lnc-Rps4l-encoded peptide RPS4XL regulates RPS6 phosphorylation and inhibits the proliferation of PASMCs caused by hypoxia

Abstract

Pulmonary artery smooth muscle cells (PASMCs) proliferation caused by hypoxia is an important pathological process of pulmonary hypertension (PH). Prevention of PASMCs proliferation can effectively reduce PH mortality. Long non-coding RNAs (lncRNAs) are involved in the proliferation process. Recent evidence has demonstrated that functional peptides encoded by lncRNAs play important roles in cell pathophysiological process. Our previous study has demonstrated that lnc-Rps4l with high coding ability mediates the PASMCs proliferation under hypoxic conditions. We hypothesize in this study that a lnc-Rps4l-encoded peptide is involved in hypoxic-induced PASMCs proliferation. The presence of peptide 40S ribosomal protein S4 X isoform-like (RPS4XL) encoded by lnc-Rps4l in PASMCs under hypoxic conditions was confirmed by bioinformatics, immunofluorescence, and immunohistochemistry. Inhibition of proliferation by the peptide RPS4XL was demonstrated in hypoxic PASMCs by MTT, bromodeoxyuridine (BrdU) incorporation, and immunofluorescence assays. By using the bioinformatics, coimmunoprecipitation (coIP), and mass spectrometry, RPS6 was identified to interact with RPS4XL. Furthermore, lnc-Rps4l-encoded peptide RPS4XL inhibited the RPS6 process via binding to RPS6 and inhibiting RPS6 phosphorylation at p-RPS6 (Ser240+Ser244) phosphorylation site. These results systematically elucidate the role and regulatory network of Rps4l-encoded peptide RPS4XL in PASMCs proliferation. These discoveries provide potential targets for early diagnosis and a leading compound for treatment of hypoxic PH.

Keywords: Rps4l-encoded conserved peptide RPS4XL; hypoxia; long non-coding RNA Rps4l; proliferation; pulmonary arterial hypertension.

Graphical abstract

Figure 1

Rps4l encoding possibility prediction (A) Bioinformatics prediction of the coding ability of Rps4l by PhyloCSF. (B) Bioinformatics prediction of the coding ability of Rps4l by CPC2. (C) The sequence and name of the Rps4l-encoded peptide. (D) Schematic structural model of Rps4l-encoded peptide. (E and F) Local quality estimate (E) and normalized QMEAN4 score (F) of a model of Rps4l-encoded peptide by SWISS-MODEL.

Figure 2

Rps4l encodes peptide RPS4XL (A) Diagram of the FLAG fusion construct used for transfection. (B and C) Western blotting (WB) analysis (B) and immunofluorescence (IF) (C) of FLAG in PASMCs transfected with ORF-FLAG, ORFmut-FLAG, ATTORF-FLAG, or NC-FLAG under normoxia. (D) coIP of RPS4XL-FLAG complexes in PASMCs transfected with ORF-FLAG plasmid using anti-FLAG antibody. (E) Mass spectrometry of specific segments of Rps4l-encoded peptide RPS4XL in PASMCs. DAPI, 4',6-diamidino-2-phenylindole.

Figure 3

Confirmation of Rps4l-encoded peptide (A) WB analysis of RPS4XL in PASMCs transfected with lvRps4l or NC under hypoxia and normoxia. (B) Lung tissues form Rps4l^Tg and wild-type (WT) mice under hypoxia and normoxia. (C) Immunochemistry of RPS4XL in lung sections from Rps4l^Tg and WT hypoxic and control mice (scale bar, 100 μm). (D) IF of RPS4XL in lung sections from Rps4l^Tg and WT hypoxic and control mice (scale bar, 100 μm). All values are represented as the mean ± SEM (∗p < 0.05 and ∗∗p < 0.01). Nor, normoxia; Hyp, hypoxia. NC, negative control.

Figure 4

Rps4l regulates PASMC proliferation through its encoded peptide, RPS4XL (A) MTT assay in hypoxic and control PASMCs transfected with lvRps4l, ORFmut, or lv-NC. (B) BrdU incorporation assay in hypoxic and control PASMCs transfected with lvRps4l, ORFmut, or lv-NC. (C) WB analysis of PCNA in hypoxic and control PASMCs transfected with lvRps4l, ORFmut, or lv-NC. (D) IF of Ki67 (scale bar, 10 μm) in hypoxic and control PASMCs transfected with lvRps4l, ORFmut, or lv-NC. (E) WB analysis of cyclin A, cyclin D, cyclin E, CDK1, CDK2, and CDK4 in hypoxic and control PASMCs transfected with lvRps4l, ORFmut, or lv-NC. (F) Scratch-wound assays in hypoxic and control PASMCs transfected with lvRps4l, ORFmut, or lv-NC. All values are represented as the mean ± SEM (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; n ≥ 3).

Figure 5

The peptide RPS4XL inhibits the proliferation of PASMCs induced by hypoxia (A) MTT assay in PASMCs treated with RPS4XL with concentrations of 5 μg/mL, 7.5 μg/mL, or 10 μg/mL under normoxia. (B) MTT assay in PASMCs treated with RPS4XL with concentrations of 5 μg/mL, 7.5 μg/mL, or 10 μg/mL under hypoxia. (C and D) WB analysis of PCNA (C) and WB analysis of cyclin A, cyclin D, cyclin E, CDK1, CDK2, and CDK4 (D) in hypoxic and control PASMCs treated with RPS4XL with concentrations of 5 μg/mL, 7.5 μg/mL, or 10 μg/mL. All values are represented as the mean ± SEM (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and NS, no significance; n ≥ 3).

Figure 6

Rps4l-encoded peptide RPS4XL interacts with RPS6 (A) Bioinformatics prediction of network of proteins interacting with Rps4l-encoded peptide RPS4XL by STRING. (B) GO and KEGG analysis of proteins interacting with Rps4l-encoded peptide RPS4XL. (C) WB analysis of FLAG in PASMCs transfected with ORF-FLAG plasmid under normoxia after coIP using anti-FLAG antibody. Mass spectrometry of specific segments of RPS6. (D) WB analysis of EHMT2, NANOG, RPS6, and Rps4l-encoded peptide RPS4XL in PASMCs after coIP using anti-RPS4XL and anti-RPS6 antibodies under normoxia. (E and F) WB analysis of RPS6 and p-RPS6 in (E) lung tissues from Rps4l^Tg and WT mice and (F) hypoxic and control PASMCs transfected with lvRps4l, ORFmut, or NC. All values are represented as the mean ± SEM (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; n ≥ 3).

Figure 7

Interference with RPS6 can inhibit the proliferation of PASMCs caused by hypoxia (A) MTT assay in hypoxic and control PASMCs transfected with si-RPS6 or si-NC. (B) BrdU incorporation assay in hypoxic and control PASMCs transfected with si-RPS6 or si-NC. (C) WB analysis of PCNA in hypoxic and control PASMCs transfected with si-RPS6 or si-NC. (D) IF of Ki67 (scale bar, 10 μm) in hypoxic and control PASMCs transfected with si-RPS6 or si-NC. (E) WB analysis of cyclin A, cyclin D, cyclin E, CDK1, CDK2, and CDK4 in hypoxic and control PASMCs transfected with si-RPS6 or si-NC. All values are represented as the mean ± SEM (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; n ≥ 3).

Figure 8

RPS4XL and RPS6 regulates hypoxia-induced PH in vivo (A) Right ventricular systolic pressure (RVSP). (B) RV/left ventricular (LV)+S weight ratio. (C) Pulmonary artery acceleration time (PAAT). (D and E) PAVTI (D) and H&E (E) staining (scale bar, 200 μm) of hypoxic mouse model infected with AAV9-lv-NC, AAV9-lv-Rps4l, AAV9-lv-mut, and AAV9-si-NC. (F–J) RVSP (F), RV/ (LV+S) weight ratio (G), PAAT (H), PAVTI (I), and H&E (J) staining (scale bar, 200 μm) of hypoxic mouse model infected with AAV9-si-NC, AAV9-si-RPS6, and AAV9-lv-NC. All values are represented as the mean ± SEM (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; n ≥ 3).