Circulating levels and the bioactivity of miR-30b increase during pubertal progression in boys

Abstract

Background: Puberty marks the transition from childhood to adulthood and is initiated by activation of a pulsatile GnRH secretion from the hypothalamus. MKRN3 functions as a pre-pubertal break on the GnRH pulse generator and hypothalamic expression and circulating levels of MKRN3 decrease peri-pubertally. In rodents, microRNA miR-30b seems to directly target hypothalamic MKRN3 expression - and in boys, circulating levels of miR-30b-5p increase when puberty is pharmacologically induced. Similarly, miR-200b-3p and miR-155-5p have been suggested to inhibit expression of other proteins potentially involved in the regulation of GnRH secretion. Here we measure circulating levels of these three miRNAs as boys progress through puberty.

Materials and methods: Forty-six boys from the longitudinal part of the Copenhagen Puberty Study were included. All boys underwent successive clinical examinations including estimation of testis size by palpation. miR-30b-5p, miR-200b-3p, and miR-155-5p were measured in serum by RT-qPCR using a kit sensitive to the phosphorylation status of the miRNAs. Thirty-nine boys had miRNA levels measured in three consecutive samples (pre-, peri-, and post-pubertally) and seven boys had miR-30b-5p levels measured in ten consecutive samples during the pubertal transition.

Results: When circulating levels of miR-30b-5p in pre- and peri-pubertal samples were compared with post-pubertal levels, we observed a significant increase of 2.3 and 2.2-fold (p-value<6.0×10^-4), respectively, and a larger fraction of miR-30b-5p appeared to be phosphorylated post-pubertally indicating an increase in its bioactivity. We also observed a negative correlation between circulating levels of miR-30b-5p and MKRN3. The inter-individual variation in circulating miR-30b levels was substantial and we could not define a clinical threshold for miR-30b-5p suggestive of imminent puberty. Also, miR-155-5p showed significantly increasing levels from the peri- to the post-pubertal stage (p=3.0×10^-3), whereas miR-200b-3p did not consistently increase.

Conclusion: Both circulating levels of miR-30b-5p and its bioactivity increase during the pubertal transition in boys supporting its role in the activation of the HPG axis at the onset of physiologically normal puberty.

Keywords: HPG-axis; MKRN3; biomarker; miR-30b; miRNA; puberty.

Figure 1

Descriptive figure of the timing of blood sampling according to the pubertal onset of the included cohorts. (A) Blood sample timing according to the pubertal onset of the main cohort. (B) Blood sample timing according to the pubertal onset of the extended cohort. The grey bar represents pubertal onset +/- 0.5 years.

Figure 2

Circulating miR-30b levels during the pubertal transition. Circulating levels of miR-30b were measured in boys from the main cohort (n=39) and compared in groups representing pre-, peri, and post-pubertal states. (A) A statistically significant increase in circulating miR-30b levels was found when pre- and peri-pubertal states were compared with post-pubertal states (p=1.21×10^-5 and 6×10^-4, respectively). (B) The circulating miR-30b levels in the main cohort were plotted according to the estimated pubertal timing of each sample and a trendline indicating an increase in circulating miR-30b levels during the pubertal transition is shown in blue. The colors represent individual boys and matched samples from the same boy are connected with lines matching the color of the dots. (C) Correlation between measured levels of miR-30b in the main cohort and circulating protein levels of Makorin RING-finger protein 3 (MKRN3) measured in the same samples (previously described in (7)). Adjusted R²: 0.035, correlation coefficient: -0.21, p-value: 0.025. (D) Measurements of miR-30b-5p in 10 successive samples during the pubertal transition of seven boys representing the extended cohort were plotted according to the estimated pubertal timing of each sample and a trendline indicating a gradual increase in miR-30b is shown in blue. The colors represent individual boys and matched samples from the same boy are connected with lines matching the color of the dots. The plot reveals a substantial individual variation (average CV 151%) in circulating miR-30b levels during the pubertal transition. ***p < 10^-3 and ****p < 10^-4.

Figure 3

Ambiguous amplification curves and phosphorylation level of miR-30b. Amplification curves for (A) miR-30b and (B) spike-in control miR159a were analyzed in samples from the main cohort using the Advanced setup. n=39 boys with 3 samples each. Trendlines with confidence intervals indicating the average amplification curve for pre- (red), peri- (green), and post-pubertal (blue) samples are shown, and the individual curves are seen in the background with faded, matched colors according to the pubertal stage. Nine samples were measured with both (C) the Advanced miRNA setup and (D) the stem-loop miRNA setup and the amplification curves are colored according to target (grey for spike-in independent of the pubertal state, red for miR-30b in pre-pubertal samples, green for miR-30b in peri-pubertal samples, and blue for miR-30b in post-pubertal samples). (E) Amplification curves for synthetic miR-30b with and without 5’-phosphorylation measured by RT-qPCR using the Advanced miRNA setup. Different percentages of miR-30b with 5’-phosphorylation were mixed with miR-30b without 5’-phosphorylation to investigate the change in the shape of the curves depending on the percentage of fully functional and 5’-phosphorylated miRNAs in the sample. The maximum dRn value is visibly lower when only 10% of the miRNAs are 5’-phosphorylated but a rising curve is still observed when only non-phosphorylated (inactive) miRNAs are present although no exponential phase is seen. (F) Correlation plot comparing the miR-30b Cq values of the nine samples using the advanced miRNA setup vs. the stem-loop miRNA setup. The dashed blue line represents x=y, the red line represents the linear model for the correlation of the two setups. Individual dots represent the nine samples analyzed. Correlation coefficient: 0.86, Adjusted R²: 0.7033, p-value: 0.0029.

Figure 4

Schematic illustration of changes in miR-30b levels and bioactivity during pubertal transition. Circulating levels of miR-30b are low in pre-pubertal boys and a large fraction of these appears unphosphorylated at the 5’ end (inactive, shown in grey). At the same time, levels of Makorin RING-finger protein 3 (MKRN3) in circulation are high (shown in red) in pre-pubertal boys. As puberty progresses, levels of miR-30b increase and a larger fraction appear to be biologically active with a 5’-phosphorylation (shown in green). A concurrent drop in the circulating level of MKRN3 is observed. While the data builds on measurements in blood, we believe that these, at least to some extent, reflect hypothalamic activation of the kisspeptin/neurokinin B/dynorphin positive (KNDy-) neurons, which are known from studies in animals to be involved in the onset of puberty. Created with BioRender.com.