Effect Of LRP16Gene On Pancreatic β-cell Function And The Molecular Mechanism

Objective:Leukemia-related protein16(LRP16), localized on chromosome11q12.1, is a novel gene cloned from human lymphocyte cells by our group in the year2000(GenBank Accession No. AF202922). That study found that estrogen downregulated mRNA expression of LRP16by binding to the LRP16promoter, which was either the estrogen-responsive element half-site (at-246to-242bp) or the GC-rich Spl binding site (at-236to-227bp). We also found that LRP16was a co-activator of ER-a. The prevalence rate and progression of diabetes markedly increases in postmenopausal women, indicating the importance of estrogen in regulating β-cell functions. Data from clinical trials have revealed that estrogen exerts long-term beneficial effects on glucose-induced insulin secretion. Studies have also suggested that the female hormone17β-estradiol protects pancreatic β-cell function. Furthermore, the estrogen receptors alpha (ER-a) and ER-β are present in pancreatic P-cells, and estrogens can regulate proliferation and insulin secretion in these cells. This indicate that LRP16gene, as a downstream targeted gene of estrogen receptors alpha (ER-a), may regulate pancreatic β-cells function. In this study, we utilized an RNA interference and over-expression strategy to regulate the LRP16gene’s expression and thereby demonstrate effect of LRP16gene on pancreatic P-cell function and the molecular mechanism.Methods:1) Construction the MIN6cell with over/down-expression of LRP16a) The MIN6cell were seeded in6cm culture dish with density of80%,24hours later, pcDNA-3.1-16and pcDNA-3.1were transfected into these cells using lipofectamine2000according to the introduction. Another24hours later, the DMEM containing G418(800μg/ml) was used to Screening for resistance to3weeks. Then the cells was cultured with the DMEM containing G418(400μg/ml) to construct cell lines with LRP16over-expression and the control cells.b) Synthesis the oligo DNA according to the siRNA target sequence which have been identified for screening with hairpin structure of the LRP16gene. The Oligo DNA was annealed to form double-stranded DNA, then connected to pENTR/U6vector to produce lentiviral vector LV-siLRP16. The connected vector was transformed into competent cells DH5a, and Recombinant colonies with positive identification were picked for PCR and sequencing. Vector LV-siLRP16and the Plasmid packaging system were cotransfected into293T cell and produce virus particle. Cells which was infected by virus particle were cultured with DMEM containing Blasticidin (0.9μg/ml) for three weeks. Then the cell lines with LRP16down-expression were constructed.2) Effect of LRP16gene on cell cycle and proliferation in MIN6a) Cell cycle analysis with flow cytometry.b) MTT.c) The mRNA of cyclin Ds and cyclin E were measured using Real-time Quantitative PCR.d) The protein expression of cyclin Ds and cyclin E were measured using Western blot.3) Effect of LRP16gene on insulin secretion by MIN6a) Measurement of insulin content by RIA.b) Assay for glucose-stimulated insulin secretion by RIA.c) The mRNA was measured using Real-time Quantitative PCR:insulin, GLUT2, GCK, pancreatic transcription factors (PDX-1; MafA; NeuroD).d) The protein expression was measured using Western blot:GLUT2, GCK, pancreatic transcription factors (PDX-1; MafA; NeuroD).4) Effect of LRP16gene on AKT signaling in MIN6 a) Effect of LRP16gene on AKT signaling and ERK1/2. After cells were incubated in medium containing5mM glucose and2%FBS for18h followed by a treatment incubation of25mM glucose for30min, Western blot was used to detected AKT signaling and ERK1/2in over/down-expression of LRP16cells.b) The nuclear localization of PDX-1under high glucose conditions. After cells were incubated in medium containing5mM glucose and2%FBS for18h followed by a treatment incubation of25mM glucose for30min, immunocytochemical analysis was performed to locate PDX-1.5) Effect of LRP16gene on apoptosis induced by high glucose in MIN6a) Apoptosis detected by Tunel. After cells were synchronized for24h, cells were cultured in medium with33.3mM glucose for12h or96h. Apoptosis was detected by confocal laser scanning microscope.b) The alterations of apoptosis marker induced by high glucose were detected by Western blot. After cells were synchronized for24h, cells were cultured in medium with33.3mM glucose for12h or96h. Protein expression of apoptosis marker in cells induced by high glucose was detected by Western blot.Results:1) Construction the MIN6cell lines with over/down-expression of LRP16a) Lipidosome transfection was used to construct the cell lines with over-expression of LRP16. The stable transfected cells were selected with G418. Western blot indicated that LRP16protein in MIN6-OE was2.33times of that of MIN6-GFP cells.b) Lentivirus mediated siRNA technology was used to construct the cell lines with reduced-expression of LRP16. The stable transfected cells were selected with blasticidin. Western blot indicated that LRP16protein in MIN6-KO was reduced to56.7%of that of MIN6-GFP cells.2) Effect of LRP16gene on cell cycle and proliferation in MIN6 a) Flow cytometry analysis implied that fewer MEN6-KO cells were in S-phase compared to the control cells.b) To test the effect of LRP16gene silence on the viability of MIN6cells, a MTT assay was performed at different time points. Our results demonstrated that there was a remarkable decrease of cell viability in the MIN6-KO cells.c) The data from the qRT-PCR and Western blot indicated that mRNA expression of cyclin Ds in MIN6-KO cells were reduced compared by control.3) Effect of LRP16gene on insulin secretion by MIN6a) The insulin content and glucose stimulated insulin secretion (GSIS) in MIN6-KO cells was substantially reduced compared to that in the control cells (MIN6-GFP).b) The expression of insulin mRNA was reduced in MIN6-KO cells compared to that in the control cells (MIN6-GFP).c) The mRNA and protein expression of GLUT2was reduced in MIN6-KO cells compared to that in the control cells (MIN6-GFP).d) The mRNA and protein expression of insulin transcription factors was reduced in MIN6-KO cells compared to that in the control cells (MIN6-GFP).4) Effect of LRP16gene on AKT signaling in MIN6a) Cells were incubated in medium containing5mM glucose and2%FBS for18h followed by a treatment incubation of25mM glucose for30min. Reduced serine-473phosphorylation of Akt and phosphorylation of ERK1/2in MTN6-KO cells was observed. Meanwhile,increased serine-473phosphorylation of Akt and phosphorylation of ERK1/2in MIN6-OE cells was observed.b) Cells were incubated in medium containing5mM glucose and2%FBS for18h followed by a treatment incubation of25mM glucose for30min. Immunocytochemical analysis of the localization of PDX-1indicates upon treatment with high glucose, PDX-1translocated to the nucleus. The nuclear localization of PDX-1in MIN6-KO cells was apparently reduced compared with MIN6-GFP cells under high glucose conditions. Meanwhile, the nuclear localization of PDX-1in MIN6-OE cells was apparently increased compared with MIN6-3.1cells under high glucose conditions.5) Effect of LRP16gene on apoptosis induced by high glucose in MIN6a) Apoptosis detected by Tunel. After cells were synchronized for24h, cells were cultured in medium with33.3mM glucose for12h or96h. Apoptosis was detected by confocal laser scanning microscope. There were significantly more apoptotic cells when cells were cultured in medium with33.3mM glucose for96h. Apoptosis in MIN6-KO was28±0.9%, while in MIN6-GFP was23±0.1%. Apoptosis in MIN6-OE was19±0.5%, while in MIN6-3.1was22±0.4%.b) The alteration of apoptosis marker in MIN6-KO induced by high glucose. After cells were synchronized for24h, cells were cultured in medium with33.3mM glucose for12h or96h. Protein expression of apoptosis marker in MIN6-KO induced by high glucose was detected by Western blot. After cells were cultured in medium with33.3mM glucose for96h, there were the decreasing expression of Bcl-2and the increasing expression of Bax in all cells. When compared to the control MIN6-GFP cells, the expression of Bcl-2was decreased and the expression of Bax was increased in MIN6-KO. The Bax/Bcl-2ratio was increased.c) The alteration of apoptosis marker in MIN6-OE induced by high glucose. After cells were synchronized for24h, cells were cultured in medium with33.3mM glucose for12h or96h. Protein expression of apoptosis marker in MIN6-OE induced by high glucose was detected by Western blot. After cells were cultured in medium with33.3mM glucose for96h,there were the decreasing expression of Bcl-2and the increasing expression of Bax in all cells. When compared to the control MIN6-3.1cell, the expression of Bcl-2 was increased and the expression of Bax was decreased in MIN6-OE. The Bax/Bcl-2ratio was decreased.Conclusions:1) We successfully constructed LRP16over-expression, down-expression and control cell lines, and has provided a stable experimental cell model for the follow-up study.2) The LRP16gene plays a key role in regulation of pancreatic β cell proliferation. This is probably owing to the reduced mRNA and protein level of cyclin Ds.3) The LRP16gene can increase insulin synthesis by regulating insulin mRNA and insulin transcription factor and increase glucose stimulated insulin secretion by regulating GLUT2expression.4) The LRP16gene can regulate PDX-1nucleus translocation by regulated AKT signaling.5) LRP16can reduce the apoptosis of pancreatic beta cell induced by high glucose.In a word, the LRP16gene plays a key role not only in proliferation, apoptosis but also in insulin synthesis and glucose stimulated insulin secretion in pancreatic β cells. It will be a new therapy method and drug target to further understand organize particularly expression of LRP16and activation of cell signaling participated by LRP16.