The Effect And Mechanism Of LINC00665 On The Proliferation And Invasion Of Breast Cancer Cells

Background and objectiveBreast cancer(BC)is the most common malignant tumor among women in the worldwide,which seriously endangers women's life and health.The etiology and pathogenesis of breast cancer are very complicated,which are caused by genetic factors,lifestyle and environmental exposure.Long non-coding RNAs(LncRNA)are a novel type of non-coding RNAs with more than 200 nucleotides,which are crucial regulating factors in the development of various tumours.Located on human chromosome 19,LINC00665 plays a key regulatory role in the proliferation and metastasis of various malignancies such as lung,liver and gastric cancers.However,the role of LINC00665 in breast cancer and its mechanism are still unknown.This study is the first to comprehensively analyze the expression and biological role of LINC00665 in breast cancer and elucidate the molecular mechanism of its potential regulation.This study includes three parts:In the first part,the role of LINC00665 in the regulatory mechanism of breast cancer via ceRNA network and its clinical significance.In the second part,silencing LINC00665 has an effect on proliferation,apoptosis,migration and invasion of breast cancer cells.In the third part,silencing LINC00665 inhibits the proliferation and invasion of breast cancer cells via sponging miR-3619-5p.Part one:The role of LINC00665 in breast cancer regulatory mechanism by ceRNA network and its clinical significanceObjectivesTo study the expression and clinical significance of LINC00665 in breast cancer tissues,and to explore the molecular mechanism of it's regulating the progression of breast cancer.Methods1.We analyzed the expression level of the LINC00665 using the Gene Expression Profiling Interactive Analysis(GEPIA)method in BC and normal tissues.2.The expression level of LINC00665 in 106 breast cancer tissues and adjacent normal breast tissues was detected by RT-PCR,and the correlation between it's expression level and age,tumor size and TNM stage of breast cancer patients was analyzed.3.The comprehensive strategies of RNA-seq data mining,bioinformatics and experimental verification for breast cancer in TCGA database were adopted to evaluate LINC00665 related pathways and constructed ceRNA network of LINC00665 in breast cancer.Results1.The results of GEPIA analysis showed that the expression level of LINC00665 in breast cancer tissues was significantly higher than that in normal breast tissues(P<0.05).2.RT-PCR results of 106 breast cancer tissues and paired para-cancer breast tissues showed that the expression level of LINC00665 in breast cancer tissues was significantly higher than that in para-cancer breast tissues(P<0.001),which was consistent with the results of GEPIA analysis.3.The expression level of LINC00665 in breast cancer tissues was correlated with tumor size and TNM stage of breast cancer patients(P<0.001),but not with age(P>0.05).4.LINC00665 regulates breast cancer progression by targeting PRLR,QPRT and LRTM2 with miR-3619-5p.ConclusionLINC00665 is highly expressed in breast cancer tissues and is associated with tumor size and TNM stage in breast cancer patients,but not with age.Bioinformatics analysis suggested that LINC00665 might regulate breast cancer progression by targeting PRLR,QPRT or LRTM2 with miR-3619-5p.Part two:Silencing LINC00665 effects on proliferation,apoptosis,migration and invasion of breast cancer cellsObjectivesThe effects of LINC00665 on the proliferation,apoptosis,migration and invasion of breast cancer cells were investigated.Methods1.The expression levels of LINC00665 in breast cancer MCF-7 and MDA-MB-231 cells and normal breast MCF-10A cells were detected by RT-PCR.2.Lipofectamine 2000TM transfection reagent was used to transfect LINC00665 siRNA and NC siRNA into logarithmic breast cancer cells,and the expression of LINC00665 in breast cancer cells was detected by RT-PCR.3.MTT cell proliferation assay was used to detect the effect of silencing LINC00665 expression on the proliferation of breast cancer cells.4.The flow cytometry was used to detect the effect of silencing LINC00665 expression on apoptosis of breast cancer cells.5.Transwell assay was used to detect the effect silencing LINC00665 expression on the migration and invasion ability of breast cancer cells.Results1.RT-PCR results showed that the expression level of LINC00665 in breast cancer MCF-7 and MDA-MB-231 cells was significantly higher than that in normal breast MCF-10A cells(P<0.001).2.RT-PCR results showed that LINC00665 expression level for breast cancer MCF-7 and MDA-MB-231 cells transfected with LINC00665 siRNA was significant descreased compared with the NC-si control group(P<0.001).3.The results of MTT showed that the growth of MCF-7 and MDA-MB-231 cells in the LINC00665 siRNA group was significantly inhibited at 48 h and 72 h after transfection compared with the NC-si control group(P<0.001).4.The results of flow cytometry showed that the apoptosis rates of MCF-7 and MDA-MB-231 cells in LINC00665 siRNA transfected group were significantly increased compared with NC-si control group(P<0.001).5.The results of the migration experiment showed that the migration abitliy of MCF-7 and MDA-MB-231 cells in LINC00665 siRNA transfected group was significant inhibited compared with NC-si control group(P<0.001).6.The results of the invasion experiment showed that the invasion ability of MCF-7 and MDA-MB-231 cells in LINC00665 siRNA transfected group was significant inhibited compared with NC-si control group(P<0.001).ConclusionLINC00665 is highly expressed in breast cancer cells.The expression of LINC00665 in breast cancer cells was successfully inhibited by transfection of LINC00665 siRNA.Silencing LINC00665 can inhibit the proliferation,migration and invasion of breast cancer cells and promote the apoptosis of breast cancer cells.Part three:Silencing LINC00665 inhibits the proliferation and invasion of breast cancer cells by regulating miR-3169-5pObjectivesTo investigate the molecular mechanism of LINC00665 regulating the biological function of breast cancer cells.Methods1.The expression levels of miR-3619-5p in breast cancer MCF-7 and MDA-MB-231 cells and normal breast MCF-10A cells were detected by RT-PCR.2.RT-PCR was used to detect the effect of silencing LINC00665 on the expression levels of miR-3619-5p in MCF-7 and MDA-MB-231 breast cancer cells.3.The direct binding targe between miR-3619-5p and LINC00665 was determined by the dual luciferase activity assay.4.Lipofectamine 2000TM transfection reagent was used to co-transfected LINC00665 siRNA or miR-3619-5p/NC inhibitor into breast cancer MCF-7 and MDA-MB-231 cells at logarithmic growth stage,and then the proliferation,migration,invasion,apoptosis and protein expression of breast cancer cells were detected by MMT assay,transwell migration and invasion assay,flow cytometry and Western Blot,respectively.5.Lipofectamine 2000TM transfection reagent was used to transfected LINC00665 siRNA or NC siRNA into breast cancer MCF-7 and MDA-MB-231 cells at logarithmic growth stage,and then the protein expression of breast cancer cells were detected by Western Blot.Results1.RT-PCR results showed that the expression level of miR-3619-5p in breast cancer MCF-7 and MDA-MB-231 cells was significantly lower compared with that in normal breast MCF-10A cells(P<0.001).2.RT-PCR results showed that miR-3619-5p expression level for breast cancer MCF-7 and MDA-MB-231 cells transfected with LINC00665 siRNA was significant up-regulated compared with the NC-si control group(P<0.001).3.The dual luciferase activity assay detected the binding target of LINC00665 and miR-3619-5p.4.The double silencing of LINC00665 and miR-3619-5p led to increased cell proliferation,migration,and invasion but inhibited apoptosis,thereby upregulating the CDK2/?-catenin protein expression but inhibiting p21 level.5.The Silencing LINC00665 reduced the expression of CDK2/?-catenin and increased the expression of p21 in breast cancer cells.ConclusionsLINC00665 negatively regulates the expression of miR-3619-5p in breast cancer cells,and LINC00665 directly interacts with miR-3619-5p in breast cancer cells.LINC00665 regulates the expression of p21,CDK2 and ?-catenin in breast cancer cells by miR-3619-5p,thereby promoting the proliferation,migration and invasion of breast cancer cells,and inhibiting cell apoptosis.