The Study Of MiR-8069 Enhances The Radiosensitivity Of Triple Negative Breast Cancer By Targeting CCND1

Part ?: To screen the genes and mi RNAs regulating TNBC radioresistanceBackground: Triple negative breast cancer(TNBC)patients account for 15-20% of all breast cancer patients and died patients account for over 25% of all breast cancer died patients.One reason for poor prognosis of TNBC is insensitivity to radiotherapy.CRISPR-Cas9 is a powerful tool which can edit genes.Its knockout library(Ge CKO v2.0)can knock out about 20,000 genes and 2,000 micro RNAs(mi RNAs).Moreover,each cell has at most one gene or mi RNA knocked out.The aims of the study were to compare the locoregional recurrecce(LR)rate in patients received radiotherapy for breast cancer molecular subtypes and to screen genes and mi RNAs regulating TNBC radioresistance by Ge CKO v2.0.Methods: We followed up the breast cancer patients who underwent radiotherapy in our hospital from January 2008 to October 2010 to obtain their prognostic information.The Kaplan-Meier method was used to analyze the 8-year LR rate of patients received radiotherapy for breast cancer molecular subtypes.The risk factors for the 8-year LR rate of breast cancer patients received radiotherapy were analyzed by the univariate and multivariate Cox analyses.MDA-MB-231 cells,human TNBC cell line,were infected by the human Ge CKO v2.0 and then underwent radiation.The surviving cells after radiation were sequenced for g RNA.The genes and mi RNAs were screened by refering to the g RNA sequences.The mi RTar Base database was used to predict the target genes of the mi RNAs.The KEGG pathway enriched analysis was conducted on the genes and the target genes predicted by mi RNAs.Results: A total of 312 breast cancer patients received radiotherapy were included in this study.The 8-year LR rates of Luminal A,Luminal B,HER2 enriched and triple negative breast cancer patients were 3.33%,5.26%,9.52% and 13.25%,respectively.TNBC patients after radiotherapy had the highest 8-year LR rates and had statistical difference from Luminal A breast cancer patients by the Kaplan-Meier analysis(p<0.05).TNBC was an independent risk factor for 8-year LR of breast cancer by univariate and multivariate Cox analyses.It had 2,268 candidate genes and 143 candidate mi RNAs regulating TNBC radioresistance by the human Ge CKO v2.0 screening.There were 8,13 and 10 genes and 26,46 and 34 mi RNAs acted on the apoptosis pathway,cell cycle pathway,and DNA replication and repair pathway,respectively.Moreover,14 mi RNAs acted on the three pathways,such as mi R-548 s,mi R-559,mi R-569,mi R-630,mi R-3133,mi R-3161,mi R-3671,mi R-4327,mi R-4465,mi R-4635,mi R-4682,mi R-5697,mi R-8052,and mi R-8069.Conclusion: TNBC is relatively insensitive to radiotherapy compared with other breast cancer molecular subtypes.The study also provides some genes and mi RNAs regulating TNBC radioresistance,which suggests a new idea for improving radioresistance of TNBC.Part ?: mi R-8069 enhances the radiosensitivity of TNBC by targetting CCND1Background: CCND1,cyclin D1,is widely considered as an oncogene.It over-expresses in breast cancer and takes part in the occurrence and progression of breast cancer.Some studies have reported that CCND1 was involved in TNBC radioresistance.In the part ?,14 mi RNAs that simultaneously act on the apoptosis,cell cycle and DNA replication and repair pathways may regulate TNBC radioresistance,among which only mi R-8069 may target CCND1.The aim of the study was to explore the effects and mechanisms of mi R-8069 on TNBC radiosensitivity.Methods: The expression of mi R-8069 was detected in TNBC cancer tissues and adjacent normal tissues by q PCR.The mechanisms of mi R-8069 regulating TNBC radiosensitivity were tested by radiation clonogenic survival assay,dual-luciferase reporter gene assay,CCK8 assay and flow cytometer technology.The effect of mi R-8069 on TNBC radiosensitivity was also detected by tumor xenograft experiments.The association between mi R-8069 and LR of TNBC patients received radiotherapy was analyzed by the Kaplan-Meier method.Results: The expression of mi R-8069 in human TNBC cancer tissues was 0.40 times compared to adjacent normal tissues(p<0.05).The sensitive enhancement ratio(SER)of high expression of mi R-8069 in MDA-MB-231 cells was 1.34 compared with the control group.The protein and m RNA levels of CCND1,the predicted target gene of mi R-8069,were 0.66 and 0.54,respectively,in mi R-8069 high expression group compared with the control group.The dual-luciferase reporter gene assay showed that the relative fluorescence signal of CCND1 WT in the mi R-8069 mimic group was significantly reduced by 17% compared with the mi R-negative group(p<0.05).The relative fluorescence signal was recovered up to 47% when the 3?-UTR site 744-750 of CCND1 was mutated.The percentage of G0/G1 cells was higher in mi R-8069 high expression group than control group(p<0.05).The OD450 values associated with cell proliferation capacity was lower in mi R-8069 high expression group than control group(p<0.05).However,there were no significant difference between high expression of mi R-8069 and CCND1 group and control group about the percentage of G0/G1 cells,OD450 values and SER(p>0.05).The volume of tumor in nude mice was(301.6 ±10.57)mm3 in mi R-8069 high expression group and was(812.1±17.02)mm3 in control group(p<0.05).The patients suffered from LR were 9 in mi R-8069 low expression group and 2 in mi R-8069 high expression group among 83 TNBC patients received radiotherapy by the Kaplan-Meier method(p<0.05).Conclusion: The expression of mi R-8069 is lower in TNBC cancer tissues than adjacent normal tissues.Over-expressing mi R-8069 enhances the radiosensitivity of TNBC.Its mechanisms are that mi R-8069 targets CCND1 3?-UTR to down the protein level of CCND1,which inhibits the cell cycle and proliferation ability of TNBC cells.mi R-8069 has the potential to be a new target for improving radioresistance of TNBC.