The Functional Role And Regulatory Mechanism Of Long Non-coding Antisense RNA HYOU1-AS In Human Breast Cancer

Breast cancer is one of the most common malignancies affecting women worldwide,and has been the second leading cause of cancer-related death in women aged<45 years.Triple-negative breast cancer(TNBC),defined by lack of estrogen receptor(ER),progesterone receptor(PR)and human epidermal growth factor receptor 2(HER2)amplification,is the most aggressive subtype of breast cancer.Cytotoxic chemotherapy is currently the only treatment option for TNBC,but the patients frequently develop resistance.TNBC patients generally have poor prognosis due to high rates of early recurrence and distant metastases,especially to lung and brain.However,no proven approach is currently available for TNBC therapies.Therefore,there is an urgent need to identify efficient therapeutic targets of TNBC through delineating molecular mechanisms underlying TNBC development and progression.Long noncoding RNA(IncRNA)is a class of RNAs longer than 200 nucleotides with limited or no protein coding ability.In the past decade,lncRNAs have attracted increasing attentions,because they not only interact with nucleic acids through base-pairing,but also associate with proteins through secondary structural recognition.Due to these characteristics,emerging evidence demonstrates that lncRNAs are involved in a variety of epigenetic regulation mechanisms,and play a critical role in the regulation of various cellular processes.Compared to protein-coding genes,lncRNAs expression exhibited a higher tissue-and organ-specific,and close correlation with many human diseases.However,the regulatory mechanisms and functions of most lncRNAs remain largely unknown.It has been frequently demonstrated that lncRNAs show distinct expression profiles between TNBC and normal tissue samples.In this dissertation,we used microarray-based high-throughput analysis to investigate lncRNAs involves in breast development and progression.Among many differentially expressed lncRNAs,we identified an antisense IncRNA,HYOU1-AS,which has not been previously reported and is transcribed from the antisense strand of the hypoxia up-regulated 1(HYOU1)gene,Our discovery may provide clues to uncover novel biomarkers and potential therapeutic targets for TNBC,which may facilitate individualized treatment of breast cancer patients in the future.The main results obtained are as follows:1.In order to identify novel TNBC-relevant lncRNAs,we performed lncRNA microarray analysis in 5 TNBC tissue samples and their matched normal mammary tissues.With the criterion of fold-change(FC)>2.0,we discovered 891 differentially expressed lncRNAs in TNBC.Among them,HYOU1-AS was the most upregulated antisense lncRNA in TNBC,which was verified by strand-specific RT-PCR(ssRT-PCR).Cell fractionation analysis showed that HYOU1-AS was mainly localized in nucleus,suggesting that it was involved in nuclear processes of TNBC.2.To investigate the role of HYOU1-AS in breast cancer,shRNA was used to knockdown HYOU1-AS in TNBC cell line MDA-MB-231.As a result,HYOU1-AS depletion inhibited cell proliferation and migration in vivo,and also reduced xenograft tumor formation in nude mice.In addition,ectopically expressed HYOU1-AS could prevent MCF-10A cells from generating regular spheroids,and promote the formation of irregular cell aggregates.These data strongly suggested an oncogenic role of HYOU1-AS in mammary tumorigenesis.3.Based on a dataset obtained from The Cancer Genome Atlas(TCGA),increased HYOU1 levels correlated with poor clinical outcomes of breast cancer patients,while HYOU1-AS and HYOU1 mRNA expression showed a strongly positive correlation.It has been illustrated that HYOU1 could stimulate angiogenesis and inhibit apoptosis,suggesting that HYOU1-AS might be involved in the development of breast cancer through targeting HYOU1.4.To explore the mechanism underlying the positive correlation between HYO U1-AS and HYOU1,we designed luciferase reporter assay and observed elevated luciferase activity promoted by HYOU1-AS through acting on the HYOU1 3’UTR downstream of the luciferase gene.Furthermore,we identified heterogeneous nuclear ribonucleoprotein A1(hnRNPA1)as a HYOU1-AS binding protein through RNA pull-down and validated their interaction using RNA binding protein immunoprecipitation(RIP)assays.We demonstrated that hnRNPAl inhibited HYOU1 expression by post-transcriptional regulation.However,this kind of negative regulation could be antagonized by ectopically expressed HYOU1-AS,suggesting that HYOU1-AS acted as a decoy of hnRNPA1 protein to relieve its inhibition of HYOUl.Taken together,the above results revealed HYOU1-AS as an antisense lncRNA,which was enriched in the nucleus and highly expressed in TNBC.HYOU1-AS could promote the expression of HYOU1 through competitively binding to hnRNPA1,an RNA-binding protein,to relieve its post-transcriptional inhibition of the HYOU1 mRNA.In addition,high levels of HYOU1 were correlated with poor prognosis of breast cancer patients,indicating that lncRNA HYOU1-AS promoted TNBC progression through upregulating HYOU1.Overall,HYOU1-AS may serve as a promising therapeutic target and potential biomarker in the clinical treatment of TNBC patients.