Genome-wide identification of chromatin-enriched RNA reveals that unspliced dentin matrix protein-1 mRNA regulates cell proliferation in squamous cell carcinoma.
Genome-wide identification of chromatin-enriched RNA reveals that unspliced dentin matrix protein-1 mRNA regulates cell proliferation in squamous cell carcinoma.
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DOI：
10.1016/j.bbrc.2017.12.136
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发表时间：
2018-01
影响因子：
3.1
通讯作者：
Shigeki Suzuki;Hiroaki Hoshino;Kazuma Yoshida;J. Nakanishi;Shizu Tsuchiya-Hirata;Seiji Kobuke;N. Haruyama;F. Nishimura;H. Shiba
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文献类型：
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作者：
Shigeki Suzuki;Hiroaki Hoshino;Kazuma Yoshida;J. Nakanishi;Shizu Tsuchiya-Hirata;Seiji Kobuke;N. Haruyama;F. Nishimura;H. Shiba
富含染色质的非编码RNA（ncRNA）通过与染色质相关蛋白相互作用而成为表观遗传过程中的关键分子。最近，蛋白质编码的mRNA基因已被报道染色质栓系，类似于ncRNA。然而，很少有人知道是否染色质富集的mRNA参与染色质修饰过程。在这里，我们通过RNA亚细胞定位分析全面检查了鳞状细胞癌（SQCC）细胞中富含染色质的RNA，该分析是RNA分级分离和RNA-seq的组合。我们鉴定了11种mRNA作为高度染色质富集的RNA。其中牙本质基质蛋白-1（dentinmatrixprotein-1，DNT-1）基因在SQCC细胞中的表达尚未见报道。此外，我们还阐明了SQCC在体外和体内的染色质中保留了未剪接的mRNA。由于抑制未剪接的CDKN 1 mRNA（unspeaking-1）表达导致SQCC细胞增殖降低，我们进行了ChIP-qPCR以鉴定其表达被unspeaking-1表观遗传修饰的细胞周期相关基因，并发现CDKN 1B启动子通过抑制unspeaking-1表达在SQCC细胞中变得活跃。这一结果进一步证实了在用siRNA处理的细胞中增加了针对unspiral-1的CDKN 1B基因表达，并且通过同时抑制SQCC细胞中CDKN 1B表达来恢复降低的细胞增殖率。此外，为了检测unsports-1是否能够与CDKN 1B启动子区结合，用与PP 7 RNA茎环的24个重复融合的unsports-1（unsports-1- 24 xPP 7）瞬时转染稳定表达PP 7-mCherry融合蛋白的SQCC细胞，我们发现unsports-1- 24 xPP 7与抗mCherry抗体有效沉淀，并在CDKN 1B启动子区显著富集。因此，unspectin-I是一种新的富含染色质的RNA，其在表观遗传学上调节SQCC的细胞增殖。
Chromatin-enriched noncoding RNAs (ncRNAs) have emerged as key molecules in epigenetic processes by interacting with chromatin-associated proteins. Recently, protein-coding mRNA genes have been reported to be chromatin-tethered, similar with ncRNA. However, very little is known about whether chromatin-enriched mRNA is involved in the chromatin modification process. Here, we comprehensively examined chromatin-enriched RNA in squamous cell carcinoma (SQCC) cells by RNA subcellular localization analysis, which was a combination of RNA fractionation and RNA-seq. We identified 11 mRNAs as highly chromatin-enriched RNAs. Among these, we focused on the dentin matrix protein-1 (DMP-1) gene because its expression in SQCC cells has not been reported. Furthermore, we clarified that DMP-1 mRNA was retained in chromatin in its unspliced form in SQCCin vitroandin vivo. As the inhibition of the unspliced DMP-1 mRNA (unspDMP-1) expression resulted in decreased cellular proliferation in SQCC cells, we performed ChIP-qPCR to identify cell cycle-related genes whose expression was epigenetically modified by unspDMP-1, and found that the CDKN1B promoter became active in SQCC cells by inhibiting unspDMP-1 expression. This result was further validated by the increasedCDKN1Bgene expression in the cells treated with siRNA for unspDMP-1 and by restoration of the decreased cellular proliferation rate by simultaneously inhibitingCDKN1Bexpression in SQCC cells. Further, to examine whether unspDMP-1 was able to associate with theCDKN1Bpromoter region, SQCC cells stably expressing PP7-mCherry fusion protein were transiently transfected with the unspDMP-1 fused to 24 repeats of the PP7 RNA stem loop (unspDMP-1-24xPP7) and we found that unspDMP-1-24xPP7 was efficiently precipitated with the antibody against mCherry and was significantly enriched in theCDKN1Bpromoter region. Thus, unspDMP-1 is a novel chromatin-enriched RNA that epigenetically regulates cellular proliferation of SQCC.