CircRNA₀025202 Regulates Tamoxifen Sensitivity And Tumor Progression Via Regulating The MiR-182-5p/FOXO3a Axis In Breast Cancer

Background and objectives:Breast cancer has the most frequent incidence and the second highest mortality in women worldwide.As a highly heterogeneous cancer type,breast cancer is divided into several different subtypes according to the expression of estrogen receptor,progesterone receptor,human epidermal growth factor receptor-2 and ki-67 index detected by immunohistochemical（IHC）.Notably,three quarters of patients with breast cancer are hormone receptor positive.For those patients,apart from surgery and adjuvant chemotherapy,anti-hormone therapy is recognized as the standard treatment.Tamoxifen,a pioneer selective estrogen receptor modulator,is the most commonly used endocrine therapy for patients with hormone receptor-positive breast cancer for decades.Although,tamoxifen was reported to largely decline the recurrence rate and mortality of the hormone receptor-positive breast cancer patients,many of them could develop drug resistance which leads to cancer relapse during the medication.Therefore,tamoxifen resistance largely limits the effectiveness of endocrine management and is regarded as the leading cause of therapeutic failure for patients with hormone receptor-positive breast cancer.In recent years,numerous studies have confirmed that the epigenetic modification are of great importance in the regulation of the tumor progression and metastasis of patients with hormone receptor-positive breast cancer.Among the various mechanism of epigenetic regulation,non-coding RNA is demonstrated to play a pivotal role in tumor progression.Circular RNA,as a new member of endogenous noncoding RNAs,was characterized by lack of free ends,resistance to the exonucleolytic and high cellular stability.It was reported as a more abundant RNA isoform in various cell types and organisms.In recent years,due to the temporal and spatial specificity of its expression,more and more studies have focused on the biological functions of circular RNA.Studies have confirmed that the development of tumors is closely related to the abnormal expression of some circular RNAs.Therefore,this paper further explores the mechanism of action in tamoxifen resistance by screening circular RNAs that are differentially expressed in tamoxifen-resistant and non-resistant cell lines,which might provide a novel marker or therapeutic target for tamoxifen-resistant breast cancer.Methods:1.Establishment of tamoxifen-resistant MCF-7 cell lineTo establish tamoxifen-resistant MCF-7 cell line（MCF-7/TR）,our team treated MCF-7 cell with low concentration of tamoxifen for one year.The cytotoxicity assay was used to detect the IC50 of the parental MCF-7（MCF-7/P）and MCF-7/TR against tamoxifen,and the resistance of MCF-7/TR was verified.Subsequently,MTT,clone formation,transwell,and cell apoptosis assays were used to detect the differences between MCF-7/P and MCF-7/TR in biological functions.2.Screening of tamoxifen resistance-related circular RNAWe extracted total cellular RNA of MCF-7/P and MCF-7/TR.The different expression profiling of MCF-7/TR and MCF-7/P was analyzed by RNA sequencing（RNA-seq）analysis.According to the abundance and differential expression,five upregulated and five downregulated circRNAs were chosen for further confirmation in MCF-7/P and MCF-7/TR cells using real-time quantitative PCR（qRT-PCR）.Hsa_circ₀025202,as the most significant down-regulated circular RNA,was selected as the target molecule for the study.Moreover,the expression levels of hsa_circ₀025202 were evaluated in hormone receptor-positive breast cancer tissues and adjacent normal tissues using qRT-PCR.To explore the correlation between hsa_circ₀025202 and clinicopathological parameters,230 patients pathological diagnosed with hormone receptor-positive breast cancer were involved in our study.We categorized all patients with breast cancer into hsa_circ₀025202 high-and low-expression groups using the median expression as cut-off threshold,and analyzed the correlation between hsa_circ₀025202 and the demographic and pathological parameters of patients with hormone receptor-positive breast cancer.3.Exploring the biological function of hsa_circ₀025202PCR,agarose gel electrophoresis,and sanger sequencing were conducted to confirm the existence of hsa_circ₀025202.The properties of hsa_circ₀025202 as a circular RNA were verified by actinomycin D treatment and RNase R digestion experiments.Cytoplasmic and nuclear RNA isolation and qRT-PCR was utilized to verify the subvocalization of hsa_circ₀025202 in breast cancer cell lines.Gain-and loss-functional assays were performed in hormone receptor-positive breast cancer cell lines T47D and MCF-7 and MCF-7/TR respectively.The efficiency was confirmed by qRT-PCR.Biological functions of hsa_circ₀025202,such as cell proliferation,migration,apoptosis,and tamoxifen drug sensitivity,were detected by MTT,transwell,cytotoxicity,and cell apoptosis assays.4.Confirmation of hsa circ 0025202 acts as an efficient micro RNA（miRNA）spongeCircInteractome and starBase V2.0 were used to explore the miRNA response elements（MREs）harbored by hsa circ 0025202.After intersecting the two database results,the miRNAs which rank in the foremost were chose as candidate miRNAs.verified by qRT-PCR,RNA immunoprecipitation and luciferase activity assays,miR-182-5p was identified as the target gene of hsa circ 0025202 for further investigation.After co-transfection of hsa_circ₀025202 overexpression vector and miR-182-5p mimics in T47D and MCF-7 cells,functional studies,such as MTT and transwell,were conducted to investigate whether miR-182-5p can reverse the biological function of hsa circ 0025202.5.Identification the target gene of miR-182-5p and its mechanismBioinformatics method was used to predict the downstream target gene of miR-182-5p,and the candidates were selected and verified by qRT-PCR,western blot and luciferase reporter assay.After a series of functional experiments,we confirmed the biological function and mechanism of hsa_circ₀025202 in the progression of hormone receptor-positive breast cancer patients and tamoxifen resistance.6.Identification the biological function of hsa circ 0025202 in vivowe established MCF-7 cells which were stably overexpressed hsa circ 0025202（M7/circ 0025202 ove）and the corresponding control cells（M7/PLCDH）.The overexpression efficiency was detected by qRT-PCR.Two weeks after 5-week-old female BALB/c mice implanted subcutaneously with estrogen sustained-release tablets,M7/circ₀025202 ove and M7/PLCDH cells were subcutaneously injected.Subsequently,tamoxifen was administered intragastrically.Tumor volume and weight were measured every three days until the end point.The expression of hsa circ 0025202 and downstream mRNA was detected by immunohistochemistry.Results:1.The results of RNA-sequencing showed that hsa_circ₀025202 level was significantly down-regulated in MCF-7/TR compared with MCF-7/P.Further,we detected the expression level of hsa_circ₀025202 in tissue specimens.The results showed that hsa circ 0025202 was significant down-regulated and negatively correlated with histological grade（p=0.005）and lymph nodes metastasis（p=0.048）in patients with hormone receptor-positive breast cancer.2.The specific divergent primer that specially amplified the back splicing point of hsa circ 0025202 were designed and used in PCR.The product of PCR was applied to agarose gel electrophoresis.And the sequence was finally determined by sanger sequencing,which confirmed the existence of hsa_circ₀025202.Amphotericin D assay indicated no significant change in the level of hsa circ 0025202.And hsa circ 0025202 showed resistance to RNase R treatment.In summary,hsa circ 0025202 is more stable than its linear form.qRT-PCR analysis of nuclear and cytoplasmic RNAs revealed that hsa_circ₀025202 was predominantly resided in the cytoplasm in MCF-7 and T47D cells.3.MTT,cytotoxicity,transwell,flow cytometry results showed that besides the stronger tamoxifen resistance,MCF-7/TR presented enhanced proliferation,colony formation,migration ability and reduced apoptosis compared with MCF-7/P cells.Whereas,hsa circ 0025202 overexpression could reverse the malignant phenotype of MCF-7/TR by inhibiting cell proliferation,clone formation and migration,inducting cell apoptosis,and sensitizing breast cancer cells to tamoxifen.4.Bioinformatics,luciferase activity assays and functional experiment confirmed that hsa_circ₀025202 could act as an efficient miRNA sponge for miR-182-5p,thereby regulating the expression and activity of its downstream target gene FOX03a.Therefore,hsa circ 0025202 could inhibit cancer progression and increase tamoxifen sensitivity in hormone receptor-positive breast cancer.5.The animal experiment results showed that M7/hsa_circ₀025202 ove group combined with tamoxifen treatment had smaller and lighter tumor.Immunohistochemical analysis also showed that decreased ki67 level,and increased FOXO3a,cleaved caspase-3 level in M7/circ 0025202 ove group comparing with M7/PLCDH group,as well as in tamoxifen group comparing with PBS group.Conclusions:In conclusion,our study demonstrated that hsa circ 0025202 could act as a potent suppressor gene for hormone receptor-positive breast cancer,and play a noticeable role in regulation of tamoxifen sensitivity.Based on the above data,we suggest that increasing level of hsa_circ₀025202 could exert beneficial therapeutic effects in hormone receptor-positive breast cancer patients,especially for those patients received tamoxifen therapy.Moreover,our result implied that hsa_circ₀025202 could be an important and promising biomarker for tamoxifen resistance during medication.