2.1. Plasmids, gene cloning, and reagents
Two different mouse Pnky shRNA cDNA fragments were cloned downstream of the U6 promoter in the pLVshRNA-Puro plasmid (#VL3102), which was purchased from Inovogen Tech. Co. (China). A mouse Pnky gene was cloned downstream of the CMV promoter in the dual expression lentiviral vector pLV-U6-CMV-Puro that was constructed by Deng's lab (Huang et al. 2019). The lentiviral particles expressing control shRNA (sc-108080) were purchased from Santa Cruz Biotech (USA). Restriction enzymes for gene cloning, such as BamHI (#R0136S), NotI (#R0189S) and HindIII (#R0104S), were purchased from New England Biolab (England). RNA miniprep kits (#AP-MN-MS-RNA-50) were obtained from Axygen (USA). RT-qPCR reagents (#4913914001) were purchased from Roche (Germany). Cy3-Pnky FISH probes and RiboTM Fluorescent In Situ Hybridization Kit (#C10910) were purchased from RiboBio Co., Ltd (China). All general chemicals were obtained from the SinoPharm Chemical Reagent Co. (China). Details about the key resource were listed in Table S1.
2.2. Cell culture and generation of stable cell lines
Murine immortalized NSC line C17.2 was maintained in DMEM (#SH30022.01) with the supplement of 10% fetal bovine serum (FBS, #SV30087), 5% horse serum (HS, #SH30074.02) and 1% penicillin/streptomycin (#SV30010) from Hyclone, Logan, UT, while NE4C cells were cultured in MEM medium (#SH30024.01, Hyclone) supplemented with 10% FBS, 2 mM L-glutamine and 1% penicillin/streptomycin.
C17.2 and NE4C cell lines that stably express Pnky shRNA were generated by transduction with the lentiviral particles expressing Pnky shRNA. In brief, when HEK 293T cells reached 70~80% confluence, they were co-transfected with the lentiviral packaging plasmids (PH1, PH2, and Pnky shRNA) using Lipofectamine 3000 (#L3000001, Invitrogen, MA, USA), and the lentiviral particles (expressing Pnky shRNA) were collected and concentrated 48 h later. C17.2 and NE4C cells were infected with the lentiviral particles, and cell lines stably expressing Pnky shRNA were screened from single-cell colonies using 96-well plates as described previously (Peng et al. 2020). Using the same strategy, we generated C17.2 and NE4C cell lines stably expressing Pnky gene as well as the cell lines expressing control shRNA.
2.3. RNA isolation and RT-qPCR
Total RNA was extracted with the RNA miniprep kit (Axygen), followed by cDNA synthesis using HiScript III 1st Strand cDNA Synthesis Kit (#R312-01, Vazyme, China) for detecting Pnky expression, or RevertAid first-strand cDNA synthesis kit (#K1622, Thermo Scientific) for determining the expression of related coding genes, according to the manufacturer's instructions. Quantitative PCR reactions were carried out using FastStart Universal SYBR Green Master (#4913914001, Roche, Germany) and gene-specific primers on a Bio-Rad CFX-96 detection system (Bio-Rad, CA, USA). qPCR data were analyzed with CFX Manager 3.1 software (Bio-Rad), and relative mRNA expression levels were calculated using the 2−ΔΔCt method. The housekeeping gene (GAPDH) was used for normalization.
2.4. Immunofluorescence staining
Immunofluorescence staining for Nestin in the above stable cell lines were performed with a standard protocol. Briefly, cells grown on the 14 mm (diameter) coverslips were fixed with 4% paraformaldehyde (PFA, Beyotime, China) for 20 min, followed by permeabilization with 0.2% TritonX-100 for 10 min at room temperature. Samples were then incubated with 10% goat serum for 1 h to block any non-specific interactions. Antibody against Nestin (#ab6142, Abcam, Cambrige, CA, USA) was added to the samples for incubation at 4 °C overnight. After washing with PBS, cells were further incubated with Cy3-conjuated secondary antibody (#A0521, Beyotime) for 2 h at room temperature. Nuclei were counterstained with mounting medium (#ab104139, containing DAPI, Abcam). Finally, the samples were imaged using an Olympus fluorescence microscopy (CKX53, Olympus, Japan).
2.5. Cell proliferation assays
Cell counting and 5-Ethynyl-2'-deoxyuridine (EdU) incorporation assay were performed to determine the cell proliferation rates of the stable cell lines. For cell counting assays, an equal number of cells (~3*104) were seeded onto 12-well plates where each cell type was grown in triplicate. Cell samples were collected every 24 hours and differential cell counts were obtained using a Countstar IC1000 cell counter (Countstar, China).
EdU is a thymidine analogue that can be incorporated into cellular DNA during DNA replication, and is usually adopted in the study of cell proliferation (Sun et al. 2016). As for EdU incorporation assay, the BeyoClick™ EdU cell proliferation kit with Alexa Fluor 555 (# C0075S, Beyotime) was used, according to the manufacturer's instructions. Briefly, cells in the logarithmic phase were seeded onto 12-well plates at a concentration of 3*105 and incubated overnight. The nest day, 10 μM EdU solution was added for 2 h incubation. Subsequently, cells were fixed with 4% PFA for 15 min and permeabilized with 0.3% Triton X-100 for 10 min at room temperature. After washing with PBS, 250 μl click reaction solution was added, followed by incubation for 30 min in dark. Finally, nuclei were stained with mounting medium (containing DAPI). At least 10 fields were selected randomly in each group and photos were taken under the Olympus fluorescence microscopy. The percentage of EdU-positive cells was compared between groups.
2.6. Wound healing assay
The effect of lncRNA Pnky in NSC migration was explored using wound healing assay. Corresponding cells were seeded onto 6-well plates and allowed to grow to 80% confluences in complete medium. Three parallel scratches were made using sterile pipette tips, and cell debris were washed off using PBS. Then, cells were further incubated in medium with 2% FBS for 48 h. The magnitude of wound healing was captured randomly using the Olympus fluorescence microscopy under bright field at the designated time points of 0 h, 24 h and 48 h, respectively. Widths of the wounds were measured and analyzed using ImageJ software.
2.7. Transwell migration assay
Transwell assay was also used to evaluate the migration of the obtained stable cell lines. In brief, 1*105 cells were suspended in 200 μl of serum-free medium and transferred to the upper chambers (# 3422, 8.0 μm pores, Corning, USA) that placed into 24-well plates. The bottom chambers were filled with 600 μl medium containing 10% FBS. After 24 h of incubation, the chambers were washed gently with cold PBS and replaced to new 24-well plates. Cells that migrated to the lower surface of the membrane were fixed with 1 ml methanol for 10 min, followed by staining with 1% crystal violet for 20 min. Whereas, cells that on the upper membrane surface were wiped off using fluffy swabs that were soaked with PBS. The numbers of migrated cells were photographed and calculated from 5-8 randomly selected fields under microscope. Experiment was performed six times with triplicate samples within each individual experiment.
2.8. Western blot analysis
Western blot analysis was performed as reported previously (Li et al. 2020b). 30 μg of cell lysates per lane were separated by 12% SDS-PAGE and transferred onto nitrocellulose membrane (Millipore, USA). After blocking with 5% nonfat dried milk, the membranes were incubated with the corresponding antibodies as follows. The antibodies against Paxillin (sc-365379), MMP2 (sc-13595), MMP9 (sc-393859), Connexin 43 (sc-13558) and GAPDH (sc-32233) were obtained from Santa Cruz, CA, USA. The antibodies recognizing ERK1/2 (#9102S), p-ERK (Thr202/Tyr204) (#9106S), AKT (#9272S), p-AKT (Ser473) (#4060S), p38 MAPK (#8690S) and p-p38 MAPK (#9216S) were obtained from Cell Signaling (MA, USA). The antibodies against SARNP (#ab225694), Aly/Ref (#ab202894), U2AF1 (#ab172614), THOC7 (#ab155218) and U2AF1L4 (#ab188582) were ordered from Abcam. HRP-conjugated goat anti-mouse or goat anti-rabbit IgG (Santa Cruz) were used to detect the primary antibodies above. Detection of the protein bands was performed with an enhanced chemiluminescence solution (Millipore, USA) using ChemiDocXRS + (Bio-Rad, CA, USA).
2.9. Colocalization of lncRNA Pnky and protein
As described elsewhere (Liu et al. 2015), cells on coverslips were first hybridized with Pnky probes conjugated with Cy3 (RiboBio), followed by immunofluorescence staining using antibodies of interest. Briefly, cells were gently rinsed in PBS and fixed in 4% PFA for 10 min at room temperature. Subsequently, cells were permeabilized in PBS containing 0.5% Triton X-100 for 10 min at 4℃, washed with PBS 3 times for 5 min, and pre-hybridizated in pre-hybridization buffer for 30 min at 37℃. Then, using anti-Pnky oligodeoxynucleotide probes, hybridization was performed in hybridization solution overnight at 37°C in dark moist chamber. On the second day, after sequential washing with 4ÎSSC，2ÎSSC and 1ÎSSC at 42°C in dark, cells were further fixed in 4% PFA for 5 min and incubated with SARNP (#ab225694), Aly/Ref (#ab202894), U2AF1 (#ab172614), THOC7 (#ab155218) and U2AF1L4 (#ab188582) antibodies at 4°C overnight, respectively. On the next day, cells were incubated with specific Alexa Fluor 488 conjugated secondary antibody (#A0428, #A0423, Beyotime) and counterstained with DAPI. A minimum of 12 fields were selected randomly in each group to analyze the colocalization of Pnky and the corresponding proteins. Images were taken using an Olympus confocal laser-scanning microscope (FV3000, Olympus, Japan).
2.10. RNA Immunoprecipitation (RIP)
RIP experiments were performed using a Magna RIPTM RNA-Binding Protein Immunoprecipitation Kit (#17-704, Millipore) according to the manufacturer’s instructions. Cells were scraped off from plates, collected, and lysed with complete RIP lysis buffer for 30 min on ice. Magnetic beads were incubated with 5 μg of antibodies, as SARNP (#ab225694), Aly/Ref (#ab202894), U2AF1 (#ab172614), THOC7 (#ab155218), U2AF1L4 (#ab188582) or normal Mouse/ Rabbit IgG (obtained from the above kit), respectively, with rotation for 40 min at room temperature. Then, beads-antibody complexes were washed adequately with RIP washing buffer, and cell lysates were added into the complexes and incubated at 4°C overnight. 10% of cell lysates (input) were stored at -80°C until starting RNA purification. The next day, with the help of magnetic separator, the RNAs associated with the corresponding proteins were pulled down. Immunoprecipitates and input were then subjected to protease K and heated at 55°C for 30 minutes to digest the proteins. Subsequently, RNAs were purified with phenol:chloroform:isoamyl alcohol, followed by precipitation using absolute ethanol. cDNAs were synthesized using HiScript III 1st Strand cDNA Synthesis Kit and the enrichment of Pnky was detected using RT-qPCR method as described above. Fold enrichment of Pnky was calculated relative to the percentage of input. The RNA and RNA-binding proteins complexes were also treated with SDS lysis buffer for further Western blot analysis to test the efficiency of immunoprecipitation.
2.11. Bioinformatics analysis
catRAPID (http://s.tartaglialab.com/catrapid/omics) was used to analyze the RNA-protein interactions (Agostini et al. 2013).
2.12. Statistical analysis
Throughout our research, appropriate sample size was determined based on the effect size, α error (0.05) and the Power (0.95), using the program GPower3.1 as reported previously (Serdar et al. 2021). Statistical analysis was carried out using IBM SPSS Statistics (version 20.0, IBM Corp., Armonk, NY, USA). Accordingly, normality and variance homogeneity were firstly assessed using Kolmogorov-Smirnov Test and Levene's Test. As for parametric data, Student's t-test or one-way ANOVA were used to assess the statistical significance, as appropriate, and results were expressed as mean ± SD of six independent experiments. If the data were not normally distributed or variance homogeneity was not met, nonparametric tests (Mann-Whitney U tests) were performed and data were displayed as median and interquartile range. Values of P < 0.05 were considered to indicate a statistically significant difference (*p < 0.05, **p < 0.01). GraphPad Prism 5 software (GraphPad Software, Inc., CA, USA) were used to make the statistical charts. As for the statistical analysis of wound healing assay, repeated measures tests were carried out using IBM SPSS Statistics. In brief, Mauchly's Tests of sphericity were firstly performed. If P ＞0.05, sphericity was met and sphericity assumed tests were used to assess the variances between the pairs. In case P < 0.05, sphericity was violated and Greenhouse-Geisser correction would further come into play.
2.13. Blind study statement
In all our experimental research, study participants, data collectors and data analysts are kept unaware of group assignment (control vs intervention).