Primary analysis and screening of microRNAs in gastric cancer side population cells

Table 1 MicroRNA microarray and quantitative real-time-polymerase chain reaction analyses results

Down-regulated genes	Fold change microarray	ΔCT	-ΔΔCT	Upregulated genes	Fold change microarray	ΔCT	-ΔΔCT
		qRT-PCR				qRT-PCR
hsa-miR-3175	7.29	21.82	-1.32	hsa-miR-25-star	27.35	6.63	4.47
hsa-miR-1246	3.46	14.07	-0.64	hsa-miR-1275	5.13	13.77	0.48
hsa-miR-663	3.12	10.69	-0.13	hsa-miR-339-5p	4.03	15.63	0.89
hsa-miR-1281	2.93	14.72	-0.74	hsa-miR-140-3 p	3.56	13.30	0.41
hsa-miR-422a	2.56	14.41	-0.69	hsa-miR-197	3.36	14.08	0.63
hsa-miR-1228-star	2.41	16.62	-0.98	hsa-miR-362-5p	3.01	15.25	0.86
hsa-miR-1975	2.10	12.84	-0.38	hsa-miR-324-5p	2.75	17.85	1.06
hsa-miR-1826	2.09	14.18	-0.67	hsa-miR-34a	2.70	23.46	2.32
hsa-miR-1915	2.08	10.83	-0.14	hsa-miR-210	2.17	13.30	0.44
hsa-miR-4284	2.07	14.34	-0.65	hsa-miR-500-star	2.06	11.06	0.18
hsa-miR-203	1.57	26.13	-3.84	hsa-miR-130a	1.77	22.49	2.13
				hsa-miR-130b	1.61	17.34	0.85
				hsa-miR-18a	1.60	13.41	0.46
				hsa-miR-29a	1.57	15.46	0.80
				hsa-miR-149	1.54	11.96	0.32

Differentially expressed microRNAs (miRNAs) in SP and MP cells of the gastric cancer cell line MKN-45 as determined by miRNA microarray and quantitative real-time-PCR analyses. These results showed that the miRNA profiles of SP cells differed from the miRNA profiles of MP cells. Moreover, downregulated miRNAs, such as hsa-miR-3175 and hsa-miR-203, and upregulated miRNAs, including hsa-miR-130a, hsa-miR-324-5p, hsa-miR-34a, and hsa-miR-25-star, showed significant differences in expression between SP and MP cells when tested by quantitative real-time PCR. SP: Side population; MP: Major population; PCR: Polymerase chain reaction.

Table 2 Differentially expressed microRNAs and their target number and known targets

microRNA	Sp/Mp (FC)	Target number	Validated target number	Known targets
hsa-miR-130a	1.77	406	3	MEOX2; TAC1; HOXA5
hsa-miR-29a	1.57	402	3	DNMT3A; BACE1; DNMT3B
hsa-miR-130b	1.61	392	0
hsa-miR-203	0.64	275	0
hsa-miR-34a	2. 70	260	3	E2F3; CCND1; CDK6
hsa-miR-149	1.54	129	0
hsa-miR-18a	1.60	104	0
hsa-miR-324-5p	2.75	33	0
hsa-miR-210	2.17	5	1	EFNA3

Bioinformatic analysis of differentially expressed microRNAs indicated that differentially expressed microRNAs had predicted or experimentally verified target genes. Moreover, there are multiple targets for many miRNAs, but most of these targets have not been studied or validated.

Table 3 Genes that are regulated by more than 4 microRNAs

Gene ID	Gene name	microRNA number	All microRNAs
80218	NAA50	5	hsa-miR-203; hsa-miR-18a; hsa-miR-34a; hsa-miR-130a; hsa-miR-130b
164	AP1G1	5	hsa-miR-324-5p; hsa-miR-130b; hsa-miR-29a; hsa-miR-130a; hsa-miR-203
7803	PTP4A1	4	hsa-miR-203; hsa-miR-130a; hsa-miR-130b; hsa-miR-29a
90355	C5orf30	4	hsa-miR-130b; hsa-miR-18a; hsa-miR-130a; hsa-miR-203
80829	ZFP91	4	hsa-miR-130b; hsa-miR-130a; hsa-miR-324-5p; hsa-miR-29a
28514	DLL1	4	hsa-miR-149; hsa-miR-130a; hsa-miR-130b; hsa-miR-34a
23013	SPEN	4	hsa-miR-203;hsa-miR-130a; hsa-miR-29a; hsa-miR-130b
57659	ZBTB4	4	hsa-miR-18a; hsa-miR-149; hsa-miR-130a; hsa-miR-130b
23261	CAMTA1	4	hsa-miR-34a; hsa-miR-203; hsa-miR-130b; hsa-miR-130a
9444	QKI	4	hsa-miR-18a; hsa-miR-130b; hsa-miR-29a; hsa-miR-130a
166336	PRICKLE2	4	hsa-miR-130b; hsa-miR-29a; hsa-miR-130a; hsa-miR-203
5156	PDGFRA	4	hsa-miR-130a; hsa-miR-149; hsa-miR-130b; hsa-miR-34a

Predicted target genes of key microRNAs. Some microRNAs, such as hsa-miR-324-5p, hsa-miR-130b, hsa-miR-29a, hsa-miR-130a, and hsa-miR-203, modulate multiple genes; these results indicate that differentially expressed microRNAs and their targets have a reticular network.