Abstract
Mammalian Sirtuin 6 (SIRT6) plays an important role in the transcriptional silencing of silent mating-type loci, telomeres, and rDNA. However, the roles of porcine SIRT6 in cell proliferation are poorly understood, and a better knowledge of these will help improve our understanding of the biological mechanisms of cell growth and development. In this study, a novel variant of porcine SIRT6 (SIRT6 V2) identified by reverse transcription-polymerase chain reaction and BLAST analysis showed a 124-bp deletion compared to wild-type SIRT6 mRNA (SIRT6 V1). Two recombinant plasmids overexpressing SIRT6 V1 and SIRT6 V2 were produced and their roles in the proliferation of porcine fetal fibroblasts (PFFs) were compared. Cells transfected with SIRT6 V1 proliferated significantly faster than those transfected with SIRT6 V2 (P<0.01), though both proliferated significantly faster than cells expressing an empty vector (P<0.01). These results might be caused by altered proportions of α helices and β sheets in the SIRT6 V2 structure relative to SIRT6 V1. These results indicate that overexpression of SIRT6 V1/V2 was involved in promoting PFF proliferation. Deletion of a 124-bp sequence attenuated the effects of SIRT6 on cell proliferation, possibly as a result of changes in the proportions of α helices and β sheets in the protein secondary structure.
Highlights
Yeast silent information regulator 2 (SIR2) possesses NAD+-dependent deacetylase and ADP-ribosyltransferase activities, and plays essential roles in transcriptional silencing of silent mating-type loci, telomeres, and rDNA
We identified a novel porcine Sirtuin 6 (SIRT6) variant and constructed plasmids to express the wild-type and novel SIRT6 variants, allowing the effects of both variants on porcine fetal fibroblast (PFF) proliferation to be compared
Fragments of 1490 and 1366 bp were subcloned into pcDNA3.1 (−) to produce pcDNA3.1(−)-SIRT6 V1 and pcDNA3.1(−)-SIRT6 V2
Summary
All animal procedures were approved by the Shanghai Care and Use Committee, China. Total RNA was isolated from each tissue using Trizol reagent (Invitrogen, USA). CDNA was synthesized for reverse transcription-PCR from less than 5 μg of each total RNA, according to the manufacturer’s protocol (TaKaRa, Japan). CDNA samples (5 L) of the same concentration were pooled together to use as a template. Porcine SIRT6 cDNA containing a complete or partial open reading frame (ORF) was cloned by reverse transcription-PCR using the primer S1 (Table 1). CDNAs containing complete or partial ORFs were named SIRT6 variant 1 (SIRT6 V1, wild-type SIRT6 mRNA) and SIRT6 variant 2 (SIRT6 V2), respectively. All PCR products were purified using a MinElute Gel Extraction Kit (Qiagen, USA) and cloned into the pMD18-T vector (TaKaRa) for sequencing
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