Eight modules were constructed for miRNAs in PCNSL.
Co-expressed miRNAs in PCNSL were clustered into eight modules. Among them, the yellow and the turquoise modules constituted one subgroup, and the green, the black, the brown, the red, the blue, and the pink modules constituted another subgroup (Fig. 1A). Module clustering indicated that the yellow module had the highest correlation with the turquoise module (Fig. 1B). Additionally, higher miRNA expression overall was observed in the yellow and the turquoise modules (Fig. 1C).
miR-432 is closely associated with the yellow and the turquoise modules.
A miRNA-miRNA network was constructed for the top 20 miRNAs in the turquoise and the yellow modules to identify key miRNAs in PCNSL (Fig. 2A and Fig. 2B). A higher number of nodes was observed between miR-330-3p and miR-432 compared to those in other miRNAs in these two modules. Meanwhile, MM values of all miRNAs from these two modules were calculated, and only miR-432 was significantly associated with both modules (Table 1). These results suggested that miR-432 may be a key link between the yellow and the turquoise modules.
Table 1
MM value of miRNAs from yellow and turquoise.
miRNA symbols | Module Color | MM turquoise | p.value MMturquoise | MM yellow | p.value MMyellow |
hsa-miR-1180_st | turquoise | 0.831533932 | 7.73424E-08 | 0.755493074 | 5.21944E-06 |
hsa-let-7e_st | turquoise | 0.898647822 | 1.97129E-10 | 0.725194359 | 1.87477E-05 |
hsa-miR-181c-star_st | turquoise | 0.701275949 | 4.60196E-05 | 0.674294639 | 0.000114912 |
hsa-miR-181d_st | turquoise | 0.906062921 | 7.947E-11 | 0.673373502 | 0.000118364 |
hsa-miR-1301_st | turquoise | 0.83909774 | 4.54851E-08 | 0.672131088 | 0.000123166 |
hsa-miR-125b_st | turquoise | 0.857983461 | 1.06368E-08 | 0.666302517 | 0.000148065 |
hsa-miR-195_st | turquoise | 0.767768656 | 2.94865E-06 | 0.642688908 | 0.000300314 |
hsa-miR-30a-star_st | turquoise | 0.788139208 | 1.05519E-06 | 0.623979705 | 0.000505083 |
hsa-miR-497_st | turquoise | 0.876727826 | 2.01544E-09 | 0.614743479 | 0.000645076 |
hsa-miR-99b_st | turquoise | 0.949820568 | 3.98807E-14 | 0.613891849 | 0.000659542 |
hsa-miR-23b_st | turquoise | 0.959223459 | 3.13691E-15 | 0.601286905 | 0.000909187 |
hsa-miR-99b-star_st | turquoise | 0.883789448 | 1.00311E-09 | 0.596595108 | 0.001021139 |
hsa-miR-99a_st | turquoise | 0.886858157 | 7.30358E-10 | 0.590983623 | 0.001170574 |
hsa-miR-30a_st | turquoise | 0.866518967 | 5.14406E-09 | 0.590517276 | 0.001183804 |
hsa-miR-145_st | turquoise | 0.616227391 | 0.00062053 | 0.584296649 | 0.001373067 |
hsa-miR-26a_st | turquoise | 0.668821007 | 0.000136809 | 0.571434137 | 0.001848806 |
hsa-miR-324-3p_st | turquoise | 0.682108684 | 8.90203E-05 | 0.541161641 | 0.003558269 |
hsa-miR-143_st | turquoise | 0.551927072 | 0.00283888 | 0.526984653 | 0.004738315 |
hsa-miR-138-1-star_st | turquoise | 0.685351317 | 7.98916E-05 | 0.526916151 | 0.004744737 |
hsa-miR-361-5p_st | turquoise | 0.880178016 | 1.44107E-09 | 0.515253615 | 0.005951521 |
hsa-let-7b_st | turquoise | 0.956827603 | 6.32129E-15 | 0.505320544 | 0.007174781 |
hsa-miR-125b-2-star_st | turquoise | 0.966099589 | 3.23665E-16 | 0.501463927 | 0.007703607 |
hsa-miR-30c_st | turquoise | 0.715206616 | 2.75744E-05 | 0.487804116 | 0.009847628 |
hsa-miR-128_st | yellow | 0.531724602 | 0.004311482 | 0.978676951 | 1.05338E-18 |
hsa-miR-487b_st | yellow | 0.556569317 | 0.002569442 | 0.978064376 | 1.49594E-18 |
hsa-miR-432_st | yellow | 0.524728939 | 0.004953713 | 0.9768701 | 2.88352E-18 |
hsa-miR-127-3p_st | yellow | 0.60785983 | 0.000770341 | 0.972053892 | 2.98886E-17 |
hsa-miR-138_st | yellow | 0.544909925 | 0.00329193 | 0.970790774 | 5.1586E-17 |
hsa-miR-134_st | yellow | 0.541974761 | 0.003498982 | 0.91302436 | 3.15475E-11 |
hsa-miR-409-3p_st | yellow | 0.507996635 | 0.006826073 | 0.906233706 | 7.77564E-11 |
hsa-miR-149_st | yellow | 0.753431143 | 5.7264E-06 | 0.863601855 | 6.62984E-09 |
hsa-miR-31_st | yellow | 0.652996218 | 0.000222187 | 0.813879544 | 2.4279E-07 |
hsa-miR-383_st | yellow | 0.57484895 | 0.001710419 | 0.792046667 | 8.5547E-07 |
hsa-miR-769-5p_st | yellow | 0.75825826 | 4.60278E-06 | 0.789553108 | 9.78532E-07 |
hsa-miR-125a-5p_st | yellow | 0.819511057 | 1.70836E-07 | 0.783915088 | 1.31748E-06 |
hsa-miR-874_st | yellow | 0.748878696 | 7.00504E-06 | 0.774319902 | 2.1433E-06 |
hsa-miR-138-2-star_st | yellow | 0.508984239 | 0.006701044 | 0.591676986 | 0.00115114 |
hsa-miR-339-3p_st | yellow | 0.601119059 | 0.000913 | 0.587562573 | 0.001270677 |
miR-432 can discriminate between PCNSL and non-cancer profiles robustly.
To identify whether abnormal miR-432 was also expressed in the peripheral circulation, serum miR-432 was determined in patients with PCNSL and non-cancer samples. The results confirmed that serum miR-432 was higher in patients with PCNSL compared with that in the non-cancer control (Fig. 3A; P < 0.0001). The ROC curve for PCNSL generated with serum miR-432 has an AUC of 0.77 with a 95% confidence interval (CI) of 0.6923 to 0.8550 (P < 0.0001), which suggests that miR-432 may be a potential discriminator between PCNSL and non-cancer profiles.
miR-432 may be involved in the regulation of cell adhesion in PCNSL.
To explore the potential biological function of miR-432 in PCNSL, down-regulated miR-432 target genes were screened. The miR-432 target gene set was downloaded from the TargetScan database, and genes down-regulated in PCNSL were analyzed in GSE25297 (Fig. 4A). Shared genes from these two databases were considered candidate target genes. They included RASGRF, DGKG, SMIM22, SPOCD1, NRCAM, CNTN2, PTPRD, POTED, IGSF3, SLC24A2, CTNND2, AIF1L, TMEM229A, GLDN, and MOBP (Fig. 4B). Among the genes, protein-protein interactions were found for GLDN, CNTN2, NRCAM, MOBP, PTPRD, CTNND2, and RASGRF1 (Fig. 4C). The GO analysis of these seven genes was performed using the “Analysis” function in STRING, and BP, MF, and CC and have been shown in Tables 2–4. Furthermore, we observed that four target genes, CTNND2, GLDN, NRCAM, and PTPRD were involved in regulating cell-cell adhesion.
Table 2
Biological process of main target genes of miR-432.
No. | term description | FDR | matching proteins |
1 | neuronal ion channel clustering | 7.39E-05 | CNTN2,GLDN,NRCAM |
2 | neuron differentiation | 0.00053 | CNTN2,CTNND2,DGKG,GLDN,NRCAM,PTPRD,RASGRF1 |
3 | neuron development | 0.0013 | CNTN2,CTNND2,DGKG,GLDN,NRCAM,RASGRF1 |
4 | synapse organization | 0.0013 | CNTN2,CTNND2,NRCAM,PTPRD |
5 | clustering of voltage-gated sodium channels | 0.0015 | GLDN,NRCAM |
6 | presynaptic membrane organization | 0.0023 | CNTN2,PTPRD |
7 | nervous system development | 0.0037 | CNTN2,CTNND2,DGKG,GLDN,MOBP,NRCAM,PTPRD,RASGRF1 |
8 | axonal fasciculation | 0.0051 | CNTN2,NRCAM |
9 | heterotypic cell-cell adhesion | 0.0071 | GLDN,NRCAM |
10 | regulation of synaptic plasticity | 0.0071 | CNTN2,RASGRF1,SLC24A2 |
11 | cell-cell adhesion | 0.0071 | CTNND2,GLDN,NRCAM,PTPRD |
12 | cell adhesion | 0.0078 | CNTN2,CTNND2,GLDN,NRCAM,PTPRD |
13 | learning or memory | 0.0144 | CNTN2,RASGRF1,SLC24A2 |
14 | regulation of neuronal synaptic plasticity | 0.0144 | CNTN2,RASGRF1 |
15 | plasma membrane bounded cell projection organization | 0.0144 | CNTN2,CTNND2,GLDN,NRCAM,RASGRF1 |
16 | regulation of cell morphogenesis involved in differentiation | 0.0166 | CNTN2,NRCAM,PTPRD |
17 | neuron projection development | 0.0166 | CNTN2,CTNND2,NRCAM,RASGRF1 |
18 | system development | 0.0183 | CNTN2,CTNND2,DGKG,GLDN,IGSF3,MOBP,NRCAM,PTPRD,RASGRF1 |
19 | synapse assembly | 0.0236 | NRCAM,PTPRD |
20 | membrane organization | 0.0252 | CNTN2,GLDN,NRCAM,PTPRD |
21 | memory | 0.0409 | RASGRF1,SLC24A2 |
22 | cell morphogenesis involved in neuron differentiation | 0.0409 | CNTN2,CTNND2,NRCAM |
23 | neuron migration | 0.0439 | CNTN2,NRCAM |
24 | regulation of neuron projection development | 0.0498 | CNTN2,NRCAM,PTPRD |
25 | neuron projection morphogenesis | 0.0498 | CNTN2,CTNND2,NRCAM |
Table 3
Molecular function of main target genes of miR-432.
No. | term description | FDR | matching proteins |
1 | protein binding involved in heterotypic cell-cell adhesion | 0.0036 | GLDN,NRCAM |
Table 4
Cell component of main target genes of miR-432.
No. | term description | FDR | matching proteins |
1 | cell periphery | 0.0203 | AIF1L,CNTN2,DGKG,GLDN,IGSF3,MOBP,NRCAM,POTED,PTPRD,RASGRF1,SLC24A2 |
2 | axon | 0.0256 | CNTN2,GLDN,NRCAM,RASGRF1 |
3 | plasma membrane | 0.0362 | AIF1L,CNTN2,DGKG,GLDN,IGSF3,NRCAM,POTED,PTPRD,RASGRF1,SLC24A2 |
4 | membrane | 0.0362 | AIF1L,CNTN2,DGKG,GLDN,IGSF3,NRCAM,POTED,PTPRD,RASGRF1,SLC24A2,SMIM22,TMEM229A |
5 | axon part | 0.0362 | CNTN2,NRCAM,RASGRF1 |
6 | neuron projection | 0.0362 | CNTN2,CTNND2,GLDN,NRCAM,RASGRF1 |
7 | main axon | 0.0362 | CNTN2,NRCAM |
8 | plasma membrane bounded cell projection | 0.0362 | AIF1L,CNTN2,CTNND2,GLDN,NRCAM,RASGRF1 |
9 | plasma membrane bounded cell projection part | 0.0362 | AIF1L,CNTN2,CTNND2,NRCAM,RASGRF1 |
10 | myelin sheath | 0.043 | CNTN2,MOBP |