After transfection of normal SH-SY5Y cells with the 7 selected miRNAs (10 nM, 72 h), the 3 miRNAs predicted to target hsc70 and the 4 miRNAs predicted to target lamp-2a all decreased the levels of the respective proteins relative to actin, but these were only statistically significant for hsa-miR-106a* and hsa-miR-301 (hsc70 protein) and hsa-miR-224, hsa-miR-373* and hsa-miR-379 (LAMP-2A protein).
HeLa cells were transfected with the siRNA targeting LAMP-1, LAMP-2 or nonspecific control RNA (100 pmol/ml) using Oligofectamine according to the manufacturer's instructions.
(b) Immunoblots and respective quanticiation from N2a cells either non-transfected (non) or stably transfected with scramble shRNA (scmbl) or shRNA targeting LAMP-2 mRNA.
(c) Quantitative RT-PCR of samples from N2a cells either non-transfected (non) or stably transfected with scmbl shRNA or shRNA targeting LAMP-2 mRNA.
The luciferase reporter constructs confirmed that four and three of these miRNAs effectively targeted lamp-2a and hsc70 3′UTrespectivelyvely, suggesting they could underpin the decrease in hsc70 and LAMP-2A reported in PD brains.
(b) Immunoblots of lysates from N2a cells either non-transfected (non) or stably transfected with scramble shRNA (Scmbl) or shRNA targeting LAMP-2 mRNA showing protein levels of LAMP-1.
Similar but milder changes were observed in PD amygdala where there was a significant increase in the two miRNAs targeting lamp-2a (hsa-miR-224 and hsa-miR-373*) and a nonsignificant increase in the two miRNAs targeting hsc70 (hsa-miR-26b and hsa-miR-106a* Figure 4b).
The levels of the three miRNAs targeting lamp-2a (hsa-miR-21* hsa-miR-224; and hsa-miR-373*) and the three miRNAs targeting hsc70 (hsa-miR-26b; hsa-miR-106a* and hsa-miR-301b) were significantly increased in PD SNc relative to actin mRNA levels.
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Also the lysosomal targeting of LAMP-1 and LAMP-2 remains an interesting open question, since these proteins are predominantly located on the limiting membranes of late endosomes and lysosomes, and incorporation into ILVs seems therefore not involved.
