| Objective: To determine the role of the key circRNA in the production of beta-AP and confirmed that Panax notoginseng saponins can affect the production of the beta-AP by regulating the expression of circRNA.It provides theoretical and experimental basis for the research and development of new drugs with the target of circRNA.Methods: 1.Screening and validation of key circRNAs: Using circRNA microarray to screen the difference expression of circRNAs in the hippocampal tissues of SAMP8 and SAMR1 in the same month.The key circRNAs that were related to the production of beta-AP were predicted by bioinformatics.RT-q PCR was used to verify the key circRNA screening in the expanded sample.2.The function verification of mmu-circ-000692 and the mechanism to control the generation of the beta-AP: The PCR products of mmu-circ-000692 were detected by RNA digestion and nucleotide sequencing.Build the model of Adeno-Associated Viral Vector of overexpression particles to infected mice hippocampal neurons HT-22 cells and mouse brain neuroma cell N2 a cell.The expression of A?1-42 in the blank plasmid group and the overexpression group was detected by enzyme-combined immunoassay.RT-q PCR was used to detect the effect of the beta-AP on the expression of mmu-circ-000692.Mi RNA vector regression(mir SVR)was used to analyze the target mi RNAs combined with mmu-circ-000692.m RNA of the target mi RNA was predicted via Mirwalk and Targetscan online.RT-q PCR and western blot were used to detect the influence of mmu-circ-000692 on IDE expression.RT-q PCR detected the effects ofmmu-circ-000692 on mi R-298-5p and mi R-346-3p expression.3.PNS regulation of key circRNA expression: 3 months-old SAMP8 AD model mice were randomly divided into model group,high-dose PNS group(200mg/Kg)and low-dose PNS group(100mg/Kg),All the mice were fed adaptively for one week,the PNS groups were treated with the designed drugs per day by intragastric administration for 8 consecutive weeks,and the same volume of normal saline was given to the model group.Morris water maze test was used to assess the change of learning and memory abilities of each group mice.RT-q PCR detect the expression of circRNA.Bioinformatics is used to predict the possible binding transcription factors of circRNA promoter region.Using western blot to detect the protein expression of transcription factor in mouse brain tissue.Results: 1.circRNA microarray showed that: there were 85 differentially expressed circRNAs in hippocampal tissues of SAMP8 and SAMR1.Among these circRNAs,45 upregulated and 40 downregulated.Then the mi RNA of circRNA with different expression was predicted by bioinformatics method.The results showed that of the 85 differentially expressed circrnas,a total of 352 mirnas may interact directly with circRNA.mi RNA was then predicted to bind to these genes using mi RNA target gene online software(mi Rwalk).There were 34 key mi RNAs identified with the above gene.According to these key mi RNAs to determine the key circRNA that is related to the production of beta-AP.According to the above method,we screened 30 key circRNAs that were involved in the production of beta-AP.Then we examined the expression of circRNAs in the hippocampus of SAMP8 mice.The results showed compared with SAMR1(normal mice of the same origin as SAMP8)of the same month.The expression of mmu-circ-000692 andmmu-circ-017963 in the hippocampus of SAMP8 mice decreased significantly and the expression of mmu-circ-013636,mmu-circ-013699 was significantly increased.Compared with 3 months old SAMP8 mice,the mmu-circ-000692,mmu-circ-017963,mmu-circ-013636 and mmu-circ-013699 were significantly reduced in the hippocampus of SAMP8 mice in the 6 months.This suggests that there is a large number of circRNAs in AD that are differentially expressed.2.According to the above results,We chose mmu-circ-000692 for further study.The results of RNA enzyme digestion experiment showed.The content of mmu-circ-000692 in the RNA enzyme digestion group was not significantly different from that of the undigested group and the expression of beta-actin in the RNA enzyme digestion group was significantly lower than that in the undigested group.This indicates that the circRNA in the above PCR products is resistance to RNA enzyme digestion.The results of RT-q PCR product of mmu-circ-000692 sequencing were showed the nucleotide sequence of the product is completely coincident with the mmu-circ-000692 sequence in circbase.confirmed that the product of primer amplification is mmu-circ-000692.In order to explore the role of mmu-circ-000692 in the production of beta-AP.Build the model of Adeno-Associated Viral Vector of overexpression particles to infected mice hippocampal neurons HT-22 cells and mouse brain neuroma cell N2 a cell.Elisa kit was used to measure the contents of intracellular beta-AP.The results showed that compared to the control group,Overexpression of mmu-circ-000692 can significantly reduce the content of beta-AP in N2 a and HT22 cells.This suggests that mmu-circ-000692 can significantly inhibit the generation of beta-AP.In order to determine the effect of the beta-AP on the expressionof mmu-circ-000692.We added 20?M A?25-35 to the HT22 cells for 24 h.The expression of mmu-circ-000692 was detected by RT-q PCR.The results showed that compared to the control group,The expression of mmu-circ-000692 can be significantly inhibited by beta-AP.Mi RNA vector regression(mir SVR)analysis and mmu-circ-000692 combined target mi RNAs.The mi RNAs with the highest scores were respectively mi R-6940-5p?mi R-3074-5p?mi R-298-5p?mi R-7001-5p and mi R-346-3p.The online site predicts the m RNA of these five target mi RNAs.The intersection is found that,In these 5 mi RNAs,there were IDE binding sites for mir-298-5p and mir-346-3p and the scores of prediction were highest.This suggests that the IDE may be the target m RNA of mir-298-5p and mir-346-3p.IDE gene and protein expression were detected in N2 a and HT22 cells were expressed in mmu-circ-000692.The results showed that compared to the control group,The expression of IDE genes and protein expression in overexpression group were significantly increased and there was no significant change in the expression of mir-298-5p and mir-346-3p in overexpression group N2 a and HT22 cells.This suggests that mmu-circ-000692 may play a role in adsorbing mir-298-5p and/or mir-346-3p through mi RNA "sponge".However,the binding effect of mmu-circ-000692 and mir-29-5p and mir-346-3p needs further study.3.To investigate whether PNS improves AD learning and memory ability.We used water maze to detect the learning and memory ability of SAMP8 mice after PNS intervention.The results showed that,compare with the model group,the escape latency of the PNS groups were decreased and the percentage of time spent in target quadrant were increased with significant difference and the times of the platform crossing were increased significantly.It is suggested that PNS cansignificantly improve AD learning and memory impairment.Previous research findings that,PNS can significantly inhibit the production of beta-AP in AD mice.In order to investigate whether PNS inhibited the formation of beta-AP by regulating the key circRNA.Using circRNA microarray to screen the difference expression of circRNAs in the hippocampal tissue between PNS intervention mice and non-intervention mice.circRNA microarray showed that: there were 9 differentially expressed circRNAs in hippocampal tissues of PNS intervention SAMP8 and non-intervention SAMP8.Among these circRNAs,3 upregulated and6 downregulated.The results of the effect of PNS on the expression of circRNA in the hippocampus of SAMP8 mice was showed that compared with the model group,the gene expression of mmu-circ-001456 ?mmu-circ-000692 ? mmu-circ-017963 mmu-circ-013636 ?mmu-circ-003540 ? mmu-circ-006173 ? mmu-circ-006229 ?mmu-circ-013699 ? mmu-circ-012180 in the brains of high-dose PNS group were significantly increased,the gene expression of mmu-circ-013636 in the brains of low-dose PNS group were significantly increased.In order to explore the mechanism of PNS regulation the key circRNA.We predicted the transcription factors that regulate the circRNA by bioinformatics.The results showed that the five transcription factors with higher scores were JUN,NFKB1,NFIC,SP1,and STAT1.Then we used western blot to verify the expression of these transcription factors in the expanded sample.The results of the effect of PNS on the expression of predicted transcription factors was showed that compared with the model group,the protein expression of SP1 in high-dose PNS group and low-dose PNS group were significantly increased while the STAT1 proteins expression were significantly decreased.The protein expressionof JUN,NFKB1 and NFIC was not significantly different from that of the model group.The results suggest that PNS may influence the expression of mmu-circ-000692 by regulating the activity of transcription factors.Conclusion: 1.mmu-circ-000692 influences gene regulation and expression by playing the role of mi R-298-5p and/or mi R-346-3p "sponge" to inhibit the formation of beta-AP.2.PNS could improve the learning and memory abilities of SAMP8 mice and PNS Regulation of the expression of circRNAs by regulating the expression of transcription factors SP1 and STAT1 in order to inhibit the formation of beta-AP. |
PDF Full Text Request