Interplay between microRNA-17-5p, insulin-like growth factor-II through binding protein-3 in hepatocellular carcinoma

Table 1 Characteristic features of non-metastatic hepatocellular carcinoma patients and healthy controls

	Average ± SD
HCC and cirrhotic patient parameters
Mean age	49 ± 13.5
Sex: Male/female	22/1
Ethanol abuse	None
AST (U/L)	100.5 ± 65.8
ALT (U/L)	85.6 ± 95.6
Alkaline phosphatase (U/L)	110.2 ± 60.7
Serum albumin (g/dL)	4.6 ± 1.5
Serum AFP (ng/mL)	155.7 ± 22.3
HCV Ab	100% (23/23 HCC patients)
HBV Ab	17.3% (4/23 HCC patients)
Healthy control (liver donor) parameters
Mean age	31 ± 10.5
Sex: Male/female	7/3
Ethanol abuse	None
HCV Ab	None
HBV Ab	None

HCC: Hepatocellular carcinoma; HCV: Hepatitis C virus; AFP: Alpha fetal protein; HBV: Hepatitis B virus; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; SD: Standard deviation.

Table 2 Number/sizes of focal lesions according to Milan criteria

Patients	No. of focal lesions	Size of focal lesions (cm)
Patient 1	3 focal lesions	1.5, 1 and 1
Patient 2	Unifocal	2.5
Patient 3	3 focal lesions	2, 2.5 and 3
Patient 4	3 focal lesions	2, 2 and 3.5
Patient 5	Unifocal	1.5-2
Patient 6	3 focal lesions	3-4, 1 and 1
Patient 7	Unifocal	4
Patient 8	3 focal lesions	4, 1 and 1
Patient 9	3 focal lesions	1, 1 and 1.5
Patient 10	Unifocal	2.5
Patient 11	2 focal lesions	1 and 1.7
Patient 12	3 focal lesions	1, 1 and 1
Patient 13	Unifocal	3
Patient 14	3 focal lesions	3, 1.5 and 2
Patient 15	3 focal lesions	1, 1 and 4
Patient 16	2 focal lesions	3 and 1.5
Patient 17	2 focal lesions	1.5 and 3
Patient 18	3 focal lesions	2.5, 2.5 and 1.5
Patient 19	3 focal lesions	1.5, 1 and 1
Patient 20	Unifocal	2
Patient 21	Unifocal	1.5
Patient 22	3 focal lesion	3, 2.5 and 1
Patient 23	Unifocal	3

Table 3 The forward and reverse primer sequences used in the wild type 1 and 2, and the mutant type 1 and 2 insulin-like growth factor binding protein-3 3’UTR constructs

Primer name	Primer sequence
WT1 forward	5’-CAATGGTAAACTTGAGCATCTTTTCACTTTCCAGTAGT-3’
WT1 reverse	5’-CTAGACTACTGGAAAGTGAAAAGATGCTCAAGTTTACCATTGAGCT-3’
WT2 forward	5’-CGTCGAAGCGGCCGACCACTGACTTTGTGACTTT-3’
WT2 reverse	5’-CTAGAAAGTCACAAAGTCAGTGGTCGGCCGCTTCGACGAGCT-3’
MUT1 forward	5’-CAATGGTAAACTTGAGCATCTTTTCATCCAGTAGT3’
MUT1 reverse	5’-CTAGACTACTGGATGAAAAGATGCTCAAGTTTACCATTGAGCT-3’
MUT2 forward	5’-CGTCGAAGCGGCCGACCACTGACGTGACTTT-3’
MUT2 reverse	5’-CTAGAAAGTCACGTCAGTGGTCGGCCGCTTCGACGAGCT-3’

WT: Wild type; MUT: Mutant type.

Table 4 Predicted target region-seed sequence binding for miR-17-5p on the 3’UTR of insulin-like growth factor binding protein-3

Target region	hsa-miR-17-5p (seed sequence) binding to IGFBP-3(target sequence)	Target sequence position on3’UTR of IGFBP-3	6mer/7mer/8mer
Region 1	miR-17-5p 3’gaUGGACGUG-ACAUUCGUGAAAc 5’ | | : | | : | | | | | | | | | IGFBP-3 5’aaACUUGAGCAUCUUUUCACUUUc 3’	196-204	6mer
Region 2	miR-17-5p 3’GAUGGAC- GUGACAUUCGUGAAAC 5’ | ||__ | | || |___ | | | | || IGFBP-3 5’ CGGCCGACCACUG-----------ACUUUG 3’	335-343	6mer

IGFBP-3: Insulin-like growth factor binding protein-3; miR-17-5p: MicroRNA-17-5p.