The Effects Of LRP16 Gene On The Function Of MIN6 Cell Line

LRP 16 is a human gene which was firstly cloned by HAN Wei-dong,et al in the general hospital of PLA in 1999.It locates on 11q12.23 chromosome,and encodes a nuclear protein.Previous studies showed that,as a target gene of estrogen,LRP16 can be up-regulated by estrogen through estrogen receptorα(ERα).And as a co-activator of ERα,LRP16 can enhance the transcriptional activation induced by ERα.In resent years,many studies showed that estrogen had the effects of protecting pancreaticβcells and increasing insulin synthesis and secretion.Whether LRP16 engage in the processes is an interesting hypothesis.Our resent studies found that the level of LRP16 protein in human insulinoma was dramatically higher than that in normal pancreaticβcells.These results suggested that LRP16 gene may play an important role in the development,insulin synthesis and secretion of pancreaticβcells.In this study,we established LRP16 gene over-expression cell models in MIN6 cell line.By using the models,we detected glucose-stimulated insulin secretion(GSIS) and the expression levels of insulin mRNAs in order to find out potential mechanisms.The study was divided into two parts:1,The effects of LRP16 gene on the proliferation and insulin secretion in MIN6 cell lineObjective:To explore the effects of LRP16 gene on the proliferation and insulin secretion in MIN6 cell line.Methods:1.Western blot was used to detect the expression level of LRP16 protein in MIN6 cell line;2.Stable over-expression of LRP16 cell models were established by SuperFect strategies;3.The effect of over-expression of LRP 16 on MIN6 cell proliferation was examined by MTT methods; 4.0 mm/L,3 mm/L and 30 mm/L glucose were used to observe the effects of GSIS on the transfected cells;5.The expression level of Glucose Transporter type2(Glut-2) protein in MIN6 cell line was determined by Western blot.Results:1.MIN6 cell line expressed LRP16 protein;2.Cell proliferation in Over-expression of LRP16 group had not significant differences compared with control group(p＞0.05);3. Over-expression of LRP16 can increase GSIS by 2.26-fold(p＜0.05),2.19-fold (p＜0.05) and 2.16-fold(p＜0.05)(p＞0.05 between the 3 groups) compared with control group when under the 0 retool/L,3 mmol/L and 30 mmol/L glucose concentrations,respectively;4.Western blot analysis showed that the level of Glut-2 protein in LRP16 over-expression group was 1.76-fold of control group(p＜0.05). Conclusions:The pancreaticβcells of mice express LRP16 protein;Over-expression of LRP16 cannot increase MIN6 cell proliferation,but can increase glucose-stimulated insulin secretion(GSIS).These stimulative effects may depend on the up-regulation of Glut-2 protein and do not depend on the glucose concentations in MIN6 cell line.2,The effects of LRP16 gene on the synthesis of insulin mRNAs and related transcription factors in MIN6 cell lineObjective:To investigate the effects of LRP16 on the synthesis of insulin mRNAs and related transcription factors in MIN6 cell line.Methods:1.Stable over-expression of LRP16 cell models were established by SuperFect strategies;2. Using Real-Time PCR to measure the levels of insulin mRNAs and Pancreatic-Duodenal homeobox factor-1(Pdx-1),MafA and NeuroD1 mRNAs.3.The expression levels of Pdx-1,MafA and NeuroD1 proteins were detected by Western blot.Results: 1.Real-Time PCR showed that the levels of InsulinⅠmRNA and InsulinⅡmRNA in LRP16 over-expression group were 1.6-fold and 1.8-fold of control group, respectively(p＜0.05);2.The expression level of Pdx-1 mRNA in LRP16 over-expression group was 1.62-fold of control group(p＜0.05),but the levels of MafA and NeuroD1 mRNAs had not significant differences between the two groups (p＞0.05);3.The expression level of Pdx-1 protein in LRP16 over-expression group was 2.04-fold of control group(p＜0.05),but the levels of MafA protein and NeuroD1 protein had not significant differences between the two groups(p＞0.05).Conclusions: Over-expression of LRP16 gene can increase the synthesis of insulin mRNAs.This effect may depend on the up-regulation of Pdx-1 protein in MIN6 cell line.