| Triple-negative breast cancer(TNBC)is characterized by the absence of estrogen receptor(ER),progesterone receptor(PR)and human epidermal growth factor receptor 2(HER2)expressions.TNBC accounts for 15%-20%of all breast cancers.It is prone to early recurrence,distant metastas,and has poor prognosis and no targeted standard treatment options.TNBC has the characteristics of early onset age,high degree of malignity and lacking the effective treatment medicine.Because it is not sensitive to endocrine therapy and molecular targeted therapy,chemotherapy is the main systemic therapy for TNBC.However,TNBC is prone to chemotherapy resistance,leading to chemotherapy failure,tumor recurrence,and higher risk of metastasis and death than other subtypes of breast cancer.The development and drug resistance mechanism of TNBC are not clear,which greatly hampers the research process of molecular targeted therapy and chemotherapy drugs for TNBC,and effective treatment strategies for TNBC are desperately needed.Non-coding RNA(ncRNA)refers to RNA that does not encode protein.In the past,ncRNA has been regarded as "junk RNA" in genome.In recent years,with the development of genomics and bioinformatics,especially the rise of high-throughput sequencing technology,ncRNA has been found to play an important role in many life activities and diseases.ncRNA is involved in all stages of tumor development.Studying the molecular mechanism of ncRNA in tumor can not only understand the pathogenesis of tumor from a new perspective,but also expand the new direction of tumor diagnosis and treatment.In this thesis,we take miR-4306 and lncRNA-712 as research objects to analyze the molecular mechanism of their role in TNBC,providing a new idea for the diagnosis and treatment of TNBC.In the first part of this thesis,we found that transcriptional downregulation of miR-4306 serves as a new therapeutic target for TNBC.MicroRNAs are widely distributed in eukaryotes and participate in various physiological and pathological processes.It is a kind of highly conserved,tissue-specific expressed,stable in tissue and blood,and low molecular weight non-coding RNAs.The detection means of microRNAs are flexible and convenient,and it is easy to make nucleic acid drugs targeted for microRNAs.MicroRNAs are potentical diagnostic biomarkers and therapeutic targets for tumor.Both estrogen receptor and progesterone receptor are steroid receptors,which act as transcription factors to induce specific gene expression and affect the life activities of target cells.The most well-known function of human epidermal growth factor receptor 2 is a transmembrane receptor with tyrosine kinase activity.However,studies have also shown that it can enter the nucleus to play the role of transcription factors.At present,there are many researches about microRNA involved in the development of TNBC,but it is not clear whether there is a certain microRNA that can be transcriptionally regulated by the above three factors together.In this study,we first used microRNA array combined with bioinformatics analysis to screen microRNAs that may be regulated by ER,PR and HER2 together.We also used luciferase reporter assay and ChIP experiment to verify that miR-4306 can be transcriptionally regulated by ER,PR and HER2.Therefore,the lack of ER,PR,and HER2 in TNBC is the main reason for the low expression of miR-4306.Next,we detected the expression of miR-4306 in 325 pairs of breast cancer tissues by qRT-PCR.The results show that miR-4306 is downregulated in TNBC compared with other subtypes of breast cancer.Clinically,low expression levels of miR-4306 are strongly associated with lymph node metastasis and a poor prognosis for TNBC.Cell experiment showed that miR-4306 could inhibit the formation of malignant phenotype of TNBC(especially lymphangiogenesis and angiogenesis,which is closely related to metastasis),and meanwhile,miR-4306 could promote the apoptosis of TNBC cells induced by cisplatin.In animal experiments,miR-4306 inhibited the growth,lymph node metastasis and lung metastasis of TNBC.The Chick embryo chorioallantoic membrane(CAM)assay showed that miR-4306 could inhibit the formation of microvasculature.Mechanistic analyses indicate that miR-4306 directly targets SIX1,Cdc42 and VEGFA to inactivate the angiogenesis pathway,EGF receptor signaling pathway,TGF-βpathway and Ras pathway.miR-4306 decreases the expression of VEGFC by targeting SIX1,thus inhibiting the lymphangiogenesis and the migration of lymphatics endothelial cells,and ultimately leading to the lymph node metastasis of tumors.miR-4306 negatively regulates the expression of cyclinDl and E-cadherin,which are downstream genes of SIX1 and Cdc42,so as to inhibit the proliferation,invasion and migration of TNBC.miR-4306 also can inhibit angiogenesis by targeting VEGFA.In conclusion,miR-4306 is expected to be the therapeutic target for TNBC.TNBC patients are more sensitive to platinum drugs,so cisplatin and carboplatin are commonly used in clinical treatment for TNBC.The orthotopic mouse model of TNBC reveals that miR-4306 mimic can be used for TNBC treatment in combination with cisplatin.Our findings suggest that miR-4306 is a key effector of TNBC metastasis pathways and a potential therapeutic target for TNBC treatment.In the second part of this thesis,we found that gene amplification of lncRNA-712 induces autophagy and metastasis in TNBC.Long noncoding RNA(lncRNA)refers to RNA with a length of more than 200nt and no protein coding ability.It can participate in gene regulation and modification in the levels of epigenetics,transcription and post transcription.LncRNA is widely involved in a variety of physiological and pathological processes.In recent years,studies have shown that lncRNA is closely associated with the occurrence,development and chemosensitivity of tumors,and can be used as a diagnostic marker and therapeutic target for tumors.Identification of key lncRNA and elucidating the mechanism in metastasis would be of great value for TNBC diagnosis and therapy.In this study,using TCGA copy number amplification data and lncRNA array data,we found that the genes of lncRNA-712,lncRNA-836 and lncRNA-246 were significantly amplified in TNBC genome.We further selected lncRNA-712,which was the most obvious amplification in TNBC genome for the further study.Next,we detected the expression of lncRNA-712 in 125 pairs of breast cancer tissues using qRT-PCR.The results showed that the expression of lncRNA-712 in TNBC tissues was significantly higher than that in adjacent breast cancer tissues,and its expression level was positively correlated with lymph node metastasis and poor prognosis.The expression of lncRNA-712 in luminal subtype and HER2 subtype breast cancer tissues was no significant difference with that in matched adjacent breast cancer tissues.We identified that the full length of lncRNA-712 was 1108nt in breast cancer by RACE.Nuclear plasma fractionation showed that lncRNA-712 was distributed in both nucleus and cytoplasm.Cell experiments revealed that lncRNA-712 promoted the proliferation,invasion,migration and autophagy of TNBC cells.Then,protein chips,RNA immunoprecipitation assay and RNA pull down assay revealed that lncRNA-712 interacted with autophagy associated protein USP14 and VPS34.LncRNA-712 lncreases USP14 protein stability,which leads to the upregulation of Beclinl expression.LncRNA-712 enhances the lipase activity of VPS34 by inhibiting the acetylation of VPS34.Moreover,we observed that lncRNA-712 can be secreted out of tumor cells by exosome,and promoted normal mammary epithelial cells autophagy.The above studies showed that lncRNA-712 was abnormally upregulated in TNBC,and lncRNA-712 induced autophagy by binding to the autophagy related proteins USP14 and VPS34.In the following study,we will further deeply explore the biological roles and underlying mechanisms in which lncRNA-712 promotes TNBC metastasis via exosomes and autophagy,which will provide new biomarkers and targets for the diagnosis and treatment for TNBC. |
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