae-Kwan Yoon等[21]的研究中,AKAP12基因还可以通过影响细胞生长周期蛋白CDKI-cyclin D1的方式使肿瘤细胞生长停滞,并且阻碍细胞核分裂,促进细胞自身凋亡机制的启动。在前列腺癌的研究发现,AKAP12能够起到较为明显的抑制癌细胞生长的作用[22]。与AKAP12基因具有同源性的大鼠SSeCKS基因在人类的NIH3T3型纤维母细胞瘤中可以因过度表达而使得肿瘤细胞生长停滞在G1期[23;24]。AKAP12在血脑屏障中具有抑制血管内皮细胞生长因子(VEGF)活性的功能,从而抑制肿瘤的发展[25]。这些研究都慢慢揭示了AKAP12抑癌作用。
(1)AKAP12在结直肠癌组织中的表达及其甲基化
结直肠癌中多个抑癌基因失活的致癌模式逐渐为人们所认识,目前已明确与结直肠癌有关的抑癌基因包括ApC、p53、p16、p15、MCC、hMLH1等。为了探究结直肠癌中AKAP12是否存在表达下降或者缺失,如果存在下调,其AKAP12启动子区域的甲基化是不是失活的重要原因。因此我们检测了45例结直肠组织及其癌旁对照中的表达及其启动子区域甲基化状态,并同时分析其与临床病理资料的相关性。
结果显示,有68.9%的标本中检测到该基因的表达下降或缺失,在不同分级和分化的结直肠癌中并没有统计学意义。经过MSP检测有26份标本(57.8%)检测出AKAP12基因启动子区域的甲基化,其中在表达下调的31份标本中,有20份标本(64.5%)检测出甲基化,且与结直肠癌分期相关(P=0.02),在Dukes'C+D的标本中发现更多甲基化,提示我们甲基化可能与病程进展相关。统计分析发现,AKAP12的表达下降与甲基化发生之间没有相关性,是不是还存在甲基化之外的其他可能原因影响该基因的转录表达?我们将在细胞实验中做初步的探索。亚硫酸盐测序的结果显示不同的位点发生甲基化的频率也不同,不同标本17个位点发生甲基化的频率并不相同,其中9、10、11、12位点高发可能与肿瘤发生发展的关系更为密切。
(2)AKAP12在结直肠癌细胞中的表达和甲基化研究
我们尝试在细胞层次上加以深入,检测了5株结直肠癌细胞株(LoVo,Colo205,SW480,LS174T和Colo320)的mRNA和蛋白表达情况及甲基化状态。通过甲基化酶的抑制剂(5-吖-2-脱氧胞苷酸,5-Aza-dC)和组蛋白乙酰化的抑制剂(Trichostatin A,TSA)作用,观察去甲基化和乙酰化能否恢复其基因和蛋白水平的表达。
结果显示SW480,LoVo和Colo320中,AKAP12在基因和蛋白水平均表达缺失。同时在LoVo和Colo320中检测到完全甲基化,Colo205、SW480和LS174T检测到部分甲基化。亚硫酸盐测序结果也与此基本相符。在三株存在表达缺失的细胞株(Colo320、LoVo和SW480)中,经5-Aza-dC作用后表达上升分别5.4倍,6.3倍和3.6倍,TSA作用后表达分别上升6.0倍,9.3倍和21.7倍,共同作用后表达分别上升35.2倍,13.8倍和120.9倍,佐证甲基化是该基因表达缺失的重要原因,并且存在一些其他原因(如组蛋白的乙酰化)也协同作用导致该基因的表达改变,并可通过去甲基化和乙酰化恢复其表达。
(3)AKAP12抑制结直肠癌体内体外生长和转移
无限增殖和凋亡受阻是肿瘤细胞的重要特征,作为一种潜在的肿瘤抑制基因,其抑制肿瘤细胞增殖和诱导其凋亡的能力至关重要。同时,在肿瘤特别是结直肠癌这种以侵润方式生长的肿瘤中,降低肿瘤细胞的迁徙、侵袭能力,也是抑癌基因发挥作用的重要方面。本研究将构建pCMV6-NEO-AKAP12真核表达载体,转染未表达AKAP12,且具有高转移能力的结直肠癌细胞系LoVo[26],AKAP12恢复表达后,观察体内和体外研究中的影响,研究其在肿瘤细胞中生长、凋亡、迁徙、侵袭、转移等方面的作用,探讨其对肿瘤生物学特性的影响。
结果显示,LoVo细胞转染表达AKAP12后,细胞生长曲线和凋亡检测可以明显看出,表达AKAP12蛋白能够抑制肿瘤细胞生长、迁徙、侵袭、非依赖生长能力,并能诱导其凋亡。体内实验发现接种LoVo-AKAP12转染组的裸鼠肿瘤生长和转移明显受到抑制。
(4)甲基化特异性高分辨熔解曲线(Methylation specific high-resolutionmelting,MS-HRM)快速定量检测结直肠癌患者外周血中AKAP12甲基化
我们立足于组织细胞学实验的基础上,检测体液(主要是外周血)中AKAP12异常甲基化的DNA,如果能实现定量检测,将为结直肠的诊断、筛查和检测复发转移提供另一种思路。本研究旨在前期研究的基础上,通过MS-HRM法初步探索检测AKAP12启动子区域的甲基化情况,探讨外周血中AKAP12高甲基化与肿瘤发生发展之间的潜在关系。
结果发现,80例结直肠癌标本中有38例(47.5%)出现不同程度甲基化,我们发现外周血中AKAP12的甲基化程度与结直肠癌的病理分级和恶性程度相关。同时用MSP方检测,只有＞10%的标本都出现甲基化条带,而采用HRM方法可检测低至1%甲基化水平,比MSP方法更敏感。并具有较好的灵敏度、特异性和重复性,为结直肠癌高危人群的筛选、病程的评估,特别是转移和复发提供更多的信息,也可能发展成为一种新的检测标志物。
综上所述,本研究发现AKAP12在结直肠癌组织中表达下调,并存在启动子区域高甲基化,且与肿瘤的病理分级相关。LoVo、Colo320和SW480中发现AKAP12表达缺失,发现LoVo和Colo320完全甲基化,LoVo、Colo320和SW480经去甲基化和去乙酰化处理能部分恢复其表达。同时,本研究构建了稳定表达的真核载体pCMV6-NEO-AKAP12,经体内体外实验证实,恢复AKAP12表达能抑制结直肠癌细胞的生长、迁徙、侵袭、悬浮生长能力,并能诱导肿瘤细胞凋亡。裸鼠模型中,AKAP12能抑制结直肠癌的生长和转移,显示了其肿瘤抑制作用。最后本研究还首次建立了甲基化特异的高分辨融解曲线法,用于检测外周血中AKAP12甲基化程度,发现直肠癌外周血中AKAP12的高甲基化,且与结直肠癌的病理分级相关。
Colorectal cancer is the third most common form of cancer and the second leading cause of cancer mortality in the world.[2;14;27]To establish more effective diagnostic and therapeutic strategies against this deadly disease,it is essential to understand its molecular pathology.One of the key molecular pathogenic elements underlying human cancer is inactivation of tumor suppressor genes.
A kinase anchor protein 12(AKAP12/AKAP250/Gravin) was first isolated as a protein recognized by serum from myasthenia gravis patients.It is one of the A kinase anchoring proteins(AKAPs),which belongs to a family of scaffolding proteins, and organizes the complex of PKA and PKC.It is also an important regulator of theβ_2-adrenergic receptor complex,which controls cell signaling,cell adhesion, mitogenesis and differentiation.AKAP12 has been mapped to chromosome 6q24-25.2.Accumulating evidence indicates that DNA hypermethylation in the AKAP12 promoter region and concurrent underexpression of the gene was noted in a variety of human cancers,including gastric cancer,esophageal cancer,lung cancer, myeloma cells and myeloid malignancies[10;29-32]Downregulation of AKAP12 expression suggests that the inactivation of AKAP12 expression may be linked to oncogenesis.A recent report using microarray data in silico genetic searches suggests that AKAP12 methylation is associated with the downregulation of AKAP12 in colon caner and suggests that AKAP12 may be a potential tumor suppressor gene candidate.However more information regarding the methylation and expression status of this gene and it's inhibiting progression and metastasis remains to be determined in this solid tumor.
(1) AKAP12 expression and promoter methylation in colorectal cancer tissues
To determine whether AKAP12 is downregulated in colorectal cancer tissues,we examined the expression of this gene.AKAP12 expression levels were determined by real-time RT-PCR in 45 tumor-normal pairs.AKAP12 mRNA was decreased in 31 of 45(68.9%) colorectal cancer tissues.The frequency of AKAP12 downregulation did not correlated with patients' age,gender,Duke's stage and differentiation.To determine whether the decreasing of AKAP12 expression was mediated by promoter hypermethylation,the methylation status of the 5'CpG island of AKAP12 gene was determined by MSP.The CpG islands of AKAP12 were methylated in 26 of 45(57.8%) tumor cases.Hypermethylation of AKAP12 mRNA was significantly associated with more advanced Duke's stage(P=0.02).The frequency of AKAP12 downregulation did not correlated with hypermethylation.To gain more information on the methylation status,particularly for the upstream region of the basic promoter and around the translation start sites,a 228bp fragment of the AKAP12 promoter region,containing 17 CpG dinucleotides,was sequenced.
(2) AKAP12 expression and promoter methylation in colorectal cancer cell lines
By RT-PCR,we first determined the expression of AKAP12 gene in five colorectal cell lines.Complete loss of AKAP12 transcript occurred in SW480,LoVo and Colo320 cells.In contrast,AKAP12 transcript remained in LS174T and Colo205 cells.Consistent with RT-PCR assay,Western blot analysis showed the lack of AKAP12 protein expression in SW480,LoVo and Colo320 and positive expression in Colo205 and LS174T.To determine if methylation was responsible for down regulation of AKAP12 in carcinoma cancer cell lines,we performed MSP analysis. Only methylated DNA was seen in LoVo and Colo320 cells whereas LS174T, Colo205 and SW480 showed partial methylation.To assess whether methylation of AKAP12 promoter was associated with transcriptional silencing,we analyzed AKAP12 mRNA expression in the presence or absence of the demethylating agent 5-Aza-dC and the histone deacetylase inhibitor TSA.Treatment of Colo320,LoVo and SW480 cells harboring extensive methylation with 5-Aza-dC and TSA upregulated the expression of AKAP12 by 5.4-fold,6.3-fold,3.6-fold and 6.0-fold, 9.3-fold,21.7fold,respectively.Interestingly the highest levels(35.2-fold,13.8-fold and 120.9-fold) of gene expression were observed when the cell lines were cultured with both agents,suggesting that aberreant histone deacetylation is also an important mechanism for inactivation of this gene and 5-aza-dC and TSA act synergistically to enhance transcriptional reactivation.
(3) AKAP12 inhabit the progression growth and invasion in vitro and in vivo
Cell proliferation and apoptosis assays were performed by WST and flow-cytometric analysis.AKAP12 inhibited colorectal cells growth,proliferation and apoptosis.We also to detect the local invasion of the cell lines and the results suggested that AKAP12 could decrease the invasive,migratory and transformation capacity of the colorectal cells in vitro.
We have found that AKAP12 have the inhibitory ability of growth and invasion on colorectal cancer in vitro,we considered that if the function of AKAP12 could be maintained in vivo? So we conducted successfully the subcutaneous xenotransplantation of colorectal in nude severe combined immunodeficient mice. Tumor size of the LoVo-AKAP12 animals was significantly smaller than that of animals from control groups(P＜0.01).The results also suggested that AKAP12 could decrease the metastasis capacity by detection the Alu sequence.
(4) Rapid determination of AKAP12 methylation levels in peripheral blood using methylation-sensitive high resolution melting(MS-HRM) analysis
The aim of this study was to apply methylation specific high resolution melting (MS-HRM) technology to detect quantitatively AKAP12 methylation in peripheral blood from 80 colorectal cancer patients and 20 healthy volunteers and in three colorectal cancer cell lines.In this study 38 of the 80 colorectal cancer samples (47.5%) were found to be methylated at the AKAP12 promoter region.AKAP12 methylation was significantly higher in the colorectal cancer samples with differentiation(P=0.03).We also compared the results generated by MS-HRM with a traditional methylation-specific PCR(MSP) assay.We found that intra-assay variability ranged from 6.14-9.90%and inter-assay variability ranged from 14.5-17.2%.The AKAP12 MS-HRM assay was able to reproducibly detect 1% methylated DNA,whereas the MSP method was unable to detect less than 10% methylation in our this study.We demonstrate here for the first time,the utility of quantitative AKAP12 MS-HRM analysis of promoter methylation in peripheral blood samples.
引文
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