Background
Methods
Patients and specimens
Immunostaining
Cell culture and cell growth
Construction of the SLC22A18 expression vector
Determination of the optimal concentration of G418
Transfection of U251 cells with pcDNA3.1-SLC22A18 and PCR confirmation of SLC22A18 expression in U251 cells
Measurement of cell growth
Measurement of apoptosis by flow cytometry
Adhesion assay
Tumor cell adherence to ECV304
Murine xenograft model
Immunohistochemical staining
DNA extraction and MSP
Total RNA isolation and reverse-transcriptase polymerase chain reaction
Western blotting
Data analysis
Results
SLC22A18 protein expression is downregulated in glioma
Downregulation of SLC22A18 protein expression is associated with malignancy in glioma
Aberrant promoter methylation contributes to SLC22A18 downregulation
Patient no. | Age (yr) | Sex | Tumor size(cm) | Recurrence within six months | Pathological grade | Methylation status | SLC22A18 protein expression rate (%) | |
---|---|---|---|---|---|---|---|---|
Glioma | Adjacent brain tissue | |||||||
1 | 53 | M | 3.5 × 4 × 3 | + | IV | + | 4 | 33 |
3 | 42 | M | 3 × 4.5 × 5 | + | III | + | 25 | 58 |
8 | 38 | F | 3.5 × 3 × 3 | + | III | + | 35 | 62 |
9 | 49 | M | 3 × 4 × 3 | + | IV | + | 3 | 42 |
10 | 58 | M | 5.5 × 4 × 4 | - | II | + | 39 | 67 |
15 | 69 | M | 3.5 × 4 × 3 | + | IV | + | 8 | 38 |
17 | 32 | M | 5 × 6 × 4 | - | II | + | 37 | 65 |
19 | 45 | F | 4 × 5 × 6 | - | II | + | 34 | 59 |
20 | 51 | M | 4.5 × 5 × 6 | + | IV | + | 0 | 25 |
21 | 43 | M | 3.5 × 4 × 5 | + | IV | + | 9 | 35 |
24 | 48 | M | 3.5 × 4 × 4 | - | II | + | 32 | 61 |
25 | 55 | F | 3.5 × 4 × 5 | - | II | + | 30 | 59 |
27 | 62 | F | 4 × 4 × 3 | + | IV | + | 0 | 30 |
29 | 34 | M | 5 × 4 × 3 | - | II | + | 37 | 65 |
30 | 39 | M | 3.5 × 2 × 3 | + | IV | + | 6 | 38 |
2 | 71 | M | 2 × 3 × 4 | - | I | - | 45 | 68 |
4 | 33 | M | 3 × 4 × 5 | - | II | - | 32 | 61 |
5 | 43 | M | 3 × 4 × 6 | - | I | - | 46 | 62 |
6 | 46 | F | 3.5 × 4 × 4 | + | III | - | 25 | 57 |
7 | 48 | M | 4.5 × 4 × 5 | - | II | - | 37 | 68 |
11 | 47 | F | 5.5 × 5 × 6 | + | IV | - | 12 | 54 |
12 | 57 | M | 5 × 6 × 5 | - | II | - | 32 | 70 |
13 | 56 | M | 3.5 × 5 × 6 | - | I | - | 52 | 75 |
14 | 46 | M | 3.5 × 5 × 4 | - | I | - | 45 | 72 |
16 | 57 | M | 3 × 3 × 4 | + | IV | - | 14 | 62 |
18 | 53 | M | 3 × 4 × 5 | - | I | - | 52 | 78 |
22 | 54 | F | 5.5 × 6 × 5 | - | I | - | 48 | 73 |
23 | 65 | M | 3 × 4 × 3 | + | III | - | 34 | 65 |
26 | 46 | F | 3.5 × 5 × 4 | - | I | - | 52 | 82 |
28 | 62 | M | 3 × 4.5 × 5 | + | III | - | 29 | 62 |
Promoter demethylation increases SLC22A18 expression and reduces U251 cell growth
PCR analysis of SLC22A18 in U251 cells
SLC22A18 protein is expressed in U251-SLC22A18 cells
Cell proliferation assay
Induction of apoptosis by the SLC22A18 expression
Group (n = 6) | 24 h | 48 h |
---|---|---|
Normal U251 | 1.95 ± 0.32 | 3.69 ± 0.19 |
U251-EV | 1.95 ± 0.38 | 3.71 ± 0.21 |
U251-SLC22A18 | 25.25 ± 4.28 | 30.84 ± 4.72 |