Long non-coding RNA LINC-01572:28 inhibits granulosa cell growth via a decrease in p27 (Kip1) degradation in patients with polycystic ovary syndrome

Abstract

Background: Disordered folliculogenesis is a key feature of polycystic ovary syndrome (PCOS), but the underlying molecular mechanism remains unclear.

Methods: Long non-coding RNA (lncRNA) expression in luteinized granulosa cells (hLGCs) derived from women with and without PCOS were analyzed using microarray and qRT-PCR. Immortalized human granulosa cell lines were cultured for proliferation assays after transfection with the LINC-01572:28 over-expression vector in the presence or absence of p27 siRNA. Protein expression analysis, rescue assays, and RNA immunoprecipitation (RIP) were used to confirm the LINC-01572:28 substrate.

Findings: LINC-01572:28 and p27 protein were elevated whereas proliferating cell nuclear antigen protein was decreased in the hLGCs of women with PCOS. LINC-01572:28 expression was positively correlated with basal testosterone levels. Over-expression of LINC-01572:28 inhibited cell proliferation and impeded G1/S transition, which were partially reversed by siRNA-mediated p27 knockdown.

Interpretation: Our findings, therefore, suggest that LINC-01572:28 suppresses cell proliferation and cell cycle progression by reducing the degradation of p27 protein via SKP2 binding.

Keywords: Granulosa cells; Polycystic ovary syndrome; Proliferation; lncRNA.

Fig. 1

LINC01572:28 is upregulated in granulosa cells of the PCOS patients. (A) Heat-map showing the hierarchical clustering of differentially expressed genes in patients with and without PCOS. red: upregulated genes; green: downregulated genes. (B) Graph showing the biotypes of non-coding RNAs profiled. (C) The differential expression of lncRNAs from microarrays and qRT-PCR respectively. (D) Graph showing the levels of the selected lncRNAs in 10 PCOS patients and 10 healthy controls. The expression levels were detected via qRT-PCR and normalized againstβ-actin. (E) Graph showing the level of the LINC01572:28 in 30 PCOS patients and 30 healthy controls. The expression level was detected via qRT-PCR and normalized againstβ-actin. (F) Graph showing the correlation between the expression level of LINC01572:28 in hLGCs and the serum concentration of testosterone in patients with PCOS. The expression level was detected via qRT-PCR. The first ∆Ct normalization was to β-actin, and the 2nd ∆Ct normalization was to the control group in cohort 3. Error bars represent SDs of at least 3 independent experiments. ***: p < .001, **: p < .01, *: p < .05, and ns (not significant; P > .05) correspond to two–tailed Student's tests.

Fig. 2

Enforced expression of LINC01572:28 inhibited granulosa cell proliferation in vitro. (A) Graph showing the level of LINC01572:28 in granulosa cells (human primary granulosa cells, KGN, and SVOG cells) treated with LINC01572:28 overexpression vector (p-LNC) or negative control vector (p-Enter). The expression level was detected via qRT-PCR and normalized againstβ-actin. (B) Cells were transfected with either LINC01572:28 overexpression vector (p-LNC) or negative control vector (p-Enter); protein samples were collected 48 h after transfection, and then subjected to immunoblot analysis. Data are representative of at least 3 independent experiments. (C) Graphs showing the proportion of proliferation cells via Edu assay in granulosa cells treated as in (A). The amount of cells was detected by staining with Hochest (blue), as the proliferated cells were detected by staining with EdU (red). The result was analyzed by fluorescence microscope. Percentages of EdU-positive cells were graphed. (D) Cell proliferation assays determined by cell counting Kit-8 at the indicated time points after transfection were performed from granulosa cells treated as in (A) Error bars represent SDs from at least 3 independent experiments. ***: p < .001, **: p < .01, *: p < .05, and ns (not significant; P > .05) correspond to two–tailed Student's tests.

Fig. 3

LINC01572–28 induced cell cycle arrest at G0-G1 phase in granulosa cells. (A) The granulosa cells were transfected with p-LNC or p-Enter for 48 h; the effect on cell cycle was assessed by PI staining followed by Modfit analysis. (B) qRT-PCR analysis for CCNE1 and CCND1 mRNA level in p-LNC and p-Enter KGN and SVOG cells. The expression level was normalized againstβ-actin. (C) Western blot analysis using Cyclin E1 and Cyclin D1 antibodies on protein extracts form granulosa cells after transfected with vectors for 48 h. (D and E) The levels of phosphorylated CDK2 and Rb in p-LNC and p-Enter granulosa cells were assessed by immunoblotting. Error bars represent SDs from at least 3 independent experiments. ***: p < .001, **: p < .01, *: p < .05, and ns (not significant; P > .05) correspond to two–tailed Student's tests.

Fig. 4

Involvement of p27 in LINC01572:28-induced proliferation arrest of granulosa cells. (A and B) The mRNA and protein level of p27 and p21 in granulosa cells after transfection with vectors for 48 h. (C) p-LNC and p-Enter cells were treated with si-Ctrl or si-p27 for 48 h; the proportion of proliferation cells was assessed by EdU assay in granulosa cells. (D) p-LNC and p-Enter cells were treated with si-Ctrl or si-p27 for 48 h; the effect on cell cycle was assessed by PI staining followed by Modfit analysis. Error bars represent SDs from at least 3 independent experiments. ***: p < .001, **: p < .01, *: p < .05, and ns (not significant; P > .05) correspond to two–tailed Student's tests.

Fig. 5

The Over-expressed LINC01572:28 induced cell proliferation arrest is partially rescued by knocking down p27 in granulosa cells. (A) p-LNC and p-Enter cells were treated with si-Ctrl or si-p27 for 48 h; and immunoblotting for p27 (a), PCNA (d), cyclin E1 (d), and the level of phosphorylated CDK2 and Rb (b and c) protein was performed. Error bars represent SDs from at least 3 independent experiments. ***: p < .001, **: p < .01, *: p < .05, and ns (not significant; P > .05) correspond to two–tailed Student's tests.

Fig. 6

LINC01572:28 stabilize p27 by interacting with SKP2 in granulosa cells. (A) qRT-PCR for LINC01572:28 from nuclear and cytoplasmic fractions of untreated granulosa cells. The cytoplasmic β-Actin and nuclear U6 were used as controls. (B) The enrichment of LINC01572:28 was measured by qRT-PCR from SKP2 RNA IPs (RIP) performed from formalde-crosslinked granulosa cells. Immunoglobulin G (IgG) IP was used as negative controls and EZH2 IP was used as positive controls. (C) SKP2-p27 interaction was examined in KGN and SVOG cells by immunoprecipitation SKP2 from whole-cell lysates followed by immunoblotting for p27 and SKP2. About 5% of cell lysis used for coIP were loaded as the inputs. IgG IP was used as control. Error bars represent SDs from at least 3 independent experiments. ***: p < .001, **: p < .01, *: p < .05, and ns (not significant; P > .05) correspond to two–tailed Student's tests.

Fig. 7

Decrease in PCNA expression while increase in p27 expression in granulosa cells of the PCOS patients. (A) The protein level of PCNA and p27 in the luteinized granulosa cells of PCOS women (n = 11) and healthy controls (n = 11). (B) Scheme graph of LINC01572:28 induced proliferation arrest in granulosa cells. LINC-01572:28 suppresses cell proliferation and cell cycle progression by reducing the degradation of p27 protein via competitive SKP2 binding. Error bars represent SDs from at least 3 independent experiments. ***: p < .001, **: p < .01, *: p < .05, and ns (not significant; P > .05) correspond to two–tailed Student's tests.