The Roles And Mechanisms Of Proten-coding Circ-EIF6 In Triple Negative Breast Cancer

BackgroundBreast cancer is one of the most common malignant tumors in women worldwide,the morbidity and mortality of breast cancer are increasing in recent years,which is a serious threaten to the health of women.Breast cancer is a heterogeneous disease,and approximately 15%-20%of patients with breast cancer are diagnosed with the triple-negative subtype(triplenegative BC[TNBC]).TNBC has been reported as a subtype with relatively poor prognosis due to rapid proliferation,early metastasis,a high recurrence rate,and a lack of molecular targets for treatment.The special biological behaviours and clinicopathological features of TNBC are the key factors that contribute to the high mortality of TNBC,and an understanding of the molecular mechanisms associated with TNBC tumorigenesis is vital for clinical treatment and improving the prognoses of TNBC patients.CircRNA(circular RNA)is characterized by the nonsequential backsplicing of pre-mRNA transcripts,resulting in the formation of covalently closed continuous loops.Endogenous circRNAs are widely expressed throughout the eukaryotic transcriptome.A large number of studies have shown that the abnormal expression of circRNAs could play crucial roles in the epigenetic regulation of malignant tumors,and circRNA has become a hot research area in cancer research.It has been reported that some circRNAs could act as ’microRNA sponges’ and adsorb microRNAs through endogenous competitive binding mechanism,thus blocking the biological functions of microRNAs.However,most circRNAs are generated from coding exons of genes,they are the most studied circRNAs and typically reside in the cytoplasm.Exonic circRNAs potentially contain specific ORFs or similar ORFs that overlap with related mRNAs,and certain so-called noncoding circRNAs might be translated into proteins.It has been proved that some exon-derived circRNAs could be used as templates to translate novel proteins in colon cancer,liver cancer and other cancers.However,researchers have not clearly determined whether protein-coding circRNAs are involved in TNBC,and their functional products remain elusive.To identify circRNAs that related to TNBC progression and metastasis,RNA-seq and bioinformatics analysis were performed and circ-EIF6 was selected for its high expression in metastatic breast cancer tissues and 231_M cell line,which was futher reaserched in this study.The functions and molecular mechanisms of circ-EIF6 were further discussed by in vitro and in vivo experiments and clinical samples.Part Ⅰ Screening and expression analysis of circRNAs associated with progression and metastasis of triple negative breast cancerObjective1.Screen circRNAs related to TNBC progression and metastasis.2.Evaluate the expression of circ-EIF6 in breast cancer tissues and cell lines.3.Discuss the correlation between circ-EIF6 expression and clinicopathologic features and prognoses of TNBC patients.MethodsRNA-seq was first used to filter circRNAs that were closely related to the progression and metastasis of TNBC,and the expression of circ-EIF6 was further evaluated in breast cancer cell lines by qRT-PCR.Moreover,98 samples of TNBC pateints were selected and the expression of circ-EIF6 was detected.Finally,the correlation between circ-EIF6 expression and clinicopathologic features and prognoses of TNBC patients were analyzed.ResultsCirc-EIF6 expression was significantly up-regulated in breast cancer tissues and 231_M cell lines,and its expression was remarkable overexpressed in TNBC cell lines.Further clinicopathological features and prognosis analysis found that the expression of circ-EIF6 was was closely related to the histological grade and distant metastasis of TNBC patients,and high expression of circ-EIF6 indacated poor prognosis of TNBC patients.Conclusions1.Circ-EIF6 was significantly overexpressed in metastatic breast cancer tissues and 231_M cell line.2.The expression of circ-EIF6 in TNBC cell lines was significantly higher than that in nonTNBC cell lines.3.The expression of circ-EIF6 was closely related to the clinicopathological features and prognosis of TNBC patients.Part Ⅱ Verification of circular properties and protein-coding abilities of circ-EIF6Objective1.Verify the basic circular properties of circ-EIF6.2.Investigate whether circ-EIF6 could be translated into novel protein EIF6-224aa.3.Determine whether EIF6-224aa was generally expressed in TNBC cells and tissues.MethodsThe genomic origin of circ-EIF6 was firstly analyzed and the existence of circ-EIF6 in TNBC cells was verified by PCR,Southern Blot,Sanger sequencing and other methods.RNase R,actinomycin D and PCR were used to verify the circular properties of circ-EIF6.RNA-FISH and subcellular fractionation were used to explore the cellular sublocation of circ-EIF6.The ribosome-binding ability of circ-EIF6 was verified by polysome profiling.Dual-Luciferase and western blot assays were performed to validate the protein-coding abilities of circ-EIF6.Coomhas staining,LC-MS and specific antibody proved the generally expression of EIF6224aa in TNBC cells and tissues.ResultsCirc-EIF6 arose from exons 3-7 of the EIF6 gene on chr20:33866724-33872064(20q11.22)and ultimately formed the mature sequence with a length of 906 nt.The specific splicing sequence of circ-EIF6 could be amplified in TNBC cells by using divergent primers.Circ-EIF6 was resistant to RNase R digestion,have a longer half-time,and had no poly(A)tail,which indicated the circular properties of circ-EIF6.Circ-EIF6 was mainly located in the cytoplasm of TNBC cells and could interact with intracellular ribosomes.The sequence of circ-EIF6 contained an IRES element and a 675nt ORF region and the IRES element could initiate the translation of circ-EIF6,which suggested that circ-EIF6 might be translate in to a novel protein with a length of 224 amino acids(EIF6224aa).Further LC-MS and western blot assays confirmed that EIF6-224aa was endogenous and universal existed in TNBC tissues and cells.Conclusions1.Circ-EIF6 has the basic circular properties of circRNA.2.Circ-EIF6 could be translated into a novel protein termed EIF6-224aa.3.EIF6-224aa is generally expressed in TNBC cells and tissues.Part Ⅲ Circ-EIF6 promotes the progression and metastasis of TNBC via encoding EIF6-224aaObjective1.Investigate the effects of circ-EIF6 knockdown on the proliferation,migration and invasion of TNBC cells.2.Determine whether circ-EIF6 could promote the proliferation,migration and invasion of TNBC cells by encoding EIF6-224aa.MethodsMTT,colony formation,EDU and flow cytometry were used to verify the effects of circEIF6 and its protein product EIF6-224aa on the proliferation of TNBC cells.Trasnwell and wound-healing assays were performed to validate the effects of circ-EIF6 and EIF6-224aa on TNBC cells migration and invasion.In vivo experiments were performed to validate the effects of circ-EIF6 on TNBC cell progression and metastasis.ResultsIn vitro assays found knockdown of circ-EIF6 could significantly inhibit the proliferation,migration and invasion abilities of TNBC cells.Overexpression assays further proved that circEIF6 could promote the proliferation,migration and invasion of TNBC cells by encoding EIF6224aa.In vivo animal model also showed that overexpression of circ-EIF6 could promote the progression and metastasis of TNBC cells.Conclusions1.Knockdown of circ-EIF6 could inhibit the proliferation,migration and invasion of TNBC cells.2.Circ-EIF6 could promote the proliferation,migration and invasion of TNBC cells via encoding EIF6-224aa.Part Ⅳ The molecular mechanisms of circ-EIF6 encoded EIF6-224aa in promoting the progression and metastasis of TNBCObjective1.Screen and verify the downstream targets of EIF6-224aa.2.Explore the effects of EIF6-224aa on MYH9 and its molecular mechanisms.3.Clarify the molecular mechanisms of EIF6-224aa in affecting MYH9 to promote the progression and metastasis of TNBC cellsMethodsThe downstream targets of EIF6-224aa were screened by Co-IP and LC-MS analysis,and the binding of EIF6-224aato MYH9 was verified by immunofluorescence,software prediction and Co-IP.CHX,MG 132 and Co-IP assays were performed to explore the effects of EIF6224aa on MYH9 protein stability and ubiquitination level.The MYH9 was further knockdown,then MTT and Transwell assay were used to detect the effects of MYH9 on EIF6-224aa functions.Finally,western blot and qRT-PCR assays were used to detect the changes of downstream Wnt/beta-catenin pathway.ResultsBy using Co-IP and LC-MS analysis,we found that MYH9 might bind to EIF6-224aa.Immunofluorescence proved that EIF6-224aa has the same celluar sublocation with MYH9 in the cytoplasm,and the prediction software also suggested EIF6-224aa could bind to MYH9 protein.Then Co-IP assay was performed and proved that EIF6-224aa could directly bind to MYH9.Furthermore,we found tha EIF6-224aa could stabilize the expression of MYH9 protein by decreasing the ubiquitination level of MYH9.Finally,we found knockdown of MYH9 could inhibit the biological functions of EIF6-224aa,and overexpression of EIF6-224aa could activate the downstream Wnt/beta-catenin pathway of MYH9.Conclusions1.MYH9 is the direct downstream target of EIF6-224aa.2.EIF6-224aa could reduce the ubiquitination level and stabilize the protein expression of MYH9.3.EIF6-224aa could activate the downstream Wnt/beta-catenin pathway of MYH9,thus promoting the progression and metastasis of TNBC cells.