水牛PPARG来源环状RNA的鉴定及表达谱分析

doi:10.16431/j.cnki.1671-7236.2019.12.001

摘要/Abstract

摘要： 为鉴定前期转录组测序分析结果的可靠性，并从中挖掘水牛脂肪沉积潜在环状RNA（circular RNA，circRNA），本研究对10个来源于过氧化物酶体增殖物激活受体γ（PPARG）基因的circRNA进行鉴定，根据测序获得的候选circRNA序列信息，设计对向引物进行半定量检测和普通测序以获得真实存在的circRNA，采用实时荧光定量PCR分析其在水牛不同组织和不同部位脂肪组织中的表达谱，半定量检测其在不同物种间的保守性。结果显示，10个候选circRNA中，有5个circRNA可以检测到，测序验证序列与转录组测序分析获得的序列一致，其中2个circRNA可设计出特异性的定量引物，这2个circRNA主要在脂肪组织中表达，且在不同部位脂肪组织中的表达量存在差异；另外3个circRNA在黄牛、牦牛、猪和小鼠的脂肪组织中均有表达，序列长度一致，序列相似度高。以上研究结果提示，转录组测序分析获得的结果可靠，其中有2个circRNA主要在脂肪组织中表达，且物种间保守，可作为研究水牛脂肪沉积的候选基因。

关键词: 水牛; PPARG基因; 环状RNA; 鉴定; 表达谱

Abstract: To evaluate the results of transcriptome sequencing analysis and explore circular RNA(circRNA) with potential function in fat deposition,10 circRNA derived from the peroxisome proliferator activated receptor γ (PPARG) were identified.Based on the obtained sequence,divergent primers were designed for RT-PCR and normal sequencing to obtain real circRNA.Real-time PCR was used to analyze the expression profile of circRNA in different tissues and adipose tissue of different parts in buffalo,RT-PCR was used to identify their expression in different species.In total,5 of the 10 PPARG-derived circRNA were identified,and their sequences were consist with those in transcriptome sequencing analysis.Specific primers for Real-time PCR were available for only 2 of the 5 circRNA,and the 2 circRNA were mainly expressed in adipose tissue and with different expression levels among adipose tissue depots.Further,3 circRNA were detectable in adipose tissues of cattle,yak,pig and mouse.Their length were consist with those in buffalo,and they were with high similarity in sequence among species.These results indicated that the results of transcriptome sequencing analysis were reliable,2 circRNA were mainly expressed in adipose tissue and conserved among species,which could be candidate circRNA for fat deposition of buffalo.

Key words: buffalo; PPARG gene; circular RNA; identification; expression profile

中图分类号:

Q786

冯雪, 洪慧颖, 孙晓菲, 亐开兴, 黄必志, 魏雪锋, 马云, 黄洁萍. 水牛PPARG来源环状RNA的鉴定及表达谱分析[J]. 中国畜牧兽医, 2019, 46(12): 3457-3465.

FENG Xue, HONG Huiying, SUN Xiaofei, QU Kaixing, HUANG Bizhi, WEI Xuefeng, MA Yun, HUANG Jieping. Identification and Expression Profile Analysis of PPARG-derived Circular RNA in Buffalo[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(12): 3457-3465.

参考文献

[1] YANG B,LIANG X W,JING Q,et al.Brief introduction to the development of Chinese dairy buffalo industry[J].Buffalo Bulletin,2013,32(3):111-120.
[2] IBRAHIM H M A.Acceleration of curing period of pastrami manufactured from buffalo meat:Ⅱ-fatty acids,amino acids,nutritional value and sensory evaluation[J].Grasas Y Aceites,2001,52(2):115-122.
[3] LOWE C E,O'RAHILLY S,ROCHFORD J J.Adipogensis at a glance[J].Journal of Cell Science,2011,124:2681-2686.
[4] 王鹤洁,秦本源,王媛媛,等.DNA甲基化与去甲基化调控脂肪沉积的研究进展[J].中国畜牧兽医,2019,46(2):519-525. WANG H J,QIN B Y,WANG Y Y,et al.Research advances on DNA methylation and demethylation to regulate adipose deposition[J].China Animal Husbandry & Veterinary Medicine,2019,46(2):519-525.(in Chinese)
[5] ZHAO R,LI F Q,TIAN L L,et al.Comprehensive analysis of the whole coding and non-coding RNA transcriptome expression profiles and construction of the circRNA-lncRNA co-regulated ceRNA network in laryngeal squamous cell carcinoma[J].Functional & Integrative Genomics,2019,19(1):109-121.
[6] 郑圆媛,惠太宇,郭丹,等.非编码RNA调节羊绒细度候选基因的研究进展[J].中国畜牧兽医,2018,45(11):3176-3184. ZHENG Y Y,HUI T Y,GUO D,et al.Research progress on cashmere fineness candidate genes regulating by non-coding RNA[J].China Animal Husbandry & Veterinary Medicine,2018,45(11):3176-3184.(in Chinese)
[7] SALZMAN J,CHEN R E,OLSEN M N,et al.Cell-type specific features of circular RNA expression[J].PLoS Genetics,2013,9(9):e1003777.
[8] MEMCZAK S,JENS M,ELEFSINIOTI A,et al.Circular RNAs are a large class of animal RNAs with regulatory potency[J].Nature,2013,495(7441):333-338.
[9] JECK W R,SORRENTINO J A,WANG K,et al.Circular RNAs are abundant,conserved,and associated with ALU repeats[J].RNA,2012,19(2):141-157.
[10] WANG P L,BAO Y,YEE M C,et al.Circular RNA is expressed across the eukaryotic tree of life[J].PLoS One,2014,9(3):e90859.
[11] ZHANG Y,ZHANG X O,CHEN T,et al.Circular intronic long noncoding RNAs[J].Molecular Cell,2013,51(6):792-806.
[12] LI F,ZHANG L,LI W,et al.Circular RNA ITCH has inhibitory effect on ESCC by suppressing the Wnt/β-catenin pathway[J].Oncotarget,2015,6(8):6001-6013.
[13] LI Z,HUANG C,BAO C,et al.Exon-intron circular RNAs regulate transcription in the nucleus[J].Nature Structural & Molecular Biology,2015,22(3):256-264.
[14] HUANG J,ZHAO J,ZHENG Q,et al.Characterization of circular RNAs in Chinese buffalo (Bubalus bubalis) adipose tissue:A focus on circular RNAs involved in fat deposition[J].Animals,2019,9(7):E403.
[15] IPARRAGUIRRE L,MUÑOZ-CULLA M,PRADA-LUENGO I,et al.Circular RNA profiling reveals that circular RNAs from ANXA2 can be used as new biomarkers for multiple sclerosis[J].Human Molecular Genetics,2017,26(18):3564-3572.
[16] LIU Y C,HONG H C,YANG C D,et al.Ouroboros resembling competitive endogenous loop (ORCEL) in circular RNAs revealed through transcriptome sequencing dataset analysis[J].BMC Genomics,2018,19(S2):171.
[17] ZHENG J,LIU X B,XUE Y X,et al.TTBK2 circular RNA promotes glioma malignancy by regulating miR-217/HNF1β/Derlin-1 pathway[J].Journal of Hematology & Oncology,2017,10(1):52.
[18] WEI X,LI H,YANG J,et al.Circular RNA profiling reveals an abundant circLMO7 that regulates myoblasts differentiation and survival by sponging miR-378a-3p[J].Cell Death & Disease,2017,8(10):e3153.
[19] QU S,YANG X,LI X,et al.Circular RNA:A new star of noncoding RNAs[J].Cancer Letter,2015,365(2):141-148.
[20] ASHWAL-FLUSS R,MEYER M,PAMUDURTI N R,et al.circRNA biogenesis competes with pre-mRNA splicing[J].Molecular Cell,2014,56(1):55-66.
[21] KRUGLIKOV I L,SCHERER P E.Dermal adipocytes:From irrelevance to metabolic targets?[J].Trends in Endocrin Metabolism,2016,27(1):1-10.
[22] ATZMON G,YANG X M,MUZUMDAR R,et al.Differential gene expression between visceral and subcutaneous fat depots[J].Hormone and Metabolic Research,2002,34(11/12):622-628.
[23] VOHL M,SLADEK R,ROBITAILLE J,et al.A survey of genes differentially expressed in subcutaneous and visceral adipose tissue in men[J].Obesity,2012,12(8):1217-1222.
[24] MISRA A,GARG A,ABATE N,et al.Relationship of anterior and posterior subcutaneous abdominal fat to insulin sensitivity in nondiabetic men[J].Obesity Research,1997,5(2):93-99.
[25] SNIJDER M B,ZIMMET PZ,VISSER M,et al.Independent and opposite associations of waist and hip circumferences with diabetes,hypertension and dyslipidemia:The AusDiab study[J].International Journal of Obesity and Related Metabolic Disorders,2004,28(3):402-409.
[26] WAJCHENBERG B L,GIANNELLA-NETO D,DA SILVA M E,et al.Depot-specifc hormonal characteristics of subcutaneous and visceral adipose tissue and their relation to the metabolic syndrome[J].Hormone and Metabolic Research,2002,34(11/12):616-621.
[27] BOYKO E J,FUJIMOTO W Y,LEONETTI D L,et al.Visceral adiposity and risk of type 2 diabetes:A prospective study among Japanese Americans[J].Diabetes Care,2000,23(4):465-471.
[28] HAYASHI T,BOYKO E J,LEONETTI D L,et al.Visceral adiposity and the risk of impaired glucose tolerance:A prospective study among Japanese Americans[J].Diabetes Care,2003,26(3):650-655.
[29] KUK J L,KATZMARZYK P T,NICHAMAN M Z,et al.Visceral fat is an independent predictor of all-cause mortality in men[J].Obesity,2006,14(2):336-341.
[30] 沈林汉,李宏卫,詹甜甜,等.miRNA-448在肿瘤中作用的研究进展[J].口腔医学,2015,35(10):889-892. SHEN L H,LI H W,ZHAN T T,et al.The research progress of functions of microRNA-448 in the tumor[J].Stomatology,2015,35(10):889-892.(in Chinese)
[31] 周凤燕,杨青,朱熙春,等.环状RNA的分子特征、作用机制及生物学功能[J].农业生物技术学报,2017,25(3):485-501. ZHOU F Y,YANG Q,ZHU X C,et al.Molecular feature,action mechanism and biology function of circular RNA[J].Journal of Agricultural Biotechnology,2017,25(3):485-501.(in Chinese)