Current evidence on circRNAs as potential theranostic markers for detecting chemoresistance in breast cancer: a systematic review and meta‑analysis

This study assessed the value of circRNAs (circular RNAs) as prognostic markers in BC (breast cancer). We searched pertinent studies on the PubMed, Embase, and Web of Science online databases published according to PRISMA guidelines. A random-effects model for meta-analysis was used to assess the combined effect size of the HRs (hazard ratios) of the included studies. The heterogeneity test used Cochran's Q-test and I2 statistics. Thirty of the 520 trials retrieved were included in the systematic review. A total of 11 chemotherapeutic agents were used in the included studies. A total of 30 studies on 30 circRNAs were included in the systematic review. Of the 30 relevant circRNAs, 28 were upregulated and two were downregulated in breast cancer versus normal samples, and both were associated with increased drug resistance. Nine of 30 studies were used for the meta-analysis. The results of the meta-analysis showed that the groups with circRNA upregulation and circRNA downregulation showed the same prognostic risk (HR = 1.37, 95% Cl: 0.80–2.36, I2 = 63.7%). The results of subgroup analysis showed that both upregulated circRNAs (HR = 2.24, 95% Cl: 1.34–3.75, I2 = 0%) and downregulated circRNAs (HR = 0.61, 95% Cl: 0.45–0.83, I2 = 0%) were associated with poor BC prognosis. Collectively, the results of all relevant articles collected indicated that circRNAs showed good potential as possible clinical biomarkers of chemoresistance in BC patients.

Inclusion and exclusion criteria. The selection criteria for inclusion in the literature were as follows: (1) studies that involved the effect of circRNAs on drug resistance or drug sensitivity in BC; (2) studies that collected clinical samples or involved in vitro preclinical analysis; and (3) studies that involved the effect of circRNA on the prognosis of BC. The criteria for exclusion were as follows: (1) duplicate studies; (2) reviews, editorials, opinions, case studies, and reports; unpublished materials, uninterpretable data, conference proceedings, or theses; (3) articles without complete information; (3) studies that did not indicate whether circRNA expression was upregulated or downregulated; (4) studies that did not include specific drug resistance changes; and (5) studies in languages other than English. Data extraction. Two researchers (Z.Z. and H.J.) extracted the data independently. When necessary, divergences were resolved by a third investigator (J.X.). The extracted information was as follows: (1) first author, publication year, circRNA, number of patients, detection methods for circRNAs, HR, CI; (2) follow-up time and outcomes; and (3) clinicopathological features, including TNM stage and T classification. When the results were not directly shown in the articles for HRs and 95% CIs, survival data were extracted from Kaplan-Meier plots using Engauge Digitizer 4.1 software. The Excel program file of Tierney et al 34 . was then be used to calculate the HRs and 95% CIs. Quality assessment. Two independent investigators (Z.Z. and H.J.) used the Newcastle Ottawa Scale (NOS) to assess the quality of the articles for meta-analysis. If one study had a total score of > 6 points, it was considered high quality. When necessary, divergences were resolved by a third investigator (J.X.). Statistical analysis. Statistical analysis was performed using Stata 15.0. Data in the form of Kaplan-Meier survival curves were converted to HRs and 95% CIs. Pooled outcome data were generated for forest plots to assess the prognostic value of circRNAs in BC. Heterogeneity tests were obtained by Cochran's Q test and Higgins I 2 . According to the rule, a random-effects model was used to generate pooled results if the I 2 value was > 50%, and a fixed-effects model was used if the I 2 value was < = 50%. A p value < 0.05 was used to determine statistical significance.
Study characteristics and quality assessment.  used in 30 studies to explore circRNA expression and its association with drug resistance and associated pathways or proteins/axes. MCF-7 was the most commonly used cell line, while the U343 and U251 cell lines were the least commonly used. The experimental methods used in these studies include western blot, transfection and vector construction, flow cytometry, Transwell, ELISA, cytotoxicity assay, dual-luciferase reporter assay, RNA pull-down, RIP assay, IHC assay, RNase R treatment assay, 5-ethynyl-2′-deoxyuridine (EdU) assay, fluorescence in situ hybridization (FISH) assay, exosome tracing and blockade of exosome secretion. After excluding duplicate circRNAs, our systematic review included a total of 30 different circRNAs, 28 of which were associated with increased drug resistance and a poor prognosis in breast cancer patients when their expression was upregulated, while only 2 were associated with increased drug resistance and a poor prognosis in breast cancer patients when their expression was downregulated.
BC chemoresistance and drug-regulated genetic pathways. In these 30 studies, a total of 32 circR-NAs were reported, and excluding duplicate circRNAs, a total of 30 circRNAs were reported, and these circRNAs led to resistance to 11 drugs through 28 pathways or associated proteins/axes. (Table 1).
Findings of prognosis analysis. Nine circRNAs were used for meta-analysis. Seven circRNAs were upregulated, and two were downregulated (Table 2 demonstrates details of prognostic research). The Newcastle-Ottawa Scale (NOS) was used to evaluate the quality of the included research ( Table 3). The results showed that they all qualified for meta-analysis. The results of the meta-analysis showed that both the upregulated and downregulated groups were at risk for poor prognosis (HR = 1.37, 95% Cl: 0.80-2.36, I 2 = 63.7%). There was significant heterogeneity between the studies. Therefore, we classified all circRNAs into "enhanced resistance"-related cir-cRNAs and "attenuated resistance"-related circRNAs according to the expression of circRNAs. Subgroup analysis was performed according to the upregulation or downregulation of circRNAs. Interestingly, the heterogeneity was significantly reduced after performing a subgroup analysis (Fig. 2), which suggested that circRNAs could be used to determine the prognosis of BC patients (upregulated circRNAs (HR = 2.24, 95% Cl: 1.34-3.75, I 2 = 0%) www.nature.com/scientificreports/ and downregulated circRNAs (HR = 0.61, 95% Cl: 0.45-0.83, I 2 = 0%) were associated with poor BC prognosis.). All four circRNAs in the upregulated group were highly expressed in tumor tissues, and they affected gene pathways that promoted drug resistance in breast cancer cells, while the two circRNAs in the downregulated group were expressed at low levels in tumor tissues and affected gene pathways that inhibited proliferation, metastasis and drug resistance in breast cancer cells.

Sensitivity analysis and publication bias.
We also performed a sensitivity analysis for OS. No significant changes were observed compared to previous results after each study was removed (Fig. 3). In addition, we used funnel plots to assess publication bias. Each dot represents one study. Nine studies fell within the 95% confidence interval. The reason for the poor symmetry may be due to the inconsistent effect of circRNAs in the upregulated and downregulated groups (Fig. 4). Finally, we performed Begg's test, which showed P = 0.004 (< 0.05), and Egger's test suggested publication bias, which may be because far more circRNAs were upregulated than downregulated among the nine circRNAs (Fig. 5).

Discussion
Studies have shown that abnormal circRNA expression is important in tumor cell proliferation, metastasis and cancer recurrence in BC patients [10][11][12][20][21][22][23][24][25][26][27][28][29][30][31][32][35][36][37][38][39] . Many studies have also confirmed that several specific circRNAs are consistently expressed in human tissues and blood. Therefore, circRNAs have the chance to be excellent biomarkers for BC diagnosis, prognosis and drug resistance assessment 3,33,48,49 . Some studies have concentrated on the effects of circRNAs on chemoresistance in breast, cervical 50 , colorectal 51,52 , gastric 53,54 , lung 55 , oral 56 , ovarian 57 , pancreatic 58 and prostate 59 cancers. In this study, we collected relevant articles before 25 October 2021 and conducted a systematic review and meta-analysis, hoping to find clues about the value of circRNAs as biomarkers for BC prognosis. In the systematic review, studies incorporating 30 circRNAs, including 28 upregulated circRNAs and two downregulated circRNAs, were included. Most studies investigated only one circRNA, while only one study focused on more than one circRNA 12 . Our systematic  Several studies have shown that target genes of upregulated circ-00006528 play a role in the MAPK and PI3K/ AKT gene pathways. Further validation showed that the expression of circ-0006528 showed a negative correlation  www.nature.com/scientificreports/ with miR-7-5p in adriamycin resistance. Another study showed a significant increase in both phosphorylated and total AKT protein in some circ-AMOTL1-overexpressing cells, suggesting that AKT might be a key factor in adjusting the resistance effect. Thus, circ-AMOTL1 affected the expression of proapoptotic (BAX and BAK) and antiapoptotic (BCL-2) factors associated with AKT 59 . This suggested that circ-AMOTL1 might be important in paclitaxel resistance in BC cells by affecting the AKT pathway, promoting antiapoptotic proteins and inhibiting proapoptotic proteins. In addition, data from a study showed that circ-ABCB1, circ-cEPHA3.1 and circ-EPHA3.2 might sponge several significantly expressed miRNAs related to drug resistance through the PI3K-AKT and AGE signaling pathways and lead to doxorubicin resistance 59 . They also found that the expression of RNA molecules  www.nature.com/scientificreports/ transcribed from this region might be due to DNA amplification in doxorubicin-treated cells. These results are beneficial for subsequent research on the mechanisms of drug resistance in BC. Nine circRNAs related to prognosis were included in the meta-analysis and were critical to the development of drug resistance. Among them, seven were upregulated (circ-MMP11, circ-WAC, circ-UBE2D2, circ-0006528, circTRIM28, circCDYL2, circ-0001598), and two were downregulated (circ-LARP4, circ-KDM4C). Certain cancer-related genes could increase susceptibility to breast cancer, leading to poorer survival rates. In our analysis, the results showed that the overall HR (95% CI) of upregulated circRNAs was 2.24 (1.34, 3.75), and that of downregulated circRNAs was 0.61 (0.45, 0.83), suggesting that both upregulated circRNAs and downregulated circRNAs could predict poorer cancer prognosis. It is worth noting that if circRNAs could be used as prognostic biomarkers of breast cancer, their clinical application prospects would be very broad. Other typical clinical indicators of tumor status are susceptible to change, but the expression of circRNAs is stable 60 . Steps should be taken to comprehensively assess the role of circRNAs as biomarkers for BC prognosis and drug-resistance assessment.
Some shortcomings must be acknowledged. First, in our study, all of the samples we collected were from Asian populations. Samples collected from a single source may not be able to distinguish between regional and racial differences and ethnic differences. Second, the method used to detect circRNAs was RT-PCR, except for one study that used raw sequencing reads. The relative homogeneity of the methods used to detect circRNAs may affect the value of the assay results. Third, the meta-analysis involved a relatively small sample size and was limited by the number of available articles. In addition, the number of studies included in the meta-analysis was so low that the results of publication bias using funnel plots may not be meaningful.
Overall, circRNAs, as stably expressed molecules, are expected to be biomarkers for breast cancer prognosis. The relationship between circRNA expression and breast cancer features needs to be further investigated, and the practical value of circRNAs in evaluation BC drug resistance and prognosis needs to be further explored.

Conclusion
Currently available evidence suggests that circRNAs might be considered potential prognostic biomarkers for BC patients and that there is a significant association between the expression of circRNAs and the prognosis of breast cancer patients. We anticipate that our findings might contribute to BC treatment.

Data availability
Data are available in a public, open access repository. The corresponding author of this paper can provide relevant information supporting the conclusions of this study if needed.