| BackgroundThe expression of three important receptors in triple negative breast cancer(TNBC)is missing,namely estrogen,progesterone and human epidermal growth factor receptor-2.Compared with other subtypes of breast cancer,TNBC patients have shorter survival time and higher mortality within 5 years after diagnosis.TNBC is highly invasive and prone to distant metastasis,often involving brain and internal organs.TNBC is not sensitive because of the lack of some of its targeted receptors.At present,TNBC treatment can only rely on systemic treatment,but conventional postoperative adjuvant chemoradiotherapy shows poor curative effect.Residual metastatic lesions will eventually lead to tumor recurrence.Therefore,it is of great value to reveal the mechanism of TNBC so as to provide more effective intervention targets.The interaction of lncRNA,miRNA and mRNA in TNBC and the related molecular mechanisms remain unclear.Therefore,it is necessary to further explore the mechanisms and interactions in gene regulatory networks,including lncRNA,miRNA,gene mutation and TNBC progression.LINC00514 is a newly discovered lncRNA,abnormal high expression appears in some malignant tumors,it plays a promoting and regulating role in the occurrence and development of cancer.However,the biological function and regulatory mechanism of LINC00514 in TNBC have not been reported.and its biological function and mechanism are not clear.Therefore,this study aims to explore the biological function and molecular mechanism of LINC00514 in TNBC through a series of biochemical techniques.ObjectivesThe core purpose of this study is to elucidate the biological regulation of LINC00514 in triple negative breast cancer(TNBC)and its related signaling mechanism,which is mainly divided into three parts:(1)To reveal the expression significance and biological function of LINC00514 in TNBC.To explore the expression of LINC00514 in TNBC clinical tissue samples and cell lines,and to analyze the correlation between the expression of LINC00514 and the clinical indicators of patients;By changing the expression of LINC00514 in cell lines,the effects of LINC00514 on the biological characteristics of TNBC cell proliferation,invasion and apoptosis were analyzed;(2)To reveal that LINC00514 targets miR-6504-5p and miR-3139 to regulate biological functions in breast cancer.The downstream targets of LINC00514 in TNBC were predicted and verified to explore the effects of miR-6504-5p and miR-3139 downstream targets of LINC00514 on proliferation,invasion and apoptosis of breast cancer cells;(3)To reveal the mechanism of the regulation of TNBC by the signaling axis of LINC00514/miR6504-5p and miR-3139/CCDC71L.By predicting and verifying the downstream targets of miR-6504-5p and miR-3139,the effects of LINC00514/miR-6504-5p and miR-3139/CCDC71L signal axis on the biological function of TNBC were explored.LncRNA-miRNA-mRNA interaction network provides a new direction for further experimental study and biomarker mining of TNBC,it provides a more meaningful target for the improvement of clinical efficacy of breast cancer.MethodsIn this study,52 patients with TNBC were enrolled and a variety of TNBC cell lines were used.RT-qPCR was used to measure the relative expression of LINC00514,miR-6504-5p,miR-3139 and CCDC71L mRNA in TNBC tissues and cells;Western blot was used to measure the expression of apoptosis related proteins Bcl-2,Bax and CCDC71L in TNBC tissues and cells;TNBC cells were transfected with shRNA to interfere the expression of lncrna.TNBC cells were transfected with miRNA mimics or inhibitor to change the expression of miRNA.TNBC cells were transfected with pcDNA3.1 expression vector to up regulate the expression of CCDC71L.The proliferation of TNBC cells was measured by colony formation assay and Edu labeling assay.The apoptosis rate of TNBC cells was measured by flow cytometry.The migration ability of TNBC cells was measured by wound healing assay.The invasion ability of TNBC cells was measured by Transwell assay,the downstream miRNA target and downstream mRNA of LINC00514 were predicted by online database Starbase,and the interaction between LINC00514 and downstream miRNA,miRNA and target mRNA was measured by luciferase reporter gene assay.Results1.The expression of LINC00514 in TNBC tissue was significantly increased to 3.98±1.25,which was statistically different compared with adjacent tissues(n=52,P<0.01).The high expression of LINC00514 was significantly correlated with lymph node metastasis(P=0.039),Ki-67 positive rate(P=0.0043)and clinical stage(P=0.008).The expression of LINC00514 in patients with advanced TNBC(stage ?-?)was significantly higher than that in patients with early TNBC(stage ?-?)(n=52,P<0.01).2.The relative expression of LINC00514 in TNBC cell line was significantly up-regulated compared with that in MCF-10A cell line(P<0.001),and MDA-MB-468 cell line(6.54± 1.03)and HCC1937 cells(5.52±1.01),as a follow-up study model.3.Sh-LINC00514 was constructed and stably transfected into TNBC cells,resulting in significant down-regulation of LINC00514.On this basis,through a series of biochemical tests,the results showed that the transfection of sh-LINC00514 significantly inhibited the colony forming ability of MDA-MB-468 cells and HCC1937 cells,and the proportion of EDU positive cells decreased significantly.The apoptosis rate of MDA-MB-468 and HCC1937 cells increased,the expression of Bcl-2 decreased,and the expression of Bax increased.The migration and invasion ability of MDA-MB-468 cells and HCC1937 cells were significantly inhibited.There were significant differences in the above functional analysis results.4.Starbase predicted that miR-6504-5p and miR-3139 were downstream miRNA targets of LINC00514.Luciferase reporter gene experiment showed that overexpression of miR-6504-5p or miR-3139 could reduce the luciferase activity of LINC00514-wt reporter,but no significant change was found in LINC00514 mut reporter,indicating that both miR-6504-5p and miR-3139 have direct interaction with LINC00514.Mir-6504-5p and miR-3139 were significantly down regulated in TNBC clinical samples,which was significantly different from the adjacent tissues.Correlation analysis showed that the expression of LINC00514 was negatively correlated with the expression of miR-6504-5p or miR-3139.The correlation coefficient between LINC00514 and miR-6504-5p was 0.4577(P<0.001),and the correlation coefficient between LINC00514 and miR-3139 was 0.2637(P<0.001).It was preliminarily confirmed that LINC00514 targeted the inhibition of miR-6504-5p and miR-3139 in TNBC.5.Overexpression of miR-6504-5p or miR-3139 was induced by transfection of TNBC cells with miRNA mimics.On this basis,colony formation assay showed that miR-6504-5p or miR-3139 mimics significantly inhibited the colony formation ability of TNBC cells.EdU assay showed that the proportion of EdU positive cells decreased significantly after up-regulation of miR-6504-5p or miR-3139 mimics.The results of flow cytometry and Western blot confirmed that he increased expression of miR-6504-5p or miR-3139 mimics led to an increase in the proportion of TNBC cell apoptosis,the expression of apoptosis marker protein Bcl-2 was inhibited,and the expression of Bax was enhanced.Wound healing assay and Transwell test confirmed that the up regulation of miR-6504-5p or miR-3139 mimics significantly inhibited the migration and invasion of TNBC cells.There were significant differences in the above functional analysis results.6.Starbase predicted that CCDC71L was the common target of miR-6504-5p and miR-3139.The luciferase reporter gene experiment confirmed that the luciferase activity of CCDC71L-wt reporter was significantly decreased after transfection of TNBC cells with miR-6504-5p or miR-3139 mimics,while the luciferase activity of CCDC71L mut reporter remained unchanged.Meanwhile,overexpression of LINC00514 reversed the decrease of CCDC71L-wt luciferase activity induced by miR-6504-5p mimics or miR-3139 mimics,while CCDC71L mut luciferase activity was not affected.RT-qPCR and western blot results confirmed that the silencing of LINC00514 could significantly reverse the up regulation of CCDC71L expression induced by down-regulation of miR-6504-5p and miR-3139,which further confirmed the existence of LINC00514/miR-6504-5p and miR-3139/CCDC71L regulatory signal axis in TNBC.7.CCDC71L overexpression vector was constructed and transfected into TNBC cells to induce CCDC71L overexpression.On this basis,colony formation and edu experiments showed that overexpression of CCDC71L or inhibition of miR-6504-5p and miR-3139 could significantly reverse the proliferation of TNBC cells inhibited by LINC00514 silencing.The results of flow cytometry showed that the increase of TNBC apoptosis induced by LINC00514 silencing was reversed by CCDC71L overexpression or inhibition of miR-6504-5p and miR-3139.Accordingly,Western blot results also confirmed that the changes of Bax and Bcl-2 expression induced by LINC00514 silencing were reversed by CCDC71L overexpression or inhibition of miR-6504-5p and miR-3139.At the same time,wound healing assay and trasnwell test confirmed that the inhibition of TNBC cell migration and invasion mediated by LINC00514 silencing was reversed by up regulating CCDC71L or silencing miR-6504-5p and miR-3139.Conclusions1.LINC00514 was highly expressed in TNBC,which promoted the proliferation,migration and invasion of TNBC cells,inhibited the apoptosis of TNBC cells,and functioned as an oncogene;2.MiR-6504-5p and miR-3139 were downstream regulatory targets of LINC00514,and their expressions were inhibited by LINC00514.MiR-6504-5p and miR-3139 can inhibit the progression of TNBC;3.The expression of oncogene CCDC71L is inhibited by miR-6504-5p and miR-3139.LINC00514 can enhance the expression of CCDC71L by inhibiting the expression of miR-6504-5p and miR-3139,which can promote the proliferation,migration and invasion of breast cancer,and inhibit apoptosis.LINC00514/miR-6504-5p and miR-3139/CCDC71L axis,is an important mechanism to promote the progress of TNBC and is expected to become a potential target for the diagnosis and treatment of TNBC. |
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