BKS.Cg-Dock7m+/+Leprdb/JNju (db/db) mice and identical genetic background BKS heterozygote db/+ mice were all obtained from the Model Animal Research Center of Nanjing University (Jiangsu, China). 16-week-old homozygous mice were housed individually in sterile microisolators for the duration of the experiment. The db/db mice were maintained for 8 weeks with hyperglycemia (random blood glucose level ≥ 16.7 mmol/l). db/+ mice (random blood glucose level < 11.1 mmol/l) were used as the controls .
All experimental procedures were performed by the Animal Care Committee of Qingdao University.
Reverse transcription-quantitative PCR (RT-qPCR) analysis
Total RNA was extracted from cell lines and tissue samples with a TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's manual. Then, RT-qPCR was performed with PrimeScript™ RT Master mix and qPCR SYBR® Premix Ex Taq™ (Takara Biotechnology Co., Ltd.). The following thermocycling conditions were used for qPCR: denaturation at 95°C for 7 min; followed by 40 cycles of denaturation at 95°C for 15 sec and 60°C for 1 min. miRNA levels were performed by SYBR PrimeScript™ miRNA RT-PCR kit (Takara Biotechnology Co., Ltd.). The following primers were used in Table 1. The RNA levels were calculated using the the 2-CqΔΔ method .
MiRNAs potentially bind to the 3’UTR of Rspo mRNA were predicted using 2 different algorithms between TargetScan 7.2 (http://www.targetscan.org/) and miRanda (http://www.miranda.org).
Culture of cell lines
293T cells were obtained from American Type Culture Collection and were cultured in DMEM (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), and 1% penicillin/streptomycin and streptomycin (0.1 mg/ml; Sigma-Aldrich; Merck KGaA) at 37°C with 5% CO2.
Primary IEC isolation
The small intestine was dissected out from mice to expose the crypts and villi through sliced longitudinally. All procedures as described previously [12, 14, 15, 16].
The siRNAs targeting Rspo3, miRNA mimic (agomiR-380-5p) and inhibitor (antagomiR-380-5p) were synthesized and purchased from Guangzhou RiboBio Co., Ltd. (China). Cells (2×105 cells/well) were plated into six-well plates and incubated overnight prior to transfection. At 40–60% confluence, cells were transfected with siRNAs (15 nM), miRNA mimic (15 nM) or inhibitor (15 nM) into cells using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.).
Dual-luciferase reporter plasmid transfection
The wild-type (WT) or mutant-type (MUT) of the 3’UTR of Rspo3 sequences was cloned into the pmiR-RB-REPORT™ plasmid (Guangzhou RiboBio Co., Ltd., Guangzhou, China). After incubation for 48 h, the cells were collected and firefly and Renilla luciferase activity were was measured using the Dual-Luciferase Reporter Assay system (Promega Corporation, Madison, WI, USA). Firefly luciferase activity was normalized to Renilla luciferase activity. The luciferase efficiency was evaluated at 2 min following Stop & Glo® reagent using SpectraMAX Multifunctional Microplate Reader (Molecular Devices).
Downregulating the expression of Rspo3 in vivo
Mice were randomly divided into four groups, with 24 in each group. All mice received a tail vein injection once a day for 3 days. The db/+-NS group comprised control mice receiving saline (0.9%; same volume as the experimental group) injections [14, 15, 17, 18], and the db/db-NS group comprised DM mice receiving saline (0.9%; same volume as the experimental group) injections [14, 15, 17, 18]; the db/db-si-Rspo3 mice received injections of Rspo3 siRNA (80 mg/kg body weight, 14,15, 17,18), and the db/db-agmiR-380-5p mice received injections of agmiR-380-5p (80 mg/kg body weight, 14,15, 17,18). In each group, six mice were euthanized with an intraperitoneal injection of ketamine/xylazine (100/10 mg/kg body weight) on day 0 (prior to injection), day 2, day 4, and day 6 for further study.
In situ hybridization
A DIG-labeled LNA-miR-380-5p probe was synthesized following the manufacturer’s instructions by RiboBio (Guangzhou, China.) In brief, a 5-mm section of paraffin-embedded tissues was incubated with methanol in PBST solution, then fixed with 4% formaldehyde solution and washed with SSC buffer, and then permeabilized with Triton X-100 solution. The tissues was incubated with DIG-labeled LNA-miR-380-5p probe for hybridization at 37°C overnight. Then, miR-380-5p expression was determined using diaminobenzidine solution (1:900; Boster Biological Technology), following the staining intensity was observed using a microscope BX51 (Olympus Corporation). The staining was quantified by counting the number of positive cells at a magnification of x400.
Immunohistochemistry and immunofluorescence.
The paraffin-embedded mice tissue samples were sectioned at a thickness of 5 µm sections. The sections were deparaffinized in xylene and rehydrated in an ethanol gradient. Tissue sections was quenched by a peroxidase-blocking solution (Dako), and then incubated in milk for 5 min, and overnight at 4℃ with primary antibodies, including anti-SI antibody (1:100; cat no. ab84977), anti-Tff3 antibody (1:150; C cat no. sc398651), anti-Lyz1 antibody (1:150; cat no. ab189937), anti-ChgA antibody (1:100; cat no. ab15160), anti-Rspo1 antibody (1:200; cat no. ab106556), anti-Rspo2 antibody (1:100; cat no. ab132836), anti-Rspo3 antibody (1:150; cat no. ab233113), anti-Rspo4 antibody (1:100; cat no. ab189515), and anti-β-actin antibody (1:100; cat no. ab8226) (all from Abcam, Inc.).
For immunohistochemistry, the section was incubated with anti-HRP rabbit/mouse secondary antibody (Dako, Glostrup, Denmark) at room temperature for 2h, and color developed by DAB (Dako). The sections were stained with Mayer's hematoxylin solution and dehydrated by xylene, and mounted under a microscope.
For immunofluorescence, the section was incubated with Alexa Fluor 488 conjugated anti-rabbit (1:1000; cat no. 4412; CST) and 546 conjugated anti-mouse (1:1000; cat no. A11030; Thermo) secondary antibody after the primary antibody. The nucleus was stained DAPI (1:5000; cat no. D8417; Sigma, D8417). And the fluorescent pictures were detected by the FV1200 laser scanning microscope (Olympus).
Protein extraction and western blotting
Total protein from tissues was using RIPA Buffer (Thermo Fisher Scientific, Inc.) supplemented with protease inhibitor cocktail (Roche Applied Science). Protein samples (40 µg/sample) for each group was loaded and resolved on a 10% SDS-PAGE gels, and subsequently transferred to the polyvinylidene fluoride (PVDF) membranes membrane (EMD Millipore). Then, the membranes were blocked with 5% skim milk at room temperature for 1 h and incubated at 4˚C overnight with primary antibodies: anti-SI antibody (1:400; cat no. ab84977), anti-Tff3 antibody (1:400; C cat no. sc398651), anti-Lyz1 antibody (1:250; cat no. ab189937), anti-ChgA antibody (1:200; cat no. ab15160), anti-Rspo1 antibody (1:1,000; cat no. ab106556), anti-Rspo2 antibody (1:1,000; cat no. ab132836), anti-Rspo3 antibody (1:1,000; cat no. ab233113), anti-Rspo4 antibody (1:1,000; cat no. ab189515), and anti-β-actin antibody (1:1,000; cat no. ab8226) (all from Abcam, Inc.). The blots were incubated with the horseradish peroxidase-conjugated secondary antibodies (cat no. 7074S; Cell Signaling Technology, Inc.) at 37˚C for 1 h at room temperature and visualized using an enhanced chemiluminescence Ultra Western HRP Substrate kit (cat no.WBULS0100; EMD Millipore). Signals were analyzed by Quantity One software version 4.6.2 (Bio-Rad Laboratories, Inc.) and the intensity values were normalized to β-actin.
Results data are represented as the mean ± standard deviation and analyzed using the statistical software package (SAS 8.0 for Windows; SAS Institute, Inc., Cary, NC, USA). Comparisons between groups were analyzed with a Student’s test, and one-way ANOVA with Tukey’s post hoc test used for multiple group comparisons. P < 0.05 was considered as the criterion for statistical significance.