Nucleic-acid Nanoflowers’ Optimization In Preparation Technology And Application In Analysis Of Molecular Mechanism Of Inflammation

Objective: In this study,Nucleic acid nanoflowers(NFs)and RNAa technology were used to investigate the mechanism of miR-155 in the process of inflammation and the effect of miR-155 on the occurrence of damage after co-expression of target genes at the promoter level,so as to realize early warning of major diseases and provide new ideas for prevention of inflammation,treatment of related diseases and prevention of their deterioration.Methods: 1.By optimizing the conditions of the ligation reaction and the rolling circle transcription reaction process,characterizing the morphology and size of NFs,detecting the toxic effects of NFs on cells,the efficiency of entry into cells,and the effect of NFs,construct NFs with RNAa effect.2.Finding the sequence that can activate miR-155 according to the principles of saRNA design,CCK-8 method was used to detect cytotoxicity,RT-qPCR method was used to detect miR-155 expression,and saRNAs that efficiently activated miR-155 were screened out.Cell morphological changes,cell migration ability,and expression of inflammation-related effectors confirmed the mechanism of miR-155 in the process of inflammation.3.Based on the predicted miR-155 target genes and their enrichment pathways,exploring the effects of the interaction between miR-155 and its target genes on the cells after co-expression.Results: 1.The reaction system of looping and amplification of NFs was optimized.T4 DNA ligase with high ligation efficiency,template to primer concentration ratio was 1: 1,hybridization time was optimized to 2 h and ligation time.Optimized to 3 h,the concentration of the raw rNTP was 2 mM,and the results of gel electrophoresis showed that there were no bands of non-target fragments in the final optimized product.SEM results showed that the surface of NFs was petal-like,the size was about 200 nm,and relatively uniform in size;the results of CCK-8 method showed that NFs had no inhibitory effect on cell proliferation,and the flow cytometry results showed that the infiltration efficiency was about 70 %.The results from related literature’s method and nanoflowers acting on cell-activated gene expression showed that there was no significant difference in expression between the two methods.It confirmed that NFs were successfully constructed and had RNAa effects,which can be used for subsequent experiments in this subject.2.We found 7 saRNAs that can effectively activate the expression of miR-155;CCK-8 methods confirmed that changing the template sequence of NFs has no inhibitory effect on cell proliferation;RT-qPCR methods detected 7 saRNAs to activate miR-155 gene expression,and in this case,we screened out the miRNA-155-5,which is a saRNA sequence that can efficiently activate miR-155.Cell morphology evaluation and scratch experiments confirmed that miR-155 overexpression can promote cell proliferation and enhance cell migration ability.Examining the expression of inflammation-related effector molecules,it was found that miR-155 over-expression caused inflammatory reactions by increasing the expression of pro-inflammatory factor(IFN-γ)and inhibiting the expression of anti-inflammatory factor(SHIP1).3.MiRNA target prediction results showed that miR-155 may promote PI3K-AKT signaling pathway and cause inflammation by targeting SHIP1,and We found 8 saRNAs that can effectively activate SHIP1 expression.CCK-8 methods were used to detect the inhibition of cell proliferation after activation of SHIP1 gene by NFs;RT-qPCR method was used to detect the expression of SHIP1 gene by 8 saRNAs,and a saRNA sequence that efficiently activated SHIP1 expression was selected: SHIP1-4.By comparing cell morphological changes and RT-qPCR method to detect the expression of miR-155 and SHIP1 after activating SHIP1 and miR-155,respectively,the results showed that miR-155 and SHIP1 expression did not change and the cells maintained a stable state.Conclusions: 1.Optimizing the reaction system of NFs,successfully construct NFs with RNAa function,and provide a feasible solution for the next step in screening saRNA;2.The saRNA sequences that can efficiently activate the expression of miR-155 had been screened out,and initially proved that increased expression of miR-155 will trigger inflammation;3.The saRNA sequence that can effectively activate SHIP1 expression was screened out.After SHIP1 overexpression,it inhibits cell proliferation and promotes apoptosis by preventing the PI3K-AKT signaling pathway.It can preliminary descript that the increase in SHIP1 expression has nothing to do with the occurrence of inflammation,and the expression of miR-155 and its target gene SHIP1 did not change after simultaneous activation,and the interaction maintained the homeostasis of the cell.