Wei Min, Shi Rong, Zeng Jun, Wang Nisha, Zhou Jueyu, Ma Wenli
Institute of Genetic Engineering, Southern Medical University, Guangzhou, Guangdong, People's Republic of China,
Tumour Biol. 2015 Jan;36(1):291-302. doi: 10.1007/s13277-014-2614-5. Epub 2014 Sep 25.
Aquaporin genes are differentially expressed in primitive versus definitive erythropoiesis. Our previous research results showed that over-expression of aquaporin-1 (AQP1) gene greatly promotes the erythroid differentiation of erythroleukemia K562 cells, using benzidine staining and quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) analysis for representative erythroid-related genes, including γ-globin. But the molecular mechanisms underlying erythroid-specific gene regulation remain unknown. In this study, we demonstrated that AQP1 induced hemoglobins expression and altered erythroid gene expression by microarray analysis in K562 cells. The retroviral expression vector of AQP1 (pBABE-puro-AQP1) was constructed and infected K562 cells to establish a stable AQP1 over-expression cell line (K562-AQP1). AQP1 over-expression effectively inhibited cell proliferation and induced cell growth arrest in G1 phase of K562 cells. Then microarray profile was applied to analyze the differentially expressed genes which involved the mechanism of AQP1 in erythroid differentiation induction. The DAVID functional annotation clustering tool was used to identify biological functions enriched with the differentially expressed genes (n = 466 genes) and to group genes into clusters based on their functional similarity. Significant enrichment of genes involved in "oxygen transporter activity" (p = 3.8E-7) including hemoglobins (HBD, HBG, HBB, HBE1, and HBQ1), HEMGN, and EBP42 were validated by qRT-PCR. Moreover, silencing of HEMGN by RNA interference in K562-AQP1 cells resulted in down-regulation of these genes. These data provide a better understanding of the role of AQP1 in erythroid differentiation, by promoting HEMGN induction and other potential signaling pathways associated with hemoglobin induction.
水通道蛋白基因在原始红细胞生成与定型红细胞生成中存在差异表达。我们之前的研究结果表明，水通道蛋白-1（AQP1）基因的过表达极大地促进了红白血病K562细胞的红系分化，采用联苯胺染色以及对包括γ-珠蛋白在内的代表性红系相关基因进行定量逆转录聚合酶链反应（qRT-PCR）分析。但是红系特异性基因调控的分子机制仍然未知。在本研究中，我们通过微阵列分析证明AQP1在K562细胞中诱导血红蛋白表达并改变红系基因表达。构建了AQP1的逆转录病毒表达载体（pBABE-puro-AQP1）并感染K562细胞，以建立稳定的AQP1过表达细胞系（K562-AQP1）。AQP1的过表达有效抑制了K562细胞的增殖并诱导细胞在G1期生长停滞。然后应用微阵列图谱分析参与AQP1诱导红系分化机制的差异表达基因。使用DAVID功能注释聚类工具来鉴定富含差异表达基因（n = 466个基因）的生物学功能，并根据基因的功能相似性将其分组。通过qRT-PCR验证了参与“氧转运活性”（p = 3.8E-7）的基因的显著富集，这些基因包括血红蛋白（HBD、HBG、HBB、HBE1和HBQ1）、HEMGN和EBP42。此外，在K562-AQP1细胞中通过RNA干扰使HEMGN沉默导致这些基因的下调。这些数据通过促进HEMGN诱导以及与血红蛋白诱导相关的其他潜在信号通路，为更好地理解AQP1在红系分化中的作用提供了依据。
