MicroRNA exhibit altered expression in the inflamed colonic mucosa of ulcerative colitis patients

Table 1 Demographic features of study subject n (%)

Characteristics	UC patients	Non IBD control
No. of patients (total)	8	8
Sex (M/F)	3/5	2/6
Age (mean ± SD, range, yr)	39.75 ± 10.29 (25-62)	40.75 ± 11.86 (24-60)
Disease duration (mean ± SD, Range, yr)	6.35 ± 6.52 (1.5-21)	NA
Disease extent
Proctitis	3 (37.5)
Left sided colitis	5 (62.5)
Medication
Mesalamine	5 (62.5)	0
Azathioprine	3 (37.5)	0
Steroids	0	0

UC: Ulcerative colitis; IBD: Inflammatory bowel disease.

Table 2 List of primers used for reverse transcription and qRT-PCR

Name	Primer	Sequence (5’-3’)
Reverse Universal Primer		GTGCAGGGTCCGAGGT
hsa-miR-125b-5p	RT	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGTCACAAGT
	Forward	TCCCTGAGACCCTAACTTG
hsa-miR-223-3p	RT	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGTGGGGTAT
	Forward	TGTCAGTTTGTCAAATACCC
hsa-miR-155-5p	RT	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCCCTAT
	Forward	TTAATGCTAATCGTGATAGG
hsa-miR-138-5p	RT	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGCGGCCTGA
	Forward	AGCTGGTGTTGTGAATCAG
hsa-miR-200a-3p	RT	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACATCGTT
	Forward	TAACACTGTCTGGTAACGAT
hsa-miR-378d	RT	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGTTTCTGTC
	Forward	ACTGGACTTGGAGTCAGAAA
Sno RNA U6	RT	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAAAATATG
	Forward	CAAATTCGTGAAGCGTTCCA

RT primers were used to sythesize miRNA specific cDNA. For real time PCR miRNA specific forward primer and reverse universal primers were used.

Table 3 List of top 44 differentially expressed miRNAs in the inflamed colonic mucosal biopsies of ulcerative colitis patients determined by microarray

MicroRNA	F.C1	Expression	MicroRNA	F.C	Expression
hsa-miR-138-5p	35.16	Upregulated	hsa-miR-148a-5p	2.42	Upregulated
hsa-miR-708-5p	34.7	Upregulated	hsa-miR-155-5p	2.33	Upregulated
hsa-miR-212-3p	24.06	Upregulated	hsa-miR-21-5p	1.81	Upregulated
hsa-miR-4538	26.12	Upregulated	hsa-miR-196b-3p	1.50	Upregulated
hsa-miR-4521	17.69	Upregulated	hsa-miR-552-3p	-7.82	Downregulated
hsa-miR-4417	16.63	Upregulated	hsa-miR-196b-5p	-7.60	Downregulated
hsa-miR-17-3p	15.28	Upregulated	hsa-miR-378d-5p	-4.06	Downregulated
hsa-miR-424-3p	14.17	Upregulated	hsa-miR-141-3p	-3.03	Downregulated
hsa-miR-874-3p	13.47	Upregulated	hsa-miR-10b-5p	-2.85	Downregulated
hsa-miR-25-5p	13.41	Upregulated	hsa-miR-215-5p	-2.56	Downregulated
hsa-miR-223-3p	13.01	Upregulated	hsa-miR-192-5p	-2.52	Downregulated
hsa-miR-1271-5p	11.96	Upregulated	hsa-miR-194-3p	-2.31	Downregulated
hsa-miR-148b-3p	11.31	Upregulated	hsa-miR-422a	-2.22	Downregulated
hsa-miR-501-5p	11.26	Upregulated	hsa-miR-200a-3p	-2.14	Downregulated
hsa-miR-4486	10.86	Upregulated	hsa-miR-378a-3p	-2.04	Downregulated
hsa-miR-224-3p	10.46	Upregulated	hsa-miR-6732-5p	-1.94	Downregulated
hsa-miR-21-3p	8.46	Upregulated	hsa-miR-147b	-1.78	Downregulated
hsa-miR-146b-5p	6.83	Upregulated	hsa-miR-200b-3p	-1.7	Downregulated
hsa-miR-149-5p	6.24	Upregulated	hsa-miR-572	-1.63	Downregulated
hsa-miR-31-5p	4.04	Upregulated	hsa-miR-4649-5p	-1.61	Downregulated
hsa-miR-491-5p	3.92	Upregulated	hsa-miR-638	-1.6	Downregulated
hsa-miR-125b-5p	2.56	Upregulated	hsa-miR-299-5p	-1.59	Downregulated

¹F.C denoted the fold change in the expression of respective miRNA in the inflamed biopsies compared to the non inflamed biopsy.

Table 4 List of potential targets and the pathway involved

MicroRNA	Target gene	Pathway involved
hsa-miR-155-5p	TAB2, CFLAR, CARD11,	NF-κB signaling.
	ReLA, TRAF3
hsa-miR-155-5p	CARD6, ERBB2IP, MAPK10	Nod like receptor signaling
hsa-miR-155-5p	S °CS1, KRAS, PAK2, RAP1B,	MAPK signaling
	RFLA, TAOK1, CACNB4, BDNF
hsa-miR-155-5p	FOS, S °CS1, IKBKE, TRAF3,	TLR signaling
	MAPK10, TAB2
has-miR-155	NFAT5, GSK3B, INPP5D, VAV3, PIK3CA	B cell receptor signaling
hsa-miR-138-5p	CSNK2A2, RELA	NF-κB signaling
hsa-miR-138-5p	CXCL8, MAP2K7	TLR signaling
hsa-miR-138-5p	CLDN19, CD274, ITGAL	Cell Adhesion Molecules.
hsa-miR-125b-5p	MAPK14, JUN.AKT1, IRAK1, TRAF6, IRF5	TLR signaling
hsa-miR-125b	TNFAIP3, CSNK2A1, IRAK1	NF-κB signaling
hsa-miR-223-3p	STAT1, RELA	TLR signaling
hsa-miR-223-3p	TAB3, ERC1, PARP1, RELA	NF-κB signaling
hsa-miR-378d	MAPK1	MAPK signaling
hsa-miR-378d	TRAF3	NF-κB signaling
hsa-miR-200a	TAB2, TNFAIP3	NF-κB signaling