MicroRNA-17-5p's Regulation Of Circadian Rhythm And Its Mechanism

Background As an endogenous time-keeping system rooted in organisms,circadian rhythm helps the living things to adjust their physiology and metabolism to the alternating day and night with a period of 24h.It's an adaptation to the changing world and a protection for themselves.CLOCK and BMAL1 are important positive regulatory factors in core molecular clock feedback loops which promote the transcription of downstream gene and drive the circadian clock to run at an appropriate speed.The molecular mechanisms underlying circadian clock are multiple interlocking transcription-translation feedback loops.And reportedly several microRNAs(miRNAs)are involed in the regulatory loops.MiRNAs are single-strand small RNAs of about 22 nucleotides which can bind to target mRNAs to regulate lots of physiological processes.Although more and more microRNAs have been characterized in recent years,the researchs on detailed function of single miRNA are insufficient,especially those related to circadian rhythm.So we try to find the miRNAs associated to core clock genes and further search for the regulatory mechanisms in molecular levels and the influence on the rhythm of animals'activities.Methods We first utilized the bioinformatic method to predict microRNAs targeting clock.Dual luciferase reporter assay system were used for the validation of microRNAs which really bind to the 3'-untranslational region of clock mRNA.Overexpression and inhibition of mir-17-5p in cell line helped us to know its influence on clock expression.We also tested the expression rhythm of mir-17-5p in synchronized cells and the suprachiasmatic nuclei(SCN)of mice by using real-time quantitative PCR and in situ hybridization.It is possible that mir-17-5p was a clock-controlled gene,so overexpression plasmids were constructed and lenti-viruses for knocking down clock were packed.Chromatin immunoprecipitation assay were performed for the confirmation of CLOCK's binding to the promoter region.In addition,we montorized the wheel-running rhythm of mice before and after the lateral ventricular injection of agomir and antagomir of mir-17-5p.Extracellular neuron discharges in vivo were recorded and immunohistochemical assays were used to test the expression of PER1 and PK2 in SCN.The effects of light on the expression of mir-17-5p were also exhibited in cell line.Results Mir-1 7-5p bound to the 3' UTR of clock mRNA at two sites.The overexpression of mir-17-5p can decrease the mount of CLOCK,and the inhibition of mir-17-5p led to an increase of the expression of CLOCK.Mir-17-5p inhibited the translation of clock but not the transcriptional process.In synchronized cells,mir-17-5p expressed rhythmically with a phase different from CLOCK.Mir-17-5p exhibited different levels with circadian time in the SCN of mice,either in constant dark or light dark cycles.It reached a peak value near CT12 and decreased to bottom value in the late night and its phase was opposite with cytoplasmic CLOCK.In the cortex of mice,mir-17-5p was expressed not in a rhythmic pattern.The transient coexpression of CLOCK and BMAL1 could not promote the expression of mir-17-5p,however,the mount of mir-17-5p was decreased in cells which was stablely transfection with interfering sequences of clock.CLOCK could bind to the transcriptional regulatory region of mir-17-5p.Injection of antagomir-17-5p decreased the amount of mature mir-17-5p and shortened the free-running period of mice.The average delt period was 15.72±1.722 min,with significance of difference compared to the negative control.The decrease of mir-17-5p in SCN contributed to increased extracellular discharge of neurons.Agomir-17-5p cut down the period by 16.27±3.01 min.Agomir and antagomir of mir-17-5p both changed the period of locomotor acitivity and the expression of phase-related gene such as PER1 and PK2.Otherwise,mir-17-5p responsed to forskolin,an agonist of cAMP cyclase which is activatd by light signal pathway.In addition to mir-17-5p,mir-384-5p can regulate the expression of clock,but its function was opposite to mir-17-5p.Mir-142-3p was an important miRNA which regulated BMA11 and its decrease in SCN led to the phase delay of mice locomotor acitivity.Conclusions The results of molecular,cellar and in vivo experiments revealed that mir-17-5p,a miRNA expressed rhythmically in the SCN of mice,modulates the free-running period of mice.CLOCK was an important target of mir-17-5p in molecular levels.Mir-17-5p participates in the regulation of circadian clock by repressing the translation of CLOCK and ultimately its transcription was affected by CLOCK proteins.The study provides a new factor for stabilizing circadian clock operation.The finding adds new information to the feedback circuits of classic molecular clock.Mir-17-5p may act as an output molecule of circadian clock.