Effects of lncRNA OIP5-AS1 regulating miR-25-3p/SOX4 axis on the biological process of human renal tubular epithelial cells induced by high glucose

doi:10.11958/20220849

Abstract

Abstract:

Objective To investigate the impact and molecular mechanism of long non-coding RNA Opa-interacting protein 5-antisense transcript 1 (lncRNA OIP5-AS1) on the proliferation, apoptosis and oxidative stress damage of human renal tubular epithelial cells induced by high glucose. Methods Human renal cortical proximal tubule epithelial cells HK-2 were cultured in vitro. HK-2 cells were transfected with lncRNA OIP5-AS1 small interfering RNA (si-OIP5-AS1), miR-25-3p mimic, miR-25-3p inhibitor and their negative controls si-NC, miR-NC and inhibitor-NC. Cells were divided into the normal glucose group (NG group), the high glucose group (HG group), the HG+si-NC group, the HG+si-OIP5-AS1 group, the HG+miR-NC group, the HG+miR-25-3p group, the HG+si-OIP5-AS1+inhibitor-NC group and the HG+si-OIP5-AS1+miR-25-3p inhibitor group. Forty-eight hours after transfection, real-time quantitative PCR (qPCR) was performed to detect levels of lncRNA OIP5-AS1, miR-25-3p and sex-determining region Y-box protein 4 (SOX4) mRNA in cells. CCK-8 assay was performed to detect cell viability and lactate dehydrogenase (LDH) activity in the cell culture supernatant. Flow cytometry was performed to analyze apoptosis, malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) activities in cells. DCFH-DA fluorescent probe was implemented to detect intracellular reactive oxygen species (ROS) production. Western blot experiment was performed to detect the protein expression of SOX4, B-cell lymphoma factor 2 (Bcl-2), Bcl-2-associated X protein (Bax), caspase-3 (Caspase-3) and cleaved-Caspase-3 (Cleaved-Caspase-3) in cells. Dual luciferase reporter assay confirmed the targeting relationship between lncRNA OIP5-AS1 and miR-25-3p, and between miR-25-3p and SOX4. Results Compared with the NG group, the expression levels of lncRNA OIP5-AS1 and SOX4 were significantly increased in the HG group, and the level of miR-25-3p was significantly decreased (P<0.05). Knockdown of lncRNA OIP5-AS1 was able to significantly down-regulate SOX mRNA and protein levels, and up-regulate miR-25-3p level, increase HK-2 cell viability, SOD, CAT activities and Bcl-2 protein level, and reduce apoptosis rate, LDH activity, MDA, ROS levels, Bax protein level and Cleaved-Caspase-3/Caspase-3 ratio (P<0.05). The up-regulating miR-25-3p expression was consistent with that of knocking down lncRNA OIP5-AS1. On the basis of knockdown of lncRNA OIP5-AS1, down-regulation of miR-25-3p significantly attenuated the protective effect of lncRNA OIP5-AS1 knockdown on high glucose-induced oxidative stress damage in HK-2 cells (P<0.05). Dual luciferase reporter assay showed binding sites between lncRNA OIP5-AS1 and miR-25-3p, as well as between miR-25-3p and SOX4. Conclusion The lncRNA OIP5-AS1 may promote high glucose-induced HK-2 cell damage through the miR-25-3p/SOX4 axis.

Key words: diabetic nephropathies, kidney tubules, epithelial cells, apoptosis, cell proliferation, miR-25-3p, OIP5-AS1, sex-determining region Y-box 4

CLC Number:

R587.24

Figures/Tables 9

References 23

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基因名称	引物序列（5'→3'）	产物大小（bp）
lncRNA OIP5-AS1	上游：TGCGAAGATGGCGGAGTAAG	285
lncRNA OIP5-AS1	下游：TAGTTCCTCTCCTCTGGCCG	285
miR-25-3p	上游：GCGCATTGCACTTGTCTCG	139
miR-25-3p	下游：AGTGCAGGGTCCGAGGTATT	139
SOX4	上游：GTGAGCGAGATGATCTCGGG	216
SOX4	下游：CAGGTTGGAGATGCTGGACTC	216
U6	上游：GCTTCGGCAGCACATA	89
U6	下游：ATGGAACGCTTCACGA	89
GAPDH	上游：CACCCACTCCTCCACCTTTG	197
GAPDH	下游：CCACCACCCTGTTGCTGTAG	197

基因名称	引物序列（5'→3'）	产物大小（bp）
lncRNA OIP5-AS1	上游：TGCGAAGATGGCGGAGTAAG	285
lncRNA OIP5-AS1	下游：TAGTTCCTCTCCTCTGGCCG	285
miR-25-3p	上游：GCGCATTGCACTTGTCTCG	139
miR-25-3p	下游：AGTGCAGGGTCCGAGGTATT	139
SOX4	上游：GTGAGCGAGATGATCTCGGG	216
SOX4	下游：CAGGTTGGAGATGCTGGACTC	216
U6	上游：GCTTCGGCAGCACATA	89
U6	下游：ATGGAACGCTTCACGA	89
GAPDH	上游：CACCCACTCCTCCACCTTTG	197
GAPDH	下游：CCACCACCCTGTTGCTGTAG	197

组别	lncRNA OIP5-AS1	miR-25-3p	SOX4 mRNA
NG组	1.00±0.00	1.00±0.00	1.00±0.00
HG组	3.25±0.41^a	0.37±0.06^a	4.05±0.52^a
HG+si-NC组	3.40±0.38	0.34±0.05	4.11±0.49
HG+si-OIP5-AS1组	1.56±0.25^bc	0.82±0.10^bc	2.26±0.30^bc
HG+miR-NC组	3.37±0.42	0.35±0.06	4.08±0.54
HG+miR-25-3p组	3.12±0.39	0.85±0.09^be	2.15±0.28^be
HG+si-OIP5-AS1+ inhibitor-NC组	1.48±0.23	0.86±0.10	2.20±0.36
HG+si-OIP5-AS1+ miR-25-3p inhibitor组	1.61±0.27^b	0.40±0.05^df	3.74±0.42^df
F	30.168^＊＊	47.386^＊＊	26.692^＊＊

组别	lncRNA OIP5-AS1	miR-25-3p	SOX4 mRNA
NG组	1.00±0.00	1.00±0.00	1.00±0.00
HG组	3.25±0.41^a	0.37±0.06^a	4.05±0.52^a
HG+si-NC组	3.40±0.38	0.34±0.05	4.11±0.49
HG+si-OIP5-AS1组	1.56±0.25^bc	0.82±0.10^bc	2.26±0.30^bc
HG+miR-NC组	3.37±0.42	0.35±0.06	4.08±0.54
HG+miR-25-3p组	3.12±0.39	0.85±0.09^be	2.15±0.28^be
HG+si-OIP5-AS1+ inhibitor-NC组	1.48±0.23	0.86±0.10	2.20±0.36
HG+si-OIP5-AS1+ miR-25-3p inhibitor组	1.61±0.27^b	0.40±0.05^df	3.74±0.42^df
F	30.168^＊＊	47.386^＊＊	26.692^＊＊

组别	细胞活力（%）	LDH（U/L）	凋亡率（%）
NG组	100.00±0.00	116.25±18.83	4.39±0.76
HG组	39.32±6.05^a	304.17±32.50^a	25.08±3.10^a
HG+si-NC组	36.81±5.74	312.80±33.26	27.12±3.39
HG+si-OIP5-AS1组	71.40±8.62^bc	145.35±20.49^bc	14.50±2.08^bc
HG+miR-NC组	37.65±5.94	308.17±31.36	26.45±2.93
HG+miR-25-3p组	74.33±9.15^be	139.65±19.04^be	12.87±1.86^be
HG+si-OIP5-AS1+ inhibitor-NC组	72.92±8.76	140.80±22.75	13.29±1.92
HG+si-OIP5-AS1+ miR-25-3p inhibitor组	45.08±6.35^df	269.42±30.28^df	21.64±2.45^df
F	34.194^＊＊	32.929^＊＊	33.303^＊＊