Role of Ribonuclease-L in Adipogenesis and Association with Metabolic Syndrome

• Main Authors: Yi-Ting Wang, 王議霆
• Other Authors: 楊偉勛
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/km9t8z

博士 === 國立臺灣大學 === 臨床醫學研究所 === 105 === Ribonuclease L (RNase-L) participates in the oligoadenylate synthetase (OAS)-RNase-L pathway in response to the infection of RNA virus. However, recently, RNase-L also has been shown to regulate various cellular functions, including myogenesis, cell proliferation, and even carcinogenesis, through its ribonuclease specificity. Herein, the aim of this thesis is to investigate the role of RNase-L in adipogenesis and metabolism. In the beginning, it was shown that knockdown of RNase-L reduced 3T3-L1 adipocyte differentiation and lipid accumulation. When RNase-L was silenced, the expression of pre-adipocyte factor-1 (Pref-1), an early repressor in adipogenesis, and its downstream pathway, focal adhesion kinase (FAK)-extracellular signal-regulated kinases (ERK)-sex determining region Y-box 9 (Sox9), both were induced by RNase-L suppression. Hence, it was hypothesized that RNase-L destabilizes Pref-1 mRNA to influence adipogenesis. Using RT-PCR, the presence of Pref-1 mRNA was detected in the messenger ribonuleoprotein (mRNP) complexes of RNase-L precipitated with anti-RNase-L antibody. The decay rate of Pref-1 mRNA was also increased in the 3T3-L1 pre-adipocytes stably over-expressing wild-type RNase-L ribonuclease compared with that of mutants. In stable cell clones with RNase-L knockdown, the further suppression of Pref-1 mRNA by specific siRNAs can partially recover the impairment of adipocyte differentiation and lipid accumulation capacity. Secondly, the meta-analyses of public mouse expression array data based from 11 independent studies were performed to determine the expression relationship between RNase-L and Pref-1. The robust multi-array average (RMA) algorithm utilizing statistical software R was used to normalize the expression distribution of each sample. The meta-analysis among 56 arrays showed a negative correlation between RNase-L and Pref-1 mRNA levels in murine adipose tissue. Interestingly, higher RNase-L and lower Pref-1 mRNAs were found in the adipose tissues of high-fat diet-fed rodents compared with those fed by normal diet, whether in this in silico meta-analysis or in vivo rat data. Over the past decades, the prevalence of metabolic syndrome (MetS) has been increasing worldwide. Since RNase-L is related to adipogenesis and obesity, our next goal was to measure the serum levels of RNase-L in humans and analyze the relationship with metabolic status. An in-house enzyme-linked immunosorbent assay (ELISA) was developed to measure human serum RNase-L levels. In a total of 396 subjects, the levels of serum RNase-L of the subjects with MetS were lower than those without (16.5±6.4 μg/ml vs. 18.4±8.0 μg/ml, P=0.018). The subjects with central obesity, elevated blood pressure, or impaired fasting glucose (IFG) had significantly lower serum RNase-L levels compared with that without. Diastolic blood pressure (β=-0.124, P=0.031) and high-density lipoprotein cholesterol (HDL-C) (β=0.131, P=0.038) was related to serum RNase-L in multivariate linear regression analysis. Risk of MetS (OR, 0.83, 95% CI, 0.71-0.98, P=0.028), central obesity (OR, 0.82, 95% CI, 0.71-0.94, P=0.005), or low HDL-C (OR, 0.86, 95% CI, 0.74-1.00, P=0.042) was reduced with every 5 μg/ml increase in serum RNase-L level. Moreover, age is also inversely associated to serum RNase-L levels in various analyses. Taken together, it was demonstrated in this thesis that RNase-L is involved in adipogenesis through destabilizing Pref-1 mRNA, a novel substrate of RNase-L. These multiple approaches, including in vitro cell model, in silico array meta-analysis, and in vivo animal data, were applied to reveal the negative relationship between RNase-L and Pref-1 in mammals. Furthermore, the negative relation between serum RNase-L levels and MetS was also proposed and look forward to better understanding the pathogenesis of the MetS.