Abstract
BackgroundMiR-1 (microRNA-1) has been used as a positive control in some microRNA experiments. We found that miR-1 transfection of nasopharyngeal carcinoma cells reveals a typical apoptotic process as shown by time-lapse microscopy so we investigated the mechanisms of miR-1 inducing apoptosis.MethodsTo confirm that miR-1 induces apoptosis, we used Annexin V and TUNEL staining and caspase assay. To determine that miR-1 directly targets genes that involve in apoptosis, we analyzed microRNA and pathway databases, and cDNA expression microarrays from miR-1 transfected cells. To demonstrate candidate miR-1 targeted genes, we used qRT-PCR analysis and luciferase reporter vector assays. To assess the miR-1 target gene PTMA (prothymosin alpha, ProTalpha) involves in apoptosis, we used PTMA siRNA to knock down PTMA.ResultsAnnexin V and TUNEL staining and caspase assay confirm that miR-1 induces nasopharyngeal carcinoma cell apoptosis. MiR-1 transfection of HeLa, Cal-27, KYSE30 and NPC-TW06 cell lines which express low levels of endogenous miR-1 also induces apoptosis. However, miR-1 transfection of cell lines such as SW620, HepG2, HEK-293T, SAS and PC-13 which express high levels of endogenous miR-1 does not result in apoptosis. MiR-1 directly targets PTMA gene. PTMA siRNA and miR-1 accelerate the apoptotic process in cells treated with apoptosis inducers.ConclusionsThe exogenous expression of miR-1 induces apoptosis in a number of cell lines. This is a model of microRNA-induced cell apoptosis. The PTMA is one of miR-1 target genes which involve in miR-1 inducing apoptosis. The apoptotic inducers including actinomycin D, camptothecin and etoposide are also the chemotherapeutic drugs in clinical cancer therapy and PTMA siRNA can accelerate apoptotic progression in cells treated with those apoptosis inducers. Therefore PTMA siRNA may have potential applications as an adjuvant in cancer chemotherapy.
Highlights
MiR-1 has been used as a positive control in some microRNA experiments
We used the Promega Caspase-Glo caspase 3/7 assay kit to measure caspase 3/ 7 activities in the nasopharyngeal carcinoma (NPC)-TW01 cells transfected with miR-1. 48 hours after transfection, for miR-1 transfected cells demonstrated a 4.6-fold increase in caspase 3/7 activities compared to the untransfected cells (p < 0.01)
MiR-1 transfection of NPC-TW01 cells induced apoptosis, whereas NPC-TW01 cells transfected with other microRNAs did not induce cellular apoptotic morphology as observed by time-lapse
Summary
MiR-1 (microRNA-1) has been used as a positive control in some microRNA experiments. We found that miR-1 transfection of nasopharyngeal carcinoma cells reveals a typical apoptotic process as shown by timelapse microscopy so we investigated the mechanisms of miR-1 inducing apoptosis. MicroRNAs are endogenous non-coding RNAs that are approximately 21 nucleotides long. They are evolutionarily conserved and found in many animals, plants, fungi and viruses. In previous investigation of pathogenic microRNAs in nasopharyngeal carcinoma (NPC) cells, we used miR-1 (microRNA-1) to transfect NPC cells as a positive control. We observed a typical apoptotic response in miR-1 transfected NPC cells with time-lapse microscopy. Negative control microRNA and other microRNAs did not induce cellular apoptosis under identical transfection conditions. Transfection of miR-1 caused apoptosis in other cancer cell lines such as HeLa, Cal-27 and KYSE30
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