Heme status affects human hepatic messenger RNA and microRNA expression

Table 1 Confirmation and comparison of selected messenger RNA array results with those of quantitative real time polymerase chain reaction for selected genes

	Array		qRT-PCR
Gene	Fold change	P value	Fold change	P value
Heme vs DHA-6 h time point
HMOX1	59.9	9.97 × 10-9	87.9	10-35
IL-8	20.50	3.92 × 10-5	13.7	0.016
ANXA1	7.18	1.87 × 10-5	32.3	0.015
BACH1	3.04	3.26 × 10-5	3.1	0.0019
Heme vs DHA-24 h time point
HSPB8	77.1	2.66 × 10-6	92	0.010
ANXA1	45.7	5.98 × 10-6	138	0.015
IL-8	33.2	4.19 × 10-5	31.7	0.016
HMOX1	25.6	2.50 × 10-5	27.6	1.33 × 10-7
BACH2	4.50	7.24 × 10-5	22.6	0.011
PNPLA3	-6.48	1.74 × 10-4	-6.65	4.59 × 10-7

P values are for comparison of results following treatment with iron protoporphyrin (heme) vs 4, 6-dioxoheptanoic acid (DHA). Listing of all messenger RNAs with ≥ 1.5 fold differences in expression at 6 h, 24 h after treatment of Huh-7 cells with heme (10 μmol/L) vs DHA (500 μmol/L). Cells were cultured, treated, harvested and total RNA prepared and assays as described in Materials and Methods. qRT-PCR: Quantitative real time polymerase chain reaction; HMOX1: Heme oxygenase 1; IL-8: Interleukin-8; ANXA1: Annexin A1; BACH: Basic leucine zipper transcription factor; HSPB8: Heat shock 22kDa protein 8; PNPLA3: Patatin-like phospholipase domain-containing protein 3.

Table 2 Summary of data analysis with use of ingenuity pathway

	6 h		24 h
Name	P value	Ratio	P value	Ratio
Top canonical pathways
Nrf2-mediated oxidative stress response	2.64 × 10-11	28/193 (0.145)	1.58 × 10-9	61/193 (0.316)
Protein ubiquitination pathway	1.44 × 10-9	31/274 (0.133)	3.26 × 10-8	75/274 (0.274)
Aldosterone signaling in epithelial cells	8.31 × 10-8	21/170 (0.124)	-	-
Glucocorticoid receptor signaling	1.6 × 10-5	24/295 (0.081)	-	-
P53 signaling	1.75 × 10-3	10/96 (0.104)	-	-
Biosynthesis of steroids	-	-	3.22 × 10-7	14/121 (0.116)
Propanoate metabolism	-	-	1.45 × 10-6	24/121 (0.198)
Urea cycle and metabolism of amino groups	-	-	5.8 × 10-6	16/78 (0.205)
Top toxicity lists
Nrf2-mediated oxidative stress response	3.03 × 10-11	30/237 (0.127)	5.41 × 10-9	66/237 (0.278)
Renal necrosis/cell death	1.05 × 10-4	25/314 (0.08)	1.06 × 10-5	79/314 (0.252)
Liver necrosis/cell death	1.79 × 10-3	14/166 (0.084)	-	-
P53 signaling	2.06 × 10-3	10/95 (0.105)	1.54E × 10-4	30/95 (0.316)
Liver proliferation	2.54 × 10-3	12/133 (0.09)	-	-
Cholesterol biosynthesis	-	-	2.26 × 10-13	16/16 (1)
FXR/RXR activation	-	-	5.84 × 10-4	26/86 (0.302)

P values are for comparison of results following treatment with iron protoporphyrin (heme) vs 4, 6-dioxoheptanoic acid (DHA). Listing of all microRNAs with ≥ 1.5 fold differences in expression at 6 h after treatment of Huh-7 cells with heme (10 μmol/L) vs DHA (500 μmol/L). Cells were cultured, treated, harvested and total RNA prepared and assays as described in Materials and Methods. FXR/RXR: Farnesoid X receptor/retinoic X receptor; Nrf2: Nuclear erythroid-2 related factor.

Table 3 Summary of analysis with ingenuity pathway analytical tools

	6 h		24 h
Name	P value	No. molecules	P value	No. molecules
Hepatotoxicity
Liver proliferation	6.71 × 10-4 - 6.26 ×10-1	12	4.24× 10-3 - 1.63× 10-1	36
Liver necrosis/cell death	2.08 × 10-3 - 3.77 × 10-1	15	-	-
Liver damage	3.31 × 10-3 - 1.25 × 10-1	12	-	-
Liver hemorrhaging	7.28 × 10-3 - 7.28 × 10-3	2	-	-
Hepatocellular peroxisome proliferation	3.57 × 10-2 - 3.57 × 10-2	1	-	-
Liver cholestasis	-	-	1.11 × 10-5 - 6.55 × 10-1	32
Hepatocellular carcinoma	-	-	2.46 × 10-4 -2.46 × 10-4	54
Liver steatosis	-	-	2.59 × 10-3 - 1.00 × 10-0	34
Liver fibrosis	-	-	1.51 × 10-2 - 4.43 × 10-1	19
Nephrotoxicity
Renal necrosis/cell death	7.22 × 10-5 - 4.21 × 10-1	25	7.44 × 10-7 - 1.00E × 10-0	79
Kidney failure	1.27 × 10-3 - 2.24 × 10-1	10	2.64 × 10-2 - 6.55 × 10-1	26
Renal enlargement	1.27 × 10-3 - 1.03 × 10-1	2	2.64 × 10-2 - 4.13 × 10-1	2
Renal damage	3.09 × 10-2 - 5.83 × 10-1	4
Glomerular	3.57 × 10-2 - 5.17 × 10-1	2
Renal tubule injury			4.27 × 10-5 - 1.63 × 10-1	30
Renal proliferation			2.17 × 10-3 - 4.83 × 10-1	24