The potential role of differentially expressed tRNA-derived fragments in high glucose-induced podocytes

Figures & data

Table 1. Primer sequences used for RT-qPCR.

Primer	Forward and reverse primers (5′–3′)
Nephrin	F: ATGGGAGCTAAGGAAGCCACA R: GATGGAGAGGATTACGCTGGG
Podocin	F: GCATCAAGCCCTCTGGATTAG R: AGACGGAGATCAACCTTGTGATA
Desmin	F: GTGGATGCAGCCACTCTAGC R: TTAGCCGCGATGGTCTCATAC
β-Actin	F: CATCCGTAAAGACCTCTATGCCAAC R: ATGGAGCCACCGATCCACA
U6	F: CTCGCTTCGGCAGCACATATACT R: ACGCTTCACGAATTTGCGTGTC

Reverse transcription primers and forward primers for tRFs and universal reverse primers were purchased from RiboBio.

Figure 1. Construction of the podocyte injury model. (A) RT-qPCR was used to measure the mRNA expression levels of nephrin, podocin and desmin in mouse podocytes in the control, HG, and mannitol groups. (B) Protein expression levels of related indexes in cells were assessed by western blotting. The data are expressed as the mean ± SD. *p < .05, ***p < .001 vs. the control group. NS: not significant; HG: high glucose; RT-qPCR: real-time quantitative polymerase chain reaction.

Figure 2. Expression profile of tRFs in podocytes of the HG group and control group. (A) Clustered heatmap of all tRFs in the HG and control groups. (B) Volcano plots of tRFs in the HG group compared to the control group. (C) tRFs commonly expressed and specifically expressed in the two groups. (D) Distribution of tRF subtypes in the two groups. (E) Distribution of tRFs in different tRNAs in the two groups. HG: high glucose; tRFs: tRNA-derived fragments.

Table 2. Differentially expressed tRFs.

tRF	tRF sequence	Type	Log2(fold change)	p Value
tRF-1:14-Cys-GCA-15	GGTGGTATAGCTCA	tRF-5a	4.43	.02
tRF-1:15-chrM.Ser-GCT	AAGAAAGATTGCAAG	tRF-5a	2.72	.00
tRF-1:15-Thr-AGT-6	GGCTCTGTGGCTTAG	tRF-5a	2.27	.00
tRF-1:14-Ala-AGC-1-M3	GGGGGTGTAGCTCA	tRF-5a	2.22	.00
tRF-1:14-Thr-TGT-2	GGCCCTATAGCTCA	tRF-5a	2.20	.00
tRF-1:15-Thr-TGT-3	GGCTCCATAGCTCAG	tRF-5a	2.07	.00
tRF-61:77-Thr-AGT-1-M2	TCCCAGCGGTGCCTCCA	tRF-3a	1.95	.01
tRF-50:70-chrM.Trp-TCA	ACACAAGTTTAACTTCTGCCA	tRF-3b	1.92	.01
tRF-1:30-Val-AAC-5	GTTTCCGTAGTGTAGTGGTTATCACATTCG	tRF-5c	1.77	.02
tRF-1:15-Thr-TGT-2	GGCCCTATAGCTCAG	tRF-5a	1.73	.00
tRF-+1:T20-Thr-CGT-2	GCGTTTGGAAGAGATATTTT	tRF-1	1.56	.01
tRF-1:28-Cys-GCA-1-M3	GGGGGTATAGCTCAGTGGTAGAGCATTT	tRF-5c	1.45	.00
tRF-1:29-Cys-GCA-1-M3	GGGGGTATAGCTCAGTGGTAGAGCATTTG	tRF-5c	1.35	.00
tRF-1:30-Gly-CCC-3-M2	GCATTGGTGGTTCAATGGTAGAATTCTCGC	tRF-5c	1.34	.02
tRF-1:29-Glu-TTC-2	TCCCATATGGTCTAGCGGTTAGGATTCCT	tRF-5c	1.32	.00
tRF-1:29-Glu-TTC-1	TCCCACATGGTCTAGCGGTTAGGATTCCT	tRF-5c	1.31	.00
tRF-1:30-Cys-GCA-1-M3	GGGGGTATAGCTCAGTGGTAGAGCATTTGA	tRF-5c	1.28	.00
tRF-1:29-Asp-GTC-3	TCCTCGTTAGTATAGTGGTGAGTATCCCT	tRF-5c	1.22	.01
tRF-1:32-chrM.Gln-TTG	TAGGATAAGGTGTTTAGGTAGCACGGAGAATT	tRF-5c	1.20	.04
tRF-1:29-Gly-CCC-3-M2	GCATTGGTGGTTCAATGGTAGAATTCTCG	tRF-5c	1.11	.05
tRF-+1:T17-Leu-AAG-2	AGGCAGGCTTTTTCTTT	tRF-1	1.10	.04
tRF-30:43-Gln-TTG-1-M4	GACTTTGAATCCAG	tRF-2	1.07	.04
tRF-1:28-Glu-TTC-2	TCCCATATGGTCTAGCGGTTAGGATTCC	tRF-5c	1.06	.04
tRF-1:15-SeC-TCA-1	GCCCGGATGATCCTC	tRF-5a	1.03	.00
tRF-1:29-Val-CAC-4	GTTTCTGTAGTGTAGTGGTTATCACGTTC	tRF-5c	1.01	.05
tRF-1:29-Glu-CTC-1	TCCCTGGTGGTCTAGTGGTTAGGATTCGG	tRF-5c	0.96	.01
tRF-1:31-Val-AAC-5	GTTTCCGTAGTGTAGTGGTTATCACATTCGC	tRF-5c	0.96	.01
tRF-1:29-Gly-TCC-1	GCGTTGGTGGTATAGTGGTGAGCATAGCT	tRF-5c	0.95	.01
tRF-1:29-Ala-CGC-5	TCCCTGGTAGTCTAGTGGTTAGGATTCGG	tRF-5c	0.95	.04
tRF-1:14-Cys-GCA-1-M16	GGGGGTATAGCTCA	tRF-5a	0.94	.03
tRF-1:29-Val-TAC-1	GGTTCCATAGTGTAGCGGTTATCACGTCT	tRF-5c	0.94	.04
tRF-1:23-Val-AAC-1-M4	GTTTCCGTAGTGTAGTGGTTATC	tRF-5b	0.93	.00
tRF-30:43-Gln-CTG-1-M5	GACTCTGAATCCAG	tRF-2	0.88	.03
tRF-1:31-Glu-CTC-2	TCCCTGGTGGTCTAGTGGTTAGGATTTGGCG	tRF-5c	0.88	.03
tRF-1:30-Gly-GCC-2-M3	GCATTGGTGGTTCAGTGGTAGAATTCTCGC	tRF-5c	0.87	.01
tRF-1:31-Glu-TTC-3	TCCCTGGTGGTCTAGTGGCTAGGATTCGGCG	tRF-5c	0.86	.05
tRF-1:30-Asp-GTC-1-M2	TCCTCGTTAGTATAGTGGTGAGTATCCCCG	tRF-5c	0.86	.03
tRF-1:31-Glu-CTC-1	TCCCTGGTGGTCTAGTGGTTAGGATTCGGCG	tRF-5c	0.84	.02
tRF-1:29-Glu-TTC-3	TCCCTGGTGGTCTAGTGGCTAGGATTCGG	tRF-5c	0.82	.04
tRF-1:28-Gly-GCC-2-M3	GCATTGGTGGTTCAGTGGTAGAATTCTC	tRF-5c	0.81	.02
tRF-1:23-Val-AAC-2-M3	GTTTCCGTAGTGTAGTGGTCATC	tRF-5b	0.80	.01
tRF-1:31-Lys-CTT-2	GCCCGGCTAGCTCAGTCGGTAGAGCATGGGA	tRF-5c	0.80	.05
tRF-1:30-Lys-CTT-3	GCCCGGCTAGCTCAGTCGGTAGAGCATGAG	tRF-5c	0.79	.03
tRF-1:29-Val-AAC-2-M3	GTTTCCGTAGTGTAGTGGTCATCACGCTC	tRF-5c	0.79	.04
tRF-1:30-Gly-GCC-1	GCATGGGTGGTTCAGTGGTAGAATTCTCGC	tRF-5c	0.78	.01
tRF-1:28-Lys-CTT-2-M2	GCCCGGCTAGCTCAGTCGGTAGAGCATG	tRF-5c	0.78	.03
tRF-1:31-Lys-CTT-3	GCCCGGCTAGCTCAGTCGGTAGAGCATGAGA	tRF-5c	0.77	.02
tRF-55:76-Arg-ACG-1-M2	TCGACTCCTGGCTGGCTCGCCA	tRF-3b	0.77	.02
tRF-1:28-Glu-CTC-1	TCCCTGGTGGTCTAGTGGTTAGGATTCG	tRF-5c	0.75	.02
tRF-1:28-Gly-GCC-1	GCATGGGTGGTTCAGTGGTAGAATTCTC	tRF-5c	0.75	.03
tRF-1:30-Ala-CGC-5	TCCCTGGTAGTCTAGTGGTTAGGATTCGGT	tRF-5c	0.74	.04
tRF-1:31-Glu-TTC-2	TCCCATATGGTCTAGCGGTTAGGATTCCTGG	tRF-5c	0.68	.03
tRF-58:75-Gln-CTG-1-M7	ATCTCGGTGGGACCTCCA	tRF-3a	0.63	.05
tRF-1:16-chrM.Met-CAT	AGTAAGGTCAGCTAAT	tRF-5a	−4.00	.05
tRF-+1:T16-iMet-CAT-1-7	TCTGGACTTTTTTTTT	tRF-1	−3.25	.01
tRF-+1:T15-Asp-GTC-1	ACTAGTGAGTTTTTA	tRF-1	−2.87	.03
tRF-1:16-Ala-AGC-1-M3	GGGGGTGTAGCTCAGT	tRF-5a	−2.72	.05
tRF-54:74-Gly-CCC-2-M4	CGATTCCCGGCCAATGCACCA	tRF-3b	−2.48	.02
tRF-60:76-Ala-AGC-6-M2	GCCCGCATTCTCCACCA	tRF-3a	−2.37	.00
tRF-1:24-Glu-CTC-1-M2	TCCCTGGTGGTCTAGTGGTTAGGA	tRF-5b	−2.36	.03
tRF-+1:T14-Asp-GTC-1	ACTAGTGAGTTTTT	tRF-1	−2.34	.00
tRF-+1:T17-Asp-GTC-1-13	AGTGATCTCTATTTTTA	tRF-1	−2.29	.02
tRF-54:75-Glu-TTC-2	TCGACTCCCGGTATGGGAACCA	tRF-3b	−2.08	.00
tRF-54:75-Asp-GTC-1	TCGATTCCCCGACGGGGAGCCA	tRF-3b	−2.01	.02
tiRNA-1:34-Lys-TTT-1	GCCCGGATAGCTCAGTCGGTAGAGCATCAGACTT	tiRNA-5	−1.95	.01
tRF-69:86-Leu-TAA-1	ACCCCACTTCTGGTACCA	tRF-3a	−1.67	.02
tRF-+1:T15-Asp-GTC-1-5	ATACAGTAGCTTTTT	tRF-1	−1.66	.01
tRF-+1:T20-Asp-GTC-1-11	TCTGGACACATGTGGCTTTT	tRF-1	−1.58	.04
tRF-+1:T15-Asp-GTC-1-6	ATTGTTAAGAATTTT	tRF-1	−1.56	.00
tRF-28:41-Gln-CTG-1-M5	TGGACTCTGAATCC	tRF-2	−1.49	.04
tRF-58:75-Ala-CGC-4-M6	TTCCCGGCATCTCCACCA	tRF-3a	−1.49	.03
tRF-1:16-Ser-AGA-1-M3	GTAGTCGTGGCCGAGT	tRF-5a	−1.45	.00
tRF-+1:T16-Trp-CCA-3	TCTGTGTTCTTTATTT	tRF-1	−1.45	.00
tiRNA-1:34-Lys-CTT-2	GCCCGGCTAGCTCAGTCGGTAGAGCATGGGACTC	tiRNA-5	−1.34	.00
tRF-1:15-Gly-GCC-2-M3	GCATTGGTGGTTCAG	tRF-5a	−1.14	.03
tRF-+1:T15-Asp-GTC-1-13	AGTGATCTCTATTTT	tRF-1	−1.14	.01
tRF-+1:T15-iMet-CAT-1-7	TCTGGACTTTTTTTT	tRF-1	−1.11	.03
tRF-+1:T16-Asp-GTC-1-13	AGTGATCTCTATTTTT	tRF-1	−1.08	.01
tRF-54:75-Glu-TTC-1	TCGACTCCCGGTGTGGGAACCA	tRF-3b	−1.02	.01
tiRNA-1:34-Lys-CTT-3	GCCCGGCTAGCTCAGTCGGTAGAGCATGAGACTC	tiRNA-5	−1.00	.00
tRF-1:32-Gly-CCC-1	GCGCCGCTGGTGTAGTGGTATCATGCAAGATT	tRF-5c	−0.99	.01
tRF-69:86-Leu-TAA-2	ACCCCACTCCTGGTACCA	tRF-3a	−0.95	.05
tRF-+1:T14-Ser-AGA-2-2	TCTAGGATTTCTTT	tRF-1	−0.94	.00
tRF-1:14-Lys-TTT-1	GCCCGGATAGCTCA	tRF-5a	−0.93	.05
tRF-1:16-Tyr-GTA-3-M2	CCTTCGATAGCTCAGC	tRF-5a	−0.81	.01
tRF-+1:T15-Ser-AGA-2-2	TCTAGGATTTCTTTT	tRF-1	−0.71	.03
tRF-+1:T19-Arg-ACG-1-3	TTGGGACTGGTACTCCTTT	tRF-1	−0.67	.03
tRF-+1:T24-Arg-ACG-1-3-M1-23:G > T	TTGGGACTGGTACTCCTTTATTTT	tRF-1	−0.62	.04
tiRNA-1:33-Gly-GCC-2-M3	GCATTGGTGGTTCAGTGGTAGAATTCTCGCCTG	tiRNA-5	−0.62	.03

Figure 3. Validation of the expression levels of selected tRFs. (A) The expression levels of tRF-1:15-chrM.Ser-GCT, tRF-1:15-Thr-AGT-6, tRF-1:28-Cys-GCA-1-M3, tRF-1:29-Glu-TTC-2, and tRF-1:30-Cys-GCA-1-M3 were measured by RT-qPCR. (B) The expression levels of tRF-1:16-chrM. Met-CAT, tRF-1:24-Glu-CTC-M2, tRF-54:75-Asp-GTC-1, tRF-54:75-Glu-TTC-2, and tRF-58:75-Ala-CGC-4-M6 were measured by RT-qPCR. The data are expressed as the mean ± SD. *p < .05, **p < .01 vs. control group. NS: not significant; HG: high glucose; tRFs: tRNA-derived fragments; RT-qPCR: real-time quantitative polymerase chain reaction.

Table 3. Target genes in top 20.

tRF	Target genes	Log2(fold change)	p Value
tRF-1:15-chrM.Ser-GCT	Nufip2, Tnfaip8, Kcne1, Bclaf3, Slfn4, Slc12a2, Hspa5, Nipal1, Echdc1, Zfp366, Rfx1, A1cf, Fchsd1, Rabl3, Abca13, Alox5ap, Clec16a, Tomm22, Fbxl4, Dbt	2.72	<.001
tRF-1:15-Thr-AGT-6	Gfap, Gm9918, Ttl, Ube3b, Pax5, Zfp456, Ptges, Nfat5, Zfp236, Zfp641, Arl3, Lcp1, Ppm1h, Col9a2, Vipr2, Sesn2, Tmed9, Olfr544, Tmem178, Ankrd11	2.27	<.001
tRF-1:28-Cys-GCA-1-M3	Gmps, Abhd11, Nr1i2, Ncl, Zfpm1, Socs7, Tpgs1, Patz1, Tmc6, Eps8l2, Hcfc1, Mxra8, Ppme1, Nnmt, Capns1, Dyrk1b, Kcnh1, Abcd1, Plekhg2, Echdc3	1.45	.005
tRF-1:29-Glu-TTC-2	AI467606, Zrsr2, Smoc2, Cplx4, Lemd3, Ralgapa2, Zfp738, Ube2n, Thumpd1, Gpr45, Zfp811, Nr5a1, Hemgn, Tert, Ddit4l, Olfr775, Nlrp6, Tdo2, Tmem30a, Olfr303	1.32	<.001
tRF-1:30-Cys-GCA-1-M3	Abhd11, Gmps, Nr1i2, Ncl, Zfpm1, Tpgs1, Socs7, Echdc3, Hcfc1, Tmc6, Nnmt, Mxra8, Gm49322, Scamp4, Patz1, Gle1, Ppme1, Eps8l2, Dyrk1b, Ush1g	2.43	.003
tRF-1:16-chrM.Met-CAT	Hoxb1, Pcdha11, Pcdha4, Pcdha2, Pcdha6, Pcdha1, Prss12, Gstcd, Adh4, Zfp90, Ttll10, Nrg4, Olfr802, Otud6b, Msr1, Zfp735, Pgr, Zfp810, Lonrf2, Tenm1	−4.00	.049
tRF-1:24-Glu-CTC-M2	Mfsd4a, Ss18l2, Slc4a3, Apopt1, Bahcc1, Mfsd4a, Slc22a15, Fam83f, Lmx1b, Paqr5, Arih2, Cotl1, Znf76, 1700102P08Rik, Lrrc10b, Prkce, B3gntl1, Gpr55, Olfr1167, Aacs	−2.36	.030
tRF-54:75-Asp-GTC-1	Snapc5, Pbx2, Sik1, Srek1, Nipal2, Lnx2, Olfr802, Ptger3, Fbln1, Frat2, Ncam1, Sel1l3, Cbln4, Prickle2, Wdr26, Ecscr, Olfr1307, Tmcc3, 2610528J11Rik, Nop16	−2.01	.022
tRF-54:75-Glu-TTC-2	Gm42669, St8sia2, Zfp641, Zfp319, Zyg11b, Bcl2, Myo1h, Plekhd1, Vkorc1, Myl6, Tmbim6, Gad2, Foxe1, Slc25a51, AI464131, Lhfpl4, Prdm10, Cd37, Uqcc3, Nfatc2	−2.08	.003
tRF-58:75-Ala-CGC-4-M6	Bnip1, Zfand2b, Vsir, Crat, Slc35b4, Ffar2, Casz1, Ktn1, D1Ertd622e, Ccr5, Pias4, Rnf169, Syt5, Slc43a2, C87436, Dtnbp1, Aph1a, Adam11, Col11a2, Zfp94	−1.49	.028

Figure 4. GO and KEGG analysis of target genes. GO analysis of target genes of downregulated tRFs; BP (A), CC (B), and MF (C) terms are included in the analysis. GO analysis of the target genes of upregulated tRFs, including BP (D), CC (E), and MF (F) terms. (G, H) KEGG pathway analysis of the target genes of selected differentially expressed tRFs. GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; tRFs: tRNA-derived fragments; BP: biological process; CC: cellular component; MF: molecular function; Sig: significant.

Figure 5. Overexpression of tRF-1:24-Glu-CTC-1-M2 contributes to podocyte injury mitigation. (A) Successful transfection of tRF-1:24-Glu-CTC-1-M2 in podocytes. (B) Expression levels of nephrin, podocin, and desmin mRNAs. (C) Expression levels of nephrin, podocin, and desmin proteins. The data are expressed as the mean ± SD. ***p < .001, ^#p < .05, ^##p < .01, and ^###p < .001 vs. the indicated group. HG: high glucose; tRFs: tRNA-derived fragments.

Data availability statement

The study data are available from the corresponding authors upon reasonable request.