Expression Characteristics And Preliminary Function Study Of Small Non-coding RNA In Extracellular Vesicles From Non-small Cell Lung Cancer

Extracellular vesicles(EVs)are membrane-surrounded structures released by cells,and play important roles in intercellular communication between cancer cells and other cells in tumor micro-environment through the delivery of their cargoes,which include proteins,lipids,and RNAs.Intriguingly,multiple studies have reported that EVs are closely associated with the genesis,development and metastasis of cancer.Small non-coding RNAs(sncRNA)are small RNAs shorter than 200nt and can not be translated into proteins.SncRNAs play important roles in various biological process,such as gene silence,transcription regulation,chromosome replication,RNA processing,transporting and degrading of protein,and so on.SncRNAs also regulate the genesis and development process of cancer.Y RNA fragments are small non-coding RNAs generated by cleavage from full-length Y RNA,and the abnormal expression of Y RNA fragments in EVs also have an intimate connection with various tumors.Firstly,we have characterized and analyzed the expression of non-coding small RNAs in plasma EVs from non-small cell lung cancer(NSCLC)patients.And we found that Y RNA fragments are significantly up-regulated in NSCLC patients-derived EVs compared with healthy control group,tRNA fragments are evidently up-regulated in lung squamous carcinomas patients-derived EVs compared with healthy control and lung adenocarcinoma groups.While rRNA,piRNA,and snRNA fragments are obviously up-regulated in NSCLC patients-derived EVs compared with healthy control.The whole genome distribution and sequences annotation results suggested that the up-regulated Y RNA fragments are hY4F and hY4rF.And it was also found that hY4rF may not be a pseudogene of hY4 RNA.Addtionally,we have also found that hY4F and hY4rF are both significantly downregulated in lung cancer cells,and can inhibit the proliferation and migration of lung cancer A549 cells.We also found that a potential mechanism that lung cancer cells can release the hY4 RNA fragments-containing EVs,which inhibit the proliferation of normal lung cells and in turn promote the growth of lung cancer cells.The hY4 RNA fragments are down-regulated in NSCLC cells,while up-regulated in NSCLC EVs.The inversed trends indicated that the hY4 RNA fragments may be sorted into EVs by selective manners.It has been reported that several RNA binding proteins function as regulatory factors in the sorting of RNAs into EVs.So,combining RNA pulldown assay with identification by MS and Western Blot assay,we found the potential RNA binding protein of hY4F and hY4rF(such as YBX l and LRPPRC).In the future research,we will decrease the expression of hY4 RNA fragments-binding protein,such as YBXl,in lung cancer cells,and study its influence on the sorting of hY4 RNA fragments into EVs.And we will use animal experiments to study the function of hY4 RNA fragments and their binding protein in the genesis and metastasis process of lung cancer.In conclusion,we have found many different small RNAs,including Y RNA fragments,tRNA,piRNA,rRNA,and snRNA fragments,are abnormal exoressed in NSCLC EVs.And we ffound hY4F and hY4rF are tumor-suppress factors,and are up-regulated in NSCLC EVs,while down-regulated in NSCLC cells.And the releasing of hY4rF-containing NSCLC EVs may inhibit the proliferation of normal lung cells and in turn promote the growth of lung cancer cells.Moreover,we have identified several binding proteins of hY4 RNA fragments,which may contribute to the study of selective sorting mechanism of hY4 RNA fragments into NSCLC EVs.