Prenatal cadmium exposure alters proliferation in mouse CD4+ T cells via LncRNA Snhg7

Prenatal cadmium (Cd) exposure leads to immunotoxic phenotypes in the offspring. Long non-coding RNAs (lncRNAs) are integral to T cell regulation. Here, we identified genes and pathways altered in CD4+ T cell by prenatal Cd exposure. We investigated the role of long non-coding RNA small nucleolar RNA hostgene 7 (lncSnhg7) in T cell proliferation: LncSnhg7 expression increases in CD4+ T cells following stimulation with anti-CD3/CD28 beads. LngSnhg7 and a downstream protein, GALNT7, are upregulated in T cells from offspring exposed to Cd during gestation. Overexpression of miR-34a, a regulator of lnhcSnhg7 and GALNT7, suppresses GALNT7 protein levels in primary T cells, but not in a mouse T lymphocyte cell line. The T cells isolated from Cd-exposed offspring exhibit increased proliferation after activation in vitro, but Treg suppression and CD4+ T cell apoptosis are not affected by prenatal Cd exposure. In conclusion, prenatal Cd exposure alters the expression of lncRNAs during T cell activation. The induction of lncSnhg7 is enhanced in splenic T cells from Cd offspring resulting in the upregulation of GALNT7 protein and increased proliferation following activation. miR-34a overexpression decreased GALNT7 expression suggesting that the lncSnhg7/miR-34a/GALNT7 is an important pathway in primary CD4+ T cells. These data highlight the need to understand the consequences of environmental exposures on lncRNA functions in non-cancerous cells. Overall design: Male and female C.Cg-Foxp3tm2Tch/J mice were mated with continuous exposure to 10 ppm CdCl2 (or unspiked water for the controls) via their drinking water. Female mice were exposed throughout pregnancy. Spiked water was replaced with normal water within 12 hours of the birth of the pups. Control and Cd-exposed offspring were aged to 8 weeks. CD4 T Cells were isolated from the splenocytes using negative selection. CD4 T cells were stimulated in vitro with anti-CD3/CD28 beads for 16h. RNA were purified from the unstimulated and stimulated cells. Gene expression was analyzed by RNA-Sequencing.

产前镉（cadmium, Cd）暴露可对子代产生免疫毒性表型。长链非编码RNA（long non-coding RNAs, lncRNAs）在T细胞调控中发挥不可或缺的作用。本研究明确了产前镉暴露后CD4+ T细胞中发生改变的基因与通路。
我们探究了长链非编码RNA小核仁RNA宿主基因7（long non-coding RNA small nucleolar RNA hostgene 7, lncSnhg7）在T细胞增殖中的作用：经抗CD3/CD28磁珠刺激后，CD4+ T细胞内的lncSnhg7表达水平显著升高。
妊娠期间镉暴露的子代T细胞中，lncSnhg7及其下游蛋白GALNT7的表达均被上调。作为lncSnhg7与GALNT7的调控因子，miR-34a的过表达可抑制原代T细胞内GALNT7的蛋白水平，但对小鼠T淋巴细胞系无此作用。
从镉暴露子代分离的T细胞在体外活化后增殖能力增强，但产前镉暴露并未影响调节性T细胞（Treg）的抑制功能以及CD4+ T细胞的凋亡水平。
综上，产前镉暴露可改变T细胞活化过程中长链非编码RNA的表达模式。镉暴露子代的脾脏T细胞中lncSnhg7的诱导表达增强，进而导致GALNT7蛋白表达上调，并在活化后促进细胞增殖。miR-34a过表达可降低GALNT7的表达水平，提示lncSnhg7/miR-34a/GALNT7通路在原代CD4+ T细胞中发挥重要调控作用。本研究结果凸显了探究环境暴露对非肿瘤细胞内长链非编码RNA功能的影响的必要性。
实验设计：将雌雄C.Cg-Foxp3tm2Tch/J小鼠进行交配，通过饮水持续暴露于10 ppm氯化镉（CdCl2）（对照组给予未添加镉的正常饮水）。雌鼠在整个妊娠期均保持暴露状态，于仔鼠出生后12小时内更换为正常饮水。对照组与镉暴露组子代小鼠均饲养至8周龄。采用阴性筛选法从脾脏淋巴细胞中分离CD4+ T细胞，经抗CD3/CD28磁珠体外刺激16小时；分别收集未刺激与刺激后的细胞，纯化总RNA，通过RNA测序（RNA-Sequencing）分析基因表达水平。