NEAT1 regulates cell proliferation and apoptosis of ovarian cancer by miR-34a-5p/BCL2

Figures & data

Figure 1 Effects of alteration of NEAT1 expression on OC cell proliferation.

Notes: (A) Expression of NEAT1 was estimated by qRT-PCR in human OC cell lines (OVCAR3, SKOV3, HO8910, and OV90) and normal ovarian epithelial cell line HOSEpiC. (B) qRT-PCR was conducted to assess the expression level of NEAT1 in SKOV3 and HOSEpiC cells transfected with NEAT1 or Vector. (C) qRT-PCR was used to evaluate the expression level of NEAT1 in OVCAR3 cells transfected with si-NEAT1 or si-NC. MTT assay was performed to detect the relative viability at 0, 24, 48, or 72 h in (D) SKOV3 and (E) HOSEpiC cells after transfection with NEAT1 or Vector and in (F) OVCAR3 cells after treatment with si-NEAT1 or si-NC. Flow cytometric analysis of cell cycle distribution was performed in (G) SKOV3 and (H) HOSEpiC cells after transfection with NEAT1 or Vector and in (I) OVCAR3 cells after treatment with si-NEAT1 or si-NC. ^*P<0.05.
Abbreviations: NEAT1, nuclear enriched abundant transcript 1; OC, ovarian cancer; qRT-PCR, quantitative real-time polymerase chain reaction; si-NC, scrambled siRNA negative control.

Figure 2 Effects of alteration in NEAT1 expression on OC cell apoptosis.

Notes: (A) Flow cytometry analysis was carried out to determine the apoptotic rate in OVCAR3 cells transfected with si-NEAT1 or si-NC and SKOV3 cells transfected with NEAT1 or Vector. (B) Caspase-3 activity assay kit was used to measure Caspase-3 activity in si-NEAT1-transfected OVCAR3 cells or NEAT1-transfected SKOV3 cells. ^*P<0.05.
Abbreviations: NEAT1, nuclear enriched abundant transcript 1; OC, ovarian cancer; PI, propidium iodide; si-NC, scrambled siRNA negative control.

Figure 3 Relationship between NEAT1 and miR-34a-5p.

Notes: (A) Diagram of the putative miR-34a-5p–binding sites in NEAT1. (B) Luciferase reporter assay was performed to measure luciferase activity in OVCAR3 cells cotransfected with WT-NEAT1 or MUT-NEAT1 reporter and miR-34a-5p mimic or miR-NC. (C) qRT-PCR analysis of miR-34a-5p expression in human OC cell lines (OVCAR3, SKOV3, HO8910, and OV90), compared with normal ovarian epithelial cell line HOSEpiC. (D) qRT-PCR analysis of miR-34a-5p in NEAT1-transfected SKOV3 cells or si-NEAT1-transfected OVCAR3 cells. ^*P<0.05.
Abbreviations: miR-34a-5p, miR-34a-5p mimic; miR-NC, miRNA negative control; MUT-NEAT1, mutant reporter plasmid-NEAT1; NEAT1, nuclear enriched abundant transcript 1; OC, ovarian cancer; qRT-PCR, quantitative real-time polymerase chain reaction; WT-NEAT1, wild reporter plasmid-NEAT1.

Figure 4 Effect of NEAT1 on proliferation and apoptosis of OC cells was mediated by miR-34a-5p.

Notes: (A) miR-34a-5p expression was evaluated by qRT-PCR in SKOV3 cells after treatment with miR-34a-5p mimic and in OVCAR3 cells after transfection with anti-miR-34a-5p. MTT assay was used to assess the proliferation ability of (B) SKOV3 cells with miR-34a-5p transfection and (C) OVCAR3 cells with anti-miR-34a-5p treatment. (D) Apoptotic rate was determined and (E) caspase-3 activity analysis performed in miR-34a-5p-introduced SKOV3 cells and anti-miR-34a-5p-treated OVCAR3 cells. (F) MTT assay of cell viability in SKOV3 cells transfected with NEAT1, or in combination with miR-34a-5p mimic. (G) MTT assay of cell viability in OVCAR3 cells transfected with si-NEAT1, or in combination with anti-miR-34a-5p. (H) Apoptotic rate and (I) caspase-3 activity analysis in SKOV3 cells transfected with NEAT1 or NEAT1+miR-34a-5p and in OVCAR3 cells transfected with si-NEAT1 or si-NEAT1+anti-miR-34a-5p. ^*P<0.05.
Abbreviations: miR-34a-5p, miR-34a-5p mimic; NEAT1, nuclear enriched abundant transcript 1; OC, ovarian cancer; qRT-PCR, quantitative real-time polymerase chain reaction.

Figure 5 Effect of miR-34a-5p on proliferation and apoptosis of OC cells was modulated by BCL2.

Notes: (A) Predicted miR-34a-5p binding sites in the 3′-UTR of BCL2. (B) Luciferase reporter assay was conducted to detect luciferase activity of SKOV3 and OVCAR3 cells cotransfected with miR-34a-5p or miR-NC and WT-BCL2–3′-UTR or MUT-BCL2–3′-UTR. (C) Western blot analysis was performed to determine the protein level of BCL2 in SKOV3 cells transfected with miR-34a-5p or miR-NC and in OVCAR3 cells transfected with anti-miR-34a-5p or anti-miR-NC. (D, E) MTT assay was performed to detect proliferation in miR-34a-5p-transfected SKOV3 cells with or without BCL2 overexpression, as well as in anti-miR-34a-5p-treated OVCAR3 cells with or without BCL2 knockdown. (F) Flow cytometry analysis of apoptosis in miR-34a-5p-transfected SKOV3 cells with or without pcDNA-BCL2 transfection, as well as in anti-miR-34a-5p-treated OVCAR3 cells with or without si-BCL2 transfection. (G) Caspase-3 activity analysis in SKOV3 cells treated with miR-34a-5p or in combination with pcDNA-BCL2, and in OVCAR3 cells treated with anti-miR-34a-5p or in combination with si-BCL2. ^*P<0.05.
Abbreviations: BCL2, B-cell lymphoma-2; miR-34a-5p, miR-34a-5p mimic; miR-NC, miRNA negative control; NEAT1, nuclear enriched abundant transcript 1; OC, ovarian cancer; UTR, untranslated region.

Figure 6 si-NEAT1-induced decrease in BCL2 expression was abated after introduction of miR-34a-5p in OVCAR3 cells.

Notes: Western blot was performed to analyze the protein level of BCL2 in OVCAR3 cells transfected with si-NEAT1, or along with anti-miR-34a-5p. ^*P<0.05.
Abbreviations: BCL2, B-cell lymphoma-2; miR-34a-5p, miR-34a-5p mimic; NEAT1, nuclear enriched abundant transcript 1; si-NC, scrambled siRNA negative control; si-NEAT1, siRNA against NEAT1.