Study On The Effect Of TRPC1 On Vascular Smooth Muscle Contraction And Breast Cancer Apoptosis

BackgroundThe TRPC channel is the first channel to be found in the TRP channels,which has the highest homology with the TRP channel and is referred to as the traditional TRP channel.Different members of the subfamily TRPC also have different distributions,and their functions are different when activated.It is involved in the activation of transcription factors,the myocardial hypertrophy and platelet activation.TRPC1 is one of the first cloned mammal transient receptor potential channels,its distribution is widespread,which has higher levels of expression in the brain,heart,kidney,lung,skeletal muscle,testis,ovary,salivary glands.Current research shows that TRPC1 not only participates in receptor mediated,but also plays an important role in the secretion and contraction of calcium dependence.And the changes in the intensity of interaction of TRPC1,TRPC4,TRPC5 and BKCa on vascular smooth muscle contraction and the role of TRPC1,TRPC4 and TRPC5 on vascular smooth muscle contraction is not fully understood.ObjectiveStudy on the the changes in the intensity of interaction of TRPC1,TRPC4,TRPC5 and BKCa on vascular smooth muscle contraction and the role of TRPC1,TRPC4 and TRPC5 on vascular smooth muscle contraction.Methods1 Transiently transfected si RNA The specific TRPC1 si RNA was transfected into vascular smooth muscle and the vascular smooth muscle was followed up experiment after 24 h.2 Preparation of vascular smooth muscle and determination of tension The isolated vascular smooth muscle was rapidly put in Krebs solution and equidistant long vessels(about 2 mm in length).The ends of the vascular smooth muscle were fixed in the microvascular apparatus.The vascular smooth muscle contraction capacity was analyzed by the analysis software data acquisition and analysis system.3 Immunoprecipitation The interaction of TRPC1,TRPC4,TRPC5 and BKCa protein in vascular smooth muscle contraction was detected by the immunoprecipitation of TRPC1,TRPC4 and TRPC5 antibody.Results1 In the vascular smooth muscle tension test,the expression of TRPC1 protein in vascular smooth muscle was significantly decreased after TRPC1 si RNA treatment,and endothelin-1-induced contraction was significantly enhanced compared with the control group.2 In the vascular smooth muscle tension test,the expression of TRPC4 protein in vascular smooth muscle was significantly decreased after TRPC4 si RNA treatment,and endothelin-1-induced contraction was significantly attenuated compared with the control group.3 In the vascular smooth muscle tension test,the expression of TRPC5 protein in vascular smooth muscle was significantly decreased after TRPC5 si RNA treatment,and endothelin-1-induced contraction was significantly enhanced compared with the control group.4 In immunoprecipitation,TRPC1 can co-precipitate TRPC5 and TRPC4,TRPC4 can co-precipitate TRPC1,TRPC5,TRPC5 can co-precipitate TRPC1,BKCa,TRPC4 can not co-precipitate TRPC5.ConclusionTRPC1 and TRPC4,TRPC5 form different polymers channel,respectively,TRPC1,TRPC5 and BKCa form calcium signaling complex to regulate vascular smooth muscle contraction.BackgroundBreast cancer is one of the common malignancies in women,which seriously affects the physical and mental health of women,and even endanger the lives of patients.In Europe and the United States,the incidence of breast cancer is the highest in female malignant tumors.Although China is a low incidence area,Rising year by year.Breast cancer is mainly due to malignant changes in the ductal epithelium of the breast,the performance of breast swelling,pain,lumps and so on.At present,the treatment of breast cancer,including five blocks: respectively,surgical treatment,chemotherapy,endocrine therapy,radiotherapy and molecular targeted therapy.The development of molecular biology and the understanding of the pathogenesis from cell and molecular levels makes tumor therapy into a new era of molecular targeted therapy.Compared with chemotherapy and radiotherapy,molecular targeted therapy is effective and selective to kill tumor cells,making treatment more targeted and less side effects.At present,targeted delivery of si RNA to specific cells in the field of drug development has great potential,si RNA can not only as a research tool to inhibit the expression of target genes,but also as a treatment for the treatment of various diseases.To target the delivery of si RNAs,we have designed a targeting peptide that can encase si RNA and form nanoparticles to deliver si RNA to breast cancer cells,which can not only pass through the mammalian cell surface receptor target delivery of si RNA and damage to normal cells is very small,this feature greatly improved the practicality of this technology.ObjectiveIn this study,we constructed the supramolecular nanoparticles by wrapping si RNAs using the peptide 1 to achieve specific delivery of TRPC1 si RNA into breast cancer cells.In addition,we use this targeted peptide to deliver TRPC1 si RNA can also cause breast cancer cell apoptosis.Methods1 Breast cancer cell cultureThe breast cancer cells MDA-MB-231 were cultured in DMEM medium supplemented with 10% fetal bovine serum,100 units/ml penicillin,100 g/m L streptomycin,and cultured in 37 °C,5% CO2 incubator.2 Peptide 1/si RNA binding assayThe gel retardation assay was performed with polypeptides 1 and si RNA at different concentrations.And treated with heparin(de-polyanionic complex stabilizer)on the complex,and the results were observed.3 Peptide 1/si RNA nanoparticle stability testThe stability of the si RNA 1/si RNA nanoparticles was tested in the environment of RNA nuclease and mouse serum incubation,and the stability of the si RNA was tested by gel blocking assay after heparin was released.4 Experimental study on apoptosis of breast cancer cellsThe polymorphism of apoptotic index bax was detected by Western blot.The advantages and disadvantages of peptide 1/si RNA nanoparticles were observed and compared with the control group.5 In vitro silencing of breast cancer cell-specific genesWestern blot was used to detect the changes of TRPC1 bands after transfection with peptide 1/si RNA nanoparticles,and compared with the control group to observe the advantages and disadvantages of peptide 1/si RNA nanoparticles.6 Statistical processingAll experimental data were expressed as mean ± standard error and misplaced t test using Sigma Plot 12.5 software.P <0.05 was considered statistically significant.Results1 When peptide 1 and si RNA ratio greater than 100: 1,the peptide 1 and si RNA binding capacity gradually increased.2 The stability of the peptide 1/si RNA nanoparticles in the nuclease-containing solution is significantly higher than that of the exposed si RNA.3 The stability of peptide 1/si RNA nanoparticles in mouse serum was significantly higher than that of exposed si RNA.4 Peptide 1/TRPC1 si RNA nanoparticles into breast cancer cells had a tendency to promote apoptosis.5 Peptide 1/TRPC1 si RNA nanoparticles into breast cancer cells had a tendency to promote downregulation of TRPC1 compared.ConclusionThis study validates the feasibility of introducing the newly synthesized peptide 1 into TRPC1 si RNA,and provides a new theoretical basis and method for the treatment of breast cancer.