The effect and underlying mechanism of hippocampal specific knockdown of RNA methyltransferase Pcif1 on spatial learning and memory

doi:10.11958/20220477

Abstract

Abstract:

Objective To investigate the effect and underlying molecular mechanism of RNA methyltransferase Pcif1 on hippocampus-dependent spatial learning and memory. Methods A total of twenty-seven 8-week-old male C57BL/6J Nifdc mice were selected for animal experiments. Animals were randomly divided into 3 groups (9 animals per group): the Pcif1 knockdown group (1 μL adeno-associated virus packaged shRNA targeting Pcif1 was stereotactic injected into mouse hippocampi), the sham-operation group (1 μL contrast adeno-associated virus was stereotactic injected into mouse hippocampi) and the wild-type group (none treatment). After treatment for 3 weeks, Morris water maze (MWM) was used to test the spatial learning and memory of mice, and Western blot assay was performed to detect the expression of related proteins in hippocampal tissues. HT22 cell line, a mouse hippocampal neuronal cell line, was selected for in vitro experiment. Cells were divided into two groups: the HT22-shPcif1 group (cellular Pcfi1 was knocked down by lentivirus packaged shRNA targeting Pcif1) and the HT22-Ctrl group (cells infected by contrast lentivirus). The whole-transcriptome sequencing (RNA-seq), cell-count-kit-8 (CCK8) assay, cell scratching assay and flow cytometry were used to study the mechanism of Pcif1 knockdown on learning and memory of mice. Results Comparing to the wild type group and the sham-operation group, spatial learning and memory were promoted in the Pcif1 knockdown group, and anti-apoptosis protein Bcl-2 in hippocampi was upregulated. Comparing to HT22-Ctrl cells, differentially expressed genes of HT22-shPcif1 cells were enriched in pathways of cell apoptosis, migration and proliferation. The cell migration of HT22-shPcif1 cells was enhanced. The cell proliferation of HT22-shPcif1 cells was enhanced under 400 μmol/L H₂O₂, and the cell apoptosis under 400 μmol/L H₂O₂ was reduced. Conclusion The selective knockdown of hippocampal Pcif1 facilitates spatial learning and memory of mice, and its mechanisms may be related to the upregulation of anti-apoptosis proteins and the enhancement of cell migration and proliferation.

Key words: tRNA methyltransferases, hippocampus, spatial learning, spatial memory, apoptosis, cell proliferation, Pcif1

CLC Number:

R338.6

Figures/Tables 14

References 31

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组别	第1天游泳速度（cm/s）	第2天逃避时间（s）		第3天逃避时间（s）	第4天逃避时间（s）
野生组	20.48±1.71	54.95±6.06		51.71±6.04	46.36±9.36
假手术组	20.56±1.10	52.58±8.36		55.56±5.03	53.37±7.00
敲低组	20.28±1.75	49.63±9.90		46.67±16.68	43.02±14.32
F	1.440	0.749		1.263	1.764
组别	第5天逃避时间（s）		第6天逃避时间（s）	第7天目的象限时间（s）	第7天跨平台次数
野生组	44.16±6.26		39.89±7.19	24.67±5.93	1（0,2）
假手术组	49.28±5.91		38.77±8.18	23.44±3.46	1（0,1）
敲低组	34.04±17.01^b		28.71±9.69^a	30.43±4.06^ab	1（0,1.5）
F或H	3.573^＊		3.849^＊	5.243^＊	0.027

组别	第1天游泳速度（cm/s）	第2天逃避时间（s）		第3天逃避时间（s）	第4天逃避时间（s）
野生组	20.48±1.71	54.95±6.06		51.71±6.04	46.36±9.36
假手术组	20.56±1.10	52.58±8.36		55.56±5.03	53.37±7.00
敲低组	20.28±1.75	49.63±9.90		46.67±16.68	43.02±14.32
F	1.440	0.749		1.263	1.764
组别	第5天逃避时间（s）		第6天逃避时间（s）	第7天目的象限时间（s）	第7天跨平台次数
野生组	44.16±6.26		39.89±7.19	24.67±5.93	1（0,2）
假手术组	49.28±5.91		38.77±8.18	23.44±3.46	1（0,1）
敲低组	34.04±17.01^b		28.71±9.69^a	30.43±4.06^ab	1（0,1.5）
F或H	3.573^＊		3.849^＊	5.243^＊	0.027

染色体	基因名称	基因差异倍数	表达变化	P_adj
chr10	Gm48128	5.91	上调	5.19E-04
chr6	Gm44248	3.00	上调	4.72E-13
chrX	Foxr2	2.79	上调	4.63E-12
chr7	Nlrp4e	2.28	上调	4.59E-08
chr5	Rundc3b	2.24	上调	2.46E-09
chr18	Slc27a6	2.13	上调	3.95E-05
chrX	Rnf128	2.12	上调	1.52E-12
chr5	Steap4	2.11	上调	6.19E-11
chr15	Cdh10	2.11	上调	2.34E-05
chr7	Fgf21	1.86	上调	1.41E-06
chrX	Armcx2	-2.12	下调	6.47E-146
chr19	Ifit3	-2.33	下调	3.54E-14
chr5	Oas2	-2.57	下调	8.54E-14
chr12	Cmpk2	-2.84	下调	3.98E-10
chr5	Gbp6	-2.86	下调	2.52E-09
chr2	Cdh26	-3.36	下调	1.97E-13
chrY	Gm10256	-3.45	下调	8.25E-09
chr5	Oas1a	-3.46	下调	1.52E-17
chr12	Rsad2	-3.58	下调	4.80E-09
chrY	Gm3376	-3.93	下调	3.34E-20

染色体	基因名称	基因差异倍数	表达变化	P_adj
chr10	Gm48128	5.91	上调	5.19E-04
chr6	Gm44248	3.00	上调	4.72E-13
chrX	Foxr2	2.79	上调	4.63E-12
chr7	Nlrp4e	2.28	上调	4.59E-08
chr5	Rundc3b	2.24	上调	2.46E-09
chr18	Slc27a6	2.13	上调	3.95E-05
chrX	Rnf128	2.12	上调	1.52E-12
chr5	Steap4	2.11	上调	6.19E-11
chr15	Cdh10	2.11	上调	2.34E-05
chr7	Fgf21	1.86	上调	1.41E-06
chrX	Armcx2	-2.12	下调	6.47E-146
chr19	Ifit3	-2.33	下调	3.54E-14
chr5	Oas2	-2.57	下调	8.54E-14
chr12	Cmpk2	-2.84	下调	3.98E-10
chr5	Gbp6	-2.86	下调	2.52E-09
chr2	Cdh26	-3.36	下调	1.97E-13
chrY	Gm10256	-3.45	下调	8.25E-09
chr5	Oas1a	-3.46	下调	1.52E-17
chr12	Rsad2	-3.58	下调	4.80E-09
chrY	Gm3376	-3.93	下调	3.34E-20

组别	细胞迁移率	细胞凋亡率
HT22-Ctrl组	21.4±4.1	28.55±0.11
HT22-shPcif1组	47.2±5.5	24.80±0.13
t	8.893^＊＊	30.893^＊＊