Mice and antibiotic treatment
Female C57BL/6 mice, 4- to 5-week-old, were acquired from the Shanghai Experimental Center of the Chinese Science Academy (Shanghai, China). As mentioned above [21], mice were treated with neomycin sulfate (1 g/L), ampicillin (1 g/L), vancomycin (0.5 g/L), and metronidazole (1 g/L) in their drinking water for 4–6 weeks, which was replaced twice per week. Mice were anesthetized with isoflurane, their eyeball was removed for blooding, then the mice were killed by severing the spinal cords. Animal experiments were approved by the Animal Care and Use Local Ethics Committee. The procedure was carried out according to the Guide for the Care and Use of Laboratory Animals issued by Anhui Medical University.
Segregation of lung mononuclear cells
As mentioned above [22], by density gradient centrifugation, and using 40% and 70% Percoll solution, mononuclear cells were isolated from the lungs.
Purification of AMs
Segregated lung mononuclear cells were stained, subsequently, AMs (F4/80+ CD11c+) were sorted using a FACS Aria II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). The purity of separated cells was more than ninety-five percent.
Microarray analysis
Microarray analysis was conducted as previously described [12]. Microarray experiments were implemented by Shanghai Biotechnology Company (Shanghai, China). Principal component analysis was performed using ClustVis [23], with Pareto scaling applied to rows, and singular value decomposition with imputation was used for principal components calculation. Each sample was from sorted AMs from 15 mice.
GO and KEGG pathway analysis
The target genes of lncRNAs were predicted in trans and cis to reveal the potential function of lncRNAs. To predict cis roles (referring to the effect of lncRNAs on adjacent target genes) coding genes 10 kb downstream and upstream of the lncRNA were searched. For prediction of trans functions (referring to the impact of lncRNAs on the expression of other genes) GO and KEGG pathway analysis of target genes regulated by lncRNAs were performed as previously mentioned [24, 25]. By using R Bioconductor genomewide mouse annotations and the library GO stats 2.34.0 from the package org.Mm.eg.db (version 3.3.0), target genes (fold-change ≥ 2) were converted to Entrez-IDs for GO and KEGG pathway analysis in R 3.2.3. Results were sorted according to p-value (p < 0.05).
Quantitative Real-time PCR
Total RNA was extracted and purified using an miRNeasy Mini Kit (Cat. #217004, Qiagen, GmBH, Germany). Reverse transcription reactions were performed in 80 µl volumes: 5 µg of total RNA, 8 µl 5 × buffer, 5 µmol/L oligo dT, 5 µmol/L random primers, 10 mmol/L DTT, 0.5 mmol/L dNTPs, 800 units of M-MLV, 160 units of RNase inhibitor, and distilled water. Reverse transcription reactions were conducted at 25 °C for 5 min, 37 °C for 50 min, heating at 70 °C for 15 min. For PCR, 50 µl volume reactions contained 0.4 µmol/L forward and reverse primers, 25 µl 2 × PCR buffer, and 2 µl cDNA template. Quantitative real-time PCR was implemented using a Roche Light Cycler 480 II (Roche Diagnostics, Germany). PCR reactions were conducted using a total of 45 cycles, including a 25 sec melting step at 95 °C, a 30 sec annealing step at 60 °C, and 50 sec of extension at 72 °C. Information of the gene-specific primers is provided in Table 1.
Table 1
The primers for each gene detected by real-time PCR
Gene | Forward primer (5’-3’) | Reverse primer (5’-3’) | Amplicon length (bp) | Ref |
LncRNA-30691 | TCCTCTACCACTCTCTGCCT | GGGTGGGAATGGAAGGAAGA | 282 | This work |
LncRNA-2705 | CCTGGGTAGAGAGAGATGCG | GTAGCATGTCCAACAGCAGG | 191 | This work |
LncRNA-73745 | TACACTGGTGCCTGCCTTTA | TGGTATCAGGGATGCTGCAT | 180 | This work |
LncRNA-92004 | TTGGTGGCTTTCTGGATTGC | CTTCCCTGTTGTCCCTGGAT | 275 | This work |
LncRNA-1605 | GCTCGCTCATCAAGAAACGT | CTTCCTGGTTCAAGCTCTGC | 257 | This work |
LncRNA-13019 | TGCATCATCGACTCAGGGAA | ATCTCTGTCTCACCCACGAG | 168 | This work |
LncRNA-65144 | TCTCTTTTCCCAAGGCCGAA | CATTCCCAAAGCTGAGCGAT | 208 | This work |
LncRNA-87138 | AAGCCACCTACTACTGTGCA | TCTTCAGCGTATCCCCTTCC | 279 | This work |
LncRNA-87135 | TGGGGAAAGAGCACAGTTGT | AGAGAAGCTGATGATGGCCA | 185 | This work |
LncRNA-12429 | GCCAACAGATTCAGCTAGCC | TGTGAATCGCTGTCCTCCTT | 211 | This work |
LncRNA-87049 | CCTGGCTGTTCCCCTATGAA | GCACGGAACATTTCACGGAT | 301 | This work |
LncRNA-29008 | AAGTGGCTCTGTCTGGTCTC | CCCCATTGCTGTGCTGAAAT | 172 | This work |
LncRNA-51618 | AGGAAGCCGAGGGAAAGAAA | GGGGAAAGAAGGAAAGGGGA | 165 | This work |
LncRNA-54499 | GCATTTGTGAGCACTGACCA | TCCAGTGTTCCCAGATGTCC | 212 | This work |
LncRNA-10921 | CTGGGCAGTAAAACGAACGT | GGGACCCAGCCATCATCTTA | 282 | This work |
LncRNA-44232 | GGTCTCGTTTTCTGCTGTGG | GTTAGCCCTGGTGTCTTTGC | 248 | This work |
LncRNA-35328 | GAGGCCAGGAAGAGGAGAAG | AATAAGCAGCCCACCAGACT | 323 | This work |
LncRNA-75509 | TCTGTGCACGGCTCTATAGG | ATGAAGCCTTGGTGAGAGCA | 179 | This work |
LncRNA-64449 | TCTTGATGAGCAGCTTGGGA | CAGGCTGTGATGGAAGATGC | 224 | This work |
LncRNA-5252 | CGCTATGAGTCGTGTGTGTG | CCAGTGGAAGCAGAGACAGA | 175 | This work |
LncRNA-58315 | TGGTTCAGTTCTCCAGCCAT | AGTTGCAATTGACAGCCCAG | 240 | This work |
LncRNA-22486 | CCACCTGCTGCATTTGAGTT | TGCCACATAGATCCACTGGG | 285 | This work |
LncRNA-73020 | TCAGAGGGCCCAGTTTCATT | CACACATGCACCTACAGTCG | 294 | This work |
LncRNA-74533 | TGCATTATGTGAGCCTCCCA | AAGGCCTCCATGATTCACCA | 211 | This work |
LncRNA-22855 | AAGGGATGTGTGCAGCTTTG | TGATTACTGGAGTGGCTGCT | 157 | This work |
LncRNA-78402 | AGACCTACATTGAGCCCACC | GGGTAGCAGAGTGTGGATGT | 244 | This work |
LncRNA-42066 | GTGTGCCCTTCATCTCGTTC | AAGAGTGGTAGGGAAAGGGC | 288 | This work |
LncRNA-30162 | TATTTCACCAAGCACCGCAC | TGCTGACACATAACGTTGCA | 275 | This work |
LncRNA-4821 | GAAACTTCCGTGTCCTGTAGG | GAGACTGTGAGCCTGACTGA | 194 | This work |
CCL24 | CTCCTTCTCCTGGTAGCCTG | ATGGCCCTTCTTGGTGATGA | 183 | [12] |
Arg1 | GCTGGGAAGGAAGAAAAGGC | TGCCGTGTTCACAGTACTCT | 232 | [12] |
IGF1 | ACTGGAGATGTACTGTGCCC | CAAAGGATCCTGCGGTGATG | 253 | [12] |
RBP4 | TTCTGTGGACGAGAAGGGTC | GTGCCATCCAGATTCTGCAG | 249 | [12] |
Timp1 | TATGCCCACAAGTCCCAGAA | ACTCTCCAGTTTGCAAGGGA | 210 | [12] |
β-actin | TGACGTTGACATCCGTAAAGACC | CTCAGGAGGAGCAATGATCTTGA | 148 | [33] |
GAPDH | TGCACCACCAACTGCTTAG | GGATGCAGGGATGATGTTC | 177 | [34] |
FISH analysis
For FISH assays, fluorescence-conjugated lncRNA30162 probes were acquired from Shanghai Gene Pharma Co. Ltd. (Shanghai, China). RAW264.7 macrophages were acquired from the Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China), and were treated in nondenaturing conditions, followed by hybridization with lncRNA 30162 probes. The slide was hybridized at 37 °C for 16 h. After RNA hybridization, samples were incubated in 4′,6-diamidino-2-phenylindole (1:1000) for 5 min of counterstaining, and observed by confocal microscopy (TCS SP5, Leica).
RNA interference
RAW264.7 cells (1 × 105) were plated in 6-well plates and transfected with small interfering RNA (siRNA), including si-NC, si-GAPDH, and si-lncRNA30162 sequences, using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). The siRNA sequences were provided in Table 2. After 48 h, cells were harvested for total RNA extraction. Knockdown efficiency was assessed using RT-qPCR. All siRNA sequences were supplied by Shanghai GenePharma Company (Shanghai, China).
Table 2
The siRNA sequences for RNA interference
Gene | sense (5’-3’) | antisense (5’-3’) |
silncRNA-30162 | GCCGUGAAUUAUCUAACAATT | UUGUUAGAUAAUUCACGGCTT |
siGAPDH | CACUCAAGAUUGUCAGCAATT | UUGCUGACAAUCUUGAGUGAG |
siNC | UUCUCCGAACGUGUCACGUTT | ACGUGACACGUUCGGAGAATT |
ELISA
Culture supernatants from RAW264.7 cells were collected and centrifuged to remove cellular debris. ELISAs for detection of ARG1 (KB13668, Shanghai Jiang Lai Biotechnology Co., China) and CCL24 (ab100681, Abcam, UK) were conducted following the manufacturer’s instructions.