Clinical Characteristics
A total of five patients with idiopathic achalasia were included in the chip analysis, including two males and three females. The average age of patients was 46.4 ± 14.1 years, and the average age of onset was 32 ± 10.7 years old. Major clinical manifestations included dysphagia (100%), reflux (100%), respiratory reactions (20%), chest pain (40%), heartburn (20%), and weight loss (80%). The average Ercard score was 6.4 ± 1.3.
Microarray Gene Expression Analysis
Microarray analysis revealed a total of 14,025 differentially expressed genes, including 7,133 differentially expressed mRNA (3,136 upregulated and 3,997 downregulated) (Fig. 1) and 6,892 lncRNA (4,900 up-regulated and 1,992 down-regulated) (Fig. 2).
Among the 3,136 elevated mRNAs, expression of 83 mRNAs increased considerably by more than 10-fold, expression of 459 mRNAs increased moderately by 5- to 10-fold, expression of 1,235 mRNAs increased mildly by 3- to 5-fold, and expression of 1,359 mRNAs increased slightly by a 2- to 3-fold change (Fig. 1B). Of the 3,997 decreased mRNAs, expression of 239 mRNAs increased considerably by more than 10-fold, expression of 1,255 mRNAs increased moderately by 5- to 10-fold, expression of 1,714 mRNAs increased mildly by 3- to 5-fold, and expression of 789 mRNAs increased slightly by a 2- to 3-fold change (Fig. 1B).
Of the 4,900 elevated lncRNAs, expression of 135 mRNAs increased considerably by more than 10-fold, expression of 758 mRNAs increased moderately by 5- to 10-fold, expression of 2,040 mRNAs increased mildly by 3- to 5-fold, and expression of 1,967 mRNAs increased slightly by a 2- to 3-fold change (Fig. 2B). Additionally, expression of 1,992 decreased lncRNAs, including 73 lncRNAs, increased considerably by more than 10-fold, expression of 425 mRNAs increased moderately by 5- to 10-fold, expression of 902 mRNAs increased mildly by 3- to 5-fold, and expression of 593 mRNAs increased slightly by a 2- to 3-fold change (Fig. 2B).
The most upregulated and downregulated genes are reported in Table 1 and Table 2. mRNAs in patients included LMO7 (FC + 26.7, P = 6.03− 5), ITGA8 (FC − 55.6, P = 2.58− 3), RPS4Y2 (FC – 47.8, P = 1.10− 3), and so on. LncRNAs included TCONS_0001118 (FC + 72.6, P = 3.01− 5), ENST00000447880.1 (FC + 45.1, P = 4.72− 5), ENST00000457658.1 (FC – 30.9, P = 8.91− 5), ASO3473 (FC – 28.3, P = 1.45− 4), and so on.
Table 1
The most up-regulated and down-regulated mRNA
Gene Symbol | p | FC (abs) |
LMO7 | 6.03318E-05 | 26.71425695 |
TMEM200C | 3.28108E-05 | 25.94489757 |
C22orf39 | 3.66968E-05 | 25.87756057 |
LHX5 | 5.35747E-06 | 24.72500133 |
CRNN | 0.027830519 | 24.03878997 |
SPRR3 | 0.017117696 | 23.78862026 |
WI2-2373I1.2 | 0.000100648 | 23.66951699 |
ENST00000442466 | 0.001293632 | 22.33677558 |
TBR1 | 7.6705E-05 | 21.22063752 |
AAK1 | 7.93936E-05 | 19.70644678 |
ITGA8 | 0.002581062 | -55.60137848 |
RPS4Y2 | 0.001103543 | -47.82133021 |
BRWD3 | 0.023419339 | -31.10210048 |
FLRT2 | 0.000220524 | -30.55600013 |
FOXP2 | 0.013681404 | -29.06785335 |
SCARA5 | 0.000218602 | -28.83943377 |
PTAR1 | 0.026496723 | -27.49359787 |
P2RY14 | 0.000304615 | -26.79329943 |
SLC31A1 | 0.001766187 | -26.33664678 |
DDX3Y | 4.30241E-05 | -26.26270772 |
Table 2
The most up-regulated and down-regulated lncRNA
lncRNA ID | p | FC (abs) |
TCONS_00011185 | 3.01402E-05 | 72.64201265 |
ENST00000447880.1 | 4.71544E-05 | 45.08069048 |
RNA147445|p0549_imsncRNA190 | 8.44416E-05 | 33.92913857 |
ENST00000512637.1 | 0.000197358 | 32.89494311 |
XR_158741.1 | 3.81046E-06 | 32.63342097 |
XR_245991.1 | 1.32945E-05 | 31.70148851 |
ENST00000507924.1 | 2.82601E-05 | 29.59564156 |
ENST00000605571.1 | 0.000329898 | 29.01344488 |
ENST00000511301.1 | 3.32355E-05 | 28.39455549 |
XR_110047.1 | 2.72823E-05 | 27.21027354 |
ENST00000457658.1 | 8.91147E-05 | -30.89314361 |
ASO3473 | 0.000145592 | -28.27190349 |
TCONS_00029197 | 3.81342E-06 | -25.04219798 |
ENST00000582940.1 | 0.008796621 | -24.34463314 |
TCONS_00017647 | 0.003232599 | -23.52995468 |
ENST00000422971.1 | 0.001210922 | -21.38524635 |
XR_427728.1 | 0.001271232 | -20.83603776 |
ENST00000604219.1 | 0.011846474 | -19.2863216 |
ENST00000434656.1 | 0.006403102 | -17.25903289 |
uc.480- | 0.010963956 | -16.26472502 |
Go And Kegg Pathway Analyses Of Differentially Expressed Genes
To help interpret the biological functions of altered mRNA and lncRNA profiles, GO and KEGG pathway analyses were performed. Among the different mRNA biological processes, GO analysis showed that anatomical structure morphogenesis, positive regulation of JNK cascade, positive regulation of stress-activated MAPK cascade, nervous system development, cell projection organization, and fatty acid derivative biosynthetic processes might be related to the disease (Fig. 3). To gain further insights into the pathogenesis of idiopathic achalasia, signaling pathway enrichment analysis was performed using the KEGG database. The following pathways showed significant transcriptional changes: Staphylococcus aureus infection (P = 0.0033), complement and coagulation cascades (P = 0.0076), cell adhesion molecules (CAMs) (P = 0.0351), DNA replication (P = 0.0402), and glycosphingolipid biosynthesis - lacto and neolacto series (P = 0.0446) (Fig. 3).
GO analysis of differentially expressed lncRNAs revealed that the following biological processes were altered in idiopathic achalasia: positive regulation of excitatory postsynaptic membrane potential, cell differentiation in kidney development, pyrimidine deoxyribonucleoside metabolic process, and positive regulation of membrane potential (Fig. 4). Significant KEGG analysis changes in lncRNAs were identified as CAMs (P = 0.0023), basal cell carcinoma (P = 0.0024), RIG-I-like receptor signaling pathway (P = 0.0065), NF-kappa B signaling pathway (P = 0.0279), ether lipid metabolism (P = 0.0360), and arachidonic acid metabolism (P = 0.0396) (Fig. 4).
Common Pathways Of Differentially Expressed Lncrnas And Corresponding Mrnas
Analyses of the functions of the differentially expressed mRNA and lncRNA target genes revealed that the CAM pathway was a common pathway. The mRNA pathway analysis found that 55 genes were inputted into the CAM pathway for annotation along with 142 background genes. The total input and background genes were 6783 and 23740, respectively. In the lncRNA target gene pathway analysis, the number of background genes annotated into the CAM pathway was 142 and the number of input genes was 14; CLDN11 (Down FC: 3.42), NRXN1 (Down FC: 9.07), NCAM2 (UP FC: 3.11), HLA-DQB1 (Down FC: 5.06), NTNG2 (UP FC: 2.75), HLA-DOA (Down FC: 2.31), CADM1 (Down FC: 2.26), CLD-N18 (Down FC: 3.94), CD22 (Down FC: 5.62), ITGA6 (Down FC: 10.43), NEGR1 (Down FC: 2.335), MPZL1 (Down FC: 4.31), CNTNAP2 (Down FC: 5.88), and NLGN1 (Down FC: 4.60).