结直肠腺瘤和癌中 MAL、PRIMA1、PTGDR 和 SFRP1 的 DNA 高甲基化及 mRNA 表达降低
DNA hypermethylation and decreased mRNA expression of MAL, PRIMA1, PTGDR and SFRP1 in colorectal adenoma and cancer.
作者信息
Kalmár Alexandra, Péterfia Bálint, Hollósi Péter, Galamb Orsolya, Spisák Sándor, Wichmann Barnabás, Bodor András, Tóth Kinga, Patai Árpád V, Valcz Gábor, Nagy Zsófia Brigitta, Kubák Vivien, Tulassay Zsolt, Kovalszky Ilona, Molnár Béla
机构信息
2nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary.
Molecular Medicine Research Group, Hungarian Academy of Sciences, Budapest, Hungary.
出版信息
BMC Cancer. 2015 Oct 19;15:736. doi: 10.1186/s12885-015-1687-x.
BACKGROUND
Colorectal cancer (CRC) development is accompanied by changes in expression for several genes; but the details of the underlying regulatory procesess remain unknown. Our aims were to assess the role of epigenetic processes in tumour formation and to identify characteristic DNA methylation and miRNA alterations in the colorectal adenoma-carcinoma sequence.
METHODS
Whole genome expression profiling was performed on colonic biopsy samples (49 healthy normal, 49 colorectal adenoma (AD), 49 CRC); on laser capture microdissected (LCM) epithelial and stromal cells from 6 CRC-normal adjacent tissue (NAT) samples pairs, and on demethylated human CRC cell lines using HGU133 Plus 2.0 microarrays (Affymetrix). Methylation status of genes with gradually altering expression along the AD-CRC sequence was further analysed on 10-10 macrodissected and 5-5 LCM samples from healthy colon, from adenoma and from CRC biopsy samples using bisulfite-sequencing PCR (BS-PCR) followed by pyrosequencing. In silico miRNA prediction for the selected genes was performed with miRWALK algorithm, miRNA expression was analysed on 3 CRC-NAT sample pairs and 3 adenoma tissue samples using the Human Panel I + II (Exiqon). SFRP1 immunohistochemistry experiments were performed.
RESULTS
A set of transcripts (18 genes including MAL, SFRP1, SULT1A1, PRIMA1, PTGDR) showed decreasing expression (p < 0.01) in the biopsy samples along the adenoma-carcinoma sequence. Three of those (COL1A2, SFRP2, SOCS3) showed hypermethylation and THBS2 showed hypomethylation both in AD and in CRC samples compared to NAT, while BCL2, PRIMA1 and PTGDR showed hypermethylation only in the CRC group. miR-21 was found to be significantly (p < 0.01) upregulated in adenoma and tumour samples compared to the healthy colonic tissue controls and could explain the altered expression of genes for which DNA methylation changes do not appear to play role (e.g. BCL2, MAL, PTGS2). Demethylation treatment could upregulate gene expression of genes that were found to be hypermethylated in human CRC tissue samples. Decreasing protein levels of SFRP1 was also observed along the adenoma-carcinoma sequence.
CONCLUSION
Hypermethylation of the selected markers (MAL, PRIMA1, PTGDR and SFRP1) can result in reduced gene expression and may contribute to the formation of colorectal cancer.
背景
结直肠癌（CRC）的发生伴随着多个基因表达的变化；但其潜在调控过程的细节仍不清楚。我们的目的是评估表观遗传过程在肿瘤形成中的作用，并确定结直肠腺瘤-癌序列中特征性的DNA甲基化和微小RNA（miRNA）改变。
方法
使用HGU133 Plus 2.0微阵列（Affymetrix）对结肠活检样本（49例健康正常样本、49例结直肠腺瘤（AD）、49例CRC）、6对CRC-正常相邻组织（NAT）样本的激光捕获显微切割（LCM）上皮和基质细胞以及去甲基化的人CRC细胞系进行全基因组表达谱分析。使用亚硫酸氢盐测序PCR（BS-PCR）随后进行焦磷酸测序，对来自健康结肠、腺瘤和CRC活检样本的10-10宏观解剖样本和5-5 LCM样本进一步分析沿AD-CRC序列表达逐渐改变的基因的甲基化状态。使用miRWALK算法对选定基因进行计算机miRNA预测，使用人类I + II组（Exiqon）对3对CRC-NAT样本和3例腺瘤组织样本进行miRNA表达分析。进行了分泌型卷曲相关蛋白1（SFRP1）免疫组织化学实验。
结果
一组转录本（包括MAL、SFRP1、磺基转移酶1A1（SULT1A1）、原癌基因1（PRIMA1）、前列腺素D2受体（PTGDR）在内的18个基因）在活检样本中沿腺瘤-癌序列表达降低（p < 0.01）。其中三个基因（I型胶原α2链（COL1A2）、SFRP2、细胞因子信号转导抑制因子3（SOCS3））在AD和CRC样本中与NAT相比均显示高甲基化，而血小板反应蛋白2（THBS2）在AD和CRC样本中显示低甲基化，而B细胞淋巴瘤2（BCL2）、PRIMA1和PTGDR仅在CRC组中显示高甲基化。与健康结肠组织对照相比，发现miR-21在腺瘤和肿瘤样本中显著上调（p < 0.01），并且可以解释DNA甲基化变化似乎不起作用的基因的表达改变（例如BCL2、MAL、前列腺素内过氧化物合酶2（PTGS2））。去甲基化处理可上调在人CRC组织样本中发现高甲基化的基因的基因表达。沿腺瘤-癌序列也观察到SFRP1蛋白水平降低。
结论
选定标志物（MAL、PRIMA1、PTGDR和SFRP1）的高甲基化可导致基因表达降低，并可能有助于结直肠癌的形成。
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