Senescence-associated lncRNAs: senescence-associated long noncoding RNAs

Abstract

Noncoding RNAs include small transcripts, such as microRNAs and piwi-interacting RNAs, and a wide range of long noncoding RNAs (lncRNAs). Although many lncRNAs have been identified, only a small number of lncRNAs have been characterized functionally. Here, we sought to identify lncRNAs differentially expressed during replicative senescence. We compared lncRNAs expressed in proliferating, early-passage, 'young' human diploid WI-38 fibroblasts [population doubling (PDL) 20] with those expressed in senescent, late-passage, 'old' fibroblasts (PDL 52) by RNA sequencing (RNA-Seq). Numerous transcripts in all lncRNA groups (antisense lncRNAs, pseudogene-encoded lncRNAs, previously described lncRNAs and novel lncRNAs) were validated using reverse transcription (RT) and real-time, quantitative (q)PCR. Among the novel senescence-associated lncRNAs (SAL-RNAs) showing lower abundance in senescent cells, SAL-RNA1 (XLOC_023166) was found to delay senescence, because reducing SAL-RNA1 levels enhanced the appearance of phenotypic traits of senescence, including an enlarged morphology, positive β-galactosidase activity, and heightened p53 levels. Our results reveal that the expression of known and novel lncRNAs changes with senescence and suggests that SAL-RNAs play direct regulatory roles in this important cellular process.

Keywords: noncoding; post-transcriptional gene regulation; proliferation; senescence-associated gene expression patterns; transcriptome.

Figure 1. Characterization of proliferating and senescent WI-38 human diploid fibroblasts

(A) Micrographs to visualize senescence-associated β-galactosidase activity in early-passage, proliferating (P) [population doubling (PDL) 20], and late-passage, senescent (S) (PDL 52) WI-38 cells. (B) Detection of F-actin to visualize the cytoskeleton using rhodamine phalloidin in fixed and permeabilized WI-38 cells. (C) Western blot analysis of the levels of p21, p53, HuR, Sirt1, and loading control β-Actin in P and S cells. The even loading and transfer of the samples was confirmed by Coomassie blue and Ponceau red staining of the gel and membrane, respectively (not shown). (D) Naturally occurring antisense transcripts (NA) lncRNAs differentially expressed in P relative to S WI-38 fibroblasts, as identified by using RNA-Seq analysis; NA-SAL-RNAs elevated in S cells (top) and in P cells (bottom) are listed. (E,F) Individual validation of NA-SAL-RNAs displaying higher abundance in S cells (E) or in P cells (F).

Figure 2. Pseudogene-encoded transcripts differentially expressed in proliferating and senescent WI-38 fibroblasts

(A) Pseudogene-encoded (PE)-SAL-RNAs transcript lncRNAs differentially expressed in P relative to S WI-38 fibroblasts, as identified by using RNA-Seq analysis; PE-SAL-RNAs elevated in S cells (top) and in P cells (bottom) are listed. (B,C) Individual validation of PE-SAL-RNAs displaying higher abundance in S cells (B) or in P cells (C).

Figure 3. Known lncRNAs differentially expressed in proliferating and senescent WI-38 fibroblasts

(A) LncRNAs differentially expressed in P relative to S WI-38 fibroblasts, identified by using RNA-Seq analysis; lncRNAs elevated in S cells (top) and in P cells (bottom) are listed. (B,C) Individual validation of lncRNAs displaying higher abundance in S cells (B) or in P cells (C). (D) Northern blot analysis of the levels of lncRNAs MALAT1, MIAT, and loading control 18S rRNA; the position of 28S rRNA is indicated for size reference. (E,F) Ten days after transfection with siRNAs to silence MALAT1 or MIAT, the levels of remaining lncRNA were monitored by RT-qPCR analysis (E), and the senescent phenotype was assessed by assessing SA-βgal-positive cells (quantified below) and protein senescence markers p21 and p53 (upregulated), as well as HuR and Sirt1 (downregulated) (F).

Figure 4. Novel SAL-RNAs preferentially expressed in proliferating WI-38 fibroblasts

(A) SAL-RNAs more abundant in P than in S WI-38 fibroblasts, identified by using RNA-Seq analysis. (B) Example of a novel SAL-RNA discovered by RNA-Seq analysis of WI-38 fibroblasts; coverage plot illustrating the increased presence of XLOC_023166 (SAL-RNA1) in P compared with S cells. (C) Validation of seven novel SAL-RNAs preferentially expressed in P cells. (D) Relative abundance in nucleus and cytoplasm of the SAL-RNAs measured in (C). The mainly cytoplasmic lncRNA 7SL and the mainly nuclear lncRNA 7SK were included as controls for sample fractionation.

Figure 5. Novel SAL-RNAs preferentially expressed in senescent WI-38 fibroblasts

(A) SAL-RNAs more abundant in S than in P WI-38 fibroblasts, identified by using RNA-Seq analysis. (B) Example of a novel lncRNA discovered by RNA-Seq analysis of WI-38 fibroblasts; coverage plot illustrating the increased presence of XLOC_025931 (SAL-RNA2) in S compared with P cells. (C) Validation of seven novel SAL-RNAs preferentially expressed in S cells. (D) Relative abundance in nucleus and cytoplasm of the SAL-RNAs measured in (C). The mainly nuclear lncRNA XIST was included as a control for sample fractionation.

Figure 6. Novel SAL-RNAs preferentially expressed in senescent WI-38 fibroblasts

(A) Degree of silencing achieved 5 days after transfecting WI-38 cells with siRNAs directed at SAL-RNA1, SAL-RNA2, and SAL-RNA3, as assessed by RT-qPCR analysis. (B–D) In cells treated as explained in (A), F-actin was detected using rhodamine phalloidin in order to visualize the cytoskeleton (B), SA-βgal activity was assessed in order to monitor cellular senescence (B), proliferation was measured by monitoring changes in cell number (C) and apoptosis was examined by measuring the level of cleaved PARP using Western blot analysis (D). (E) Western blot analysis to detect the levels of p53 in WI-38 10 days after silencing SAL-RNAs preferentially expressed in senescent cells. (F,G) By 10 days after transfecting WI-38 fibroblasts (P), with SAL-RNA1-directed siRNA, the levels of TP53 mRNA and p53 were measured by RT-qPCR and Western blot analyses, respectively (F), and senescent cells were visualized by assessing SA-βgal-positive cells (G, left) and protein senescence markers p21 and p53 (upregulated), as well as HuR and Sirt1 (downregulated) (G, right).