Promoting Insul In Secretion In Island Of Langerhans Cell By Silencing The PMR1 Expression

In recent years,diabetes incidence is rapidly soaring all over the world,diabetes have already become the third-largest serious chronic disease threatening to human health after the tumor and cardiovascular disease.Diabetes mellitus is a syndrome with disordered metabolism and inappropriate hyperglycemia due to either a deficiency of insulin secretion or to a combination of insulin resistance and inadequate insulin secretion to compensate.Insulin secretion mechanism is not very clear,it may mainly be through the intracellular calcium signal system or ATP-sensitive potassium channel and so on.There are varieties of therapies to diabetes at present,including oral diabetes medications,insulin,insulin analogues, stem cells,islet cell transplantation and so on.But for the lack of causes to the disease,it is unsatisfactory now.Therefore to clarify the mechanism of insulin secretion and to seek a better treatment for its causes have been a hotspot for biomedical research.RNA interference is a natural phenomenon in the organisms:a double-stranded RNA generated by intracellular introduction or endogenous transcription,binding to the homologous mRNA and mediates the targeted mRNA degradation,which causes the silencing of the corresponding gene after the transcription.RNA interference is a new method found for gene silencing in resent year,which specifically blocks or reduces the corresponding gene expression.So RNA interference has applications for treating diseases due to certain gene over-expression or gene mutation.Plasma membrane-related Ca²⁺-ATPase-1(PMR1),a member of the secretary pathway Ca²⁺ -ATPase family,is a Ca²⁺ and Mn²⁺ transporting ATPase localized to the golgi,or a golgi sub compartment,where it appears to be of importance for the normal functioning of the insulin secretary pathway.Studies have found that siRNA specificitied to anti-PMR1 can silence the expression of PMR1 mRNA and reduce protein expression of endogenous PMR1,which causing increased calcium influx, resulting in insulin secretion.Inhibiting the expression of PMR1 can helpβ-cells to secreting insulin in patients with diabetics or islet transplantation,which is expected to become the new target for treatment of diabetes.This experiment improve the gene silencing of siPMR1 synthesized molecules using RNAi technology in vitro isletβcell line NIT-1,and to observe the impact of insulin secretion in islet cells. It provides a theoretical foundation for the treatment of diabetic by using RNAi technology to silence the target of PMR1 expression.Objectives:1.To identify PMR1 gene expression in NIT-1 cells.2.To improve the gene silencing efficiency of the siPMR1 using of RNA interference.3.To observe the impact of silencing PMR1 gene on insulin secretion.Methods:1.Cell cultureMouseβcell derived NIT-1 cells were cultured in dulbecco's modified eagle's medium(Sigma) containing 25 mmol/l glucose and 2 mmol/l pyruvate, supplemented with 10%FBS,75 mg/L of penicillin and 50 mg/L streptomycin. The conditions were 37℃,5%CO₂,saturated humidity in the cell incubator.Used in this experiment,isletβcell line NIT-1 cells were provided by endocrine laboratory in second affiliated hospital of Zhongshan medical university.2.Detection of PMR1 mRNA in pancreaticβcells NIT-1 by PCRThe total mRNA of NIT-1 cells was extracted by trizol reagent,measured its OD260 and OD280 by ultraviolet spectrophotometer then calculated OD260/OD280,if the ratio greater than 1.8,indicating the quality of extraction of RNA reliable for the next step.Reverse-transcriptase according to the kit provided by TOYOBO.Taking lug total RNA in 20ul total reaction volume then reverse transcriptase to synthesis of cDNA.According to the cDNA sequence of internal GAPDH and the cDNA sequence of PMR1,two pairs of specific oligonucleotide primers were designed and synthesized.The PCR primers for control GAPDH were as follows:Sense sequence(GF):5'-ACCACAGTCCATGCCATCAC-3';Antisense sequence(GR):5'-TCCACCACCCTGTTGCTGTA-3'.The PCR primers for PMR1 were as follows:Sense sequence(FF):5'-GTTAGTCATAGGCGAGC-3';Antisense sequence(FR):5'-GTCCATGCTCTTCTGCAG-3'To amplify a 450 bp fragment of GAPDH and a 640bp fragment of PMR1.PCR reaction system as follows:the template cDNA 1.5 ul,PMR1 on the downstream primer(10μM) of the 0.5 ul,GAPDH upstream and downstream primers(10μM) of the 0.25 ul,ddH20 16.75 ul,the remaining group were according to the instructions of PCR kit,annealing temperature 56.5℃.PCR product was detected with 1.2% agarose gel electrophoresis and imaging analysis by gel imaging system.Each samplewere duplicate reactions two times.3.Transfection of siRNA Cells were seeded onto 6cm diameter and 96-well plates,and grown to 50-80% confluences.Then changing the culture medium for non-double-antibiotics and fetal bovine serum,containing final concentration of 100nM siRNA of DMEM culture medium,exchanging with 10%FBS DMEM culture medium after 6 hours,and continue to cultivate for the subsequent detection indicators.The sequences of siPMR1 were as follows:Sense:5'-GUUAGUCAUAGGCGAGCCUdTdT-3';Ant sense:5'-AGGCUCGCCUAUGACUAACdTdT-3'.The sequences of Negative control siRNA were as follows:Sense:5'-CGUGAUUGCGAGACUCUGAdTdT-3';Ant sense:5'-UCAGAGUCUCGCAAUCACGdTdT-3'.Experiment is divided into three groups:firstly,group of blank transfection (transfected with liposome composite liquid-only)l;Secondly,group of control siRNA transfection(transected with leptosomes containing composite liquid and negative control siRNA);Finally,group of siPMR1 transfection(transfected with composite liquid containing leptosomes and siPMR1).Did it according to the instructions of Lipofectamine 2000.4.Detection of gene silencing of PMR1 in mouse NIT-1 cellsA small interfering RNA(siRNA) duplex corresponding to nucleotides of mouse PMR1 cDNA was generated.This siRNAs were introduced into NIT-1 cells in vitro by lipid-mediated transfection using oligofectamine2000.We detected the gene silencing function of siPMR1 by semi-quantitative RT-PCR.Steps as follows: Collected cells after transfection 24h,wash 2 times with PBS,to extract total RNA accordance to the method with 2.3.2 provided and reverse transcription.PCR conditions are the same as 2.3.2,PCR product was separated with 1.2%agars gel electrophoresis and imaging analysis by gel imaging system.interference for the target gene.5.Determination of insulinAfter cells in the 96-well transfected with siRNA molecules(see 1.2.3) 24h, 36h,48h,collecting cell culture medium hole 100 ul in each 96-well,home stand by -30℃refrigerator.Measure the insulin levels according to the instruction of iodine [¹²⁵I]insulin radioimmunoassay kit.6.Statistical methodsData express by mean±standard deviation(X±S),we did statistical analysis by using factorial analysis and one-way ANOVA of statistical software SPSS13.0. Did homogeneity of variance first,then comparing the inter-group differences use of LSD or Dunnett T3 based on the test of homogeneity of variance,toα=0.05 level as significant.Results:1.The total mRNA of NIT-1 cells was extracted by trizol reagent in this experiment, then to identify PMR1 gene by using semi-quantitative RT-PCR method,there were two specific bands of about 450 bp and 640 bp could be seen after separating PCR products with 1.2%agarose gel electrophoresis,these bands with the expected negative control of GAPDH gene band and the band in line with the PMR1.2.The total mRNA of NIT-1 cells was extracted after transfection 24 hours,then detection abundance of PMR1 mRNA in cells through the method of Semi-quantitative RT-PCR,GAPDH as a reference,there were two specific DNA fragments(PMR1 and GAPDH) could be seen in each group,whose size in line with expectations;Test gray-scale of DNA fragment in each group lane for statistical analysis,the results showed that there was a significant difference(p＜0.05) of the relative expression of PMR1 mRNA in each group;there was a significant difference (p＜0.05) of the relative expression of PMR1 mRNA between siPMR1 transfection group and blank transfection group and control siRNA transfection group but there was not significant difference(P=0.563) of the relative expression of PMR1 mRNA between blank transfection group and control siRNA transfection group.3.The insulin levels measured in each experimental group was dealt with analysis of variance designed of factorial,the results show that there was a significant difference (p＜0.05) between different transfection methods and different transfection times, and interactive effects were existence between transfection methods and transfection times;The insulin results from different methods and times in each group was processed by one-way ANOVA,results show that amount of insulin secretion in group of siPMR1 was significantly higher than which in the blank group and control siRNA group;Insulin secretion in group of 24h after transfection was significantly higher than which in groupies of 36h and 48h after transfection;there was no significant difference about insulin secretion in each group 48h after transfection.Conclusions:1.siPMR1 can silence expression of PMR1 gene.2.PMR1 gene silencing can significantly promote insulin secretion of isletβcells, using RNAi technology to target the treatment of diabetic PMR1 silencing provides a theoretical foundation.