MiR‐17 family‐mediated regulation of Pknox1 influences hepatic steatosis and insulin signaling

Abstract The aberrant expression of Pknox1 is associated with hepatic glucose and lipid dysmetabolism status of type 2 diabetes mellitus (T2DM) and nonalcoholic fatty liver disease (NAFLD). However, the underlying mechanism causing Pknox1 overexpression in this pathological status remains unclear. By using miRNA target prediction programs, we found that the 3′‐UTR of the Pknox1 mRNA sequence contains highly conserved target sites of miR‐17 family. In a rat model of streptozotocin and high‐fat diet‐induced T2DM and NAFLD complication, the increased hepatic expression of Pknox1 was consistent with decreased expressions of miR‐17 family, especially miR‐17 and miR‐20a. Furthermore, an inverse correlation was observed between Pknox1 and miR‐17 and miR‐20a in free fatty acids‐induced hepatocyte steatosis. Dual‐luciferase reporter assay further showed that Pknox1 was a valid target gene of miR‐17 family. The ectopic expression of miR‐17 or miR‐20a could markedly suppress Pknox1 expression in hepatocytes. MiR‐17 or miR‐20a overexpression also resulted in significantly enhanced insulin sensitivity and reduced hepatocyte steatosis in HepG2 and L02 cells, which were determined by altered phosphorylation on insulin receptor signaling pathway proteins and decreased intracellular triglyceride and lipid accumulation, respectively. These data implicate the upregulated hepatic expression of Pknox1 in T2DM complicated with NAFLD may be caused by the reduced expression of miR‐17 family, indicating that developing miRNA‐mediated regulation strategies on Pknox1 may provide new therapeutic options for metabolic disease.

Transcription factor Pknox1 belongs to the three-amino acid loop extension class of homeodomain proteins and is ubiquitously expressed both in fetal and adult mice and can form tripartite DNAbinding complexes with members of the Hox and Pbx protein families. 3 Pknox1 is essential at multiple stages of embryonic development. 4 In mouse, Pknox1-null embryos die before gastrulation, because epiblast cells undergo p53-dependent apoptosis. 5 Whereas, mouse embryos carrying a hypomorphic Pknox1 i/i mutation (expressing about 5% protein compared with WT) show a leaky embryoniclethal phenotype and defects in angiogenesis, hematopoiesis, and eye development. 4,6,7 Thus, Pknox1 is previous known as a tumor suppressor associated with the maintenance of genomic stability.
Recently, several studies have shown that Pknox1 was also involved in hepatic lipogenesis and insulin-dependent glucose homeostasis. 8 The Pknox1 i/i mice feature a complex phenotype characterized by increased insulin sensitivity and protection from streptozotocin (STZ)-induced diabetes, accompanied with reduced hepatic lipogenesis and protection from methionine-and choline-deficient diet (MCDD)-induced steatohepatitis. While, hepatic expression of Pknox1 was significantly higher in the high-fat diet (HFD)-treated or T2DM (db/db) mice, suggesting aberrant Pknox1 expression may be associated with glucose and lipid dysmetabolism status of T2DM complicated with NAFLD. 9 However, the underlying mechanism causing Pknox1 overexpression in this pathological status remains unclear.
MicroRNAs (miRNAs) are small noncoding RNAs that can cause mRNA degradation or translation inhibition by interacting with the 3′-untranslated region (3′-UTR) of the target gene mRNA. 10,11 Accumulated evidences have shown that miRNAs play crucial roles in the development of metabolic disease through regulating the expression of glucose and lipid metabolism and IR-related genes. 12 For example, miR-122, as the predominant liver miRNA, has been proposed to play a central role in the maintenance of lipid and glucose homeostasis, it regulates hepatic fatty acid oxidation and cholesterol metabolism by way of downregulation of genes involved in cholesterol biosynthesis such as HMG-CoA reductase 13 ; miR-149 decreases lipogenesis in hepatocytes by targeting fibroblast growth factor-21  in the presence of free fatty acids (FFA) treatment 14 ; miR-34a inhibits very low density lipoprotein (VLDL) secretion and lipid accumulation, and improved hepatic steatosis in an HNF 4α-dependent manner 15 ; miR-96 promotes the pathogenesis of hepatic IR through the suppression of insulin receptor (INSR) and insulin receptor substrate (IRS). 16 However, certain miRNA, which can target and regulate the hepatic expression of Pknox1 has not been identified.
In this study, we used miRNA target prediction programs to explore Pknox1-targeted miRNAs and identified miR-17 seed family (miR-17 family) as endogenous regulators of Pknox1. We then aimed to identify the role of miR-17 family-mediated regulation of Pknox1 in hepatic lipogenesis and IR, in order to seek new intervention targets for the prevention and treatment of progressive liver disease caused by T2DM and NAFLD complication.

| Rat model of T2DM complicated with NAFLD
Six-week-old male Wistar rats that weighed approximately 150 g were used for studies. The animals were reared under a specific pathogenfree condition, and all procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Zhejiang University.
The induction of T2DM with NAFLD complication was performed according to Antony et al 17 with some modifications. The rats were fed with HFD for 1 week and then intraperitoneally injected with 40 mg/kg STZ (Sigma-Aldrich, St. Louis, MO, USA) after an overnight fast and fed with HFD until the end of the study. At the 2nd, 4th, and 8th week after STZ administration, the rats were killed and the blood and the liver samples were collected for subsequent analyses. Agematched rats were included as controls. For testing the insulin receptor signaling, mice were injected intraperitoneally with insulin solution (1 mU/g body weight) at 10 minutes prior to being killed.

| Histological analysis
Formalin-fixed, paraffin-embedded specimens were cut into 8 μ mol L −1 serial sections and subjected to standard hematoxylin and eosin (H&E) staining for histological examination. HepG2 and L02 cells grown in 6-well plates were washed with PBS and fixed with 10% neutral formalin, followed by staining with Oil Red O and hematoxylin. Sections and cells were imaged at 400× magnification (Olympus, Japan).

| Cell culture and treatments
Human hepatic cell line HepG2 and L02 cells were grown in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin in 5% CO 2 at 37°C. In order to establish a cellular model of hepatic steatosis, cells were treated for 48 hours with a mixture of FFA including oleate and palmitate in a final ratio of 2:1, at a final concentration of 1 m mol L −1 . To assess IR, IRS1, and ATK phosphorylation, cells were cultured with serum-free DMEM for 12 hours, and then stimulated with insulin (200 n mol L −1 ) for 10 minutes.

| Cellular triglyceride assay
Hepatocytes were collected for intracellular TG determination using a commercial kit (Applygen Technologies, China) according the manufacturer's instructions. TG values were normalized by total protein contents.

| MiRNA analysis
For quantitative expression analysis of miRNAs, total RNAs enriched with miRNAs were isolated by mirVana ™ miRNA Isolation Kit (Thermo fisher scientific, USA) from liver samples or HepG2 and L02 cells according to the manufacturer's instructions. Thereafter, cDNA was synthesized using PrimeScript ® RT reagent Kit (Takara, Japan) with specific miR-17 family RT primers (RiboBio, China) according to the manufacturer's instructions. Relative quantitative real time PCR (qPCR) was performed, using the SYBR Premix Ex Taq kit (Takara) in the ABI Prism 7900 (Applied Bio systems, USA) with the primers of the above miRNAs (RiboBio). The comparative cycle threshold (Ct) method was applied to quantify the expression levels of miRNAs.
Data are normalized over the average CT value of U6, and 2 −ΔΔCT method was used to determine relative miRNA expression.
SiRNA and plasmid transfection was carried out using lipofectamine ™ 3000 (Thermo fisher scientific), miRNA transfection was carried out using lipofectamine ™ RNAiMAX (Thermo fisher scientific) according to the manufacturer's instructions. At 24 hours after transfection, the effects of siRNA-or miRNA-mediated gene silencing and translational regulation were measured by western blot analysis.

| Statistical analysis
Differences between groups were analyzed, using conventional Student's t test or ANOVA. Each experiment was repeated at least three times, and the data are presented as mean ± SD. A P < 0.05 was considered to be statistically significant.  Figure 1B,D). Moreover, the serine phosphorylation of the insulin receptor substrate 1 (IRS1) was significantly increased, indicating that insulin receptor signaling was also impaired in this model ( Figure 1D). However, there were no significant differences in the levels of these metabolic parameters and hepatic PKNOX1 among the age-matched control rats (8-, 10-, 14-week-old) with normal feeding ( Figure S1). These data suggest that the increased hepatic Pknox1 may be specifically associated with the aberrant metabolism status in T2DM/NAFLD model. Subsequently, by using three publicly available algorithms (Tar-getScan, miRanda, and PicTar), we found that the 3′-UTR of the  The miR-17 family (also known as miR-17-92 cluster) maps to human chromosome 13 and consists of 6 individual miRNAs (miR-17, miR-18a, miR-19a, miR-19b, miR-20a, and miR-92a). 20 The organization and sequences of the miR-17-92 family is highly conserved among vertebrates, and gene duplication and deletion events during evolution have resulted in two mammalian paralogs: the miR-106b-25 cluster and the miR-106a-363 cluster. The miRNAs encoded by miR-17-92 and its two paralogs can be grouped into four seed families (miR-17, miR-18, miR-19 and miR-92). Among them, miR-17, miR-20a, miR-20b, miR-93, miR-106a, and miR-106b belonged to the miR-17 seed family, which share the same seed sequence. 21 As shown in Figure S2A,B; the 3′-UTR of the Pknox1 mRNA sequence contains two highly conserved target sites of miR-17 seed family and one conserved target site of miR-19 seed family. And it has been validated that Pknox1 as a target of both miR-17 and miR-19a in a previous study on mixed lineage leukemia. 22 However, the hepatic miR-19 level was slightly altered in the rat model of T2DM/ NAFLD ( Figure S2C). While, the hepatic levels of miR-17 seed family, especially miR-17 and miR-20a, were significantly downregulated and inversely correlated with those of Pknox1 in this model. The inversed correlation between miR-17 and Pknox1 was also confirmed in human samples (r = −0.981, P < 0.01). We found that Pknox1 expression levels were increased in the liver sections of NAFLD patients as compared to those in the healthy subjects, especially in the patients complicated with T2DM. While, the expression levels of miR-17 were decreased in the liver samples of NAFLD patient ( Figure S3). Therefore, we focused on miR-17-and miR-20amediated regulation of Pknox1 on hepatic glucose and lipid metabolism in the follow-up experiments.
Besides miR-17 family, miR-223 was shown to target and inhibit Pknox1 gene expression. 23 We also found that the hepatic level of miR-223 was gradually decreased by HFD-feeding and STZ administration. However, there is no obvious change of miR-223 level in the hepatocytes cultured by FFA containing medium as compared to those by normal medium ( Figure S4). It may be due to the specific expression tendency of miR-223, which is preferentially expressed in the hematopoietic system and is considered to play important roles in the differentiation of hematopoietic stem cells, myeloid, erythroid and lymphoid cells. 24 Thus, the dysregulation of miR-223 maybe presented mainly in hepatic macrophage and affects its function by targeting Pknox1. The study by Zhuang et al. support and complement our speculation. They revealed that miR-223 was a crucial regulator of macrophage polarization by way of inhibiting Pknox1 and then protected against diet-induced adipose tissue inflammatory response and systemic IR. 23 In contrast to macrophages, the expression of Pknox1 in hepatocytes was shown to be regulated mainly by miR-17 family. In FFA-induced hepatocyte steatosis, the hepatic expression level of Pknox1 was inversely correlated with miR-17 and miR-20a.
In addition, mutation of either miR-17 family-targeted MREs or of both two MREs in the Pknox1 mRNA 3′-UTR resulted in an increase in luciferase expression as compared to the wild-type reporter construct, which further verified an indeed regulatory relationship between the miR-17 family and the Pknox1 mRNA.
Moreover, supplement the level of miR-17 or miR-20a in hepatocytes by transfection with their mimics was shown to suppress Pknox1 expression and then enhanced insulin receptor signaling and reduced intracellular TG and lipid accumulation in hepatocytes.
These observations indicate that it is possible that miR-17 family mimics would serve as a novel approach for prevention and treatment of T2DM and NAFLD complication. However, to further determine the therapeutic effectiveness of miR-17 family-mediated regulation of Pknox1 in glucose and lipid metabolism disorders, the results from in vitro studies were verified in in vivo studies.
In conclusion, this study reveals that the downregulation of miR-17 family may cause the aberrant Pknox1 expression in glucose and lipid dysmetabolism status of T2DM complicated with NAFLD. Furthermore, we provide evidence that upregulation of miR-17 family can redress the insulin sensitivity and lipid metabolism in hepatocytes via targeting Pknox1. This evidence suggests a new pathogenic mechanism of NAFLD complicated with T2DM and may develop potential therapeutic strategies for metabolic disease.

ACKNOWLEDG EMENTS
The work was supported by the National Natural Science Fund

CONFLI CT OF INTEREST
The authors declare that they have no competing interests.