All animal experiments were carried out in strict accordance with the regulations for the Administration of Laboratory Animals of Guangdong Province. These experiments were handled in compliance with, and were approved by, the Institutional Animal Care and Use Committee at the South China Agricultural University (Guangzhou, China) with approval number SCAU#0011. All efforts were made to minimize animal suffering.

Six 7-week-old XH female chickens were obtained from the Avian Farm of the South China Agriculture University (Guangzhou, China). Chickens were euthanized, and organs and tissues were collected after rapid dissection, then immediately frozen in liquid nitrogen and stored at −80°C. The samples included the cerebrum, cerebellum, hypothalamus, pituitary, heart, liver, spleen, lung, kidney, muscular stomach, glandular stomach, breast muscle, leg muscle, abdominal fat, and subcutaneous fat.

Total RNA was extracted from each tissue using Trizol reagent (TaKaRa, Japan) following the manufacturer's protocol. The integrity and concentration of all obtained RNA samples were assayed by 1.5% agarose gel electrophoresis, and evaluated by ultraviolet spectroscopy using the 260/280 nm ratio.

A PARIS Kit (Ambion, Life Technologies, USA) was used to harvest the cytoplasmic and nuclear cell lysates. Fresh cultured cells were placed on ice, after being collected and washed once in PBS. Subsequently, cells were resuspended in 500 μL ice-cold Cell Fractionation Buffer and incubated on ice for 10 min. After centrifugation for 5 min at 4°C and 500 × g, the cytoplasmic fraction was carefully aspirated away from the nuclear pellet; thereafter, the nuclear pellet was washed in ice-cold Cell Fractionation Buffer and lysed in Cell Disruption Buffer. Samples were split for RNA isolation, following the manufacturer's protocol.

The PrimeScript RT Reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Japan) was used to synthesize cDNA. The iTaq Universal SYBR Green Supermix Kit (Toyobo, Japan) was used for cDNA quantification, according to the manufacturer's protocol, in a BioRad CFX96 Real-Time Detection instrument. The chicken β-actin gene was used as an internal control. Data analysis was carried out using the comparative 2^−ΔΔCT method (Livak and Schmittgen, 2001).

Primers were designed using Premier Primer 5.0 software (Premier Biosoft International, Palo Alto, CA, USA) or OLIGO Primer Analysis Software Version 7 (Molecular Biology Insights, USA), and synthesized by Sangon Biotech (Shanghai, China). The major primers used in this study are listed in Table 1. Of these primers, lncRNA-Six1 5′ RACE-outer, lncRNA-Six1 5′ RACE-inner and lncRNA-Six1-3′ RACE were used to clone the full-length of chicken lncRNA-Six1. The other 7 primers (lncRNA-Six1-ORF-1, lncRNA-Six1-ORF-2, lncRNA-Six1-ORF-3, lncRNA-Six1-ORF-4, lncRNA-Six1-ORF-5, lncRNA-Six1-ORF-6, and lncRNA-Six1-ORF-7) were used for the open reading frame (ORF) amplification of lncRNA-Six1, and pSDS-β-actin was used as a positive control. Moreover, primers pSDS-lncRNA-Six1 and pSDS-Six1 were used to amplify the coding sequences, and then construct the overexpression vector. The last 4 primers (pGL3-basic-P1, pGL3-basic-P2, pGL3-basic-P3, and pGL3-basic-P4) were used for the dual-luciferase reporter assay for the promoter region of Six1. Primers for RT-quantitative PCR (qPCR) are shown in Table 2. The siRNAs used for the knockdown of Six1 were synthesized by Guangzhou RiboBio (Guangzhou, China) and are listed in Table 3. LncRNA-Six1 is an RNA molecule present in the cytoplasm and nucleus. The siRNA and ASO that were used for the specific knockdown of lncRNA-Six1 in the cytoplasm and nucleus, respectively, were designed and synthesized by Guangzhou RiboBio (Guangzhou, China), and are listed in Table 3.

The partial lncRNA-Six1 sequence was obtained from our previous lncRNA-seq data (accession number GSE58755). RACE PCR was performed to obtain the full-length sequence of the lncRNA-Six1. Total RNA from breast muscle tissue was used as the template for nested-PCR reactions using a SMARTer RACE cDNA Amplification Kit (Clontech, Osaka, Japan), following the manufacturer's instructions. The products of the RACE PCR were cloned into the pJET 1.2/blunt cloning vector (CloneJET PCR Cloning Kit; Fermentas, Glen Burnie, MD, USA) and sequenced by Sangon Biotech (Shanghai, China).

For lncRNA-Six1 overexpression plasmid construction, the full-length sequence of lncRNA-Six1 was amplified by PCR, and cloned into the expression plasmid pSDS-20218 (SiDanSai, Shanghai, China) by using BsaI restriction enzyme. Six1 overexpression constructs were generated by amplifying the Six1 coding sequence, which was then subcloned into the overexpression plasmid vector, pSDS-20218 (SiDanSai, Shanghai, China). Seven ORFs of lncRNA-Six1 were also amplified and cloned into pSDS-20218 (SiDanSai, Shanghai, China). A region from the GFP gene was amplified and cloned into pSDS-20218 as a negative control, and was named pSDS-Control.

Luciferase reporter vectors including different sized Six1 promoter fragments were constructed from the chicken genome using XhoI and HindIII restriction sites. The PCR products were excised with XhoI and HindIII restriction endonucleases and ligated into plasmid vector pGL3-Basic (Promega, Wisconsin, USA). The recombinant constructs were named pGL3-basic-P1 (−2247/+216), pGL3-basic-P2 (−1595/+216), pGL3-basic-P3 (−1129/+216), and pGL3-basic-P4 (−495/+216). These were numbered relative to the first base of the initiation codon of the Six1 gene.

DF-1 cells were cultured in DMEM (Gibco, USA) supplemented with 10% (v/v) fetal bovine serum (Hyclone, USA) and 0.2% penicillin/streptomycin (Invitrogen, USA). QM-7 cells were cultured in high-glucose M199 medium (Gibco, USA) with 10% fetal bovine serum, 10% tryptose phosphate broth solution (Sigma, USA), and 0.2% penicillin/streptomycin. All cells were cultured at 37°C in a 5% CO₂, humidified atmosphere. In this study, DF-1 cells were used to study the function of lncRNA-Six1, lncRNA-Six1-ORF-2, and Six1 in chicken genome. For QM-7 cells, were used to study the influence of Six1 on muscle growth.

All transient transfections used Lipofectamine 3000 reagent (Invitrogen, USA) following the manufacturer's protocol. The concentrations used for plasmid transfections were as follows: 6-well plate, 2.5 μg/well; 12-well plate, 1 μg/well; 24-well plate, 0.5 μg/well; 48-well plate, 0.25 μg/well; 96-well plate, 0.1 μg/well. For siRNAs and ASO, a concentration of 100 nM were used. si-lncRNA-Six1 and ASO-lncRNA-Six1 were co-transfected for interference with lncRNA-Six1, and named as si-ASO-lncRNA-Six1 in this study. The PRL-TK vector (Promega, USA) was used as an internal control plasmid for Renilla luciferase expression and was co-transfected with the pGL3 luciferase reporter vectors. pSDS-Control and pGL3 basic were used as the empty vector control plasmids. In this study, except for Cell Counting Kit (CCK)-8 assays, all cells were harvested or analyzed after 48 h transfection.

The recombinant plasmids (pGL3-basic-P1, pGL3-basic-P2, pGL3-basic-P3, and pGL3-basic-P4), used to analyze the Six1 promoter region, were co-transfected with pRL-TK in DF-1 cells. The pGL3-basic was also co-transfected with pRL-TK as a control. Firefly and Renilla luciferase activities were measured at 48 h post-transfection using a Dual-GLO Luciferase Assay System Kit (Promega, USA), following the manufacturer's instructions. Luminescence was measured using a Fluorescence/Multi-Detection Microplate Reader (BioTek, USA) and firefly luciferase activities were normalized to Renilla luminescence in each well.

DF-1 cellular proteins were extracted using radioimmunoprecipitation assay (RIPA) buffer with phenylmethanesulfonyl fluoride protease inhibitor. Western blot analysis was performed as previously reported (Feng et al., 2016). The antibodies used for Western blots were as follows: Flag tag polyclonal antibody (20543-1-AP; Proteintech, USA; 1:1,000), goat anti-rabbit IgG (H&L)-HRP (BS13278; Bioworld, USA; 1:5,000), rabbit anti-Six1 (sc-9127; Santa Cruz, USA; 1:100), myogenin antibody (orb6492; Biorbyt, UK; 1:100), B103 (DHSB, USA; 0.5 μg/ml), goat anti-rabbit IgG-HRP (BA1054; Boster, China; 1:5,000), peroxidase-goat anti-mouse IgG (BA1051; Boster, China; 1:2,500), and HRP-conjugated monoclonal mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (KC-5G5; Kangchen, China; 1:10,000).

At 48 h after transfection, DF-1 cells were incubated at 37°C for 2 h in the presence of 50 μM EdU (RiboBio, China). The cells were then fixed in 4% paraformaldehyde for 30 min and neutralized using 2 mg/mL glycine solution. The cells were permeabilized by adding 0.5% Triton X-100. A solution containing EdU (Apollo Reaction Cocktail; RiboBio, China) was added and the cells were incubated at room temperature for 30 min. The nuclear stain Hoechst 33342 was then added and incubation was continued for another 30 min. The number of EdU-stained cells was determined using images of three randomly selected fields obtained with a fluorescence microscope (TE2000-U; Nikon, Japan).

Flow cytometry analysis was performed on a BD AccuriC6 flow cytometer (BD Biosciences, USA) and data was processed using FlowJo7.6 software. The cell cultures in growth media were collected after a 48 h transfection and fixed in 70% ethanol overnight at −20°C. Propidium iodide (Sigma, USA) (50 μg/mL) containing 10 μg/mL RNase A (Takara, USA) was added and incubated for 30 min at 37°C in the dark and then the samples were sent for detection.

For the cell growth assays, cells were seeded in a 96-well plate. After being transfected with (a) pSDS-lncRNA-Six1, (b) si-ASO-lncRNA-Six1, (c) pSDS-Six1, or (d) si-Six1, the proliferation of the cell culture was monitored at 24 h, 48 h, 72 h, and 96 h using the TransDetect CCK (TransGen Biotech, Beijing, China). Every 24 h, 10 μL of CCK solution was added and the absorbance at 450 nm was measured using a Model 680 Microplate Reader (Bio-Rad) after a 1 h incubation.

DF-1 cells were seeded in 12-well plates. After a density of 50% confluence was reached, cells were transfected. Subsequently, a linear wound was generated by scratching the monolayer of cells with a 1 mL pipette tip, when the cells reached a density of 100% confluence. The proliferation and migration of cells were captured at 0, 24, 48, and 72 h by using a microscope (TE2000-U; Nikon, Japan). The width of the scratches was measured by Nano Measurer software. The formula to calculate the cell migration rate was as follows: (W0h – W × h) × 100%/ W0h, where W0h represents the mean wound width at 0 h, and W × h represents the mean wound width at 24, 48, and 72 h.

After transfection for 48 h, cells were fixed in 1% formaldehyde for 10 min at 37°C, and quenched with 125 mM glycine for 5 min. Samples were then washed twice with cold PBS and placed in SDS lysis buffer. After sonication process [(5 s pulse + 5 s interval) × 25% of maximum power, for 14 times], chromatin was immunoprecipitated with the DYKDDDDK Tag (D6W5B) rabbit monoclonal antibody (14,793; Cell Signaling, USA; 1:50), as previously reported (Harms et al., 2015). The relative quantity of the immunoprecipitated factor was calculated by qPCR.

All results are presented as the mean ± S.E.M, based on at least three independent experiments for each treatment. Statistical significance of differences between means was assessed by performing an unpaired Student's t-test and P < 0.05 or less was considered significant.

We identified the 5′ and 3′ ends of lncRNA-Six1 by RACE analysis (Figure 1A and Supplementary Data 1). Predictions of the identity of lncRNA-Six1 by the Coding Potential Calculator suggested a low coding potential and low evolutionary conservation consistent with a non-coding RNA (Kong et al., 2007; Figure 1B). In order to verify this prediction, we analyzed the coding ability of seven potential ORFs of lncRNA-Six1 by Western blot. We found that only ORF-2 generated a micropeptide (7.26 kDa), suggestive that lncRNA-Six1 is an lncRNA with low protein-encoding potential (Figure 1C and Supplementary Data 2). The National Center for Biotechnology Information's Basic Local Alignment Search Tool (BLAST) showed that the lncRNA-Six1 was 2,350 bp long, located on chromosome 5 and spanned from 54,483,370 to 54,487,175, and comprised two exons. The micropeptide was also located at chromosome 5 from 54,484,978 to 54,485,172, and the 3′ end of lncRNA-Six1 was separated from Six1 by 432 bp (Figure 1D). We also investigated the intracellular localization of lncRNA-Six1, the RT-PCR results confirmed that lncRNA-Six1 is an RNA molecule present in the cytoplasm and nucleus (Figure 1E).

We measured lncRNA-Six1 expression from different tissues from 7-week-old XH chickens using RT-qPCR. LncRNA-Six1 was expressed in all 15 tissues, and breast muscle had the highest levels (Figure 1F). This suggested that lncRNA-Six1 may function in chicken skeletal muscle development.

To unveil functions for lncRNA-Six1, we performed lncRNA-Six1 overexpression and knockdown experiments to assess its role in cell proliferation and viability. The plasmid pSDS-lncRNA-Six1 was constructed for lncRNA-Six1 overexpression, and siRNA and ASO for lncRNA-Six1 knockdown (si-ASO-lncRNA-Six1) were designed and synthesized. After 48 h of transfection, the relative RNA expressions of lncRNA-Six1 were detected (Figures 2A,B). LncRNA-Six1 overexpression significantly promoted cell proliferation, as judged by EdU incorporation (Figures 2C,E). Conversely, proliferation was significantly inhibited after lncRNA-Six1 knockdown (Figures 2D,F). LncRNA-Six1 overexpression also resulted in a slight decrease in the number of G0/G1 cells, and a slightly increase in the number S phase cells. LncRNA-Six1 knockdown reversed this trend, and G0/G1 cells increased significantly and S phase cells decreased significantly (Figures 2G,H). In addition, we also used the CCK-8 assay and found that lncRNA-Six1 overexpression promoted an increase in cell number. However, with lncRNA-Six1 interference, the opposite was true (Figures 2I,J). Together, these data were indicative that lncRNA-Six1 has a positive regulatory effect on cell growth and proliferation, and induces cell division.

In order to further understand the molecular mechanism of lncRNA-Six1, we predicted its cis-regulated target genes on the UCSC Genome Browser (http://genome.ucsc.edu/) (The differentially expressed genes at 100 Kb upstream or downstream of differentially expressed lncRNAs were selected as the potential cis target genes of lncRNAs) (Li et al., 2017; Figure 3A). The results showed that the lncRNA-Six1 is located upstream of Six1, which suggests that Six1 may be a potential cis-regulatory target gene for lncRNA-Six1.

To investigate whether lncRNA-Six1 targeted regulate Six1, we cloned individual segments from the Six1 promoter in front of firefly luciferase. These constructs were used to transiently transfect DF-1 cells to determine the region required for Six1 promoter activity. Deletions extending from −1595 to +216, −1129 to +216, and −495 to +216 showed no significant difference in luciferase activities compared with the pGL3-basic control group. However, a significant increase of luciferase activity was observed with the longest construct (−2247 to +216) when compared with the region from −1595 to +216. This was indicative that an essential region of the Six1 promoter was located at −2247 to −1595 bp (Figure 3B). Interestingly, the 3′ end of lncRNA-Six1 was separated from Six1 by 432 bp and the whole RNA overlaps the key promoter regions of Six1 (Figure 3C). This was suggestive that this lncRNA is the regulatory factor of Six1.

More importantly, after overexpression of lncRNA-Six1, the mRNA and protein levels of Six1 increased, while inhibition of lncRNA-Six1 down-regulated Six1 mRNA and protein (Figures 3D,E). Therefore, we confirmed that Six1 is a direct target gene of lncRNA-Six1 in chickens.

To further investigate whether the micropeptide was involved in the cis-regulation of Six1, the relative mRNA expression of the lncRNA-Six1-ORF-2 was detected after overexpression or interference of lncRNA-Six1 (Figures 4A,B). The intracellular localization and tissue expression profiles of lncRNA-Six1-ORF-2 were also analyzed (Figures 4C,D). The results showed that lncRNA-Six1-ORF-2 was present in the cytoplasm and nucleus, and highly expressed in breast muscle. Overexpression of lncRNA-Six1-ORF-2 was performed to study its physiological effects (Figure 4E). A cell cycle analysis after overexpression of lncRNA-Six1-ORF-2 was carried out (Figure 4F). Moreover, we also used the scratch-wound assay and found that lncRNA-Six1-ORF-2 overexpression promoted cell proliferation and migration (Figures 4G,H).

The mRNA and protein levels of Six1 were significantly increased after overexpression of lncRNA-Six1-ORF-2 (Figure 4I). Meanwhile, a dual-luciferase reporter assay was developed to measure the Six1 promoter activity, after co-transfection with pSDS-lncRNA-Six1, pSDS-lncRNA-Six1-ORF-2, and si-ASO-lncRNA-Six1 with pGL3-basic-P1 (Figures 4J,K). We found that the relative luciferase activity was significantly increased with the overexpression of lncRNA-Six1 or lncRNA-Six1-ORF-2, while the knockdown of lncRNA-Six1 caused the relative luciferase activity to be decreased significantly. In the chromatin immunoprecipitation assay, it was found also that the micropeptide was enriched by the Six1 promoter region (Figures 4L,M). These results indicate that the micropeptide may be a key factor required for lncRNA-Six1 to act in cis.

We examined tissue expression profiles for Six1 and found high expression in breast and leg muscle (Figure 5A). Six1 overexpression and knockdown in DF-1 and QM-7 cells verified the positive regulatory function of Six1 in myogenic proliferation (Figures 5B,C). In DF-1 cells, overexpression of Six1 significantly promoted cell proliferation (Figures 5D,F). This also resulted in a significant decrease in the number of cells in G0/G1, and a significant increase in the number of S-phase cells (Figure 5H). However, cell proliferation was inhibited after interference of Six1 expression (Figures 5E,G), and resulted in a larger number of G0/G1 cells and fewer S phase cells (Figure 5I).

We found similar results for transfected QM-7 myoblasts. Six1 overexpression significantly reduced the number of cells that progressed to G0/G1 and increased the number of S phase cells (Figure 5J). These observations coincided with enhanced cell viability (Figure 5L). Conversely, Six1 knockdown significantly inhibited myoblast proliferation and reduced cell viability (Figures 5K,M).

To identify the role of Six1 in the chicken skeletal muscle growth, the mRNA expression level of growth-related genes, such as MYOG, MYHC, MYOD, IGF1R, and INSR, were detected by qPCR after overexpression or knockdown of Six1. Furthermore, the protein levels of MYOG and MYHC were also detected by Western blot. The results showed that overexpression of Six1 could promote the upregulation of growth-related genes (Figure 5N). In contrast, growth-related gene expression was inhibited after interference with Six1 (Figure 5O). This data demonstrated that Six1 promoted the expression of muscle-growth-related genes and further induced chicken muscle growth.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.