Long Intergenic Non-Protein Coding RNA 02381 Promotes the Proliferation and Invasion of Ovarian Endometrial Stromal Cells through the miR-27b-3p/CTNNB1 Axis

Abstract

Purpose: Catenin Beta 1 (CTNNB1) is a key regulator of cell proliferation and invasion in endometriosis; however, its upstream factor is not clear. Long noncoding RNAs may participate in endometriosis. The aim of this study was to investigate the mechanism of interaction between LINC02381 and CTNNB1 in endometriosis.

Method: Screening and validation of RNAs were completed by whole transcriptional sequencing and qRT-PCR. The subcellular localization of LINC02381 was determined by RNA in situ hybridization and nucleo-cytoplasmic separation. Plasmids were transfected for functional experiments. Luciferase assay was used to verify the binding relationship.

Results: The expression of LINC02381 and CTNNB1 was significantly increased in ovarian ectopic endometrial tissues (OSAs) and ectopic endometrial stromal cells (ESCs). When LINC02381 was downregulated in ESCs, the expression of CTNNB1, metallopeptidase 9 (MMP9) and cyclinD1, as well as ESCs invasion and proliferation, decreased. LINC02381 was mainly present in the cytoplasm of ESCs, indicating that it may act as a competitive endogenous RNA. Bioinformatic analysis revealed that microRNA-27b-3p (miR-27b-3p) is a downstream target of LINC02381. miR-27b-3p decreased in OSAs and ESCs. Moreover, when miR-27b-3p was upregulated in ESCs, the expression of CTNNB1, MMP9 and cyclinD1, as well as the invasion and proliferation ability of ESCs, were reduced. Additionally, rescue experiments demonstrated that the expression of CTNNB1, MMP9 and cyclinD1, as well as the invasion and proliferation ability, were significantly increased in the group transfected with both sh-LINC02381 and a miR-27b-3p inhibitor.

Conclusion: LINC02381 upregulated CTNNB1 by adsorbing miR-27b-3p, causing increased proliferation and invasion of ESCs.

Keywords: CTNNB1; LINC02381; endometriosis; invasion; miR-27b-3p; proliferation.

Figure 1

The lncRNA/miRNA/mRNA interaction network. Five pairs of ectopic endometrial tissue (OSA) and eutopic endometrial tissue (EM) samples from women with endometriosis were sent for whole-transcriptome sequencing. sRNA: small RNA. (A–C) Heatmap of the differentially expressed lncRNAs, sRNAs, and mRNAs. (D) The lncRNA/miRNA/mRNA regulatory interactions were formed according to the bioinformatics analysis.

Figure 2

Expression and distribution of LINC02381. (A) Paired OSA and EM samples were harvested for real-time quantitative polymerase chain reaction (qRT-PCR) to quantify the RNA expression of LINC02381 (n = 21, P < 0.001). (B) Paired ectopic endometrial stromal cells (ESCs) and eutopic endometrial stromal cells (EMs) were collected for qRT-PCR to quantify the RNA expression of LINC02381 (n = 17, P < 0.001). (C) ESCs were harvested for cytoplasmic and nuclear fractionation experiments and qRT-PCR (n = 3, P = 0.000). (D) ESCs were harvested for RNA fluorescent in situ hybridization (FISH) to determine the distribution of LINC02381. *** P < 0.001 vs. individual controls.

Figure 3

LINC02381 enhances CTNNB1 expression and the proliferation and invasion of ESCs. (A) Paired OSA and EM samples were collected for qRT-PCR to quantify the mRNA expression of CTNNB1 (n = 15, P < 0.05). (B) Paired ESCs and EMs were collected for qRT-PCR to quantify the mRNA expression of CTNNB1 (n = 13, P < 0.001). (C) Paired OSA and EM samples were collected for Western blotting to determine the protein expression of CTNNB1 (n = 9, P < 0.001). (D) Paired ESCs and EMs were collected for Western blotting to determine the protein expression of CTNNB1 (n = 5, P < 0.001). (E,F) ESCs were cultured for qRT-PCR after LINC02381 knockdown to quantify the RNA expression of LINC02381 (n = 3, P < 0.001) and CTNNB1 (n = 3, P < 0.05). (G) ESCs were cultured for Western blotting after LINC02381 knockdown to test the protein expression of CTNNB1 (n = 3, P < 0.01); (H,I) ESCs were cultured for qRT-PCR to quantify the mRNA expression of cyclinD1 (n = 3, P < 0.01) and MMP9 (n = 3, P < 0.05) when LINC02381 was knocked down. (J) ESCs were cultured for Western blotting to detect the protein expression of cyclinD1 (n = 3, P < 0.05) and MMP9 (n = 3, P < 0.01) following the decrease in LINC02381. (K) ESCs were cultured for Transwell assays after LINC02381 was knocked down (n = 3, P < 0.05). (L) ESCs were cultured for CCK-8 assays after LINC02381 was knocked down (from 24 h to 72 h, n = 3, P < 0.05). * P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 4

The LINC02381/miR-27b-3p/CTNNB1 regulatory axis. (A,B) Paired OSA and EM samples and paired ESCs and EMs were collected for qRT-PCR to quantify the RNA expression of miR-27b-3p (n = 15, P < 0.001; n = 12, P < 0.001, separately). (C) ESCs were harvested for qRT-PCR after LINC02381 knockdown (n = 3, P < 0.05). (D) The miRanda software was used to predict the binding relationship between LINC02381 and miR-27b-3p. (E) 293T cells were collected for Dual-Luciferase Reporter Gene Assay after transfection with indicated plasmids (n = 3, P < 0.01). (F,G) ESCs were collected for qRT-PCR after transfection with mimic-miR-27b-3p and mimic-NC. The efficiency of miR-27b-3p overexpression (n = 3, P < 0.001); the mRNA expression of CTNNB1 decreased after overexpression of miR-27b-3p (n = 3, P < 0.01). (H) The protein expression of CTNNB1 in ESCs decreased after miR-27b-3p overexpression (n = 3, P < 0.001). (I) The miRanda software was used to predict the binding relationship between CTNNB1 and miR-27b-3p. (J) 293T cells were collected for Dual-Luciferase Reporter Gene Assay after transfection with indicated plasmids (n = 3, P > 0.05). (K) ESCs were collected for qRT-PCR to quantify the mRNA expression of CTNNB1 after transfection with the indicated plasmids (n = 3, P < 0.05, both). (L) ESCs were cultured for Western blotting to determine the protein expression of CTNNB1 after transfection with the indicated plasmids (n = 3, P < 0.01; n = 3, P < 0.001, separately); * P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 5

The LINC02381/miR-27b-3p/CTNNB1 regulatory axis. (A,B) ESCs were harvested for qRT-PCR after miR-27b-3p expression was increased to quantify the mRNA expression of cyclinD1 (n = 3, P < 0.05) and MMP9 (n = 3, P < 0.01). (C) ESCs were harvested for Western blot analysis after mimic-miR-27b-3p/mimic-NC was added to determine the protein expression of cyclinD1 (n = 3, P < 0.01) and MMP9 (n = 3, P < 0.001). (D) ESCs were harvested for Transwell assays after the expression of miR-27b-3p increased to detect the invasive ability (n = 3, P < 0.001). (E) ESCs were harvested for CCK-8 assays after the expression of miR-27b-3p increased to detect the proliferative ability (n = 3, P < 0.01). (F,G) ESCs were harvested for qRT-PCR after the indicated plasmids were added to quantify the mRNA expression of cyclinD1 (n = 3, P < 0.01; n = 3, P < 0.05, separately) and MMP9 (n = 3, P < 0.05, both). (H) Cells were collected for Western blotting after transfection with the indicated plasmids. LINC02381 knockdown reduced cyclinD1 levels by 37.25% (n = 3, P < 0.05) and MMP9 levels by 52.38% (n = 3, P < 0.01). After the rescue experiment, cyclinD1 increased 1.61-fold (n = 3, P < 0.05) and MMP9 1.52-fold (n = 3, P < 0.05). (I) The indicated plasmids were transfected into ESCs, and cell invasion was evaluated (n = 3, P < 0.05; n = 3, P < 0.001, separately). (J) ESCs were harvested and subjected to CCK-8 assays after transfection (from 24 to 72 h, n = 3, P < 0.05; n = 3, P < 0.01, separately). * P < 0.05, ** P < 0.01, *** P < 0.001.