Inhibition of the Growth of Raji Cells by Precursor MicroRNA-15a

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1 22 Clin Oncol Cancer Res (2010) 7: DOI /s Inhibition of the Growth of Raji Cells by Precursor MicroRNA-15a Dong-mei HE Qin CHEN Institute of Hematology, Medical College of Jinan University,Guangzhou , Guangdong Province, China. Correspondence to: Dong-mei HE Tel: This work was supported by the Constructing Key Disciplines Foundation of State Council Overseas Chinese Affairs (No ). Received January 7, 2010; accepted February 12, Tel (Fax): OBJECTIVE To investigate the effects of microrna-15a (mir- 15a) on the growth of the lymphoma Raji cell line. METHODS A eukaryotic expression vector of precursor mir-15a (pre-mir-15a) was constructed. Paired oligonucleotides for premir-15a expression were designed and synthesized chemically. Annealed oligonucleotides were inserted into a pgcsil- GFP vector under a U6 promoter to construct a pre-mir-15a expression plasmid. Oligonucleotides with a scrambled sequence which were used as a negative control were also constructed into the pgcsil-gfp vector. A recombinant expression vector was identified through PCR and sequencing. A pre-mir-15a expression plasmid and a negative control plasmid were all transferred into Raji cells with lipofectamine Quantitative reverse-transcription polymerase chain reaction (qrt-pcr) was employed to explore expression of mir-15a. The expression levels of the protein were assayed via immunofluorescence. The growth effect of Raji cells was measured by CCK8 assay. Apoptosis was determined using Hoechst dyeing and flow cytometry. The growth-inhibitory effect on Raji cells was measured using a CCK8 assay. RESULTS The results showed that the insertion sequence was correct. The expression level of mir-15a was obviously higher in Raji cells transferred with pre-mir-15a plasmid than in the blank group and in the negative control group. After Raji cells were transferred with the pre-mir-15a expression plasmid for 48 h, Bcl-2 protein expression levels were obviously decreased,which indicated that there was a significant difference as compared with the negative control group and blank group (P < 0.05). The CCK8 assay showed that transfection of pre-mir-15a expression plasmid decreased the cell growth at 24, 48 and 72 h post-transfection. After Raji cells were transferred with pre-mir-15a expression plasmid, apoptotic cells can be seen with Hoechst dyeing, and the cell apoptotic rate was obviously higher than that in blank group and negative control group. CONCLUSION Pre-miR-15a can induce apoptosis and inhibit the growth of Raji cells. KEY WORDS: hsa-mir-15 microrna, lymphoma, cell line, Bcl-2 protein. Copyright 2010 by Tianjin Medical University Cancer Institute & Hospital and Springer

2 Clin Oncol Cancer Res (2010) 7: Introduction MicroRNAs (mirnas) are short noncoding 19-24nt RNA molecules and can regulate gene expression by imperfect base-pairing with complementary sequences located mainly in the 3 UTRs of target mrnas [1-3]. MiRNAs represent one of the major regulatory families of genes in eukaryotic cells mainly by inducing translational repression [4-6]. MiR-15a is located at chromosome 13q14 and is frequently deleted in B-cell chronic lymphocytic leukemia (CLLs) [7-9]. A few reports have shown that deregulated expression of mir-15a induces apoptosis by targeting the antiapoptotic protein Bcl-2 [10,11]. Previously, we found that mir-15a and mir-16 oligonucleotides can induce apoptosis of lymphoma cells. In this study, the expression vector of precursor mir- 15a (pre-mir-15a) was constructed and transformed into human lymphoma cell line Raji. Materials and Methods Reagents The plasmid pgcsil-gfp and Hairpin-it TM mirnas Real-Time PCR Quantitation Kit was purchased from Shanghai Genechem Biotechnology Co. Ltd. Antibody against Bcl-2 was purchased from Santa Cruz. RPMI1640 and newborn calf serum was purchased from Gibco BRL. Cell culture The Raji cell line was cultured in RPMI 1640 with 5% CO 2, at 37 C. The medium was augmented with 10% newborn calf serum and 100 μ/ml of penicillin plus 100 μg/ml of streptomycin. A subculture was performed 3-4 days later and the cells in logarithmic growth phrase were used in all experiments. Construction of the pre-mir-15a expression plasmids The oligonucleotides of pre-mir-15a were designed and synthesized. As a negative control for sequence specificity was needed, we synthesized a negative control oligonucleotide which was a nonsense sequence. The oligonucleotides were annealed and ligated into the Age I and EcoR I site of linearized pgcsil. The ligation products were transformed into E. coli. The clones were selected and plasmid DNA was isolated. The plasmid was digested and clones with the pre-mir- 15a insert were selected. The purification of plasmid for transfection was performed using a DNA purification system. Transfection with the pre-mir-15a expression vector Raji cells in the exponential phase of growth were grown for 24 h and then transferred with 1 μg aliquots of premir-15a expression plasmids using Lipofectamine 2000 (Invitrogen) following the instructions of the manufacturer. Negative control cells were treated with a negative control plasmid consisting of a plasmid encoding a nonsense sequence whose sequence is not found in the mouse, human, or rat genome databases. Assay of mir-15a mrna by qrt-pcr qrt-pcr was performed using the Hairpin-it TM mir- NAs Real-Time PCR Quantitation Kit following the manufacturer s instructions at 24 h after pre-mir-15a transfection. Briefly, total RNA was isolated using Trizol reagent (Invitrogen Corporation, USA). cdnas were synthesized from total RNA, using specific primers for mir-15a. qrt-pcr was performed in a final volume of 20 μl mixture and based on the amplification parameters: 95 C for 10 min, followed by 40 cycles at 95 C for 12 s, 55 C for 25 s, and 72 C for 25 s. The threshold cycle (C t ) was determined using default threshold settings. The expression of mir-15a was determined according to standard curve of a mir-15a reference sample. All experiments were performed in triplicate and repeated 3 times. Quantification of Bcl-2 protein using immunofluorescence by flow cytometry Transferred cells with pre-mir-15a and untreated Raji cells were collected by centrifugation (2000 g) for 5 min. The cells were washed twice and fixed in 4% formaldehyde (Sigma) for 30 min on ice, followed by permeabilization with 0.1% Triton X in phosphate-buffered albumin for 10 min at 4 C. Cells were then washed in cold phosphate-buffered saline (PBS) and were subsequently added to 10 μl of mouse anti-human Bcl-2 monoclonal antibody (1:100) for 30 min, at room temperature in the dark. After washing twice with PBS, 10 μl of Cy3- conjugated goat anti-mouse secondary antibody IgG was added. The secondary antibody (monoclonal antibody, Zhongshan Bio Corp, China) was also used at a 1:100 dilution. After washing twice again with PBS, the cells were examined by flow cytometry (Becton Dickinson) and the percentage of cells with positive staining for Bcl-2 protein was determined. Quantifying cell proliferation with an CCK8 assay A CCK8 assay was performed to determine cell viability. The Raji cells were then washed, counted and seeded at a density of cells per well in 96-well plates. Six hours later, pre-mir-15a and the negative control plasmid were added to the cells at 1 μg aliquots. At 24, 48, 72 h, CCK8 solution was added 4 h before the end of incubation. Cell viability was measured with a spectrophotometer at an absorbance of 450 nm. Assays of cell apoptosis The transferred Raji cells were harvested after transient transfection and then fixed in methanol. Morphology was determined through Hoechst dyeing. Flow cytometry was performed in order to evaluate the cell-cycle status and apoptosis. Briefly, cells were centrifuged and

3 24 Clin Oncol Cancer Res (2010) 7: washed in PBS followed by a fixation procedure in 70% ethanol. The tubes containing the cell pellets were stored at 20 C for at least 24 h. Subsequently, the cells were centrifuged at 800 g for 15 min, and the supernatant was discarded so as to remove the ethanol completely. The pellets were resuspended in 0.5 ml PBS followed by incubation with 20 μg/ml RNase A (Sigma, USA) for 30 min, at 37 C, and then stained with propidium iodide solution (20 μg/ml, Sigma, USA) for 30 min. The DNA content was analyzed using a FACScan flow cytometer. Cells with a DNA content below that indicative of G1 phase were regarded as apoptotic cells. The percentage of the cells in apoptotic sub-g1 was analyzed by Multi- Cycle software. Statistical analysis Results are shown through mean ± SD. Statistical comparisons were made by ANOVA. Differences were deemed significant if a real alpha was All statistical analyses were carried out using SPSS Results Identification of pre-mir-15a expression plasmids The pre-mir-15a expression plasmids were identified through PCR, and a fragment of 393 bp was seen upon 1.5% agarose gel electrophoresis (Fig.1). Whereas, the unligate plasmids produced a fragment of 306 bp. DNA sequencing confirmed that the ligation reaction of the pre-mir-15a insert was correct. 306 bp bp Fig.1. Identification of pre-mir-15a expression plasmids. 1, ddh 2 O; 2, PGCSIL; 3, DNA Mark; 4, 5, 7, 8: PGCSIL-pre-miR-15a; 6, unligated plasmids. The expected size of the product of the pre-mir-15a expression by PCR is 393 bp; the unligated plasmids produced a fragment of 306 bp. mir-15a expression assay The qrt-pcr data included at least 3 independent experiments with 3 replicates in each experiment. After 24 h of transient transfection, the Ct value of qrt-pcr in the cells treated with the pre-mir-15a vector, the negative control vector and in the untransferred Raji cells were ± 0.35, ± 0.42, and ± 0.38, respectively. This indicated that mir-15a expression was increased. There was significant difference in the expression level of mir-15a in Raji cells transferred with pre-mir-15a plasmid when compared with the negative controls (P < 0.05), whereas in the negative controls mir-15a expression was not altered. Inhibition of Bcl-2 protein expression using a premir-15a expression vector Indirect immunofluorescence staining was performed. By flow cytometry, the Bcl-2 protein level was (97.8 ± 0.6)% in the untransferred Raji cells. The level of Bcl-2 was (58.4 ± 1.1)% at 48 h after transient transfection with the pre-mir-15a expression vector. Expression of Bcl-2 was significantly decreased (P < 0.05) in the cells treated with the pre-mir-15a expression vector as compared with either the negative control vector group (96.8 ± 0.9)% or the untransferred Raji cell group. There was no significant difference in Bcl-2 levels between the negative control group and the untransferred cells (P > 0.05). Cell survival A CCK8 assay was performed to examine the effects of pre-mir-15a expression on the growth of Raji cells. Seventy-two hours after transient transfection, pre-mir- 15a expression significantly inhibited the viability of the Raji cells (P < 0.05). Thus, compared with either the negative control vector group or the untransferred cell group, transfection of pre-mir-15a could effectively reduce the viability of Raji cells. There were no differences in the viability of Raji cells between the negative control vector group and the untransferred cell group (P > 0.05, Table1). Induced apoptosis of Raji cells by pre-mir-15a The Raji cells exhibited the features characteristic of apoptosis, such as membrane blebbing, nuclear condensation, fragmentation and formation of apoptotic bodies (not shown). The rate of apoptosis was 12.43% at 48 h Table 1. OD values after transfection of pre-mir-15a vectors at different times using CCK8 assay (x ± s, n = 3). Groups 24 h 48 h 72 h Blank control ± ± ± Negative control plasmid ± ± ± Pre-miR-15a plasmid ± 0.007* ± 0.005* ± 0.004* *P < 0.05

4 Clin Oncol Cancer Res (2010) 7: A B C Fig.2. Apoptosis rate of Raji cells at 48 h after transfection with pre-mir-15a. Results are from a representative experiment. A, Raji cells; B, Negative control plasmid; C, Pre-miR-15a group. A significant increase of apoptosis rate was observed after transfection with pre-mir-15a (C), P < after transfection with pre-mir-15a (Fig.2). However, significantly decreased apoptosis (P < 0.05) was observed in the blank group (1.26%) and in the negative control group (4.69%). Discussion MiRNAs are reported to be abnormally expressed in many human malignancies [12-19]. Recently, the mir- 15a/16-1 cluster was found down-regulated in CLL [10-12]. We present evidence from our study that mir-15a was significantly down-regulated in human lymphoma Raji cells and that overexpression of mir-15a suppressed the growth of Raji cells. In initial experiments, we confirmed that linearized pre-mir-15a oligonucleotides were inserted into the plasmid vector through PCR and sequencing methods. We further assessed whether premir-15a had an effect on mir-15a mrna, Bcl-2 protein levels and cell viability and/or cell proliferation. The results from our study demonstrated that after transfection with the pre-mir-15a vector, mir-15a mrna expression was significantly increased. The negative control group had no effect on mir-15a mrna. The results confirmed that the mir-15a precursor was transcribed into mir-15a in Raji cells. Bcl-2 was a putative target for mir-15a; therefore, we examined the protein levels of Bcl-2 in Raji cells transferred with mir-15a precursors and found that there was a significant decrease in Bcl-2 protein compared with cells transferred with the negative control plasmid. The results of the study confirmed that mir-15a may target Bcl-2 at the level of translation in Raji cells. The viability of Raji cells was decreased after being treated with pre-mir-15a, when compared with the controls (P < 0.05). Moreover, the viability of both cells transferred with the negative control vector and cells that were not transferred was not decreased. After transient transfection with pre-mir-15a, Raji cells showed the characteristic features of apoptosis and an increased apoptotic rate. Thus, the pre-mir-15a expression vector caused specific inhibition of cell growth through cell apoptosis but caused no nonspecific toxic effects. Our observation is in agreement with the findings of other studies, in which mirna mimics can inhibit the growth of tumor cells [20-25]. The data from this study suggest that mir-15a may hold significant promise as a novel molecular therapy for human lymphomas. Further research and work to improve the efficacy of the pre-mir-15a delivery and action, to help stably express mir-15a, and to establish the in vivo conditions and the clinical applicability of mirna mimics are still needed. Conflict of interest statement No potential conflicts of interest were disclosed. References 1 Calin GA, Croce CM. MicroRNA signatures in human cancers. Nat Rev Cancer 2006; 6: Li N, Flynt AS, Kim HR, et al. Dispatched Homolog 2 is targeted by mir-214 through a combination of three weak microrna recognitionsites. Nucleic Acids Res. 2008; 36: Nahvi A, Shoemaker CJ, Green R. An expanded seed sequence definition accounts for full regulation of the hid 3' UTR by bantam mirna. RNA 2009; 15: Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microrna targets. Cell 2005; 120: Steitz JA, Vasudevan S. mirnps: versatile regulators of gene expression in vertebrate cells. Biochem Soc Trans 2009; 37: Wakiyama M, Takimoto K, Ohara O, et al. Let-7 microrna-mediated mrna deadenylation and translational repression in a mammalian cell-free system. Genes Dev 2007; 21: Calin GA, Dumitru CD, Shimizu M, et al. Frequent dele-

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