MicroRNA‐351 eases insulin resistance and liver gluconeogenesis via the PI3K/AKT pathway by inhibiting FLOT2 in mice of gestational diabetes mellitus

Abstract Gestational diabetes mellitus (GDM) is known as different degree glucose intolerance that is initially identified during pregnancy. MicroRNAs (miRs) may be a potential candidate for treatment of GDM. Herein, we suggested that miR‐351 could be an inhibitor in the progression of GDM via the phosphoinositide 3‐kinase/protein kinase B (PI3K/AKT) pathway. Microarray analysis was used to identify differentially expressed genes and predict miRs regulating flotillin 2 (FLOT2). Target relationship between miR‐351 and FLOT2 was verified. Gestational diabetes mellitus mice were treated with a series of mimic, inhibitor and small interfering RNA to explore the effect of miR‐351 on insulin resistance (IR), cell apoptosis in pancreatic tissues and liver gluconeogenesis through evaluating GDM‐related biochemical indexes, as well as expression of miR‐351, FLOT2, PI3K/AKT pathway‐, IR‐ and liver gluconeogenesis‐related genes. MiR‐351 and FLOT2 were reported to be involved in GDM. FLOT2 was the target gene of miR‐351. Gestational diabetes mellitus mice exhibited IR and liver gluconeogenesis, up‐regulated FLOT2, activated PI3K/AKT pathway and down‐regulated miR‐351 in liver tissues. Additionally, miR‐351 overexpression and FLOT2 silencing decreased the levels of FLOT2, phosphoenolpyruvate carboxykinase, glucose‐6‐phosphatase, fasting blood glucose, fasting insulin, total cholesterol, triglyceride, glyeosylated haemoglobin and homeostasis model of assessment for IR index (HOMA‐IR), extent of PI3K and AKT phosphorylation, yet increased the levels of HOMA for islet β‐cell function, HOMA for insulin sensitivity index and glucose transporter 2 expression, indicating reduced cell apoptosis in pancreatic tissues and alleviated IR and liver gluconeogenesis. Our results reveal that up‐regulation of miR‐351 protects against IR and liver gluconeogenesis by repressing the PI3K/AKT pathway through regulating FLOT2 in GDM mice, which identifies miR‐351 as a potential therapeutic target for the clinical management of GDM.


| INTRODUCTION
In Asia, it is reported that the incidence of diabetes is rapidly increasing and that no less than 60% of the patients diagnosed with diabetes is alive. 1 Diabetes is one of the most common diseases among Chinese population which has been specially analysed in Chinese reports. 2,3 Gestational diabetes mellitus (GDM) is a cumulatively obstetric disorder and a complication with variable severity of glucose intolerance when pregnancy is initially recognized. 4 Insulin resistance (IR) is associated with GDM, and gluconeogenesis is also involved in GDM due to correlation with IR. 5,6 Multiple gestation is a factor associated with rising risk of GDM. 7 Varying other factors, including genetic factors and environmental ones, may impact the pathogenesis of GDM, and a prevalent theory is that GDM is arisen from IR in pregnancy and the metabolic defects of β-cell dysfunction. 8 Both the mother and her children may be negatively affected by both short-and long-term adverse health outcome brought by GDM. 3 Nowadays, women still desire for intensive counselling and treatment for GDM around the world, 9 so it is urgent to elucidate the molecular mechanisms underlying GDM development and identify novel prognostic markers and molecular therapeutic targets for improving the diagnosis, prevention and treatment of GDM.
Flotillins are known to be correlated with vesicular invaginations of the plasma membrane and regulation of signal transduction. 10 Recently, flotillin 2 (FLOT2) was identified to be a newly discovered target for miR-34a in melanoma. 11 Evidence showed that FLOT2 was in interaction with signalling molecules such as receptors, kinases, adhesion molecules and G proteins in a direct manner. 12 In addition, a previous study revealed that FLOT2 was an insulin pathway in type 2 diabetes mellitus (T2DM). 13 Furthermore, GSE87295 database revealed that FLOT2 was up-regulated in GDM patients compared with the normal individuals. The association between FLOT2 and phosphoinositide 3-kinase/protein kinase B (PI3K/AKT3) pathway has been explored and the mechanism was dependent on MMPs expression, E-cadherin expression, Foxo1 activity and cell cycle arrest. 14 Besides, a earlier finding indicated the role of PI3K/ AKT pathway in diabetes. 15 The participation of PI3K/AKT pathway in GDM was pointed out in a previous paper and sex hormone-binding globulin exerts a function during the process by affecting the insulin signal transduction pathway. 16 MicroRNAs (miRs) are recognized as small endogenous RNAs which account for altering gene-expression post-63 transcriptionally and different biological process. 17,18 Furthermore, miR-351 was presumed to bind to the 3'untranslated region (3'-UTR) of FLOT2 in microRNA.org. MicroRNA-351 is a family member of the interferon β (INFβ)-inducible mRNAs which plays a role in accelerating cellular antiviral activities. 19 Micro-RNA-351 level is temporarily elevated in the period of muscle regeneration and implicated in muscle atrophy. 20 Based on the aforementioned literature, we could suggest that miR-351 regulated GDM by mediating FLOT-dependent PI3K/AKT pathway. In this current study, we aimed to identify the role of miR-351 in GDM by down-regulating FLOT via PI3K/AKT pathway.

| Ethic statement
This animal experiment was approved by the animal ethics committee of Linyi People's Hospital. In addition, best efforts were made to minimize the suffering of animals.

| Microarray analysis
The GDM related gene expression chip GSE87295 was downloaded from the Gene Expression Omnibus (GEO) database (https://www. ncbi.nlm.nih.gov/geo/), which included vascular endothelial cell gene expression data of five GDM samples and five control samples. The annotated platform for GSE87295 was the GPL10558-Illumina HumanHT-12 V4.0 expression beadchip. The differential analysis was carried out with R. Software "limma" package 21  with standard food with free access to water and food. The experiment was conducted after adaptive feeding for 1 week.

| Establishment of GDM mouse model
The GDM mouse model was established by intraperitoneal injection of streptozotocin (STZ). Before the experiment, venous blood of mouse tail was collected to determine the fasting blood glucose (FBG), with 3-5 mmol/L as the normal blood glucose value. The vaginal smear method was used to determine the oestrus cycle of mice.
The female and male mice in the early stage of oestrus were mated.  with 20 μg of each were dissolved in 2.5 mL of saline, respectively, and then rapidly injected into the tail vein of mice, and mice in the normal and GDM groups were injected with the equal amount of saline.

| Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
The liver tissues of mice in each group were grounded into uniform fine powder. The total RNA was extracted using Trizol (Invitrogen Inc., Carlsbad, CA, USA). The ratio of optical density (OD) 260 nm /OD 280 nm was measured using an ultraviolet spectrophotometer to determine the purity of the RNA and to ensure that the OD value was between 1.8 and in each group after transfection were determined.

| Western blot analysis
The total protein was extracted from grinding liver tissues in each group using radio-immunoprecipitation assay (RIPA) kit (R0010, Bei-   SPSS 21.0 (IBM Corp., Armonk, NY, USA) was employed for statistical analysis. The experiment was repeated three independent times. The measurement data were displayed as mean ± SD. All data were tested for normal distribution and homogeneity of variance. Comparison between two groups was performed with t test, and comparisons among multiple groups with one-way ANOVA. P < 0.05 was considered to be statistically significant. network was plotted ( Figure 1B). The differentially expressed genes that interacted with disease-related genes in the interaction network were FLOT2, CD44 and IGFBP2, which revealed that these three genes may affect GDM. There were studies showing that CD44 26 and IGFBP2 27,28 were related to GDM. Therefore, we mainly focused on the role of FLOT2 in GDM. As shown in Figure 1A, the expression of FLOT2 was higher in GMD samples than that in control samples. Moreover, it was demonstrated that the alteration of the PI3K/AKT pathway could influence GDM, 29 and other studies revealed that FLOT2 could regulate the PI3K/AKT pathway. 14,30 With this respect, we speculated that the differential expression of
There was a specific binding region between the sequences of miR-351 and FLOT2 (Figure 2A). Further, dual luciferase report gene assay was performed to verify that miR-351 could bind to FLOT2. As shown in Figure 2B, compared with the NC group, the luciferase activity of 3'UTR in FLOT2-Wt was significantly inhibited by miR-351 mimic (P < 0.05), while the luciferase activity of 3ʹUTR in FLOT2-Mut showed no significant difference (P > 0.05). These findings demonstrated that miR-351 could specifically bind to FLOT2-3ʹUTR, and down-regulated the expression of FLOT2 after transcription.

| miR-351 overexpression and FLOT2 silencing reduce pancreatic cell apoptosis and alleviate GDM in mice
Haematoxylin-eosin staining was performed to observe the pathological changes of the pancreas under different transfection. As shown in Figure 3A  group. 31 All above, it was cleared that the pathological changes of liver cells could be alleviated by overexpressing miR-351 and silencing of FLOT2 in GDM mice.

| Overexpression of miR-351 and silencing of FLOT2 promote insulin sensitivity and rescue islet β-cell function in GDM mice
Glucose oxidase method was used to measure the content of FBG, ELISA for FINS and end-point method for TC and TG. And the content of FBG, FINS, TC and TG was used for evaluating the level of HOMA-IRI, HOMA-β% and HOMA-ISI. As shown in Table 2, com-

| MiR-351 inhibits the PI3K/AKT pathway by down-regulating FLOT2 in GDM mice
Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis were applied to determine the levels of PI3K, AKT and FLOT2 as well as the extent of PI3K and AKT phosphorylation after different transfection. As shown in  Notes. One-way ANOVA was performed for statistical analysis, and the experiment was repeated three times (n = 10).

| DISCUSSION
Gestational diabetes mellitus is known as a severity of glucose intolerance which is initially diagnosed during pregnancy, and it is increasingly prevalent among Chinese female population as a pivotal public health problem. 32,33 Significantly, GDM without recognition and treatment is likely to bring maternal and foetal consequences, so it needs to be early detected and treated. 34 Treatment to reduce blood glucose concentration alone or together with special obstetric care seems to effectively avoid the possibility of some perinatal complications, thus ameliorating GDM. 35   the invasion and migration of lung adenocarcinoma, miR-133 targeted with FLOT2. 39 Flotillins, also named as reggie proteins, consist of FLOT1 and FLOT2, two commonly expressed and highly conserved proteins. 40 FLOT2, initially found to occur in goldfish, is commonly expressed in tissues of mammals and encodes a membrane protein correlated with caveolae. 38 FLOT2 is shown to be regulated by miR-802 and miR-449a. 41,42 Besides, a prior study revealed that FLOT2 was an insulin pathway in T2DM. 13 More importantly, FLOT2 has been proved as a factor related with β cell function, while IR and β cell function are known as two important pathological factors for diabetes mellitus occurrence. 43 Furthermore, GSE87295 database in our study revealed that FLOT2 was up-regulated in GDM patients compared with the normal people.
Furthermore, our study provided evidence that miR-351 overexpression inhibits the PI3K/AKT pathway by suppressing FLOT2. As an inhibitor in a glucose uptake pathway, 44 the AKT pathway has been reported to regulate the invasion and migration of lung adenocarcinoma cells by targeting FLOT2. 39 In meantime, FLOT2 exerts a cancerous role through the PI3K/AKT3 pathway, thus accelerating the nasopharyngeal carcinoma cell cycle. 14 Similar to our finding, miR-485 exerts a negative function on metastasis and epithelial-mesenchymal transition in lung adenocarcinoma by targeting FLOT2 via the PI3K/AKT pathway. 45 Epidermal growth factor-regulated activation of the PI3K/AKT pathway has been verified to be of great importance in promoting placenta development and foetal growth in humans and rodents. 29 PI3K/AKT pathway activation reverses IR and promotes glycometabolism by a novel formula Sang-Tong-Jian (STJ) in T2DM. 46 Liuwei Dihuang decoction, a traditional Chinese medicine formula, intervenes IR by down-regulating the PI3K/AKT pathway of T2DM rats in liver. 47 Moreover, PI3K/AKT signalling has been proved to play a role in cardiomyocyte apoptosis induced by high glucose. 48 In this study, insulin stimulated PI3K/AKT pathway.
Then diabetes PI3K/AKT pathway was activated in GDM (due to increased insulin level) yet did not translate to improved glycemic effect, suggesting the incidence of IR. These findings came to a demonstration that miR-351 negatively regulated PI3K/AKT pathway by down-regulating FLOT2. Another intriguing finding emerging in this study is that miR-351 overexpression attenuates IR and gluconeogenesis in liver according to the decrease of PEPCK and G-6-Pase and increase of GLUT2. The close relationship between PEPCK and IR has been well investigated and identified. 49 PEPCK exerts a role in development of IR in mice and confers high susceptibility to det-induced IR, 50,51 which is identified to be a significant indicator for diagnosing and treating diabetes regulated by the PI3K/AKT pathway. 52 Both PEPCK and G-6-Pase are in tight and positive association with gluconeogenesis. 53,54 Similar to our finding, miR-877-5p is inversely correlated with PEPCK expression and miR-451 negatively modulated G-6-Pase expression. 55,56 The primary hepatic liver transporter GLUT2 is a transporter with low affinity and high capacity which is closely related to insulin receptor. 57 AKT, a significant downstream node of the PI3K/AKT pathway is responsible for altering PEPCK and G-6-Pase expression levels via Forkhead box protein O1. 58 Besides, the PI3K/AKT pathway is also implicated in up-regulation of the expression of GLUT2 and GLUT4 via STJ. 46 Taken together, we can make a conclusion here that miR-351 overexpression decreases PEPCK and G-6-Pase, and increases GLUT2, thus improving insulin sensitivity and inhibiting gluconeogenesis in liver.
To be concluded, these preliminary results found that miR-351 eases IR and liver gluconeogenesis in GDM via PI3K/AKT pathway by down-regulating FLOT2 ( Figure S1). With the aim to overcome some of the limitations of our study, further work needs to be done on larger samples and on treatment of patients with GDM.
Studies focusing on mechanisms of action underlying would better characterize the role of miR-351 targeted FLOT2 via PI3K/AKT pathway.

ACKNOWLEDG EMENTS
We would like show sincere appreciation to the reviewers for critical comments on this article.