High expression of ATM in human glioma cell lines and tissues. Expression of ATM was determined in HEB, the human normal glial cell line, and U87, U251, LN229 and U373, the human glioma cell lines, by qRT-PCR (Fig. 1A) and western blot (Fig. 1B). All glioma cell lines expressed significantly higher levels of ATM as compared to HEB (P < 0.001) (Fig. 1A&B). Further, the expression of ATM in glioma tissue and its adjacent normal tissue was also detected by qRT-PCR and western blot. Following the trend, the mRNA level of ATM was found significantly increased in glioma tissue as compared to its adjacent tissue (P < 0.001) (Fig. 1C&D).
ATM silencing induced TMZ chemotherapy resistance of glioma in vitro. U251-LV-ATM-KD and U87-LV-ATM-KD stable cell lines were constructed by lentivirus infection and the efficiency was verified by measuring the expression of ATM mRNA and protein level (Fig. 2A&B). These cells were used to investigate the role of ATM in TMZ chemoresistance. The 50% inhibitory concentration (IC50) of TMZ for U87 and U251 cells, as determined by MTT assay, was found to be 53.8 µmol/ml and 113.9 µmol/ml for U87 and U251 cells, respectively (Fig. 2C&D). Further, we detected the effect of ATM knockdown on the resistance of U87 and U251 cells to TMZ chemotherapy. As shown in Fig. 2E, after ATM knockdown, the resistance of U87 and U251 cells to TMZ chemotherapy was significantly decreased, thus significantly reducing the percentage of cellular apoptosis in response to TMZ treatment (p < 0.01) (Table 2). Cell cycle analysis of ATM knockdown U87 and U251 cells in response to TMZ (IC50) chemotherapy was done by flowcytometry. Results revealed a significant decrease in percentage of G1 phase cells (P < 0.01) and increase in S phase cells (P < 0.05) in both the cell lines. Besides, a significantly higher percentage of U251 cells were found in G2/M phase as compared to U87 cells (P < 0.05) (Table 3 and Fig. 2F). ATM knockdown significantly inhibited the apoptosis of U87 and U251 cells in response to TMZ (IC50) chemotherapy (p < 0.01) (Fig. 2G). The above results suggests that the ATM knockdown in glioma cells may promote their DNA synthesis and cell division, while inhibiting apoptosis, in response to TMZ chemotherapy.
Table 2
Effects of ATM knockdown on the cell cycle of glioma cell TMZ (IC50) chemotherapy
Groups
|
G1(%)
|
P
|
S(%)
|
P
|
G2/M(%)
|
P
|
U87NC + TMZ
|
50.30 ± 1.58
|
|
28.55 ± 1.87
|
|
21.15 ± 1.83
|
|
U87siATM-1 + TMZ
|
43.63 ± 0.13
|
0.0019
|
32.91 ± 0.82
|
0.021
|
23.46 ± 0.69
|
0.110
|
U251NC + TMZ
|
48.57 ± 0.54
|
|
37.09 ± 0.69
|
|
18.53 ± 0.48
|
|
U251siATM-1 + TMZ
|
43.42 ± 0.38
|
0.0055
|
38.68 ± 0.49
|
0.043
|
20.12 ± 0.58
|
0.042
|
Table 3
The first 10 signaling pathways and the number of genes involve after ATM knockdown
Gene Set
|
No. of Genes
|
Significance Probability
|
KEGG_FOCAL_ADHESION
|
21
|
2.67E-09
|
KEGG_ECM_RECEPTOR_INTERACTION
|
13
|
1.05E-07
|
KEGG_PATHWAYS_IN_CANCER
|
20
|
2.99E-05
|
KEGG_LYSOSOME
|
11
|
2.79E-04
|
KEGG_NEUROTROPHIN_SIGNALING_PATHWAY
|
11
|
2.98E-04
|
KEGG_MAPK_SIGNALING_PATHWAY
|
16
|
2.98E-04
|
KEGG_RENAL_CELL_CARCINOMA
|
8
|
6.80E-04
|
KEGG_REGULATION_OF_ACTIN_CYTOSKELETON
|
13
|
1.65E-03
|
KEGG_CELL_ADHESION_MOLECULES_CAMS
|
10
|
1.96E-03
|
KEGG_ETHER_LIPID_METABOLISM
|
5
|
5.54E-03
|
ATM silencing induced TMZ chemotherapy resistance of glioma, in vivo. A subcutaneous xenograft tumor model of ATM knockdown glioma cells was constructed in nude mice, to further study the molecular mechanism in vivo. ATM knockdown could significantly inhibit the tumor growth rate in nude mice, as compared to control group (Fig. 3A-C). Further, in response to TMZ chemotherapy, 3 days after stopping the treatment (the 32nd day after injecting cells), an increase in tumor growth was observed. Tumor volume in the control group decreased to the lowest level on the 10th day (the 39th day after injecting cells) after stopping the chemotherapy, but started to increase from the 13th day (the 42nd day after injecting cells). However, nude mice in ATM knockdown group showed little response to TMZ chemotherapy, demonstrated by continued proliferation of the tumor reaching a significantly increase in volume on 10th and 13th day (the 39th day and 42nd day after injecting cells) after stopping TMZ chemotherapy, as compared to the control group (p < 0.05), (Fig. 3D).
Screening for ATM knockdown-induced TMZ chemotherapy-resistant genes in glioma. In order to further explore the molecular mechanism of ATM knockdown induced glioma resistance to TMZ chemotherapy, we used human genome-wide oligonucleotide microarray to detect the changes of gene expression profile after ATM knockout in U251 cells. Further, using the High Content Screening Cellomics system, the role of differentially expressed genes in U251 cells in response to TMZ chemotherapy and the target genes related to the chemotherapy-resistance caused by ATM knockdown, especially those capable of inducing apoptosis and improving chemotherapy-sensitivity, were screened out. As shown in Fig. 4A, B, C, D, and E, the quality of RNA samples used for gene chip detection and the coincidence degree of each signal distribution curve of the chip data was good, indicating the reliability of the experiment. The interchip correlation coefficients in each group were very close to 1.0, indicating that the data had good repeatability and reliability, and could be used for the screening of downstream differentially expressed genes. The screening criteria for genes with significant differences must be in line with |FC| >1.5, and P < 0.05, and according to this standard, 324 up-regulated genes and 402 down-regulated genes were screened out. The two factors of p value and FC value obtained by T test analysis were used to draw the volcano map that highlighted the significant difference between the two groups of sample data. In the volcano map, the y-coordinate shows the -lg p value calculated by T-test, and the x-coordinate shows the FC value after log2 conversion. In the upper left red region, down-regulated genes were expressed, while in the upper right red region, up-regulated genes were expressed (Fig. 4F). Cluster analysis was performed on all differentially expressed genes (Fig. 4G ). In the figure, there was a gene in each row and a specimen in each column. Different colors represented the expression levels of different genes; red represents up-regulation and green represents down-regulation of gene expression, while black represents no difference in gene expression. The expression profiles of the three samples in the U251-NC and U251ATM-KD groups were highly consistent, and the differences between the samples in the U251-NC and U251ATM-KD groups could be clearly distinguished by the color gradient change, indicating the high reliability of these differentially expressed genes screened. We also conducted pathway, molecular function, biological process and cellular component GO analysis of differentially expressed genes after ATM knockdown, sort by p value. Pathway analysis showed the possibility of high correlation of MAPK and tumor-related pathways with this study (Fig. 4H, Table 4). Molecular function GO analysis showed that ATM is mainly related to the function of DNA binding, transcription-related factor activity, transcription factor binding, receptor binding, G protein and small G protein regulatory active molecules (Fig. 4I, Table 5). Biological process GO analysis showed that ATM is mainly involved in biological processes, including intracellular signal transduction, tissue and organ development, negative regulation of cellular biological processes, and gene transcription (Fig. 4J, Table 6). Cellular component GO analysis showed that ATM knockdown induced differentially expressed genes were composed of cellular components, including cytoplasmic proteins and membrane related proteins (Fig. 4K, Table 7).
Table 4
The first 10 Molecular functional gene set and the number of genes involve after ATM knockdown
Gene Set
|
No. of Genes
|
Significance Probability
|
DNA_BINDING
|
37
|
5.40E-10
|
TRANSCRIPTION_FACTOR_ACTIVITY
|
24
|
3.53E-07
|
TRANSCRIPTION_REPRESSOR_ACTIVITY
|
14
|
1.40E-05
|
TRANSCRIPTION_COREPRESSOR_ACTIVITY
|
11
|
2.15E-05
|
RECEPTOR_BINDING
|
21
|
3.92E-05
|
SMALL_GTPASE_REGULATOR_ACTIVITY
|
9
|
5.58E-05
|
HEPARIN_BINDING
|
6
|
5.74E-05
|
GTPASE_REGULATOR_ACTIVITY
|
11
|
1.88E-04
|
TRANSCRIPTION_FACTOR_BINDING
|
17
|
2.83E-04
|
GLYCOSAMINOGLYCAN_BINDING
|
6
|
4.31E-04
|
Table 5
The first 10 biological process gene set and the number of genes involve after ATM knockdown
Gene Set
|
No. of Genes
|
Significance Probability
|
SIGNAL_TRANSDUCTION
|
79
|
2.21E-16
|
MULTICELLULAR_ORGANISMAL _DEVELOPMENT
|
61
|
3.32E-16
|
ANATOMICAL_STRUCTURE_DEVELOPMENT
|
57
|
1.17E-14
|
NEGATIVE_REGULATION_OF_BIOLOGICAL _PROCROCESS
|
46
|
1.17E-14
|
NEGATIVE_REGULATION_OF_CELLULAR _PROCESCESS
|
44
|
4.14E-14
|
SYSTEM_DEVELOPMENT
|
50
|
1.84E-13
|
NUCLEOBASENUCLEOSIDENUCLEOTIDE_AND_NUCNUCLEIC_ACID_METABOLIC_PROCESS
|
60
|
9.39E-13
|
BIOPOLYMER_METABOLIC_PROCESS
|
71
|
2.10E-12
|
TRANSCRIPTION
|
44
|
5.28E-12
|
ESTABLISHMENT_OF_LOCALIZATION
|
44
|
6.14E-10
|
Table 6
The first 10 cellular component gene set and the number of genes involve after ATM knockdown
Gene Set
|
No. of Genes
|
Significance Probability
|
CYTOPLASM
|
102
|
4.08E-22
|
MEMBRANE
|
91
|
2.02E-18
|
NUCLEUS
|
72
|
1.05E-16
|
INTEGRAL_TO_MEMBRANE
|
64
|
6.22E-14
|
INTRINSIC_TO_MEMBRANE
|
64
|
9.32E-14
|
MEMBRANE_PART
|
71
|
5.26E-13
|
CYTOPLASMIC_PART
|
62
|
2.41E-12
|
PLASMA_MEMBRANE
|
63
|
2.50E-12
|
INTEGRAL_TO_PLASMA_MEMBRANE
|
46
|
7.07E-10
|
INTRINSIC_TO_PLASMA_MEMBRANE
|
46
|
1.02E-09
|
Table 7
Effects of target gene knockdown on TMZ (IC50) chemotherapy in U251 cells
Groups
|
proliferation rate
|
Chemotherapy proliferation rate
|
Chemotherapy inhibition rate(%)
|
U251NC + TMZ
|
2.41
|
0.60
|
40.05
|
U251NC
|
4.02
|
U251siATM-1 + TMZ
|
3.40
|
0.90
|
10.29
|
U251siATM-1
|
3.79
|
U251siCPA4 + TMZ
|
1.13
|
0.76
|
24.67
|
U251siCPA4
|
1.50
|
U251siHKDC1 + TMZ
|
2.54
|
0.86
|
14.48
|
U251siHKDC1
|
2.97
|
U251siMPZL1 + TMZ
|
1.89
|
0.74
|
26.17
|
U251siMPZL1
|
2.56
|
U251siNME4 + TMZ
|
1.67
|
0.82
|
17.73
|
U251siNME4
|
2.03
|
U251siPGM2L1 + TMZ
|
2.53
|
0.80
|
20.44
|
U251siPGM2L1
|
3.18
|
U251siTNFAIP2 + TMZ
|
3.15
|
0.72
|
28.57
|
U251siTNFAIP2
|
4.41
|
U251siTNFRSF21 + TMZ
|
2.10
|
0.74
|
26.32
|
U251siTNFRSF21
|
2.85
|
U251siUSP18 + TMZ
|
1.15
|
0.63
|
37.16
|
U251siUSP18
|
1.83
|
U251siUSP25 + TMZ
|
2.28
|
0.68
|
31.32
|
U251siUSP25
|
3.32
|
U251siRBFOX2 + TMZ
|
2.61
|
0.55
|
55.18
|
U251siRBFOX2
|
4.73
|
U251siARHGAP29 + TMZ
|
2.86
|
0.65
|
34.85
|
U251siARHGAP29
|
4.39
|
U251siZNF404 + TMZ
|
3.07
|
0.65
|
35.23
|
U251siZNF404
|
4.74
|
U251siIFIT1 + TMZ
|
2.08
|
0.91
|
8.77
|
U251siIFIT1
|
2.28
|
U251siTNFSF18 + TMZ
|
2.13
|
0.76
|
23.93
|
U251siTNFSF18
|
2.80
|
U251siDCP2 + TMZ
|
2.64
|
0.55
|
45.11
|
U251siDCP2
|
4.81
|
U251siBNIP3L + TMZ
|
2.43
|
0.82
|
17.63
|
U251siBNIP3L
|
2.95
|
Screening and identification of genes related to ATM knockdown induced TMZ chemotherapy resistance of glioma. After ATM knockdown, differentially expressed genes were screened to enter the downstream test according to the following criteria: differentially expressed new genes with p < 0.05 and |FC|>1.5, whose functions were not fully defined, remove multiple transmembrane protein genes, and remove genes with unknown functions. For the 402 down-regulated genes, 19 genes were preliminarily screened by the screening criteria, and the relative expressions of 19 candidate genes in U251 cells were detected by qRT-PCR (Fig. 5A). In order to identify the role of 19 candidate genes in glioma TMZ chemotherapy, RNA interference of multiple targets was performed on each of them, and stable lentivirus knockdown cell lines were constructed. CDC23, RAC1 and CD164, which were less than 70% of the fluorescence efficiency after lentivirus infection, were not considered further and a total of 16 genes were finally detected downstream. High content screening Cellomics system (HCS) was used to detect the effect of candidate gene RNA interference on TMZ treated U251 cell, the cell proliferation, chemotherapy proliferation rate and chemotherapy inhibition rate after 5 days of chemotherapy (Table 8); U251-NC was used as negative control and U251-ATM-KD-1 was used as positive control. Among the 16 candidate genes, 14 had lower chemotherapy inhibition rate than the negative control (40.05%). Among them, the candidate genes that were close to the positive control U251-ATM-KD-1 (10.29%) and might be related to apoptosis are: IFIT1 (the inhibition rate of TMZ chemotherapy was 8.77%), BNIP3L (the inhibition rate of TMZ chemotherapy was 17.63%), and NME4 (the inhibition rate of TMZ chemotherapy was 17.73%). The 96-well plate was scanned and read at the same time point and in the same field for 5 consecutive days, image collection and data analysis were conducted, and the scan images (Fig. 5B) and cell count values of the 5 days of TMZ (IC50) chemotherapy of the three candidate gene knockdown cells were obtained. U251-NC was used as the negative control, and U251-ATM-KD-1 was used as the positive control. As shown in Fig. 5C, D and E, after knockdown of IFIT1, BNIP3L and NME4 respectively, the proliferation of U251 cells in TMZ (IC50) were all higher than those in U251-NC group. Of these, IFIT1 showed the highest response, which was equivalent to the positive control U251-ATM-KD-1. Therefore, in order to reveal the molecular mechanism of ATM knockdown induced resistance to TMZ chemotherapy of glioma, we choose IFIT1 for in-depth study.
Table 8
Effects of IFIT1 knockdown on the cell cycle of glioma cell TMZ (IC50) chemotherapy
Groups
|
G1(%)
|
p
|
S(%)
|
p
|
G2/M(%)
|
p
|
U251NC + TMZ
|
28.38 ± 3.9
|
|
44.49 ± 4.52
|
|
27.13 ± 2.52
|
|
U251siIFIT1 + TMZ
|
5.77 ± 0.86
|
0.000013
|
56.54 ± 5.31
|
0.0022
|
37.69 ± 3.46
|
0.0004
|
U87NC + TMZ
|
31.12 ± 4.3
|
|
45.56 ± 3.8
|
|
23.32 ± 3.52
|
|
U87siIFIT1 + TMZ
|
10.25±
|
0.0001
|
57.53 ± 5.1
|
0.00355
|
32.22 ± 3.87
|
0.00108
|
Role of IFIT1 in glioma cell TMZ chemotherapy. In order to further verify the role of IFIT1 gene in glioma’s resistance to TMZ chemotherapy, we used single-target RNA to interfere with the expression of IFIT1 in U251 cells. At first, we detected the expression of IFIT1 in U87, U251, LN229, U373 and HEB cell lines by qRT-PCR and western blot, which revealed an increased expression in all the tested glioma cell lines, as compared to HEB cell line (Fig. 6A and B). Then, with lentivirus interference, we constructed U251-IFIT1-KD and U87-IFIT1-KD stable cell lines (Fig. 6C and D). Both regular and knockdown versions of U251 cells were treated with TMZ for 5 days and then assessed for changes in Cell proliferation, clone formation, cell cycle and apoptosis. MTT assay was used to detect the changes in cell proliferation, and as shown in Fig. 6E and F, the proliferation of U251-IFIT1-KD cells was higher than that of the control U251-NC cells. On the 5th day of TMZ treatment, the inhibition rate of chemotherapy on U251-IFIT1-KD group (15.3 ± 3.2 %) was significantly lower than that of the negative control group (29.3 ± 6.6 %), (p < 0.05). Meanwhile, a similar suppression in the inhibition rate of TMZ chemotherapy was also seen in U87 cells (Fig. 6G, P < 0.05). Further, the number of clonal formation of U251-IFIT1-KD + TMZ (IC50) and U87-IFIT1-KD + TMZ (IC50) group was significantly less than their respective control cells, (Fig. 6H, p < 0.05). FACS method was used to detect cell cycle stage and the results showed that when IFIT1 was knockdown, cells at G1 phase and S phase increased significantly (p < 0.001), while proportionally decreasing cells at G2/M phase. This indicates that the IFIT1 knockdown can promote DNA synthesis and cellular proliferation while inducing resistance to TMZ chemotherapy in U251 and U87 cells (Fig. 6I and Table 9). Annexin v-apc single staining apoptosis analysis showed that the rate of apoptosis of U251/U87-IFIT1-KD + TMZ (IC50) group (6.61 ± 0.28%) was significantly lower than that of the control group (7.43 ± 0.41%) (Fig. 6J, p < 0.001). The above results showed that IFIT1 knockdown can significantly promote the proliferation of glioma cells, while inhibiting their apoptosis, thus imparting resistance in glioma cells to TMZ chemotherapy. Thus, IFIT1 may serve as a molecular marker indicating the sensitivity of glioma to TMZ chemotherapy.
Table 9 Correlation of p-ATM, IFIT1 and MGMT expression in glioblastoma
Groups
|
n
|
p-ATM
|
MGMT
|
Low
|
High
|
P
|
Low
|
High
|
P
|
IFIT1
|
|
Low
|
13
|
13
|
0
|
|
7
|
6
|
|
High
|
57
|
42
|
15
|
0.037
|
48
|
9
|
0.016
|
Expression of p-ATM, IFIT1 and MGMT in glioblastoma tissues. Immunohistochemical staining was used to evaluate the expression of p-ATM, IFIT1 and MGMT in tissues obtained from 70 cases of glioblastoma. p-ATM was expressed in the nucleus and/or cytoplasm, and the median expression was 0 % (range 0–30%), including 55 cases with low expression (Fig. 7A) and 15 cases with high expression (Fig. 7B), IFIT1 was primarily expressed in the cytoplasm, with a median expression of 24% (range 0–65%), including 13 cases with low expression (Fig. 7C) and 57 cases with high expression (Fig. 7D). Further, with 55 cases of low expression and 15 cases of high expression, the median expression of MGMT was found to be 10% (range 0–50%). In addition, the expression of IFIT1 was observed in endothelial cells of certain specimens (Fig. 7E), tumor tissue necrosis area (Fig. 7F), edematous area (Fig. 7G), and around vascular endothelial cells (Fig. 7H). Spearman's analysis revealed a positive correlation between p-ATM and IFIT1 expression (r = 0.249, p < 0.05), and a negative correlation between IFIT1 and MGMT expression (r=-0.288, p < 0.05) (Table 10).
Table 10 Correlation between expression of p-ATM and clinical characteristics
Groups
|
n
|
p-ATM
|
Low
|
High
|
P
|
Gender
|
|
Male
|
42
|
32
|
10
|
|
Female
|
28
|
23
|
5
|
0.555
|
Surgery Age
|
|
> 60
|
29
|
26
|
3
|
|
≤ 60
|
41
|
29
|
12
|
0.059
|
KPS Score
|
|
> 70
|
44
|
37
|
7
|
|
≤ 70
|
26
|
18
|
8
|
0.146
|
The relationship between the clinical characteristics and expression of p-ATM, IFIT1 and MGMT. Mann-whitney test found no correlation between p-ATM expression and the clinical characteristics, including gender, age and KPS (Table 11). In order to analyze the relationship between the prognosis and expression of p-ATM, we used progression free survival (PFS) and overall survival (OS) as evaluation indicators, by following up 70 patients for a period of 11–154 weeks, median follow-up time was 45 weeks. During this period, tumor progression was observed in 64 cases (91.4%) and death in 43 cases (61.4%). Univariate analysis indicated (Table 12) no correlation of ATM expression with neither PFS (Fig. 8A) nor OS (Fig. 8B). Further, we verified the expression of p-AKT and IFIT1 in glioblastoma tissues and their relationship with prognosis. Results found that the IFIT1 was widely expressed in glioblastoma, and the extent of its expression was positively correlated with the expression of p-ATM, and negatively correlated with the expression of MGMT. Increased expression of IFIT1 generally indicates a better prognosis, while the combined analysis of IFIT1 and MGMT is more accurate in predicting the prognosis.
Table 11
Univariate analysis of factors affecting patients' PFS and OS(log-rank test)
Groups
|
cases
|
PFS
|
OS
|
Median
|
95% CI
|
P
|
Median
|
95% CI
|
P
|
Gender
|
|
|
|
|
|
|
|
Male
|
42 (60.0)
|
27
|
16.4–37.6
|
|
56
|
30.7–81.3
|
|
Female
|
28 (40.0)
|
24
|
11.9–36.1
|
0.934
|
44
|
28.9–59.1
|
0.550
|
ATM
|
|
|
|
|
|
|
|
Low
|
55 (78.6)
|
28
|
19.1–36.9
|
|
53
|
36.8–69.2
|
|
High
|
15 (21.4)
|
23
|
14.1–31.9
|
0.730
|
38
|
25.8–50.2
|
0.762
|
Table 12
The primer sequences for PCR.
Names
|
primers
|
Sequences (5’- 3’)
|
GAPDH
|
Forward
|
TGACTTCAACAGCGACACCCA
|
Reverse
|
CACCCTGTTGCTGTAGCCAAA
|
ATM
|
Forward
|
CCACCAGAATCTCAAGGAATCAC
|
Reverse
|
AGTAGCAGCCAAGGACACC
|
ARHGAP29
|
Forward
|
GACTTTCATCGAAAACTTCCACG
|
Reverse
|
AATTTGCGAAACTTGTGTGTGAG
|
BNIP3L
|
Forward
|
TTGGATGCACAACATGAATCAGG
|
Reverse
|
TCTTCTGACTGAGAGCTATGGTC
|
CDC23
|
Forward
|
CATGGCTGCAATAGCAAGAAAG
|
Reverse
|
CGCCTCATTTTTCACTTGTCCT
|
DCP2
|
Forward
|
TTGTGCTGCTAGAGAGGTCTT
|
Reverse
|
GAGAACCACTCAATGTTCCGAAT
|
IFIT1
|
Forward
|
GCGCTGGGTATGCGATCTC
|
Reverse
|
CAGCCTGCCTTAGGGGAAG
|
RAC1
|
Forward
|
ATGTCCGTGCAAAGTGGTATC
|
Reverse
|
CTCGGATCGCTTCGTCAAACA
|
RBFOX2
|
Forward
|
GACGCAATGGTTCAGCCTTTT
|
Reverse
|
GCGTACTTCCGTAGAGTGTCAG
|
TNFSF18
|
Forward
|
AGCCATTCAAGAACTCAAGGAG
|
Reverse
|
AGGAGGTTCAGAAGATGCCATT
|
ZNF404
|
Forward
|
AAGTAAATGCGTACCATCAGGAG
|
Reverse
|
TCCCACTTTAGGTCTCTGTTGT
|
CD164
|
Forward
|
CAGTGCCTTAGAAGACAGTGAA
|
Reverse
|
AAGTTACATCCTCTGACCAATCC
|
CPA4
|
Forward
|
AGGTGGATACTGTTCATTGGGG
|
Reverse
|
TTGCTGATCTCGTCTCCATTTC
|
HKDC1
|
Forward
|
TGAGCCGTCTGACCAAAGC
|
Reverse
|
TAGGGGTCGTCATAGGCACA
|
MPZL1
|
Forward
|
ACGCCAAAAGAAATCTTCGTGG
|
Reverse
|
TCAACCCGCCAGTCGTACTA
|
NME4
|
Forward
|
AGGGTACAATGTCGTCCGC
|
Reverse
|
GACGCTGAAGTCACCCCTTAT
|
PGM2L1
|
Forward
|
CGAGATCGTCTTTGTTGCCGA
|
Reverse
|
AGCCTCTCTGCTTGAAGTCTG
|
TNFAIP2
|
Forward
|
GGCCAATGTGAGGGAGTTGAT
|
Reverse
|
CCCGCTTTATCTGTGAGCCC
|
TNFRSF21
|
Forward
|
TTGACTGACCGAGAATGCACT
|
Reverse
|
TTCATCACACTAGAAGGCACATC
|
USP18
|
Forward
|
CAGTCTGGAGGGCAGTATGAG
|
Reverse
|
GGCATTTCCATTAGCACTCC
|
USP25
|
Forward
|
GCACCAGCAGACGTTTTTGAA
|
Reverse
|
AGCATTCTTCGCAGTAAGGAAA
|