Mitochondrial ncRNA targeting induces cell cycle arrest and tumor growth inhibition of MDA-MB-231 breast cancer cells through reduction of key cell cycle progression factors

Abstract

The family of long noncoding mitochondrial RNAs (ncmtRNAs), comprising sense (SncmtRNA), and antisense (ASncmtRNA-1 and ASncmtRNA-2) members, are differentially expressed according to cell proliferative status; SncmtRNA is expressed in all proliferating cells, while ASncmtRNAs are expressed in normal proliferating cells, but is downregulated in tumor cells. ASncmtRNA knockdown with an antisense oligonucleotide induces massive apoptosis in tumor cell lines, without affecting healthy cells. Apoptotic death is preceded by proliferation blockage, suggesting that these transcripts are involved in cell cycle regulation. Here, we show that ASncmtRNA knockdown induces cell death preceded by proliferative blockage in three different human breast cancer cell lines. This effect is mediated by downregulation of the key cell cycle progression factors cyclin B1, cyclin D1, CDK1, CDK4, and survivin, the latter also constituting an essential inhibitor of apoptosis, underlying additionally the onset of apoptosis. The treatment also induces an increase in the microRNA hsa-miR-4485-3p, whose sequence maps to ASncmtRNA-2 and transfection of MDA-MB-231 cells with a mimic of this miRNA induces cyclin B1 and D1 downregulation. Other miRNAs that are upregulated include nuclear-encoded hsa-miR-5096 and hsa-miR-3609, whose mimics downregulate CDK1. Our results suggest that ASncmtRNA targeting blocks tumor cell proliferation through reduction of essential cell cycle proteins, mediated by mitochondrial and nuclear miRNAs. This work adds to the elucidation of the molecular mechanisms behind cell cycle arrest preceding tumor cell apoptosis induced by ASncmtRNA knockdown. As proof-of-concept, we show that in vivo knockdown of ASncmtRNAs results in drastic inhibition of tumor growth in a xenograft model of MDA-MB-231 subcutaneous tumors, further supporting this approach for the development of new therapeutic strategies against breast cancer.

Fig. 1. ASncmtRNA knockdown (ASK) induces inhibition of cell proliferation and apoptotic cell death of MDA-MB-231 breast cancer cells.

MDA-MB-231 cells were transfected with 200 nM Andes-1537 (1537) or ASO-C or left untreated (NT). a Knockdown of ASncmtRNA-1 and −2 at 24 h post-transfection was corroborated by RT-PCR, using 16 S and 18 S rRNA as controls. Numbers on the left represent size in bp of MW standards. b Viability was determined by MTT assay at 24, 48, and 72 h post-transfection. c At 48 h post-transfection, Andes-1537 induced over 60% death, determined by Trypan blue (Tb) exclusion assay, compared to 3–5% in controls (two-tailed Student’s t-test; Mean ± S.E.M. ****p < 0.0001; ASO-C vs. Andes-1537; n = 3). d Representative image of fluorescent TUNEL assay for determination of apoptosis, at 48 h post-transfection. Bars = 50 µm. e Ten fields per sample of the experiment shown in d were photographed and quantified. The graph shows % of TUNEL-positive cells (two-tailed Student’s t-test; Mean ± S.E.M. ****p < 0.0001; ASO-C vs. Andes-1537; n = 3). f Apoptosis was further confirmed at 48 h post-transfection by Annexin V-binding and co-staining with PI and analyzed by flow cytometry. g A triplicate analysis of the experiment in f shows increased Annexin V binding in Andes-1537-treated cells, compared to controls (two-tailed Student’s t-test; Mean ± S.E.M.****p < 0.0001; ASO-C vs. Andes-1537; n = 3)

Fig. 2. ASK induces cell cycle arrest through downregulation of key cell cycle progression factors.

a MDA-MB-231 cells were transfected with 200 nM Andes-1537, ASO-C or left untreated (NT) for 24 h, stained with PI and cell cycle distribution was analyzed by flow cytometry. b A triplicate analysis of the experiment in a shows accumulation of cells in S phase and a decrease in the G2/M population, compared to controls (ASO-C vs. Andes-1537; two-tailed Student’s t-test, ***p < 0.001). c Representative results of Western blot analysis of cells treated as in a for 24 h. Surv, survivin; β-act, β-actin. Quantification of three independent experiments (n = 3) is shown for cyclin B1 (d), cyclin D1 (e), cyclin A1 (f), cyclin E1 (g), CDK1 (h), CDK4 (i) and survivin (j). d–j two-tailed Student’s t-test; (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ASO-C vs. Andes-1537). All graphs represent Mean ± S.E.M.

Fig. 3. The miRNAs hsa-miR-4485-5p, hsa-miR-4485-3p and hsa-miR-1973 are upregulated by ASK.

a Scheme depicting sequence and position of hsa-miR-4485-5p (pink), hsa-miR-4485-3p (teal), and hsa-miR-1973 (green) on the IR of ASncmtRNA-2. The A in brown represents a base that is contained in hsa-miR-4485-3p as well as in hsa-miR-1973. b Relative quantification of miRNAs by RT-qPCR (Taqman assay) is shown for hsa-miR-4485-5p, hsa-miR-4485-3p and hsa-miR-1973, in a triplicate analysis of three independent experiments (n = 3), in untreated cells (NT) or cells transfected with Andes-1537 or ASO-C at 24 h post-transfection (two-tailed Student’s t-test; Mean ± S.E.M. **p < 0.01; ****p < 0.0001; ASO-C vs. Andes-1537)

Fig. 4. Transfection of MDA-MB-231 cells with hsa-miR-4485-3p mimic induces downregulation of cyclin B1 and cyclin D1.

MDA-MB-231 cells were transfected for 48 h with control mimic (C), hsa-miR-1973 mimic (1973) or hsa-miR-4485-3p mimic (4485-3p) (n = 3). a miRNA upregulation was confirmed by Taqman RT-qPCR assays (two-tailed Student’s t-test; ****p < 0.0001; C vs. each specific mimic) (n = 3). b Relative total number of cells (two-tailed Student’s t-test; **p < 0.01, ***p < 0.001; C vs. each specific mimic; n = 3). c Representative results of Western blot analysis in mimic-transfected cells. Surv, survivin; β-act, β-actin. d–h, Graphical representation of the results of three independent experiments (n = 3) for cyclin B1 (d), cyclin D1 (e), survivin (f), CDK1 (g) and CDK4 (h) (two-tailed Student’s t-test; *p < 0.01, ***p < 0.001; C vs. each specific mimic). All graphs represent Mean ± S.E.M.

Fig. 5. ASK induces increase of nuclear-encoded miRNAs, which regulate CDK1 expression.

a Forty-eight hour after transfection of ASO-C or Andes-1537 or no treatment (NT), relative levels of hsa-miR-5096 and hsa-miR-3609 were determined by RT-qPCR (Taqman assays) (two-tailed Student’s t-test; **p < 0.01; ASO-C vs. Andes-1537; n = 3). b Putative binding sites for hsa-miR-5096 and hsa-miR-3609 on the mRNA 3’UTR of CDK1 and hsa-miR-5096 on CDK4, determined by TargetScanHuman. Bold lines represent potential 5’ seed region binding; solid lines depict Watson–Crick base pairing, broken lines show G:U wobble. c Relative quantification of hsa-miR-5096 and hsa-miR-3609 after transfection of mimics (two-tailed Student’s t-test; **p < 0.01; C vs. each specific mimic; n = 3). d Representative results of Western blot analysis for CDK1 and CDK4, using β-actin as loading control, in mimic-transfected cells at 48 h. e, f Graphical representation of triplicate analyses of the results shown in d, for CDK1 (e) and CDK4 (f) (two-tailed Student’s t-test; *p < 0.01; C vs. each specific mimic; n = 3). All graphs represent Mean ± S.E.M.

Fig. 6. ASK retards tumor growth and precludes primary tumor relapse in an MDA-MB-231 subcutaneous xenograft assay.

a Eleven Balb/c NOD/SCID mice were inoculated subcutaneously with 2.5 × 10⁶ cells and were then injected ip, in a blinded fashion, with ASO-C (six mice) or Andes-1537 (five mice) on days 12, 14, 16, 19, 21, 23, and 26 post-cell inoculation. Tumor size was monitored and the experiment was terminated on day 27. One mouse from the ASO-C group was sacrificed on day 23 for having reached the ethical limit for tumor size (#). **p < 0.005, two-tailed Student’s t-test, ASO-C vs. Andes 1537, day 27. b Fifteen mice were inoculated as in a and injected ip with ASO-C (five mice) or Andes-1537 (10 mice) on days 21, 23, 26, 30, 33, 35, 37, 40, 42, and 44 post-cell inoculation. Primary tumors were surgically resected on day 29. The experiment was terminated on day 51. **p < 0.005, ****p < 0.0001, two-tailed Student’s t-test, ASO-C vs. Andes-1537, day 51. Graphs represent Mean ± S.E.M.

Fig. 7. Hypothetical model for induction of proliferation blockage by ASK. ASncmtRNAs are produced in mitochondria.

The antisense oligonucleotide Andes-1537 binds to the single-stranded loop region of ASncmtRNA-2, creating a substrate for RNase H, which cleaves the transcript in this region. After processing by Dicer, mitochondrial miRNA hsa-miR-4485, and possibly others, are released. By an unknown mechanism, possibly an indirect effect of hsa-miR-4485 expression, several nuclear-encoded miRNAs are increased, mainly hsa-miR-5096 and hsa-miR-3609. In conjunction, all these miRNAs block translation of key cell cycle progression factors, resulting in a drastic inhibition of proliferation. Triggering of apoptosis is mediated by miRNAs targeting survival factors such as survivin