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Objective To explore the impacts of long non-coding RNA(LncRNA)GNAS antisense RNA1(GNAS-AS1)on the proliferation and migration of gastric cancer(GC)cells by regulating the miR-449a/Notch1 axis.Method Tumor tissue and adjacent tissue samples were collected from 30 patients diagnosed with GC at Sichuan Provincial People's Hospital from September 2013 to September 2017;GC cells AGS were randomly divided into Control group,si-NC group,si-GNAS-AS1 group,si-GNAS-AS1+inhibitor NC group,and si-GNAS-AS1+miR-449a inhibitor group.Real-time fluorescence quantitative PCR method was applied to detect the expres-sion of GNAS-AS1,miR-449a,and Notch1 mRNA;MTT experiments and plate cloning experiments were applied to detect the proliferation;wound healing test was applied to detect cell migration;Transwell experiment was applied to detect cell invasion.Western Blot was applied to detect the expression of Notch1,E-cadherin,Vimentin,and N-cadherin proteins.Double Luciferase reporter gene experiment was applied to verify the relationship between GNAS-AS1 and miR-449a,between miR-449a and Notch1,respectively.Results Compared with adjacent tissues,the expression of GNAS-AS1 and Notch1 mRNA in tumor tissue was increased,the expression of miR-449a was reduced(P<0.05).Compared with the Control group and si-NC group,the expression of GNAS-AS1,OD490 value,number of clones formed,scratch healing rate,number of cell invasions,and the expression of Notch1,Vimentin,and N-cadherin proteins in AGS cells in the si-GNAS-AS1 group reduced,the expression of miR-449a and E-cadherin protein increased(P<0.05).Compared with the si-GNAS-AS1 group and the si-GNAS-AS1+inhibitor NC group,the OD490 value,scratch healing rate,number of cell invasions,Notch1,Vimentin,and N-cadherin expression in the si-GNAS-AS1+miR-449a inhibitor group increased,the expression of miR-449a and E-cadherin protein reduced(P<0.05).GNAS-AS1 targeted and negatively regulated miR-449a expression,while miR-449a targeted and negatively regulated Notch1 expression.Conclusion Silencing GNAS-AS1 may inhibit the expression of Notch1 protein by up-regulating miR-449a,thereby inhibiting the proliferation,migration,and invasion pro-cesses of GC cells.
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Objective To investigate the effects of knockdown of miR-449a on breast cancer xenograft in nude mice.Methods In this study,we established a model of breast cancer xenograft in nude mice with breast cancer MCF-7 cell line,then stably transfected MCF-7 cells with plasmids containing shRNA-miR-449a precursors;the stable transfected cells were injected subcutaneously into the nude mice by multi-point injection.We used negative plasmid as negative control and PBS as blank control.We detected tumor volume after the treatment of nude mice and tumor growth inhibition rate was calculated.The expressions of miR-449a and Notch1 in xenograft in nude mice were detected by real-time PCR;the levels of Notch 1,β-catenin and E-cadherin in the xenograft tumor tissues were detected by Western blot; and the expression of E-cadherin in tumor tissues was detected by immunohistochemistry.Results In the process of xenograft tumor formation,no nude mice died.Knockdown of miR-449a could significantly inhibit the growth of tumor volume after tumor detection (P <0.05 ).miR-449a was significantly down-regulated in xenograft tumor tissues,and the expressions of Notch 1 and β-catenin were also down-regulated,while the level of E-cadherin was increased.The results of immunohistochemistry showed that the expression of Notch1 protein was decreased and that of E-cadherin protein was increased (P <0.05).Conclusion Knockdown of miR-449a inhibited the normal growth of breast cancer xenografts in nude mice by down-regulating the expressions of Notch 1 andβ-catenin and promoting the expression of E-cadherin.
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Objective To investigate the effects of knockdown of miR-449a on breast cancer xenograft in nude mice.Methods In this study,we established a model of breast cancer xenograft in nude mice with breast cancer MCF-7 cell line,then stably transfected MCF-7 cells with plasmids containing shRNA-miR-449a precursors;the stable transfected cells were injected subcutaneously into the nude mice by multi-point injection.We used negative plasmid as negative control and PBS as blank control.We detected tumor volume after the treatment of nude mice and tumor growth inhibition rate was calculated.The expressions of miR-449a and Notch1 in xenograft in nude mice were detected by real-time PCR;the levels of Notch 1,β-catenin and E-cadherin in the xenograft tumor tissues were detected by Western blot; and the expression of E-cadherin in tumor tissues was detected by immunohistochemistry.Results In the process of xenograft tumor formation,no nude mice died.Knockdown of miR-449a could significantly inhibit the growth of tumor volume after tumor detection (P <0.05 ).miR-449a was significantly down-regulated in xenograft tumor tissues,and the expressions of Notch 1 and β-catenin were also down-regulated,while the level of E-cadherin was increased.The results of immunohistochemistry showed that the expression of Notch1 protein was decreased and that of E-cadherin protein was increased (P <0.05).Conclusion Knockdown of miR-449a inhibited the normal growth of breast cancer xenografts in nude mice by down-regulating the expressions of Notch 1 andβ-catenin and promoting the expression of E-cadherin.
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Objective The purpose of this study is to investigate the effect of miR-449a on pancreatic cancer cells and the molecular mechanism. Methods The expression levels of miR-449a in pancreatic cancer cells treated or untreated with radiation was detected by qRT-PCR.High expression of miR-449a was achieved by transfecting miR-449a mimics into SW1990 cells. The cell growth,apoptosis and colony formation ability was assessed by MTT assay,flow cytometry and colony formation assay,respectively. The relationship of miR-449a and Cyclin D1 was determined by the TargetScan and dual luciferase reporter. Immunohistochemistry was used to examine protein levels of Cyclin D1 in pancreatic cancer and normal pancreas tissues. Si-Cyclin D1 was used to detecte the effect of Cyclin D1 on radiosensitivity of pancreatic cancer cells. Results The expression levels of miR-449a in pancreatic cancer cells with radiation treatment were decreased significantly. Mir-449a mimics increased the cell proliferation rates and apoptosis rates obviously,and decreased the colony formation ability in SW1990 cells treated with radiation. Results from the TargetScan and dual luciferase reporter showed that Cyclin D1 was the target of miR-449a. The positive staining rates of Cyclin D1 in pancreatic cancer tissue(85.7%,30/35)was higher than those in normal pancreas tissue(20%,2/10).Knockdown of Cyclin D1 enhanced the radiosensitivity of pancreatic cancer cells.Conclusion MiR-449a enhances the radiosensitivity of pancreatic cancer cells by targeting Cyclin D1.
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OBJECTIVE: Lung cancer has become the primary cause of cancer‑related death now. New therapies targeting the molecular regulatory machinery were required imperatively. MicroRNAs and long noncoding RNAs can respectively or cooperatively function as oncogenes or tumor suppressor genes in human cancers. The present study identified that miR‑449a was down‑regulated in tissue of human lung cancer. In this study, we aimed to investigate the function of miR‑449a in NL9980 and L9981 lung carcinoma cells lines and the relationship with lncRNA nuclear enriched abundant transcript 1 (NEAT1). MATERIALS AND METHODS: miR‑449a was profiled in several lung carcinoma cell lines by quantitative reverse transcription‑polymerase chain reaction RT‑PCR. We analyzed the effects of miR‑449a overexpression on proliferation, apoptosis and cell cycle in L9981 cells. The regulatory relationship between miR‑449a and NEAT1 was predicted in silico and further studied by miR‑449a inhibitor and mimics assay. RESULTS: miR‑449a was expressed in four cell lines, which we selected, however miR‑449a was in high level in NL9980 and in low level in L9981 (P < 0.05). When the miR‑449a was the overexpression in L9981 cells, the cell growth was suppressed, and the apoptosis cells were promoted compared with the control group (P < 0.05). The G1/G0 became longer and S, G2/M became shorter (P < 0.05) by miR‑449a overexpression. Further study of the interaction between miR‑449a and NEAT1 show that NEAT1 was up‑regulated when cells were transfected with miR‑449a inhibitor, and NEAT1 was down‑regulated when cells transfected with miR‑449a mimics. CONCLUSIONS: Our data indicate that miR‑449a may function as a suppressor of lung cancer, and affects the expression of NEAT1 in lung cancer cells.