| Aim: Long noncoding RNA-differentiation antagonizing nonprotein coding RNA(DANCR)plays a role in the differentiation of synovial mesenchymal stem cells into chondrocytes,dentin-like differentiation of dental pulp cells,liver cancer,prostate cancer,colon cancer.DANCR is closely related to tumorigenesis,colonization,tumor invasion,metastasis,proliferation,migration,apoptosis,disease progression and prognosis.However,there are few reports on the relationship between DANCR and breast cancer,and the specific mechanism has not been elucidated.This study aimed to investigate the biological function of DANCR in breast cancer.Methods: Fresh primary breast cancer samples and paired adjacent non-tumor soft tissues were obtained from 26 patients who underwent surgical resection at our department from May 2017 to November 2018.None of the patients has received chemotherapy,radiotherapy and targeted therapy before surgery.The expression of DANCR in breast cancer tissues and cell lines were detected by qRT-PCR.Furthermore,the expression of DANCR in breast cancer cell lines MCF-7(HR+,Her2-)? MDA-MB-231(HR-,Her2-)? SK-BR-3(HR-,Her2+)and normal epithelial cell line MCF-10 A were detected by qRT-PCR.We chose the MDAMB-231 cell line for further study,lentivirus-mediated knockdown of DANCR by using 2 distinct shRNAs(381,766).qRT-PCR analysis also confirmed that DANCR was significantly down-regulated in MDA-MB-231 breast cancer cells which were transfected with shRNAs.MTT and Transwell assays were used to detect the effect of DANCR on the proliferation and migration of MDA-MB-231 cells.Flow cytometry was used to detect whether lncRNA DANCR affected cell cycle and apoptosis.Western blot analysis was used to detect cell cycle related proteins.SPSS was used to analyze the expression level of DANCR and the clinical characteristics of patients: age,tumor stage,histological grade,Ki-67,hormone receptor status.Results: 1?The expression of DANCR in breast cancer patient tissues and cell lines were detected by qRT-PCR.Results showed that RNA level of DANCR were significantly higher in breast cancer tissues than that in normal breast tissues,P<0.05.2?The RNA level of DANCR in breast cancer cell lines MCF-7,SK-BR-3,MDAMB-231 and normal epithelial cell line MCF-10 A were detected by qRT-PCR.We found that DANCR was significantly up-regulated in HER2 positive SK-BR-3 cell line and TNBC MDA-MB-231 cell lines compared with MCF-10A(normal epithelial cell line),P<0.05.3?Lentivirus-mediated knockdown of DANCR by 2 distinct shRNAs(381,766)were performed in MDA-MB-231 cell line.GFP signals were detected to validate infection rate and DANCR knockdown stable cells were successfully constructed.RNA level of DANCR were detected in these cell lines.Compared with the negative control cells,qRT-PCR results confirmed that DANCR was significantly down-regulated in both shRNA-381 and shRNA-766,P<0.05.4?We performed MTT assays to detect the effect of DANCR on the proliferation of MDA-MB-231 cells.Viability and proliferation were detected by MTT assay.Compared with the negative control cells,both shRNA-381 and shRNA-766 caused significant decrease of viability in MDA-MB-231,P<0.05.5?Transwell assays was used to detect the effect of DANCR on the migration of MDA-MB-231 cells.Compared with the negative control cells,both shRNA-381 and shRNA-766 caused significant decrease of viability in MDA-MB-231.Number of migrated cells in shRNA groups were all significantly decreased as well,P<0.05.The results showed that knockdown of DANCR significantly inhibited the migration of MDA-MB-231 breast cancer cells.6?Using flow cytometry to detect the effect of DANCR on cell cycle,the ratio of G0/G1 phase cells was 34.2%(NC),48.38%(381),40.17%(766),respectively.DANCR knockdown markedly increased the G0/G1 proportion as compared with negative control groups.7 ? Apoptosis of these cells were also detected,compared with the negative control cells,no obvious changes in apoptosis were observed in shRNA groups.Knockdown of DANCR had no effect on the apoptosis of MDA-MB-231 breast cancer cells.8?The expression of cyclin D1 and CDK4 were analyzed by Western blot.Compared with the negative control cells,the depletion of DANCR partially inhibited the expression of Cyclin D1 and CDK4 in MDA-MB-231 breast cancer cells,P<0.05.9?To further investigate the relationship between lncRNA DANCR and clinic pathological characteristics,we divided all patients into 2 groups(high DANCR and low DANCR)by using the median value of the expression of DANCR(1.344)as a cut off value.The correlation between DANCR and various clinical features were analyzed.In high DANCR group,0%,31% and 69% patients were TNM I,II,III,respectively,while in low DANCR group the proportions were 61%,31% and 8%.In high DANCR group,0%,54%,46% patients were histological grade 1,2,3 respectively,while in low DANCR group the proportion were 8%,38% and 54%.In addition,69% and 23% patients were ER negative in high DANCR and low DANCR groups,respectively.High DANCR was significantly associated with lower TNM stage and ER status.However,there was no significant association between DANCR expression and patient age,histological grade and ki-67,P>0.05.Conclusions: our results showed that DANCR was markedly increased in breast cancer tissues and breast cancer cell lines.Knocking down of DANCR inhibited cell proliferation and migration capacity in MDA-MB-231 breast cancer cells,and induced cell cycle arrest in the G0/G1 phase by reducing the expression of Cyclin D1 and CDK4.Thus,our results demonstrated that DANCR could promote viability,migration and cell cycle in TNBC.We analyzed the association between DANCR expression and clinicopathological characteristics,and found that high lncRNA DANCR level was significantly related with TNM stages and the status of ER.This study would provide theological support for studies in TNBC and suggested that DANCR bears significant potential as diagnostic biomarkers and therapeutic targets in breast cancer.However,more studies are needed for further investigation. |
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