Mechanism study of EIF4A3-induced circ_0022382 regulation of breast cancer cell biology

doi:10.21203/rs.3.rs-4016797/v1

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Mechanism study of EIF4A3-induced circ_0022382 regulation of breast cancer cell biology

https://doi.org/10.21203/rs.3.rs-4016797/v1

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Breast cancer is one of the most common cancers in women and poses a serious threat to women's health. In this paper, we examined the role of circular (circ)RNA in breast cancer, and screened out circ_0022382 for high expression in breast cancer by differential analysis of breast cancer and adjacent tissues in the GEO database. The high expression of circ_0022382 in breast cancer cell lines MDA-MB-231, MCF-7 as well as breast cancer tissues was confirmed by qRT-PCR. The proliferation, migration and apoptosis of MDA-MB-231 and MCF-7 breast cancer cells were significantly increased by transfection of siRNA targeting circ_0022382, and dual luciferase reporter assays showed that circ_0022382 bound to microRNA (miR) let-7a-5p. Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis predicted that the mammalian target of rapamycin (mTOR) and phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathways were the main pathways regulated by let-7a-5p. The addition of let-7a-5p inhibitor reduced the proliferation and migration of breast cancer cells caused by circ_0022382 knockdown by rescuing the expression of p-AKT. Let-7a-5p also directly regulates the expression of cystine transporter solute carrier family 7 member 11 (SLC7A11) proteins, and through examination of glucose in cell supernatants and the addition of cystine inhibitors (dithiothreitol), we found that this process was closely related to disulfidptosis. In addition, eukaryotic translation initiation factor 4A3 (EIF4A3) promoted increased expression of circ_0022382 in breast cancer cells. In conclusion, EIF4A3-induced circ_0022382 regulates the expression of SLC7A11 proteins and related proteins in the PI3K-AKT signaling pathway by sponging let-7a-5p, which affects glucose metabolism and induces the death of breast cancer cells. Therefore, circ_0022382 may be useful as a marker or therapeutic agent in breast cancer treatment.

circ_0022382

PI3K-AKT

SLC7A11

proliferation

migration

EIF4A3

Breast cancer has high incidence and mortality among women[1]. Various treatment methods have been employed in clinical practice to address breast cancer at different stages. These methods encompass surgical intervention, postoperative adjuvant therapy and neoadjuvant therapy[2, 3]. However, patients often develop drug resistance following multiple treatment lines. This resistance can be attributed to the intricate nature of the tumor microenvironment during treatment and the genomic instability inherent to the tumor itself[4]. For example, the phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha gene of early and middle stage hormone receptor-positive and hormone epidermal receptor-negative breast cancer patients is prone to mutation during treatment, which will lead to drug resistance to endocrine therapy[5, 6], causing difficulties in the treatment of breast cancer. Therefore, it is necessary to find stable and specific molecular markers of breast cancer. Circular RNA (circRNA), as a non-coding RNA, was first thought to be meaningless and a byproduct of the gene-splicing process. However, since next-generation sequencing has sequenced the human genome in 2012, circRNA has been found to be specifically expressed in many cancers and has biological functions affecting tumor proliferation[7], metastasis[8, 9], drug resistance[10] and immune response[11, 12]. At present, our understanding of circRNA is relatively well developed, including its cell localization, mechanism and source. Most of circRNA is localized in the cytoplasm and functions by spongiforming micro (mi)RNAs[13–15], while AUF1[16], eukaryotic translation initiation factor 4A3 (EIF4A3)[17] and other RNA-binding proteins related to post-transcriptional regulation increase the circRNA formed by alternative splicing by binding pre-mRNA. In addition, circRNA can be detected in body fluids such as blood[18, 19], urine[20] and breast milk[21]. Therefore, circRNA has become a popular biomarker in cancer research.

In the present study, bioinformation analysis was carried out in the Gene Expression Omnibus (GEO) database (breast cancer tissue sequencing results), which found that circ_0022382 was highly expressed in breast cancer. The biological characteristics and mechanism of action of circ_0022382 regulating breast cancer were then determined by in vitro and in vivo experiments, and these results should inform the choice of breast cancer treatment.

1.Breast tissue samples and cells

Three paired human breast cancer tissues and adjacent tissues were obtained from the Department of Oncology Surgery(thyroid&breast), Affiliated Hospital of Jiangsu University. The study was approved by the Ethics Committee of Affiliated Hospital of Jiangsu University. Breast cancer cells including MDA-MB-231, MCF-7 and normal breast epithelial cells MCF-10A were purchased from ATCC (ATCC; Manassas, VA, USA).

2.GEO database analysis of circRNA and miRNA expression profiles

We first searched for the keywords “breast cancer”, “circRNA” and “miRNA” in the GEO database, selected the corresponding datasets for “circRNA” and “miRNA”, then preprocessed the datasets using GEO2R(https://www.ncbi.nlm.nih.gov/geo/geo2r/). At the same time, we examined the samples using boxplots and principal component analysis, then used R studio's limma package to draw volcano and heat maps for differentially expressed genes.

3.Bioinformatics analysis

We used the ENCORI (https://starbase.sysu.edu.cn/) online website to predict miRNAs involved in circRNA interactions, and the online website Draw Venn Diagram (https://bioinformatics.psb.ugent.be/webtools/Venn/) to find intersecting miRNAs. We used the online tool DAVID v6.8 (https://david-d.ncifcrf.gov/) to perform KEGG analysis on the mRNAs predicted by ENCORI and the WeChat platform (https://www.bioinformatics.com.cn/login/) to visualize the results of KEGG enrichment analysis. CircInteractome (https://circinteractome.nia.nih.gov/)was used to predict the RNA-binding proteins bound by circRNA.

4.RNA extraction and quantitative real-time PCR

Total RNA was extracted from cells according to the instructions for Trizol reagent (Thermo Fisher Scientific, Carlsbad, CA) and we measured the concentration using a Nanodrop 1000 ultramicro spectrophotometer (NanoDrop, Wilmington, DE, USA). For quantitative analysis of mRNA, we first reverse transcribed total RNA into cDNA according to the mRNA reverse transcription reagent instructions (Vazyme Biotech, Nanjing, China; R323-01), and then amplified the cDNA according to the qPCR instructions (Vazyme; Q711). Using GAPDH as the internal reference, the primer sequences are as follows, circ_0022382: forward (F) 5'-GATTCTACCCATCACGGC-3', reverse (R) 5'-GAAGGCGGGAAGGCAT-3'; GAPDH: F 5'-ATGGGGCAGCCGTTAGGAAA-3', R 5'-GCGCCAAATACACAATC-3'; cystine transporter solute carrier family 7 member 11 (SLC7A11): F 5'-CTTTGTTGCCCTCTCCTGCTTC-3', R 5'-CAGAGGAGTGTGCTTGTGGACA-3'. For quantitative analysis of miRNA, the miRNA was first specifically reverse transcribed into cDNA using the stem loop method according to the miRNA reverse transcription reagent instructions (Vazyme; MR101-02). Then, the cDNA was amplified according to the qPCR instructions (Vazyme; MQ101-02) using U6 as the internal reference, and three complex wells were designed. The primer sequences used were as follows: let-7a-5p: F 5'-GCCGTGAGGTAGTAGGTT-3', R 5'-AGTGGGGGTCCGGGTATT-3'; U6: F 5'-CTCGTTCGAGCACA-3', R 5'-ACGCTTCACGAATTGCGT-3'.

5.DNA extraction and PCR

Genomic DNA was extracted from breast cancer MDA-MB-231 cells according to the instructions of the DNA extraction kit (Vazyme; DC112-01). Then, the extracted genomic DNA was amplified according to the three-step PCR method (high temperature denaturation, low temperature annealing and temperature-appropriate extension). Finally, the amplified product was electrophoresed and imaged. The molecular location and content were judged according to a DNA ladder (Applied Biological Materials, Nanjing; G016).

6.Cell transfection

On the day before transfection, MDA-MB-231 and MCF-7 breast cancer cells in the logarithmic growth phase were inoculated into a 6-well plate at a density of 1 × 105 cell/mL. The next day, when the cell density had reached approximately 70%, transfection was carried out according to the instructions for LipofectamineTM 2000 (Thermo Fisher Scientific).

7.Cell Counting Kit-8 experiments

MDA-MB-231 and MCF-7 breast cancer cells after transfection for 24 h were digested and inoculated into 96-well plates at a cell density of 5 × 104 cells/mL and incubated in a 37℃ incubator containing 5% CO2. After 24 h, adding 10µL Cell Counting Kit-8 (Biosharp, Beijing) to each well and then measured by enzyme-linked immunosorbent assay reader at a wavelength of 450nm.

8.Flow cytometry apoptosis detection

Transfected cells were digested with trypsin without EDTA, centrifuged at 1000 g, then washed twice with PBS. The cells were then stained according to the manufacturer instructions (Transgen Biotech, Beijing) and fluorescence was detected by flow cytometry. Higher FITC values indicated, more cell apoptosis, and higher PI values indicated more advanced apoptotic and necrotic cells.

9.Scratch wound healing experiment

Transfected BC cells were seeded in 6-well plates. A scratch was produced over the cells with a 200-µL pipette tip, and detached cells were removed by washing with PBS. All cells used in the wound-healing assay were cultured with DMEM with 2% FBS. The area of each scratch was assessed with a microscope at 0 and 24 h for the MDA-MB-231 cell line and MCF-7 cell line. Cell healing rate=(0h scratch area − 24h scratch area)/0h scratch area * 100%.

10.Transwell experiment

Breast cancer MDA-MB-231 and MCF-7 cells were transfected for 24 h, digested with trypsin and resuspended in serum-free high-glucose DMEM cell culture medium. The cells were inoculated into Transwell chambers at a density of 1 ×105 cells/well, and the cells were placed in 600 µL of high-sugar DMEM cell culture medium containing 10% fetal bovine serum. After 12 h of cultivation, the Transwell chambers were removed and washed 2–3 times with PBS. We used a cotton swab to wipe-off any cells that had not penetrated through the Transwell chamber, then fixed the inserts in a 4% paraformaldehyde solution for 30 min. The fixative was removed and the cells were washed 2–3 times with PBS. Finally, crystal violet solution was used to stain the cells at room temperature for 30 min before washing 2–3 times with PBS, cleaning the remaining substances and wiping the upper chamber cells clean with a cotton swab. After drying at room temperature, we observed the migration of cells under a microscope in random fields at the bottom of the chamber, took photos and counted corresponding groups of chamber cells. The obtained data was subjected to statistical analysis in the next step.

11.Dual luciferase reporter assay

We followed the experimental instructions for the Dual Luciferase Reporter Assay Kit (Vazyme; DL 101-01).

12.RNA immunoprecipitation

We performed RNA immunoprecipitation according to the instructions of the RIP reagent kit (Merck, DARMSTADT; 17–701).

13.Western blotting

Total proteins were extracted by using RIPA lysis buffer (Epizyme, Shanghai, China). Protein lysates were separated by 10% SDS-PAGE gels and then transferred to a PVDF membrane (Merck). After incubation with 5% non-fat milk for more than 1 h to block nonspecific binding, the membranes were immunoblotted overnight at 4°C with primary antibodies, i.e., anti-mTOR_(Cell Signaling Technology, Danvers, MA; 2972S), anti-PTEN_(AiFang Biological, Hunan;AF300857), anti-p-PI3K_(Cell Signaling Technology; 4228T), anti-PI3K_(Cell Signaling Technology; 11889S), anti-p-AKT_(Cell Signaling Technology; 4060T), anti-AKT_(AiFang; AF300966), anti-SLC7A11_(Abclonal, Wuhan; A2413), anti-EIF4A3_(AiFang; AF301768), anti-Bcl2_(Affinity;AF6139), anti-Bax_(Cohesion; CQA8019) and anti-β-actin_(Santa Cruz; sc-47778). Then, the membranes were incubated with diluted secondary antibodies for 1 h at room temperature. The membranes were scanned by an Odyssey infrared scanning system (LI-COR Biosciences, Lincoln, NE).

14.Lentiviral transfection and subcutaneous tumor inoculation

Transfected MDA-MB-231 cells with LV-circ_0022382 or LV-vector (GenePharma, Suzhou, China)were injected into the 4-week-old female athymic nude mice (n = 5, each group). Tumor growth was monitored every week. 4 weeks later, all mice were euthanatized by cervical dislocation. The collected tumors volumes were measured and calculated according to the formula (width2×length)/2. All experimental and animal care procedures complied with the rules of the Ethics Committee of Jiangsu University.

15.Immunohistochemistry

After removing the tumors, preparing slices and sequentially dehydrating, embedding and sealing, the slices were stained with anti-Ki-67 (AiFang; AF300540), anti-SLC7A11 (Proteintech, Wuhan, China; 26864-1-AP), or anti-p-AKT (Cell Signaling Technology).

16.Cellular immunofluorescence

Cells were seeded on cover slips in 24-well plates. and cultured for 24 h. Thereafter, the medium was discarded and the cells were washed three times with PBS. The cells were fixed in 4% paraformaldehyde for 15 min and then washed three times in PBS. Then, the cells were permeabilized with 0.5% Triton X-100 for 20 min. The cells were washed three times in PBS and blocked with goat serum at room temperature for 30 min. After discarding the blocking solution, primary antibody, i.e., anti-p-AKT (Cell Signaling Technology), or anti-SLC7A11 (Proteintech) was added. The cells were incubated overnight at 4℃, the primary antibody was removed and the cells were washed three times with PBS, then incubated with secondary antibody (Abclonal; AS007) at room temperature for 1 h. The cell slides were inverted onto anti-fluorescence quencher containing DAPI, and images were captured under a confocal microscope.

17.Glucose concentration detection

The glucose concentration in the cell supernatant was detected according to the glucose kit (Nanjing Jiancheng Bioengineering Institute, Nanjing; A154-2-1).

18.Statistical analysis

All experiments were performed in triplicate, and GraphPad Prism 8 software (GraphPad Software, San Diego, CA, USA) was used to analyze the data. All data are expressed as means ± Standard error of the mean(SEM). The significance of differences between the two groups was determined using the Student's t-test, and one-way analysis of variance was used to compare multiple groups.

1.Screening the GEO database identified circ_0022382 as highly expressed in breast cancer.

In the GSE165884 dataset of the GEO database, by performing differential analysis on normal breast tissue and corresponding breast cancer tissue from four samples, and using GEO2R analysis with thresholds of |log fold change (FC)|>2 and adj. p<0.05, we found 70 differentially expressed circRNAs, of which 37 were upregulated. Of these, 33 differentially expressed genes were visualized by the ggplot2 package in R studio, and volcano and heat maps were drawn (Fig.1a and b). Next, we analyzed the database GSE45498, taking 39 normal breast tissues and 55 triple negative in situ breast cancer tissues as the study focus. Using GEO2R, with thresholds |logFC|>1.4 and adj. p<0.05, we found 32 differentially expressed miRNAs, including six downregulated miRNAs, and visualized these six differentially expressed genes by the ggplot2 package in R studio, then plotted volcano and heat maps (Fig.1c and d). The 33 upregulated circRNAs obtained in the first step were used by the Encyclopedia of RNA Interactomes (ENCORI) database to predict the corresponding bound miRNAs, which intersected with the six miRNAs obtained in the second step that were downregulated in breast cancer. The intersecting miRNAs included hsa-let-7 (Fig.1e) and circRNAs bound to hsa-let-7, namely, circ_0022382. Next, we designed divergent primers for circ_0022382 and convergent primers for its parent gene fatty acid desaturase 2 (FADS2). The cDNA and genomic DNA from MDA-MB-231 cells were amplified by PCR using the divergent and convergent primers, respectively, and the agarose gel results showed that using convergent primers, fragments were amplified from both cDNA and genomic DNA; in contrast, using divergent primers, fragments were amplified from cDNA but not genomic DNA, demonstrating that the product formed by divergent primers was indeed circRNA with a circular structure (Fig.1f). After verifying the specificity of the divergent primers, real-time PCR analysis showed that the expression of circ_0022382 in breast cancer cells MDA-MB-231 and MCF-7 was higher than in normal breast epithelial MCF-10A cells (p<0.001), and the expression of circ_0022382 in breast cancer tissues was also higher than in the corresponding paracancerous tissues (p<0.05; Fig.1g).

2.Circ_0022382 regulates the proliferation, migratory capacity and apoptosis of breast cancer cells.

In transfection experiments, circ_0022382 was knocked-down in breast cancer cell lines MDA-MB-231 and MCF-7 using silencing RNA (siRNA). Transfection efficiency was detected by quantitative (q)-PCR, which found that the transfection primers si-circ_0022382-1 and si-circ_0022382-2 could effectively knock-down circ_0022382 expression in MDA-MB-231 and MCF-7 cells (Fig.2a). We chose si-circ_0022382-1 for follow-up experiments. Cell Counting Kit-8 experiments showed that the proliferative capacity of MDA-MB-231 and MCF-7 cells was significantly reduced after 72 h of transfection (Fig.2b). In addition, clonal proliferation results showed that knockdown of circ_0022382 caused a significant decrease in the number of breast cancer clones (Fig.2c). Scratch injury experiments showed a decrease in scratch wound repair after knocking down circ_0022382 (Fig.2d); Transwell experiments also confirmed a decrease in cell migratory capacity (Fig.2e). Breast cancer cells were then double stained with Annexin V and propidium iodide (PI). Flow cytometry analysis showed that apoptosis increased (Fig.2f), and western blot results showed that the ratio of Bax/Bcl2 increased (SI.a), indicating that knockdown of circ_0022382 promoted cell apoptosis.

3. Circ_0022382 regulates the proliferation and migration of breast cancer cells by affecting the PI3K-AKT signaling pathway via sponging of let-7a-5p.

Screening of circ_0022382 in the online ENCORI database indicated that it targeted the hsa-let-7 family of miRNAs, and knockdown of circ_0022382 in MCF-7 and MDA MB-231 breast cancer cells showed by q-PCR that hsa-let-7a-5p could sponge levels of circ_0022382 (Fig.3a). The binding of let-7a-5p and circ_0022382 was further validated through dual luciferase reporter assay (Fig.3b). Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis was performed on the downstream genes of let-7a-5p, and it was found that downstream target genes were enriched in the mammalian target of rapamycin (mTOR), phosphatidylinositol 3-kinase/AKT (PI3K-AKT), and mitogen activated protein kinase (MAPK) signaling pathways (Fig.3c). Western blot results showed that after knocking-down circ_0022382, the phosphorylation of PI3K and AKT was significantly reduced. As a downstream target gene of PI3K-AKT, mTOR expression also significantly decreased, and immunofluorescence results showed a decrease in the extent of AKT phosphorylation (Fig.3d-e). Co-transfection of si-circ_0022382 and let-7a-5p inhibitor significantly restored the weakened cell proliferative and migratory capacity caused by knocking down circ_0022382, as well as the decreased amount of AKT phosphorylation (Fig.3f-h).

4.Circ_0022382 directly regulates SLC7A11 through let-7a-5p.

The ENCORI database showed that let-7a-5p bound SLC7A11 (Fig.4a). After overexpression of let-7a-5p in breast cancer cells, the expression of SLC7A11 mRNA and SLC7A11 protein was significantly decreased according to q-PCR, western blot and immunofluorescence detection (Fig.4b-d). Co-transfection of si-circ_0022382 and let-7a-5p inhibitor restored the decrease in SLC7A11 caused by knocking-down circ_0022382 (Fig.4e).

5.Circ_0022382 regulates the processing of SLC7A11 and is closely related to disulfidptosis.

The ENCORI database showed that SLC7A11 mRNA was more highly expressed in BRCA cancer tissues (Fig.5a), and SLC7A11 protein was also more highly expressed in breast cancer cells MDA-MB-231 and MCF-7 than in normal breast epithelial MCF-10A cells (Fig.5b). Therefore, SLC7A11 may be a cancer promoting factor in breast cancer, given that knocking-down circ_0022382 caused a decrease in the expression of SLC7A11, which is closely related to ferroptosis and disulfidptosis. The addition of ferroptosis inhibitor failed to rescue the decreased cell proliferative and migratory capacity caused by knocking-down circ_0022382 (SI.c-d), while the addition of dithiothreitol (a drug that inhibits the accumulation of cysteine in cells) rescued the cells’ proliferation and scratch wound healing (Fig.5d-e). Meanwhile, after knocking-down circ_0022382, the extent of AKT phosphorylation decreased, and AKT is related to glucose metabolism. After measuring the cell supernatant, it was found that there were more glucose residues in the cell supernatant of the si-circ_0022382 group (Fig.5c), which may be due to lower cellular uptake. Therefore, although the decrease in SLC7A11 expression leads to a decrease in cysteine entry into cells mediated by a decrease in NADPH consumption, knocking-down the circ_0022382 led to a decrease in cellular glucose uptake, making it difficult for cells to obtain supplementary NADPH from the pentose phosphate pathway to compensate for the NADPH consumed by cysteine.

6.EIF4A3 promotes FADS2 gene variable splicing to produce more circ_0022382.

The CircInteractome database predicted that the upstream sequences of eukaryotic translation initiation factor 4A3 (EIF4A3) and circ_0022382 were bound (Fig.6a). At the same time, the ENCORI database showed that compared with normal tissues, EIF4A3 was highly expressed in breast cancer tissues (Fig.6b). Western blot results also showed that EIF4A3 was strongly expressed in breast cancer cells (Fig.6c). Therefore, EIF4A3 may be a potential oncogenic protein in breast cancer. However, after knocking down circ_0022382, the expression of EIF4A3 protein did not change (SI.b), so EIF4A3 may not be a downstream gene of circ_0022382. However, after knockdown of EIF4A3, the expression of circ_0022382 in breast cancer cells decreased significantly (Fig.6d), and the results of RNA Immunoprecipitation (RIP) further showed the binding of circ_0022382 and EIF4A3 protein (Fig.6e). Therefore, EIF4A3, as an upstream gene of circ_0022382, induced the FADS2 gene to produce more circular RNA through variable splicing during transcription.

7.Effects of circ_0022382 on tumor proliferation in vivo

MDA-MB-231 cells were transfected with either negative control sequence lentivirus (LV-NC) or stable-knockdown circ_0022382 sequence lentivirus (LV-circ shRNA),. Through subcutaneous tumor loading, we found that knockdown of circ_0022382 significantly reduced tumor volume and mass (Fig.7a-c). Immunohistochemical results showed a decrease in the expression of p-AKT, SLC7A11 and Ki-67 after knockdown of circ_0022382 (Fig.7d).

CircRNA is an important molecular marker in tumors, playing a crucial role in tumor growth and progression. In breast cancer, many circRNAs have been found to be related to tumor metabolism, recurrence and metastasis, drug resistance and anti-tumor immunotherapy, providing a direction for clinical treatment of breast cancer.

The abnormal expression of the PI3K-AKT signaling pathway can drive cancer progression[22, 23], and is related to the drug resistance produced by multi-line chemotherapy[24], so it has been widely studied as a mechanism of cancer. The present study found that circ_0022382 affected the proliferative and migratory capacity of breast cancer cells by sponging miRNA let-7a-5p. In addition, circ_0022382 can also affect the expression of SLC7A11 protein. Database analysis and cell protein detection suggest that SLC7A11 may be a tumor promoting factor in breast cancer. Therefore, we suspected that the decline of SLC7A11 protein expression may be related to ferroptosis, because ferroptosis will also lead to the decline of SLC7A11 protein expression in System Xc-[25, 26]. However, after adding Ferroptosis inhibition (Fer-1), the proliferation, migration, and proliferation of cells did not recover. This may be due to the complex regulatory mechanism of SLC7A11 in tumor cells. Gan proposed that the expression level of SLC7A11 determined the sensitivity of tumor cells to oxidative stress, and that the amount of cysteine transferred by cells indirectly reflected the expression level of SLC7A11. Therefore, the expression level of SLC7A11 in cells can be divided into low, medium and high levels. The addition of H2O2 induces oxidative stress in cells with low or high expression of SLC7A11, while cells with moderate expression of SLC7A11 are insensitive to H2O2-induced oxidative stress[27]. Therefore, the decrease in cell proliferation and migration caused by knocking down circ_0022382 may not be related to oxidative stress. When glucose is consumed normally, NADPH is supplemented through the pentose phosphate pathway, which can compensate for the consumption of NADPH by cysteine. When glucose is lacking, the supply of NADPH is insufficient. At this time, tumor cells with high expression of SLC7A11 will also consume a large amount of NADPH, leading to NADPH deficiency and cell disulfidptosis[28]. The addition of dithiothreitol to cells with knocked-down circ_0022382 restored their proliferation and migration. This is because dithiothreitol can inhibit the accumulation of cysteine in cells, thereby reducing the consumption of NADPH. Therefore, even if the cell intake of glucose is insufficient, it will not lead to disulfidptosis caused by the lack of NADPH. Therefore, circ_ 0022382 may indirectly cause disulfidptosis by affecting glucose intake. As a possible mechanism of apoptosis induced by knocking-down circ_0022382, the decrease in AKT phosphorylation in the PI3K-AKT signaling pathway may induce an increase in cell apoptosis. In addition, the upregulation of the Bax/Bcl2 ratio can also explain the phenomenon of apoptosis in breast cancer cells. Interestingly,we found that in MCF-7 cells, the negative regulatory protein Phosphatase and Tensin Homolog (PTEN) of AKT significantly increases after knocking down circ_0022382, while in MDA-MB-231 cells, PTEN did not show a significant increase from Figure.3d. This may be because AKT phosphorylation is not only regulated by PTEN, but also by protein phosphatase 2A[29]. The EIF4A3 protein is upregulated in many cancers and is associated with poor prognosis. As an RNA helicase that plays a regulatory role after transcription, the interaction between EIF4A3 and RNA has been widely studied. For mRNA, EIF4A3 can affect the stability and cellular sub-localization of Bcl2 mRNA[30]. For non-coding RNA, EIF4A3 can act as an upstream gene to influence the generation of lncRNA or circRNA[31, 32], as well as act as a sponge-like gene competing with non-coding RNA to bind or recruit non coding RNA to regulate the expression of downstream target genes[33, 34].

Using in vivo experiments, we conducted subcutaneous tumor-bearing experiments on immunocompromised nude mice to further verify the effect of the expression level of circ_0022382 on the proliferation of breast cancer cells, but we have not studied its effect on the migration of breast cancer cells in vivo. At the same time, the tumor microenvironment is quite complex, composed of immune cells and stromal cells such as myeloid-derived suppressor cells[35, 36], CD8 + T[37] and cancer-associated fibroblasts[38], which are also closely related to tumor progression. Further experiments are needed to explore the relationship between circ_0022382 and the tumor microenvironment.

In conclusion, we found that circ_0022382 may affect the growth of breast cancer cells by affecting the expression of PI3K-AKT-mTOR signaling pathway-related molecules and SLC7A11 through sponging let-7a-5p. By screening for circ_0022382 in the GEO database, and through in vitro and in vivo experiments. we have found that circ_0022382 may be useful as a molecular marker of breast cancer.

Acknowledgments

We would like to thank the staff at our laboratory for the assistance they have provided in this study.

Statement and delcarations

Funding

This work was supported by [Yongjiang Innovation Team in the Field of Science and Technology](Grant numbers [2022A-003-C])

Competing Interests

The authors declare that they have no competing interests.

Author information

Wei Liu and Jun Zhang have contributed equally to this work and share first authorship. Xiaochun Sun and Huabiao Chen are co-corresponding authors.

Authors and Affiliations

School of Medicine, Jiangsu University, Zhenjiang, 212013 , China

Xiaochun Sun, Wei Liu, Yu Ye, Jiawen Zhang, Jianqin Zhu & Qiwen Yu

Department of Laboratory, The Affiliated Hospital of Yangzhou University, Yangzhou, 225001, China

Jun Zhang

HEALTH BioMed Research & Development Center, Health BioMed Co., Ltd., Ningbo, Zhejiang, China; School of Medicine, Ningbo University, Ningbo, 315211, Zhejiang, China

Tao Li & Huabiao Chen

Author Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Wei Liu, Yu Ye, Jiawen Zhang, Jianqin Zhu, Qiwen Yu and Tao Li. The first draft of the manuscript was written by Wei Liu and Jun Zhang. Huabiao Chen and Xiaochun Sun supervised the study. All authors read and approved the final manuscript.

Corresponding authors

Xiaochun Sun and Huabiao Chen are co-corresponding authors. Correspondence to Xiaochun Sun, [email protected] or Huabiao Chen, [email protected].

Data availability statements

Differentially expressed circRNAs and miRNAs in breast cancer patients were obtained by analyzing the GEO dataset (https://www.ncbi.nlm.nih.gov/geo/) with GEO2R (https://www.ncbi.nlm.nih.gov/geo/geo2r/).

Ethical approval

All procedures performed in studies involving human participants were approved by Ethics Committee of the Affiliated Hospital of Jiangsu University . All methods of animal experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals. All methods are reported in accordance with ARRIVE guidelines for the reporting of animal experiments. The study was carried out in accordance with the relevant guidelines and regulation.

Consent to participate

The study was conducted in accordance with the Declaration of Helsinki.Written informed consent was obtained from the parents.

Consent to publish

The authors affirmed that human research participants provided informed consent for publication of the images in Fig.1g.

Fan L, Strasser-Weippl K, Li JJ et al (2014) Breast cancer in China [J]. Lancet Oncol 15(7):e279–e289
Phadke S (2022) Optimization of Neoadjuvant Therapy for Early-Stage Triple-Negative and HER2 + Breast Cancer [J]. Curr Oncol Rep 24(12):1779–1789
Xie K, Ren X, Hong X et al (2022) Platinum-based adjuvant therapy was efficient for triple-negative breast cancer: a meta-analysis from randomized controlled trials [J]. Bioengineered 13(6):14827–14839
Sadeghi F, Asgari M, Matloubi M et al (2020) Molecular contribution of BRCA1 and BRCA2 to genome instability in breast cancer patients: review of radiosensitivity assays [J]. Biol Proced Online 22:23
Hu H, Zhu J, Zhong Y et al (2021) PIK3CA mutation confers resistance to chemotherapy in triple-negative breast cancer by inhibiting apoptosis and activating the PI3K/AKT/mTOR signaling pathway [J]. Ann Transl Med 9(5):410
Fujimoto Y, Morita TY, Ohashi A et al (2020) Combination treatment with a PI3K/Akt/mTOR pathway inhibitor overcomes resistance to anti-HER2 therapy in PIK3CA-mutant HER2-positive breast cancer cells [J]. Sci Rep 10(1):21762
Chen ZG, Zhao HJ, Lin L et al (2020) Circular RNA CirCHIPK3 promotes cell proliferation and invasion of breast cancer by sponging miR-193a/HMGB1/PI3K/AKT axis [J]. Thorac Cancer 11(9):2660–2671
Yang M, Hu H, Wu S et al (2022) EIF4A3-regulated circ_0087429 can reverse EMT and inhibit the progression of cervical cancer via miR-5003-3p-dependent upregulation of OGN expression [J]. J Exp Clin Cancer Res 41(1):165
Shu C, Xu P, Han J et al (2022) Upregulation of circRNA hsa_circ_0008726 in Pre-eclampsia Inhibits Trophoblast Migration, Invasion, and EMT by Regulating miR-345-3p/RYBP Axis [J]. Reprod Sci 29(10):2829–2841
Yi J, Wang L, Hu GS et al (2023) CircPVT1 promotes ER-positive breast tumorigenesis and drug resistance by targeting ESR1 and MAVS [J]. EMBO J 42(10):e112408
Lei J, Zhu J, Hui B et al (2023) Circ-HSP90A expedites cell growth, stemness, and immune evasion in non-small cell lung cancer by regulating STAT3 signaling and PD-1/PD-L1 checkpoint [J]. Cancer Immunol Immunother 72(1):101–124
Li J, Xu J, Wu G et al (2022) Circular RNA hsa_circ_0068252 Functions in Cisplatin Resistance and Immune Response via miR-1304-5p/PD-L1 Axis in Non-Small Cell Lung Cancer [J]. Chemotherapy 67(4):223–233
Zhang YQ, Qi WB, Wu YL (2023) EIF4A3-induced circular RNA SCAP facilitates tumorigenesis and progression of non-small-cell lung cancer via miR-7/SMAD2 signaling [J]. Environ Sci Pollut R 30(24):65237–65249
Zhou X, Zhao Q, Xiao M (2023) Hsa_circ_0137652 Regulates miR-1205/CCNB1 Axis to Accelerate the Malignancy of Breast Cancer [J]. Mol Biotechnol 65(11):1824–1835
Zhuang Y, Li L, Wu H et al (2022) CircRNA ACVR2A Sponges miR-1290 to Modulate Cell Progression in Gastric Cancer [J]. J Oncol, 2022: 9461054
Wu Y, Xu M, Feng Z et al (2023) AUF1-induced circular RNA hsa_circ_0010467 promotes platinum resistance of ovarian cancer through miR-637/LIF/STAT3 axis [J]. Cell Mol Life Sci 80(9):256
Wang RJ, Zhang S, Chen XY et al (2018) EIF4A3-induced circular RNA MMP9 (circMMP9) acts as a sponge of miR-124 and promotes glioblastoma multiforme cell tumorigenesis [J]. Mol Cancer 17(1):166
Zhang K, Wan X, Khan MA et al (2022) Peripheral Blood circRNA Microarray Profiling Identities hsa_circ_0001831 and hsa_circ_0000867 as Two Novel circRNA Biomarkers for Early Type 2 Diabetic Nephropathy [J]. Diabetes Metab Syndr Obes 15:2789–2801
Sun X, Kong S, Jiang C et al (2022) Diagnostic Value of Circular RNA hsa_circ_0002874 Expression in Peripheral Blood of Patients with Gastric Cancer [J]. Lab Med 53(1):65–70
He YD, Tao W, He T et al (2021) A urine extracellular vesicle circRNA classifier for detection of high-grade prostate cancer in patients with prostate-specific antigen 2–10 ng/mL at initial biopsy [J]. Mol Cancer 20(1):96
Wang D, Chen Z, Zhuang X et al (2020) Identification of circRNA-Associated-ceRNA Networks Involved in Milk Fat Metabolism under Heat Stress [J]. Int J Mol Sci 21(11):4162
Cai M, Li H, Chen R et al (2021) MRPL13 Promotes Tumor Cell Proliferation, Migration and EMT Process in Breast Cancer Through the PI3K-AKT-mTOR Pathway [J]. Cancer Manag Res 13:2009–2024
Hu GF, Wang C, Hu GX et al (2020) AZD3463, an IGF-1R inhibitor, suppresses breast cancer metastasis to bone via modulation of the PI3K-Akt pathway [J]. Ann Transl Med 8(6):336
Nicolini A, Ferrari P, Kotlarova L et al (2015) The PI3K-AKt-mTOR Pathway and New Tools to Prevent Acquired Hormone Resistance in Breast Cancer [J]. Curr Pharm Biotechnol 16(9):804–815
Chen Q, Zheng W, Guan J et al (2023) SOCS2-enhanced ubiquitination of SLC7A11 promotes ferroptosis and radiosensitization in hepatocellular carcinoma [J]. Cell Death Differ 30(1):137–151
Shen L, Zhang J, Zheng Z et al (2022) PHGDH Inhibits Ferroptosis and Promotes Malignant Progression by Upregulating SLC7A11 in Bladder Cancer [J]. Int J Biol Sci 18(14):5459–5474
Yan Y, Teng H, Hang Q et al (2023) SLC7A11 expression level dictates differential responses to oxidative stress in cancer cells [J]. Nat Commun 14(1):3673
Liu T, Ren Y, Wang Q et al (2023) Exploring the role of the disulfidptosis-related gene SLC7A11 in adrenocortical carcinoma: implications for prognosis, immune infiltration, and therapeutic strategies [J]. Cancer Cell Int 23(1):259
Liu CY, Hu MH, Hsu CJ et al (2017) Correction: Lapatinib inhibits CIP2A/PP2A/p-Akt signaling and induces apoptosis in triple negative breast cancer cells [J]. Oncotarget 8(6):10760
Yin Y, Xu W, Song Y et al (2022) Icariin Regulates the hsa_circ_0003159/eIF4A3/bcl-2 Axis to Promote Gastric Cancer Cell Apoptosis [J]. Evid Based Complement Alternat Med, 2022:1955101
Wang XH, Song HM, Fang L et al (2022) EIF4A3-mediated circPRKCI expression promotes triple-negative breast cancer progression by regulating WBP2 and PI3K/AKT signaling pathway [J]. Cell Death Discovery 8(1):92
Xu QQ, Zhao TT, Han HH et al (2023) EIF4A3 stabilizes the expression of lncRNA AGAP2-AS1 to activate cancer-associated fibroblasts via MyD88/NF-kappa b signaling [J]. Thorac Cancer 14(5):450–461
Ren LH, Fang X, Shrestha SM et al (2022) LncRNA SNHG16 promotes development of oesophageal squamous cell carcinoma by interacting with EIF4A3 and modulating RhoU mRNA stability [J], vol 27. Cellular & Molecular Biology Letters, p 89. 1
Wang XJ, Liu S, Xu B et al (2021) circ-SIRT1 Promotes Colorectal Cancer Proliferation and EMT by Recruiting and Binding to eIF4A3 [J]. Anal Cell Pathol, 2021:5739769
Khaki Bakhtiarvand V, Ramezani-Ali Akbari K, Amir Jalali S et al (2022) Myeloid-derived suppressor cells (MDSCs) depletion by cabozantinib improves the efficacy of anti-HER2 antibody-based immunotherapy in a 4T1-HER2 murine breast cancer model [J]. Int Immunopharmacol, 113(Pt B): 109470
Zhang R, Dong M, Tu J et al (2023) PMN-MDSCs modulated by CCL20 from cancer cells promoted breast cancer cell stemness through CXCL2-CXCR2 pathway [J]. Signal Transduct Target Ther 8(1):97
Dong H, Xie C, Yao Z et al (2022) PTPRO-related CD8(+) T-cell signatures predict prognosis and immunotherapy response in patients with breast cancer [J]. Front Immunol 13:947841
Wen S, Hou Y, Fu L et al (2019) Cancer-associated fibroblast (CAF)-derived IL32 promotes breast cancer cell invasion and metastasis via integrin beta3-p38 MAPK signalling [J]. Cancer Lett 442:320–332

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Mechanism study of EIF4A3-induced circ_0022382 regulation of breast cancer cell biology

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