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Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes
Published on: April 12, 2015
Expression and Function of hsa-miR-6165 in Human Cell Lines and During the NT2 Cell Neural Differentiation Process
Maryam Hassanlou1, Bahram Mohammad Soltani2, Seyed Javad Mowla3
1Molecular Genetics Department, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
Abstract:
MicroRNAs are small non-coding RNAs that posttranscriptionally regulate mRNA expression. hsa-miR-6165 which was previously discovered in our group is located in the forth intron of p75NTR gene and its function is still under investigation. As P75NTR has diverse cellular functions, some of the complexity of its function could be attributed to the internally located microRNA. Our analysis revealed that treatment of HCT116 cells with 5-azacytidine promoted differential expression of hsa-miR-6165 from its host gene which is consistent with the bioinformatic prediction of an independent promoter for hsa-miR-6165. In addition, hsa-miR-6165 promoter is capable of driving GFP reporter gene in HeLa cells. The putative target gene expression level which was detected using RT-qPCR is inversely proportional to the expression level of hsa-miR-6165 during NT2 cell neural differentiation. Furthermore, hsa-miR-6165 overexpression resulted in significant downregulation of ABLIM-1, PVRL1, and PDK1 target genes, while it attenuates NT2 neural differentiation. Hsa-miR-6165 overexpression in SW480 cells also resulted in significant downregulation of PKD1, DAGLA, and PLXNA2 putative target genes, while it increases the sub-G1 cell population of SW480 and HEK293T cells as detected by flow cytometry. Overall, in this study, we report an independent promoter for hsa-miR-6165 which is active in HeLa cells. Additionally, hsa-miR-6165 targets ABLIM-1, PVRL1, PKD1, PLXNA2, and PDK1 genes, and unlike in HEK293T and SW480 cells, hsa-miR-6165 overexpression does not affect HeLa cells while its downregulation reduces sub-G1 cell population. Our results validate that hsa-miR-6165 affects the cell cycle progression and could increase apoptosis in human cell lines.
Insights
MicroRNAs (miRNAs) are small non-coding RNAs. This study identifies an independent promoter for hsa-miR-6165, revealing its role in regulating target genes and affecting cell cycle progression and apoptosis in human cells.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- MicroRNAs (miRNAs) are key regulators of gene expression.
- hsa-miR-6165, located within the p75NTR gene, has an uncharacterized function.
- The p75NTR gene has diverse cellular roles, potentially influenced by its intronic miRNA.
Purpose of the Study:
- To investigate the promoter activity and function of hsa-miR-6165.
- To identify target genes regulated by hsa-miR-6165.
- To determine the effect of hsa-miR-6165 on cell cycle progression and apoptosis.
Main Methods:
- 5-azacytidine treatment of HCT116 cells to induce differential miRNA expression.
- Reporter gene assays in HeLa cells to assess promoter activity.
- RT-qPCR to measure target gene expression during NT2 cell neural differentiation.
- Flow cytometry to analyze cell cycle and apoptosis in SW480 and HEK293T cells.
Main Results:
- An independent promoter for hsa-miR-6165 was identified and shown to be active in HeLa cells.
- hsa-miR-6165 expression inversely correlates with target gene expression during neural differentiation.
- Overexpression of hsa-miR-6165 downregulates ABLIM-1, PVRL1, PDK1, PKD1, DAGLA, and PLXNA2.
- hsa-miR-6165 overexpression affects cell cycle progression and increases apoptosis in specific cell lines.
Conclusions:
- hsa-miR-6165 possesses an active, independent promoter.
- hsa-miR-6165 directly targets and downregulates several genes, including ABLIM-1, PVRL1, PDK1, PKD1, DAGLA, and PLXNA2.
- hsa-miR-6165 influences cell cycle progression and can induce apoptosis in human cell lines.
