The effect of miR-493-3p on high glucose-induced myocardial cell injury and apoptosis via the lncRNA CCAT2/TCF7L2 axis

doi:10.11958/20252647

Abstract

Abstract:

Objective To investigate the impact of miR-493-3p on high glucose-induced cardiomyocyte injury and apoptosis by regulating long noncoding RNA (lncCCAT2)/transcription factor 7-like 2 (TCF7L2) axis. Methods Diabetic cardiomyopathy (DCM) cell model (AC16) was established in vitro with high glucose (HG, 55 mmol/L glucose). Experimental grouping:the control group, the HG group, the si-RNA negative control group, the si-CCAT2 group, the RNA mimics negative control group, the miR-493-3p-mimics group, the miR-493-3p-mimics +Lentivirus negative control group and the miR-493-3p-mimics +LV-CCAT2 group. Cell viability and apoptosis rate were detected by CCK8 assay and flow cytometry. The levels of lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) in cell culture medium and superoxide dismutase (SOD) in cells were detected by the kit methods. Dual luciferase reporter gene assay was used to verify the targeting relationship between miR-493-3p and lncRNA CCAT2. The expression of miR-493-3p, lncRNA CCAT2, β?catenin, TCF7L2, Caspase-3 and CyclinD1 genes were detected by RT-PCR. Western blot assay was used to detect the expressions of β-catenin (nuclear), β-catenin (Cytoplasm), TCF7L2, Caspase-3 and CyclinD1 proteins. Results Compared with the control group, miR-493-3p, OD₄₅₀and SOD were decreased in the HG group, and levels of lncRNA CCAT2, LDH, AST, apoptosis rate, the expression levels of TCF7L2 mRNA and protein increased. Up-regulating miR-493-3p or knocking down the expression of lncRNA CCAT2 can antagonize the above changes (P<0.05). The double luciferase reporter gene experiment verified the relationship between miR-493-3p and CCAT2 and TCF7L2. Compared with the HG+miR-493-3p-mimics group, the TCF7L2, β-catenin (nuclear), downstream Caspase-3 and CyclinD1 mRNA and protein expression were increased in the HG+miR-493-3p-mimics+LV-CCAT2 group (P<0.05). Conclusion miR-493-3p can alleviate myocardial cell injury and apoptosis under high glucose condition by inhibiting lncRNA CCAT2 expression, and there is a competitive relationship between the two regulation of TCF7L2.

Key words: diabetic cardiomyopathies, myocytes, cardiac, transcription factor 7-like 2 protein, apoptosis, miR-493-3p, LncRNA CCAT2

CLC Number:

R542.2，R587.1

Figures/Tables 13

References 18

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基因名称	引物序列（5'→3'）	产物大小/bp
miR-493-3p	上游：GTGAAGGTCTACTGTGTGC	97
miR-493-3p	下游：GTCCAGTTTTTTTTTTTTCCTG	97
CCAT2	上游：CTTCCAGCTCCACCTCTG	158
CCAT2	下游：GAGCTCAAAGGACGATGAGG	158
β-catenin	上游：AAAGCGGCTGTTAGTCACTGG	215
β-catenin	下游：CGAGTCATTGCATACTGTCCAT	215
TCF7L2	上游：GAAGGAGCGACAGCTTCATA	200
TCF7L2	下游：GGGGGAGGCGAATCTAGTAA	200
Caspase-3	上游：GAAATTGTGGAATTGATGCGTGA	164
Caspase-3	下游：CTACAACGATCCCCTCTGAAAAA	164
CyclinD1	上游：GGAGGAGGGTTGTGCTACAG	289
CyclinD1	下游：GCCTAGAACCCCACTACAGC	289
U6 snRNA	上游：CTCAACTGGTGTCGTGGAGTCGGC AATTC	102
U6 snRNA	下游：ACACTCCAGCTGGGTCACCGGGTGT AAATC	102
GAPDH	上游：TGTTGCCATCAATGACCCCTT	202
GAPDH	下游：CTCCACGACGTACTCAGCG	202

基因名称	引物序列（5'→3'）	产物大小/bp
miR-493-3p	上游：GTGAAGGTCTACTGTGTGC	97
miR-493-3p	下游：GTCCAGTTTTTTTTTTTTCCTG	97
CCAT2	上游：CTTCCAGCTCCACCTCTG	158
CCAT2	下游：GAGCTCAAAGGACGATGAGG	158
β-catenin	上游：AAAGCGGCTGTTAGTCACTGG	215
β-catenin	下游：CGAGTCATTGCATACTGTCCAT	215
TCF7L2	上游：GAAGGAGCGACAGCTTCATA	200
TCF7L2	下游：GGGGGAGGCGAATCTAGTAA	200
Caspase-3	上游：GAAATTGTGGAATTGATGCGTGA	164
Caspase-3	下游：CTACAACGATCCCCTCTGAAAAA	164
CyclinD1	上游：GGAGGAGGGTTGTGCTACAG	289
CyclinD1	下游：GCCTAGAACCCCACTACAGC	289
U6 snRNA	上游：CTCAACTGGTGTCGTGGAGTCGGC AATTC	102
U6 snRNA	下游：ACACTCCAGCTGGGTCACCGGGTGT AAATC	102
GAPDH	上游：TGTTGCCATCAATGACCCCTT	202
GAPDH	下游：CTCCACGACGTACTCAGCG	202

组别	miR-493-3p	lncRNA CCAT2
Control组	1.01±0.15	1.01±0.12
HG组	0.30±0.02^a	2.24±0.38^a
HG+mimics-NC组	0.31±0.03	2.07±0.21
HG+miR-493-3p-mimics组	0.92±0.08^bc	1.37±0.05^bc
HG+Smart Silencer-NC组	0.36±0.07	2.08±0.23
HG+Smart Silencer-CCAT2组	1.14±0.16^bd	0.94±0.21^bd
F	130.960^＊＊	58.594^＊＊

组别	miR-493-3p	lncRNA CCAT2
Control组	1.01±0.15	1.01±0.12
HG组	0.30±0.02^a	2.24±0.38^a
HG+mimics-NC组	0.31±0.03	2.07±0.21
HG+miR-493-3p-mimics组	0.92±0.08^bc	1.37±0.05^bc
HG+Smart Silencer-NC组	0.36±0.07	2.08±0.23
HG+Smart Silencer-CCAT2组	1.14±0.16^bd	0.94±0.21^bd
F	130.960^＊＊	58.594^＊＊

组别	OD₄₅₀值			LDH/（U/L）
Control组	0.89±0.03			89.47±17.99
HG组	0.63±0.03^a			396.68±43.54^a
HG+mimics-NC组	0.66±0.04			374.51±78.11
HG+miR-493-3p-mimics组	0.76±0.04^bc			176.56±35.66^bc
HG+Smart Silencer-NC组	0.62±0.04			330.88±36.32
HG+Smart Silencer-CCAT2组	0.75±0.05^bd			234.03±18.54^bd
F	585.245^＊＊			20.861^＊＊
组别		AST/ （U/L）	SOD/ （U/mgprot）		凋亡率/ %
Control组		13.90±3.48	16.58±1.98		2.09±0.46
HG组		76.49±18.98^a	3.73±0.82^a		13.62±1.12^a
HG+mimics-NC组		71.89±11.50	3.98±0.82		13.26±1.12
HG+miR-493-3p-mimics组		26.79±6.50^bc	9.63±0.56^bc		7.24±0.56^bc
HG+Smart Silencer-NC组		69.03±12.08	4.09±0.96		12.77±1.10
HG+Smart Silencer-CCAT2组		36.69±4.91^bd	11.39±1.93^bd		8.04±0.51^bd
F		7.050^＊	52.465^＊＊		78.865^＊＊