Alternative Splicing Variants Of SIRT1 Are Involved In The Differentiation Of Rat Neural Stem Cells

Objective:Neural stem cells are distributed in the nervous system, and has the ability to self-renewal and multi-directional differentiation potential. Because of its low immunogenicity and has a good migration performance, it has been widely used in regeneration after nerve injury and repair. Neural stem cells can differentiate into neurons, astrocytes and oligodendrocytes, but its differentiation is affected by many factors, including the extracellular environment, nutrients, signal pathways,transcription factors and cytokines. In nerve injury and neurodegenerative diseases, neural stem cells are often divided into glial cells in the local microenvironment, how to control the differentiation of neural stem cells in a certain environment for the expected end cell is a key problem to be solved.Silent information regulator 2 homolog 1(SIRT1) is a kind of NAD+ dependent deacetylate enzymes, through deacetylate histone and a variety of nonhistones regulate cell aging and cell differentiation, apoptosis, cell glucolipid metabolism and other important physiological processes. Studies have found that SIRT1 plays a regulatory role in the process of neural stem cells differentiation, but these studies of the regulation direction have mixed results. For example, research shows that SIRT1 level decreased significantly after induction of neural stem cells, overexpression of SIRT1 decreased the differentiation of neural stem cells into neurons; But there are also studies have shown that inhabitation of SIRT1 make the differentiation of neural stem cells improved. Therefore, the researches show that SIRT1 has a regulatory role in the differentiation of neural stem cells, but whether it is to promote the differentiation of neural stem cell factor or inhibiting factor, there is no unified conclusion.Alternative Splicing(AS) refers to the m RNA precursors(Pre-m RNA) by selecting the combination of different Splicing sites, cut off different introns or exons, combine exons in different way to produce different mature m RNA molecule. Different m RNA alternative splicing isomer can further translation to different or mutual antagonism protein, or lead to different phenotypes due to the different expression levels in the same cell. Different alternative splicing variants in the specific cell function may be play roles independently or work together. In recent years, studies have shown that SIRT1 exist alternative splicing, human SIRT1 have discovered two alternative splicing variants, lack exon 8 and lack exon 2-9,named SIRT1-△Exon8 and SIRT1-△Exon2/9 respectively, the two different alternative splicing variants each play specific functions, and has the obvious difference with SIRT1- FL in cell functions.SIRT1 is widely distributed in the nervous system of rats, and whether alternative splicing variants of SIRT1 also exist in its nervous system is not clear; If so, what is the function of the alternative splicing variants and whether it is involved in the differentiation of neural stem cells remain to be unknown. We hypothesized that SIRT1 may exist in the rat nervous system of alternative splicing variants, perhaps the existence of SIRT1 alternative splicing was overlooked before, led to the contradictory conclusions, that means, the possible presence of SIRT1 alternative splicing may provide a reasonable explanation to the before conflicting views about the role of SIRT1.The current studies of SIRT1 alternative splicing most concentrated in anthropogenic and mice cells. Rat is the second commonly used experiment mammals, with SIRT1 is widely distributed in the nervous system of rats, to explore whether SIRT1 exist alternative splicing in rat will lay the foundation of subsequent research on alternative splicing variants in physiological and pathological functions. SIRT1 gene in mammalian is highly conservative, but due to the differences between species, rat and human SIRT1 gene is not the same. Through genetic comparisons, we speculated that the rat SIRT1 gene is likely to lack exon 7, forms the SIRT1-△Exon7.This research first observed the rat nervous system exists SIRT1-△Exon7, second we compared the full length of the SIRT1(SIRT1-FL) and alternative splicing variants of SIRT1(SIRT1-△Exon7) in hippocampus(the source of neural stem cells) and cortex(enrichment area of mature nerve cells), provide a preliminary exploration for whether SIRT1 is involved in the differentiation of neural stem cells; And then by comparing the SIRT1-FL and SIRT1-△Exon7 in the expression of neural stem cells before and after differentiation, clear the role of SIRT1 in neural stem cells differentiation. Our study will provide new ideas for the exploration of SIRT1 regulate the differentiation of neural stem cells research. Methods:(1) PCR detection of SIRT1-FL and SIRT1-△Exon7: take the newborn SD rat brain tissue, separate hippocampus and cortex. Extract total RNA, ordinary reverse transcription PCR detect SIRT1-FL and alternative splicing variants SIRT1-△Exon7 expression. Then detect the expression of SIRT1-FL and SIRT1-△Exon7 in hippocampus and cortex respectively by RT-PCR.(2) Neural stem cells culture, identification and differentiation:Adopt the serum-free cell culture method,the newborn rat hippocampal neural stem cells proliferate to suspended nerve ball, identify neural stem cells with immunofluorescence technique and detect its marker protein expression of Nestin. With DMEM/F-12 medium containing 10% FBS induct neural stem cells differentiation, then with neuron specificity of microtubule associated protein β-Tubulin antibodies and glial cell specificity of collagen fibre acidic protein(glial fibrillary acidic protein, GFAP) antibodies immunofluorescence staining respectively, appraisal the differentiation of neurons and glial cells.(3) RT-PCR detect SIRT1-FL and SIRT1-△Exon7:To detect the proliferated nerve and induced differentiated nerve ball SIRT1- FL and SIRT1-△Exon7 m RNA levels with RT-PCR method.(4)SIRT1 enzyme activity detection: Using SIRT1 activity detection kits check SIRT1 acetylation enzyme activity changes before and after neural ball differentiation. Results:(1) SIRT1-△Exon7 and SIRT1-FL m RNA were expressed in both hippocampus and cortex of rats;(2)The hippocampus SIRT1-△Exon7 m RNA level was 62.02% lower than the SIRT1-FL(P < 0.05), the cortex SIRT1-△Exon7 m RNA level was 83.33% lower than the SIRT1-FL(P < 0.05); cortex SIRT1-FL m RNA level was 5.81% higher than the hippocampus content(P < 0.05), whereas the cortex SIRT1-△Exon7 is 56.68% lower than the hippocampus(P < 0.05);(3)Before the differentiation of neural stem cells SIRT1-△Exon7 m RNA level was 44.12% lower than the SIRT1-FL(P < 0.05), after the differentiation SIRT1-△Exon7 m RNA level was 74.62% lower than SIRT1-FL(P < 0.05); SIRT1-FL m RNA content after differentiation increased by 30.21% versus Neural stem cells, SIRT1-△Exon7 m RNA content after differentiation reduction 41.02% versus Neural stem cells(P < 0.05); After Differentiation the total SIRT1 acetylation enzyme activity increased 30.08% versus before differentiation(P < 0.05). Conclusion:(1)We discovered the existence of SIRT1-△Exon7 in the nervous system of rats;(2)The hippocampus and cortex expresses lower SIRT1-△Exon7 than SIRT1-FL, SIRT1-△Exon7 in cortex is lower than hippocampus, but SIRT1-FL in cortex is more than hippocampus. The two brain regions respectively represent the enrichment area of neural stem cells and differentiation cells. It denotes that SIRT1-FL may promote the differentiation of neural stem cells, while the SIRT1-△Exon7 plays a opposite function in neural stem cells.(3)The expression of SIRT1-△Exon7 is lower than the expression of SIRT1-FL before and after differentiation of neural stem cells. The expression of SIRT1-△Exon7 is lower after differentiation of neural stem cells, but the expression of SIRT1-FL is higher before differentiation of neural stem cells, this finding accords with the text result of the hippocampus and cortex. The increase extent of deacetylase enzyme activity is equal to the SIRT1-FL after differentiation of neural stem cells, It implies that SIRT1-FL promotes differentiation of neural stem cells by enhance the activity of the deacetylase enzyme, the activity of SIRT1 decreased after lost Exon7, so SIRT1-△Exon7 has little impact on activity of SIRT1. Above all, the inhibition SIRT1-△Exon7 to differentiation of neural stem cells has little relation with SIRT1-FL.