Study of Resistin gene expression in peripheral blood mononuclear cell and its gene polymorphism in a small range population

Study of Resistin gene expression in peripheral blood mononuclear cell and its gene polymorphism in a small range population 1 first-author 1 2 3 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:gbbf2000@sina.com †E-mail:gbbf2000@sina.com gbbf2000@sina.com Department of Very Important People, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Department of Very Important People, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Institute of Infectious Disease, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Institute of Infectious Disease, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Department of Emergency, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Department of Emergency, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Objective: 2 To observe the expression of Resistin mRNA in peripheral blood mononuclear cells and its gene polymorphism in coding region in a small range population in Zhejiang Province of China. Methods: 2 Eighty-three cases of type 2 diabetes mellitus and 53 healthy people were included. The expression of Resistin mRNA in peripheral blood mononuclear cells was detected by RT-PCR and semi-quantitative PCR assay. The sequencing work was done in Resistin cDNA and gene polymorphism was analyzed. Results: 2 At the same condition, in 83 diabetes patients, Resistin mRNA was detected in 23 cases (11 males and 12 females). There was no Resistin mRNA expression in 53 healthy people. The ratio of PCR products between Resistin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was from 0.564 to 1.238, averaging 0.804±0.436. The sequence of Resistin cDNA is almost identical with each other and with that in GenBank with no single nucleotide polymorphism being found. Conclusion: 2 Resistin mRNA is expressed in human peripheral blood mononuclear cells in some type 2 diabetes mellitus, but its expression is at a low level. Among the experiment population we did not find polymorphism phenomenon in Resistin coding region. The different individual’s Resistin coding region is highly coincident. Resistin mRNA Peripheral blood mononuclear cells Coding region Gene polymorphism INTRODUCTION 1 Recently discovered as an endocrine organ, adipose tissue can secrete many cytokines such as Resistin, adiponectin and free fatty acids, and receives much attention in the study of insulin resistance and type 2 diabetes mellitus (T2DM). As a peptide hormone secreted by adipocyte, Resistin is considered to be the linkage between obesity and insulin resistance (Steppan et al., 2001 2001 ). Most studies on Resistin investigate adipose tissue. In this experiment we detected the expression of Resistin mRNA in human peripheral blood mononuclear cells (PBMC) in a small range population in Zhejiang Province of China and analysed the polymorphism of the Resistin gene. MATERIALS AND METHODS 1 Sample selection 2 Serum samples were taken with permission from 83 cases of T2DM patients [48 males and 35 females, mean age (54.1±16.3) years] who fulfilled the World Health Organization criteria for T2DM, and 53 healthy people [33 males and 20 females, mean age (58.6±9.7) years] from June, 2005 to Dec., 2005. Detection of Resistin mRNA in human PBMC with semi-quantitative RT-PCR analysis 2 Total RNA was extracted with Trizol reagent (BD Bioscience) from human PBMC isolated from 3 ml peripheral blood using lymphocyte separating reagent (Sigma Co., USA), and RNA was treated with RNase-free Dnase. First-strand cDNA was generated from 10 µl of total RNA in a 20 µl volume containing 10×dNTP, 50 mmol/L oligo (dT) primer, M-MLV (200 μl −1 ) etc. Oligo (dT) primer was 5′-TTTTTTTTTTTTTTTTTT-3′, and the reaction duration was for 1 h in a 42 °C water bath. First-strand cDNA from reverse transcription was used for conventional PCR. Conventional PCR was carried out in mixtures containing 10 μl of template DNA, 10×dNTP, 50 mmol/L primers, 5 U/μl Taq DNA polymerase, 2 mmol/L MgCl 2 etc. Denaturation was at 94 °C for 1 min, annealing at 55 °C for 45 s, polymerization at 72 °C for 30 s with 30 cycles, and ending with a single 5-min extension step at 72 °C. Primers were 5′-TCTAGCAAGACCCTGTGC-3′ and 5′-CAGGTTTATTTCCAGCTCC-3′. To quantify mRNAs, we employed the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal standard and determinated the relative levels of specific mRNAs by comparing the ratio of PCR products. The PCR primers for GAPDH were 5′-TGGGGAAGGTGAAGGTCGGA-3′ and 5′-GGGATCTCGCTGCTCGAAGA-3′. Quantitative analyses of mRNA levels were performed using GelScan V. 5.02 (BioSciTec GmbH, Giessen, Germany) image analysing system. Sequencing work 2 Resistin cDNA sequence was assayed by the Sanger dideoxy-mediated chain termination method using Automated DNA Sequencers ABI3700. Statistical analysis 2 All data were expressed in as mean± SD with software SPSS11.0 being used to analyse the data. t test and Pearson correlation analysis were adopted. RESULTS 1 There were 23 T2DM patients (11 males and 12 females) who showed expression of Resistin mRNA among the 83 cases (Fig. 1 1 ). No Resistin mRNA was detected in the 53 healthy people. Semi-quantitative PCR showed that the ratio between Resistin gene and GAPDH (Fig. 2 2 ) was from 0.564 to 1.238 and that the average was 0.804±0.436. Sequencing work was done in 23 positive cases. No single nucleotide polymorphism (SNP) was found after blastn. The detected Resistin gene order was almost identical with that in GenBank, with coincidence rate being as high as 98%. M: Maker; 1~7: Resistin gene; 8: Negative control; 9: Blank control M: Maker; 1~6: GAPDH; 7: Negative control; 8: Blank control DISCUSSION 1 Human Resistin gene is located in No. 19 chromosome. Resistin mRNA has 476 basic radicals with their coding region having 326 basic radicals which code 108 amino acids (Steppan et al., 2001 2001 ). Human Resistin gene was mainly expressed in various kinds of adipose tissue (Nogueiras et al., 2003 2003 ; Minn et al., 2003 2003 ), with its expression being regulated by many kinds of factors. There are reports that androgen (Ling et al., 2001 2001 ) and growth hormone (Delhanty et al., 2002 2002 ) can stimulate the expression of Resistin mRNA, while free fatty acid (Juan et al., 2001 2001 ), rosiglitazone (Li and Lazar, 2002 2002 ) and tumour necrosis factor (Fasshauer et al., 2001 2001 ; Yang et al., 2005 2005 ) can inhibit its expression. In 3T3-L1 adipocytes, Chung et al.( 2006 2006 ) found that PPARgamma activation represses the expression of the Resistin gene by modulating Sp1 activity, Sp1 binding site of the Resistin promoter (−122/−114 bp) was necessary and the level of O-glycosylation of Sp1 was decreased in this repression. We detected 136 people’s expression of Resistin mRNA from PBMC in Zhejiang Province in China. Among the 136 people, there were 83 T2DM patients and 53 healthy people, 81 males and 55 females. Only 23 T2DM patients had expression of Resistin mRNA. The result of semi-quantitative PCR showed that the level of expression differed greatly between patients. Further, the expression of Resistin mRNA was at a low level among experimental people and most people had no expression. Those who had Resistin mRNA expression all were T2DM patients, and this might demonstrate that Resistin has a close relation with T2DM. Moreover, we also found that females had a higher ratio of Resistin mRNA expression than males (22%/13%). As a type of cytokine secreted by adipose cells, Resistin is distributed mainly in various kinds of adipose tissue. But it also can be found in pancreatic islets (Minn et al., 2003 2003 ), trophoblastic cells in placental tissue (Yura et al., 2003 2003 ), human macrophages (Patel et al., 2003 2003 ) and leukemia cells (Yang et al., 2003 2003 ). In the most recent study, Wiesner et al.( 2006 2006 ) put forward that brain injury, or an inflammatory stimulus, regulates the central expression of Resistin gene normally considered to be adipose tissue-specific. Kaser et al.( 2003 2003 ) found that human PBMC had expression of Resistin mRNA, and he even suggested human PBMC may be a major source of Resistin. Although we detected Resistin mRNA expression in human PBMC, the expression in our experiment is low. Some factors, such as the method adopted, difference of region and race, drugs that perhaps can influence Resistin, may contribute to this finding. Studies of Resistin expression in other regions and races are needed to learn more about Resistin. Regarding Resistin gene polymorphism, our study found that in Japanese people, some SNPs such as −638G > A, −420C > G, −358G > A, −167C > T, +157C > T and +299G > A were detected in flanking region (Osawa et al., 2004 2004 ), promoter region (Azuma et al., 2004 2004 ) and introns (Osawa et al., 2002 2002 ) of the Resistin gene. This SNP can increase RETN promoter activity and influence Resistin gene transcriptional activity, and thereby influence the expression of Resistin gene and protein (Osawa et al., 2004 2004 ; Azuma et al., 2004 2004 ). Korea scholar Cho et al.( 2004 2004 ) screened −537A > C and −420C > G in the promoter region of Resistin gene, and found that polymorphisms are major determinants of plasma Resistin concentrations in human. In Taiwan Province of China, Tan et al.( 2003 2003 ) studied 1102 Chinese type 2 diabetes patients and 743 subjects without diabetes. The Resistin 3′-untranslated region (UTR) +62G→A polymorphism was determined by PCR. They found that type 2 diabetes subjects had a lower frequency of Resistin gene 3′-UTR+62A allele (GG:GA/AA, 83.5%:16.5%) than the controls (GG:GA/AA, 75.1%:24.9%). These findings suggest that Resistin may play a role in the pathogenesis of type 2 diabetes and insulin resistance-related hypertension. In non-Asian race studies, Gouni-Berthold et al.( 2005 2005 ) repeated Tan’s experiment. He found that in a German Caucasian population the +62G→A polymorphism of the Resistin gene is associated with hypertension but not with T2DM. Our experiment selected some people from Zhejiang Province of China and sequenced the coding region of Resistin gene. We found that the sequence of coding region was highly conservative although polymorphism might exist in other region such as promoter region, introns and 3′-UTR. This point is similar with that of Osawa et al.( 2002 2002 ). In his experiment, sequences for 24 Japanese type 2 diabetic patients were initially analyzed using PCR direct sequencing. Three SNPs (−167C > T, +157C > T and +299G > A) were found in the introns, but none were present in the coding regions. In another study, Sentinelli et al.( 2002 2002 ) analyzed the coding sequence of the three exons of the Resistin gene, together with its 5′ regulatory region and 3′-UTR, by single-strand conformation polymorphism (SSCP) in 58 type 2 diabetic subjects, 59 obese subjects, and 60 normal subjects. Only one sequence variant (G1326C) was detected in the 3′-UTR of exon 3. As a promising candidate gene for type 2 diabetes, Resistin is not fully recognized by us. A lot of work remains for us to understand its physiological functions with more large scale experiments being needed to confirm the conclusion we put forward. References K Azuma , S Oguchi , Y Matsubara , T Mamizuka , M Murata , H Kikuchi , K Watanabe , F Katsukawa , H Yamazaki , A Shimada , 等 . Novel resistin promoter polymorphisms: association with serum resistin level in Japanese obese individuals . Horm Metab Res , 2004 . 36 ( 8 ): 564 - 570 . 15326567 DOI: 10.1055/s-2004-825762 http://doi.org/10.1055/s-2004-825762 . Novel resistin promoter polymorphisms: association with serum resistin level in Japanese obese individuals YM Cho , BS Youn , SS Chung , KW Kim , HK Lee , KY Yu , HJ Park , HD Shin , KS Park . Common genetic polymorphisms in the promoter of resistin gene are major determinants of plasma resistin concentrations in humans . Diabetologia , 2004 . 47 ( 3 ): 559 - 565 . 14740159 DOI: 10.1007/s00125-003-1319-x http://doi.org/10.1007/s00125-003-1319-x . Common genetic polymorphisms in the promoter of resistin gene are major determinants of plasma resistin concentrations in humans SS Chung , HH Choi , YM Cho , HK Lee , KS Park . Sp1 mediates repression of the resistin gene by PPARgamma agonists in 3T3-L1 adipocytes . Biochem Biophys Res Commun , 2006 . 348 ( 1 ): 253 - 258 . 16876120 DOI: 10.1016/j.bbrc.2006.07.048 http://doi.org/10.1016/j.bbrc.2006.07.048 . Sp1 mediates repression of the resistin gene by PPARgamma agonists in 3T3-L1 adipocytes PJ Delhanty , D Mesotten , F McDougall , RC Baxter . Growth hormone rapidly induces resistin gene expression in white adipose tissue of spontaneous dwarf (SDR) rats . Endocrinology , 2002 . 143 ( 6 ): 2445 - 2448 . 12021211 DOI: 10.1210/en.143.6.2445 http://doi.org/10.1210/en.143.6.2445 . Growth hormone rapidly induces resistin gene expression in white adipose tissue of spontaneous dwarf (SDR) rats M Fasshauer , J Klein , S Nermann , M Eszlinger , R Paschke . Tumor necrosis factor alpha is a negative regulator of resistin gene expression and secretion in 3T3-L1 adipocytes . Biochem Biophys Res Commun , 2001 . 288 ( 4 ): 1027 - 1031 . 11689013 DOI: 10.1006/bbrc.2001.5874 http://doi.org/10.1006/bbrc.2001.5874 . Tumor necrosis factor alpha is a negative regulator of resistin gene expression and secretion in 3T3-L1 adipocytes I Gouni-Berthold , E Giannakidou , M Faust , J Kratzsch , HK Berthold , W Krone . Resistin gene 3′-untranslated region +62G→A polymorphism is associated with hypertension but not diabetes mellitus type 2 in a German population . J Intern Med , 2005 . 258 ( 6 ): 518 - 526 . 16313475 DOI: 10.1111/j.1365-2796.2005.01566.x http://doi.org/10.1111/j.1365-2796.2005.01566.x . Resistin gene 3′-untranslated region +62G→A polymorphism is associated with hypertension but not diabetes mellitus type 2 in a German population CC Juan , LC Au , VS Fang , SF Kang , YH Ko , SF Kuo , YP Hsu , CF Kwok , LT Ho . Suppressed gene expression of adipocyte resistin in an insulin-resistant rat model probably by elevated free fatty acids . Biochem Biophys Res Commun , 2001 . 289 ( 5 ): 1328 - 1333 . 11741341 DOI: 10.1006/bbrc.2001.6132 http://doi.org/10.1006/bbrc.2001.6132 . Suppressed gene expression of adipocyte resistin in an insulin-resistant rat model probably by elevated free fatty acids S Kaser , A Kaser , A Sandhofer , CF Ebenbichler , H Tilg , JR Patsch . Resistin messenger-RNA expression is increased by proinflammatory cytokines in vitro . Biochem Biophys Res Commun , 2003 . 309 ( 2 ): 286 - 290 . 12951047 DOI: 10.1016/j.bbrc.2003.07.003 http://doi.org/10.1016/j.bbrc.2003.07.003 . Resistin messenger-RNA expression is increased by proinflammatory cytokines in vitro Y Li , MA Lazar . Differential gene regulation by PPARgamma agonist and constitutively active PPARgamma2 . Mol Endocrinol , 2002 . 16 ( 5 ): 1040 - 1048 . 11981038 DOI: 10.1210/me.16.5.1040 http://doi.org/10.1210/me.16.5.1040 . Differential gene regulation by PPARgamma agonist and constitutively active PPARgamma2 C Ling , J Kindblom , H Wennbo , H Billig . Increased resistin expression in the adipose tissue of male prolactin transgenic mice and in male mice with elevated androgen levels . FEBS Lett , 2001 . 507 ( 2 ): 147 - 150 . 11684088 DOI: 10.1016/S0014-5793(01)02961-1 http://doi.org/10.1016/S0014-5793(01)02961-1 . Increased resistin expression in the adipose tissue of male prolactin transgenic mice and in male mice with elevated androgen levels AH Minn , NB Patterson , S Pack , SC Hoffmann , O Gavrilova , C Vinson , DM Harlan , A Shalev . Resistin is expressed in pancreatic islets . Biochem Biophys Res Commun , 2003 . 310 ( 2 ): 641 - 645 . 14521959 DOI: 10.1016/j.bbrc.2003.09.061 http://doi.org/10.1016/j.bbrc.2003.09.061 . Resistin is expressed in pancreatic islets R Nogueiras , R Gallego , O Gualillo , JE Caminos , T Garcia-Caballero , FF Casanueva , C Dieguez . Resistin is expressed in different rat tissues and is regulated in a tissue-and gender-specific manner . FEBS Lett , 2003 . 548 ( 1-3 ): 21 - 27 . 12885401 DOI: 10.1016/S0014-5793(03)00708-7 http://doi.org/10.1016/S0014-5793(03)00708-7 . Resistin is expressed in different rat tissues and is regulated in a tissue-and gender-specific manner H Osawa , H Onuma , A Murakami , M Ochi , T Nishimiya , K Kato , I Shimizu , Y Fujii , J Ohashi , H Makino . Systematic search for single nucleotide polymorphisms in the resistin gene: the absence of evidence for the association of three identified single nucleotide polymorphisms with Japanese type 2 diabetes . Diabetes , 2002 . 51 ( 3 ): 863 - 866 . 11872693 . Systematic search for single nucleotide polymorphisms in the resistin gene: the absence of evidence for the association of three identified single nucleotide polymorphisms with Japanese type 2 diabetes H Osawa , K Yamada , H Onuma , A Murakami , M Ochi , H Kawata , T Nishimiya , T Niiya , I Shimizu , W Nishida , 等 . The G/G genotype of a resistin single-nucleotide polymorphism at −420 increases type 2 diabetes mellitus susceptibility by inducing promoter activity through specific binding of Sp1/3 . Am J Hum Genet , 2004 . 75 ( 4 ): 678 - 686 . 15338456 DOI: 10.1086/424761 http://doi.org/10.1086/424761 . The G/G genotype of a resistin single-nucleotide polymorphism at −420 increases type 2 diabetes mellitus susceptibility by inducing promoter activity through specific binding of Sp1/3 L Patel , AC Buckels , IJ Kinghorn , PR Murdock , JD Holbrook , C Plumpton , CH Macphee , SA Smith . Resistin is expressed in human macrophages and directly regulated by PPAR gamma activators . Biochem Biophys Res Commun , 2003 . 300 ( 2 ): 472 - 476 . 12504108 DOI: 10.1016/S0006-291X(02)02841-3 http://doi.org/10.1016/S0006-291X(02)02841-3 . Resistin is expressed in human macrophages and directly regulated by PPAR gamma activators F Sentinelli , S Romeo , M Arca , E Filippi , F Leonetti , M Banchieri , U Di Mario , MG Baroni . Human resistin gene, obesity, and type 2 diabetes: mutation analysis and population study . Diabetes , 2002 . 51 ( 3 ): 860 - 862 . 11872692 . Human resistin gene, obesity, and type 2 diabetes: mutation analysis and population study CM Steppan , ST Bailey , S Bhat , EJ Brown , RR Banerjee , CM Wright , HR Patel , RS Ahima , MA Lazar . The hormone resistin links obesity to diabetes . Nature , 2001 . 409 ( 6818 ): 307 - 312 . 11201732 DOI: 10.1038/35053000 http://doi.org/10.1038/35053000 . The hormone resistin links obesity to diabetes MS Tan , SY Chang , DM Chang , JC Tsai , YJ Lee . Association of resistin gene 3′-untranslated region +62G→A polymorphism with type 2 diabetes and hypertension in a Chinese population . J Clin Endocrinol Metab , 2003 . 88 ( 3 ): 1258 - 1263 . 12629116 DOI: 10.1210/jc.2002-021453 http://doi.org/10.1210/jc.2002-021453 . Association of resistin gene 3′-untranslated region +62G→A polymorphism with type 2 diabetes and hypertension in a Chinese population G Wiesner , RE Brown , GS Robertson , SA Imran , E Ur , M Wilkinson . Increased expression of the adipokine genes resistin and fasting-induced adipose factor in hypoxic/ischaemic mouse brain . Neuroreport , 2006 . 17 ( 11 ): 1195 - 1198 . 16837853 DOI: 10.1097/01.wnr.0000224776.12647.ba http://doi.org/10.1097/01.wnr.0000224776.12647.ba . Increased expression of the adipokine genes resistin and fasting-induced adipose factor in hypoxic/ischaemic mouse brain RZ Yang , Q Huang , A Xu , JC McLenithan , JA Eisen , AR Shuldiner , S Alkan , DW Gong . Comparative studies of resistin expression and phylogenomics in human and mouse . Biochem Biophys Res Commun , 2003 . 310 ( 3 ): 927 - 935 . 14550293 DOI: 10.1016/j.bbrc.2003.09.093 http://doi.org/10.1016/j.bbrc.2003.09.093 . Comparative studies of resistin expression and phylogenomics in human and mouse Z Yang , M Zhang , L Xu , J Zhang , W Hongwei . Effect of tumor necrosis factor-alpha on resistin expression in 3T3-L1 adipocytes and its mechanism . J Huazhong Univ Sci Technol Med Sci , 2005 . 25 ( 2 ): 121 - 123 . 130 16116950 . Effect of tumor necrosis factor-alpha on resistin expression in 3T3-L1 adipocytes and its mechanism S Yura , N Sagawa , H Itoh , K Kakui , MA Nuamah , D Korita , M Takemura , S Fujii . Resistin is expressed in the human placenta . J Clin Endocrinol Metab , 2003 . 88 ( 3 ): 1394 - 1397 . 12629135 DOI: 10.1210/jc.2002-011926 http://doi.org/10.1210/jc.2002-011926 . Resistin is expressed in the human placenta 10.1631/jzus.2007.B0132 Project (No. 2003C33031) supported by the Science and Technology Department of Zhejiang Province, China 2006-06-22 2006-10-17 2022-01-05 Copyright © 2007, Journal of Zhejiang University Science 2007 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 02 08