Circ_RPPH1 facilitates progression of breast cancer via miR-1296-5p/TRIM14 axis

Abstract

Circular RNA Ribonuclease P RNA Component H1 (circ_RPPH1) and microRNA (miRNA) miR-1296-5p play a crucial role in breast cancer (BC), but the molecular mechanism is vague. Evidence showed that miR-1296-5p can activate tripartite motif-containing 14 (TRIM14). Clinical indications of eighty BC patients were collected and the circ_RPPH1 expression was detected using real-time quantitative PCR. MCF-7 and MDA-MB-231 cells were transfected with overexpression or knockdown of circ_RPPH1, miR-1296-5p, or TRIM14. Cell counting kit-8, cell cloning formation, wound healing, Transwell, and flow cytometry assays were performed to investigate the malignant phenotype of BC. The dual-luciferase reporter gene analyses were applied to reveal the interaction between these target genes. Subcutaneous tumorigenic model mice were established with circ_RPPH1 overexpression MDA-MB-231 cells in vivo; the tumor weight and volume, levels of miR-1296-5 and TRIM14 mRNA were measured. Western blot and immunohistochemistry were used to detect TRIM14 in cells and mice. Circ_RPPH1 levels were notably higher in BC patients and have been found to promote cell proliferation, invasion, and migration of BC cells. Circ_RPPH1 altered cell cycle and hindered apoptosis. Circ_RPPH1 knockdown or miR-1296-5p overexpression inhibited the malignant phenotype of BC. Furthermore, miR-1296-5p knockdown reversed circ_RPPH1's promotion effects on BC. Interestingly, TRIM14 overexpression counteracts the inhibitory effects of miR-1296-5p overexpression and circ_RPPH1 silencing on BC. Moreover, in BC tumor-bearing mice, circ_RPPH1 overexpression led to increased TRIM14 expression and facilitated tumor growth. Circ_RPPH1 enhanced BC progression through miR-1296-5p/TRIM14 axis, indicating its potential as a biomarker and therapeutic target in BC.

Keywords: Breast cancer; circular RNA ribonuclease P RNA component H1; microRNA-1296-5p; tripartite motif-containing 14.

Figure 1.

Circ_RPPH1 overexpression promoted malignant phenotype of BC cells. (a) Real-time quantitative PCR (RT-qPCR) was used to detect circ_RPPH1 expression in patients with BC; circ_RPPH1 mRNA expression in BC tissues was higher than in para-carcinoma tissues (n = 80). (b-c) Cell counting kit-8 assay was used to measure the cell viability of BC cells; circ_RPPH1 overexpression [circ_RPPH1(+)] increased cell viability of MCF-7 cells and MDA-MB-231 cells (n = 6). The cell viability (%) = (OD₄₅₀ of the experimental group – average OD₄₅₀ of the blank group)/(average OD₄₅₀ of the Control group – average OD₄₅₀ of the blank group) × 100%. (d-e) Cell cloning formation assay was used to detect the proliferation of BC cells; circ_RPPH1 (+) promoted proliferation of MCF-7 cells and MDA-MB-231 cells (n = 3). (f-g) Cell wound healing assay was used for cell migration detection; circ_RPPH1 (+) promoted migration of MCF-7 cells and MDA-MB-231 cells (n = 3, scale bar = 200 μm). The cell migration rate (%) = (0 h scratch width −24 h scratch width)/0 h scratch width × 100%. (h-i) Transwell assay was used to detect the invasion of BC cells; circ_RPPH1 (+) promoted invasion of MCF-7 cells and MDA-MB-231 cells (n = 3, scale bar = 50 μm). Data were presented as the mean ± SD. **p < .01 vs. the para-carcinoma tissue; ^▲▲p < .01 vs. the Control group.

Figure 2.

Circ_RPPH1 overexpression promoted BC cell entry into S phase and inhibited cell apoptosis. (a-c) Flow cytometry was used to detect the cycle of BC cells; circ_RPPH1 overexpression [circ_RPPH1(+)] decreased cells in the G0/G1 and G2/M phase, as well as promoting cells entry into the S phase in MCF-7 cells and MDA-MB-231 cells. (d-e) Flow cytometry was also used to detect the apoptosis; circ_RPPH1 (+) inhibited apoptosis in MCF-7 cells and MDA-MB-231 cells. (f) Western blot assay was used to detect TRIM14 protein expression. Circ_RPPH1 (+) increased TRIM14 protein expression in MCF-7 and MDA-MB-231 cells. Data were presented as the mean ± SD, n = 3. ^▲▲p < .01 vs. the Control group.

Figure 3.

Circ_RPPH1 silencing inhibited BC cell malignant phenotype by increasing miR-1296-5p. (a) The predicted results of the binding situation of circ_RPPH1 and miR-1296-5p. (b) The luciferase activities in BC cells co-transferred with wild-type (WT) or mutant (Mut) circ_RPPH1 plasmid together with miR-1296-5p mimic or mic-NC (n = 3). (c-d) Results of RT-qPCR verified the silencing of circ_RPPH1 [circ_RPPH1(-)] and miR-1296-5p [miR-1296-5 (-)] in BC cells (n = 3). (e-f) Circ_RPPH1 (-) inhibited cell viability, while miR-1296-5p (-) treatment reversed it (n = 6). (g, h) Results of cell cloning formation assay; (i, j) Results of wound healing assay; (k, l) Results of Transwell assay; they respectively revealed the inhibitory effects of circ_RPPH1 (-) on BC cell proliferation, migration, and invasion, while miR-1296-5p (-) treatment reversed it (n = 3, scale bar = 200 μm or 50 μm). Data were presented as the mean ± SD. ^▲p < .05, ^▲▲p < .01 vs. the Control group; ^★p < .05, ^★★p < .01 vs. the circ_RPPH1 silencing (-) group.

Figure 4.

Circ_RPPH1 silencing blocked the G0/G1 phase and inhibited apoptosis in BC cells by increasing miR-1296-5p. (a-c) the effects of circ_RPPH1 silencing [circ_RPPH1(-)] and miR-1296-5p silencing [circ_RPPH1(-)] of the cell cycle in BC cells. Circ_RPPH1 silencing blocked the G0/G1 phase. (d-e) Circ_RPPH1 (-) promoted apoptosis in BC cells, but miR-1296-5p (-) reversed it. (f) Western blot assay was used to detect TRIM14 protein expression. Circ_RPPH1 (-) inhibited TRIM14 protein expression in MCF-7 and MDA-MB-231 cells, while mir-1296-5p (-) antagonized this. Data were presented as the mean ± SD, n = 3. ^▲p < .05, ^▲▲p < .01 vs. the Control group; ^★★p < .01 vs. the circ_RPPH1 (-) group.

Figure 5.

MiR-1296-5p overexpression inhibited BC cell malignant phenotype via TRIM14. (a) The predicted results of the binding situation of miR-1296-5p and TRIM14. (b) The luciferase activities in BC cells co-transferred with WT or Mut TRIM14 plasmid together with miR-1296-5p mimic or mic-NC (n = 3). (c-d) the overexpressing miR-1296-5p [miR-1296-5 (+)] in MCF-7 and MDA-MB-231 cells inhibited cell viability while the overexpressing TRIM14 [TRIM14 (+)] reversed it (n = 6). (e, f) Results of cell cloning formation assay; (g, h) Results of wound healing assay; (i, j) Results of Transwell assay; they were respectively revealed the inhibitory effects of miR-1296-5 (+) on the proliferation, migration, and invasion of MCF-7 and MDA-MB-231 cells, as well as and TRIM14 (+) reversed it (n = 3, scale bar = 200 μm or 50 μm). Data were presented as the mean ± SD. ^▲p < .05, ^▲▲p < .01 vs. the Control group; ^★★p < .01 vs. the miR-1296-5p (+) group.

Figure 6.

MiR-1296-5p overexpression blocked the G0/G1 phase and promoted apoptosis in BC cells. (a-c) Flow cytometry was used to detect the cycle of BC cells; The overexpressing miR-1296-5p [miR-1296-5 (+)] increased percentage of GO/G1 cells in MCF-7 and MDA-MB-231 cells. (d-e) Flow cytometry was used to detect apoptosis of BC cells; miR-1296-5 (+) promoted cell apoptosis. Overexpressing TRIM14 [TRIM14 (+)] reversed them. Data were presented as the mean ± SD, n = 3. ^▲p < .05, ^▲▲p < .01 vs. the Control group; ^★★p < .01 vs. the miR-1296-5p (+) group.

Figure 7.

Circ_RPPH1 overexpression promoted BC progression in mice. (a-c) the BC tumor size and weight in mice (n = 5). The tumor volume = length × width²/2. (d, e) the levels of miR-1296-5p and TRIM14 mRNA were detected by real-time quantitative PCR (n = 3). (f, g) Immunohistochemistry used to observe TRIM14 expression in BC tumor (n = 3, scale bar = 100 μm). (h, i) Western blot was used to detect TRIM14 expression levels in BC tumor. (h) Representative protein bands of TRIM14; (i) the semi-quantitative statistical analysis results of Western blot (n = 3). Data were presented as the mean ± SD. ^▲▲p < .01 vs. the Control group. circ_RPPH1(+) group indicated circ_RPPH1 overexpression group.