Regulatory Mechanism And Function Of CircEIF3H In Triple Negative Breast Cancer

BackgroundBreast cancer is the most common female malignancy worldwide and it can be divided into different subtypes according to the histopathologic diagnosis.Triple-negative breast cancer(TNBC),which is defined as a type of breast cancer with negative expression of estrogen receptor,progesterone receptor and human epidermal growth factor receptor-2,is regarded as an aggressive subtype clinically with high recurrence,metastasis,and mortality rate.TNBC is not sensitive to endocrinotherapy or target therapy,therefore,it is critical to illustrate the molecular mechanisms of TNBC carcinogenesis and progression and develop novel therapeutic targets for better treatment.Circular RNA(circRNA)is a class of RNA formed by covalent linkage of the 3’ and 5’ends of a single-strand RNA.They are characterized by high abundance,high stability and high conservation and play essential roles in broad physiological and pathological processes.The well-established molecular mechanisms of circRNAs are listed as below:(1)Regulate the expression of miRNA-target mRNAs through sequestering of miRNAs.(2)Interact with proteins and regulate their activities or functions.(3)Serve as templates of translation and produce peptides.In recent years,the important biological functions of circRNAs in malignant tumors,including cell proliferation,invasion and metastasis,angiogenesis,cellular energy,tumor immunosurveillance and chemotherapy resistance have been widely concerned by scientists.Therefore,exploring the roles and molecular mechanisms of circRNAs in TNBC would provide a new theoretical basis for the individualized target therapy of TNBC.High-throughput RNA-seq in paired breast cancer tissues and adjacent nontumor tissues was performed to investigate the circRNAs associated with breast cancer.Among them,circEIF3H,which is one of the prominently upregulated circRNAs in breast cancer tissues,was chosen for further evaluation.Since the expression of circEIF3H in TNBC was higher than that of other breast cancer subtypes,we mainly focused on the mechanism and function of circEIF3H in TNBC.Firstly we identified the circular characteristics and potential prognostic value of circEIF3H.Then we illustrated the regulatory mechanism of circEIF3H.Finally,we determined the biological function of circEIF3H-IGF2BP2/HuR-HSPD1/RBM8A/G3BP1 axis in TNBC progression and metastasis.Our study provided a theoretical basis for circEIF3H serving as a novel prognostic biomarker and an exploitable target for individualized therapy of TNBC in the approaching future.Part I Screening and identification of breast cancer-related circRNAsObjective1.Analyse the expression profiling of circRNAs in breast cancer tissues and adjacent nontumor tissues.2.Determine the expression level of circEIF3H in breast cancer tissues.3.Explore the association between circEIF3H expression and prognosis of breast cancer.4.Identify the existence and characteristics of circEIF3H.Methods1.RNA-seq in breast cancer tissues and adjacent nontumor tissues was performed to find out the breast cancer related circRNAs.2.We collected 196 cases of breast cancer tissues with follow-up information and qRT-PCR was conducted to detect the expression level of circEIF3H in these tissues.3.Kaplan-Meier analysis was used to explore the association between circEIF3H expression and prognosis of breast cancer.4.We designed convergent primers for PCR to amplify EIF3H mRNA and divergent primers to amplify circEIF3H.Sanger sequencing of PCR product was used to confirm the back-splice site of circEIF3H.Act D treatment and RNase R digestion were used to detect the half-life and RNase resistance of circEIF3H.UCSC genome browser was utilized to analyse the conservation and possible circulization mechanism.Results1.According to the RNA-seq in paired breast cancer tissues and adjacent nontumor tissues,81 circRNAs were differentially expressed.Among them,circEIF3H was one of the prominently upregulated circRNAs in breast cancer tissues.2.Among 70 pairs of breast cancer tissues and adjacent normal breast tissues,circEIF3H was upregulated in most breast cancer tissues.We also found the highest circEIF3H expression in TNBC tissues among four subtypes.3.High expression of circEIF3H contributed to a poor overall survival in all types of breast cancers and TNBC subtype.4.The result of PCR showed that circEIF3H was only amplified by divergent primers in cDNA and the putative junction was validated by Sanger sequencing.CircEIF3H was highly conservative and more resistant to RNase R than linear EIF3H with longer half-life.The long flanking introns with complementary ALU sequence might facilitate the formation of circEIF3H.ConclusionsWe discovered a novel circRNA,circEIF3H,which was upregulated in breast cancer tissues,especially in TNBC tissues.Large cohort survival analysis confirmed the association between high circEIF3H expression and poor prognosis of TNBC,indicating that circEIF3H may serve as a biomarker for diagnosis and prognosis evaluation.Besides,circEIF3H was an stable and highly conservative circRNA and the typical long flanking introns with complementary ALU sequence in EIF3H gene might facilitate the formation of circEIF3H.Part II The molecular signal pathway of circEIF3HObjective1.Detect the subcellular localization of circEIF3H.2.Determine whether circEIF3H could act as a microRNA sponge.3.Predict and subsequently confirm the possible proteins interacting with circEIF3H.4.Illustrate the molecular mechanism of circEIF3H-IGF2BP2/HuR complex.Methods1.Subcellular localization of circEIF3H was detected through qRT-PCR after nuclear and cytoplasmic RNA separation and RNA-FISH with 3’-Cy3-modified probes.2.RIP assay with AGO2 antibody was conducted to determine whether circEIF3H could act as a microRNA sponge.3.In order to find the possible proteins interacting with circEIF3H,we performed RNA pulldown assay using biotin-labelled circEIF3H sequence and the precipitates were subjected to mass spectrometry analysis.RIP assays were conducted to further confirm these RNA-protein interactions.A series of circEIF3H truncations were used in RNA pulldown assay to map the specific regions interacting with IGF2BP2 and HuR.4.IGF2BP2 enhanced-CLIP seq data(GSE90639)and HuR enhanced-PAR-CLIP seq data(GSE29780)were analyzed to find the potential targets of IGF2BP2 and HuR,and Western blot was used for further validation.Act D treatment was performed to detect the half-life of these target mRNAs to explore the regulatory effect of circEIF3H-IGF2BP2/HuR.Results1.Both nuclear/cytosol fractionation assay and RNA-FISH assay showed that circEIF3H was predominantly localized in cytoplasm.2.RIP assay showed no significant enrichment of circEIF3H in anti-AG02 group,indicating that circEIF3H may not serve as a miRNA sponge.3.The specific peptide of IGF2BP2 and HuR were identified by mass spectrometry after RNA pulldown.Co-IP confirmed the interaction between IGF2BP2 and HuR,and RIP assay confirmed the direct binding of circEIF3H with IGF2BP2/HuR.CircEIF3H truncations were conducted to map the binding fragment of circEIF3H with IGF2BP2 and HuR and the result showed that the sequence near the back-splice site was required for these interactions.4.Six genes were selected from the intersection between IGF2BP2 and HuR CLIP seq data.The expression levels of three genes(HSPD1,RBM8A and G3BP1)were positively correlated with circEIF3H.RIP assay validated the direct interaction of IGF2BP2/HuR with them.The half-lives of HSPD1/RBM8A/G3BP1 were reduced after circEIF3H knockdown,indicating the regulatory effect of circEIF3H-IGF2BP2/HuR complex on mRNA stability.ConclusionsCircEIF3H,a circRNA predominantly localized in cytoplasm,could directly bind to IGF2BP2/HuR via its back-splicing site and serve as a scaffold to induce the formation of circEIF3H-IGF2BP2/HuR complex.This complex helped to stabilize the mRNA of HSPD1,RBM8A and G3BP1,therefore unregulated their expression levels.Part III CircEIF3H promotes TNBC progression and metastasisObjective1.Explore the biological function of circEIF3H in TNBC.2.Identify the effects of IGF2BP2 and HuR in TNBC progression and metastasis.3.Illustrate the roles of HSPD1/RBM8A/G3BP1 in TNBC progression and metastasis.4.Verify the regulatory functions of circEIF3H-IGF2BP2/HuR-HSPD1/RBM8A/G3BP1 axis in TNBC progression and metastasis.Methods1.The biological function of circEIF3H in TNBC.(1)In vitro studies:MTT assay,clone formation,cell cycle assay,transwell migration and invasion assays were performed to detect the proliferative and metastatic abilities of TNBC cells after circEIF3H overexpression or knockdown.(2)In vivo studies:In order to observe the effects of circEIF3H in TNBC proliferation and metastasis,MDA-MB-231 cells with stable circEIF3H overexpression or knockdown were injected subcutaneously or though lateral tail veins to establish nude mice xenograft tumor model and lung metastasis model.2.The regulatory functions of circEIF3H-IGF2BP2/HuR-HSPD1/RBM8A/G3BP1 axis.(1)The functions of IGF2BP2 and HuR in TNBC:After the overexpression or knockdown of IGF2BP2/HuR,qRT-PCR and immunofluorescence were used to confirm the transfection efficiency.Then MTT assay and Transwell system were utilized to detect the changes in TNBC cell proliferation and metastasis.(2)The roles of HSPD1/RBM8A/G3BP1 in TNBC:After the transfection of HSPD1/RBM8A/G3BP1 siRNAs,qRT-PCR was used to confirm the transfection efficiency.Subsequently,MTT assay,EdU and clone formation were performed to monitor cell proliferation,and Transwell system were used to analyse metastatic ability of TNBC cells.(3)Verify the functions of circEIF3H-IGF2BP2/HuR-HSPD1/RBM8A/G3BP1 axis.In vitro studies:TNBC cells were cotransfected with circEIF3H overexpression plasmid and HSPD1/RBM8A/G3BP1 siRNAs respectively or simultaneously.Afterthat,MTT assay,EdU assay,clone formation,Transwell migration and invasion assays were conducted.In vivo studies:Immunohistochemistry(IHC)was performed to detect the expression levels of HSPD1,RBM8A and G3BP1 in nude mice xenograft tumors.Tissue samples:The correlation between circEIF3H expression and HSPD1/RBM8A/G3BP1 were analysed through qRT-PCR in 49 breast cancer tissues.Results1.CircEIF3H knockdown in MDA-MB-231 and MDA-MB-468 cells induced striking inhibition of cell proliferation,migrations and clone formation abilities.Flow cytometry showed that circEIF3H knockdown led to conspicuous G0/G1 phase arrest and S phase reduction.Ectopic expression of circEIF3H played the opposite roles.2.Both si-IGF2BP2 and si-HuR could inhibit TNBC cell proliferation and metastasis.3.Significantly decrease in cell proliferation and migration were observed in HSPD1,RBM8A and G3BP1 silenced TNBC cells compared with the control groups.4.Rescue experiments showed that HSPD1/RBM8A/G3BP1 silence reversed the cancer promoting effect of circEIF3H.In vivo experiments showed higher expression levels of HSPD1/RBM8A/G3BP1 in circEIF3H overexpressing group.Positive correlations between the expression of circEIF3H and HSPD1/RBM8A/G3BP1 respectively in 49 human breast cancer tissues were also confirmed.ConclusionsBoth in vitro and in vivo studies illustrated that circEIF3H was essential for TNBC proliferation and metastasis.Furthermore,circEIF3H promoted TNBC progression via enhancing the mRNA stabilities of its downstream target genes(HSPD1,RBM8A and G3BP1)and subsequently up-regulating their expression levels.