The Absorption Mechanism Of Exogenous RNA

miRNAs are one of the most important members of the non-coding RNA family and play a wide and important regulatory role in many aspects of life activities in various species.From the definition of miRNA,it has been considered as an endogenously synthesized nucleic acid molecule that exerts regulation function on genes expression,miRNAs are encoded in the genome and form mature single-strand miRNAs through a series of processing and target on the mRNA 3'UTR sequence of the target genes,mediating their post-transcriptional regulation.As the miRNA research developing in recent years,we have gained a lot of enlightenments.The research team of Chenyu Zhang first detected the presence of plant miRNAs in serum of human and various tissues in animal,and proposed the concept of "Cross species regulation of miRNA" which is different from the traditional mechanism.This discovery has expanded the existing knowledge about miRNA regulatory functions The study of miRNAs from milk,rice and honeysuckle existing in human blood also confirmed that human can absorb dietary animal-derived and plant-derived miRNAs Based on these evidence,we want to explore those questions that have not been answered:How does human uptake miRNAs from dietary resources?Why some exogenous miRNAs are able to transfer to human circulation,but others can't?How can these exogenous miRNAs be transferred to human circulatory system.The answers to these questions may be hidden in some more basic discussions,such as the membrane selective mechanism by which cells pick up miRNAs,and the processing that the absorbed miRNAs undergo.In summary,it is the life course of exogenous miRNAs within host cell.In order to understand these issues more clearly,we designed in vitro experiments to simulate the conditions for in vivo absorption of exogenous RNA.The first thing to verify is whether exogenous miRNAs can be directly uptake by cell.We know that each cell is a delicate machine that can separate itself from the external environment and sustain its life by selectively importing and exporting essential substances according to physiological and pathological conditions.However,because of the barrier function of the cell membrane,the absorption of macromolecules such as exogenous nucleic acids and proteins by cells is greatly hindered.With the classical endocytic and non-endocidal pathways,some macromolecules can be transported into the cytoplasm.However,they need some natural or synthetic carriers in order to deliver efficiently,such as liposome-mediated transfection of RNA,plasmids and the nanoparticles which carry macromolecular targeting drugs.Based on these theoretical and research foundations,it is generally believed that exogenous miRNAs are thought to be difficult to absorb directly through the cell membrane barrier and need to be assisted by vectors.In order to verify this idea,we mixed HeLa and HEK 293T cells with various artificial fluorescence-labeled precursor and mature miRNAs(termed as exogenous RNA),observed the fluorescence signal of labeled miRNA at different time points,and found that the fluorescence signal of miRNA was observed on the edge of both cells within 0.5 hours.With the increasing of incubation time,the fluorescence signal was gradually localized in the plasma and showed a regular pattern of distribution,indicating that the exogenous RNA could be directly transported into the cell.Moreover,the location of these signals suggests that the RNA may be associated with some kind of subcellular organelles.So we labeled organelles that may be involved in miRNA processing when incubating exogenous RNAs,including endoplasmic reticulum,mitochondria,and lysosomes.We found the colocalization of RNA and mitochondria,demonstrating that mitochondria may participate in the absorption and utilization of exogenous miRNAs.At the same time,in order to explore the possible mechanism of RNA transmembrane transport,we used fluorescent tracker to label endosome and also used inhibitors involved in endocytic pathways.But RNAs' fluorescence signal still can be found clearly in cells,indicating that the translocation of exogenous RNA does not depend on endocytic pathways.And changing the temperature conditions of cell culture revealed that the absorption of exogenous miRNA is temperature sensitive process.At the same time,the results of control experiment of serum-free and exosome-remove suggest that it is the non-exosome component in serum that may mediate the absorption of exogenous RNA.For better observe the absorption of exogenous RNA,we used fluorescence confocal microscope to record the dynamic process of uptaking exogenous RNA by HeLa cell.The results showed that mitochondria are directly involved in transmembrane transporting and intracellular absorption exogenous RNA particles.Beyond these,the exact processing steps after exogenous miRNAs absorbed by cells and its meaning on life cycle of cell need to verify further.Explaining these scientific issues will provide new ideas for the application of miRNA in the Medicine.