| Breast cancer accounts for about 25% of all types of cancer in women,and Triplenegative breast cancer(TNBC)accounts for about 15% to 20% of breast cancer.TNBC is a highly heterogeneous and aggressive cancer with high mortality and poor prognosis in all breast cancer subtypes.Its molecular characteristics are the negative expression of estrogen receptor,progesterone receptor,and human epidermal growth factor receptor 2.Anthracycline-based adjuvant chemotherapy is still one of the standard treatments for TNBC.Pirarubicin(THP)is comparable to Doxorubicin in efficacy,but THP is less cardiotoxic and widely used.However,chemotherapy has not yet resulted in a particularly effective TNBC targeting strategy.Therefore,it is urgent to find new drug intervention targets to provide a new theoretical basis for the diagnosis,treatment,and prognosis of TNBC.Circular RNA(circ RNA)and other non-coding RNA(nc RNA)participate in various physiological and pathological processes of cells.Circ RNAs can play the role of competitive endogenous RNAs(ce RNAs),which sponge micro RNAs(mi RNAs)and thus reduce the inhibitory effect of mi RNAs on the m RNAs it targets.Although circ RNA has potential value as a diagnostic and prognostic biomarker of TNBC and plays an important role in the malignant progression of TNBC,the mechanism of its role in TNBC and whether it mediates the antitumor effect of THP remain unclear,and further studies are in urgent need.The purpose of this study was to screen target circ RNAs associated with TNBC tissue and THP therapy.Through in vitro and in vivo experiments,various experimental methods were used to provide theoretical support for the malignant progression of TNBC and the anti-TNBC mechanism of THP from the perspective of circ RNA.This study consists of three parts:(1)Bioinformatics analysis of circ RNA gene chips and cascade and validation of circ EGFR/mi R-1299/EGFR pathwayIn this study,the results of circ RNA sequencing in TNBC tissues reported in the literature intersected with the results of circ RNA sequencing in MDA-MB-231 cells interfered by THP in our research group.circ EGFR was significantly upregulated and ranked first in TNBC tissues but significantly downregulated and ranked first in MDAMB-231 cells with THP intervention.Sanger sequencing,agarose gel electrophoresis,ribonuclease R assay,and actinomycin D assay proved that circ EGFR has a ring structure and is real existence.circ EGFR was mainly distributed in the cytoplasm,suggesting that circ EGFR mainly plays a role in the cytoplasm.At the cellular level,circ EGFR expression was significantly downregulated after THP intervention in MDA-MB-231 cells,consistent with THP intervention in MDAMB-231 cell chips.circ EGFR was specifically highly expressed in TNBC cells(MDAMB-231,HCC1806,and MDA-MB-468).Thirty-eight clinical TNBC tissue samples were collected,and it was confirmed that circ EGFR was highly expressed in TNBC tissues,consistent with the results of TNBC tissues sequencing.High circ EGFR expression was associated with poor overall survival and disease free survival.The receiver operating characteristic curve showed that circ EGFR expression could sensitively distinguish between TNBC tissue and paired normal breast tissue.These results indicate that circ EGFR in TNBC patients has potential value as a biomarker for the diagnosis and prognosis of TNBC.Plasma exosomes of TNBC patients and non-TNBC patients were extracted and separated,and circ EGFR was highly expressed in the plasma exosomes of TNBC patients.In 49 TNBC patients,high expression of circ EGFR in plasma exosomes was only associated with poor disease-free survival.circ EGFR expression was downregulated in plasma exosomes of TNBC patients treated with anthracyclines.These results suggest that circ EGFR in plasma exosomes of TNBC patients has potential value as a prognostic biomarker for TNBC.Circ Bank and Circular RNA Interactome were then used to predict mi RNAs with binding sites to circ EGFR,and three candidate mi RNAs were obtained by intersection screening.However,only mi R-1299 was significantly associated with the prognosis of TNBC patients.Therefore,mi R-1299 is determined as the following research object.The Gene Ontology and Kyoto Encyclopedia of Genes and Genome signal pathway enrichment analysis were used to analyze the target genes of mi R-1299.The results showed that the EGFR tyrosine kinase inhibitor resistance signal pathway had the lowest p value.Further,the intersection of the mi R-1299 target genes in the EGFR tyrosine kinase inhibitor resistance signaling pathway with THP intervention in MDAMB-231 cells m RNAs sequencing results yielded 14 candidate m RNAs.Epidermal Growth Factor Receptor(EGFR),the parent gene of circ EGFR,is included,and EGFR plays a vital role in the EGFR tyrosine kinase inhibitor resistance signaling pathway.Therefore,EGFR is selected as the object of further study.Binding sites between the3’-untranslated region of EGFR m RNA and mi R-1299 were found using the Targetscan database.The circ EGFR/mi R-1299/EGFR pathway is thus cascaded.Dual luciferase reporter gene assay confirmed a binding site between circ EGFR and mi R-1299 and a binding site between mi R-1299 and EGFR.In 38 TNBC tissues,mi R-1299 was significantly underexpressed.At the same time,EGFR was significantly overexpressed,and circ EGFR expression was negatively correlated with mi R-1299 expression and positively correlated with EGFR expression.In contrast,mi R-1299 expression was negatively correlated with EGFR expression,consistent with ce RNA mechanism characteristics.It is further indirectly proved that the three have a mutual regulation relationship.(2)THP inhibited the proliferation,migration,invasion,and epithelialmesenchymal transformation of MDA-MB-231 cells through the circ EGFR/mi R-1299/EGFR pathwayMDA-MB-231 cells were transfected with stable overexpression and stable silencing circ EGFR lentiviruses,and stable expression cell lines were successfully constructed.Overexpression of circ EGFR can promote proliferation,migration,invasion,and Epithelial-mesenchymal transition(EMT)of MDA-MB-231 cells and reduce cell susceptibility to THP intervention.Silencing circ EGFR had the opposite effect.This study confirmed that THP could inhibit the proliferation,migration,invasion,and EMT of MDA-MB-231 cells,and circ EGFR overexpression can partially block the above effects of THP.These results suggested that THP could inhibit the malignant phenotype of MDA-MB-231 cells by downregulating circ EGFR.In order to confirm the regulatory relationship between circ EGFR,mi R-1299,and EGFR,the effects of circ EGFR on downstream mi R-1299 and EGFR were detected by RT-q PCR and Western blot,and it was found that overexpression of circ EGFR could downregulate the expression of mi R-1299 and upregulated EGFR m RNA and protein expression.The effect of silencing circ EGFR was the opposite.In addition,mi R-1299 mimics can partially block the upregulation of EGFR by overexpressing circ EGFR.It was proved that there were targeted regulatory relationships between circ EGFR and mi R-1299 and between mi R-1299 and EGFR in MDA-MB-231 cells,which was consistent with the results of dual luciferase reporter gene assay.In order to further confirm the role of circ EGFR in promoting the malignant phenotype of MDA-MB-231 cells through mi R-1299/EGFR,the rescue experiment in this study found that overexpression of circ EGFR could promote the proliferation,migration,invasion,and EMT of MDA-MB-231 cells.mi R-1299 mimics can inhibit the malignant phenotype of MDA-MB-231 cells and partially block the effect of circ EGFR overexpression on promoting the malignant phenotype.This study also found that mi R-1299 inhibitor can promote the proliferation,migration,invasion,and EMT of MDA-MB-231 cells.si-EGFR could inhibit the malignant phenotype of MDAMB-231 cells and partially block the effect of mi R-1299 inhibitor on malignant phenotype promotion.These results suggest that circ EGFR promotes the malignant phenotype of MDA-MB-231 cells through mi R-1299/EGFR.(3)The effect of circ EGFR on the inhibitory effect of THP on TNBC xenograftsBALB/c nude mice were selected as the study objects.The xenograft tumor model was established by subcutaneous injection of MDA-MB-231 cells in the axilla.Nude mice inoculated with stable overexpression circ EGFR MDA-MB-231 cells showed the fastest tumor growth and heaviest tumor weight.Ki67 immunohistochemical staining had the highest positive rate.In tumor tissues,circ EGFR and EGFR expressions were significantly upregulated,mi R-1299 expression was significantly downregulated,Ecadherin protein expression was significantly decreased,and Vimentin and Snail protein expression were significantly increased.The results were the opposite for nude mice inoculated with stable silenced circ EGFR MDA-MB-231 cells.These results indicated that overexpression of circ EGFR could promote the malignant progression of TNBC xenografts,while silencing circ EGFR had the opposite effect.THP can inhibit the growth of TNBC xenografts and reduce tumor volume and weight.The positive rate of Ki67 immunohistochemical staining was decreased.The expression of circ EGFR and EGFR was significantly downregulated,the expression of mi R-1299 was significantly upregulated,the expression of E-cadherin protein was significantly increased,and the expression of Vimentin and Snail protein were significantly decreased.However,overexpression of circ EGFR can partially block the above effect of THP.These results indicate that circ EGFR not only mediates THP to inhibit the malignant progression of TNBC xenograft tumors but also reduces the sensitivity of TNBC xenograft tumors to THP treatment.In summary,this study reached the following conclusions through in vivo and in vitro experiments:(1)Circ EGFR(hsa＿circ＿0080220)was screened and verified by bioinformatics analysis,proving it was highly expressed in TNBC cell lines,patient tissues,and plasma exosomes,and was associated with poor prognosis of patients.The expression level of circ EGFR in patient tissue has potential diagnostic value to distinguish TNBC tissue from normal breast tissue.(2)The circ EGFR/mi R-1299/EGFR pathway was cascaded and verified by bioinformatics analysis and dual luciferase reporter gene assay;(3)In vitro studies confirmed that overexpression of circ EGFR promoted the proliferation,migration,invasion,and EMT of MDA-MB-231 cells and decreased the sensitivity of THP treatment while silencing circ EGFR showed the opposite effect.THP can inhibit the malignant phenotype of MDA-MB-231 cells by downregulating the expression of circ EGFR,which may be realized through the circ EGFR/mi R-1299/EGFR pathway.(4)In vivo studies confirmed that overexpression of circ EGFR can promote tumor growth and EMT and reduce tumor sensitivity to THP treatment.Silencing circ EGFR inhibited the malignant progression of the tumor. |
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