| The incidence of breast cancer in China has been growing at a rate of two times the global rate of growth since 1990s,especially in urban areas.Based on the statistics in 2014,the breast cancer has been ranked 6th in the death rate of women in China.Triple negative breast cancer accounts for about 15-20%of all breast cancer types.It has the highest degree of malignancy in all breast cancer subtypes,with the characteristics of poor prognosis,easy metastasis and short survival time.Because of the lack of effective drug targets,the clinical treatment of triple negative breast cancer is usually the surgery combined with chemotherapy.The risk of recurrence is very high and it is urgent to find new effective drug targets.LncRNAs is a large group of transcripts with complicated functions that have been discovered in recent years.Because of the lack of open reading frame,the cannot encode proteins and they have been considered as?transcriptional noise?in the early research.With the development of research,lncRNAs‘diverse regulatory mechanisms in biological processes have been discovered and they have been thought to play extremely important role in cellular biological processes.In a variety of malignant tumors,including triple negative breast cancer,many lncRNAs expression level have been found to be abnormal.Their abnormal expression level is closely related to the occurrence and development of malignant tumors,which has become a hotspot in current cancer research.By targeting the over-expressed lncRNAs in tumor tissues,the antisense oligonucleotides?ASO?delivered into the tumor cells would be able to combine to the lncRNAs and induce the degradation or block their functions,which have been proved to be effective in anti-tumor effect.However,the biggest problem of ASO being utilized in drug research is the delivery.The small nucleic acid molecules are easily degraded and excessive chemical modification would cause non-specific cytotoxicity.The development of nano-delivery carriers provide small nucleic acid molecules a favorable technical support.By packaging in the nanoparticles,small nucleic acid molecules can effectively resist nuclease degradation in vivo,increase half-life and entry the tumor tissues to play anti-tumor effect.In order to overcome the multidrug resistance of tumor cells,we designed nanoparticles to co-encapsulate two kinds of anti-tumor drugs,which can co-delivery drugs with different working pathways,resulting in synergistic anti-tumor effect.The co-delivery of traditional chemotherapeutic drugs and gene therapy drugs is an important research direction in the design of anti-tumor nano-drugs.In this paper,we take lncRNA ASBEL as the research object and design a series of ASOs targeting ASBEL.By effectively suppress ASBEL function and MDA-MB-231 cells proliferation,antago3 was selected.By performing a series of experiments in vivo and in vitro,the anti-tumor effect of antago3 was evaluated.Then,we designed the nano-delivery system based on hyaluronic acid and chitosan and managed to co-deliver antago3 and curcumin.The anti-tumor ability of the nano-delivery system was evaluated by experiments in vivo and in vitro.The specific experimental work includes the following aspects:1.We performed the secondary structure prediction of the complementary region of ASBEL and BTG3 mRNA by software.15 different candidate antagoNATs were selected to reduce ASBEL expression level and suppress its functions.Antago3 was finally selected by evaluating the change of expression level of ASBEL and the change of cell proliferation after the transfection.We further verified the effectiveness of antago3 by performing the anti-tumor ability detection with the different chemical modification and concentration gradient of antago3 used in the experiments.Antago3was identified as a candidate ASO drug for triple negative breast cancer therapy by targeting ASBEL.2.The anti-tumor effects of antago3 were evaluated by a series of in vitro cellular experiments and in vivo nude mice experiments.The experiments in vitro showed that the proliferation ability and migration ability were remarkably suppressed and the cell apoptosis was induced upon transfection with antago3.The targets and anti-tumor mechanisms of antago3 were determined by the detection of differential expression levels of ASBEL and BTG3 mRNA in the nucleus and cytoplasm respectively after the transfection of antago3 with different concentrations.In vivo animal experiments showed that antago3 delivered by liposome was able to remarkably inhibit the tumor growth.3.The hyaluronic acid-chitosan nanoparticles were constructed by the principle of nano self-assembly.Curcumin and antago3 were further encapsulated in the nanoparticles,resulting in CNP?curcumin encapsulated?,ANP?antago3 encapsulated?and CANP?curcumin and antago3 co-encapsulated?.The TEM observation,the size and charge detection were performed for CANP and the empty nanoparticles.The protective effect of nanoparticles on antago3 was evaluated.4.In vitro and in vivo anti-tumor effect of CNP,ANP and CANP were evaluated.The three nanoparticles exhibited efficient cell transfection efficiency in vitro.The proliferation,invasion and migration ability of cells were strongly suppressed and the cell apoptosis was induced upon transfection.The detection of RNA level and protein level of BTG3 gene,and the protein level of several curcumin targets,indicated that the drug encapsulated in the nanoparticles played a role.In animal experiments,the three nanoparticles were injected through tail vein respectively in three treatment groups.The results showed that CANP injection achieved the best anti-tumor effect.Further analysis of the extract tumor tissues found that the expression of ASBEL was decreased and BTG3 mRNA was increased.H&E staining and TUNEL staining showed that there was a large range of apoptosis in the tumor tissues.H&E staining of normal organs and the record of body weight of nude mice indicate that the nano-delivery system had high biosafety. |
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