miR-342-3p, localized to 14q32, is a tumor suppressor miRNA implicated in carcinogenesis. Given the presence of a promotor-associated CpG island for its host gene, EVL, we hypothesized that intronic miR-342-3p is a tumor suppressor co-regulated with host gene by promoter DNA methylation in B cell lymphoma. By bisulfite pyrosequencing-verified methylation-specific PCR (MSP), EVL/MIR342 methylation was detected in five (50%) lymphoma cell lines but not normal peripheral blood and tonsils. EVL/MIR342 methylation correlated with repression of both miR-342-3p and EVL in cell lines. In completely methylated SU-DHL-16 cells, 5-AzadC treatment resulted in promoter demethylation and re-expression of miR-342-3p and EVL. In 132 primary lymphoma samples, EVL/MIR342 was preferentially methylated in B cell lymphomas (N = 68; 68.7%) than T cell lymphoma (N = 8; 24.2%) by MSP (P < 0.0001). Moreover, EVL/MIR342 methylation was associated with lower miR-342-3p expression in 79 primary NHL (P = 0.0443). In SU-DHL-16 cells, the tumor suppressor function of miR-342-3p was demonstrated by the inhibition of cellular proliferation and increase of cell death upon over-expression of miR-342-3p. Mechanistically, overexpression of miR-342-3p resulted in a decrease of LC3-II, a biomarker of autophagy, which was pro-survival for SU-DHL-16. Pre-treatment with 3-methyladenine, an autophagy inhibitor, abrogated tumor suppression associated with miR-342-3p overexpression. By luciferase assay, MAP1LC3B, a precursor of LC3-II, was confirmed as a direct target of miR-342-3p. Finally, in SU-DHL-16 cells, overexpression of miR-342-3p downregulated the known target DNMT1, with promoter demethylation and re-expression of tumor suppressor E-cadherin. Intronic miR-342-3p is co-regulated with its host gene EVL by tumor-specific promoter DNA methylation in B cell lymphoma. The tumor suppressor function of miR-342-3p was mediated via inhibition of pro-survival autophagy by targeting MAP1LC3B and downregulation of DNMT1 with demethylation and re-expression of tumor suppressor genes.

中文翻译：

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B细胞淋巴瘤中miR-342-3p的表观遗传沉默及其对自噬的影响

定位于 14q32 的 miR-342-3p 是一种与致癌作用有关的肿瘤抑制因子 miRNA。鉴于其宿主基因 EVL 存在启动子相关的 CpG 岛，我们假设内含子 miR-342-3p 是一种肿瘤抑制因子，通过 B 细胞淋巴瘤中的启动子 DNA 甲基化与宿主基因共同调节。通过亚硫酸氢盐焦磷酸测序验证的甲基化特异性 PCR (MSP)，在五个 (50%) 淋巴瘤细胞系中检测到 EVL/MIR342 甲基化，但在正常外周血和扁桃体中未检测到。EVL/MIR342 甲基化与细胞系中 miR-342-3p 和 EVL 的抑制相关。在完全甲基化的 SU-DHL-16 细胞中，5-AzadC 处理导致启动子去甲基化和 miR-342-3p 和 EVL 的重新表达。在 132 个原发性淋巴瘤样本中，EVL/MIR342 在 B 细胞淋巴瘤（N = 68；68.7%）中比 T 细胞淋巴瘤（N = 8；24.2%) 由 MSP (P < 0.0001)。此外，EVL/MIR342 甲基化与 79 例原发性 NHL 中较低的 miR-342-3p 表达相关（P = 0.0443）。在 SU-DHL-16 细胞中，miR-342-3p 的肿瘤抑制功能通过 miR-342-3p 过表达后细胞增殖的抑制和细胞死亡的增加来证明。从机制上讲，miR-342-3p 的过表达导致 LC3-II（一种自噬的生物标志物）减少，这有助于 SU-DHL-16 的存活。用 3-甲基腺嘌呤（一种自噬抑制剂）预处理可以消除与 miR-342-3p 过表达相关的肿瘤抑制。通过荧光素酶测定，LC3-II 的前体 MAP1LC3B 被确认为 miR-342-3p 的直接靶标。最后，在 SU-DHL-16 细胞中，miR-342-3p 的过表达下调了已知靶标 DNMT1，启动子去甲基化和肿瘤抑制因子 E-钙粘蛋白的重新表达。在 B 细胞淋巴瘤中，内含子 miR-342-3p 通过肿瘤特异性启动子 DNA 甲基化与其宿主基因 EVL 共同调节。miR-342-3p 的肿瘤抑制功能是通过靶向 MAP1LC3B 抑制促存活自噬和通过去甲基化和肿瘤抑制基因的重新表达下调 DNMT1 来介导的。