Gene expression profile of mitogen-activated kinases and microRNAs controlling their expression in HaCaT cell culture treated with lipopolysaccharide A and cyclosporine A

Figures & data

Table 1. Nucleotide sequence of primers used in RT-qPCR.

mRNA	Starter	Sequence	Product size (bp)	Tm (°C)
DUSP1	Forward	5’- GGATACGAAGCGTTTTCGGC-3’	1531	85.5
	Reverse	5’-AGAGGTCGTAATGGGGCTCT-3’
DUSP4	Forward	5’-GACCGGCAAAAATACACGGG-3’	520	91.6
	Reverse	5’-GAACCTAGGATGTAGCCCGC-3’
MAP2K2	Forward	5’- AGCTGGAGGAGCTGGAACTTG-3’	817	89.5
	Reverse	5’- CTATCCATCCCGTGACCGC-3’
MAP2K7	Forward	5’-GACAGTTTCCCTACAAGAAC-3’	146	82.5
	Reverse	5’-CCTGTGATCTTTAGTAAGGC-3’
MAP3K2	Forward	5’-TCTGTTTTATCTTCTCAGGCCA-3’	958	80.5
	Reverse	5’- CCCTGGGTCCTTCTAGCTCT-3’
MAPK9	Forward	5’-AGTCATCCTGGGTATGGGCT-3’	570	81.3
	Reverse	5’-GCGTTGCTACTTACTGCTGC-3’
ACTB	Forward	5’-TCACCCACACTGTGCCCATCTACGA-3’	295	87.8
	Reverse	5’-CAGCGGAACCGCTCATTGCCAATGG-3’

DUSP1, dual specificity phosphatase 1; DUSP4, dual specificity phosphatase 4; MAP2K2, mitogen-activated protein kinase kinase 2; MAP2K7, mitogen-activated protein kinase kinase 7; MAP3K2, mitogen-activated protein kinase kinase kinase 2; MAPK9, mitogen-activated protein kinase 9; ACTB, beta actin; bp, base pair; Tm, melting temperature.

Figure 1. Examples of melting curves confirming the specificity of the reaction.

Figure 2. The Venn diagram of microarray results.

Table 2. Expression pattern of MAPK-related genes in the HaCaT cell line exposed to LPS and then cyclosporine for 2, 8, and 24 hours compared to the control (p < 0.05).

ID	mRNA	LPS	LPS+CsA
		log2 Fold Change
		H_8 vs C	H-2 vs C	H-8 vs C	H-24 vs C
201041_s_at	DUSP1	(−)3.14 ± 0.34	(+)5.11 ± 0.37	(+)5.53 ± 0.55	(+)4.16 ± 0.13
201044_x_at		(−)3.11 ± 0.21	(+)6.88 ± 0.43	(+)5.55 ± 0.47	(+)4.22 ± 0.17
204014_at	DUSP4	(−)4.08 ± 0.43	(+)3.87 ± 0.24	(−)1.88 ± 0.11	(+)1.01 ± 0.18
204015_s_at		(−)4.09 ± 0.34	(+)2.12 ± 0.31	(−)2.09 ± 0.09	(−)1.01 ± 0.03
213490_s_at	MAP2K2	(−)3.51 ± 0.13	(+)2.80 ± 0.18	(+)4.08 ± 0.22	(+)3.90 ± 0.19
209952_s_at	MAP2K7	(−)4.70 ± 0.16	(+)2.31 ± 0.12	(+)2.17 ± 0.34	(+)3.50 ± 0.14
221695_s_at	MAP3K2	(−)4.09 ± 0.48	(+)4.01 ± 0.31	(+)3.47 ± 0.22	(+)3.51 ± 0.71
210570_x_at	MAPK9	(−)4.56 ± 0.31	(+)2.88 ± 0.21	(+)2.16 ± 0.22	(+)2.20 ± 0.31
203218_at		(−)4.43 ± 0.28	(+)2.71 ± 0.31	(+)2.19 ± 0.28	(+)2.12 ± 0.19

(+) – overexpression compared to the control; (−) – downregulated compared to the control; LPS – liposaccharide A; CsA – cyclosporine A; C – control culture; H_2, H_8, H_24 – culture exposed to cyclosporine A for 2, 8, 24 hours; DUSP1 – dual specificity phosphatase 1; DUSP4 – dual specificity phosphatase 4; MAP2K2 – mitogen-activated protein kinase kinase 2; MAP2K7 – mitogen-activated protein kinase kinase 7; MAP3K2 – mitogen-activated protein kinase kinase kinase 2; MAPK9 – mitogen-activated protein kinase 9.

Figure 3. Expression profile of mRNA encoding selected MAPK-related genes in HaCaT treated with LPS and then with cyclosporine a for 2, 8, 24 hours compared to the control culture.

Table 3. Results of ANOVA and Tukey’s post-hoc test for MAPK genes, the expression of which was determined by the qRT-PCR technique.

mRNA	p-value ANOVA	Adjusted p-value of Tukey’s post-hoc test
		LPS vs. H_2	LPS vs. H_8	LPS vs. H_24	H_2 vs. H_8	H_2 vs. H_24	H_8 vs. H_24
DUSP1	<0.01	<0.001	<0.001	<0.001	0.504	<0.001	<0.001
DUSP4	<0.01	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
MAP2K2	<0.01	<0.001	<0.001	<0.001	<0.001	<0.001	0.040
MAP2K7	<0.01	<0.001	<0.001	<0.001	<0.001	<0.001	0.666
MAP3K2	<0.01	<0.001	<0.001	<0.001	<0.001	<0.001	0.900
MAPK9	<0.01	<0.001	<0.001	<0.001	<0.001	<0.001	0.900

LPS – lipopolysaccharide A; C – control culture; H_2, H_8, H_24 – culture exposed to cyclosporine A for 2, 8, 24 hours; DUSP1 – dual specificity phosphatase 1; DUSP4 – dual specificity phosphatase 4; MAP2K2 – mitogen-activated protein kinase kinase 2; MAP2K7 – mitogen-activated protein kinase kinase 7; MAP3K2 – mitogen-activated protein kinase kinase kinase 2; MAPK9 – mitogen-activated protein kinase 9.

Figure 4. miRnas potentially modulating the expression of MAP kinase-related genes in keratinocytes exposed to cyclosporine a compared to the control.

Table 4. Expression pattern of miRnas potentially regulating the expression of DUSP1, DUSP4, MAP2K2, MAP2K7, MAP3K2 and MAPK9 of the HaCaT cell line exposed to LPS and then cyclosporine for 2, 8, and 24 hours compared to the control.

miRNA	Expression
	LPS vs. C	H-2 vs C	H-8 vs C	H-24 vs C	p-value ANOVA	p-value Tukey’s post-hoc test
miR-34a	(+)2.17 ± 0.32	(+)2.98 ± 0.21	(+)2.98 ± 0.18	(+)2.54 ± 0.22	0.6521	0.690^1 0.690^2 0.820^3 1^4 0.995^5 0.995^6
miR-1275	(+)1.87 ± 0.43	(+)2.98 ± 0.11	(+)3.13 ± 0.18	(+)3.19 ± 0.21	<0.001	<0.001^1,2,3,4,5 0.514^6
miR-3188	(+)1.99 ± 0.22	(+)1.22 ± 0.32	(+)2.10 ± 0.19	(+)2.19 ± 0.09	<0.001	<0.001^1,4,5 0.097^2 0.238^3 0.912^6
miR-382	(+)4.98 ± 0.99	(+)2.19 ± 0.82	(+)2.88 ± 0.71	(+)2.18 ± 0.21	<0.001	<0.001^1,2,3 0.419^4 0.346^5 0.0376^6
miR-200a-5p	(+)3.09 ± 0.21	(+)2.19 ± 0.34	(+)2.09 ± 0.12	(+)2.18 ± 0.54	<0.001	<0.001^1,2 0.036^3 0.419^4 0.900^5 0.031^6

(+) – overexpression in comparison to the control; (−) – downregulated in comparison to the control; LPS – liposaccharide A; C – control culture; H_2, H_8, H_24 – culture exposed to cyclosporine A for 2, 8, 24 hours; statistically significant differences between:¹LPS vs. H_2; ²LPS vs. H_8; ³LPS vs. H_24; ⁴H_2 vs. H_8; ⁵ H_2 vs. H_24; ⁶H_8 vs. H_24.

Table 5. Concentration of MAP kinase-related genes at the protein level in HaCaT culture exposed to LPS and CsA.

Protein	C [ng/mL]	LPS [ng/mL]	H-2 [ng/mL]	H-8 [ng/mL]	H-24 [ng/mL]	p-value Student’s t-test or ANOVA	p-value Tukey’s post-hoc test
DUSP1	0.92 ± 0.09	0.32 ± 0.11	below the detection threshold	below the detection threshold	below the detection threshold	<0.001^a	-
DUSP4	1.06 ± 0.11	0.56 ± 0.01	below the detection threshold	below the detection threshold	below the detection threshold	<0.001^a	-
MAP2K2	2.11 ± 0.12	1.87 ± 0.34	0.34 ± 0.09	0.21 ± 0.12	0.20 ± 0.02	<0.001^b	<0.001^1,2,3,4,5,6,7 1^10 0.004^8,9
MAP2K7	1.79 ± 0.19	1.56 ± 0.12	0.87 ± 0.03	0.81 ± 0.09	0.80 ± 0.12	<0.001^b	<0.001^1,2,3,4,5,6,7 1^10 0.004^8,9
MAP3K2	0.98 ± 0.07	0.54 ± 0.05	below the detection threshold	below the detection threshold	below the detection threshold	<0.001^a	-
MAPK9	4.12 ± 0.78	2.19 ± 0.34	below the detection threshold	below the detection threshold	below the detection threshold	<0.001^a	-

LPS – lipopolysaccharide A; C – control culture; H_2, H_8, H_24 – culture exposed to cyclosporine A for 2, 8, 24 hours; DUSP1 – dual specificity phosphatase 1; DUSP4 – dual specificity phosphatase 4; MAP2K2 – mitogen-activated protein kinase kinase 2; MAP2K7 – mitogen-activated protein kinase kinase 7; MAP3K2 – mitogen-activated protein kinase kinase kinase 2; MAPK9 – mitogen-activated protein kinase 9; statistically significant differences between: ¹C vs. LPS; ²C vs. H_2; ³C vs. H_8; ⁴ C vs. H_24; ⁵LPS vs. H_2; ⁶LPS vs. H_8; ⁷LPS vs. H_24; ⁸H_2 vs. H_8; ⁹H_2 vs. H_24; ¹⁰H_8 vs. H_24.

Data availability statement

All data were included in the paper.