AnWei-sci (Shanghai, China) provided human breast cancer cell lines MCF-7 (AW-CELLS-H0213) and MDA-MB-231 (AW-CELLS-H0217). Short tandem repeat (STR) analysis was leveraged to validate the purity and ensure cell line stability. All cells underwent mycoplasma testing and were mycoplasma free. Cells were cultured in Dulbecco’s Modified Eagle Medium (C0891-100 mL) supplemented with 10% fetal bovine serum (C0251), 100 µg/mL streptomycin (C0222), and 100 U/mL penicillin. All reagents were provided by Beyotime (Shanghai, China).

MCF-7 and MDA-MB-231 cells were initially sorted for CD44⁺ cells using magnetic cell sorting (MACS; Miltenyi Biotech, Auburn, CA, USA) before executing the side population (SP) sorting in order to separate CD44⁺/CD24^– cell populations [20]. In MACS, cells were revived in 0.1% phosphate-buffered saline (PBS; C0221A)-bovine serum albumin (BSA, ST2254) from Beyotime (Shanghai, China). They were then labeled with phycoerythrin (PE)-conjugated CD24 antibody (555428, BD-PharMingen, San Diego, CA, USA) and fluorescein isothiocyanate (FITC)-conjugated CD44 antibody (555478, BD-PharMingen, San Diego, CA, USA)-phosphate-buffered saline (PBS; C0221A, Beyotime, Shanghai, China). Employing the “DOUBLE POSITIVE SORT” program on the AutoMACS system (Miltenyi Biotech, Auburn, CA, USA), cell suspension was filtered and sorted. The positive and negative fractions for SP were evaluated and sorted with a FACSCalibur flow cytometer (Becton Dickenson, Mountain View, CA, USA).

Following the directions of the maker, TRIzol reagent (R0016, Beyotime, Shanghai, China) was utilized to extract RNA. With a reverse transcription kit (D7168S, Beyotime, Shanghai, China), 500 mg RNA was reversely transcribed into cDNA (10 µL). qPCR was conducted to quantitate lncRNAs MIR155HG and RelA on the Step One Plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using SYBR Premix Ex Taq™ II (Q331-02/03, Vazyme, Nanjing, China) in three independently repeated experiments. Expression analyses were achieved by 2^{-Δ⁢Δ⁢Ct} method. Sangon Biotech (Shanghai, China) created primers, with the sequences below: lncRNA MIR155HG forward (F), 5^′-ACGGTTGTGCGAGCAGAGAATCTA-3^′ and reverse (R), 5^′-CTCATCTAAGCCTCACAACAACCT-3^′; RelA F, 5^′-CTTCCTCAGCCATGGTACCTCT-3^′ and R, 5^′-CAAGTCTTCATCAGCATCAAACTG-3^′; Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) F, 5^′-CCATGGGGAAGGTGAAGGTC-3^′ and R, 5^′-AGTGATGGCATGGACTGTGG-3^′.

MCF-7 and MDA-MB-231 stem cells were divided into the following groups: Blank, short hairpin RNA negative control (shNC), short hairpin RNA targeting MIR155HG (shMIR155HG), vector, RelA, shNC+vector, shMIR155HG+vector, shNC+RelA, and shMIR155HG+RelA. GenePharma (Shanghai, China) supplied shMIR155HG and shNC. The pcDNA3.1-RelA (RelA) and control empty vector plasmids (vector) were acquired from GeneChem (Shanghai, China). Cell transfection with 1 µg shRNA or 2 µg plasmid was completed utilizing Lipofectamine 2000 (11668019, Invitrogen, Carlsbad, CA, USA) (48 h, 37 ℃) for later trials. The sequences were as follows: shMIR155HG-1, 5^′-GCAGATAACTTGTCTGCATTTCAAGAGAATGCAGACAAGTTATCTGCTTTTTT-3^′; shMIR155HG-2, 5^′-GCATTCACATGGAACAAATTTCAAGAGAATTTGTTCCATGTGAATGCTTTTTT-3^′; shNC, 5^′-GCACCCAGTCCGCCCTGAGCAAATTCAAGAGATTTGCTCAGGGCGGACTGGGTGCTTTTT-3^′.

Equal densities of cells were planted onto 60 mm cell culture dishes, and cultured for 4–10 h (37 ℃) to promote adhesion. Following that, cells received X-ray radiation (0, 2, 4, and 6 Gy) utilizing an RS2000 X-ray biological research irradiator (Rad Source Technologies, Suwanee, GA, USA) [21]. To facilitate colony development, cell incubation was conducted for 10–14 days. The colonies underwent fixation (methanol, 322415, Sigma-Aldrich, St. Louis, MO, USA) and staining (0.1% crystal violet; C0121, Beyotime, Shanghai, China). At least fifty cells in a colony were counted. Surviving fraction = the number of colonies/(cells inoculated $\times$ plating efficiency).

Using lysis buffer (KGC4901; Keygene, Jiangsu, China), total protein from the cells was determined via BCA protein assay kit (P0010; Beyotime, Shanghai, China). For sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE, KGC4901; Keygene, Jiangsu, China), 40 µg of protein per lane was loaded and separated on 10% gels, followed by being transferred onto polyvinylidene fluoride (PVDF) membranes (KGC4806; Keygene, Jiangsu, China). Membranes experienced 1-h blockage at room temperature (RT) using 5% non-fat milk in Tris-Buffered Saline with Tween-20 (TBST, ST825; Beyotime, Shanghai, China), and cultivation with primary antibodies (4 ℃, overnight) obtainable from Abcam (Cambridge, UK) except RelA (65 kDa; 1:1000, AM06378SU-N, Origene, Rockville, MD, USA), incorporating $\beta$-catenin (85 kDa; 1:400, ab224803), Nanog (37 kDa; 1:1000, ab109250), SRY-Box transcription factor 2 (SOX2, 35 kDa; 1:1000, ab92494), phosphorylated histone variant H2A.X ($\gamma$H2A-X, 15 kDa; 1:5000, ab81299), BCL2-Associated X Protein (Bax, 21 kDa; 1:1000, ab32503), B-cell lymphoma-2 (Bcl-2, 26 kDa; 1:2000, ab182858), Cleaved Caspase 3 (17 kDa; 1:500, ab32042), Caspase 3 (35 kDa; 1:5000, ab32351), and GAPDH (36 kDa; 1:1000, ab8245). Next, membranes were incubated (1 h, RT) with horseradish peroxidase (HRP)-conjugated secondary antibodies (Abcam, Cambridge, UK), Goat Anti-Mouse Immunoglobulin G (IgG, ab205719; 1:2000)/Goat Anti-Rabbit IgG (ab6721; 1:2000). Using gel imaging equipment (Chemi Doc MP, Bio-Rad, Hercules, CA, USA), protein bands were observed through the Pierce ECL Plus Western Blotting Substrate (KGC4902; Keygene, Jiangsu, China). With GAPDH as the loading control and ImageJ 5.2.1 software (NIH, Bethesda, MD, USA), protein production was measured and normalized.

Cells were seeded at equal densities onto glass slides and incubated (37 ℃, 4–10 h) to allow for attachment. After that, cells underwent exposure to 2 Gy X-rays for 24 h. Immunofluorescence staining was carried out employing a DNA damage detection kit ($\gamma$H2A-X Immunofluorescence Assay) (C2036S; Beyotime, Shanghai, China) in compliance with the guidelines. Simply put, PBS-washed cells were fixed with fixative (15 min; C2036S-1, Beyotime, Shanghai, China), blocked with blocking solution (20 min; C2036S-3, Beyotime, Shanghai, China), and incubated with $\gamma$H2A-X antibody (1:250; C2036S-4, Beyotime, Shanghai, China) (4 ℃, overnight), and rabbit Cy3 secondary antibody (1:500; C2036S-5, Beyotime, Shanghai, China) (RT, 1 h). After washing, cell nucleus was stained with 4,6-diamino-2-phenyl indole (DAPI) (5 min, RT; C2036S-6, Beyotime, Shanghai, China), and images were obtained employing FluoView^TM FV1000 confocal laser scanning microscope (200$\times$, Olympus, Tokyo, Japan).

With the goal to create spheres, single-cell suspensions (30,000 cells/well) were cultivated in serum-free Minimum Essential Medium (MEM, 11058021) with 20 ng/mL bFGF, 20 ng/mL EGF, and B27 (all from Thermo Fisher Scientific, Waltham, MA, USA) in 100 mm ultra-low attachment plates (4615, Corning Inc., Corning, NY, USA). The development of spheres was captured by an inverted microscope (200$\times$, Olympus, Tokyo, Japan).

Following 24-h exposure to 2 Gy X-rays, cells experienced color development by Annexin V-FITC and PI (FITC Annexin V Apoptosis Detection Kit, C1062S, Beyotime, Shanghai, China), followed by cell cycle analysis exploiting TaliTM Cell Cycle Kit (A10798, Thermo Fisher Scientific, Waltham, MA, USA). Measurements of apoptosis and cell cycle were completed utilizing a flow cytometer, follows by result evaluation with CellQuest software (version 5.1, BD Biosciences, San Jose, CA, USA).

Transcription factor-binding motifs were predicted using the JASPAR database (https://jaspar.elixir.no/). MIR155HG-wild type (WT) and mutant (MUT) sequences (wild and mutated sequences of the binding site between MIR155HG and RELA) were cloned into a luciferase reporter vector (16147, Promega, Madison, WI, USA). With Lipofectamine 2000, MIR155HG-WT or MIR155HG-MUT and RELA were co-transfected into MDA-MB-231 and MCF-7 stem cells. Overnight cell lysis was followed by luciferase activity assessment employing a luminometer (Bio-Rad, Hercules, CA, USA) and a Dual-Luciferase Reporter Assay Kit (E1910, Promega, Madison, WI, USA).

ChIP Assay Kit (17-295, Millipore, Temecula, CA, USA) was purchased in advance. Briefly, cells were lysed on ice for 10 min. To extract 200–1000 bp DNA fragments from cell lysates, cells underwent seven times of 5-second pulses on ice with a Sonicator 3000 (Misonix, Farmingdale, NY, USA). An agarose bead mixture, ChIP buffer, and a protease inhibitor cocktail were used to pre-clear cell lysates for 1 h on ice. The lysates were treated (12 h, 4 ℃) with either normal rabbit IgG (2729, 1:50) or RelA antibody (8242, 1:100) from Cell Signaling Technology (Danvers, MA, USA). The supernatant containing 60 µL Protein A Agarose/Salmon Sperm DNA was added and spun (1 h, 4 °C). Wash buffers containing low salt (150 mM), high salt (500 mM), LiCl, and TE buffer were applied to wash the beads. After elution of the complexes using elution buffer, 5 M NaCl was used to reverse the cross-links, and the mixture experienced 4-h incubation (65 ℃). Following RNase A treatment, samples were collected for qPCR to gauge the levels of the MIR155HG promoter. The primer sequences used were F 5^′-GGTCTCCAGCTGATTCGGTC-3^′ and R 5^′-CCAGGAGCGTCTCCTTGGTT-3^′.

Each experiment was independently repeated three times. Statistical studies were carried out with the aid of GraphPad Prism 8.0 (GraphPad Software, Boston, MA, USA). Data were displayed as mean $\pm$ standard deviation (SD). Two-group and multi-group comparisons were accomplished utilizing an independent sample t-test, and one-way or two-way ANOVA followed by Tukey’s post-hoc test, respectively. p 0.05 reflected statistically significant differences.

Through flow cytometry, we first separated stem cells from breast cancer cell lines, and detected CD44⁺/CD24^– cells $>$80% in MDA-MB-231 cells and 10% in MCF-7 cells (Fig. 1A). Next, we performed RT-qPCR to ascertain MIR155HG expression level in stem and non-stem cells. MIR155HG mRNA expression was greater in BCSCs than non-stem cells, according to RT-qPCR data (p 0.001, Fig. 1B,C). Next, MDA-MB-231 and MCF-7 stem cells were transfected with shMIR155HG-1 and shMIR155HG-2 to knock down MIR155HG, where MIR155HG expression was confirmed to be decreased (p 0.001, Fig. 1D,E). The shMIR155HG-1, which exhibited the most evident knockdown efficacy, was selected for further experiments. Different radiation dosages were applied to these cell lines, colony formation assay confirmed knockdown of MIR155HG conferred greater sensitivity to X-rays irradiation treatment in the MIR155HG-knockout MDA-MB-231 and MCF-7 stem cells compared to their parental cells (Fig. 1F–H). Combined with reference to a previous study [21], 2 Gy X-rays was selected for the following experiment.

Fig. 1.

Long non-coding RNA MIR155HG is highly expressed in Breast cancer stem cells (BCSCs), and knockdown of MIR155HG reduces cellular radioresistance. (A) FITC-CD44- and PE-CD24-stained MDA-MB-231 and MCF-7 cells (flow cytometry). (B,C) MIR155HG messenger RNA (mRNA) levels in MDA-MB-231 and MCF-7 stem and non-stem cells (reverse transcription quantitative polymerase chain reaction (RT-qPCR)). (D,E) MIR155HG mRNA levels in short hairpin RNA targeting MIR155HG (sh-MIR155HG)-transfected MCF-7 and MDA-MB-231 stem cells (RT-qPCR). (F) Representative images of MIR155HG-silencing MDA-MB-231 and MCF-7 stem cells under radiation (colony formation assays). (G,H) Survival curves of MIR155HG-silencing MDA-MB-231 and MCF-7 stem cells under radiation. n = 3. ^*⁣**p 0.001 compared to non-stem cells. ⁺p 0.05, ^+⁣++p 0.001 compared to short hairpin RNA negative control (shNC).

One day after 2 Gy X-ray irradiation, we detected the expression of $\gamma$H2A-X, a significant effector of DNA damage, in MDA-MB-231 and MCF-7 stem cells to clarify how MIR155HG increases radioresistance in stem cells. Based on Western blot data, MIR155HG knockdown resulted in higher $\gamma$H2A-X protein levels (p 0.01, Fig. 2A,B), as well as lower levels of Bcl-2/Bax (p 0.05, Fig. 2C) and higher levels of Cleaved Caspase 3/Caspase 3 proteins (p 0.001, Fig. 2D,E). Immunofluorescence staining results suggested that MIR155HG knockdown was associated with higher amounts of $\gamma$H2A-X (red) (p 0.05, Fig. 2F,G).

Fig. 2.

Knockdown of MIR155HG reduces DNA damage repair and promotes apoptosis protein expression in BCSCs. (A–E) Phosphorylated histone variant H2A.X ($\gamma$H2A-X), BCL2-Associated X Protein (Bax), B-cell lymphoma-2 (Bcl-2), Caspase 3 and Cleaved Caspase 3 protein levels in sh-MIR155HG-transfected MCF-7 and MDA-MB-231 stem cells after 2 Gy X-ray irradiation for 24 h (Western blot, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control). (F,G) $\gamma$H2A-X levels in sh-MIR155HG-transfected MCF-7 and MDA-MB-231 stem cells after 2 Gy X-ray irradiation for 24 h (Immunofluorescence). Red fluorescence represents $\gamma$H2A-X and blue fluorescence indicates DAPI cell nuclear staining. Magnification 200$\times$, scale bar 100 µm. n = 3. ⁺p 0.05, ⁺⁺p 0.01, ^+⁣++p 0.001 compared to shNC.

Next, following 2 Gy X-ray irradiation, we assessed the development of tumor spheres in MDA-MB-231 and MCF-7 stem cells. The tumor sphere-forming ability was weaker in sh-MIR155HG group than shNC group (p 0.01, Fig. 3A–C). Additionally, $\beta$-catenin, Nanog, and SOX2 protein levels were lower in sh-MIR155HG group versus shNC group (p 0.05, Fig. 3D–G).

Fig. 3.

Knockdown of MIR155HG reduces stemness and inhibits Wnt-related protein expression in BCSCs. (A–C) Stemness in sh-MIR155HG-transfected MCF-7 and MDA-MB-231 stem cells after 2 Gy X-ray irradiation for 24 h (Sphere formation assay). Magnification 200$\times$, scale bar 100 µm. (D–G) $\beta$-catenin, Nanog, and SRY-Box transcription factor 2 (SOX2) protein levels in sh-MIR155HG-transfected MCF-7 and MDA-MB-231 stem cells after 2 Gy X-ray irradiation for 24 h (western blot, GAPDH as an internal control). n = 3. ⁺p 0.05, ⁺⁺p 0.01, ^+⁣++p 0.001 compared to shNC.

To further assess whether MIR155HG regulates cellular apoptosis and cycle induction in response to radiation, we examined apoptosis and cell cycle profiles in MDA-MB-231 and MCF-7 stem cells after X-ray irradiation (2 Gy). Sh-MIR155HG group exhibited increased apoptosis (p 0.001, Fig. 4A–C), as well as a decrement in G2 phase cells and increment in G0/G1 phase cells (p 0.001, Fig. 4D–F).

Fig. 4.

Knockdown of MIR155HG promotes apoptosis and induces G1 phase arrest in BCSCs. (A–C) Apoptosis in sh-MIR155HG-transfected MCF-7 and MDA-MB-231 stem cells after 2 Gy X-ray irradiation for 24 h (flow cytometry). (D–F) Cell cycle in sh-MIR155HG-transfected MCF-7 and MDA-MB-231 stem cells after 2 Gy X-ray irradiation for 24 h (flow cytometry). n = 3. ^+⁣++p 0.001 compared to shNC.

By means of JASPAR analysis, we identified four potential binding sites for the NF-$\kappa$B subunit RelA within the MIR155HG promoter region. Among them, the site located at -1828/-1837 showed higher score in JASPAR analysis and was used for subsequent analysis (Fig. 5A,B). Furthermore, RelA significantly enhanced luciferase activity in MIR155HG-WT cells (p 0.001, Fig. 5C,D). ChIP analysis confirmed the binding of RelA to MIR155HG promoter (p 0.001, Fig. 5E). Overexpression of RelA markedly upregulated RelA levels in stem cells (p 0.001, Fig. 5F–I).

Fig. 5.

Nuclear factor kappa B (NF-$\kappa$B) subunit RelA transcriptionally activates MIR155HG to promote radioresistance in BCSCs. (A) RelA binding motifs (JASPAR analysis). (B) Potential binding sequences between RelA and the MIR155HG promoter (JASPAR prediction). (C,D) Luciferase activity in MCF-7 and MDA-MB-231 stem cells with RelA overexpression at the MIR155HG promoter (Dual-luciferase reporter gene assay). (E) Interaction between RelA and MIR155HG promoter (Chromatin immunoprecipitation (ChIP) assay). (F–I) RelA expression in MCF-7 and MDA-MB-231 stem cells overexpressing RelA (RT-qPCR and western blot). n = 3. *p 0.05, ***p 0.001 compared to NC. ^+⁣++p 0.001 compared to immunoglobulin G (IgG). ^^^p 0.001 compared to vector.

Following 2 Gy X-ray treatment for 24 h, colony survival rates were reduced in the shMIR155HG+vector group, but were elevated in the shNC+RelA group (p 0.05, Fig. 6A–C). In the shMIR155HG+RelA group, colony survival rates were greater than those in the shMIR155HG+vector group but lower than those in the shNC+RelA group (p 0.05, Fig. 6A–C). Following 2 Gy X-ray treatment for 24 h, compared to the shNC+vector group, MDA-MB-231 and MCF-7 stem cells in the shMIR155HG+vector group had upregulated $\gamma$H2A-X and Cleaved Caspase 3/Caspase 3 protein, and downregulated Bcl-2/Bax (p 0.05, Fig. 6D–H). Conversely, in the shNC+RelA group, protein levels of $\gamma$H2A-X and Cleaved Caspase 3/Caspase 3 declined, while Bcl-2/Bax levels rose (p 0.05, Fig. 6D–H). In the shMIR155HG+RelA group, $\gamma$H2A-X and Cleaved Caspase 3/Caspase 3 proteins levels were lower than those in the shMIR155HG+vector group but greater than those in the shNC+RelA group, while the opposite trends were detected in the levels of Bcl-2/Bax (p 0.05, Fig. 6D–H). Immunofluorescence staining results confirmed $\gamma$H2A-X (red) in stem cells was increased in the shMIR155HG+vector group (p 0.001, Fig. 7A–C), yet decreased in the shNC+RelA group (Fig. 7A–C), compared to the shNC+vector group. In the shMIR155HG+RelA group, $\gamma$H2A-X levels were higher than those in the shNC+RelA group (p 0.05), but lower than those in the shMIR155HG+vector group (p 0.001, Fig. 7A–C).

Fig. 6.

NF-$\kappa$B subunit RelA upregulates MIR155HG to inhibit $\gamma$H2A-X and apoptosis-related protein expression in BCSCs. (A–C) Representative images of survival rates of MDA-MB-231 and MCF-7 stem cells in shNC+vector, shMIR155HG+vector, shNC+RelA, and shMIR155HG+RelA groups after 2 Gy X-ray treatment for 24 h (colony formation assay). (D–H) $\gamma$H2A-X, Bax, Bcl-2, Caspase 3, and Cleaved Caspase 3 protein expressions in MDA-MB-231 and MCF-7 stem cells in shNC+vector, shMIR155HG+vector, shNC+RelA, and shMIR155HG+RelA groups after 2 Gy X-ray treatment for 24 h (western blot). n = 3. ^#p 0.05, ^#⁢#p 0.01, ^#⁢#⁢#p 0.001 compared to shNC+vector. ^Δp 0.05, ^Δ⁢Δ⁢Δp 0.001 compared to shMIR155HG+vector. ^&p 0.05, ^&⁣&&p 0.001 compared to shNC+RelA.

Fig. 7.

NF-$\kappa$B subunit RelA upregulates MIR155HG to promote DNA damage repair in BCSCs. (A–C) $\gamma$H2A-X levels in MDA-MB-231 and MCF-7 cells in shNC+vector, shMIR155HG+vector, shNC+RelA, and shMIR155HG+RelA groups after 2 Gy X-ray treatment for 24 h (Immunofluorescence). Red fluorescence represents $\gamma$H2A-X and blue fluorescence indicates DAPI cell nuclear staining. Magnification 200$\times$, scale bar 100 µm. n = 3. ^#⁢#⁢#p 0.001 compared to shNC+vector. ^Δ⁢Δ⁢Δp 0.001 compared to shMIR155HG+vector. ^&p 0.05 compared to shNC+RelA.

Sphere formation assays revealed that following 2 Gy X-ray treatment for 24 h, tumor sphere was reduced in shMIR155HG+vector group yet increased in shNC+RelA group (p 0.05, Fig. 8A–C). Tumor sphere formation ability was potentiated in shNC+RelA group relative to shNC+vector group, but weakened in shMIR155HG+RelA group compared to shNC+RelA group (p 0.05, Fig. 8A–C). Western blot data revealed in contrast to shNC+vector group, the expressions of $\beta$-catenin, Nanog, and SOX2 were decreased in shMIR155HG+vector group yet increased in shNC+RelA group (p 0.05, Fig. 8D–G). These protein levels in shMIR155HG+RelA group were higher than those in shMIR155HG+vector group, but lower than those in shNC+RelA group (p 0.05, Fig. 8D–G).

Fig. 8.

MIR155HG Knockdown reverses NF-$\kappa$B subunit RelA-induced stemness and regulates Wnt-related protein expression in BCSCs. (A–C) Stemness in MDA-MB-231 and MCF-7 cells in shNC+vector, shMIR155HG+vector, shNC+RelA, and shMIR155HG+RelA groups after 2 Gy X-ray treatment for 24 h (sphere formation assay). Magnification 200$\times$, scale bar 100 µm. (D–G) $\beta$-catenin (E), Nanog (F), and SOX2 (G) protein expressions in MDA-MB-231 and MCF-7 stem cells in shNC+vector, shMIR155HG+vector, shNC+RelA, and shMIR155HG+RelA groups after 2 Gy X-ray treatment for 24 h (western blot, GAPDH as an internal control). n = 3. ^#p 0.05, ^#⁢#p 0.01, ^#⁢#⁢#p 0.001 compared to shNC+vector. ^Δp 0.05, ^Δ⁢Δp 0.01, ^Δ⁢Δ⁢Δp 0.001 compared to shMIR155HG+vector. ^&p 0.05, ^&&p 0.01, ^&⁣&&p 0.001 compared to shNC+RelA.

Flow cytometry analysis showed that following 2 Gy X-ray treatment for 24 h, apoptosis was facilitated in stem cells in shMIR155HG+vector group, but dampened in shNC+RelA group (p 0.01, Fig. 9A–C). Relative to shMIR155HG+RelA group, shNC+RelA group had weaker while shMIR155HG+vector group had stronger apoptosis ability (p 0.001, Fig. 9A–C). MDA-MB-231 stem cells in the shMIR155HG+vector group had more G0/G1 phase cells and less G2/M phase cells in comparison to the shNC+vector group; in contrast, the shNC+RelA group displayed more S phase cells (p 0.05, Fig. 9D,E). Compared to shMIR155HG+RelA group, shMIR155HG+vector group had more cells in the G0/G1 phase and less cells in the G2/M phase (p 0.001, Fig. 9D,E); similarly, cells in the S phase were more in shNC+RelA group (p 0.05, Fig. 9D,E). In the shMIR155HG+vector group, MCF-7 stem cells displayed more G0/G1 phase cells and less G2/M phase cells relative to the shNC+vector group; in contrast, the shNC+RelA group displayed more S and G2/M phase cells and less G0/G1 phase cells (p 0.01, Fig. 9D,F). Compared to shMIR155HG+RelA group, shNC+RelA group had less cells in the G0/G1 phase and more cells in the S phase and G2/M stage (p 0.05, Fig. 9D,F), while shMIR155HG+vector group had more cells in the G0/G1 phase and less cells in G2/M stage (p 0.001, Fig. 9D,F).

Fig. 9.

MIR155HG knockdown reverses NF-$\kappa$B subunit RelA-suppressed apoptosis and G1 phase arrest in BCSCs. (A–C) Apoptosis in MDA-MB-231 and MCF-7 cells in shNC+vector, shMIR155HG+vector, shNC+RelA, and shMIR155HG+RelA groups after 2 Gy X-ray treatment for 24 h (flow cytometry). (D–F) Cell cycle in MDA-MB-231 and MCF-7 stem cells in shNC+vector, shMIR155HG+vector, shNC+RelA, and shMIR155HG+RelA groups after 2 Gy X-ray treatment for 24 h (Flow cytometry). Each experiment was independently repeated three times. ^#p 0.05, ^#⁢#p 0.01, ^#⁢#⁢#p 0.001 compared to shNC+vector. ^Δ⁢Δ⁢Δp 0.001 compared to shMIR155HG+vector. ^&p 0.05, ^&⁣&&p 0.001 compared to shNC+RelA.