NCX2 Regulates Intracellular Calcium Homeostasis and Translocation of HIF-1α into the Nucleus to Inhibit Glioma Invasion

Abstract

Glioma is the most common tumor of the central nervous system, and its poor prognosis can be linked to hypoxia and gene inactivation. Na⁺/Ca²⁺ exchanger 2 (NCX2) is expressed only in the normal brain and not in other tissues or glioma. We constructed a hypoxic microenvironment to more accurately understand the effect of NCX2 in glioma. Our previous experiments confirmed that NCX2 inhibited the growth of U87 cells in nude mice, indicating that NCX2 is a potential tumor suppressor gene. Malignant tumor cells are often exposed to an anoxic environment. To more accurately understand the effect of NCX2 in glioma, we constructed a hypoxic microenvironment. To detect the localization of NCX2 in transfected U87 cells, immunofluorescence was used. We tested the function of NCX2 in glioma, i.e., how it contributes to the cytosolic Ca²⁺ homeostasis by X-Rhod-1. We tested the cell proliferation of NCX2 in glioma in hypoxic using Cell counting kit-8 (CCK8). Cell migration and invasion were evaluated in 24-well transwell matrigel-coated or non-matrigel-coated in hypoxia. NCX2 promoted the proliferation of U87 cells in the hypoxic microenvironment. It inhibited the invasion and migration abilities of U87 cells. We demonstrated that NCX2 was located on the cell membrane and that it reduced intracellular Ca²⁺ levels and reactivated P53 and PTEN. We further demonstrated that NCX2 impaired cell invasion through the HIF-1α pathway in glioma. The results indicated that NCX2 plays a key role in glioma formation and tumor invasion functionality.

Keywords: Glioma; HIF-1α; Hypoxic; Invasion; NCX2.

Fig. 1

NCX2 cell sublocalization dimensional views. Na⁺ and Ca²⁺ are transported bidirectionally by NCX2

Fig. 2

Lentiviral vector transduction and NCX2 expression. A Lentiviral vector-transduced cells. U87-NC and U87-NCX2 were imaged under fluorescence microscope at × 400. B RT-PCR for NCX2 mRNA expression in U87MG. GAPDH was used as an internal reference. C Western blot analysis for NCX2. After transfection with NCX2, proteins from U87-NC and U87-NCX2 were collected and western blotting showed an increase in NCX2 protein. GAPDH was used as a loading control. *P < 0.01

Fig. 3

HIF-1α knockdown and HIF-1α expression. A small interfering RNAs (siRNAs) cells. Control-siRNA and HIF-1α-siRNA were imaged under fluorescence microscope at × 400. B RT-PCR for HIF-1α mRNA expression in U87MG. GAPDH was used as an internal reference. C Western blot analysis for HIF-1α. After transfection with siRNAs, proteins from Control-siRNA and HIF-1α-siRNA were collected and western blotting showed a decrease in HIF-1α protein. GAPDH was used as a loading control. *P < 0.01

Fig. 4

NCX2 detected on cell membrane and regulated Ca²⁺. To detect the localization of NCX2 in transfected U87 cells, immunofluorescence was used. A U87-NCX2 showed the upregulation of NCX2 (red fluorescence). The cells were counterstained with DAPI (blue fluorescence) to make the nucleus visible. B NCX2 reduced intracellular calcium ions

Fig. 5

Growth curve of U87-NC and U87-NCX2 under hypoxic conditions. Hypoxia insignificantly inhibited U87-NCX2 cell proliferation in the early 48 h and slightly promoted cell proliferation after 48 h. (*P < 0.01, n = 5)

Fig. 6

NCX2 and HIF-1α inhibited invasion and migration in vitro under hypoxic conditions. A Cells migration after hypoxia for 36 h. B Cells invasion after hypoxia for 36 h. C, D Comparison of cells migration (left) and invasion (right) under hypoxia for 36 h (migration) and 36 h (invasion) (*P < 0.01)

Fig. 7

The effect of NCX2 on the protein expression levels of PKCα, PKCβ, PTEN, and P53. To determine the effect of NCX2 on the HIF-1α pathway, total HIF-1α and nucleus HIF-1α protein were extracted. There was a significant difference between U87-NCX2 and U87-NC. β-tubulin was used as control (*P < 0.01)

Fig. 8

VEGF96, product length = 96 bp; VEGF168, product length = 168 bp; VEGF193, product length = 193 bp. U87-NC and U87-NCX2 cells were subjected to hypoxia. The mRNA expression levels of MMP2, MMP7, MMP14, TIMP1, VEGF, and PAI-1 were analyzed by RT-PCR. GAPDH was used as an internal control (*P < 0.05, **P < 0.01)

Fig. 9

From KEGG and Biocarta database and our study, we established a cell pathway model of NCX2 regulation in U87 cells. Ca²⁺ efflux/influx through NCX2 to increase/decrease the Ca²⁺ in the cell. PKCs or wild-type P53 are activated by increased intracellular Ca²⁺. PKC expression to achieve a regulation of the ERK pathway. When P53 is activated, it competes with HIF-1α to bind p300 and inhibit HIF-1α nuclear translocation affecting cell proliferation, energy metabolism, invasion, and angiogenesis. Ca²⁺ can also regulate the expression of PTEN by other ways to affect the HIF-1 pathway