Expression profiles of microRNAs in familial or sporadic AIMAH cases compared with normal control
MicroRNA microarray analysis was respectively performed on tissue samples from 3 cases of familial AIMAH patients (Group1), 2 sporadic cases (Group2) as well as 3 normal control tissues (Group3) to compare the difference on microRNA expression levels. The differences on microRNAs expression were analyzed between familial AIMAH patients and normal controls to identify potential microRNA which may play important roles in the pathogenesis of familial AIMAH. 16 microRNAs passed the FDR cutoff value of 0.05. 7 miRNAs were upregulated (hsa-miR-4306,hsa-miR-130a༌hsa-miR-20b༌hsa-miR-20a༌hsa-miR-15a༌hsa-miR-106a༌hsa-miR-17) and 3 miRNAs were downregulated (hsa-miR-197, hsa-miR-3656, hsa-miR-3196) (Table 1). However, the following microRNAs exhibit differentially expression levels with statistical significance but signal intensities were very low, such as: hsa-miR-17*, hsa-miR-18b, hsa-miR-1976, has-miR-454, has-miR-629*, has-miR-629. Although we could detect some difference on microRNAs expression levels between the group of familial AIMAH patients and normal controls, expression levels of these microRNAs were statistically insignificant with p-value more than 0.05. After filtering low-intensity miRNAs, raw signal intensities were normalized by median. The differentially expressed miRNAs passed volcano plot filtering(Fig. 2A).
Table 1
MiRNAs with signifificantly different expression (P < .05) between familial AIMAH patients (Group1) and normal control tissues (Group3)
miRNAs | Fold change | P value | Case/control |
hsa-miR-4306 hsa-miR-130a hsa-miR-20b hsa-miR-20a hsa-miR-15a hsa-miR-106a hsa-miR-17 hsa-miR-197 hsa-miR-3656 hsa-miR-3196 hsa-miR-17* hsa-miR-18b hsa-miR-1976 has-miR-629* has-miR-629 has-miR-454 | 1.43 3.05 3.11 2.07 1.97 1.98 1.98 0.52 0.53 0.51 | 0.003223 0.03120 0.003463 0.005487 0.04733 0.01258 0.01607 0.02572 0.04458 0.006570 0.01237 0.02489 0.03086 0.03518 0.03823 0.03496 | Up Up Up Up Up Up Up Down Down Down Up Up Down Down Up Down |
Similar analysis was performed on the differences of microRNAs expression levels between 2 sporadic AIMAH patients and 3 normal controls. Through filtering according to FDR cutoff value of 0.05 and expression signal intensities, 8 microRNAs were differentially expressed between above 2 groups, 1 miRNAs were upregulated (hsa-miR-3196) and 2 miRNAs were downregulated (hsa-miR-342-3p, hsa-miR-532-3p) (Table 2). However there was 5 microRNAs meeting requirements of statistical significance but their signal intensities were not strong enough, including hsa-miR-1976, hsa-miR-485-3p, hsa-miR-3195, hsa-miR-3180 and hsa-miR-445. After filtering low-intensity miRNAs, raw signal intensities were normalized by median. The differentially expressed miRNAs passed volcano plot filtering (Fig. 2B). The other microRNAs were found no expression difference in sporadic AIMAH patients compared to that of normal controls.
Table 2
MiRNAs with signifificantly different expression (P < .05) between sporadic cases (Group2) and normal control tissues (Group3)
miRNAs | Fold change | P value | Case/control |
hsa-miR-342-3p hsa-miR-532-3p hsa-miR-3196 hsa-miR-1976 hsa-miR-485-3p hsa-miR-3195 hsa-miR-3180 hsa-miR-445 | 0.21 0.65 1.41 | 0.009685 0.04271 0.02156 0.0125 0.0241 0.0272 0.0288 0.0370 | Down Down Up Down Down Up Up Up |
In order to identify the microRNAs which were differentially expressed between familial and sporadic AIMAH patients, we compared the microRNAs expression profiles of these two independent groups of samples. The same criteria of FDR cutoff value and expression intensities were applied to filter the candidate micro RNAs. Results showed that, 2 miRNAs were upregulated (hsa-miR-342-3p, hsa-miR-532-3p) and 9 miRNAs were downregulated (hsa-miR-4306, hsa-miR-20b, hsa-miR-20a, hsa-miR-106a, hsa-miR-17, hsa-miR-101, hsa-miR-16, hsa-miR-26a, hsa-miR-106b) (Table 3). After filtering low-intensity miRNAs, raw signal intensities were normalized by median. The differentially expressed miRNAs passed volcano plot filtering (Fig. 2C).
Table 3
MiRNAs with signifificantly different expression (P < .05) between familial AIMAH patients (Group1) and sporadic cases (Group2)
miRNAs | Fold change | P value | Case/control |
hsa-miR-342-3p hsa-miR-532-3p | 3.6 2.1 | 0.01188 0.03911 | Up Up |
hsa-miR-4306 hsa-miR-20b hsa-miR-20a hsa-miR-106a hsa-miR-17 hsa-miR-101 hsa-miR-16 hsa-miR-26a hsa-miR-106b | 0.71 0.35 0.46 0.44 0.44 0.24 0.64 0.81 0.46 | 0.006598 0.009508 0.006258 0.04284 0.02335 0.01947 0.02747 0.02877 0.02046 | Down Down Down Down Down Down Down Down Down |
Validation Of Microrna Arrays By Qrt-pcr
With the purpose of validating the results from microRNA microarrays, qRT-PCR was performed on total RNA extracted from familial and sporadic AIMAH surgical tissues compared with the normal controls respectively. We evaluated microRNA level of has-miR-20b that, based on our microarray data, was differentially expressed in tissues from familial AIMAH patients compared with normal controls. The expression level of has-miR-20b in familial AIMAH patients was 1.56 fold higher than normal controls with p-value of 0.01165 which was in accordance with the results from microRNA arrays (3.11 fold higher with p-value = 0.003463). The expression levels of has-miR-342-3p (0.68 fold downregulated with p-value = 0.005814) were determined by qRT-PCR respectively in sporadic AIMAH patients and normal control samples to validate the comparison of microRNA expression profile between these 2 groups, which was consistent with the result from microRNA array (hsa-miR-342-3p was 0.21 fold downregulated in sporadic AIMAH patients versus normal controls). Similarly, hsa-miR-342-3p and hsa-miR-101 were selected to validate the different microRNA expression levels between familial and sporadic AIMAH patients, qRT-PCR results showed that, hsa-miR-342-3p was 1.58 fold upregulated while hsa-miR-101 was 0.40 fold downregulated in familial AIMAH patients compared to sporadic patients. These qRT-PCR test results were consistent with those from microRNA arrays in which hsa-miR-342-3p was 3.6 fold upregulated and hsa-miR-101 was 0.24 fold downregulated in familial AIMAH patients versus sporadic patients.
Hierarchical Clustering Of Micrornas With Different Expression Levels
In order to identify the special microRNAs expression signatures among different groups of patients, hierarchical clustering analysis was performed on all microRNAs expression levels in the form of signal intensities included in microarrays. This hierarchical clustering analysis clustered microRNAs together according to their expression levels and samples from different groups based on similarity of the investigated microRNA expression profiles[24]. Supervised hierarchical pairwise comparison among these 3 groups of samples, we could always identify a special group of microRNAs which could discriminate one group from the other. The 16 differential expressed microRNAs signature from the results in microarray could clearly discriminate between familial AIMAH patients and normal controls including 10 downregulated microRNAs and 6 upregulated microRNAs (Fig. 3A). Similarly, the microRNAs signature containing 8 differentially expressed microRNAs could discriminate between the sporadic AIMAH patients and normal controls with statistical significance p-value of 0.05 (Fig. 3B). The identified 17 differentially expressed microRNAs could well discriminate between familial and sporadic AIMAH patients.
Comprehensive target-network prediction of differential expressed microRNAs and Pathway analysis
Functional analysis of the altered microRNAs between the AIMAH patients and normal controls revealed that some enriched pathways such as signal transduction, signaling molecules and interaction metabolic, were potentially associated with the pathogenesis of AIMAH. When comparison analysis was performed between familial AIMAH patients and normal controls, all of the 16 altered microRNAs were imported into KEGG Pathway analysis software to reveal that Circadian Rhythm pathway was the most affected pathway by these microRNAs with 6 genes predicted as the potential targets, including NPAS2, CRY2, BHLHE40, BHLHE41, CRY1 and CLOCK. At the same time, however, renal cell carcinoma pathway, mTOR signaling pathway, glioma pathway, pancreatic cancer pathway and endocytosis pathway were ranked top most signaling pathways affected by these altered microRNAs (Fig. 4A). When KEGG Pathway analysis was similarly performed on the differentially expressed 8 microRNAs between the sporadic AIMAH patients and normal controls, renal cell carcinoma pathway, dilated cardiomyopathy pathway, axon guidance pathway, Ubiquitin mediated proteolysis, endocytosis and MAPK signaling pathway were ranked top most affected pathways on the basis of predicted targets, while renal cell carcinoma pathway contained 5 genes which was predicted as the potential downstream targets for the altered microRNAs, including CDC42, CUL2, EP300, GRB2 and SLC2A1 (Fig. 4B).
Interestingly, the renal cell carcinoma pathway was ranked at top 2 most affected pathways among all associated pathways containing potential targets of the altered microRNAs which were differentially expressed in familial or sporadic AIMAH patients compared to normal controls. In comparison of altered microRNAs between familial AIMAH patients and normal controls, renal cell carcinoma pathway contained 25 genes which were targeted by these microRNAs( including EGLN3, EGLN2, EGLN1, PAK6, PAK7, CUL2, CDC42, RAC1, SOS2, GAB1, SLC2A1, TGFA, PAK1, PIK3R1, AKT3, PIK3R2, MAP2K1, MET, RAF1, MAPK1, HIF1A, CRKL, VEGFA, RAP1A and CRK), while 5 genes (including CDC42, CUL2, EP300, GRB2 and SLC2A1) were targeted by microRNAs which were differentially expressed between sporadic AIMAH patients and normal controls. These results indicated that renal cell carcinoma pathway might play an important role in the pathogenesis of AIMAH although AIMAH was a benign hereditary endocrine abnormality without malignant characters (Fig. 5).