Analysis of Human microRNA Expression Profiling During Diquat-Induced Renal Proximal Tubular Epithelial Cell Injury

Figures & data

Table 1 Primer Sequence for Real-Time PCR

ID	Primer Sequence (from 5’ to 3’)
has-miR-1255b-5p RT	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAACCAC
has-miR-1255b-5p F	CGCGGCGATGAGCAAAGAAA
has-miR-1910-3p RT	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTGTCAT
has-miR-1910-3p F	GCGGAGGCAGAAGCAGG
has-miR-1972 RT	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTGAGCC
has-miR-1972 F	CGTCAGGCCAGGCACAGT
has-miR-2276-3p RT	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCCTCGC
has-miR-2276-3p F	CGCGTCTGCAAGTGTCAGAG
has-miR-4443 RT	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAAACC
has-miR-4443 F	CATAGCGCTTGGAGGCGTG
has-miR-598-5p RT	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGCTCAC
has-miR-598-5p F	GCGGTGATCCCGATGGT
R	AGTGCAGGGTCCGAGGTATT
U6 F	CGCTTCGGCAGCACATATAC
U6 R	TTCACGAATTTGCGTGTCATC

Table 2 Differentially Expressed miRNAs

Change	miRNA	log2	p-value	Change	miRNA	log2	p-value
Up	hsa-miR-548z	5.63	0.0052	Down	hsa-miR-26a-1-3p	−1.09	0.0198
	hsa-miR-548h-3p	5.63	0.0052		hsa-miR-6783-3p	−1.11	0.0494
	hsa-miR-1538	5.62	0.0074		hsa-miR-155-3p	−1.23	0.0348
	hsa-miR-5699-3p	5.28	0.0090		hsa-miR-548d-3p	−1.60	0.0368
	hsa-miR-6884-5p	5.22	0.0199		hsa-miR-199b-5p	−1.70	0.0224
	hsa-miR-4449	5.18	0.0269		hsa-miR-6514-5p	−1.83	0.0390
	hsa-miR-6815-5p	5.11	0.0181		hsa-miR-1292-3p	−1.84	0.0221
	hsa-miR-3175	5.05	0.0165		hsa-miR-766-5p	−1.88	0.0159
	hsa-miR-4443	3.00	0.0002		hsa-miR-1972	−2.04	0.0042
	hsa-miR-656-3p	2.73	0.0007		hsa-miR-598-5p	−2.28	0.0009
	hsa-miR-4485-3p	2.35	0.0000		hsa-miR-2276-3p	−2.32	0.0015
	hsa-miR-29a-5p	1.86	0.0375		hsa-miR-3121-3p	−4.68	0.0398
	hsa-miR-195-5p	1.74	0.0073		hsa-miR-6726-3p	−4.68	0.0395
	hsa-miR-6511a-5p	1.67	0.0005		hsa-miR-6806-3p	−5.02	0.0289
	hsa-miR-4508	1.27	0.0414		hsa-miR-6852-5p	−5.13	0.0174
	hsa-miR-1255b-5p	1.16	0.0018		hsa-miR-377-5p	−5.17	0.0207
	hsa-miR-9898	1.02	0.0336		hsa-miR-4456	−5.57	0.0467
	hsa-miR-7704	1.02	0.0000		hsa-miR-1910-3p	−5.62	0.0002

Figure 1 HK-2 cell cytotoxicity and apoptosis induced by diquat.

Notes: (A) Cell viability. HK-2 cells were cultured in a cell medium with diquat (0, 20, 40, 60, 80, 100, and 120 μmol/L) for 24 h, and cell viability was measured by CCK-8 assay. (B and C) Cell apoptosis rate. HK-2 cells were cultured in a cell medium with diquat (0, 40, 80, and 120 μmol/L) for 24 h. Flow cytometry was used to detect the apoptosis rate of HK-2 cells by double staining of Annexin V and PI. Data is presented as mean ± SD of three or more independent experiments, *P<0.05 and **P<0.001 with respect to the CTRL group, and ^#P<0.05 with respect to the diquat group, indicating statistical significance by one-way ANOVA and post-hoc Tukey’s test.

Figure 2 Inter-sample correlation, principal components, and public and unique sequence analysis.

Notes: (A) Correlation coefficient plot between samples. (B) The plot of the ratio analysis of shared and unique sequences between samples. (C) A principal component analysis algorithm used to analyze the samples.

Figure 3 Profiles of the DE miRNAs. HK-2 cells were cultured in a medium containing 80 μmol/L diquat or an equivalent amount of PBS for 24 h.

Notes: (A) Volcano plot of miRNAs. (B) Scatter plot of differences in miRNAs between samples. (C) Heat map of DE miRNAs in HK-2 cells. |log2(FoldChange)| > 1, P value <0.05 relative to the CTRL group. (D) Histogram of RT-qPCR results. U6 normalized miRNA expression. Values are expressed as the mean ± SD, with *P value <0.05, diquat treatment group vs CTRL group.

Figure 4 Results of GO and KEGG analyses visualized by imageGP platform.

Notes: (A–C) Enrichment plot of GO analysis. (D) Pathway analysis enrichment plot for KEGG. Q-value < 0.05.

Figure 5 Essential target genes and network regulation map visualized by Cytoscape_v3.6.1 software.

Notes: The regulatory network was drawn based on the previous target gene prediction results. The Top20 candidate target genes associated with apoptosis were indicated by green circles, the differentially expressed miRNAs by pink circles, and the target gene with interaction degree over 3 by orange circles. The strength of the association was indicated by the line thickness.

Data Sharing Statement

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.