RRAS: A key regulator and an important prognostic biomarker in biliary atresia

Table 1 Microarray analysis showing clinical characteristics of biliary atresia patients

Case No.	Gender	Age	TB/DB	ALT/AST	AKP/GGT	Albumin	Disease	Group
1	Female	50 d	171/130	132/254	604/609	35.8	BA	1
2	Male	49 d	291/231	148/155	581/408	39.4	BA	1
3	Male	57 d	148/117	189/166	493/535	35.2	BA	1
4	Female	73 d	145/118	111/153	460/1460	36.3	BA	2
5	Male	84 d	160/127	100/149	713/1278	37.3	BA	2
6	Female	66 d	129/108	121/165	632/1235	39.2	BA	2
7	Female	103 d	151/112	85/62	451/360	39.4	BA	3
8	Female	97 d	118/89	74/78	522/501	34.3	BA	3
9	Male	110 d	155/121	105/97	377/912	35.4	BA	3
10	Female	77 d	102/89	201/137	234/317	39.4	Cholestasis	4
11	Male	64 d	137/108	404/267	584/1044	37.2	Cholestasis	4
12	Female	55 d	144/112	389/266	612/1339	41.0	Cholestasis	4
13	Male	4 yr	16/9	33/35	200/50	39.4	Liver trauma	5
14	Male	6 yr	10/4	20/25	192/44	40.1	Liver trauma	5
15	Male	4 yr	12/4.7	29/37	101/38	37.1	Liver trauma	5

TB: Total bilirubin; DB: Direct bilirubin; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; AKP: Alkaline phosphatase; GGT: γ-glutamyl transferase; BA: Biliary atresia.

Table 2 Sequences of primers in selected genes used in reverse-transcription polymerase chain reaction

Gene name	Primer sequences
RRAS	F: TTGGTCGGGAACAAGGCAGAT
	R: CTCGTCCACGTTGAGACGCAGT
POMC	F: GAGAGCAGCCAGTGTCAGG
	R: GAAGTGGCCCATGACGTACT
SLC26A6	F: CGGTATCCTGTGCGTGACT
	R: GGAAGTGCCAAACAGGAAGT
STX3	F: GGCAAAAAGACAACCGATGA
	R: TGTCGTGAAGCTCCTTGATG
β-actin	F: GGGAAATCGTGCGTGCATT
	R: CAGGCAGCTCGTAGCTCTT

Table 3 Significant pathways involved in pathogenesis of biliary atresia

Pathway name	P value	Profile No.
Cell adhesion molecules	0.000118	Profile49
Regulation of actin cytoskeleton	0.000739	Profile49
T Leukocyte transendothelial migration	0.001738	Profile49
Asthma	0.002023	Profile49
Allograft rejection	0.003243	Profile49
Systemic lupus erythematosus	2.44E-05	Profile74
Lysosome	0.0002109	Profile74
NF-kappa B signaling pathway	0.0036605	Profile74
MAPK signaling pathway	0.0052322	Profile74
Allograft rejection	0.0058515	Profile74
Graft-versus-host disease	0.0071277	Profile74
Type I diabetes mellitus	0.00781	Profile74
Chemokine signaling pathway	1.81E-08	Profile75
Matrix_Metalloproteinases	6.52E-07	Profile75
Cytokine-cytokine receptor interaction	3.59E-05	Profile75
T cell receptor signaling pathway	4.34E-05	Profile75
Antigen processing and presentation	0.000337	Profile75
Leukocyte transendothelial migration	0.0010211	Profile75
Lysosome	2.09E-07	Profile76
Toll-like receptor signaling pathway	0.000284	Profile76
T cell receptor signaling pathway	0.000388	Profile76
Chemokine signaling pathway	0.000755	Profile76
Asthma	0.000841	Profile76
Matrix_Metalloproteinases	0.001402	Profile76
Allograft rejection	0.001697	Profile76

Table 4 Genes with the highest degree and k-core in dynamic gene networks

Gene symbol	Definition	Degree	k-core
RRAS	Homo sapiens related RAS viral (r-ras) oncogene homolog (RRAS), mRNA	12	6
POMC	Homo sapiens POMC, transcript variant 1, mRNA	12	6
SLC26A6	Homo sapiens SLC26A6, transcript variant 3, mRNA	12	6
STX3	Homo sapiens STX3, mRNA	10	6

Table 5 Reverse-transcription polymerase chain reaction showing relative expression levels of RRAS, POMC, SLC26A6 and STX3 (mean ± SD)

Group	POMC	SLC26A6	RRAS	STX3
Biliary atresia (n = 40)	0.58 ± 0.090	0.43 ± 0.054	0.89 ± 0.103	0.61 ± 0.074
Neonatal cholestasis (n = 14)	0.41 ± 0.081	0.30 ± 0.029	0.47 ± 0.074	0.51 ± 0.045
P-value	0.031	0.023	0.004	0.017

Table 6 Fibrosis scores for different groups of biliary atresia patients at different ages

Group	Fibrosis score (n)					Total
	0	1	2	3	4
< 60 d	1	3	8	2	0	14
60-90 d	0	2	3	6	4	15
> 90 d	0	0	1	4	6	11
Total	1	5	12	12	10	40