PDRG1调节p53/p21通路促进结直肠癌发生发展的分子机制研究

Gene copy number variation (CNV) is closely related to the occurrence of colorectal cancer. Our previous studies have found that PDRG1 was overexpressed in colorectal cancer tissues,as well as the number of CNV. Moreover, PDRG1mRNA expression was positively correlated with CNV. At the same time, high expression of PDRG1 promoted the proliferation and inhibited the apoptosis of colon cancer cells. Luciferase report experiments confirmed that overexpression of PDRG1 inhibited p53/p21 activity, suggesting that PDRG1 may play a role in p53/p21 signaling pathway. However, the exact mechanism is still unclear. This project aims to: 1) confirm the role of PDRG1 in promoting the occurrence and development of colon cancer in vitro and nude mice experiments by constructing PDRG1 overexpression/knockdown cell lines ; 2) to find and verify the important target genes of p53/p21 pathway directly regulated by PDRG1 through immunoprecipitation, mass spectrometry, luciferase reporting experiment and immunofluorescence staining, finally reveal the pro-cancer mechanism; 3) and to detect the expression of PDRG1 in plasma and tissues of colorectal adenoma, colorectal cancer and normal control group to elucidate the role of PDRG1 in the development of colorectal cancer and its early diagnostic value . This study provides a theoretical basis for further understanding the function and specific mechanism of PDRG1, and suggests that PDRG1 may be a new indicator for early diagnosis of colorectal cancer.

基因拷贝数变异与结直肠癌发生密切相关，我们前期研究发现结直肠癌组织中PDRG1高表达且拷贝数增加，二者呈正相关。同时体外实验证实PDRG1过表达促进结肠癌细胞增殖，抑制细胞凋亡；荧光素酶报告实验证实PDRG1过表达抑制p53/p21活性，提示在p53/p21信号通路中发挥作用。然而具体作用机制尚不清楚。本课题拟通过构建PDRG1过表达/敲低细胞株，细胞和裸鼠实验证实PDRG1促进结肠癌发生、发展的作用；通过免疫沉淀、质谱、荧光素酶报告实验、免疫荧光染色等手段寻找和验证PDRG1直接调控的p53/p21通路重要的靶基因，揭示其作用机制；以及检测结直肠腺瘤、结直肠癌及正常对照组血浆及组织中PDRG1的表达，分析PDRG1在结直肠癌发展进程中的作用，评价其早期诊断价值。本研究为深入了解PDRG1的功能及具体作用机制提供理论依据，同时提示PDRG1可能可以作为早期诊断结直肠癌的新指标。

结直肠癌发病机制复杂，我们前期采用全基因组测序发现结直肠癌组织中PDRG1（p53 and DNA damage-regulated gene）拷贝数增加，实时PCR结果证实PDRG1在癌组织中表达上调，且mRNA表达增高与拷贝数增加呈正相关。基于前期研究，本课题通过细胞转染、Western blot、免疫组织化学染色、蛋白组学、Co-IP、生物信息学分析等方法对PDRG1在结肠癌中的功能及临床应用价值进行了研究，结果证明PDRG1在结肠癌中高表达，且与肿瘤位置、TNM 分期、肿瘤复发和无瘤生存期有关。细胞学实验发现PDRG1过表达促进结肠癌细胞增殖、抑制细胞凋亡，且促进肿瘤细胞侵袭和转移；下调PDRG1可以抑制结肠癌细胞生长和侵袭，证实PDRG1在结肠癌进展中发挥癌基因的作用。随后采用荧光素酶报告实验发现PDRG1过表达抑制p53/p21活性，cancer passway array、蛋白组学及KEGG信号通路分析均提示PDRG1影响p53信号通路。PDRG1过表达抑制了p53及其下游p21蛋白的表达，敲低PDRG1可以上调p53和p21的表达，同时影响细胞周期、凋亡相关蛋白如cleaved caspase3、cleaved caspase7、p27、PTEN的表达。Nutlin-3应用于PDRG1高表达细胞株，可以部分逆转PDRG1对p53和p21的抑制，从而改善 PDRG1对结肠癌细胞生长和增殖的促进作用。依据蛋白组学分析，PDRG1高表达组真核翻译起始因子 6（EIF6）表达增加，数据库分析证明结直肠癌中EIF6表达较正常组织明显增高，且EIF6的表达与PDRG1显著正相关。Co-IP分析结果表明PDRG1和EIF6之间存在相互作用，因此PDRG1可能通过与癌基因EIF6作用发挥促癌作用。临床组织及血浆标本检测结果证实结直肠癌患者组织和血浆中PDRG1高表达，提示可能作为结直肠癌早期诊断的指标。综上，PDRG1在结直肠癌中发挥促癌作用，可促进结肠癌细胞生长、增殖、迁移和侵袭，且该作用与p53/p21信号通路有关，可作为结直肠癌干预的靶标，研究成果具有较好的转化应用前景。