Cell culture

Human glioma U87 and U251 cell lines were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. The cells were cultured in DMEM supplemented with 10% FBS and 1% antibiotics at 37°C with 5% CO₂.

The present study was approved by the Ethics Committee of China-Japan Union Hospital of Jilin University and The First Hospital of Jilin University.

CALCOCO2 expression in glioma cell lines

CALCOCO2 expression in four glioma cell lines, i.e., U87, U251, U373, and A-172, was assessed by quantitative real-time polymerase chain reaction (RT-qPCR). Briefly, total messenger RNAs (mRNAs) of cells were extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The mature mRNAs (2 µg per sample) were reverse transcribed into cDNAs using Super MMLV reverse transcriptase (BioTeke, Beijing, China). The mRNA levels of CALCOCO2 were determined by RT-qPCR using the Bio-Rad Connect Real-time PCR platform. RT-qPCR consisted of an initial denaturation step at 95°C for 15 s, 30 cycles of 95°C for 5 s, and 60°C for 30 s. The mRNA expression levels were determined by a comparative CT (2^–ΔΔCt) analysis.

Construction of lentivirus vectors targeting CALCOCO2

A short hairpin RNA (shRNA) was designed according to the sequence of CALCOCO2. The shRNA oligos were synthesised and inserted into the plasmid GV115 (GeneChem, Shanghai, China), and then recombinant lentiviruses were constructed by plasmid co-transfection of 293T cells according to the manufacturer’s instructions. The viral supernatant was collected and filtered through a 0.45-µm filter (Millipore, Billerica, MA, USA) at 72 h post-transfection, and the viral titre was determined. Subsequently, the viral supernatant was added to the U87 and U251 cell lines, and the expression of CALCOCO2 in cells was observed under a fluorescence microscope at 48 h (Olympus America, Melville, NY, USA). The cells infected with shCALCOCO2 and control shRNA were termed shCALCOCO2 and shControl, respectively.

Silencing efficiency assessment

The silencing efficiency of CALCOCO2 at the protein level was assessed by western blotting. Briefly, after U87 and U251 cells were infected with shCALCOCO2 or shControl for 5 days, they were collected and lysed with protein lysate (100 mM tris(hydroxymethyl)aminomethane hydrochloride (pH 6.8), 10 mM ethylenediaminetetraacetic acid, and 4% sodium dodecyl sulphate) for 20 min. The lysates were centrifuged, and the supernatants were collected. The total protein was measured by a BCA protein assay (HyClone-Pierce, Rockford, IL, USA), separated by 12.5% sodium dodecyl sulphate polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and blocked for 1 h at room temperature (25°C). The membranes were then incubated with rabbit anti-GAPDH or rabbit anti-CALCOCO2 primary antibodies (1 : 500; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and incubated at 4°C overnight. The membranes were washed with Tris-buffered saline and Tween, and a moderate volume of secondary antibody (goat anti-mouse IgG, 1 : 5000; Santa Cruz Biotechnology) was added and incubated for 3 h at room temperature. The membranes were then detected using enhanced chemiluminescence (ECL) reagent (ECL-Plus/Kit; Amersham, Piscataway, NJ, USA).

Cell counting

Multiparametric high-content screening (HCS) was utilised to determine the cell growth status. Briefly, U87 and U251 cells in the logarithmic phase in shCALCOCO2 or shControl groups were seeded on 96-well plates at a density of 4000 cells/well. Subsequently, the cells were incubated for 5 days, and every day the living cells exhibiting green fluorescence in each plate were recognised and counted using ArrayScan™ HCS software (Cellomics Inc., Pittsburgh, PA, USA).

MTT assay

A 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetra-zoniumbromide (MTT) assay was performed to assess cell viability. Briefly, the exponential growth cells infected with shCALCOCO2 or shControl were seeded on 96-well plates at a density of 4000 cells/well and incubated for 1, 2, 3, 4, or 5 days. At a predetermined timepoint, 20 µl of MTT was added to the cells, followed by incubation for 4 h. The supernatants were removed, and 100 µl of dimethyl sulphoxide (DMSO) was added to decompose formazan. The viability of cells was analysed by detecting absorbance at 490 nm using a microplate reader (BioTek Instruments, Winooski, VT, USA).

Flow cytometry

U87 and U251 cells infected with shCALCOCO2 or shControl were seeded on six-well plates after lentivirus infection for 5 days at a density of 3 × 10⁵ and cultured for 48 h. Subsequently, the cells were harvested, centrifuged, washed with PBS twice, and then resuspended using staining buffer at a cell concentration of 1.0 × 10⁶/ml. The cell suspensions were then stained with Annexin and PI at room temperature for 15 min in the dark and evaluated by flow cytometry (FCM, FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA).

Caspase-Glo 3/7 assay

Caspase-Glo 3/7 reagent was prepared by mixing caspase-Glo 3/7 with the substrate and was then stored at 4°C. Cells transfected with shCALCOCO2 or shControl at the logarithmic phase were seeded on 96-well plates at a density of 4000 cells/well and then cultured for 1, 2, 3, 4, or 5 days. The caspase-Glo 3/7 reagent was added to the cells at an amount equivalent to the volume of the culture, shaken for 30 s, and cultured for 0.5–3 h at room temperature according to cell conditions. The fluorescence of each well was assessed using a microplate reader.

Gene microarray

The genome-wide effect of the silencing of CALCOCO2 in the U87 cell line was investigated using a GeneChip^® PrimeView™ Human Gene Expression Array (Affymetrix; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Briefly, after cells were treated with shControl or shCALCOCO2 for 72 h, the total mRNA was extracted, quantified, reverse-transcribed, and labelled with biotin using the GeneChip^® 3’ IVT Express Kit (Thermo Fisher Scientific). Subsequently, the labelled cDNAs were used to hybridise the GeneChip^® PrimeView™ Human Gene Expression Array consisting of 20,000 genes according to the manufacturer’s protocol. After hybridisation, the gene chips were washed and scanned using a GeneChip^® Fluidics Station 450, and images were acquired using GeneChip operating software. Data were summarised, and GeneSpring software was used for data analysis. Differentially expressed genes generated from the microarray analyses were analysed by the Ingenuity Pathway Core Analysis (IPA^®, QIAGEN, Redwood City, CA, USA) to interpret the underlying molecular mechanisms. The enrichment of gene networks was analysed based on the overlap score (p-value and z-score). Three main analyses were performed using IPA, i.e. analyses of diseases and functions, gene networks, and downstream targets.

Assessment of downstream target proteins

To investigate the role of CALCOCO2 in the pathogenesis of glioma, the expression levels of related proteins in U87 and U251 cells infected with shCALCOCO2 or shControl were assessed by western blotting. The specific methods were as described above, and the protein levels were measured.

Statistical analyis

All experiments were repeated thrice and the results expressed as means ± standard deviation. Statistical differences were evaluated using paired Student’s t-tests implemented in SPSS 23.0 (SPSS Inc., Chicago, IL, USA). P < 0.05 indicated statistical significance, and p < 0.01 and p < 0.001 were considered highly significant.

CALCOCO2 expression in glioma cells and the silencing of CALCOCO2 in U87 and U251 cell lines

As shown in Figure 1 A, CALCOCO2 mRNA was overexpressed in all four cell lines, and the expression was highest in U87 and U251 cells. Moreover, as shown in Figures 1 B and C, most U87 and U251 cells were positive for green fluorescent protein under a microscope, indicating the efficient silencing of CALCOCO2 in both cell lines. The relative CALCOCO2 mRNA levels for shCALCOCO2-treated cells were 0.138 ±0.014 and 0.375 ±0.025 for the U87 and U251 cell lines, respectively, which were significantly lower than those of shControl cells (1.002 ±0.071 and 1 ±0.032, respectively, p < 0.001; Figures 1 D, E). Consistent with these findings, the protein levels of CALCOCO2 were significantly downregulated compared with those in shControl U87 and U251 cells (Figures 1 F, G).

Figure 1

CALCOCO2 silencing in the U87 and U251 cell lines. A – Expression of CALCOCO2 in glioma cell lines. qRT-PCR was performed to evaluate the expression levels of CALCOCO2 in four glioma cell lines (U87, U251, U373, and A-172). B, C – Microscopic images of U87 and U251 cell lines in the shControl and shCALCOCO2 groups. D, E – qRT-PCR analysis of the efficiency of CALCOCO2 silencing at the mRNA level. F, G – Western blot analysis of the efficiency of CALCOCO2 silencing at the protein level

Data are shown as means ± SD (n = 5; *p < 0.05, **p < 0.01, and ***p < 0.001).

Effects of CALCOCO2 silencing on cell growth

As shown in Figure 2 A, CALCOCO2 silencing significantly inhibited U87 cell growth compared to that of the shControl group (p < 0.05). The cell counting results showed that the proliferation fold change values for the shCALCOCO2 group in the U87 cell line at days 4 and 5 were 2.8 ±0.07 and 4.1 ±0.11, respectively, which were obviously lower than those for shControl cells (4.13 ±0.05 and 6.42 ±0.2, respectively). Similarly, there was a significant difference in cell counts between the shCALCOCO2 and shControl groups in the U251 cell line, especially on days 4 and 5, indicating the inhibitory effect of CALCOCO2 silencing on cell growth. An MTT assay also showed that both U87 and U251 cells exhibited slower proliferation and growth after the silencing of CALCOCO2, and these effects were even more pronounced on day 5, when the proliferation fold changes in the shCALCOCO2 group were 2.163 ±0.0068 and 1.696 ±0.0672 in U87 and U251 cells, respectively, while the proliferation fold changes in the shControl group were 3.882 ±0.0547 and 3.117 ±0.0793, respectively.

Figure 2

Silencing of CALCOCO2 inhibited the proliferation of both U87 and U251 cells. A – Representative images of HCS analysis of U87 cell lines in the shControl and shCALCOCO2 groups at various timepoints after lentivirus infection. B, C – Statistical analysis of cell numbers for the U87. D – Representative images of HCS analysis of U251 cell lines in the shControl and shCALCOCO2 groups at various timepoints after lentivirus infection. E, F – Statistical analysis of cell numbers for the U87 cell lines

HCS – multiparametric high-content screening.

Effects of CALCOCO2 silencing on cell apoptosis

As shown in Figures 3 A–D, the percentages of cell apoptosis in shCALCOCO2-infected U87 and U251 cell lines, as detected by FCM, were 11.44 ±0.1178% and 9.32 ±0.0955%, respectively, on day 4 post-CALCOCO2 silencing, while the shControl group exhibited significantly decreased apoptotic percentages of 4.13 ±0.1308% and 4.3 ±0.1%, respectively (p < 0.001). In addition, caspase3/7 measurements showed that the expression levels of caspase3/7 were approximately 1.72 and 2.07 times greater than those in the shControl group for U87 and U251 cells after infection with shGATAD2A for 3 days (Figures 3 E, F).

Figure 3

Silencing of CALCOCO2 promoted apoptosis in both U87 and U251 cells. A – Cell apoptosis in U87 was analysed by FCM. B – Quantified results of A were presented. C – Cell apoptosis in U251 was analysed by FCM. D – Quantified results of A were presented. E, F – Caspase 3/7 activity assays of U87 and U251 cells

Data are shown as mean ± SD (n = 3; *p < 0.05, **p < 0.01, and ***p < 0.001).

Molecular mechanisms underlying the effects of CALCOCO2 in gliomas

In the gene microarray analysis, there were 586 differentially expressed genes, including 357 genes that were downregulated and 229 genes that were upregulated (Figure 4 A). These discriminative genes were functionally analysed by IPA. As shown in Figure 4 B, 17 CALCOCO2-related functions and diseases were detected by IPA, and infectious diseases, cancer, organismal injury, and abnormalities were the highest ranked categories. The gene interaction network confirmed these results (Figure 4 C). The silencing of CALCOCO2 markedly upregulated CASP1, FMR1, GSK3B, BECR5, CHEK1, and FAS and downregulated the other loci (Figure 4 D). The expression levels of BBECN1, CASP1, FAS, GSK3B, BIRC5, and IL-1β were further analysed by western blotting (Figure 4 E).

Figure 4

Molecular mechanism underlying the effect of CALCOCO2 in glioma pathogenesis and progression. A–D – IPA analysis of differentially expressed genes obtained from the microarray analysis. A – Heat map of differentially expressed genes. B – Z-scores of related functions and diseases. C – Interaction network. Upregulated genes are shown in red, and downregulated genes are shown in green. D – Potential downstream genes influenced by CALCOCO2 by IPA. E – Western blotting analysis

Data are shown as mean ± SD (n = 3; *p < 0.05, **p < 0.01, and ***p < 0.001).