The Mechanisms Of MALAT1 Regulating Biological Functions Of Breast Cancer Through EEF1A1

Research background:Breast cancer(BC)is the most common cancer and the leading cause of cancer death among women throughout the world.It is estimated that more than 2 million female breast cancer cases is newly diagnosed in 2018,accounting for approximately 1 in 4 cancer cases among women.There are several different BC treatment options,ranging from local,such as surgery and radiotherapy,to systemic,such as chemotherapy,endocrine treatment and targeted therapy.In spite of advances in different treatment options,a substantial number of patients exhibit resistance,recurrence and metastasis.Thus,more studies are needed to investigate the underlying molecular mechanisms involved in mammary tumor progression and metastasis.These studies may help develop new biomarkers or targets for disease prognosis and treatment.Transcriptome analyses reveal that up to 80% of the human genome is transcribed into RNAs,whereas only <2% of the human genome DNA is composed of protein-coding genes.Thus,the majority of the human genome is transcribed into non-coding RNAs.LncRNAs are defined as transcripts longer than 200 nucleotides without protein-coding capacity.LncRNAs are actively involved in various biological processes and function as epigenetic regulators of gene expression at the transcriptional,post-transcriptional and protein levels.Consistent with their role in these regulatory processes,it is not surprising that lncRNAs are also involved in development of human disease.The nuclear-retained Metastasis-Associated Lung Adenocarcinoma Transcript 1(MALAT1),also referred as Nuclear-Enriched Abundant Transcript 2(NEAT2),is a highly conserved lncRNA among mammals.The gene encodes a long non-coding RNA with a length of ?8000 nt.MALAT1 was initially identified as a prognostic marker for metastasis and survival in early-stage non-small cell lung carcinoma.In the last two decades,a number of studies have shown that it is also upregulated in multiple malignancies,including liver,colon,stomach and bladder.Its upregulation is associated with tumorigenesis or disease progression.For instance,a recent study found that MALAT1 acts as a proto-oncogene in hepatocellular carcinoma by upregulating the oncogenic splicing factor SRSF1,resulting in the activation of the Wnt and mTOR pathways.However,contradictory effects of MALAT1 have been reported on tumorigenesis and development in breast cancer.It has been observed that high expression of MALAT1 is associated with poor relapse-free survival in breast cancer.MALAT1 promotes proliferation and invasion in breast cancer cells in vitro.Moreover,genetic loss or systemic delivery of antisense oligonucleotides targeting MALAT1 in mice with established mammary tumors resulted in slower tumor growth,significant differentiation into cystic tumors and decreased metastasis.In contrast,studies by Eastlack showed that MALAT1 abundance correlates with inhibition of oncogenic cell function in breast cancer.Similarly,a recent work also reported a tumor suppressive role of MALAT1 in breast cancer metastasis.Furthermore,few studies have been reported about molecular mechanisms of MALAT1 in breast cancer.Therefore,more studies are needed to identify the biological functions of MALAT1 and investigate the underlying molecular mechanisms in breast cancer.Research objectives:1.To evaluate the expression of MALAT1 in breast cancer tissues and cells2.To evaluate the biological functions of MALAT1 in breast cancer3.To investigate the possible mechanisms of MALAT1 involved in development of breast cancerResearch methods:Screen the expression level of MALAT1 in breast cancer cell lines and breast cancer tissues using Real-time Quantitative PCR(RT-PCR)and TCGA database analysis.Construct MDA-MB-231 and SKBR3 cell model with MALAT1 knockdown by short hairpin RNAs(shRNAs).Assess whether down-regulation of MALAT1 could affect cell proliferation,cell cycle and invasion by CCK-8 assay,flow cytometry and transwell assay.Reverse transcription associated trap(RAT)assay were used to identify the lncRNA–DNA interaction network associated with MALAT1.Luciferase reporter assay,ChIP and RT-PCR were performed to explore the possible mechanism of MALAT1 in regulating target gene expression.Rescue experiments were performed to confirm the impact on breast cancer by MALAT1-target gene pathway.Research results:1.Expression of MALAT1 was significantly higher in the breast cancer tissues than in paracancer tissues.MALAT1 was overexpressed in breast cancer cell lines as compared to normal mammary cell lines.2.MALAT1 knockdown vectors were constructed succsessfully.MALAT1 expression was downregulated in shMALAT1-transfected cells compared with control.Knockdown of MALAT1 leads to decreased cell proliferation,cell invasion,and cell cycle arrest.3.Using RAT-Seq,we profiled the lncRNA–DNA interaction network associated with MALAT1 and found that MALAT1 lncRNA bound to many pathway gene targets that are closely related to tumor progression.4.MALAT1 interacts with the promoter of EEF1A1 and affects promoter activity.Knockdown of MALAT1 alters the status of H3K4 methylation in the EEF1A1 promoter and inhibits the expression of EEF1A1.Moreover,ectopic over-expression of EEF1A1 reversed the inhibitory effect of MALAT1 knockdown in tumor phenotypes.Research conclusion:In this study,we have demonstrated that MALAT1,a highly conserved lncRNA,enhances proliferation and invasion by trans-targeting EEF1A1 in breast cancer cells.Using RNA reverse transcription-associated trap sequencing,we characterized the genome-wide interaction network targets for MALAT1 lncRNA,identifying the translation elongation factor 1-alpha 1 gene EEF1A1 as a critical target of MALAT1.These data expand our current knowledge of the MALAT1 interacting network and related epigenetic mechanisms,laying the foundation for developing new therapeutic strategies to treat breast cancer.