MicroRNA-1 induces apoptosis by targeting prothymosin alpha in nasopharyngeal carcinoma cells
© Wu et al; licensee BioMed Central Ltd. 2011
Received: 12 July 2011
Accepted: 7 November 2011
Published: 7 November 2011
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 time-lapse microscopy so we investigated the mechanisms of miR-1 inducing apoptosis.
To 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.
Annexin 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.
The 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.
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. MicroRNAs function as regulators of gene expression and play important roles in biological processes [1, 2]. 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. To our surprise, 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 also caused apoptosis in other cancer cell lines such as HeLa, Cal-27 and KYSE30.
Apoptosis is a form of spontaneous cell death characterized by externalization of phosphatidylserine, cell shrinkage, chromatin margination and condensation, nuclear fragmentation, activation of caspases, cellular budding and production of apoptotic bodies [3, 4]. Apoptosis is one of the important biological processes for organisms. Many diseases have been associated with abnormal apoptosis; excessive apoptosis has been linked to auto-immunity and insufficient apoptosis plays a large role in cancer transformation.
In the present study, we confirm that miR-1 directly targets PTMA mRNA. PTMA also known as prothymosin-alpha or ProTalpha is a hormone or hormone-like polypeptide precursor first isolated from the rat thymus gland . PTMA functions in transcription regulation and cell proliferation [6, 7], and acts as an apoptotic inhibitor by binding to Apaf-1 [8, 9]. This study shows that PTMA siRNA and miR-1 accelerate the apoptotic process in cells treated with apoptotic inducers in comparison to the control. Hence the PTMA siRNA and miR-1 may have a potential therapeutic application for cancer therapy.
The following cell lines were used for this study: HeLa, human cervical adenocarcinoma [ATCC CCL-2.2], Cal-27, human tongue carcinoma [ATCC CRL-2095 ], KYSE30, human esophageal carcinoma [JCRB0188 ], HepG2, human hepatocellular carcinoma [ATCC HB-8065 ], PC13, human lung large cell carcinoma [IBL], SW620, human colorectal adenocarcinoma [ATCC CCL-227 ], SAS, human tongue carcinoma [JCRB0260 ], HEK-293T, human transformed embryonal epithelial cell [ATCC CRL-11268 ], NPC-TW01 and NPC-TW06 were human nasopharyngeal carcimoma that were established in our laboratory [10–12]. All cell lines were cultured in DMEM containing 5% fetal calf serum and L-glutamine and incubated in a 10% CO2 incubator.
Transfection of microRNA and siRNA
The microRNAs in this study were purchased from Ambion (Austin, TX, USA); the microRNAs included Pre-miR™ hsa-miR-1 miRNA precursor positive control (Cat# AM17150), Pre-miR™ hsa-miR-486 miRNA precursor (Cat# PM10546), Pre-miR™ hsa-miR-429 miRNA precursor (Cat# PM10221), Pre-miR™ hsa-miR-200a miRNA precursor (Cat# PM10991) and Pre-miR™ miRNA precursor negative control #1 (Cat# AM17110). A total of 1.5 × 105 cells were seeded in each well of a 6-well culture plate in 1 ml of normal culture medium for microRNA transfection. The microRNA transfection mixture was prepared by diluting 1 μl of 50 μM microRNA in 100 μl of Opti-MEM I reduced serum medium (GIBCO, Gaithersburg, MD, USA). A total of 6 μl of HiPerFect transfection reagent (QIAGEN, Valencia, CA, USA) was added to the diluted microRNA and mixed by vortexing. After incubation for 10 minutes at room temperature, normal culture medium was added to mixture to bring the final volume to 1 ml. The microRNA transfection mixture was added to the well containing cells and mixed gently. A mock-transfected control was prepared by the same process but without the addition of microRNA. Another untransfected control (blank) was also prepared by the same process but without the addition of microRNA and transfection reagent. The culture medium in all experimental groups was changed after 24 hours to the regular medium and transfected again. The siRNA transfection conditions of PTMA Pre-design ChimeraRNAi (Abnova Cat# H00005757-R01, Taipei, Taiwan) and AllStars negative control siRNA (QIAGEN Cat#1027281) were identical to those for the microRNA transfection.
The morphological features of cell apoptosis were observed by ASTEC CCM-MULTI time-lapse microscopy (ASTEC, Fukuoka, Japan). Phosphatidylserine was stained using an Annexin V FITC assay kit (Serotec, Raleigh, USA). DNA fragments were stained using a DeadEnd Fluorometric TUNEL system (Promega, Madison, WI, USA). Caspase activity was detected by a Caspase-Glo caspase 3/7 assay kit (Promega) and Z-VAD-FMK caspase inhibitor (Promega). All of the experimental processes were performed according to manufacturer instructions.
NPC-TW01 and HeLa cells were transfected with miR-1 and miR-negative control and transfected again after 24 hours. Total RNA was extracted using Trizol (Invitrogen, Gaithersburg, MD, USA) after 42 hours. Total RNA from the miR-1 transfected cells was labeled with Cy5; total RNA from the miR-negative transfected cells was labeled with Cy3. The Cy-labeled RNAs were hybridized to a human whole genome oligo 4 × 44 K microarray (Agilent, Santa Clara, CA, USA). After washing and drying, the microarrays were processed with the Agilent microarray scanner; microarray data were analyzed by the GeneSpring GX 11 and Ingenuity Pathways analysis system. All microarray experiments were performed according to Agilent protocols. The microarray data had been submitted to NCBI GEO (http://www.ncbi.nlm.nih.gov/geo) under the accession numbers GSM706489 and GSM706490.
Primers used for qRT-PCR
Entrez Gene ID
Sequences (5' to 3')
Luciferase reporter assay
Oligonucleotides used for pmirGLO luciferase reporter vector construction
Sequences(5' to 3')
Treatment of apoptotic inducers
For assessing the apoptotic effects of PTMA siRNA or miR-1, a total of 5 × 103 NPC-TW01 cells were seeded in each well of a 96-well culture plate and were transfected with PTMA siRNA, miR-1 or control as mentioned above. After transfection for 48 hours, actinomycin D apoptotic inducer was added to the culture media to a final concentration of 5 μM (or camptothecin 10 μM, or etoposide 200 μM). Cells were subsequently observed by time-lapse microscopy (ASTEC, Fukuoka, Japan). Another 96 well plates (which experimental conditions were identical to that plate for time-lapse microscopy observation) were performed MTT assay for quantitating cell viability.
MiR-1 induces NPC cell apoptosis
MiR-1 directly targets PTMA
Candidate miR-1 targeted genes involved in apoptosis
Entrez Gene Name
Entrez Gene ID
Fold change in NPC-TW01
Fold change in HeLa
complement component 5
caspase recruitment domain family, member 8
Fas apoptotic inhibitory molecule
glutamate receptor, ionotropic, N-methyl D-aspartate 2A
tumor protein p63 (TP73L)
qRT-PCR confirming miR-1 down regulates genes in different cell lines
PTMA siRNA assists cells progression to apoptosis
MiR-1 can be used as a positive control and for optimizing transfection conditions in microRNA experiments (e.g., Ambion Pre-miR miRNA Starter Kit, QIAGEN Syn-hsa-miR-1 miScript miRNA mimic positive control). In this study, we noticed that transfection of NPC-TW01 and other cancer cell lines with exogenous miR-1 induce cell apoptosis. Endogenous miR-1 expression may be linked to apoptosis; for example, myocardial infarction increases miR-1 expression and induces apoptosis in rat H9C2 myoblast cells [13, 14]. MiR-1 expression also increases and apoptosis is induced when rat cardiomyoctes are incubated with H2O2 and treated with high glucose [16, 17]. The mir-1-1 gene is methylated and has reduced expression in human hepatocellular carcinoma cell lines compared to normal liver cells. When the mir-1-1 gene is hypomethylated, it re-expresses miR-1 and can induce apoptosis . In mouse myoblast cells, the mutation of MyoD transcription factors down regulates miR-1 expression and decreases cell apoptosis . MiR-1 expression is down regulated in human lung cancer tissues and cell lines in comparison to levels in normal human lungs. MiR-1 affects lung cancer cells in response to anti-cancer drugs . MiR-1 is down regulated in human bladder cancer  and head-neck squamous cell carcinoma (HNSCC) cells . Microarray gene expression profiles of miR-1 transfected cells revealed 17 miR-1 targeted candidate genes, they included PTMA. The authors demonstrated that miR-1 directly targets TAGLN2 and acts as a tumor suppressive microRNA [21, 22]. All reports in existing literature indicate that miR-1 plays an important role in apoptosis and cancer pathogenesis. In this study, we show that cell lines with low levels of endogenous miR-1 expression were apoptosis inducible via transfection with miR-1. Cell lines with high endogenous miR-1 expression were not apoptosis inducible by transfection with miR-1. Further testing with more cell lines is needed to obtain a definitive conclusion. MiR-1 directly down regulates PTMA mRNA levels and induces apoptosis. We used PTMA siRNA to down regulate PTMA mRNA levels and observed whether PTMA siRNA could induce apoptosis. Knocking down PTMA expression alone did not induce apoptosis but accelerated apoptotic progression in cells treated with apoptosis inducers. These results are in correlation with previous reports that PTMA is an apoptotic inhibitor and has anti-apoptotic properties [9, 23, 24]. The anti-apoptotic mechanism is that PTMA binds to Apaf-1 then prevents Apaf-1/cytochrome C apoptosome formation. Without apoptosome formation, pro-caspase-9 cannot be activated to induce the caspase cascades that lead to apoptosis [8, 9]. MiR-1 induces apoptosis and targets PTMA. PTMA is an apoptotic inhibitor, and knock down of PTMA expression levels is not enough to induce cell apoptosis. Therefore, miR-1-induced apoptosis is likely to alter the expression of other pro-apoptotic proteins. We found an up regulation of some pro-apoptotic proteins (HTRA, SMAC, p53, TNF-R1, TNF-R2, TRAILR-1, TNF-alpha, TNF-beta) expression in miR-1 transfected cells (unpublished data). MicroRNA and pathway database analysis reveals that miR-1 indirectly regulates those pro-apoptotic protein expressions. We will verify the pathway network between miR-1 and those pro-apoptotic proteins in the near future.
We have demonstrated that exogenous expression of miR-1 can induce apoptosis in some cell lines. This is a model of microRNA-induced cell apoptosis. PTMA is one of miR-1 target genes that involve in apoptosis. PTMA siRNA and miR-1 can accelerate the apoptotic process in NPC-TW01 cells when cells are treated with apoptotic inducers. The apoptotic inducers: actinomycin D, camptothecin and etoposide are also as chemotherapy drugs in clinical cancer therapy so miR-1 and PTMA siRNA may have potential applications in assisting cancer therapy.
This work was supported in part by the National Science Council (NSC99-2320-B-002-030, and NSC100-2325-B-002-034) and by National Taiwan University Hospital (NTUH-99-A102) to CTL.
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