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Abstract：
To study the differences in VASH2 expression in pediatric medulloblastoma (MB) tumor tissues of different molecular subtypes, to analyze the correlation between VASH2 and the molecular subtypes of medulloblastoma, clinicopathological data, and prognosis, and to explore the specific mechanism of VASH2\'s role in SHH medulloblastoma cell lines DAOY. We analyzed 47 pediatric medulloblastoma cases admitted to the Department of Pediatric Neurosurgery of the First Affiliated Hospital of Xinjiang Medical University from January 2011 to December 2019, and the expression levels of YAP1 and GAB1 in these tumor tissues were detected by immunohistochemistry (IHC) and molecularly typed (WNT-type, SHH-type, and non-WNT/SHH-type). The correlation between VASH2 and molecular typing of medulloblastoma was analyzed. We also analyzed the medulloblastoma dataset in the GEO database (GSE30074 and GSE202043) to explore the correlation between VASH2 and the prognosis of medulloblastoma patients, as well as performed a comprehensive GO enrichment analysis specifically for the VASH2 gene to reveal the underlying biological pathways of its complex molecular profile. We used vasopressin 2 (VASH2) as a research target and overexpressed and knocked down VASH2 in SHH medulloblastoma cell lines DAOY by lentiviral vectors in vitro, respectively, to investigate its role in SHH medulloblastoma cell lines DAOY cell proliferation, apoptosis, migration, invasion and biological roles in the cell cycle. (1) Among 47 pediatric medulloblastoma cases, 8 were WNT type, 29 were SHH type, and 10 were non-WNT/SHH type. the positive rate of VASH2 was highest in the SHH type with a 68.97% positive rate, followed by non-WNT/SHH and lowest in the WNT type. The results of the multifactorial analysis showed that positive expression of VASH2 was associated with medulloblastoma molecular subtype (SHH type), site of tumor development (four ventricles), and gender (male), P < 0.05. (2) The results of cellular experiments showed that overexpression of VASH2 increased the invasion and migration ability of medulloblast Daoy, while knockdown of VASH2 inhibited the invasion and Overexpression of VASH2 upregulated the expression of Smad2 + 3, Smad4, Mmp2 and the apoptotic indicators Bcl-2 and Caspase3, while knockdown of VASH2 suppressed the expression of Smad2 + 3 and Mmp2, and silenced the expression of Smad4 and the apoptotic indicators Bcl2, Caspase3 expression. Flow cytometric cycle analysis showed that VASH2 overexpression increased the S phase in the Daoy cell cycle, while VASH2 knockdown decreased the S phase in the SHH medulloblastoma cell lines DAOY cell cycle. Bioinformatics analysis showed that there was no statistically significant difference between the expression of VASH2 genes in the GSE30074 and GSE202043 datasets and the prognosis of the patients, but the results of this dataset analysis suggested that we need to continue to expand the sample size of the study in the future. The results of the GO enrichment analysis showed that the angiogenic pathway was the most significantly enriched, and the PPI interactions network of VASH2 was obtained from the STRING database. Using the STRING database, we obtained the PPI interaction network of VASH2, and the KEGG enrichment analysis of VASH2-related genes showed that VASH2-related genes were related to the apoptosis pathway, and therefore it was inferred that VASH2 also affects the development of tumors through apoptosis. We found for the first time that the positive expression rate of VASH2 was closely associated with SHH-type pediatric medulloblastoma and that VASH2 was involved in the invasion, migration, cell cycle, and apoptotic capacity of SHH medulloblastoma cell lines DAOY by affecting downstream indicators of the TGF-β pathway. This suggests that it is involved in the progression of pediatric medulloblastoma, and VASH2 is expected to be a diagnostic and therapeutic target for SHH-type pediatric medulloblastoma.
摘要：
研究VASH2在不同分子亚型小儿髓母细胞瘤(MB)肿瘤组织中的表达差异,分析VASH2与髓母细胞瘤分子亚型的相关性,临床病理资料,和预后，探讨VASH2在SHH髓母细胞瘤细胞株DAOY中的作用机制。我们分析了2011年1月至2019年12月新疆医科大学第一附属医院小儿神经外科收治的47例小儿髓母细胞瘤病例，采用免疫组织化学（IHC）和分子分型（WNT型、SHH型,和非WNT/SHH型）。分析VASH2与髓母细胞瘤分子分型的相关性。我们还分析了GEO数据库（GSE30074和GSE202043）中的髓母细胞瘤数据集，以探讨VASH2与髓母细胞瘤患者预后之间的相关性。以及专门针对VASH2基因进行了全面的GO富集分析，以揭示其复杂分子谱的潜在生物学途径。我们以血管加压素2（VASH2）为研究靶点，通过慢病毒载体体外在SHH髓母细胞瘤细胞株DAOY中过表达和敲低VASH2，分别,探讨其在SHH髓母细胞瘤细胞系DAOY细胞增殖中的作用,凋亡，迁移,侵袭和细胞周期中的生物学作用。(1)47例小儿髓母细胞瘤中,8是WNT型，29个是SHH型，10例为非WNT/SHH型。SHH型VASH2阳性率最高，阳性率为68.97%，其次是非WNT/SHH，在WNT类型中最低。多因素分析结果显示,VASH2阳性表达与髓母细胞瘤分子亚型(SHH型)有关,肿瘤发展部位（四脑室），和性别（男性），P<0.05。(2)细胞实验结果表明,VASH2的过表达增加了髓母细胞Daoy的侵袭和迁移能力,VASH2的敲除抑制侵袭，VASH2的过表达上调了Smad23，Smad4，Mmp2和凋亡指标Bcl-2和Caspase3的表达，而VASH2的敲除抑制了Smad23和Mmp2的表达，沉默了Smad4和凋亡指标Bcl2，Caspe3的表达。流式细胞仪分析显示VASH2过表达增加了Daoy细胞周期的S期,而VASH2敲除降低SHH髓母细胞瘤细胞系DAOY细胞周期的S期。生物信息学分析显示,GSE30074和GSE202043数据集的VASH2基因表达与患者预后无统计学差异,但是这个数据集分析的结果表明，我们需要在未来继续扩大研究的样本量。GO富集分析结果表明，血管生成途径的富集最为显著，并从STRING数据库获得VASH2的PPI相互作用网络。使用STRING数据库，我们获得了VASH2的PPI相互作用网络，对VASH2相关基因的KEGG富集分析显示，VASH2相关基因与凋亡通路有关，因此推断VASH2也通过凋亡影响肿瘤的发展。我们首次发现VASH2的阳性表达率与SHH型小儿髓母细胞瘤的侵袭密切相关,迁移,细胞周期,和SHH髓母细胞瘤细胞系DAOY的凋亡能力通过影响TGF-β途径的下游指标。这表明它与小儿髓母细胞瘤的进展有关，VASH2有望成为SHH型小儿髓母细胞瘤的诊断和治疗靶点。
