Genome-Wide Analysis of Differentially Expressed miRNAs and Their Associated Regulatory Networks in Lenses Deficient for the Congenital Cataract-Linked Tudor Domain Containing Protein TDRD7.
Genome-Wide Analysis of Differentially Expressed miRNAs and Their Associated Regulatory Networks in Lenses Deficient for the Congenital Cataract-Linked Tudor Domain Containing Protein TDRD7.
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DOI：
10.3389/fcell.2021.615761
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发表时间：
2021
影响因子：
5.5
通讯作者：
Lachke SA
中科院分区：
文献类型：
--
作者：
Anand D;Al Saai S;Shrestha SK;Barnum CE;Chuma S;Lachke SA
TDRD7编码Tudor结构域蛋白，参与转录后基因表达调控，TDRD7突变/缺失导致人类早发性白内障。虽然Tdrd7与关键的晶状体mRNAs的调控有关，但Tdrd7缺乏对microRNAs(MiRNAs)的影响以及这如何导致转录组错误表达和白内障，尚不清楚。我们通过研究表现出完全穿透性青少年白内障的Tdrd7靶向基因敲除(Tdrd7-/-)小鼠来解决这一关键的知识鸿沟。我们在出生后第4天对Tdrd7/-小鼠的晶状体进行了Affymetrix miRNA 3.0微阵列分析，这是白内障形成的一个阶段。这一分析确定了22个miRNA[14个过度表达(miR-15a，miR-19a，miR-138，miR-328，miR-339，miR-345，miR-378b，miR-384，miR-467a，miR-1224，miR-1935，miR-1946a，miR-3102，miR-3107)，8个下调(let-7b，miR-34c，miR-298，miR-382，miR-409，miR-1198，miR-1947，miR-3092)]显著错误表达(折叠变化≥±1.2，p<；0.05)在Tdrd7/-透镜中。为了了解这些错误表达的miRNAs如何影响Tdrd7/-白内障，我们预测了它们的mRNA靶标，并通过对P4和P30 Tdrd7/-晶状体进行比较转录分析，检测了它们在Tdrd7-/-晶状体缺乏时的错误表达。为了确定这些靶标mRNAs的优先顺序，我们使用了各种严格的过滤器(例如，Tdrd7/-LENS中的折叠变化，基于iSyTE的晶状体富集型表达)，并分别确定了98个过表达和89个高表达的miRNAs靶基因，它们被归类为“最高优先级”、“高优先级”和“有希望的”候选基因。对于Tdrd7/-Lens过表达的miRNAs，该方法确定了18个最优先的降低的靶mRNAs：ALAD、Ankrd46、Ceacam10、Dgat2、EDNRB、H2-Eb1、Klhl22、Lin7a、Loxl1、LPIN1、NPC1、Olfm1、Ppm1e、Ppp1r1a、Rgs8、Shisa4、Snx22和Wnk2。这些靶点在晶状体缺陷/白内障的其他基因特异性干扰小鼠模型(如BRG1、E2F1/E2f2/E2f3、Foxe3、HSF4、Klf4、Mafg/Mafk、Notch)中也发生了改变，表明它们在晶状体生物学中的重要性。基因本体论(GO)提供了对它们与晶状体病理学相关性的进一步洞察。例如，Tdrd7缺陷的晶状体囊膜缺陷可以通过与“基底膜”相关的mRNA靶标(例如，Col4A3、Loxl1、Timp2、TIMP3)的减少来解释。GO分析还发现了最近与晶状体生物学/病理学相关的新基因(如CASZ1、RASGRP1)。总之，这些分析定义了一个新的Tdrd7下游miRNA-mRNA网络，进而发现了几个新的与晶状体生物学相关的mRNA靶点及其相关途径，并为先天性白内障的病理提供了分子洞察力。
Mutations/deficiency of TDRD7, encoding a tudor domain protein involved in post-transcriptional gene expression control, causes early onset cataract in humans. While Tdrd7 is implicated in the control of key lens mRNAs, the impact of Tdrd7 deficiency on microRNAs (miRNAs) and how this contributes to transcriptome misexpression and to cataracts, is undefined. We address this critical knowledge-gap by investigating Tdrd7-targeted knockout (Tdrd7-/-) mice that exhibit fully penetrant juvenile cataracts. We performed Affymetrix miRNA 3.0 microarray analysis on Tdrd7-/- mouse lenses at postnatal day (P) 4, a stage preceding cataract formation. This analysis identifies 22 miRNAs [14 over-expressed (miR-15a, miR-19a, miR-138, miR-328, miR-339, miR-345, miR-378b, miR-384, miR-467a, miR-1224, miR-1935, miR-1946a, miR-3102, miR-3107), 8 reduced (let-7b, miR-34c, miR-298, miR-382, miR-409, miR-1198, miR-1947, miR-3092)] to be significantly misexpressed (fold-change ≥ ± 1.2, p-value < 0.05) in Tdrd7-/- lenses. To understand how these misexpressed miRNAs impact Tdrd7-/- cataract, we predicted their mRNA targets and examined their misexpression upon Tdrd7-deficiency by performing comparative transcriptomics analysis on P4 and P30 Tdrd7-/- lens. To prioritize these target mRNAs, we used various stringency filters (e.g., fold-change in Tdrd7-/- lens, iSyTE-based lens-enriched expression) and identified 98 reduced and 89 elevated mRNA targets for overexpressed and reduced miRNAs, respectively, which were classified as “top-priority” “high-priority,” and “promising” candidates. For Tdrd7-/- lens overexpressed miRNAs, this approach identified 18 top-priority reduced target mRNAs: Alad, Ankrd46, Ceacam10, Dgat2, Ednrb, H2-Eb1, Klhl22, Lin7a, Loxl1, Lpin1, Npc1, Olfm1, Ppm1e, Ppp1r1a, Rgs8, Shisa4, Snx22 and Wnk2. Majority of these targets were also altered in other gene-specific perturbation mouse models (e.g., Brg1, E2f1/E2f2/E2f3, Foxe3, Hsf4, Klf4, Mafg/Mafk, Notch) of lens defects/cataract, suggesting their importance to lens biology. Gene ontology (GO) provided further insight into their relevance to lens pathology. For example, the Tdrd7-deficient lens capsule defect may be explained by reduced mRNA targets (e.g., Col4a3, Loxl1, Timp2, Timp3) associated with “basement membrane”. GO analysis also identified new genes (e.g., Casz1, Rasgrp1) recently linked to lens biology/pathology. Together, these analyses define a new Tdrd7-downstream miRNA-mRNA network, in turn, uncovering several new mRNA targets and their associated pathways relevant to lens biology and offering molecular insights into the pathology of congenital cataract.
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