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. 2020 Nov 17;29(3):1279–1293. doi: 10.1016/j.ymthe.2020.11.018

Figure 2.

Figure 2

lnc-MAP3K13-7:1 Upregulation Decreased DNMT1 Protein Levels and Inhibited GC Proliferation In Vitro

(A) Quantitative real-time PCR analysis of lnc-MAP3K13-7:1 overexpression using lentiviral vectors in KGN cells. Expression of lnc-MAP3K13-7:1 in stably transfected cells in comparison with that in empty vector-transfected cells (left, n = 9, p < 0.0001). Quantitative real-time PCR analysis of DNMT1 and DNMT3A expression after lnc-MAP3K13-7:1 overexpression (middle and right, DNMT1, n = 9, p = 0.614; DNMT3A, n = 3, p = 0.433). (B) Global DNA methylation of KGN cells after lnc-MAP3K13-7:1 overexpression compared with that in empty vector-transfected cells (n = 3, p = 0.0864, Mann-Whitney U test). (C) Western blot analysis of DNMT1 and DNMT3A protein expression in KGN cells after lnc-MAP3K13-7:1 overexpression. The quantification of protein levels is presented at the bottom (n = 3 for each group; DNMT1, p < 0.0001; DNMT3A, p = 0.682). (D) Quantitative real-time PCR detection of cyclin-dependent kinase inhibitor 1A (CDKN1A) expression in GCs from 42 patients with PCOS and 42 normal controls (p = 0.0004). (E and F) Effects of lnc-MAP3K13-7:1 overexpression on KGN cell growth measured using the Cell Counting Kit-8 assay (E, n = 3, p < 0.0001) and cell cycle distribution determined using flow cytometry (F, n = 4, p < 0.0001 for each group). (G) Quantitative real-time PCR analysis of changes in proliferation-related marker expression (proliferating cell nuclear antigen [PCNA] as a positive regulator and CDKN1A and CDKN1B as negative regulators) 72 h after lnc-MAP3K13-7:1 lentivirus transfection (n = 6; PCNA, p = 0.019; CDKN1A, p = 0.004; CDKN1B, p = 0.339). (H) Western blot analysis of changes in proliferation-related marker expression (PCNA, p21, cyclin E1, CDK2, p-CDK2 [Thr160]), retinoblastoma protein [Rb], and p-Rb [Ser807/Ser811]) normalized to β-actin protein expression in lnc-MAP3K13-7:1-overexpressing KGN cells. The quantification of protein levels is presented on the right (n = 3; Rb, p = 0.156; p-Rb, p = 0.003; cyclin E1, p = 0.923; PCNA, p = 0.003; CDK2, p = 0.663; p-CDK2, p < 0.0001; p21, p = 0.007). In (A)–(C) and (E)–(H), mean ± SD. In (A) and (C)–(H), ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (Student’s t test).