The long non-coding RNA MIR31HG regulates the senescence associated secretory phenotype

Abstract

Oncogene-induced senescence provides a barrier against malignant transformation. However, it can also promote cancer through the secretion of a plethora of factors released by senescent cells, called the senescence associated secretory phenotype (SASP). We have previously shown that in proliferating cells, nuclear lncRNA MIR31HG inhibits p16/CDKN2A expression through interaction with polycomb repressor complexes and that during BRAF-induced senescence, MIR31HG is overexpressed and translocates to the cytoplasm. Here, we show that MIR31HG regulates the expression and secretion of a subset of SASP components during BRAF-induced senescence. The SASP secreted from senescent cells depleted for MIR31HG fails to induce paracrine invasion without affecting the growth inhibitory effect. Mechanistically, MIR31HG interacts with YBX1 facilitating its phosphorylation at serine 102 (p-YBX1^S102) by the kinase RSK. p-YBX1^S102 induces IL1A translation which activates the transcription of the other SASP mRNAs. Our results suggest a dual role for MIR31HG in senescence depending on its localization and points to the lncRNA as a potential therapeutic target in the treatment of senescence-related pathologies.

Fig. 1. MIR31HG knock-down decreases the induction of SASP components during BRAF-induced senescence.

a Relative MIR31HG expression normalized to housekeeping genes (HPRT1 and RPLP0) in BJ ER:BRAF cells were treated with ethanol (Control) or 1 μM 4-OHT for 48 h (n = 4). b Box plot showing MIR31HG expression in log2 (fpkm + 1) units in thyroid carcinoma (THCA) and colorectal carcinoma (CRC) comparing BRAF mutant and BRAF wild-type tumours. The box plot represents the median (middle line), the box indicates the first and third quartiles and the whiskers indicate ±1.5 × interquartile range. Two-tailed Wilcoxon test was performed to compare the expression differences in the BRAF mutant and BRAF wild-type tumours (CRC BRAF_wt = 309; CRC BRAF_mutant = 49; THCA BRAF_mutant = 290; THCA BRAF_wt = 199). c Heat map showing relative expression of differentially expressed genes in BJ ER:BRAF cells (control or siMIR31HG), treated with ethanol (Control 1–3) or 1 μM 4-OHT for 48 h (Sen 1–3 and Sen siMIR31HG 1–3). d Heat map showing relative expression in RPKMs of a subset of SASP genes previously defined from the data provide in a, where differentially expressed genes (‘DE’, FDR < 0.01) are indicated in red, unchanged (non-significant, ‘ns’) in white. e qRT-PCR analysis of selected components of the SASP normalized to housekeeping genes (HPRT1 and RPLP0) in BJ ER:BRAF cells transfected with the indicated siRNAs (Control or siMIR31HG1-2), treated with ethanol (Control) or 1 μM 4-OHT for 48 h. The graphs show results compared to control ethanol-treated set to 1 (n = 4). f Mass spectrometry-based secretome analysis of senescent BJ ER:BRAF (1 μM 4-OHT) treated with siMIR31HG or control siRNA. The volcano plot shows differentially secreted proteins in MIR31HG knock-down senescent cells (Sen siMIR31HG) compared to control senescence cells (Sen) (−log10 p value along y-axis and log fold change along x-axis). Names are displayed for significantly changed proteins between secretomes (pval < 0.01) and SASP proteins are marked in red. g Secreted IL6, CXCL1, IL8 and MMP3 (pg/ml) measured by ELISA in the CM of BJ ER:BRAF (Control or siMIR31HG1-2) treated for 72 h with ethanol (Control) or 1 μM 4-OHT (n = 3–5). All statistical significances were calculated using two-tailed Student’s t-tests, except b where two-tailed Wilcoxon test was used. *p < 0.05; **p < 0.01; ***p < 0.001; ns non-significant. All error bars represent means ± s.d. Source data are provided as a Source Data file.

Fig. 2. The SASP of senescent MIR31HG knock-down cells induces senescence but not invasion in a non-autonomous manner.

a Western blot analysis for p53 and GAPDH. in WT BJ cells incubated for 72 h with the CM collected from BJ ER:BRAF cells (Control or siMIR31HG1-2) treated with ethanol (Control) or 1 μM 4-OHT for 72 h. Molecular weight marker is shown in kDa. b Quantification of the western blot band intensities for p53 relative to GAPDH (n = 3). c Representative images of β-galactosidase staining from the same condition as indicated in a (n = 3). Scale bar: 50 μm. d Quantifiaction of the% of β-galactosidase-positive cells from c (n = 3). e Relative growth by crystal violet staining dissolved in acetic acid and measured at 590 nm in WT BJ cells incubated for 72 h with the CM collected from BJ ER:BRAF cells (Control or siMIR31HG1-2) treated with ethanol (Control) or 1 μM 4-OHT for 72 h. The graph shows the absorbance relative to the control cells set as 1 (n = 3). f qRT-PCR analysis of a subset of components of the SASP normalized to housekeeping genes (HPRT1 and RPLP0) in total RNA extracted from cells described in a. The graph shows the RNA expression relative to control ethanol-treated cells set to 1 (n = 4). g MDA-MB-231 invading cells through a matrigel membrane in contact with the CM collected from BJ ER:BRAF cells (Control or siMIR31HG) treated for 72 h with ethanol (Control) or 1 μM 4-OHT. Representative images are shown in the figure (n = 4). Scale bar: 50 μm. h Quantification of the invading cells relative to control ethanol-treated cells (n = 3). All statistical significances were calculated using two-tailed Student t-tests, *p < 0.05; **p < 0.01, ns, non-significant. All error bars represent means ± s.d. Source data are provided as a Source Data file.

Fig. 3. MIR31HG knock-down decreases IL1A translation.

a Western blot for p-RELA, RELA, CEBPB and GAPDH of BJ ER:BRAF cells (Control or siMIR31HG1-2) treated with ethanol (Control) or 1 μM 4-OHT for 72 h. Molecular weight marker is shown in kDa. b Quantification of the p-RELA and CEBPB western blot band intensities relative to GAPDH (n = 3). c Chromatin immunoprecipitation followed by qPCR for CEBPB binding to the IL6 promoter and to an unrelated region in BJ ER:BRAF cells (Control or siMIR31HG1) treated with ethanol (Control) or 1 μM 4-OHT for 48 h. The graph shows the percentage of the input that binds CEBPB (n = 3). d Immunofluorescence analysis for RELA and DAPI of BJ ER:BRAF cells (Control or siMIR31HG1-2) treated with ethanol (Control) or 1 μM 4-OHT for 72 h. Representative images are shown in the figure (n = 3). Scale bar: 20 μm. e Western blot for IL1A in BJ ER:BRAF cells (Control or siMIR31HG1-2) were treated with ethanol (Control) or 1 μM 4-OHT for 72 h. Molecular weight marker is shown in kDa (n = 3). f Quantification of IL1A western blot band intensities relative to Vinculin (n = 3). g Immunofluorescence of IL1A and DAPI in BJ ER:BRAF cells (Control or siMIR31HG1-2) treated with ethanol (Control) or 1 μM 4-OHT for 72 h. Representative merged images are shown in the figure (n = 3). Scale bar: 50 μm. h Relative RNA expression for the indicated SASP factors in BJ ER:BRAF cells transfected with the indicated siRNAs (Control or siMIR31HG1-2), treated with ethanol (Control) or 1 μM 4-OHT for 48 h, in the absence or presence of 10 ng/ml of h-rIL1A for 2 h before RNA extraction (n = 3). i Western blot for IL6 from precipitated protein from the media in the conditions indicated in h in the absence or presence of 10 ng/ml of h-rIL1A for 24 h before harvesting the CM (n = 3). S.e. short exposure, l.e. long exposure. j Polysome profile performed by sucrose gradient separation in senescent BJ ER:BRAF control cells (Sen Control, black) or siMIR31HG cells (Sen siMIR31HG, red) treated with 1 μM 4-OHT for 72 h. Y-axis shows the absorbance at 260 nM and X-axis shows the number of the fractions collected. Red area shows the heavy polysome fractions and grey area shows the light polysome fractions. One representative experiment is shown in the figure (n = 3). k Distribution of the amount of IL1A mRNA in the heavy polysome (red) light polysome (grey) or other fractions (white) from the experiment in g. l Relative amount of IL1A, IL6 and ACTB mRNA present in the heavy polysome fractions in the experiment described in j. m Western blot analysis of newly synthesized IL1A, Vinculin and GAPDH levels in control senescent cells and MIR31HG- depleted senescent cells purified by AHA pulse‐labelling and coupling to biotin followed by streptavidin pull‐down. As control, proliferating cells without AHA labelling is shown. HRP-streptavidin shows the uniform labelling of newly synthesized proteins coupled to biotin. A representative experiment is shown (n = 3). n Quantification of IL1A, Vinculin and GAPDH western blot band intensities from pulldown samples in senescence (Sen, white) and senescence MIR31HG KD cells (Sen-MIR31HG KD, grey) (n = 3). All statistical significances were calculated using two-tailed Student’s t-tests, *p < 0.05; **p < 0.01; ns non-significant. All error bars represent means ± s.d. Source data are provided as a Source Data file.

Fig. 4. MIR31HG interacts with YBX1 and YBX1 knock-down phenocopies MIR31HG depletion.

a Schematic representation of the pulldown procedure. b Cellular extracts from BJ ER:BRAF (Control or siMIR31HG) treated with 1 μM 4-OHT for 72 h were incubated with antisense oligos, enriched proteins extracted and subjected to LC-MS analysis (see ‘Methods'). The volcano plot highlights proteins enriched in the MIR31HG pulldown analysis compared to a luciferase control pull down. Marked in red are protein with p value <0.05. c Heat map showing the relative expression of significant differentially expressed SASP genes in BJ ER:BRAF cells (control or siMIR31HG) treated with ethanol (Control 1-3) or 1 μM 4-OHT for 48 h (Sen 1-3, Sen siMIR31HG 1-3, Sen YBX1-KD1-3). d Relative RNA expression of selected components of the SASP normalized to housekeeping genes (HPRT1 and RPLP0) in BJ ER:BRAF cells transfected with the indicated siRNAs (Control or siYBX1, 1–2), treated with ethanol (Control) or 1 μM 4-OHT for 48 h. The graph shows results compared to control ethanol-treated set to 1 (n = 4). e Secreted IL6 and CXCL1 (pg/ml) measured by ELISA in the CM of BJ ER:BRAF (Control or siYBX1, 1–2) treated for 72 h with ethanol (Control) or 1 μM 4-OHT (n = 3). f Western blot for IL1A in BJ ER:BRAF cells (Control or siYBX1, 1–2) were treated with ethanol (Control) or 1 μM 4-OHT for 72 h. Molecular weight marker is shown in kDa (n = 3). g Quantification of the IL1A western blot band intensities relative to Vinculin (n = 3). h Relative RNA binding using GFP-YBX1 in 4-OHT-treated BJ ER:BRAF, represented as the percentage of the input bound relative to an empty GFP cell line. YBX1 was used as a positive control (blue), MALAT1, ND1, and ND4 binding as negative controls (green) and cytokines binding (pink). The results are shown as the percentage of input relative to an empty GFP cell line treated in the same conditions (n = 3). i Top, schematic representation of MIR31HG with the putative YBX1-binding sites (BS) found in published iCLIP data. BS1-3 were found in Goodarzi et al. iCLIP data and BS5-4 were retrieved from Wu et al. dataset. Bottom, representation of MIR31HG truncations (Mut1 to Mut5). j EMSA showing the in vitro binding of 2 nM of the corresponding radiolabelled transcript and the indicated amounts of recombinant YBX1. BSA at the highest concentration (400 ng) was used as a negative control (n = 3). k EMSA showing the in vitro binding of 400 ng of YBX1 to 2 nM of the radiolabelled MIR31HG wild type in competition with the indicated amounts of unlabelled Mut5 or Mut3 (n = 3). All statistical significances were calculated using two-tailed Student’s t-tests, *p < 0.05; ns non-significant. All error bars represent means ± s.d. Source data are provided as a Source Data file.

Fig. 5. Phosphorylated YBX1 induces IL1A translation.

a Western blot for p-RSK1, RSK1, p-YBX1, total YBX1 and Vinculin in BJ ER:BRAF cells treated with 1 μM 4-OHT for the indicated time. Molecular weight marker is shown in kDa (n = 3). b Western blot for p-YBX1, total YBX1 and Vinculin in BJ ER:BRAF cells untreated (control) or treated with 1 μM 4-OHT and the indicated inhibitor at 1 μM for the indicated time. DMSO is used as vehicle control. Molecular weight marker is shown in kDa (n = 3). c Western blot for p-YBX1, total YBX1, IL1A and Vinculin in BJ ER:BRAF cells untreated (control) or treated with 1 μM 4-OHT and BI-D1780 at 1 μM for 72 h. Molecular weight marker is shown in kDa (n = 4). d Relative luciferase measurement in BJ ER:BRAF cells expressing a doxycycline-induced empty, wild type (WT), mutant S102A or mutant S102D versions of YBX1 were transfected with a siRNA against YBX1 and reporter constructs pGL3-promoter (control) or pGL3-promoter-3′UTR (3′UTR) containing the 3′UTR of IL1A mRNA and treated with ethanol (control) or with 1 μM 4-OHT for 48 h. The graph shows the Luciferase values normalized to renilla relative to the empty cell line in the absence of 4-OHT set as 1 (n = 4). e RIP analysis using a GFP-tagged version of WT YBX1, S102A mutant or S102D mutant in formaldehyde crosslinked cells induced with doxycycline and treated with 1 μM 4-OHT for 72 h. The graph shows YBX1 binding to MIR31HG, IL1A and MALAT as a negative control. The results are shown as the percentage of input relative to an empty GFP cell line treated in the same conditions (n = 4). f MDA-MB-231 invading cells through a matrigel membrane in contact with the CM from BJ ER:BRAF cells expressing a doxycycline-induced empty, wild type, mutant S102A or mutant S102D treated for 72 h with ethanol (Control) or 1 μM 4-OHT. Representative images are shown in the figure. Scale bar: 50 μm. g Quantification of the invading cells from (f) relative to control ethanol-treated cells (n = 3). All statistical significances were calculated using two-tailed Student t-tests, *p < 0.05; **p < 0.01; ***P < 0.001; ns non-significant. All error bars represent means ± s.d. Source data are provided as a Source Data file.

Fig. 6. MIR31HG promotes YBX1 phosphorylation by facilitating YBX1 interaction with its kinase RSK.

a Western blot analysis for p-YBX1, total YBX1, GAPDH and H3 in total and cytoplasmic fractions of BJ ER:BRAF cells (Control or siMIR31HG1-2) treated with ethanol (Control) or 1 μM 4-OHT for 72 h (n = 3). b Quantification of the pYBX1 band intensities relative to total YBX1 and Vinculin from three independent experiments. c PLA representative images showing the interaction (red dots) between YBX1 and RSK in BJ ER:BRAF cells (Control or siMIR31HG1) were treated with ethanol (Control) or 1 μM 4-OHT for 72 h. DAPI staining (blue) shows the nucleus. d Quantification of the number of interactions (dots) per cell in the conditions indicated in a representative experiment from four independent replicates (n = 4). Scale bar: 20 μm. e Left, in vitro kinase experiment incubating recombinant YBX1 in the presence of ³²P-ATP and recombinant RSK1 in the presence of the indicated in vitro-transcribed RNA, run in 4–12% NuPAGE Bis–Tris gel and exposed to an Amersham Hyperfilm ECL film. Right, quantification of the band intensities represented as p-YBX1 related to p-RSK (n = 4). f Western blot for p-YBX1, total YBX1 and Vinculin in BJ ER:BRAF cells expressing a doxycycline-induced empty, MIR31HG WT, Mut3 or Mut5 treated for 36 h with ethanol (Control) or 1 μM 4-OHT. g Quantification of the p-YBX1 band intensities relative to total YBX1 and Vinculin (n = 3). h Scheme of the working model: (1) During OIS MIR31HG is induced and locates in the cytoplasm. (2) At the same time, transcription of IL1A mRNA is induced. (3) IL1A mRNA is exported to the cytoplasm where it interacts with YBX1. MIR31HG facilitates YBX1 interaction with the kinase RSK promoting its phosphorylation which in turns promotes IL1A translation. (4) IL1A signalling induces the transcription of other SASP mRNAs through CEBPB and NF-κB. (5) These RNAs will be translated in the cytoplasm and the SASP components will be secreted (6). Statistical significance was calculated using two-tailed Student’s t-tests, *p < 0.05; **p < 0.01; ***p < 0.001. Error bars represent means ± s.d. Source data are provided as a Source Data file.