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Global MicroRNA Expression Profiling Reveals Differential Expression of Target Genes in 6-Hydroxydopamine-injured MN9D Cells

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Abstract

Recent evidence indicates that microRNAs (miRNAs) play a key role in neurodegenerative diseases. However, little is known about how these small RNAs contribute to dopaminergic neuronal apoptosis. Here, we profiled the expression of miRNAs in MN9D cells with and without 6-hydroxydopamine (6-OHDA) treatment by miRCURY™ LNA microRNA arrays. We identified six miRNAs (miR-668-3p, let-7d-3p, miR-3077-3p, miR-665-5p, miR-99b-3p, and miR-323-3p) that were significantly lower and five miRNAs (miR-875, miR-207, miR-425-5p, miR-19b-3p, and miR-338-3p) that were significantly higher after 6-OHDA treatment. Among them, five have been demonstrated to be implicated in neurodegenerative diseases. Consistent with our prediction, the deregulated miRNA’s target mRNAs, such as peroxiredoxin III (Prx III) and Myc, also showed changes in their expression levels. Furthermore, using a dual-luciferase reporter assay, we confirmed that Prx III was a direct target gene of miR-875. Taken together, these findings demonstrate that changes in miRNA expression occur after 6-OHDA treatment and suggest that miRNAs and their predicted targets have a potential role in apoptosis of MN9D cells.

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (81101899) and a project funded by the Priority Academic Program Development of Higher Education Institution (PAPD).

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Department of Human Anatomy and Neurobiology, Xuzhou Medical College, Tongshan Road 209#, Xuzhou, 221009, China

    Li Li, Hui-Zhen Chen, Fang-Fang Chen, Feng Li, Meng Wang, Li Wang & Dian-Shuai Gao

  2. Department of Human Anatomy, Histology and Embryology, School of the Basic Medicine, The Fourth Military Medical University, No. 17, Changle West Road, Xi’an, 710032, Shanxi Province, China

    Li Li, Yun-Qing Li & Dian-Shuai Gao

  3. Department of Pathophysiology, Xuzhou Medical College, Tongshan Road 209#, Xuzhou, 221002, China

    Li Li

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  1. Li Li
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12017_2013_8244_MOESM1_ESM.jpg

6-OHDA decreases viability of MN9D cells. Cells were incubated for 30 min in different concentrations of 6-OHDA followed by 24 h resting period. The data are expressed as a percentage of control cells ± standard errors of the mean (n = 5) * p < 0.05 versus control cells. Control: cells without treatment. Vehicle: cells treated with physiological saline containing 0.15 % ascorbic acid. (JPEG 130 kb)

12017_2013_8244_MOESM2_ESM.jpg

6-OHDA treatment induces apoptotic nuclear morphology in MN9D cells. Cells were incubated for 30 min in different concentrations of 6-OHDA followed by 24 h resting period. Then cells were stained with the DNA-binding stain fluorochrome Hoechst 33258. White arrows represent the condensed and fragmented nucleus of apoptotic cells. The percentage of Hoechst-positive apoptotic cells per visual field was determined (the number of Hoechst-positive cells per field/total number of cells per field). Approximately 100 cells were counted in each of 5 randomly selected fields. Scale bars represent 20 μm. A, Control: cells without treatment. B, Vehicle: cells treated with physiological saline containing 0.15 % ascorbic acid, C~H, 6-OHDA treated cells (100~600 μM) (n = 5) (JPEG 3475 kb)

12017_2013_8244_MOESM3_ESM.jpg

6-OHDA induces cell apoptosis as measured by flow cytometry assay. Cells were incubated for 30 min in different concentrations of 6-OHDA and followed by 24 h resting period. Then the apoptotic proportion of cells was analyzed by Annexin V-FITC/PI flow cytometry. A, Control: cells without treatment. B, Vehicle: cells treated with physiological saline containing 0.15 % ascorbic acid C~H, 6-OHDA treated cells (100~600 μM) (n = 5) (JPEG 380 kb)

12017_2013_8244_MOESM4_ESM.jpg

6-OHDA induces the cleavage of caspase-3. Cells were incubated with 6-OHDA 100 μM for 30 min and followed by 24 h resting period. Western blotting analysis indicated that the 6-OHDA treatment of MN9D cells could increase the expression of cleaved caspase-3. Levels of cleaved caspase-3 were normalized to β-actin levels. * p < 0.05 versus control cells. (n = 3) (JPEG 675 kb)

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Li, L., Chen, HZ., Chen, FF. et al. Global MicroRNA Expression Profiling Reveals Differential Expression of Target Genes in 6-Hydroxydopamine-injured MN9D Cells. Neuromol Med 15, 593–604 (2013). https://doi.org/10.1007/s12017-013-8244-z

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  • DOI: https://doi.org/10.1007/s12017-013-8244-z

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