LncRNA NEAT1 Recruits SFPQ to Regulate MITF Splicing and Control RPE Cell Proliferation

Abstract

Purpose: Retinal pigment epithelium (RPE) cell proliferation is precisely regulated to maintain retinal homoeostasis. Microphthalmia-associated transcription factor (MITF), a critical transcription factor in RPE cells, has two alternatively spliced isoforms: (+)MITF and (-)MITF. Previous work has shown that (-)MITF but not (+)MITF inhibits RPE cell proliferation. This study aims to investigate the role of long non-coding RNA (lncRNA) nuclear-enriched abundant transcript 1 (NEAT1) in regulating MITF splicing and hence proliferation of RPE cells.

Methods: Mouse RPE, primary cultured mouse RPE cells, and different proliferative human embryonic stem cell (hESC)-RPE cells were used to evaluate the expression of (+)MITF, (-)MITF, and NEAT1 by reverse-transcription PCR (RT-PCR) or quantitative RT-PCR. NEAT1 was knocked down using specific small interfering RNAs (siRNAs). Splicing factor proline- and glutamine-rich (SFPQ) was overexpressed with the use of lentivirus infection. Cell proliferation was analyzed by cell number counting and Ki67 immunostaining. RNA immunoprecipitation (RIP) was used to analyze the co-binding between the SFPQ and MITF or NEAT1.

Results: NEAT1 was highly expressed in proliferative RPE cells, which had low expression of (-)MITF. Knockdown of NEAT1 in RPE cells switched the MITF splicing pattern to produce higher levels of (-)MITF and inhibited cell proliferation. Mechanistically, NEAT1 recruited SFPQ to bind directly with MITF mRNA to regulate its alternative splicing. Overexpression of SFPQ in ARPE-19 cells enhanced the binding enrichment of SFPQ to MITF and increased the splicing efficiency of (+)MITF. The binding affinity between SFPQ and MITF was decreased after NEAT1 knockdown.

Conclusions: NEAT1 acts as a scaffold to recruit SFPQ to MITF mRNA and promote its binding affinity, which plays an important role in regulating the alternative splicing of MITF and RPE cell proliferation.

Figure 1.

(−)MITF was expressed at low levels relative to (+)MITF in proliferative RPE cells. (A) RPE cells from 2-month-old mice were isolated and cultured in vitro for 7 days. BrdU-positive signals (red) could be detected in primary cultured RPE cells but not in isolated RPE tissue. (B) Expression of (−)Mitf and (+)Mitf analyzed by RT-PCR. (C) qRT-PCR analysis of the percentages of (−)Mitf and (+)Mitf in the isolated RPE tissue and primary cultured RPE cells. (D) Ki67 immunostaining in hESC–RPE cells. Highly pigmented RPE cells had lower Ki67-positive signals and lower pigmented RPE cells had higher Ki67-positive signals. (E) Ki67-positive percentages based on (D). (F) The expression of (−)MITF and (+)MITF analyzed by RT-PCR in the low-proliferative and highly proliferative hESC–RPE cells. (G) qRT-PCR analysis of the percentage of (−)MITF and (+)MITF in low-proliferative and highly proliferative hESC–RPE cells. Scale bar: 50 µm. **P < 0.01, ***P < 0.001; n = 3.

Figure 2.

LncRNA NEAT1 was highly expressed in proliferative RPE cells. (A, B) Expression of Neat1 in primary cultures of mouse RPE cells and isolated RPE tissue was analyzed by RT-PCR (A) and qRT-PCR (B). (C) qRT-PCR estimation of NEAT1 levels in high and low proliferation groups of hESC-derived RPE cells. (D) Expression of NEAT1 was detected by RT-PCR in proliferating ARPE-19 and D407 RPE cell lines; the highly proliferative hESC–RPE cells were used as positive controls. *P < 0.05, ***P < 0.001; n = 3.

Figure 3.

Knockdown of NEAT1 inhibited RPE cell proliferation. (A) qRT-PCR showing the knockdown efficiency of NEAT1 in ARPE-19 cells. (B, C) ARPE-19 cells were transfected with si-NEAT1 or negative control (NC); cells were counted 48 hours after transfection, and the cell number in the NC group was normalized as 100. (D) Cell growth curves of ARPE-19 cells after NEAT1 knockdown. (E, F) Ki67 immunostaining of ARPE-19 cells showed a decrease in the percentage of cells staining positive after knockdown of NEAT1. (G, H) Western blotting showing protein levels of MET, E2F1, P-Rb, P-AKT, and P-ERK in ARPE-19 cells 48 hours after knockdown of NEAT1. Scale bar: 50 µm. *P < 0.05, **P < 0.01, ***P < 0.001; n = 3. ns, no significant difference.

Figure 4.

Knockdown of NEAT1 changed the splicing pattern of MITF. (A) qRT-PCR was used to analyze the expression of total MITF mRNA in ARPE-19 cells after knockdown of NEAT1. (B) After knockdown of NEAT1 in ARPE-19 cells, the expression of MITF splicing isoforms was analyzed by RT-PCR. (C) qRT-PCR analysis of the percentages of (−)MITF and (+)MITF in the NEAT1 knockdown ARPE-19 cells. (D, E) qRT-PCR was used to analyze the expression levels of NEAT1 and total MITF mRNA after the si-NEAT1 transfection. (F) After knockdown of NEAT1 in D407 cells, the expression of MITF splicing isoforms was analyzed by RT-PCR. (G) qRT-PCR analysis of the percentages of (−)MITF and (+)MITF in the NEAT1 knockdown D407 cells. *P < 0.05, **P < 0.01, ***P < 0.001; n = 3.

Figure 5.

SFPQ bound directly to MITF and regulated its splicing. (A, B) RIP showing binding of SFPQ to MITF mRNA and NEAT1. (C) An amplicon of lncRNA MIR497HG was used as a negative control for RIP. (D) qRT-PCR quantitation of SFPQ in ARPE-19 cells before and after infection with lentivirus-expressing SFPQ. (E) qRT-PCR showing no change in total MITF mRNA levels in SFPQ-overexpressing ARPE-19 cells. (F, G) RIP demonstrating binding of SFPQ to NEAT1 (F) and MITF (G) mRNA in ARPE-19 cells overexpressing SFPQ. (H) SFPQ RIP showing enrichment of MITF mRNA in ARPE-19 + SFPQ compared with control ARPE-19 cells. (I) RT-PCR showing decreased (−)MITF and increased (+)MITF expression in ARPE-19 + SFPQ relative to ARPE-19 cells. (I) qRT-PCR analysis of the percentages of (−)MITF and (+)MITF in ARPE-19 + SFPQ cells. *P < 0.05, ***P < 0.001; n = 3.

Figure 6.

SFPQ regulated MITF splicing in a NEAT1-dependent manner. (A–D) RIP showed the direct binding ability of SFPQ to MITF after siRNA knockdown of NEAT1 in ARPE-19 cells (A, B) and ARPE-19 + SFPQ cells (C, D), with decreased binding in NEAT1 knockdown cells. (E) RT-PCR showing expression of (+)MITF and (−)MITF in ARPE-19 + SFPQ cells after siRNA knockdown of NEAT1. (F) qRT-PCR analysis of the percentages of (−)MITF and (+)MITF in the NEAT1 knockdown ARPE-19 + SFPQ cells. *P < 0.05, ***P < 0.001; n = 3.

Figure 7.

Graphical summary of NEAT1 recruitment of SFPQ binding to MITF mRNA and regulation of its splicing. (A) NEAT1 works as a scaffold to recruit SFPQ binding to MITF mRNA, which produces both (+)MITF and (−)MITF isoforms. (B) Knockdown of NEAT1 decreases the binding of SFPQ to MITF mRNA, which increases the splicing of (−)MITF and decreases that of (+)MITF.