Changes in the MicroRNA Profile Observed in the Subcutaneous Adipose Tissue of Obese Patients after Laparoscopic Adjustable Gastric Banding

Background. Laparoscopic adjustable gastric banding (LAGB) results in significant lasting weight loss and improved metabolism in obese patients. To evaluate whether epigenetic factors could concur to these benefits, we investigated the subcutaneous adipose tissue (SAT) microRNA (miRNA) profile before (T0) and three years (T1) after LAGB in three morbidly obese women. Case Reports. SAT miRNA profiling, evaluated by TaqMan Array, showed four downexpressed (miR-519d, miR-299-5p, miR-212, and miR-671-3p) and two upexpressed (miR-370 and miR-487a) miRNAs at T1 versus T0. Bioinformatics predicted that these miRNAs regulate genes belonging to pathways associated with the cytoskeleton, inflammation, and metabolism. Western blot analysis showed that PPAR-alpha, which is the target gene of miR-519d, increased after LAGB, thereby suggesting an improvement in SAT lipid metabolism. Accordingly, the number and diameter of adipocytes were significantly higher and lower, respectively, at T1 versus T0. Bioinformatics predicted that the decreased levels of miR-212, miR-299-5p, and miR-671-3p at T1 concur in reducing SAT inflammation. Conclusion. We show that the miRNA profile changes after LAGB. This finding, although obtained in only three cases, suggests that this epigenetic mechanism, by regulating the expression of genes involved in inflammation and lipid metabolism, could concur to improve SAT functionality in postoperative obese patients.


Introduction
Obesity is a major health problem worldwide [1]. Besides lifestyle interventions and pharmacotherapy, significant, lasting weight loss in obese patients can be obtained with laparoscopic adjustable gastric binding (LAGB) surgery [2,3]. This procedure is usually applied in morbidly obese patients (BMI greater than 40 kg/m 2 ) and/or if obesity is associated with comorbidities, such as diabetes or cardiovascular diseases [4]. We previously reported that LAGB also improved metabolism, in terms of inflammation, insulin resistance, and liver steatosis, three years after LAGB [3]. MicroRNAs (miRNAs) are short (19-22 bp) noncoding RNAs that regulate the expression of mRNA targets mainly by binding their complementary sequences at the 3 untranslated region (3 UTR) and inhibiting their translation [5]. The miRNA profile in subcutaneous (SAT) and in visceral (VAT) adipose tissues differs between obese and lean subjects [6,7]. The aim of the present study was to investigate the SAT miRNA profile in 2 Journal of Obesity three morbidly obese women before (T0) and three years (T1) after LAGB and to evaluate whether miRNAs are involved in post-LAGB metabolic improvement.

Patients. Three obese women (mean body mass index
[BMI] 42.9 kg/m 2 ; mean age 48 years) undergoing LAGB (at T0 and T1) and two lean controls (mean BMI 21.5 kg/m 2 ; mean age 37 years) undergoing laparoscopic cholecystectomy entered the study. All subjects gave informed consent to the study, which was performed according to the Helsinki II Declaration and was approved by the Ethics Committee of our School of Medicine.

Serum Determinations.
A fasted serum sample was also collected from enrolled subjects, the main biochemical parameters were measured by routine methods; leptin (L) and adiponectin (A) adipokines were measured by ELISA methods and the L/A ratio was calculated.

Adipocytes Size and Number Quantification.
Periumbilical bioptic SAT samples were obtained from all subjects. All biopsies were snap-frozen and stored in liquid nitrogen until RNA isolation. The number and diameter of adipocytes (at T0 and T1) were measured as previously reported [3]. Briefly, hematoxylin and eosin stains were used to identify the cellular components and their morphological changes. For each sample, adipocytes were counted in three fields (100 m each) and their size was measured by two operators; the average of each parameter was then calculated.

miRNAs Expression Profile. TaqMan low density arrays
Human MicroRNA Panel v1.0 (Applied Biosystems Inc., Foster City, CA, USA), containing 377 preloaded human miRNA targets and the endogenous control RNU48, were used according to the manufacturer's instructions. RT-PCR was performed with 800 ng of cDNA and the 7900 HT real-time PCR system (Applied Biosystems) as reported elsewhere [7]. miRNA expression was normalized to RNU48 and quantified using the RQ Manager 1.2 software (Applied Biosystems) with the following formula: ). miRNAs whose mean baseline RQ levels were <0.5 (downexpressed) or >2.0 (upexpressed) in all obese patients versus controls were considered differently expressed. We compared the expression of these differently expressed miRNAs with those previously reported in the SAT of other obese cohorts [6,[8][9][10][11][12][13][14]. Only miRNAs whose expression trend was confirmed were selected for evaluation after LAGB.
The levels of miR-370 and miR-487a (upexpressed) and miR-519d (downexpressed) were validated by TaqMan miRNA assays (Applied Biosystems) in accordance with the manufacturer's instructions on the 7900 HT real-time PCR system (Applied Biosystems).

2.7.
Bioinformatics. The MiRTarBase, STRING, and KEGG databases were used to select the experimentally validated miRNA/target gene interaction, to explore protein-protein interactions and to identify the pathways significantly ( < 0.001) deregulated, respectively.

Statistics.
Biochemical and clinical data are expressed as mean and standard error of the mean (SEM), whereas miRNA expression data are reported as log 10 mean RQ and SEM. The Wilcoxon test was used to compare data obtained at T0 and T1. Differences were considered statistically significant at < 0.05. Statistical analyses were carried out with the PASW Statistics (Ver.18; SPSS Inc. Headquarters, Chicago, Ill. USA).
Concerning miR-212, it was demonstrated to be upexpressed in high fat fed mice and its levels measured in liver were downregulated by physical activity [20]. miR-212 positively regulated fatty acids biosynthesis and hepatic storage by acting on fibroblast growth factor 21 (FGF-21) : Metabolic pathways predicted to change by our miRNAs after LAGB. Network identified by STRING showing a close interaction among the proteins regulated by the subset of 6 miRNAs. The above 6 miRNAs also targeted the pathways of several proteins in cancer and infection diseases (data not reported). [20]. Interestingly, FGF-21 is a target gene of PPAR-and is involved in cellular response to oxidative stress [21]. Consequently, the reduced SAT expression of both miR-212 and -519d, that we observed in our obese patients after LAGB, could exert a role in reducing oxidative stress and inflammation by acting on the PPAR--FGF-21 axis. In agreement with this hypothesis, we previously reported a reduced SAT inflammation after LAGB in a larger cohort compared to the present [3] and here we suggest that this effect could be, in part, caused by a miRNA-mediated mechanism.
Finally, although miR-370 and 487a, which were upexpressed after LAGB, were predicted to regulate the expression of genes belonging to several metabolic pathways, their contribution to surgery metabolic improvement, if any, remains to be established.

Conclusions
In conclusion, although our data were obtained in only three patients (mainly because patients were unwilling to