Background and Aims

The mammalian intestinal epithelium self-renews rapidly and homeostasis is preserved via tightly controlled mechanisms. Long noncoding RNAs transcribed from ultraconserved regions (T-UCRs) control different cell functions, but little is known about their role in maintaining the integrity of the intestinal epithelium. We searched for T-UCRs that regulate growth of the intestinal mucosa and investigated the mechanism by which T-UCR uc.173 regulates epithelial renewal.

Methods

C57BL/6J mice were deprived of food for 48 hours in fasting experiments. Some mice were given intraperitoneal injections of a plasmid encoding LNA-anti-uc.173, to knock down endogenous uc.173. For studies using organoids, primary enterocytes were isolated from the intestine and transfected with the uc.173 transgene to increase uc.173 levels. Intestinal epithelial cells (Caco-2 and IEC-6 lines) were transfected with LNA-anti-uc.173 or uc.173 transgene. We quantified intestinal epithelial renewal based on BrdU incorporation, villus height and crypt depth, and cell number. The association of uc.173 with microRNA 195 (miRNA195) was determined by RNA pull-down assays.

Results

Genome-wide profile analyses identified 21 T-UCRs, including uc.173, that were differentially expressed between intestinal mucosa of fasted vs non-fasted mice. Increasing levels of uc.173 by expression of a transgene increased growth of intestinal epithelial cells and organoids. Decreasing uc.173 levels by LNA-anti-uc.173 in mice reduced renewal of the intestinal epithelium. We found that uc.173 interacted directly with the primary transcript of miRNA195, leading to miRNA195 degradation.

Conclusions

In analyses of intestinal epithelial cells and mice, we identified uc.173 noncoding RNA that regulates growth of the intestinal mucosa and stimulates intestinal epithelial renewal by reducing levels of miRNA195.

中文翻译：

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长的非编码RNA uc.173通过诱导MicroRNA 195的降解促进肠粘膜的更新。

背景和目标

哺乳动物的肠上皮自我更新很快，并且通过严格控制的机制来保持体内稳态。从超保守区（T-UCR）转录的长非编码RNA控制着不同的细胞功能，但对其维持肠道上皮完整性的作用知之甚少。我们搜索了调节肠粘膜生长的T-UCR，并研究了T-UCR uc.173调节上皮更新的机制。

方法

在禁食实验中，将C57BL / 6J小鼠的食物剥夺48小时。给一些小鼠腹膜内注射编码LNA-anti-uc.173的质粒，以敲除内源性uc.173。对于使用类器官的研究，从肠中分离出原代肠上皮细胞，并用uc.173转基因进行转染以增加uc.173的水平。用LNA-抗uc.173或uc.173转基因转染肠上皮细胞（Caco-2和IEC-6系）。我们基于BrdU掺入，绒毛高度和隐窝深度以及细胞数量对肠上皮更新进行了定量。uc.173与microRNA 195（miRNA195）的关联是通过RNA下拉测定法确定的。

结果

全基因组概况分析确定了21个T-UCR，包括uc.173，在空腹小鼠和非空腹小鼠的肠粘膜之间差异表达。通过转基因表达增加uc.173的水平可以增加肠上皮细胞和类器官的生长。LNA-anti-uc.173在小鼠中降低uc.173水平可减少肠上皮的更新。我们发现uc.173与miRNA195的主要转录本直接相互作用，导致miRNA195降解。

结论

在对肠上皮细胞和小鼠的分析中，我们鉴定了uc.173非编码RNA，该RNA调节肠粘膜的生长并通过降低miRNA195的水平刺激肠上皮的更新。