Clinical samples
Samples were taken from CD patients with colonic and ileal inflammatory lesions. Normal tissue was taken from healthy subjects undergoing colonoscopy. All samples were obtained from the affiliated Jiangning Hospital with Nanjing Medical University between October 2019 and October 2020. All participants had signed informed consent forms. Our experiments were approved by the ethic community of the affiliated Jiangning Hospital with Nanjing medical university. The clinical characteristics of CD and control patients are presented in Table 1.
Cell culture and transfection
IEC-6 cells (ATCC, Manassas, VA) were cultured with DMEM containing contained with 10% fetal bovine serum (FBS) at 5% CO2, 37 °C. The shRNAs for LINC01272 as well as its corresponding scrambled siRNAs (sh‐NC) were purchased by Hanbio (Shanghai, China). The miRNA oligonucleotides including miR‐153-5p mimic, miR‐153-5p inhibitor, NC mimic and NC inhibitor were obtained from RiboBio (Guangzhou, China). Transient transfection was performed by using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Cells were harvested 48-h post transfection for further experiment.
Establishment of CD mouse models
We used 2,4,6-trinitrobenzenesulfonic acid (TNBS) to construct the CD mouse model following the previous study 25. Thirty female BALB/C mice were randomly divided into the control group (n=10), TNBS group (n = 10), and TNBS + sh-LINC01272 group (n = 10). The control group was fed a conventional diet, and the latter two groups were used TNBS (3.75 mg) to establish the CD model. Mice were shaved before the experiment and coated with TNBS 3.75 mg (dissolved in 48% ethanol). After fasting for 24-h, mice were narcotized using pentobarbital sodium on the 7th, 14th and 21st day after the coated TNBS, respectively. The trocar was inserted into the colon with a 20G trocar in the prone position, the top of which was about 4cm away from the anus. An ethanol solution of 100 μl TNBS was relaxedly injected into lumen immediately by a 1 ml syringe fixed with a trocar. In the third group, mice were injected intraperitoneally with 500μL sh-LINC01272. The blank control and the model group were treated with same amount of saline. After the first treatment, the disease activity index (DAI) scores were recorded according to the previous report 26.
qRT-PCR analysis
Using Trizol to isolate RNA from cells or tissues. After the RNA was completely dissolved, the 5×PrimeScript® RT Master Mix kit was used for reverse transcription (10 μL). qRT-PCR was carried out by SYBR Premix Ex Taq TM (Takara, Otsu, Japan) on StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific). Here are the conditions of PCR reaction: Maintaining 94°C for 5 min, followed by 40 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 60 s. The primers for qRT-PCR were shown in Table 2.
Western blot
Bicinchoninic Acid (BCA) kit (Wuhan Bost Biotechnology Company, Hubei, China) was used to extract the total proteins required for the experiment and detect its concentration. Then add 30 mg/well of loading buffer to the extracted proteins and boil it in a 95℃-water bath for 5 min After that, it was first separated by 10% polyacrylamide gel electrophoresis (Wuhan Bode Biotechnology, Hubei, China), and transferred to PVDF transmembrane (Millipore). Finally, the transferred transmembrane was placed in skim milk for 1 h. These bands were incubated with primary antibodies (1:1000, Abcam, MA, USA) at 4℃ overnight. Subsequently, these bands were treated with indicated secondary antibodies for 1-2 h. The dual-color infrared fluorescence scanning imaging system (Odyssey, Licor, NE, USA) was used to capture images, and then Image J 1.52v imaging analysis software (NIH, Bethesda, MD, USA) was used for analysis.
Immunofluorescence
IEC-6 cells were cultured in immunofluorescence-specific plates. Cells were washed using PBS and fixed in 4% paraformaldehyde for 20 min. Then cells were blocked with 0.1% BSA for 30 min at 23℃. Next, cells were followed by incubating with primary rabbit anti-collagen I (1:100 dilution) or rabbit anti-collagen III antibodies (1:100) overnight at 4°C. Alexa 555 secondary antibodies (Molecular Probes, Thermo Fisher Scientific, USA) and DAPI were used to incubate the cells. The cells were observed under a fluorescence microscope (Keyence, Ōsaka, Japan).
RNA Binding Protein Immunoprecipitation
The RIP experiment was carried out with the Magna RIP kit (Magna, ON, CAN). Briefly, the lysed cells were treated with the RIP buffer solution, and the magnetic beads were labeled using anti-Ago2 or IgG. The abundance of LINC01272 and miR-153-5p was verified by qRT-PCR.
Luciferase reporter assays
The promotor region of LINC01272 was cloned by PCR and inserted into the pGL3-LINC01272-WT and MUT plasmids through enzyme digestion to construct renilla luciferase vector. For the promotor region of LINC01272, the forward primer was “CGACUGACGCCG”, the reverse primer was “UCGACAUCGA”. The miR-153-5p plasmids were co-transfected with pGL3-LINC01272 WT-renilla luciferase vector or pGL3-LINC01272 MUT-renilla luciferase vector into IEC-6 cells by Lipofectamine 2000 (Invitrogen). After the transfection plasmid was added to cells by the Lipofectamine 3000 (Invitrogen) for 48 h, the renilla luciferase in cell lysate was assessed by the dual-luciferase reporter assay system (BEST, Nanjing, China).
Masson staining
The tissue sections were stained with Masson's reagent according to the following procedures: (i) fixed in Bouin's or Zenker's liquor overnight, (ii) washed in running water until the yellow color fades and rinsed in distilled water for twice, (iii) stained with hematoxylin for 5 min, (iv) placed in 0.5% hydrochloric acid in 70% alcohol for 5 s, (v) washed for 30 s in running tap water and rinsed in distilled water for twice, (vi) stained with acid ponceau for 5-10 min and rinsed in distilled water for three times. Finally, cells were observed under a microscope.
HE staining
Briefly, the samples were dewaxed and hydrated with gradient alcohol, the sections were incubated in hematoxylin solution for 15 min followed washed with PBS. Secondly, the slices were counterstained with 0.5% eosin solution for 5 min, dehydrated with gradient alcohol, cleared, and sealed. Finally, photos were observed with light microscope (Keyence, Ōsaka, Japan).
Subcellular fraction
Cells were obtained in PBS and resuspended in ice 500 μL CLB buffer for 10 min. Homogenization was implemented by applying 15 strokes using a 1 ml needle on ice. Thereafter, 50 μl of 2.5M sucrose was added to restore isotonic conditions. The first round of centrifugation was implemented at 6300g for 5 min in a tabletop centrifuge at 4°C. The pellet washed with TSE buffer at 4000g for 6 min in a tabletop centrifuge at 4°C until the supernatant was clear. The resulting supernatant discarded and the pellets were nucleus. The resulting supernatant from the first round of differential centrifugation was sedimented for 150 min at 14000 rpm in a tabletop centrifuge. The resulting pellets were membranes and the supernatant was cytoplasm.
Wound-healing assay
All instruments are sterilized on the clean bench. Use a marker pen to draw a horizontal line 0.5~1cm from the back of the 6-well plate. At least 5 lines pass through each well. Add about 5*10^5 cells to the plate. The next day, use the tip of the pipette to make the scratch on the horizontal line. The tip of the pipette should be vertical and not inclined. Washing 3 times, serum-free medium was added into 6-well plate, and we put the plate into an incubator. Taking pictures at 0, 6, 12, and 24 h.
Transwell assay
The transwell membrane was covered with invasion matrigel. Briefly, the cells were plated on upper chambers with a serum‐free medium at a density of 5 × 104 cells/well, while the bottom chamber was filled with medium with 10% FBS. After incubation for 24 h, the invaded cells were fixed with ice‐cold methanol followed by staining with crystal violet (0.1%). Finally, the cells observed under a microscope (Olympus, Tokyo, Japan).
Bioinformatic prediction of lncRNA-miRNA interaction
The bioinformatics prediction was performed through the website: http://starbase.sysu.edu.cn/mirLncRNA.php.
Statistical analysis
All the results were presented as means ± SD. The significance between two groups was assessed by Unpaired Student’s t-test or paired t-test, and multiple groups was evaluated by one-way ANOVA with Bonferroni post-hoc tests. P < 0.05 was considered significant.