LINC00461 Promoted Endometrial Carcinoma Growth and Migration by Targeting MicroRNA-219-5p/Cyclooxygenase-2 Signaling Axis

Abstract

Endometrial carcinoma (EC) ranks as the most common female genital cancer in developed countries. Lately, more and more long noncoding RNAs (lncRNAs) have been identified as vital regulators in numerous physiological and pathological processes, including EC. However, the expression pattern and precise functions of different lncRNAs in EC remain unclear. In this study, we reported LINC00461 was upregulated in EC patient tissues and cell lines. In addition, LINC00461 knockdown could remarkably suppress cell proliferation, cell cycle progression, cell migration, and promote cell apoptosis in EC cells. We discovered LINC00461 could sponge microRNA-219-5p (miR-219-5p) and suppress its expression, thereby upregulating expression level of miR-219-5p's target, cyclooxygenase-2 (COX-2). In vivo animal models, LINC00461 knockdown inhibited tumor growth by increasing miR-219-5p level and reducing COX-2 expression, thus confirming LINC00461 functions as an oncogene in EC. In this study, a novel regulatory role of LINC00461/miR-219-5p/COX-2 axis was systematically investigated in context of EC, with the aim to provide promising intervention targets for EC therapy from bench to clinic. [Formula: see text].

Keywords: COX-2; LINC00461; endometrial carcinoma; metastasis; miR-219-5p; tumor growth.

Figure 1.

LINC00461 was elevated in EC tissues and cell models. (A) Relative LINC00461 levels in EC tissues (tumor, n = 45) and control tissues (normal, n = 45), determined using qRT-PCR. (B) LINC00461 levels in EC tissues (tumor, n = 45) and control tissues (normal, n = 45), determined using ISH. (C) Kaplan–Meier curves of overall survival of 45 EC patients, stratified by LINC00461 expressions. (D) Relative LINC00461 levels in various EC cell lines (KLE, Ishikawa, HEC1-A, HEC-1-B, and AN3-CA) and hEECs, determined by qRT-PCR (mean ± SEM, **P < 0.01). EC: endometrial carcinoma; hEEC: human endometrial epithelial cell; ISH: in situ hybridization; qRT-PCR: quantitative real-time polymerase chain reaction; SEM: standard error of the mean.

Figure 2.

Effects of LINC00461 on EC cells. (A) Expression levels of LINC00461 in Ishikawa or HEC-1-B cells expressing LINC00461 shRNAs (shRNA-1#, shRNA-2#), or the corresponding control (shRNA-NC). (B) Growth curves of Ishikawa or HEC-1-B cells expressing shRNA-1#, shRNA-2#, or shRNA-NC. Cell growth rate was assessed using a CCK-8 kit. (C) Cell cycle analysis of Ishikawa or HEC-1-B cells expressing shRNA-1#, shRNA-2#, or shRNA-NC. Cell percentage in G1, S, and G2 phages was quantified. (D) TUNEL analysis of Ishikawa or HEC-1-B cells expressing shRNA-1#, shRNA-2#, or shRNA-NC. TUNEL-positive cell percentage was quantified. (E) Protein levels of p21, cyclinD1, Bcl-2, Bax, and cleaved caspase-3 in Ishikawa or HEC-1-B cells expressing shRNA-1#, shRNA-2#, or shRNA-NC, determined using western blotting. Results were quantified by ImageJ (mean ± SEM, *P < 0.05, **P < 0.01). CCK-8: Cell Counting Kit-8; EC: endometrial carcinoma; SEM: standard error of the mean; shRNA: short harpin RNA; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling.

Figure 3.

LINC00461 altered migration ability of EC cells. (A) Wound scratch healing assay of Ishikawa or HEC-1-B cells expressing shRNA-1#, shRNA-2#, or shRNA-NC. (B) Representative images of Ishikawa or HEC-1-B cells expressing shRNA-1#, shRNA-2#, or shRNA-NC. Migrated and invaded cells were quantified. (C) Protein levels of E-cadherin, Vimentin, MMP9, and MMP2 in HEC1A or Ishikawa cells expressing shRNA-1#, shRNA-2#, or shRNA-NC, determined using western blotting. Results were quantified by ImageJ (mean ± SEM, *P < 0.05, **P < 0.01). EC: endometrial carcinoma; miR-219-5p: microRNA-219-5p; SEM: standard error of the mean; shRNA: short harpin RNA.

Figure 4.

LINC00461 directly bind to miR-219-5p and negatively regulated its expression. (A) StarBase prediction identified seeds match for LINC00461 in the mature sequence of miR-219-5p. Predicted seed-recognition site on miR-219-5p sequence and the corresponding LINC00461 sequence are depicted. (B) Relative luciferase activity of the LINC00461 reporter plasmid was assayed in Ishikawa or HEC-1-B cells expressing miR-219-5p or miR-NC. Mutant LINC00461 reporter was used as a control. (C) Ishikawa or HEC-1-B cells were harvested and mixed with Ago2 antibodies to perform RIP assay. LINC00461 or miR-219-5p enrichments were tested by qRT-PCR and compared to anti-IgG control. (D) Relative miR-219-5p levels in Ishikawa or HEC-1-B cells expressing shRNA-1#, shRNA-2#, or shRNA-NC, determined using qRT-PCR. (E) Relative miR-219-5p levels in EC tissues (tumor, n = 45) and control tissues (normal, n = 45), determined using qRT-PCR. (F) Pearson correlation analysis of the relative expressions between miR-219-5p and LINC00461 (mean ± SEM, **P < 0.01). EC: endometrial carcinoma; IgG: immunoglobulin G; miR-219-5p: microRNA-219-5p; qRT-PCR: quantitative real-time polymerase chain reaction; RIP: RNA binding protein immunoprecipitation; SEM: standard error of the mean; shRNA: short harpin RNA.

Figure 5.

miR-219-5p negatively regulated COX-2 expression through targeting its 3′-UTR. (A) Intersection analysis of miR-219-5p’s potential targets using TargetScan. The predicted seed-recognition sites in COX-2 3′-UTR and corresponding miR-219-5p sequence were depicted. (B) Relative luciferase activity of the COX-2 3′-UTR reporter plasmid was assayed in Ishikawa or HEC-1-B cells expressing miR-219-5p or miR-NC. Constructs expressing mutant COX-2 3′-UTR was used as a control. (C) Relative COX-2 mRNA levels in Ishikawa or HEC-1-B cells expressing miR-NC, miR-219-5p, inh-NC, or inh-miR-219-5p, determined using qRT-PCR. (D) Protein levels of COX-2 in Ishikawa or HEC-1-B cells expressing miR-NC, miR-219-5p, inh-NC, or inh-miR-219-5p, determined using western blotting. Relatively quantitative results were analyzed by ImageJ. (E) Relative COX-2 mRNA levels in EC tissues (tumor, n = 45) and control tissues (normal, n = 45), determined using qRT-PCR. (F) IHC analysis and (G) western blot analysis of COX-2 protein levels in EC tissues (tumor, n = 45) and control tissues (normal, n = 45). (H) Pearson correlation analysis of the relative expressions between miR-219-5p and COX-2 mRNA, between LINC00461 and COX-2 mRNA (mean ± SEM, **/##P < 0.01). COX-2: cyclooxygenase-2; IHC: immunohistochemistry; miR-219-5p: microRNA-219-5p; qRT-PCR: quantitative real-time polymerase chain reaction; SEM: standard error of the mean.

Figure 6.

LINC00461/miR-219-5p affected growth rate, cell death, invasion, and migration of EC cells. (A) Growth curves of Ishikawa or HEC-1-B cells expressing shRNA-NC + inh-NC, shRNA-1# + inh-NC, and shRNA-1# + inh-miR-219-5p. Cell growth rates were assessed using a CCK-8 kit. (B) Cell cycle analysis of Ishikawa or HEC-1-B cells expressing shRNA-NC + inh-NC, shRNA-1# + inh-NC, and shRNA-1# + inh-miR-219-5p. Cell percentages in G1, S, and G2 phages were quantified. (C) TUNEL analysis of Ishikawa or HEC-1-B cells expressing shRNA-NC + inh-NC, shRNA-1# + inh-NC, and shRNA-1# + inh-miR-219-5p. TUNEL-positive cell percentage was quantified. (D) Wound scratch healing assay of Ishikawa or HEC-1-B cells expressing shRNA-NC + inh-NC, shRNA-1# + inh-NC, and shRNA-1# + inh-miR-219-5p. (E) Representative images of Ishikawa or HEC-1-B cells expressing shRNA-NC + inh-NC, shRNA-1# + inh-NC, and shRNA-1# + inh-miR-219-5p. The migrated and invaded cells were quantified (mean ± SEM, */#P < 0.05, **/##P < 0.01). CCK-8: Cell Counting Kit-8; EC: endometrial carcinoma; SEM: standard error of the mean; shRNA: short harpin RNA; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling.

Figure 7.

LINC00461 knockdown inhibited tumor growth in an EC mouse xenograft model. (A) Representative images of corresponding tumors dissected from mice 5 weeks post-implantation. (B) Xenograft tumor volumes (left) and weight (right) of nude mice derived from subcutaneous implantation of Ishikawa cells stably expressing shRNA-1# or shRNA-NC. (C) IHC staining of Ki67 and COX-2 in the xenograft tumors from shRNA-1# or shRNA-NC group. (D) Relative levels of LINC00461 and miR-219-5p in the xenograft tumors from shRNA-1# or shRNA-NC group. (E) TUNEL analysis in the xenograft tumors from shRNA-1# or shRNA-NC group. (F) Protein levels of p21, cyclinD1, Bcl-2, Bax, cleaved caspase-3, E-cadherin, Vimentin, MMP9, and MMP2 in the xenograft tumors from shRNA-1# or shRNA-NC group, determined using western blotting and quantified by ImageJ. (G) Schematic illustration of LINC00461 promotes EC proliferation and migration by inhibiting miR-219 and upregulating COX-2 (mean ± SEM, **P < 0.01). COX-2: cyclooxygenase-2; EC: endometrial carcinoma; IHC: immunohistochemistry; SEM: standard error of the mean; shRNA: short harpin RNA; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling.