The Study Of LncRNA-BC069792 Expression And Its Mechanism In Breast Cancer

Research purposes:Breast cancer is the most common malignant tumor that threatens women’s health.The statistics in recent years show that the incidence and mortality of breast cancer remains high globally.In 2020,the incidence rate of breast cancer ranked the first in women worldwide,accounting for 30%of all malignant tumors.Breast cancer is a heterogeneous disease in morphology and genetics.With the development of early diagnosis and therapy methods,the treatment effect of early breast cancer has made great progress.Nevertheless,recurrence and metastasis may also occur in some breast cancer patients diagnosed at early stage.It is difficult to cure advanced breast cancer.How to improve the life quality and survival rate of advanced breast cancer is an important issue at present.Therefore,sights into novel specific molecular markers and therapeutic targets are the key to prolong the survival time and improve the life quality of breast cancer patientsLong noncoding RNAs(lncRNAs)are noncoding transcripts that lack protein-coding potential and are larger than 200 nucleotides.lncRNAs include five types:antisense lncRNA,intergenc lncRNA,promoter-associated lncRNA,intronic transcript lncRNA,UTR associated lncRNA.Lots of evidence has implicated that lncRNAs regulate gene expression at the transcriptional,post-transcriptional,and epigenetic levels.LncRNAs may function as oncogenes or tumor suppressors in the process of carcinogenesis and progression.LncRNAs participate in cell proliferation,invasion and metastasis,all of which are involved in tumorigenesis.At present,the roles of lncRNAs in various cancers such as liver cancer,breast cancer,bladder cancer and other tumors have been reported.However,the functions and mechanisms of lncRNAs in the development of breast cancer have been less studied.Thus,gene expression microarray was used to screen differentially expressed lncRNAs between tumor and non-tumor tissues(GEO:GSE72307),lncRNA BC069792 and BM466146 were chosen for further study.RT-qPCR analysis further confirmed the downregulated expression of lncRNA BC069792 and BM466146 in breast cancer tissues.We correlated BC069792 and BM466146 expression with clinicopathologic characteristics and patients’survival of breast cancer.The location of BC069792 and BM466146 was also measured in breast cancer cells.The effects of BC069792 and BM466146 on the proliferation,migration an invasion of breast cancer cells were investigated using in vitro functional experiments.Molecular mechanism of BC069792 in breast cancer was also investigated.High throughput generation sequencing technology,Ago2-RNA immunoprecipitation(RIP),dual-luciferase activity assay,Western blot,Real-time fluorescence quantitative polymerase chain reaction and immunohistochemistry were used to identify the binding microRNA of BC069792 and signaling pathway linked to BC069792 in breast cancer.This study may provide a theoretical basis for lncRNAs as novel diagnostic and prognostic markers and therapeutic targets for breast cancer.Methods:1.Expression of lncRNAs in breast cancer compared with non-cancerous tissues and clinical significance.98 pairs of matched specimens were collected from female patients with breast cancer,the expression of BC069792 and BM466146 was detected by RT-qPCR analysis to confirm the gene expression microarray Results.We associated the BC069792 and BM466146 expression with clinicopathologic characteristics and patients’ survival of breast cancer.We also measured the location of BC069792 and BM466146 through RT-qPCR amplified with separated cytoplasm RNA and nuclear RNA in breast cancer cells.Moreover,receiver operator characteristic(ROC)curves were constructed to evaluate the diagnostic value of BC069792 and BM466146 in breast cancer2.The effects of BC069792 and BM466146 on the proliferation,migration and invasion of breast cancer cells were investigated.Overexpression plasmid or empty plasmid of BC069792 and BM466146 was transfected and the efficiency was confirmed by RT-qPCR.To explore the role of BC069792 and BM466146 on cell proliferation,CCK-8,EdU and colony formation assays were performed.The effects of BC069792 and BM466146 on breast cancer cell migration and invasion capabilities were investigated by cell migration and invasion assay.BC069792 was chosen for further study according to the previous Results.SiRNA of BC069792 was transfected and the knockdown efficiency was confirmed by RT-qPCR.The influence of BC069792 on the apoptotic rate of breast cancer cells was detected by Annexin V-FITC/PI double staining flow cytometry Actinomycin D assay was performed to evaluate the stability of BC069792 in breast cancer cells.3.In vivo tumorigenic and metastasis assay.Overexpression of BC069792 lentivirus plasmid(pcDNA 3.1-BC069792)was constructed to stabilize the breast cancer cell line MDA-MB-231,and then the infected breast cancer cells were injected into nude mice through the armpit fat pad or the lateral tail vein of 4-week-old BALB/c nude mice to observe the xenograft tumor experiments were further performed to effect of BC069792 on the proliferation,invasion and metastasis in breast cancer in vivo4.The molecular mechanism of BC069792 in breast cancer was investigated.BC069792 and its antisense RNA were in vitro transcribed and biotin-labeled.High throughput generation sequencing technology was performed to identify the target genes of BC069792.RT-qPCR and Western blot were conducted to confirm the relationship between BC069792 and its target genes KCNQ4.To determine whether BC069792 works as a competing endogenous RNA(ceRNA),miRNAs potentially bound to both BC069792 and KCNQ4 were predicted by the bioinformatics analysis.Dual-luciferase activity assay and RIP experiment were performed to confirm BC069792 functions as a ceRNA by binding to microRNA,regulating the expression of KCNQ4.Signaling pathway linked to KCNQ4 was confirmed by RT-qPCR and Western blot analysis.Results:1.The expression and clinical significance of lncRNAs in breast cancerThe expression of IncRNA BC069792 and BM466146 was downregulated in human breast cancer tissues compared with normal breast tissues.Moreover,downregulation of BC069792 and BM466146 was detected in breast cancer cases with lymph node metastasis when compared to those without lymph node metastasis.ROC curve analysis indicated that BC069792 and BM466146 expresssion could clearly distinguish between breast cancer and normal breast tissues.The expression level of BC069792 and BM466146 was correlated with histological grade,lymph node metastasis,Ki-67 index and HER-2 expression2.LncRNAs inhibited the proliferation,migration and invasion of breast cancer cells in vitroOverexpression of BC069792 and BM466146 suppressed the proliferation of breast cancer cells.Upregulated expression of BC069792 significantly decreased the migration and invasion capability of breast cancer cells,whereas BM466146 had no effect on breast cancer cell migration and invasion.BC069792 were chosen for further study.BC069792 knockdown decreased the migration and invasion abilities of MDA-MB-231 and MDA-MB-468 cells and had no influence on cell growth,apoptosis and cell cycle.3.LncRNAs suppressed the growth,invasion and metastasis of breast cancer cells in vivoIn implanted subcutaneous oncogenic animal model,the breast cancer tumor growth of xenograft in the stable lentivirus LV-BC069792 group were significantly slower than those in negative control group.After 5 weeks of growth,Tumors volumes in the LV-BC069792 group-were non-invasive or well-encapsulated.However,tumors in the negative control group invaded locally into skin accessory area,striated muscle and surrounding fat tissues.The lungs in the LV-BC069792 group had less metastatic foci than the negative control group as viewed under microscope.Furthermore,the metastatic foci in the LV-NC group had greater volumes and number than those in the LV-BC069792 group.4-Study on the molecular mechanisms of BC069792 in breast cancerThe Results of high throughput generation sequencing showed that 802 down-regulated genes and 407 up-regulated genes were detected after BC069792 was upregulated.However,363 down-regulated genes and 181 up-regulated genes were found in the BC069792 knockdown group.KCNQ4 was chosen as the target gene of BC069792 by RT-qPCR and Western blot analysis.BC069792 functioned as ceRNA by competitively binding to hsa-miR-658 to regulate the expression of KCNQ4.Moreover,Western blot analysis showed that overexpression of BC069792 resulted in the upregulation of KCNQ4 and then inhibited AKT phosphorylation.Conclusions and significance:lncRNA BC069792 and BM466146 were found to be significantly downregulated in breast cancer by lncRNAs expression array analysis and RT-qPCR further confirmed the array Results.Expression of BC069792 and BM466146 were found to be associated with TNM stage,histological grade,Ki-67 index and lymph node metastasis.Patients with low expression of BC069792 had worse prognosis.BC069792 RNA was located both in the nucleus and in the cytoplasm of breast cancer cells.BC069792 exerted tumor suppressive function in breast cancer.BC069792 inhibited the proliferation,invasion and metastasis ability of breast cancer cells in vitro and in vivo.KCNQ4 was the target gene of BC069792.BC069792 could promote the expression of KCNQ4 and inhibit AKT phosphorylation,then inactivating AKT signaling pathway which suppressed the proliferation and invasion of breast cancer cells.These findings demonstrate that BC069792 is a novel promising candidate for the therapy and prognosis of breast cancer.