S100 calcium binding protein A6 and associated long noncoding ribonucleic acids as biomarkers in the diagnosis and staging of primary biliary cholangitis

Table 1 Primer sequences used in this study

Target gene	Forward sequence (5’→3’)	Reverse sequence (5’→3’)
S100A6	AATGTGCGTTGTGTAAGC	CGGTCCAAGTCTTCCATC
LINC00312	GGAAGGAATACCACAGAAGT	TGAAGAACAGGACATTGACA
LINC00472	AGAGTTGCTGTAGAAGAAGG	AGGAGGAGAGTAGAAGAGAC
LINC01257	TGCTGCGAATGATGACTT	AGGACTTGAATCTGCTACTG
HMGB2	TTACGTTCCTCCCAAAGGTG	TCTTTGGCTGACTGCTCAGA
RC3H2	TTGCAAAGAAATGCGTTGAG	GATTGGCAGACAACTGCTGA
ADAMTS1	CCTCTGTCTGTGTGCAAGGA	GTGGCTCCAGTTGGAATTGT
SERPINE1	CTCTCTCTGCCCTCACCAAC	GTGGAGAGGCTCTTGGTCTG
PALD1	GCCGAAGTTGTTCCCATTTA	GCTGAAAGTCAGAGCCAACC
GSTA4	TCCGTGAGATGGGTTTTAGC	TGCCAAAGAGATTGTGCTTG
ACTA2	TTCAATGTCCCAGCCATGTA	GAAGGAATAGCCACGCTCAG
GAPDH	GCACCGTCAAGGCTGAGAAC	TGGTGAAGACGCCAGTGGA

ACTA2: Actin alpha 2, smooth muscle; ADAMTS1: ADAM metallopeptidase with thrombospondin type 1 motif 1; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; GSTA4: Glutathione S-transferase alpha 4; HMGB2: High mobility group box 2; PALD1: Phosphatase domain containing paladin 1; RC3H2: Ring finger and CCCH-type domains 2; S100A6: S100 calcium binding protein A6; SERPINE1: Serpin family E member 1.

Table 2 Top 10 dysregulated genes in bile duct ligation and sham mice

Gene name	Transcript	Lg fold change
Up-regulated
Hmgb2	ENSMUSG00000054717	3.53
Rc3h2	ENSMUSG00000075376	3.33
Adamts1	ENSMUSG00000022893	3.15
Serpine1	ENSMUSG00000037411	3.08
S100a6	ENSMUSG00000001025	2.98
Pald1	ENSMUSG00000020092	2.67
Gsta4	ENSMUSG00000032348	2.50
D17H6S56E-5	NM_033075	2.46
Acta2	ENSMUSG00000035783	2.39
Ifi204	ENSMUSG00000073489	2.33
Down-regulated
Mcm10	ENSMUSG00000026669	3.23
Upp2	ENSMUSG00000026839	2.85
2810043O03Rik	AK012901.1	2.59
Dnaaf5	ENSMUSG00000025857	2.41
Sva	ENSMUSG00000023289	2.40
Naca	ENSMUSG00000061315	2.35
Dhps	ENSMUSG00000060038	2.33
Cdh15	ENSMUSG00000031962	2.26
Gzmm	ENSMUSG00000054206	2.20
Alox12	ENSMUSG00000000320	2.15

2810043O03Rik: RIKEN complementary deoxyribonucleic acid 2810043O03 gene; Acta2: Actin alpha 2, smooth muscle; Adamts1: A disintegrin-like and metallopeptidase (reprolysin type) with thrombospondin type 1 motif, 1; Alox12: Arachidonate 12-lipoxygenase; Cdh15: Cadherin 15; D17H6S56E-5: Deoxyribonucleic acid segment, Chr 17, human D6S56E 5; Dhps: Deoxyhypusine synthase; Dnaaf5: Dynein, axonemal assembly factor 5; Gsta4: Glutathione S-transferase alpha 4; Gzmm: Granzyme M (lymphocyte met-ase 1); Hmgb2: High mobility group box 2; Ifi204: Interferon activated gene 204; Mcm10: Minichromosome maintenance 10 replication initiation factor; Naca: Nascent polypeptide-associated complex alpha polypeptide; Pald1: Phosphatase domain containing paladin 1; Rc3h2: Ring finger and CCCH-type domains 2; S100A6: S100 calcium binding protein A6; Serpine1: Serpin family E member 1; Sva: Seminal vesicle antigen; Upp2: Uridine phosphorylase 2.

Table 3 Demographics and clinical characteristics in the training and validation datasets

Characteristics	Training	Validation	P value
No.	140	115	-
Age, mean ± SD, yr	56.0 ± 13.9	57.2 ± 13.2	0.504
Gender, n (%)
Male	17 (12.1)	13 (11.3)
Female	123 (87.9)	102 (88.7)	1.0
Pathological stage
Ι and II	36 (45.0)	26 (40.0)
III and IV	44 (55.0)	39 (60.0)	0.614

Normally distributed data are expressed as mean ± SD. Categorical variable values are described as n (%). SD: Standard deviation.

Table 4 Demographics and clinical characteristics of primary biliary cholangitis patients and healthy controls¹

Characteristics	PBC (n = 145)	HCs (n = 110)	P value
Age, mean ± SD, yr	56.1 ± 13.4	55.3 ± 11.8	0.58
Gender, n (%)
Male	17 (12.5)	13 (16.7)
Female	128 (87.5)	97 (83.3)	1.00
ALT, U/L	78.6 ± 35.7	18.4 ± 6.5	< 0.001
AST, U/L	104.8 ± 43.5	20.2 ± 4.3	< 0.001
ALP, U/L	257.4 ± 79.9	64.7 ± 14.5	< 0.001
γGT, U/L	416.7 ± 209.2	26.3 ± 10.4	< 0.001
TBIL, μmol/L	66.8 ± 10.6	11.8 ± 4.0	< 0.001
DBIL, μmol/L	51.9 ± 11.4	6.4 ± 0.5	< 0.001
TBA, μmol/L	71.3 ± 11.6	2.8 ± 0.4	< 0.001
HA, ng/mL	146.9 (104.6-190.1)	67.0 (53.9-79.7)	< 0.001
LN, ng/mL	148.9 (76.7-182.8)	70.4 (58.7-82.9)	< 0.001
C-IV, ng/mL	154.8 (121.1-192.0)	60.1 (55.2-66.7)	< 0.001
PC-III, ng/mL	161.0 (135.1-184.5)	57.3 (49.9-63.5)	< 0.001
Pathological stage
Ι and II	62 (42.8)	-
III and IV	83 (57.2)	-

¹Normally distributed data are expressed as means ± SD, variables with a skewed distribution are presented as median (interquartile range). Categorical variable values are described as n (%). γGT: Gamma-glutamyl transpeptidase; ALT: Alanine aminotransferase; ALP: Alkaline phosphatase; AST: Aspartate aminotransferase; C-IV: Collagen type IV; DBIL: Direct bilirubin; HA: Hyaluronic acid; LN, Laminin; PC-III: Procollagen III; TBA: Total bile acid; TBIL: Total bilirubin; SD: Standard deviation; PBC: Primary biliary cholangitis; HCs: Healthy controls.

Table 5 Characteristics of primary biliary cholangitis patients based on the expression of log10 LINC00472 cutoff value

	Relative Expression of log10 LINC00472
	L1 (< 2.33) (n = 38)	L2 (≥ 2.33) (n = 42)	P value
Age, mean ± SD, years	60.3 ± 14.9	55.1 ± 14.0	0.109
Gender, n (%)
Male	5 (13.2)	5 (11.9)
Female	33 (86.8)	37 (88.1)	0.886
ALT, U/L	73.2 (52.7-100.1)	73.2 (46.2-100.7)	0.985
AST, U/L	109.2 ± 45.9	103.0 ± 44.6	0.543
ALP, U/L	264.0 ± 89.4	252.2 ± 78.3	0.532
γGT, U/L	420.2 ± 197.9	413.2 ± 237.1	0.887
TBA, μmol/L	73.2 ± 12.4	70.9 ± 13.3	0.438
TBiL, μmol/L	68.0 (63.0-73.0)	63.0 (58.0-73.0)	0.166
DBiL, μmol/L	50.6 ± 9.8	52.2 ± 11.4	0.505
LINC00312	1.51 ± 0.32	1.43 ± 0.31	0.261
S100A6	2.40 ± 1.05	3.57 ± 0.66	< 0.0001
LINC01257	4.25 ± 1.39	3.22 ± 1.78	0.005
HA, ng/mL	144.8 (101.6-208.8)	135.5 (95.4-195.4)	0.537
LN, ng/mL	126.1 (48.4-178.4)	156.1 (57.6-175.8)	0.78
C-IV, ng/mL	127.2 (100.9-170.4)	176.0 (154.7-232.0)	< 0.0001
PC-III, ng/mL	156.6 (125.8-190.1)	161.6 (128.0-184.5)	0.916

SD: Standard deviation; γGT: Gamma-glutamyl transpeptidase; ALT: Alanine aminotransferase; ALP: Alkaline phosphatase; AST: Aspartate aminotransferase; C-IV: Collagen type IV; DBIL: Direct bilirubin; HA: Hyaluronic acid; LN: Laminin; PC-III: Procollagen III; S100A6: S100 calcium binding protein A6; TBA: Total bile acid; TBIL: Total bilirubin.