Overexpression of microRNA-34a Attenuates Proliferation and Induces Apoptosis in Pituitary Adenoma Cells via SOX7

Abstract

Pituitary adenomas constitute one of the most common intracranial tumors and are typically benign. However, the role of the tumor suppressor microRNA-34a (miR-34a), which is implicated in other cancers, in pituitary adenoma pathogenesis remains largely unknown. miR-34a expression was compared between GH4C1 cancer cells and normal cells derived from the pituitary gland of Rattus norvegicus, and the effects of miR-34a on GH4C1 cell proliferation and apoptosis were examined. miR-34a target genes were identified and analyzed computationally. The mRNA levels of the miR-34a target genes were measured using qRT-PCR, and the protein levels of the differentially expressed targets were assessed by western blotting. miR-34a expression was significantly lower in GH4C1 cells, whereas miR-34a overexpression significantly inhibited GH4C1 cell proliferation and promoted cell apoptosis though SRY-box 7 (SOX7). Our data facilitate the development of a better understanding of the pathogenesis and treatment of pituitary adenomas by elucidating the crucial role of miR-34a in the development of pituitary adenomas.

Keywords: SOX7; apoptosis; cell division; microRNAs; pituitary adenoma.

Figure 1

miR-34a Different Expression miR-34a expression in GH4C1 cells before and after transfection. (A) miR-34a expression was measured by qRT-PCR in GH4C1 cells and normal rat pituitary tissues, using U6 as an internal control. (B) miR-34a expression was also assessed by qRT-PCR after transfection with miR-34a mimic oligos. *p < 0.05; **p < 0.01.

Figure 2

Effect of miR-34a on Cell Proliferation and Apoptosis Effects of miR-34a on proliferation and apoptosis in GH4C1 cells. (A) Proliferation was assessed using the Cell Counting Kit-8 at 1–6 days after transfection with the miR-34a mimic, negative control, and blank GH4C1 cell controls. (B) Colony-formation assay after transfection with the miR-34a mimic, negative control, or blank GH4C1 cell controls. (C) Statistical analysis of results of the colony-formation assay after transfection with the miR-34a mimic, negative control, or blank GH4C1 cell controls. (D) Effects of miR-34a on proliferation 5 days after transfection. Original magnification: 40×. (E and F) In (E), apoptosis was assessed by annexin V-FITC and propidium iodide staining and flow cytometry after transfection with the miR-34a mimic, negative control, and blank GH4C1 cell controls. (F) Statistical analysis of the apoptosis index after transfection with the miR-34a mimic or negative control. Con, blank control group; E, early; L, late. *p < 0.05; **p < 0.01.

Figure 3

Target Gene Identification Candidate target genes and functional enrichment. (A) Venn diagrams showing the intersection of candidate target genes identified in four microRNA databases. (B) Venn diagrams showing the intersection between target genes identified in microRNA databases and in two sets of gene array data. (C) Protein-protein interactions among miR-34a target genes, with interaction scores set to medium confidence (0.400) and disconnected nodes hidden. Pink indicates experimentally determined known interactions; blue indicates known interactions from curated databases; yellow indicates text mining; green indicates predicted interactions by gene neighborhood; black indicates co-expression. (D) Sequence complementarity in RNA22.

Figure 4

Sox7 Dual-Luciferase Reporter miR-34a targets the Sox7 gene in GH4C1 cells. (A) Target gene protein levels visualized by western blotting. (B) Luciferase reporter validation of whether miR-34a could directly bind on Bcl2, Myc, or Sox7 mRNA in GH4C1 cells. (C) Schematic representation of the Sox7 3′UTR showing putative miRNA target sites; sequence in red indicates mutated potential miR-34a binding sites. (D) GH4C1 cells were co-transfected with a luciferase reporter containing the Sox7 3′ UTR and miRNA mimics in parallel with mutated Sox7. At 48 hr after transfection, the luciferase intensity was measured. ***p < 0.01.

Figure 5

Effect of Sox7 on Cell Proliferation and Apoptosis Effects of SOX7 expression on proliferation and apoptosis in GH4C1 cells. (A and B) Knockdown of SOX7 with different SOX7 siRNA sequences showed that si-SOX7 #2 and #3 significantly inhibited SOX7 expression at the mRNA and protein levels in GH4C1 cells. Beta-actin and GAPDH were used as controls. (C and D) Proliferation was assessed using CCK-8 at 1–5 days after knockdown with siRNAs, negative control, and blank GH4C1 cell controls. Effects of siRNAs on proliferation at 4 days after SOX7 knockdown are indicated. Original magnification: 40×. (E) Apoptosis of GH4C1 cells transfected with SOX7 siRNAs and negative control was tested by annexin V-FITC and propidium iodide staining and flow cytometry. (F) Statistical analysis of the apoptosis index after transfection with SOX7 siRNAs or negative control. E, early; L, late. OD, optical density. *p < 0.05; **p < 0.01.