Transcription factor STAT4 counteracts radiotherapy resistance in breast carcinoma cells by activating the MALAT1/miR-21-5p/THRB regulatory network

Abstract

Considering the limited research and the prevailing evidence of STAT4's tumor-suppressing role in breast carcinoma (BC) or in breast radiotherapy (RT) sensitivity requires more in-depth exploration. Our study delves into how STAT4, a transcription factor, affects BC cell resistance to radiotherapy by regulating the MALAT1/miR-21-5p/THRB axis. Bioinformatics analysis was performed to predict the regulatory mechanisms associated with STAT4 in BC. Subsequently, we identified the expression profiles of STAT4, MALAT1, miR-21-5p, and THRB in various tissues and cell lines, exploring their interactions and impact on RT resistance in BC cells. Moreover, animal models were established with X-ray irradiation for further validation. We discovered that STAT4, which is found to be minimally expressed in breast carcinoma (BC) tissues and cell lines, has been associated with a poorer prognosis. In vitro cellular assays indicated that STAT4 could mitigate radiotherapy resistance in BC cells by transcriptional activation of MALAT1. Additionally, MALAT1 up-regulated THRB expression by adsorbing miR-21-5p. As demonstrated in vitro and in vivo, overexpressing STAT4 inhibited miR-21-5p and enhanced THRB levels through transcriptional activation of MALAT1, which ultimately contributes to the reversal of radiotherapy resistance in BC cells and the suppression of tumor formation in nude mice. Collectively, STAT4 could inhibit miR-21-5p and up-regulate THRB expression through transcriptional activation of MALAT1, thereby mitigating BC cell resistance to radiotherapy and ultimately preventing BC development and progression.

Keywords: Breast carcinoma; MALAT1; STAT4; THRB; long non-coding RNA; microRNA-21-5p; radiotherapy resistance; transcription factor.

Figure 1

STAT4 expression in BC tissues. A, B: STAT4 mRNA and protein levels in cancerous and normal paracancerous tissues of BC patients by RT-qPCR and Western blot, n = 66, N indicates normal paracancerous tissue, C indicates BC tissue, * indicates comparison with normal paracancerous tissue, *P < 0.05; C: Kaplan-Meier curves display the correlation of STAT4 with patients’ overall survival.

Figure 2

STAT4 expression was downregulated in radiation therapy resistant BC cells. A, B: STAT4 mRNA and protein levels in normal human MCF-10A mammary epithelial cells and four human BC cell lines (SKBR3, MDA-MB-231, MCF-7 and MDA-MB-468) by RT-qPCR and Western blot (WB); *P < 0.05 versus HBL-100 cells; C, D: RT-qPCR and WB for STAT4 mRNA and protein levels in MCF-7 at different time points after single-dose X-ray irradiation, *P < 0.05 versus 0 Gy group; E, F: STAT4 mRNA and protein levels in MCF-7 after X-ray irradiation of different doses by RT-qPCR and WB; G, H: RT-qPCR and WB for STAT4 mRNA and protein levels in MCF-7 and its corresponding radioresistant cell line MCF-7R, *P < 0.05 versus MCF-7 group; I, J: STAT4 mRNA and protein levels in MCF-7R in each group by RT-qPCR and WB, *P < 0.05 versus oe-NC group.

Figure 3

STAT4 overexpression could reverse radiation therapy resistance. A: Cell survival rate by MTT assay, * denotes P < 0.05; B: Plate cloning assay to detect cell proliferation ability, * denotes P < 0.05; C: Flow cytometry to detect apoptosis, * denotes P < 0.05; D: Plate cloning assay to detect changes in MCF-7R proliferating capacity in each group, * denotes P < 0.05.

Figure 4

Impact of STAT4 on resistance to radiotherapy through regulation of MALAT1 expression in breast carcinoma (BC) cells. A: Co-expression relationship between STAT4 and MALAT1 in BC; B: RT-qPCR to quantify MALAT1 levels in BC tissues and normal counterparts, n = 66, * indicates comparison with normal tissues adjacent to cancer, *P < 0.05; C: Correlation analysis of MALAT1 and STAT4 mRNA levels in 66 BC tissue samples; D: RT-qPCR to quantify MALAT1 in MCF-7R, *P < 0.05 versus oe-NC group; E: CHIP assay to detect STAT4-bound MALAT1 in each group of MCF-7R, *P < 0.05; F: Dual luciferase reporter assay to measure MALAT1 levels in oe-NC and oe-STAT4-treated 293T cells for MALAT1 promoter activity; G: RT-qPCR assay for knockdown efficiency of three pairs of MALAT1 interference sequences; H: RT-qPCR assay for STAT4 and MALAT1 levels in MCF-7R, *P < 0.05; I: Western blot assay for each group of MCF-7R cells, *P < 0.05; J: MTT assay to detect MCF-7R cell survival, *P < 0.05; K: Plate cloning assay to detect MCF-7R proliferation, *P < 0.05; L: Flow cytometry to detect MCF-7R cell apoptosis, *P < 0.05; M: Plate cloning assay to identify changes in the proliferative capacity of MCF-7R, *P < 0.05.

Figure 5

Validation of the targeting correlation between MALAT1 and miR-21-5p in breast carcinoma (BC) cells. A: Venn diagram of StarBase, LncBase predicted miRNAs downstream of MALAT1 and BC-associated miRNAs in GeneCards; B: Diagram of the predicted binding loci of MALAT1 and miR-21-5p by StarBase; C: RT-qPCR detection of miR-21-5p evels in BC tissues and adjacent normal counterparts, n = 66, * indicates comparison with normal counterparts adjacent to cancer, *P < 0.05; D: Correlation analysis of miR-21-5p and MALAT1 in 66 BC tissue samples; E: The targeting correlation between MALAT1 and miR-21-5p by dual luciferase reporter assay, *P < 0.05 versus mimic NC group; F: The binding of biotin-labeled miR-21-5p-WT and mutant sequence miR-21-5p-MUT to MALAT1 by RNA-pull-down assay, *P < 0.05; G: The binding of biotin-labeled MALAT1-WT and mutant sequence MALAT1-MUT to miR-21-5p by RNA-pull-down assay, *P < 0.05; H: RT-qPCR to detect MALAT1 levels in MCF-7R, *P < 0.05 versus oe-NC group; I: RIP assay to detect MALAT1 enrichment within the RISC core element Ago2 in MCF-7R, *P < 0.05; J: RIP assay to detect miR-21-5p enrichment within the RISC core element Ago2 in MCF-7R, *P < 0.05; K: RT-qPCR to quantify miR-21-5p in MCF-7R, *P < 0.05.

Figure 6

MALAT1 regulates THRB by adsorbing miR-21-5p. A: Venn diagram of the four websites (miRDB, mirT-CDS, mirDIP and StarBase) predicting the miR-21-5p target gene; B: GEPIA website analyzing THRB expression in BC patients; C: StarBase website predicting the target binding loci of miR-21-5p and THRB; D, E: RT-qPCR and Western blot to quantify THRB mRNA and protein levels in cancer tissue and adjacent counterparts, n = 66, *P < 0.05 versus adjacent counterparts; F: Correlation of miR-21-5p and THRB mRNA levels in 66 BC tissue samples; G: Targeting correlation between miR-21-5p and THRB by double luciferase reporter assay, *P < 0.05 versus mimic-NC group; H: RT-qPCR to detect miR-21-5p levels in MCF-7R, *P < 0.05; I, J: RT-qPCR and Western blot to quantify THRB mRNA and protein levels in MCF-7R, *P < 0.05; K, L: RT-qPCR to measure MALAT1 and miR-21-5p levels in MCF-7R, *P < 0.05; M, N: RT-qPCR and Western blot to determine THRB in MCF-7R, *P < 0.05. 7R cells, mRNA and protein expression levels of THRB in each group, *P < 0.05.

Figure 7

STAT4 regulation of MALAT1/miR-21-5p/THRB axis to influence breast carcinoma (BC) cell resistance to radiotherapy. A: RT-qPCR results indicate STAT4, MALAT1, miR-21-5p and THRB levels in MCF-7R, *P < 0.05; B: Western blot reveals STAT4 and THRB protein levels in MCF-7R, *P < 0.05; C: MTT assay for cell survival rate, *P < 0.05; D: Plate cloning assay on cell proliferating capacity, *P < 0.05; E: Flow cytometry about apoptosis level, *P < 0.05; F: Plate cloning assay to measure changes in MCF-7R cell proliferating capacity, *P < 0.05.

Figure 8

The in vivo validation of the STAT4-regulated MALAT1/miR-21-5p/THRB axis and its effect on breast carcinoma cell radiotherapy resistance. A: Representative images of tumors in each group, bar = 10 mm; B: Tumor change curves, *P < 0.05; C: Comparison of tumor weight, *P < 0.05; D: RT-qPCR for measuring STAT4, THRB, miR-21-5p and MALAT1 levels in solid tumors, *P < 0.05; E: Western blot reveals STAT4 and THRB protein levels in solid tumors, *P < 0.05; F: Immunohistochemistry shows STAT4 and THRB protein levels in solid tumors, *P < 0.05; G: Immunohistochemistry on ki67 levels in solid tumors, *P < 0.05; H: TUNEL staining to determine apoptosis, *P < 0.05; n = 12.

Figure 9

The molecular mechanism by which STAT4 counteracts miR-21-5p and enhances THRB expression via the transcriptional activation of MALAT1, effectively reversing radiotherapy resistance in breast carcinoma cells.