Qualitative synthesis analysis of the articles in adherence to the PRISMA guidelines

A total of 387 articles were compiled from all the searched databases, supplemented by additional 32 articles manually added through the free terms search strategy. As a result, 419 articles were considered for further assessment. Following the removal of duplicates (n = 99), 320 articles remained for subsequent assessment based on the established inclusion and exclusion criteria, undergoing initial screening based on abstract and title evaluation. Finally, 51 articles were screened for relevance and fully read. This analysis resulted in 38 full-text articles selected for qualitative analysis, using the six categories of the QUIPS tool [38]. As a result, 37 articles presented a low risk of bias and one a high risk of bias.This article was excluded considering that the outcome measurement of IR was not properly assessed (Fig. 1).

Fig. 1

PRISMA Flow Diagram. Flow diagram of the study identification and selection process, following Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines

General characteristics of the selected articles

The 37 original articles selected were published between June 2011 and September 2023. The information on the selected articles is shown in Table 1. The articles were published by research groups from eight different countries, with China presenting the highest number of publications (n = 24 articles), followed by South Korea (n = 3), USA (n = 3), Germany (n = 3), Iran (n = 1), Mexico (n = 1), Australia (n = 1), and Taiwan (n = 1). A total of 33 ncRNAs were described in the selected articles, of which 27 were miRNAs, nine lncRNAs, and two circRNAs.

Table 1 Comprehensive analysis and main characteristics of the thirty-seven selected articles

One of the aims of this systematic review was to include studies that identified and described the function of these ncRNAs in modulating the IR response in TNBC cells. However, in some studies the reported expression of a given ncRNA was determined by comparing its expression in the TNBC samples with controls, not necessarily assessing changes in expression in the TNBC samples pre- and post- IR administration. In such cases, the subsequent modulation of the ncRNAs expression was evaluated in experimental assays to determine its impact on the response to various doses of IR. For example, the ncRNAs let-7d, miR-16-5p, miR-23b-5p, miR-22, miR-33a, miR-129-5p, miR-200c, miR-218, miR-1290, and AFAP1-AS1 were downregulated in the parental TNBC cell lines compared to controls [27, 30, 32, 39,40,41,42,43,44,45]. Conversely, miR-27a, miR-33a, miR-122, miR-199a-5p, miR-205, miR-302a, miR-634, LINC00511, LINC00963, DUXAP8, PCAT6, Circ_0008500 (has_circ_0008500), and CircNCOR1 (hsa_circ_0042174) were upregulated in the parental TNBC cell lines compared to controls [28, 31, 34, 41, 46,47,48,49,50,51,52,53,54]

Further, the original articles were also evaluated based on the experimental assays and IR dosages used to determine the effects of IR on TNBC cells. A total of 17 articles used the cell viability assay as the primary methodology for assessing the effects of IR. Other assays used included: clonogenic or colony formation (n = 21), tumor volume measurement (n = 6), cell survival (n = 3), cell proliferation (n = 3), sphere formation (n = 2), ROS generation (n = 1), and tumor formation (n = 1) assays. Other articles determined the impact of IR and the role of the ncRNAs evaluating: apoptosis (n = 15), DNA damage (n = 4), autophagy (n = 2), cell migration (n = 2) and invasion (n = 1).

The studies describing the role of miR-93-5p, miR-205, miR-302a, AFAP1-AS1, DUXAP8, Circ_0008500, and CircNCOR1 also used in vivo TNBC models, specifically tumor xenografts, to evaluate their role in modulating the response to radiation [32, 34, 45, 46, 50, 51, 54, 55]. These studies included the analysis of tumor volume and weight, and histopathology. Only two studies were conducted in TNBC clinical samples [32, 45]. In these studies, the expression of AFAP1-AS1 [32] and miR-1290 [45] was evaluated in the tissue samples in relation to the response to RdT. Another interesting approach employed in nine studies [32, 45, 47, 48, 50,51,52, 56, 57] involved the development of radioresistant (RR) cell lines. The most used radioresistant cell line was the RR-MDA-MB-231 (in eight studies); the RR-MDA-MB 468 and RR-SUM159 cell lines were used in one study each [50]. Regarding the IR dosage, 34 studies used the dose below 10 Gy (with different fractionated dose ratios), while 5 studies used doses above 10 Gy. In studies involving animal models, a higher dosage of IR was applied, ranging between 4–15 Gy, either delivered as a single dose (n = 5) or as a fractionated controlled dosage (n = 4). The application of fractionated doses is consisted with RdT protocols typically applied for TNBC in clinical practice [32, 34, 45, 46, 50, 51, 53,54,55].

The information above is described for each study in Table 1.

NcRNAs modulating IR response in TNBC

The role, association, or involvement of the ncRNAs in modulating the IR response in the TNBC are summarized in Fig. 2 and Tables 2, 3. Among the 37 selected studies, 29 described the ncRNAs in the modulation of radiosensitivity (Table 2), and nine of radioresistance (Table 3). Among the studies describing the involvement of ncRNAs in modulating radiosensitivity, 21 highlighted the involvement of 18 distinct miRNAs, seven the involvement of seven distinct lncRNAs, and one the involvement of one circRNA. For the modulation of radioresistance, miRNAs were also the most reported ncRNAs, with five studies describing the role of six different miRNAs, followed by two studies on lncRNAs, and one study on circRNA. Gain- and loss-of-function strategies were employed to manipulate the levels of ncRNAs expression and determine their impact on the modulatiion of response to IR. The ectopic expression of the ncRNAs was the most common strategy used (n = 26 studies), followed by expression inhibition (n = 11).

Fig. 2

NcRNAs modulating IR response on TNBC irradiated cell lines and corresponding mechanisms. NcRNAs in green and red indicate action on radiosensitivity and radioresistance, respectively. NcRNAs in black present both radiosensitivity and radioresistance action. Image created using BioRender

Table 2 NcRNAs associated with radiosensitivity on TNBC-expression levels, mechanisms of action and biological impactTable 3 NcRNAs associated with radioresistance on TNBC-expression levels, mechanisms of action and biological impactNcRNAs modulating radiosensitivity

Most of the ncRNAs modulating radiosensitivity were miRNAs (Table 2). Among them, miR-200c was the most cited, reported in three studies [30, 43, 58]. The mechanisms of action attributed to this miRNA, involved the regulation of cell survival, particularly autophagy and apoptosis, and DNA damage repair. Apoptosis and autophagy were modulated by the ectopic expression (overexpression) of miR-200c resulting in the downregulation of the UBQLN1 and LC311 proteins (involved in several aspects of autophagy) and the upregulation of several proteins associated with apoptosis. These expression changes led to the decrease of autophagy and increase of apoptosis [30]. On the other hand, its action on DNA damage repair pathways was evidenced by the γ-H2AX foci formation, along with the downregulation of the phosphorylated DNA-dependent protein kinase catalytic subunit (DNA-PKcs) [58]. This down-regulation affected the non-homologous end joining (NHEJ) pathway, an essential pathway for the repair of IR-induced DNA damage. The TNBC cells with the impaired NHEJ pathway did not recover from the damage caused by the IR and presented diminished survival rates [58]. Additionally, the ectopic expression of miR-200c affected the DNA repair mechanism by downregulating the expression of the lncRNA LINC02582, which in turn, downregulated USP7 and CHK1 expression, rendering the RR-TNBC cells more sensitive to IR [43]. It is well known, that lncRNAs (and circRNAs) can act as miRNA sponges, competing for miRNA target binding and reducing their regulatory effect. This mechanism can impact different biological processes, including the ones modulating radioresponse [59, 60].

In the additional studies investigating the effect of ncRNAs in apoptosis, caspase-3 emerged as the most targeted apoptotic protein. Four ncRNAs, miR-27a, miR-129-5p, miR-634, and lncRNA CCAT1, were identified conferring sensitivity to the irradiated cells by up-regulating caspase-3 expression, and consequently promoting apoptosis [28, 42, 52, 61]. Other molecules such as the anti-apoptotic BCL2 and PARP proteins, a marker for apoptosis known to suppress DNA repair, were also targeted. Overexpression of miR-31 [62] and miR-3188 [63] led to a decrease of BCL2 and PARP expression protein levels, resulting in increased apoptosis and decreased cell viability, respectively, and enhancing the radiosensitivity in TNBC cells. MiR-93-5p also was described modulating radiosensitivity when overexpressed, by increasing apoptosis, inhibiting cell viability and migration in in vitro TNBC models [29]. The miR-93-5p action on this pathway that involves an Eph receptor tyrosine kinase and the transcription factor NF-κB, was evidenced by the reduction of tumor growth in the TNBC xenografts. Notably, miR-93-5p has been previously associated with deregulated expression in plasma exosomes from patients with breast cancer, in association with radiosensitivity [55]. Collectively these studies indicate the promising potential of miR-93-5p as a valuable marker for radioresponse.

Other ncRNAs were described to modulate radiosensitivity by their down-regulated expression levels. For instance, the downregulation of Circ_0008500 was shown to improve radiosensitivity and inhibit tumorigenesis in the irradiated TNBC cell line MDA-MB-468 through the miR-758-3p/PFN2 axis. This regulation led to the increase of BAX expression, a pro-apoptotic protein, and decreased expression of BCL2 [34]. Moreover, the knockdown of LINC00511 promoted apoptosis and increased the expression of STXBP4 (STXBP4) levels through competitive binding to miR-185 [53].

In addition to the induction of apoptosis, the ncRNAs also act on autophagy in TNBC cells to enhance sensitivity to IR. The upregulation of miR-129-5p reduced the expression of autophagy-related proteins, such as HMGB1, LC3-II, and p62, which in turn decreased autophagy on MDA-MB-231 irradiated cells [42]. Conversely, miR-199a-5p regulated the autophagy induced by IR. The positive regulation of this miRNA maintained low levels of the autophagy-associated proteins LC3-I, LC3-II, and low levels of DRAM1 and Beclin1 expression, resulting in a controlled IR-induced autophagy rate in TNBC cell lines [49].

Restraint of DNA damage repair has been identified as a strategic approach to enhance radiosensitivity. In general, the induction of cell death through IR requires the accumulation of a substantial DNA damage, particularly DSB. However, tumor cells can take advantage of alternative molecular mechanisms that activate and drive the DNA repair processes. The activation of DNA damage repair (DDR) cascades may reduce the effectiveness of IR, ultimately promoting cell survival. Notably, several studies have shown that the negative regulation of critical DNA repair pathways can increase radiosensitivity [64,65,66]. The knockdown of SIRT1 induced by miR-22 upregulation was demonstrated to restrain the DDR on MDA-MB-231 irradiated cell line, leading to a decrease in cell proliferation [40]. Moreover, miR-139-5p and miR-205 were associated with DDR pathways by targeting markers of these pathways. The ectopic transfection of miR-139-5p, causing a delay in DNA repair, promoted with double power the radiosensitivity potency compared to the presence of two or more DDR mutations [67]. For miR-205, its overexpression led to the downregulation of the expression of ATM, ZEB1, and UBE2N (UBC13), which code for proteins that act in the homologous recombination (HR)-mediated DNA damage repair pathway, as evaluated in the study of Zhang et al. [50] by the γ-H2AX assay. The inhibition of the HR pathway by this miRNA, enhanced the radiosensitivity of the TNBC cells [50]. MiR-142-3p and miR-302a were also reported to impact DNA repair by downregulating BRCA1/BRCA2 and RAD52 expression, which code for proteins involved in the HR pathway [51, 68].

In addition to the cellular processes cited above, the ncRNAs have been shown to modulate radiosensitivity by exerting control over the proliferation of stem cells populations. Two studies suggested that one of the mechanisms by which IR modulation occurs in TNBC cells is based on the inhibition of stem cells proliferation and self-renewal ability [39, 57, 68]. For example, let-7d downregulated the CyclinD1/Akt/Wnt1 pathway, resulting in a diminished stem cell population. Other targets related to stemness, such as BOD1 and KLF4, were downregulated by miR-142-3p [39, 68]. Conversely, lower levels of the lncRNA NEAT1 were found to be correlated with decreased stem cell renewal. The knockdown of this lncRNA, using the CRISPR-Cas9 method in a RR-TNBC cell line, led to the downregulation of key stemness genes, such as BMI1, OCT4 and SOX2, resulting in decreased stem cell renewal and enhanced radiosensitivity [57].

The EGFR signaling pathway was another pathway affected by ncRNAs in the modulation of radiosensitivity. The ectopic expression miR-7 reduced the expression levels of EGFR, AKT, ERK, and STAT3 and radiosensitized TNBC cells [69]. MiR-302a and miR-3188 were also reported to increase radiosensitivity of the TNBC cells by affecting the EGFR pathway, causing the downregulating of AKT expression [51, 63]. Similarly, miR-218 exerted a comparable action, but it targeted the primary downstream effector of EGFR, the p44/42 MAPK (ERK 1/2), decreasing cell survival upon IR exposure [44].

Finally, another described mechanism demonstrated to modulate radiosensitivity in TNBC was associated with the high-density lipoprotein (HDL). Wolfe et al. [41] showed that miR-33a negatively regulated HDL and induced radiosensitivity in TNBC cell lines composed of both inflammatory and non-inflammatory cells. These types of cells are present in several TNBC cell lines and are characterized by distinct expression patterns and clinical behavior [41]. In the cells expressing high levels of miR-33a, the transfection with anti-miR-33a decreased radioresistance, as evidenced by the reduction of colony formation in the clonogenic assays. Conversely, in cells with low levels of miR-33a, its ectopic expression reversed the HDL-induced radiosensitization. Notably, in BC patients treated with radiation, high miR-33a expression was associated with worse overall survival. This study highlights the importance of comprehensively characterizing the molecular signature and clinical characteristics of the distinct TNBC cell populations, as these differences may ultimately impact on the clinical prognosis of patients submitted to RdT protocols.

The data presented above is illustrated in Fig. 2 and described in Table 2.

NcRNAs modulating radioresistance

NcRNAs were also observed to confer radioresistance to the TNBC cells. However, a smaller number of ncRNAs were described compared to those that increased radiosensitivity (Table 3). Among these ncRNAs, one miRNA (miR-27a) and one lncRNA (LINC00963) were common to both groups