儿童髓母细胞瘤中 PRDM1/ DKK1信号调节影响肿瘤侵袭的机制研究

Medulloblastoma（MB）is the most common fatal malignant tumor in children. PRDM1 is a mutant gene screened by gene sequencing in the pre-study. The expression of PRDM1 and DKK1 was the same in the same tissue samples. After up-regulating the expression of PRDM1, the expression level of DKK was significantly increased, which significantly inhibited the invasion ability of the mother cells. The expression of DKK1 was significantly increased after down-regulation of miR-653, and its change trend was the same as that of PRDM1, and inhibited the invasion ability of medullary cells in children. Overexpression of miR-653 significantly promotes the invasiveness of medulloblastoma in children, and the mechanism of PRDM1 in the process of invasion of medulloblastoma in children has not been studied. Bioinformatics analysis and experimental validation suggest that PRDM1 may be a targeted regulatory gene of miR-653. In this study, PRDM1 was used as a clue, DKK1 and miR-653 and were used as the entry points to further explore the relationship between the three and comprehensively evaluate the mechanism of the effect on the invasion ability of medulloblastoma. Because PRDM1, DKK1 and miR-653 play a role in children's medulloblastoma and the relationship between the three has not been reported in the literature, this topic has a source of innovation may provide new ideas for intervention in children's MB invasion.

髓母细胞瘤（MB）是小儿常见恶性肿瘤。侵袭是导致肿瘤复发和影响预后的主要因素。PRDM1是本研究前期应用基因测序筛选到的突变基因。在同一组织标本中PRDM1与DKK1表达趋势相同，上调PRDM1表达后，DKK1的表达水平明显增加显著抑制了随母细胞的侵袭能力；下调miR-653后DKK1表达显著增加，其变化趋势与PRDM1变化趋势相同，并且抑制了儿童髓母细胞的侵袭能力；过表达miR-653显著促进了儿童MB的侵袭力，并且PRDM1在儿童MB侵袭过程中机制尚未研究。生物信息学分析提示PRDM1可能是miR-653的靶向调节基因。本研究以PRDM1为线索，以DKK1和miR-653为切入点探讨三者之间的关系，综合评判对MB侵袭能力的影响机制。由于PRDM1,DKK1及miR-653在儿童MB中的作用及三者之间相互关系目前均未见文献报道，本课题具有源头创新，可能为干预儿童MB侵袭提供新思路。

髓母细胞瘤(medulloblastoma，MB)是儿童最常见的恶性脑肿瘤，具有高度侵袭性，手术无法在细胞学层面全部切除肿瘤细胞，并且容易通过血液及脑脊液循环通路播散，在治疗上比较困难，术后复发率较高。。因此，探讨儿童MB的侵袭机制，寻找可能干预的措施，对于提高患儿的生活质量及生存率具有重要意义。本课题组在前期对临床儿童MB组织标本通过基因测序时发现PRDM1 基因突变率达51%，与非瘤脑组织相比，PRDM1在儿童MB中低表达，并且与DKK1的表达趋势一致，我们通过Chip-seq和Chip-PCR明确了PRDM1对DKK1的转录调控作用。本研究进一步通过上调PRDM1 表达后，DKK1 的表达水平明显增加，MB侵袭能力相应减弱，并且降低了MB中MMP-2 和MMP-9 的表达量。此外，我们通过一系列分子细胞实验明确了PRDM1和DKK1均能显著抑制MB细胞增殖，诱导细胞凋亡和细胞周期G2期阻滞。MicroRNA在肿瘤的发生发展中也发挥着重要作用，我们通过基因芯片技术分析临床儿童MB组织标本，筛选出诸多上调的microRNAs，借助生信分析预测miR-653可能调控PRDM1。通过荧光素酶报告基因实验，我们明确了miR-653对PRDM1的靶向调控作用，并且在MB细胞中上调miR-653可明显抑制PRDM1和DKK1的表达；而下调miR-653后PRDM1和DKK1的表达显著上调，MB细胞的侵袭能力显著降低。以上结果表明，MB中miR-653通过抑制PRDM1和DKK1，从而促进MB的增殖和侵袭转移。最后，我们通过MB裸鼠皮下移植实验，证实了PRDM1对MB细胞体内发生发展的抑制作用。由此，我们通过一系列体内外试验证明了miR-653通过调控PRDM1/DKK1轴促进MB增殖和侵袭转移的现象及分子机制，明确miR-653或PRDM1/DKK1作为MB药物靶标的潜在应用价值，为后续转化医学研究奠定基础。