The tissue specific regulation of miR22 expression in the lung and brain by ribosomal protein L29

Abstract

Endogenous miR22 is associated with a diverse range of biological processes through post-translational modification of gene expression and its deregulation results in various diseases including cancer. Its expression is usually tissue or cell-specific, however, the reasons behind this tissue or cell specificity are not clearly outlined till-date. Therefore, our keen interest was to investigate the mechanisms of tissue or cell-specific expression of miR22. In the current study, miR22 expression showed a tissues-specific difference in the poly(I:C) induced inflammatory mouse lung and brain tissues. The cell-specific different expression of miR22 was also observed in inflammatory glial cells and endothelial cells. The pattern of RPL29 expression was also similar to miR22 in these tissues and cells under the same treatment. Interestingly, the knockdown of RPL29 exerted an inhibitory effect on miR22 and its known transcription factors including Fos-B and c-Fos. Fos-B and c-Fos were also differentially expressed in the two cell lines transfected with poly(I:C). The knockdown of c-Fos also exerted its negative effects on miR22 expression in both cells. These findings suggest that RPL29 might have regulatory roles on tissue or cell-specific expression of miR22 through the transcription activities of c-Fos and also possibly through Fos-B.

Figure 1

The expression of miR22 in different cells and mouse tissues in response to poly(I:C). Human glioblastoma astrocytoma cells (U251) and human umbilical vein cells (EA.hy926) were transfected with poly(I:C) at the different concentrations for 6 h. The miR22 level in U251 cells (a) and EA.hy926 cells (b) was determined with qRT-PCR using U6 as an internal control. Mice were treated with 100 µg of poly(I:C) for 24 h and miR22 level of the brain (c) and the lung (d) tissues were quantified by qPCR. All qPCR data are representative of three independent experiments with three replicates. (**P < 0.01; ***P < 0.001).

Figure 2

RPL29 expression in the brain and lung tissues of mouse and in two different cell lines in response to poly(I:C). RPL29 mRNA was quantified in the lung (a) and brain (c) tissues of mice treated with poly(I:C) for 24 h through qPCR. RPL29 protein was quantified in the lung (b) and brain (d) tissues of mice with similar treatment through western blot. Full-length blots/gels are presented in Supplementary Figure S2-S5. RPL29 mRNA was also detected in poly(I:C) treated U251 (e) and EA.hy926 (f) cells through qPCR. All data were curated from three independent experiment with three replication and represented as mean ± SD (***P < 0.001).

Figure 3

The expression of miR22 in RPL29 knocked-down cells. Human glioblastoma astrocytoma cells (U251) and human umbilical vein cells (EA.hy926) were transfected with si-RPL29 for 24 h followed by poly(I:C) treatment at the concentration of 100 ng/ml for 6 h. The expression miR22 in U251 cells (a) and EA.hy926 cells (b) was determined with qRT-PCR using U6 as an internal control. All data are representative of at least three independent experiments with three replicates. (***P < 0.001).

Figure 4

Expression of transcription factors of miR22 in RPL29 knocked down cells. U251 and EA.hy926 cells were treated with RPL29 si-RNA for 24 h at the final concentration of 50 nM, and the mRNA expression of Fos-B, c-Fos, and PU.1 in U251 (a–c) and EA.hy926 (d–f) cells were determined respectively through qRT-PCR. GAPDH was used as an internal control, and all data were curated from three independent experiments having three replicates. (*P < 0.05; **P < 0.01; ***P < 0.001).

Figure 5

The expression of Fos-B and c-Fos in different cells upon poly(I:C) treatment and the expression of miR22 in c-Fos knocked down cells. Fos-B and c-Fos mRNA were measured in U251 (a–b) and EA.hy926 (c–d) cells transfected with poly(I:C) for 6 h through qRT-PCR. Later on c-Fos was knocked down with si-RNA (Final concentration 50 nM) in both types of cells and transfected with poly(I:C) (100 ng/ml) for another 6 h. The expression of miR22 was quantified in U251 (e) and EA.hy926 (f) cells. All data were curated from three independent experiment with three replications (*P < 0.05; **P < 0.01; ***P < 0.001).