Studies On The Roles Of MiR-199a-5p In Radiation-induced Autophagy In Breast Cancer

Autophagy is a self-digestive process that leads to sequestration anddegradation of intracellular material within lysosome. Autophagy plays roles inmaintaining cellular homeostasis by regulating organelle and protein turnover, whilethe underlying mechanism in carcinogenesis is still elusive. Autophagy can behaveas a tumor suppressor or oncogene. The similar paradox is exhibited during tumortherapy, in which autophagy could play pro-survival role or autophagy lead toprogrammed cell death during breast cancer treatment, mainly dependent on thetreatment strategies and cancer context. MicroRNAs (miRNAs), the short (~22nucleotides) non-coding RNAs, have emerged recently as novel endogenous generegulators. They bind by incomplimentary base pairing to the3’-untranslated region(3’-UTR) of their target mRNA to post-transcriptionally suppress gene expression.miRNAs have been shown to play important roles in virtually many cellular eventslike cell proliferation and apoptosis, and hence miRNAs are being categorized astumor suppressors and oncogenes. Recently, a few reports illustrated that miRNAscould activate rather than suppress gene expression. miR-199a-5p has been reportedto deregulated in several aggressive tumor types (hepatocellular, breast and testicularcancers), recent studies indicated that miR-199a-5p is a putative tumor suppressor.Despite all these studies, functions and the target genes of miR-199a-5p are largelyunknown especially in autophagy process and autophagy induced by ionizingradiation (IR) treatment.Objective: To explore the impact of miR-199a-5p on the process of autophagyand identify the related target genes in human breast cancer cells, the effect ofmiR-199a-5p after IR treatment in breast cancer will be also demonstrated.Methods:(1) qRT-PCR was used to detection endogenous miRNA expression;(2) Western blot was used to analyze protein expression;(3) GFP-LC3morphological assay and MAPLC3western blot assay were used to monitor autophagy process;(4) The target gene of miR-199a-5p was predicted bybioinformatics methods;(5)3’UTR fragments of target gens were synthesized byPCR and the luciferase expression vector containing3’UTR fragments wereconstructed;(6) PCR site-directed mutagenesis was used to acquire mutants of3’UTR fragments and inserted into luciferase vectors;(7) Dual-Luciferase ReporterAssay was used to demonstrate the binding of miR-199a-5p and targets;(8) Cellsviability was evaluated by Cell counting Kit-8;(9) Cell cycles were analyzed byFACS assay;(10) X-ray generator was utilized to deliver radiation;(11) Studentt-test and χ2test were used for statistical purpose;(12) ImageJ software was used tocalculate the band value.Results:1Impact of IR on miR-199a-5p expression in breast cancer cell linesmiR-199a-5p level increased to3folds after IR treatment in MCF7cells.However, miR-199a-5p expression decreased obviously after IR treatment inMDA-MB-231cells. When transfected with miR-199a-5p mimic, the expressionlevel of miR-199a-5p increased to20folds in MCF7cells and increased to65foldsin MDA-MB-231as compared with Negative Control (NC) group. IR furtherimproves the expression of miR-199a-5p besides transfection with mimic, theexpression of miR-199a-5p increased to50folds in MCF7cells and to90folds inMDA-MB-231cells.2The inhibitory effects of miR-199a-5p on radiation-induced autophagy inMCF7cellsAfter treatment with IR(8Gy), the ratio of MAPLC3II/LC3I increased to1.28and1.29at16h and32h, respectively. GFP-LC3vector was transfected into MCF7cells, after IR treatment autophagy positive cells increased to35%(vs sham control7%,**p<0.01). There was no change in the autophagy level after transfection withmimic or NC（p>0.05）. In NC+IR (8Gy) group, the autophagy positive cellsincreased more than35%(**p<0.01). However, the autophagy level in mimic+IR(8Gy) group decressed to15%, lower than IR(8Gy) group and NC+IR(8Gy) group,higher than NC group and sham-IR group(**p<0.01).Based on western blot, LC3II expression in IR(8Gy) group was higher than sham control group obviously, LC3II expression in mimic+IR group was lower thanIR(8Gy) and NC+IR(8Gy) group obviously. Transfection with NC or mimic didn’timpact LC3II protein expression. Chloroquine(CQ), a inhibitor of autophagy processwhich leads to accumulation of autophagosome, could be used to analysis autophagyflux. After transfection with mimic and NC, CQ and IR treatment were sequentiallyintroduced into MCF7cells. The LC3II/LC3I ratio in NC+IR group, mimic+IRgroup, NC+IR+CQ group, mimic+IR+CQ group increased to2.13folds,1.28folds,2.71folds and1.81folds as compared with NC group, respectively. These resultsindicated that miR-199a-5p inhibited IR-induced autophagy in MCF7cells.3The activation roles of miR-199a-5p in IR-induced autophagy inMDA-MB-231cellsBy Western blot analysis，the LC3II/LC3I ratio increased to2.16folds and1.62folds at8h and16h after radiation, respectively. After transfection withmiR-199a-5p mimic， compared with NC group, the LC3II/LC3I ratio increased to1.44folds,1.10folds,1.59folds in mimic group, NC+IR group and mimic+IR group,respectively. To monitor the autophagy flux, cells were treated with CQ, LC3II/LC3Iincreased to1.47folds in NC+CQ group compared with NC group, andLC3II/LC3I increased to1.97folds in mimic+CQ group compared with NC group,higher than that in NC+CQ group and mimic group. These results indicated thatmiR-199a-5p could increase autophagy directly and enhance IR-induced autophagyin MDA-MB-231cells.4The putative target genes of miR-199a-5p and luciferase vectorconstructionBioinformatics tools (miRBase、PicTar and Targetscan) were used to predict theputative target genes of miR-199a-5p,3’UTR sequences of two autophagy relatedgenes, DRAM1and BECN1, take on complimentary binding sites with miR-199a-5pseed. PCR was used to acquire partial3’UTR fragment of DRAM1and BECN1genes and PGL3-DRAM1and pMIR-BECN1luciferase vector were constructed.PCR site-directed mutagenesis was used to acquire mutants luciferase vectors. Allthe expression vectors were confirmed by sequencing.5The inhibitory effects of exogenous miR-199a-5p on DRAM1and Beclin1 expression in MCF7cell line.miR-199a-5p mimic(or NC) and luciferase vector were co-transfected intoMCF7cells, and pRL-SV40was used as internal control. PGL3-DRAM1andpMIR-BECN1luciferase activity decreased to70%and75%(*p<0.05), respectively,but not in the mutant3’UTR vector. The DRAM1and BECN1expression increasedafter IR treatment compared with sham-IR group in MCF7cells, but suppressed inmimic group. The expression of DRAM1and BECN1were lower in mimic+IRgroup compared with NC+IR group.6The enhancement effects of miR-199a-5p on DRAM1and Beclin1expression in MDA-MB-231cell line.MDA-MB-231cells were transfected with miR-199a-5p mimic, western blotresults showed that the expression of DRAM1and BECN1increased obviously inmimic group and NC+IR group. Dual-Luciferase Reporter Assay results showed thatmiR-199a-5p mimic increased PGL3-DRAM1and pMIR-BECN1luciferase activityto1.2folds and2.7folds, respectively (**p<0.01), but not in the mutant3’UTRvector.7Effects of miR-199a-5p on cells proliferationAfter transfection with miR-199a-5p mimic or NC into MCF7cells andMDA-MB-231cells, cells viability were detected by CCK8kit72h after differentdoses of IR (0,2Gy,4Gy,6Gy). The cells viability decreased to90%and75%inmimic+4Gy guoup and mimic+6Gy group in MDA-MB-231cells,respectively(**p<0.01). The cells viability decreased to82%in NC+6Gy group(**p<0.01). There was no change in MCF7cells after different dose IR treatment inNC group and mimic group.8The regulatory roles of miR-199a-5p in IR-induced cell cycle progressionFACS assay was used in this study. In MCF7cells, the G2/M phase cells are2.72%,2.05%,13.35%and3.2%in NC group, mimic group, IR group andmimic+IR group, respectively, illustrating that IR induced accumulation of cells atG2/M phase in MCF7cells. In MDA-MB-231cells, the G2/M phase cells are5.23%,18.56%,23.61%, and5.34%in NC group, mimic group, IR group and mimic+IRgroup, respectively. Both miR-199a-5p and IR induced accumulation of cells at G2/M phase in MDA-MB-231cell.Conclusion1miR-199a-5p regulates autophagy process in MCF7and MDA-MB-231breast cancer cell lines, but appeared different outcomes. miR-199a-5p suppressesIR-induced autophagy in MCF7cells, but increases autophagy directly and enhancesIR-induced autophagy in MDA-MB-231cells.2Two autophagy related genes DRAM1and BECN1are the target genes ofmiR-199a-5p. miR-199a-5p suppresses the expression of two targets in MCF7cellsthrough binding to3’UTR, but increases the expression of two targets inMDA-MB-231cells through binding to3’UTR.3miR-199a-5p regulates cell cycle progression, but appears different outcomesin MCF7and MDA-MB-231cells. This maybe explain the reason why miR-199a-5phave reverse effects in the regulation of two target genes.4miR-199a-5p increases the effect of IR to supress cells viability inMDA-MB-231cells.In summary, this research renewed the miRNA-autopahgy net work, discovereda new autophagy-regulated miRNA which has different outcomes on the regulationof target genes depending on different cell context, providing new sight for miRNAfunction and mechanism. However, more work should be done to further explore themechanisom of miRNAs regulate target genes, especially upregulate target genes.