MiR-338-3p regulates neuronal maturation and suppresses glioblastoma proliferation

Abstract

Neurogenesis is a highly-regulated process occurring in the dentate gyrus that has been linked to learning, memory, and antidepressant efficacy. MicroRNAs (miRNAs) have been previously shown to play an important role in the regulation of neuronal development and neurogenesis in the dentate gyrus via modulation of gene expression. However, this mode of regulation is both incompletely described in the literature thus far and highly multifactorial. In this study, we designed sensors and detected relative levels of expression of 10 different miRNAs and found miR-338-3p was most highly expressed in the dentate gyrus. Comparison of miR-338-3p expression with neuronal markers of maturity indicates miR-338-3p is expressed most highly in the mature neuron. We also designed a viral "sponge" to knock down in vivo expression of miR-338-3p. When miR-338-3p is knocked down, neurons sprout multiple primary dendrites that branch off of the soma in a disorganized manner, cellular proliferation is upregulated, and neoplasms form spontaneously in vivo. Additionally, miR-338-3p overexpression in glioblastoma cell lines slows their proliferation in vitro. Further, low miR-338-3p expression is associated with increased mortality and disease progression in patients with glioblastoma. These data identify miR-338-3p as a clinically relevant tumor suppressor in glioblastoma.

Fig 1. In vivo detection of selected miRNAs using an mCherry sensor.

(A) Construction of the lentiviral vector, using an FUCW backbone and two target-complementary sequences immediately downstream of mCherry. (B) Co-injection of control GFP-expressing and mCherry-expressing viruses (equal titer) into the dentate gyrus of adult mice results in roughly equal infection rates; sections counter-stained with DAPI. (C) Co-injection of miR137-3p sensor (red) and control GFP-expressing virus. (D) Co-injection of miR338-3p sensor (red) and control GFP-expressing virus. (E) Expression levels of 10 different miRNAs in the dentate gyrus relative to control mCherry-expressing vector. *p<0.05, **p<0.01, ***p<0.001; one-way ANOVA, analyzed post-hoc using Tukey’s range test. Results show mean ± SEM.

Fig 2. MiR-338-3p expression increases with maturity in dentate gyrus granule neurons.

(A) Co-localization of nestin (arrowheads) with miR-338-3p sensor. (B) Co-localization of nestin (arrowheads) with miR-132-3p sensor. (C) Co-localization of NeuN (arrowheads) and miR-338-3p sensor. (D) Co-localization of NeuN (arrowheads) and miR-132-3p sensor. (E) Percentage of cells co-labeled with one of the sensors and nestin as compared to control mCherry virus. (F) Percentage of cells co-labeled with one of the sensors and doublecortin as compared to control mCherry virus. (G) Percentage of cells co-labeled with one of the sensors and NeuN as compared to control mCherry virus. (H) Ratio of cells co-labeled with either the miR-338-3p or miR-132-3p sensor and nestin as compared to control mCherry virus. (I) Ratio of cells co-labeled with either the miR-338-3p or miR-132-3p sensor and doublecortin as compared to control mCherry virus. (J) Ratio of cells co-labeled with either the miR-338-3p or miR-132-3p sensor and NeuN as compared to control mCherry virus. ns p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; one-way ANOVA, analyzed post-hoc using Tukey’s range test. Results show mean ± SEM.

Fig 3. In vivo verification of miR-338-3p sponge efficacy.

(A) Design of lentiviral miR-338-3p sponge with a sensor cassette. The miR-338-3p sensor cassette contains 2 perfectly complementary miR-338-3p target sequences downstream of GFP driven by the pUbiquitin promoter and the sponge cassette consists of 6 targets downstream of both the H1 and U6 promoters for a total of 2 sensor targets to sense miR-338-3p activity and 12 sponge targets to sequester endogenous miR-338-3p. (B) Low magnification images of dentate gyrus show the mCherry control and the miR-338-3p sponge exhibit similarly high levels of expression. (C) Images from (B), but under high magnification. This demonstrates the ability of the sponge cassette to sequester ligand away from the miR-338-3p targets expressed in the sensor cassette.

Fig 4. MiR-338-3p knockdown results in abnormal granule cell morphology in neonatal dentate gyrus.

(A) Representative images of granule cells infected with the retroviral GFP control or the mCherry miR-338-3p sponge (red). (B) Granule neurons expressing the miR-338-3p sponge (red) displaying primary dendrites projecting at divergent angles from the soma compared to control neurons. (C) Granule cells infected with the control virus showing bipolar organization, while the miR-338-3p knockdown neurons (red) show multiple primary dendrites. (D) Branching angles of primary dendrites infected with the control vector or infected with the sponge. (E) Proportion of granule cells with multiple primary dendrites relative to all granule cells in both control and knockdown conditions. Each separate image was treated as an independent sample within the mouse of both the control and knockdown populations. *p<0.05, ****p<0.0001; t-test. Results show mean ± SEM.

Fig 5. MiR-338-3p knockdown results in cellular neoplasia in vivo.

Neoplasm infected with the miR-338-3p sensor (red) and sponge (green) and stained for (A) nestin (blue) as a marker for immature neurons, (B) GFAP (blue) as a marker for astrocytes, and (C) NeuN (blue) as a marker for mature neurons.

Fig 6. MiR-338-3p knockdown results in proliferation of GFAP-positive cells.

(A) Expression pattern of of miR-338-3p sponge (green) and BrdU (blue), a marker of cellular proliferation, in a dentate gyrus neoplasm following miR-338-3p knockdown. (B) Co-localization of GFAP (red) and BrdU (blue) in dentate gyrus neoplasm following miR-338-3p knockdown.

Fig 7. Overexpression of miR-338-3p decreases in vitro proliferation of GBM cells.

(A) Construction of miR-338-3p overexpressor lentivirus containing a GFP-coding region to indicate expression along with two miR-338-3p transcripts downstream of the U6 promoter. (B) Endogenous expression of miR-338-3p in U251 GBM cells, as indicated by miR-338-3p sensor lentivirus (red) expression compared to control lentivirus expressing GFP-only. (C) Expression of miR-338-3p following infection with overexpressor virus in U251 GBM cells as indicated by miR-338-3p sensor (red). (D) Endogenous expression of miR-338-3p in SF295 GBM cells, as indicated by miR-338-3p sensor lentivirus (red) expression compared to control lentivirus expressing GFP-only. (E) Expression of miR-338-3p following infection with overexpressor virus in and SF295 GBM cells as indicated by miR-338-3p sensor (red). (F) Population growth kinetics of U251 GBM cells infected with an empty vector or miR-338-3p overexpressor (7–12 DPI). (G) Population growth kinetics of SF295 GBM cells infected with an empty vector or miR-338-3p overexpressor (7–10 DPI). Dotted lines in (F) and (G) fit theoretical population growth curves to the observed data, using the equation: Y = 25 × 2^t/DT, where Y is the number of cells at time t, and DT is the doubling time. ****p<0.001; Pearson’s chi-squared test. Results show mean ± SEM.

Fig 8. Outcomes in GBM patients based on miR-338-3p expression level.

GBM patients with low mir-338-3p expression exhibit decreased overall and disease-free survival. Kaplan-Meier (A) survival and (B) disease-free survival curves of GBM patients grouped by mir-338-3p expression level. P-values determined by the log-rank test are indicated in the graphs.