Induction Of Autophagy And The Screening And Validation Of Autophagy-Related MicroRNAs

Background and ObjectiveAutophagy is a process of self-degradation and recycling of cellular constituents.Autophagy exists in eukaryotic cells, which is a common phenomenon. Autophagy hasessential roles in survival, development, homeostasis and contributes to a variety of diseases.MicroRNAs (miRNAs) are endogenous, non-protein-encoding small single-stranded RNAs.MicroRNAs can target mRNAs for degradation or inhibit their translation through sequencecomplementary. Lots of reports have shown that microRNAs are involved in various biologicprocesses, therefore, microRNAs play important roles in the development and progression ofmany diseases, including cancer, neurodegenerative diseases, infections, aging, heart disease,etc. Recent studies have observed that microRNAs can regulate Atg proteins expressions inthe formation of autophagosome. In this study, potential autophagy-related microRNAs werescreened through a series of bioinformatics methods, and then they were further verified bybiological experiments. In addition, the effects of autophagy on the neuronal cells undersimulated microgravity have not been reported. To explore whether autophagy were involvedin the effects of microgravity on the neuronal cells, the expression of autophagy-related geneLC3 in SH-SY5Y cells under simulated microgravity were investigated.MethodsMicroRNAs involved in the regulation of autophagic process may be predicted bybioinformatics methods. Rapamycin-induced MCF7 cells, and SKOV-3 cells for 12h cancauses autophagy. MicroRNAs mimics or microRNAs inhibitors were transiently transfectedinto cells, and then LC3 expression levels on MCF7 cells and SKOV-3 cells were observed.Microgravity was simulated by clinostat, SH-SY5Y cells were cultured for 12h and 24h.Static cultured cells can be as the control groups under the same conditions. GFP-LC3 puncta were examined by a fluorescence microscope. The expression levels of LC3-I, LC3-II proteinwere analysed by Western Blotting method.Results(1) Rapamycin treated cells for 12h to induce autophagy, GFP-LC3 puncta increased inbreast cancer MCF7 cells (P < 0.05) and ovarian cancer SKOV-3 cells (P < 0.001) comparedwith the control groups. In the groups with rapamycin plus NH4CL, GFP-LC3 puncta weresignificantly higher than that in the other groups on MCF7 cells. Compared with the controlgroups, the expression levels of LC3-II protein were clearly elevated using Western Blottingin MCF7 cells and SKOV-3 cells, and their expression were more significant in the groupswith rapamycin plus NH4CL.(2) MiR-142-5p was an autophagy-related microRNA through bioinformatics methods,but it could not influence LC3 expression in rapamycin-induced MCF7 cells via biologicalexperiments.(3) Four autophagy-associated microRNAs on ovarian cancer were screened, and the onlyone microRNA had an effect in rapamycin-induced SKOV-3 cells. MicroRNAs mimics ormicroRNAs inhibitors were transiently transfected into SKOV-3 cells, and the results showedthat microRNA mimic reduced the expression levels of LC3 protein. On the contrary,microRNA inhibitor accumulated LC3 protein expression. MicrroRNA mimic increased thenumber of GFP-LC3 puncta under fluorescence microscopy, but microRNA inhibitordecreased GFP-LC3 levels. The others microRNAs mimics had no effect on LC3 expression.(4) After clinorotation, the number of GFP-LC3 puncta significantly increased comparedwith the control group (P < 0.05) by fluorescence microscopy. Moreover, LC3 proteinexpression also showed a significant accumulation using Western Blotting.ConclusionsRapamycin could induce autophagy in MCF7 cells and SKOV-3 cells. The miR-142-5pwould not affect autophagy on MCF7 cells. In screening four ovarian cancerautophagy-associated microRNAs, the only one cause autophagic effect in SKOV-3 cells.Screening and identification of autophagy-related microRNAs molecules would promote understanding of autophagic molecular mechanism. Simulated microgravity with clinostatincrease autophagosomes in SH-SY5Y cells. The effects of autophagy on neuronal cellswould provide a new clue or idea for research of aerospace medicine.