circ_HIPK3 regulates function and morphology of Aβ induced hippocampal neurons by targeting miR-381-3p/ZNF217 axis

doi:10.11958/20231013

Abstract

Abstract:

Objective To analyze the influence of cyclic RNA homologous domain interacting protein kinase 3 (circ_HIPK3) on function and morphology of myloid β-protein (Aβ) induced hippocampal neurons by targeting miR-381-3p/zinc finger protein 217 (ZNF217) axis. Methods Hippocampal neurons of neonatal rats were prepared and divided into the control group, the Aβ group, the si NC1 group, the si HIPK3 group, the si HIPK3+inhibitor NC group, the si HIPK3+miR-381-3p inhibitor group, the si HIPK3+miR-381-3p inhibitor+si NC2 group and the si HIPK3+miR-381-3p inhibitor+si ZNF217 group. Except the control group, all the other groups were modeled by 40 μmol/L Aβ_1~42. qRT-PCR was used to determine the circ of hippocampal neurons circ_HIPK3, miR-381-3p and ZNF217 mRNA levels. Cell morphology was observed by transmission electron microscope, and the survival rate of hippocampal neurons was measured by CCK-8 method. Hochesst 33342 method was used to measure apoptosis of hippocampal neurons. The intracellular Ca²⁺fluorescence intensity of hippocampal neurons was detected by flow cytometry. The expression levels of P-Tau, B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), Caspase-3 and ZNF217 proteins in hippocampal neurons were measured by Western blot assay. Double luciferase reporter genes were used to analyze the targeting relationship between miR-381-3p and circ_HIPK3, ZNF217. Results In the control group, the structure of hippocampal neurons was normal, the morphology of nucleus was normal, and there were no pathological changes in mitochondria and endoplasmic reticulum. In the Aβ group, hippocampal neurons showed degenerative changes, abnormal nuclear morphology, membrane invagination, a large number of mitochondria swelling and a large number of lipid droplets vacuoles in cytoplasm. Compared with the Aβ group, the hippocampal neuronal structure was partially restored in the si HIPK3 group. Compared with the si HIPK3 group, the hippocampal neuronal structure was severely damaged in the si HIPK3+miR-381-3p inhibitor group. Compared with the si HIPK3+miR-381-3p inhibitor group, the damage of hippocampal neurons in the si HIPK3+miR-381-3p inhibitor+si ZNF217 group was reduced. Compared with the control group, the circ_HIPK3, ZNF217 mRNA and ZNF217 protein levels, apoptosis rate, Ca²⁺ fluorescence intensity, P-Tau, Bax, Caspase-3 protein expression of hippocampal neurons were increased in the Aβ group, and the miR-381-3p level, survival rate and Bcl-2 protein expression decreased (P<0.05). Compared with the Aβ group, the circ_HIPK3, ZNF217 mRNA and ZNF217 protein levels, apoptosis rate, Ca²⁺ fluorescence intensity, P-Tau, Bax and Caspase-3 protein expression of hippocampal neurons were decreased in the si HIPK3 group, and miR-381-3p level, survival rate and Bcl-2 protein expression increased (P<0.05). Compared with the si HIPK3 group, the circ_HIPK3, ZNF217 mRNA and ZNF217 protein levels, apoptosis rate, Ca²⁺ fluorescence intensity, P-Tau, Bax and Caspase-3 protein expression of hippocampal neurons in the si HIPK3+miR-381-3p inhibitor group were increased, and the miR-381-3p level, survival rate and Bcl-2 protein expression decreased (P<0.05). Compared with the si HIPK3+miR-381-3p inhibitor group, the ZNF217 mRNA and ZNF217 protein levels, apoptosis rate, Ca²⁺ fluorescence intensity, P-Tau, Bax and Caspase-3 protein expression of hippocampal neurons in the si HIPK3+miR-381-3p inhibitor+si ZNF217 group were decreased, and the survival rate and Bcl-2 protein expression increased (P<0.05). miR-381-3p targeted and combined with HIPK3 and ZNF217. Conclusion circ_HIPK3 silencing may ameliorate Aβ-induced damage of hippocampal neuronal structure and function by regulating miR-381-3p/ZNF217 axis.

Key words: hippocampus, neurons, tristetraprolin, circ_HIPK3, miR-381-3p, ZNF217

CLC Number:

R749.16

Figures/Tables 13

References 18

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基因名称	引物序列（5′→3′）	产物大小/bp
circ_HIPK3	上游：ATGGCCTCACAAGTCTTGGTC	189
circ_HIPK3	下游：GCACTACCTTTCGTGGAGGAT	189
ZNF217	上游：TCCAAATGTGCTGACTGTTC	201
ZNF217	下游：CTCTTTTGTGCCATGCTGTT	201
GAPDH	上游：AATCCCATCACCATCTTCCAG	128
GAPDH	下游：CCTTCTCCATGGTGGTGAAGAC	128
miR-381-3p	上游：TAATCTGACTATACAAGGGCAAGCT	87
miR-381-3p	下游：TATGGTTGTTCTGCTCTCTGTCTC	87
U6	上游：CTCGCTTCGGCAGCACA	96
U6	下游：AACGCTTCACGAATTTGCGT	96

基因名称	引物序列（5′→3′）	产物大小/bp
circ_HIPK3	上游：ATGGCCTCACAAGTCTTGGTC	189
circ_HIPK3	下游：GCACTACCTTTCGTGGAGGAT	189
ZNF217	上游：TCCAAATGTGCTGACTGTTC	201
ZNF217	下游：CTCTTTTGTGCCATGCTGTT	201
GAPDH	上游：AATCCCATCACCATCTTCCAG	128
GAPDH	下游：CCTTCTCCATGGTGGTGAAGAC	128
miR-381-3p	上游：TAATCTGACTATACAAGGGCAAGCT	87
miR-381-3p	下游：TATGGTTGTTCTGCTCTCTGTCTC	87
U6	上游：CTCGCTTCGGCAGCACA	96
U6	下游：AACGCTTCACGAATTTGCGT	96

组别	circ_HIPK3	miR-381-3p	ZNF217
组别	circ_HIPK3	miR-381-3p	mRNA	蛋白
对照组	1.01±0.14	1.00±0.16	1.02±0.12	0.32±0.04
Aβ组	2.97±0.42^a	0.42±0.07^a	2.54±0.31^a	0.78±0.09^a
si NC1组	2.99±0.42	0.43±0.07	2.52±0.31	0.76±0.09
si HIPK3组	1.62±0.23^b	0.62±0.10^b	2.02±0.25^b	0.41±0.05^b
si HIPK3+inhibitor NC组	1.63±0.23	0.63±0.10	2.01±0.25	0.42±0.05
si HIPK3+miR-381-3p inhibitor组	2.05±0.29^c	0.51±0.08^c	2.16±0.27^c	0.45±0.05^c
si HIPK3+miR-381-3p inhibitor+si NC2组	2.04±0.29	0.51±0.08	2.17±0.27	0.46±0.05
si HIPK3+miR-381-3p inhibitor+si ZNF217组	2.03±0.28	0.59±0.09	1.95±0.24^d	0.40±0.05^d
F	30.018^＊	21.379^＊	20.006^＊	46.846^＊

组别	circ_HIPK3	miR-381-3p	ZNF217
组别	circ_HIPK3	miR-381-3p	mRNA	蛋白
对照组	1.01±0.14	1.00±0.16	1.02±0.12	0.32±0.04
Aβ组	2.97±0.42^a	0.42±0.07^a	2.54±0.31^a	0.78±0.09^a
si NC1组	2.99±0.42	0.43±0.07	2.52±0.31	0.76±0.09
si HIPK3组	1.62±0.23^b	0.62±0.10^b	2.02±0.25^b	0.41±0.05^b
si HIPK3+inhibitor NC组	1.63±0.23	0.63±0.10	2.01±0.25	0.42±0.05
si HIPK3+miR-381-3p inhibitor组	2.05±0.29^c	0.51±0.08^c	2.16±0.27^c	0.45±0.05^c
si HIPK3+miR-381-3p inhibitor+si NC2组	2.04±0.29	0.51±0.08	2.17±0.27	0.46±0.05
si HIPK3+miR-381-3p inhibitor+si ZNF217组	2.03±0.28	0.59±0.09	1.95±0.24^d	0.40±0.05^d
F	30.018^＊	21.379^＊	20.006^＊	46.846^＊

组别	细胞存活率/%	细胞凋亡率/%	Ca²⁺荧光强度
对照组	100.00±14.28	3.24±0.46	30.22±4.31
Aβ组	47.26±6.75^a	25.66±3.66^a	76.17±10.88^a
si NC2组	46.57±6.66	25.17±3.59	75.89±10.84
si HIPK3组	79.81±11.40^b	13.22±1.88^b	42.66±6.09^b
si HIPK3+inhibitor NC组	78.66±11.23	13.50±1.92	42.57±6.09
si HIPK3+miR-381-3p inhibitor组	62.55±8.93^c	19.86±2.83^c	61.98±8.85^c
si HIPK3+miR-381-3p inhibitor+si NC2组	62.79±8.97	19.79±2.83	62.05±8.86
si HIPK3+miR-381-3p inhibitor+si ZNF217组	71.28±10.18^d	14.55±2.08^d	50.42±7.21^d
F	18.801^＊	54.344^＊	24.552^＊