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. 2020 Dec 29;13(1):1422-1439.
doi: 10.18632/aging.202376. Epub 2020 Dec 29.

Functional characterization of DLK1/MEG3 locus on chromosome 14q32.2 reveals the differentiation of pituitary neuroendocrine tumors

Yiyuan Chen  1 , Hua Gao  1 , Qian Liu  1 , Weiyan Xie  1 , Bin Li  1 , Sen Cheng  1 , Jing Guo  1 , Qiuyue Fang  1 , Haibo Zhu  2 , Zhuang Wang  3 , Jichao Wang  4 , Chuzhong Li  1   2   5   6 , Yazhuo Zhang  1   2   5   6
Affiliations

Functional characterization of DLK1/MEG3 locus on chromosome 14q32.2 reveals the differentiation of pituitary neuroendocrine tumors

Yiyuan Chen et al. Aging (Albany NY). .

Abstract

Pituitary neuroendocrine tumors (PitNETs) represent the neoplastic proliferation of the anterior pituitary gland. Transcription factors play a key role in the differentiation of PitNETs. However, for a substantial proportion of PitNETs, the etiology is poorly understood. According to the transcription data of 172 patients, we found the imprinting disorders of the 14q32.2 region and DLK1/MEG3 locus associated with the differentiation of PitNETs. DLK1/MEG3 locus promoted somatotroph differentiation and inhibited tumor proliferation in PIT1(+) patients, furthermore, the level of DLK1 played a critical role in the trend of somatotroph or lactotroph differentiation. Anti-DLK1 monoclonal antibody blockade or siMEG3 both indicated that the DLK1/MEG3 significantly promoted the synthesis and secretion of GH/IGF-1 and inhibited cell proliferation. In addition, loss of DLK1 activated the mTOR signaling pathway in high DLK1-expressing and PIT1(+) GH3 cell lines, a mild effect in the low DLK1-expressing and PIT1(+) MMQ cell lines and no effect in the PIT1(-) ATT20 cell line. These findings emphasize that expression at the DLK1/MEG3 locus plays a key role in the differentiation of PitNETs, especially somatotroph adenomas, and provide potential molecular target data for patient stratification and treatment in the future.

Keywords: DLK1/MEG3 locus; differentiation; growth hormone secreting; pituitary neuroendocrine tumors; somatotroph adenomas.

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Conflict of interest statement

CONFLICTS OF INTEREST: The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
The protein-coding RNA landscape in 172 PitNETs. (A) Heatmap of unsupervised hierarchical clustering of the top 352 most variable genes among 31 somatotroph adenomas, 17 lactotroph adenomas, 79 gonadotroph adenomas and 45 corticotroph adenomas (|log2FC|>2, adj.P.val<0.001). Pathological and clinical annotations were provided. (B–D) Volcano plots showing the significantly differentially expressed genes among somatotroph adenomas vs. 3 other subtypes of PitNETs (up: red, down: blue), all log2FC of DLK1 > 7. (E) Venn diagram showing the intersection of DEGs among somatotroph adenomas vs. 3 other subtypes of PitNETs, DEGs were identified under the cutoff of adj.P.val < 0.001. (F, G) Metascape enrichment analysis including pathways and GO term of 110 overlapping DEGs in Figure 1E. (H) Outline of bioinformatic GSEA analysis.
Figure 2
Figure 2
The lncRNA landscape in 172 PitNETs. (A) Heatmap of unsupervised hierarchical clustering of the top 63 most variable genes among 31 somatotroph adenomas, 17 lactotroph adenomas, 79 gonadotroph adenomas and 45 corticotroph adenomas (|log2FC|>2, adj.P.val<0.001). Pathological and clinical annotations were provided. (B–D) Volcano plots showing significantly differentially expressed genes somatotroph adenomas vs. 3 other subtypes of PitNETs (Up: red, Down: blue). (E) Volcano plots showing the AC126177.8, AC355974.2, LINC02475, MEG3, MEG9 and MiR7-3HG were the most significant lncRNAs, DEGs were identified by the R package limma, 31 somatotroph adenomas vs. 141 other subtype PitNETs.
Figure 3
Figure 3
Description of DLK1/MEG3 locus in 172 PitNETs. (A) The imprinting disorders of 14q32.2 region was filtered based on genes distribution in Chromosome 14. A high-resolution profile showed the genomic positions and gene trees for DLK1/MEG3 locus. (B) The TPM values of DLK1/MEG3 and characteristic molecule in 172 PitNETs. (C) DLK1 combined transcription factors and immuophenotype could distinguish somatotroph, lactotroph, gonadotroph and corticotroph PitNET patients. *compare to control group P<0.05 **P<0.01 ***P<0.001.
Figure 4
Figure 4
Clinical relevance of DLK1 in somatotroph adenomas. (A) MR images of PitNET patients. (B) Transmission electron microscope of PitNET patients. (C) Immunohistochemistry image of DLK1, PIT1 and SSTR2 in PitNET patients. 400×. (D) The TPM values of DLK1, serum GH levels and tumor volumes in 31 somatotroph adenomas. (E) Correlation of DLK1 and serum GH, tumor volume. (F) There was statistic difference of GH level between patients with high DLK1 and low DLK1, not tumor volume. (G) ROC curve. HRAS (0.825), WNT5B (0.819), Notch2 (0.799), DLK1 (0.795) and ITPR2 (0.782) were filtered in PI3K/AKT/mTOR pathway, Notch pathway and growth hormone synthesis, secretion and action. *compare to control group P<0.05 **P<0.01 ***P<0.001.
Figure 5
Figure 5
Effect of anit-DLK1 antibody on the bioactivity of PitNET cell lines. (A) Western blot assay measured the levels of DLK1 and PIT1 in GH3 cell line, MMQ cell line and ATT20 cell line. (B) Anti-DLK1 antibody inhibited the cell viability of GH3 cells in the dose- and time-dependent manner, not MMQ cells or ATT20 cells. (C) Anti-DLK1 antibody inhibited the secretion of GH/IGF-1 in GH3 cells, not PRL in MMQ cells and ACTH in ATT20 cells. (D) Clone forming experiment showed the anti-DLK1 antibody promoted the cell proliferation in GH3 cell line. (E) Confocal experiment showed DLK1 regulated the level of PIT1 in GH3 cell line. (F) Western blot experiment showed Anti-DLK1 antibody activated the mTOR pathway in GH3 cell line. *compare to control group P<0.05 **P<0.01 ***P<0.001.
Figure 6
Figure 6
Effect of siMEG3 on the bioactivity of PitNET cell lines. (A) RT-qPCR experiment measured the RNAi efficiency of siMEG3-1 and siMEG3-2. (B) Western blot experiment showed RNAi-MEG3 reduced the level of DLK1, PIT1 and GH in GH3 cell line. (C) RNAi-MEG3 obviously increased the cell viability of GH3 cell line, mildly increase in MMQ cell line, and no change in ATT20 cell line. (D) RNAi-MEG3 inhibited the secretion of GH/IGF-1 in GH3 cells, not PRL in MMQ cells and ACTH in ATT20 cells. (E) Confocal experiment showed RNAi-MEG3 inhibited the levels of DLK1 and PIT1 in GH3 cells. *compare to control group P<0.05 **P<0.01 ***P<0.001.
Figure 7
Figure 7
Differentiation and transcription factors in PitNETs base on the DLK1 expression.
See this image and copyright information in PMC

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