SDF-1α/MicroRNA-134 Axis Regulates Nonfunctioning Pituitary Neuroendocrine Tumor Growth via Targeting VEGFA

Abstract

Background: Nonfunctioning pituitary neuroendocrine tumor (NF-PitNET) is difficult to resect. Except for surgery, there is no effective treatment for NF-PitNET. MicroRNA-134 (miR-134) has been reported to inhibit proliferation and invasion ability of tumor cells. Herein, the mechanism underlying the effect of miR-134 on alleviating NF-PitNET tumor cells growth is explored.

Methods: Mouse pituitary αT3-1 cells were transfected with miR-134 mimics and inhibitor, followed by treatment with stromal cell-derived factor-1α (SDF-1α) in vitro. MiR-134 expression level: we used quantitative real-time PCR (qRT-PCR) to detect the expression of miR-134. Cell behavior level: cell viability and invasion ability were assessed using a cell counting kit-8 (CCK8) assay and Transwell invasion assay respectively. Cytomolecular level: tumor cell proliferation was evaluated by Ki-67 staining; propidium iodide (PI) staining analyzed the effect of miR-134 on cell cycle arrest; western blot analysis and immunofluorescence staining evaluated tumor migration and invasive ability. Additionally, we collected 27 NF-PitNET tumor specimens and related clinical data. The specimens were subjected to qRT-PCR to obtain the relative miR-134 expression level of each specimen; linear regression analysis was used to analyze the miR-134 expression level in tumor specimens and the age of the NF-PitNET population, gender, tumor invasion, prognosis, and other indicators.

Results: In vitro experiment, miR-134 was observed to significantly inhibit αT3-1 cells proliferation characterized by inhibited cell viability and expressions of vascular endothelial growth factor A (VEGFA) and cell cycle transition from G1 to S phase (P < 0.01). VEGFA was verified as a target of miR-134. Additionally, miR-134-induced inhibition of αT3-1 cell proliferation and invasion was attenuated by SDF-1α and VEGFA overexpression (P < 0.01). In primary NF-PitNET tumor analysis, miR-134 expression level was negatively correlated with tumor invasion (P = 0.003).

Conclusion: The regulation of the SDF-1α/miR-134/VEGFA axis represents a novel mechanism in the pathogenesis of NF-PitNETs and may serve as a potential therapeutic target for the treatment of NF-PitNETs.

Keywords: SDF-1α (CXCL12); invasion; microRNA-134(miR-134); nonfunctioning pituitary neuroendocrine tumor (NF-PitNET); proliferation; vascular endothelial growth factor A (VEGFA).

Figure 1

The expression of SDF-1α and VEGFA in invasive and non-invasive NF-PitNET. (A) Immunohistochemical staining assay of SDF-1α and VEGFA in invasive NF-PitNET and non-invasive NF-PitNET. Brain tissues from epileptic were used for the negative control. (B) The quantification of SDF-1α and VEGFA expression are shown between invasive and non-invasive NF-PitNET. Data were represented as means ± SD (n = 6). ^****p < 0.0001 vs. non-invasive NF-PitNET group.

Figure 2

SDF-1α inhibits the expression of miR-134. αT3-1 cells were treated with SDF-1α, control, miR-134 mimics + SDF-1α, miR-134 mimics + PBS, miR-134 inhibitor + SDF-1α, miR-134 inhibitor + PBS, black vector control + SDF-1α, black vector control + PBS, respectively. (A, B) MiR-134/U6 relative level in αT3-1 cells by qRT-PCR. Data were represented as means ± SD (n = 3). ***p < 0.001 vs. control group. ^#p < 0.05 vs. miR-134 mimics + PBS group. ^& p < 0.05 vs. miR-134 inhibitor + PBS group. ^*p < 0.05 vs. black vector control + PBS group.

Figure 3

Effects of SDF-1α/miR-134 on cell cycle transition, proliferation, and viability. MiR-134 mimics or inhibitor was transiently transfected into αT3-1 cells. After 48 h transfection, αT3-1 cells were treated with 20 ng/ml SDF-1α, equal volume of PBS, and black vector control respectively. (A) Cell cycle distribution using flow cytometry. (B) Cell viability using CCK-8 assay. (C) Analysis of cell invasion using transwell assay. (D) Immunohistochemical staining assay of Ki-67. (E) Positive Ki-67 cell was counted manually by Image J. Data were represented as means ± SD (n = 3). ^####p < 0.0001 and ^#p < 0.05 vs. miR-134 mimics + PBS group. ^&&&&p < 0.0001 and ^&&p < 0.01 vs. miR-134 inhibitor + PBS group. ^****p < 0.0001, ^**p < 0.01 and ^*p < 0.05 vs. black vector control + PBS group.

Figure 4

The expression of VEGFA and proliferation/invasion-related downstream protein of αT3-1 cells. (A) Schematic diagram of the target protein band. (B) The quantification of VEGFA expressions using western blot analysis. (C) The quantification of MMP2 expressions using western blot analysis. (D) The quantification of MMP9 expressions using western blot analysis. (E) The quantification of Vimentin expressions using western blot analysis. (F) The quantification of Snail expressions using western blot analysis. Data were represented as means ± SD (n = 5). ^####p < 0.0001 and ^## p < 0.01 vs. miR-134 mimics + PBS group. ^&&&& p < 0.0001 vs. miR-134 inhibitor + PBS group. ^****p < 0.0001 vs. black vector control + PBS group.

Figure 5

The schematic diagram of SDF-1α/miR-134/VEGFA axis in NF-PitNET. SDF-1α promotes VEGFA expression by inhibiting miR-134 expression in NF-PitNETs proliferation and invasiveness.