Fat metabolism in the mammary gland is a complex process that is regulated by many factors, and the molecular mechanism is not clearly understood. SIRT2 encodes a member of the sirtuin family of proteins, and it can deacetylate factors in physiological and biochemical reactions in many organisms (Kim et al., Reference Kim, Lee, Lee, Kim, Suk, Lee, Hur, Hong, Do, Park and Jeong2018). SIRT2 has been shown to play a key role in lipogenesis (Perrini et al., Reference Perrini, Porro, Nigro, Cignarelli, Caccioppoli, Genchi, Martines, Fazio, Capuano, Natalicchio, Laviola and Giorgino2020) and the expression of SIRT2 is negatively correlated with adipogenesis in the 3T3-L1 cell line. Feeding and seasonal environmental changes will both alter the expression of SIRT2 in mammalian tissue (Jing et al., Reference Jing, Gesta and Kahn2007).
MicroRNAs are a class of single-stranded, small-molecule, noncoding RNAs that are abundant in a variety of plants, animals and microorganisms and can cause the mRNA of the target gene to be degraded or blocked during translation via binding to the 3′-untranslated region (3′-UTR) of the target gene (Bartel, Reference Bartel2009). MicroRNAs are associated with the occurrence of a variety of traits and diseases (Fernandes et al., Reference Fernandes, Barretti, Phillips and Menezes Oliveira2018). Previous studies of miR-212 have mainly concerned its positive effect on inhibiting numerous types of cancer (Wanet et al., Reference Wanet, Tacheny, Arnould and Renard2012; Lin et al., Reference Lin, Wang, Jin, Shi, Lu and Qi2016) as well as regulation of the nervous system (Xie et al., Reference Xie, Fu, Cao, Liu, Wu, Li and Chen2018; Aten et al., Reference Aten, Page, Kalidindi, Wheaton, Niraula, Godbout, Hoyt and Obrietan2019). However, the functional role of miR-212 in mammary epithelial cells is not known, and there has been little research on the regulation of lipogenesis by miR-212.
This study elucidated the effect of miR-212 on the regulation of lipogenesis in a MAC-T cell line by targeting SIRT2; the results contribute to an understanding of the regulation of lipogenesis in bovine mammary glands.
Materials and methods
Ethical approval was not sought since the research reported here did not include any animal studies.
Bioinformatics screening of miRNAs targeting the SIRT2 gene in dairy cows
Two kinds of online software, TargetScan (http://www.targetscan.org/vert_72/) and miRBase (http://www.mirbase.org/), were used to predict miRNAs which have the targeted regulatory effect on the SIRT2 gene, and the predicted results are shown in Fig. S1 of the online Supplementary File.
Cells used in this study: MAC-T cells and HEK 293T cells
The bovine mammary epithelial (MAC-T) cell line is a bovine mammary epithelial cell line and HEK 293T is a human embryonic kidney cell line which is a model cell line that is very suitable for luciferase reporter gene validation experiments because of the high efficiency of plasmid transfection (Fanunza et al., Reference Fanunza, Frau, Sgarbanti, Orsatti, Corona and Tramontano2018). Both exhibit good viability and a short proliferative cycle, making them ideal for culturing in the laboratory. In this study, the MAC-T cells and HEK 293T cells were a gift of College of Veterinary Medicine, Yangzhou University. The method of cell culture is detailed in the online Supplementary File.
Overexpression of and interference by miR-212, miR-375 and miR-655 in MAC-T cells
MiRNA mimics are substances that mimic endogenous miRNAs in living organisms and which are produced by chemical synthesis to enhance the function of endogenous miRNAs. An miRNA inhibitor is a chemically modified inhibitor that specifically targets specific miRNAs in cells. Based on the bovine miR-212, miR-375 and miR-655 sequences provided in the miRBase database, each miRNA as well as mimics and inhibitors for each were designed by Gene Pharma (Suzhou, China). The sequences are given in the online Supplementary File.
The medium used for transfection was Opti-MEM (Gibco, cat: 31985088) mixed with nothing and the transfection reagent was Lipofectamine™ 2000 (Invitrogen, cat: 11668019). The transfection concentrations of miRNA mimics and inhibitor were 100 and 200 nm. Four microliters of Lipofectamine™ 2000 and one milliliter Opti-MEM medium were placed in each well of a 6-well plate and the transfection process was performed according to the instruction manual of Lipofectamine™ 2000. The MAC-T cells were plated in the 6 wells for 24 h and cultivated in complete DMEM/F-12 medium mixed with 10% FBS at 37°C, 5% CO2. When the cells occupied over 70–80% of the plate, we started the transfection step and changed the medium to Opti-MEM and after 6 h transfection, we changed the Opti-MEM medium to complete DMEM/F-12 medium mixed with 10% FBS and cultivated the cell for 48 h at 37°C, 5% CO2.
Extraction and purification of total RNA
The total RNA in the Mac-T cells was extracted by TRIzol Reagent (Invitrogen) following with the manufacturer's protocol. Spectrophotometry (Nanodrop ND-1000, Thermo Fisher, USA) and agarose gel electrophoresis were used to ensure the quantity and quality of the RNA. The mRNA and miRNA in the total RNA were reverse-transcribed into cDNA using a PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara, cat: RR047A) and a Mir-X™ miRNA First-Strand Synthesis Kit (Takara, cat: 638313).
RT-qPCR analysis
According to the miR-212, miR-375 and miR-655 sequences provided in the miRBase database, upstream primers for fluorescent quantitative PCR were designed. Details of these primers are provided in the online Supplementary File. Based on the user manual of Mir-X™ miRNA First-Strand Synthesis Kit, the RT-qPCR assays of miRNAs were performed with a 25 μl reaction system consisting of 9 μl of ddH2O, 12.5 μl of SYBR Advantage Premix (2×), 0.5 μl of ROX Dye (50×), 0.5 μl upstream and downstream primers each and 2.0 μl of cDNA. PCR conditions for miRNAs: Denaturation at 95°C for 10 s, (95°C for 5 s and 60°C for 20 s) × 40 Cycles, then dissociation curve at 95°C for 60 s, 55°C for30 s and 95°C for 30 s.
The forward primer of SIRT2 was 5′-GAAATACCGTCTTCCCTA-3′ and the reverse primer of SIRT2 was 5′-GATGAAGTAGTGGCAGAT-3′. Based on the user manual of TB Green™ Premix Ex Taq™ II (Takara, cat: RR820A) the RT-qPCR assay of SIRT2 was performed with a 20 μl reaction system consisting of 6 μl of ddH2O, 10 μl of TB Green Premix Ex Taq II (2×), 0.4 μl of ROX Dye II (50×), 0.8 μl upstream and downstream primers (10 μm) each and 2.0 μl of cDNA. PCR conditions for SIRT2: Pre-denaturation at 95°C for 30 s, (95°C for 3 s and 60°C for 30 s) × 40 Cycles, then 95°C for 3 s, 60°C for 30 s in the melt stage. The expression was normalized to GAPDH (F: 5′-GCAAGTTCCACGGCACAG-3′, R: 5′-GGTTCACGCCCATCACAA-3′).
All the primers were synthesized by Sangon Biotech (Shanghai, China) and all real-time PCR reactions, including the controls with no templates, were carried out in a Bio-Rad CFX96 real-time PCR detection system (Bio-Rad, USA) in triplicate. The relative expression was calculated using the 2−△△Ct method (Mestdagh et al., Reference Mestdagh, Van Vlierberghe, De Weer, Muth, Westermann, Speleman and Vandesompele2009).
Western blot
After 48 h of transfection, cellular total protein was lysed in RIPA buffer (Solarbio, China) mixed with 1% Phenylmethanesulfonyl fluoride (PMSF) (Beyotime, China, cat: ST505). Then the protein was mixed with protein loading buffer and denatured at 100°C for 10 min. 20μg protein was separated by 10% of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane (Millipore, USA) and probed with the primary monoclonal rabbit anti-SIRT2 antibody (Abcam, USA, cat: ab67299) and the monoclonal mouse anti-β-actin antibody (Proteintech Group, 66009-1-IG, China). Polyclonal goat anti-rabbit HRP-conjugated IgG (Tiangen, China) was used as the secondary antibody. All antibodies were applied according to the manufacturers' instructions. Signals were detected using the chemiluminescent ECL western blot system (Pierce, USA).
Luciferase reporter gene validation
To generate reporter constructs for luciferase assays, the 3′ UTR of SIRT2 gene, followed by the wild-type miR-212, miR-375 and miR-655 predicted target sites were flanked by the SacI and XhoI restriction sites that were incorporated into the amplification primers. The SIRT2 wild-type gene fragment and the lentiviral vector GP-miRGLO were digested with SacI and XhoI. DNA T4 ligase was used to ligate the double-digested SIRT2 wild-type gene fragment and the linearized vector. Mutagenic PCR was used to mutate the SIRT2 target site sequences from CCAAGG to GGUUCC, AACAAA to UUGUUU, and UGUAUUA to ACAUAAU. The dual-luciferase reporter vector and miRNA mimics were cotransfected into HEK 293T cells to detect whether the miRNAs can combine with the 3′ UTR of SIRT2. At 48 h post-transfection the fluorescence intensity of firefly luciferase was measured with the Dual-Glo luciferase assay system according to the manufacturer's instructions (Promega, cat: N1110).
Co-transfection of the siRNA of SIRT2 and miR-212 mimics into MAC-T cell lines
In order to show that miR-212 regulates lipogenesis by targeting only SIRT2 and no other genes, the siRNA of SIRT2 and miR-212 mimics were co-transfected into MAC-T cell lines. The siRNA of SIRT2 was synthesized by GenePharma (Suzhou, China) and comprised these primers:
F: 5′-GCAUGGACUUUGACUCCAATT-3′
R: 5′-UUGGAGUCAAAGUCCAUGCTT-3′
The transfection concentrations of the miR-212 mimics and siRNA were 100 nm respectively. Transfection reagents (4 μl) were added to each well in a 12-well plate. The transfection process was same as previously described.
Determining the expression levels of fatty acid synthesis related genes
Following co-transfection with miR-212 mimics and siRNA for 48 h, total RNA was obtained from the MAC-T cell and reverse-transcribed into cDNA by the PrimeScript™ RT Reagent Kit with gDNA Eraser, then the RT-qPCR assays of fatty acid synthesis related genes were performed using the same method as previously described for SIRT2. Primer details are in the Supplementary File. Primers were synthesized by Shanghai Shenggong. Detection employed a Bio-Rad CFX96 real-time PCR detection system. The expression was normalized to GAPDH. All the real-time PCR reactions, including the controls with no templates, were carried out in a Bio-Rad CFX96 real-time PCR detection system (Bio-Rad, USA) in triplicate. The relative expression was calculated using the 2−△△Ct method.
Cellular TAG content assay
Lipofectamine™ 2000 was transfected into the MAC-T cells with the miR-212 mimic. After 48 h of cell incubation, the cells were harvested with a lysis buffer (50 mmol/l Tris-HCL, pH 7.4, 150 mmol/NaCl, 1% Triton X-100). A triglyceride kit (Sigma-Aldrich, USA, cat: MAK040) and an atomic absorbance spectrophotometer (Perkin Elmer, USA) were used to measure the TAG (triglyceride) content according to the manufacturer's instructions. The values obtained were calibrated to the total protein content, which was measured with a BCA protein assay kit (Thermo Fisher, USA).
Statistical analysis
Statistical analyses were performed using the SPSS 18.0 statistics software package. Data are presented as the mean ± sd (standard deviation) of triplicate values. Significant differences between the groups were determined using a one-way analysis of variance (ANOVA) and two-way ANOVA with P < 0.05 (*) and P < 0.01 (**) indicating significant differences.
Data availability statement
The datasets generated during and/or analyzed in this study are available from the corresponding author on reasonable request.
Results
The transfection of mimics and inhibitors was successful
The agarose gel electrophoresis results of RNA extraction from MAC-T cells are shown in Fig. S2 of the online Supplementary File. The results of real-time PCR of miR-212, miR-375 and miR-655 after transfection are shown in Fig. 1(a–c).
Fig. 1. The relative expression (a) miRNA-212, (b) miRNA-375, and (c) miRNA-655 after transfecting the mimics and inhibitors and the relative expression of SIRT2 after transfecting the (e) mimics and (f) inhibitors of miRNA-212, miRNA-375 and miRNA-655. NC, negative control. Values are presented as average ± standard deviation. *P < 0.05 and **P < 0.01.
There are three distinct bands in the agarose gel electrophoresis image which means that the quality of the RNA extraction is good and can be used for subsequent experiments. The expression of the miRNAs was significantly upregulated and the levels were from two hundred to three hundred times higher than the negative control groups after transfection of the mimics. In confirmation, after transfection of the respective inhibitor of the three miRNAs the expression of the miRNAs was significantly downregulated and the levels were from ten to twenty five times lower than the negative control groups.
MicroRNA-212 interferes with the expression of the SIRT2 gene
The expression of SIRT2 after the mimics and inhibitors of miR-212, miR-375 and miR-655 were transfected into cells is shown in Fig. 1(d–e). Expression was significantly downregulated to roughly half of the negative control group after transfection with the miR-212 mimics and was significantly upregulated to approximately five times higher than the negative control group after transfection with the miR-212 inhibitor. For miR-375 and miR-655, expression of the SIRT2 gene did not change significantly after transfection compared with negative control, which indicates that miR-212 inhibits the SIRT2 gene and miR-375 and miR-655 have no direct effect on the SIRT2 gene in the bovine mammary epithelial cell line.
The results from the validation of miR-212, miR-375, and miR-655 targeting SIRT2 are shown in Fig. 2(a–c). After co-transfection with the wild-type vectors of the bta-miR-212 and bta-miR-212 mimics, the expression of luciferase was significantly decreased to 28.0 ± 2.9 percent of the negative control group. There was no significant difference in the expression of luciferase compared with the negative control (P > 0.05) after co-transfection with the SIRT2 mutant vector plasmid and bta-miR-212. The expression of luciferase activity was not significantly different after co-transfection with either the wild-type vectors of the bta-miR-375 and bta-miR-375 mimics, the mutant vector plasmid and bta-miR-375 mimics, the wild-type vectors of the bta-miR-655 and bta-miR-655 mimics or the mutant vector plasmid and bta-miR-655 mimics.
Fig. 2. Validation of (a) miRNA-212, (b) miRNA-375 and (c) miRNA-655 targeting SIRT2. (d)Western blot analyses of the expression of β-actin and SIRT2 protein in cells with miR-212 mimics. NC, negative control. Values are presented as means ± standard errors; *P < 0.05 and **P < 0.01.
After the transfection of miR-212 mimics into MAC-T cells, a change in the SIRT2 protein relative to the negative control group was detected using a western blot. As shown in Fig. 2(d), the band corresponding to the miR-212 mimic group was significantly thinner and lighter than that of the negative control.
MicroRNA-212 targets SIRT2 to promote lipogenesis
The results of the expression of SIRT2 after transfecting siRNA and the expression of fatty acid synthesis related genes after co-transfection with siRNA of SIRT2 and miR-212 mimics are shown in Fig. 3(a–d). The result of the TAG analysis is shown in Fig. 3(e). Transfection with miR-212 significantly increased the expression of FASN, SREBP1 and PPARγ (levels 2.90 ± 0.12, 2.55 ± 0.36 and 4.67 ± 0.51 times higher than the negative control groups, respectively). After co-transfecting with the siRNA and miR-212 mimics and transfecting with only siRNA of SIRT2, there was no significant differences in the expression of either FASN or SREBP1. Likewise, the expression of PPARγ was not changed significantly when the siRNA of SIRT2 was transfected alone. The triglyceride level in MAC-T cells was significantly increased after transfecting with miR-212 mimics.
Fig. 3. The expression of PPARγ, FASN and SREBP1 after cotransferring siRNA and miR-212 mimics and the triglyceride level in MAC-T after transfecting with miR-212 mimics. (a) The expression of SIRT2 after transfection with siRNA; (b) The expression of PPARγ after co-transfection; (c) The expression of FASN after co-transfection; (d) The expression of SREBP1 after co-transfection; (e) The triglyceride level in MAC-T. NC, negative control. Values are presented as means ± standard errors; *P < 0.05 and **P < 0.01.
Discussion
In the present study, we identified three miRNAs (miR-212, miR-375 and miR-655) which may have a regulatory relationship with SIRT2 gene by two online software tools. Then we experimentally verified the relative functions of the three miRNAs and the SIRT2 gene in MAC-T cells. The RT-qPCR, western blot and dual-luciferase reporter genes demonstrated that SIRT2 is the target gene of miR-212, but not of miR-375 or miR-655. The expression changes of PPARy, FASN and SREBP1 after co-transfection with miR-212 mimics and siRNA as well as the TAG determination experiment showed that miR-212 promotes lipogenesis in MAC-T by targeting SIRT2.
MicroRNA can down-regulate the target gene by inhibiting the translation of mRNA according to the principle of complementary base pairing. The purpose of understanding the relationship is to determine the target site (Friedman et al., Reference Friedman, Farh, Burge and Bartel2009), therefore, predicting the miRNA target gene is a pivotal step when studying the regulation of biological processes by miRNAs. The luciferase reporter assay is one of the most common approaches used to validate a direct miRNA target site (Campos-Melo et al., Reference Campos-Melo, Droppelmann, Volkening and Strong2014). In the present study, it was found that by binding to the SIRT2 mRNA 3′-UTR, the miR-212 mimic decreased luciferase activity. It is generally considered that the target region for miRNA is located at the 3′-UTR of the target gene. However, some studies have suggested that the 5′-UTR end of the target gene may also contain the target region (Jagtap and Shivaprasad, Reference Jagtap and Shivaprasad2014). Therefore, if miRNA can regulate the expression of gene but there is no significant change in the result of luciferase reporter gene validation, a possibility might be that the miRNA binds to the 5′-UTR region of gene, which can be determined by redesigning vector according to the 5′-UTR region of gene and conducting the experiment of luciferase reporter gene validation again.
Recent studies suggested that miRNAs play important roles in lipogenesis and triglyceride homeostasis (Wagschal et al., Reference Wagschal, Najafi-Shoushtari, Wang, Goedeke, Sinha, deLemos, Black, Ramirez, Li, Tewhey, Hatoum, Shah, Lu, Kristo, Psychogios, Vrbanac, Lu, Hla, de Cabo, Tsang, Schadt, Sabeti, Kathiresan, Cohen, Whetstine, Chung, Fernandez-Hernando, Kaplan, Bernards, Gerszten and Naar2015). For instance, one of the miR-212 family members, miR212-5p, specifically binds to the 3′ UTR of stearoyl-CoA desaturase-1 (SCD1) and fatty acid synthase (FAS) and inhibits their activity, whilst overexpression of miR-212-5p decreases the protein levels of SCD1 and FAS in vitro and in vivo, and silencing of miR-212-5p has the opposite effects in mouse primary hepatocytes (Guo et al., Reference Guo, Yu, Wang, Li, Liu, Du, Xiao, Chen and Guo2017). Furthermore, miR-375 expression was increased in the serum of high fat diet (HFD)-fed mice compared to that in healthy control and the inhibition of miR-375 can up-regulate the expression of adiponectin (Lei et al., Reference Lei, Zhou, Yang and Li2018). MiR-375 also promotes lipogenesis in mouse pre-adipocytes via regulation of ERK1/2 signaling upstream of PPARγ (Deiuliis, Reference Deiuliis2016) and MiR-146b can promote lipogenesis by the down-regulation of SIRT1, which interferes with the SIRT1-FOXO1 cascade (Ahn et al., Reference Ahn, Lee, Jung, Jeon and Ha2013). In the present study, we find a new pro-adipogenic miRNA, miR-212, which can significantly promote lipogenesis by down-regulating SIRT2.
We observed that miR-212 significantly promoted lipogenesis and increased TAG accumulation in MAC-T. Furthermore, we found miR-212 affected the mRNA expression of lipogenesis related genes, including PPARy, FASN and SREBP1. It is generally accepted that PPARγ is a critical factor for lipogenesis and has relationships with other lipogenic genes (Farmer, Reference Farmer2006; Rosen and MacDougald, Reference Rosen and MacDougald2006; Siersbaek et al., Reference Siersbaek, Mielsen and Mandrup2012). In buffalo, SREBP1 gene may act on ERK1/ERK2 signaling pathway to regulate the expression of PPARy gene (Xu et al., Reference Xu, Wang and Zhou2019a, Reference Xu, Chen, Jia, Deng, Jiang, Qin, Qiu, Wang, Yang and Jiang2019b) and, as a transcription regulator, PPARγ is able to enhance the expression of many genes related to lipogenesis including FASN (Lefterova et al., Reference Lefterova, Zhang, Steger, Schupp, Schug, Cristancho, Feng, Zhuo, Stoeckert, Liu and Lazar2008; Zhang et al., Reference Zhang, Sun, Zhong, Sun and Zhou2016). Moreover, many other studies have indicated that these genes (PPARγ, FASN, SREBP1) are associated with milk fat synthesis in mammary epithelial cells (Yang et al., Reference Yang, Guo, Zan, Wang and Tang2017). Our results revealed that miR-212 has the ability to influence lipogenesis and TAG content by affecting the expression of lipogenesis related genes in MAC-T.
Epigenetic mechanisms including DNA methylation, histone modifications and non-coding RNA regulation are related to the regulation of lipogenesis (Ronn et al., Reference Ronn, Volkov, Davegardh, Dayeh, Hall, Olsson, Nilsson, Tornberg, Nitert, Eriksson, Jones, Groop and Ling2013). Specifically, histone deacetylases and sirtuins, including SIRT1-7, have been shown to play central roles in lipid metabolism (Lomb et al., Reference Lomb, Laurent and Haigis2010; Feng et al., Reference Feng, Liu, Sun, Bugge, Mullican, Alenghat, Liu and Lazar2011; Hu et al., Reference Hu, Xia and Hou2018). Previous research on Canadian Holstein cows also found that the down expression of SIRT2 gene is associated with milk fat reduction and could affect milk fat synthesis by different pathways (Ran and Eveline, Reference Ran and Eveline2017). In this study, we found that reduced SIRT2 expression promotes lipogenesis in MAC-T cells, the result is in line with previous studies in human visceral adipose stem cells, where SIRT2 regulates lipogenesis negatively and associates with accumulation of visceral fat in human obesity (Perrini et al., Reference Perrini, Porro, Nigro, Cignarelli, Caccioppoli, Genchi, Martines, Fazio, Capuano, Natalicchio, Laviola and Giorgino2020).
Many studies have confirmed that SIRT2 is involved in lipogenesis (Perrini et al., Reference Perrini, Porro, Nigro, Cignarelli, Caccioppoli, Genchi, Martines, Fazio, Capuano, Natalicchio, Laviola and Giorgino2020; Xu et al., Reference Xu, Wang and Zhou2019a, Reference Xu, Chen, Jia, Deng, Jiang, Qin, Qiu, Wang, Yang and Jiang2019b; Lantier et al., Reference Lantier, Williams and Hughey2018) and the co-transfection experiment showed that by targeting SIRT2, miR-212 upregulates the mRNA expression of FASN and SREBP1 but not PPARγ. These are the first data to confirm that SIRT2 can directly regulate FASN and SREBP1 negatively, whilst the result of PPARγ is at odds with a recent report (Xu et al., Reference Xu, Wang and Zhou2019a, Reference Xu, Chen, Jia, Deng, Jiang, Qin, Qiu, Wang, Yang and Jiang2019b) that in bovine ovarian granular cells, SIRT2 knockdown or treatment with inhibitors produced negative effects on PPARγ. It was supposed that the mechanism might be SIRT2 mediating the transcription factors FOXO1. This is a sensible hypothesis because it is well-documented that SIRT2 acetylates this transcription factor (Armoni et al., Reference Armoni, Harel, Karni, Chen, Bar-Yoseph, Ver, Quon and Karnieli2006; Wang and Tong, Reference Wang and Tong2009) and in turn FOXO1 regulates the transcription of PPARγ negatively in adipose tissue by binding to the promoter region to inhibit lipogenesis in cells (Kohan et al., Reference Kohan, Carvajal, Gabler, Vantman, Romero and Vega2010). However, in this study, SIRT2 knockdown had no effect on the expression of PPARγ. There are two possibilities. One is that in the Mac-T cell line, PPARγ is regulated by SIRT1 and not by SIRT2, whilst SIRT1 was also targeted by miR-212 (in mice, SIRT1 was identified as a target gene of miR-212: Li et al., Reference Li, Bai, Wang, Xie, Chen, Fu and Wen2018) to inhibit the expression of PPARγ (Picard et al., Reference Picard, Kurtev, Chung, Topark-Ngarm, Senawong, Machado De Oliveira, Leid, McBurney and Guarente2004). The second possibility is that the miR-212 might have interfered with the process of SIRT2-FOXO1-PPARγ signaling. Regrettably, it is not possible to clarify this situation from our data, and further studies will be necessary to assess these hypothesizes.
In conclusion, we have identified miR-212 as a new class of regulator of lipogenesis in MAC-T cells. We have shown that miR-375 and miR-655 cannot target SIRT2, which is different from the theoretical prediction. Furthermore, our subsequent results suggest that the functions of the miR-212-SIRT2 axis could be crucial for fat synthesis in MAC-T cells by regulating lipogenesis related genes.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0022029920000229.
Acknowledgments
This research was funded by the National Natural Science Foundation of China (Nos. 31472067, 31872324, 31702080, 31802035).
