Tissue and Serum Thioredoxin System and miR-21, miR-23a/b and let-7a as Potential Biomarkers for Brain Tumor Progression

Thioredoxin system and miRNAs are potential targets for both cancer progression and treatment. However, role of miRNAs and their relation between the expression prole of thioredoxin system in brain tumor progression remains unclear. Thus, in this study we aimed to determine the expression proles of redox components Trx-1, TrxR-1 and PRDX-1, and oncogenic miR-21, miR-23a/b and let-7a and oncosuppressor miR-125 in different brain tumor tissues and their association with increasing tumor grade. We studied Trx-1, TrxR-1 and PRDX-1 mRNA expression levels by quantitative real-time polymerase chain reaction (qRT-PCR) and protein levels by Western blot and miR-23a, miR-23b, miR-125a, miR-21 and let-7a miRNA expression levels by qRT-PCR in 16 glioma, 15 meningioma, 5 metastatic and 2 benign tumor samples. We also examined Trx-1, TrxR-1 and PRDX-1 protein levels in serum samples of 36 brain tumor patients and 37 healthy volunteers by ELISA. We found that Trx-1, TrxR-1 and PRDX-1 presented high mRNA expression but low protein expression in low-grade brain tumor tissues whereas they showed higher protein expression in sera of patients with low-grade brain tumors. miR-23b, miR-21, miR-23a and let-7a were highly expressed in low-grade brain tumor tissues and positively correlated with the increase in thioredoxin system activity. Our ndings showed that Trx-1, TrxR-1 and PRDX-1 and miR-21, miR-23a/b, and let-7a might be used for brain tumor diagnosis in the clinic. Further prospective studies including molecular pathway analyses are required to validate the miRNA/thioredoxin system regulatory axis in brain tumor progression. in this study we hypothesized that redox components including Trx-1, TrxR-1 and PRDX-1 might have a relation with oncogenic miR-21, miR-23a/b and let-7a and tumor suppressor miR-125a levels which both having a codependent relationship among Nrf2/ARE and PI3K/Akt pathways. As a preliminary study, we aimed to determine the expression proles of Trx-1, TrxR-1, PRDX-1, oncogenic miR-21, miR-23a/b and let-7a and oncosuppressor miR-125 in different brain tumor tissues and their association with increasing tumor grade. Herein, we studied Trx-1, TrxR-1 and PRDX-1 mRNA expression levels by quantitative real-time polymerase chain reaction (qRT-PCR) and protein levels by Western blot and miR-23a, miR-23b, miR-125a, miR-21 and let-7a miRNA expression levels by qRT-PCR in 16 glioma, 15 meningioma, 5 metastatic and 2 benign tumor samples. We also examined Trx-1, TrxR-1 and PRDX-1 protein levels in serum samples of 36 brain tumor patients and 37 healthy volunteers by ELISA. We found that Trx-1, TrxR-1 and PRDX-1 presented high mRNA expression but low protein expression in low-grade brain tumor tissues whereas they showed higher protein expression in sera of patients with low-grade brain tumors. miR-23b, miR-21, miR-23a and let-7a were highly expressed in low-grade brain tumor tissues and positively correlated with the increase in thioredoxin system activity. Our ndings enhanced the understanding of the relation between miRNAs and thioredoxin system activity. Moreover, miR-23a/b, miR-21 and let-7a might be utilized as diagnostic and prognostic markers for brain tumor patients and potential for its in Shapiro-Wilk test. Pairwise comparison of qRT-PCR results was subjected to Student’s t-test. Western blot results were analyzed with one-way analysis of variance (ANOVA) and Tukey’s HSD tests. Two-sample Kolmogorov-Smirnov test was used for comparison of nonparametric results in ELISA. Pearson correlation test was conducted for qRT-PCR. Whole data were analyzed within 95% condence interval.


Introduction
Brain tumors are diagnosed both in adults and children which lead to high mortality and morbidity worldwide [1][2][3]. 1.6% new cases and 2.5% deaths of brain tumors globally have been reported in 2020 [4]. Malignant glioma and meningiomas are the most common types among all brain tumors [2].
Magnetic resonance imaging and various molecular diagnostic markers are in use for patients with brain tumor in the clinic [5]. However, distinguishing primary and metastatic tumors is challenging with these methods [6]. Therefore, novel diagnostic biomarkers remain under investigation.
Herein, we studied Trx-1, TrxR-1 and PRDX-1 mRNA expression levels by quantitative real-time polymerase chain reaction (qRT-PCR) and protein levels by Western blot and miR-23a, miR-23b, miR-125a, miR-21 and let-7a miRNA expression levels by qRT-PCR in 16 glioma, 15 meningioma, 5 metastatic and 2 benign tumor samples. We also examined Trx-1, TrxR-1 and PRDX-1 protein levels in serum samples of 36 brain tumor patients and 37 healthy volunteers by ELISA. We found that Trx-1, TrxR-1 and PRDX-1 presented high mRNA expression but low protein expression in low-grade brain tumor tissues whereas they showed higher protein expression in sera of patients with low-grade brain tumors. miR-23b, miR-21, miR-23a and let-7a were highly expressed in low-grade brain tumor tissues and positively correlated with the increase in thioredoxin system activity. Our ndings enhanced the understanding of the relation between miRNAs and thioredoxin system activity. Moreover, miR-23a/b, miR-21 and let-7a might be utilized as diagnostic and prognostic markers for brain tumor patients and potential therapeutic targets for its treatment in the clinic.

Ethics Statement and Sample Collection
This research was carried out with Medicana International Ankara Hospital Research Ethics Committee approval (#21102019/04). Brain tumor samples were surgically resected from 38 patients who underwent surgery at Medicana International Ankara Hospital from January 2020 to October 2020. All patients obtained written informed consents. The brain tumors were classi ed according to World Health Organization (WHO) criteria [57]. 16 of these tumors were low-and high-grade glioma (WHO grade I and II; grade III and IV, respectively), 15 were low-and high-grade meningioma (WHO grade I and II; grade III and IV, respectively), 5 were metastatic tumors and 2 were other benign tumors. Clinicopathological features of tumor samples are listed in Table 1. Serum samples of 36 brain tumor patients and of 37 healthy volunteers were also obtained. 13 of these volunteers were at the age 35 and below, 20 were ages between 36-56 and 4 were at the age of 57 and over. Minimum required number of samples and replicates were revealed by power analysis using G-Power. A randomized observative study including control and experimental groups was carried out. All control and experimental groups were independent variables whereas mRNA, miRNA and protein expression results, were de ned as dependent variables.
cDNA synthesis was accomplished using stem-loop transcriptase primers and relative miRNA expression was assessed as performed for relative mRNA expression. All mRNA and miRNA levels were normalized to house-keeping gene GAPDH[58] (n=30 in total) and relative fold change was analyzed according to 2 −ΔΔCt . Primer sequences were summed-up in Table 2.

Gene
Oligonucleotide Sequence

Enzyme-Linked ImmunoSorbent Assay (ELISA)
Human Trx-1 (#E1452Hu), TrxR-1 (#E3953Hu) and PRDX-1 (#E2924Hu) (all from Bioassay Technology Laboratory, China) protein concentrations in sera of brain tumor patients and healthy volunteers were evaluated by ELISA following manufacturer's guideline. Brie y, standard solutions and samples were added to 96-well plate and biotinylated primary antibodies and streptavidin-HRP were added to wells respectively. Plates were then incubated 60 minutes at 37 o C. Subsequently, plates were washed with wash buffer; incubated with substrate solutions for 10 minutes at 37 o C in the dark recommended in guideline. After adding stop solution to wells, color change from blue to yellow was determined and optical density of each well was measured by plate reader (SPECTROstar® Omega, BMG LABTECH, Germany) at 450 nm. Each serum sample was studied triplicate and protein levels in serum samples of brain tumor patients (n=36) were compared with that of healthy volunteers (n=37).

Statistical Analysis
Data used for qRT-PCR and Western blot data exhibited normal distribution whereas ELISA data presented non-normal distribution by Shapiro-Wilk test. Pairwise comparison of qRT-PCR results was subjected to Student's t-test. Western blot results were analyzed with one-way analysis of variance (ANOVA) and Tukey's HSD tests. Two-sample Kolmogorov-Smirnov test was used for comparison of nonparametric results in ELISA. Pearson correlation test was conducted for qRT-PCR. Whole data were analyzed within 95% con dence interval.

Results
Thioredoxin system components showed higher mRNA expression but low protein expression in low-grade brain tumor tissues TrxR-1 mRNA expression was higher in all brain tumor tissues compared to control group (Fig. 1A) by qRT-PCR. Trx-1 and TrxR-1 mRNA expressions were higher in low-grade meningioma tissues than that of high-grade meningioma (Fig. 1B) but vice versa for protein expressions by Western blot (Fig. 1C).
Similarly, high-grade glioma tissues had higher Trx-1 and TrxR-1 protein expressions than that in lowgrade glioma by Western blot (Fig. 1C). There was no signi cant difference in Trx-1, TrxR-1 and PRDX-1 mRNA expression levels between high-and low-grade glioma patients (Fig. 1B). Trx-1, TrxR-1 and PRDX-1 mRNA expression levels were lower in other benign primary tumors when compared to metastatic tumors (Fig. 1B). However, metastatic tumors had lower Trx-1 and TrxR-1 protein expression compared with highgrade glioma and high-grade meningioma by Western blot.
Thioredoxin system components showed higher protein expression in serum samples of patients with low-grade brain tumors Sera of brain tumor patients had signi cantly lower Trx-1 (Fig. 1D) and TrxR-1 (Fig. 1E) protein expressions comparing to serum samples of healthy volunteers by ELISA. No signi cant difference was observed in PRDX-1 protein expression between the two groups (Fig. 1F). Serum samples of high-grade glioma patients had signi cantly lower Trx-1 (Fig. 1G), TrxR-1 (Fig. 1H) and PRDX-1 (Fig. 1I) protein expressions than that in low-grade glioma patients. Serum samples of patients with high-grade meningioma and other benign primary tumors had signi cantly lower Trx-1 (Fig. 1G) and TrxR-1 (Fig. 1H) protein expressions when compared to low-grade meningioma and metastatic tumors, respectively. Sera of healthy volunteers had signi cantly higher Trx-1 and TrxR-1 protein expressions than all groups by ELISA (Fig. 1G, H). PRDX-1 protein expression level was signi cantly higher in healthy volunteers when compared to sera of patients with high-grade meningioma, metastatic tumor or other benign primary tumors (Fig. 1I).
miR-23a/b, miR-21 and let-7a are highly expressed in lowgrade brain tumor tissues and positively correlated with the increase in thioredoxin system activity miR-23a/b, miR-21 and let-7a expression levels were greater in all brain tumor tissues when compared to control group ( Fig. 2A) by qRT-PCR. No signi cant difference was noticed between the groups for miR-125a expression by qRT-PCR. Low-grade meningioma tissues had signi cantly higher miR-23b, miR-21, miR-23a and let-7a miRNA expression levels than that of high-grade meningioma (Fig. 2B). miR-21 and let-7a expressions were lower in other primary benign tumors when compared to metastatic tumors (Fig.  2B). There was no signi cant alteration in miR-125a miRNA expression levels among all groups by qRT-PCR.

Discussion
In this study, we showed that thioredoxin system components had higher expression in low-grade brain tumor samples also having a strong positive correlation with oncogenic miR-21, miR-23a/b and let-7a and tumor suppressor miR-125a levels for the rst time. We examined high mRNA and low protein expressions of Trx-1 and TrxR-1 in low-grade meningioma and benign primary tumor tissues when compared to high-grade meningioma and metastatic tumor tissues, respectively by qRT-PCR and high Trx-  [73,74,44]. Thus, our ndings imply that those miRNAs might be potential biomarkers for diagnosis of meningioma. Herein, we also found that miR-21 and let-7a expressions were greater in metastatic tumors when compared to other primary benign tumor tissues showing a positive correlation between miRNA expressions and tumor grade. Trx-1, TrxR-1 and PRDX-1 mRNA expression levels were positively correlated with miR-21, miR-23a/b, miR-125, and let-7a expressions in brain tumor tissues by Pearson correlation analysis. Kalinina et al demonstrated high correlation between the antioxidant protection including thioredoxins and peroxiredoxins and miRNAs [75] which also support our results. PRDX-3 was linked with miR-23b for human prostate cancer progression [49]. Similarly, miR-23a and miR-23b regulate TrxR-1 expression during skeletal muscle differentiation [76]. Our key ndings may implicate the diagnostic value of miR-21, miR-23a/b, let-7a and thioredoxin system components for brain tumors which also improve the understanding in determining the levels of biomarkers in different brain tumor grades.
In the current study, a correlation analysis has been done for the relation between thioredoxin system components and various miRNAs as oncogenes in brain tumor tissues and serum samples of patients, however, in vitro and in vivo functional studies including the association between miRNAs and redox system through Nrf2/ARE [55] and PI3K/Akt [56] pathways must be performed for the miRNA/thioredoxin system regulatory axis in brain tumor progression which generates a crucial limitation for this study. This limitation, still, does not hinder our further studies since the expression and correlation pro les in thioredoxin system and miRNAs would facilitate in-depth studies for better understanding of miRNA and redox system component functions as novel biomarkers. Since this research has been conducted with human subjects, brain tissue samples could not be obtained from healthy volunteers. To cope with this limitation, we compared levels of thioredoxin system components with metastatic and/or other benign tumors for qRT-PCR and Western blot and serum samples of healthy volunteers for ELISA.
Taken together, our ndings show that thioredoxin system components including Trx-1, TrxR-1 and PRDX-1 and miR-21, miR-23a/b, and let-7a could be potential biomarkers together for brain tumor diagnosis especially for meningioma cases. Further prospective studies are needed to validate their relation and its projection to brain tumor treatment in the clinic. Figure 1 mRNA and protein expressions of brain tumor samples of patients. a Relative mRNA fold-change of Trx-1,

Declarations
TrxR-1 and PRDX-1 of brain tumor samples of patients and control group by qRT-PCR (n=30, *p<0.05 by Student's t-test); b Relative mRNA fold-change of Trx-1, TrxR-1 and PRDX-1 of low-and high-grade glioma, low-and high-grade meningioma and metastatic and primary tumor samples by qRT-PCR (*p<0.05 by Student's t-test); c Relative protein expressions of Trx-1, TrxR-1 and PRDX-1 for low-and high-grade glioma, low-and high-grade meningioma and metastatic tumor samples (*p<0.05; **p<0.001 by One-way analysis of variance (ANOVA) and post-hoc Tukey's HSD tests); and serum d Trx-1, e TrxR-1 and f PRDX-1 protein concentrations for brain tumor patients (n=36) and healthy volunteers (n=37) by ELISA test (*p<0.05 and **p<0.001 by Two-sample Kolmogorov-Smirnov test; and bar graphs indicating serum g Trx-1 (U/L), h TrxR-1 (ng/ml) and i PRDX-1 (ng/ml) protein concentrations for low-and high-grade glioma, low-and high-grade meningioma and metastatic and primary tumor samples by ELISA test (*p<0.05 and **p<0.001 by Two-sample Kolmogorov-Smirnov test) Figure 2 miRNA expressions of brain tumor samples of patients and correlation analysis between miRNAs and thioredoxin system. a Relative miRNA fold-change of miR-125a, miR-23a/b, miR-21 and let-7a for brain tumor samples of patients and control group by qRT-PCR (n=30, *p<0.05 and **p<0.001 by Student's ttest); b Relative miRNA fold-change of miR-125a, miR-23a/b, miR-21 and let-7a of low-and high-grade glioma, low-and high-grade meningioma and metastatic and primary tumor samples by qRT-PCR