Firstly, lncRNAs related to glioma were obtained by retrieving LINCDISEASE database (http://www.rnanut.net/lncrnadisease/index.html), and finally 142 Experimental and Experimental/Predicted lncRNAs were screened after the exclusion of duplicate items. Subsequently, GEO database (https://www.ncbi.nlm.nih.gov/geo/) was searched to obtain a microarray dataset of glioma, GSE15824, which comprised of 2 normal samples and 12 tumor samples. Differential analysis was then performed using the “limma” package of R language with normal samples as the control, while |logFC|>2 and p value<0.05 were regarded as the screening criteria for the differential expressed genes. Furthermore, the GEPIA database (http://gepia2.cancer-pku.cn/#index) was explored to the expression patterns of LINC00320, MYC and PLEKHA1 in relation to glioma in TCGA. Additionally, the downstream transcription factors and regulatory gene triplets of LINC00320 in low-grade glioma were predicted by searching the lncMAP database (http://bio-bigdata.hrbmu.edu.cn/LncMAP/survival.jsp).
A total of 60 cases of glioma were confirmed by pathology and collected from January 2013 to January 2014 for inclusion in the current study. The eligible patients received operations for the first time, including 38 males and 22 females (aged 46-67 years old; average age of 55.17 years old). Of the enrolled 60 cases, 31 cases were in stage I+II, 29 cases in stage III; 21 cases with KPS ≥80 points, and 39 cases with KPS <80 points. The exclusion criteria in our study omitted patients with severe metabolic system diseases, patients complicated with other malignant tumors, patients with incomplete clinical data, and patients with severe heart, kidney and lung dysfunction; and those with severe cognitive impairment. In addition, 35 cases of normal brain tissue resected by internal decompression operation in patients with severe brain injury were taken as the control group, and none of the aforementioned patients underwent radiotherapy and chemotherapy prior to operation. The follow-up lasted for 5 years, till January 2019, and was performed by using telephone or review. The overall survival (OS) rate of enrolled patients was calculated, and it was defined as the time from the randomization of enrollment to the death of patients for any reason. The OS of each group was observed and recorded for 5 years. At the end, during the follow-up period of 3-60 months, 7 patients were lost to follow-up, with a calculated follow-up rate of 88.33%.
Cell Culture and Transfection
The human microglia cell line CHME-5, glioma cell line SHG-44, U251 and BT325 cells was purchased from the Cell Resource Center of Shanghai Institute of Life Sciences, Chinese Academy of Sciences. CHG5 cells were purchased from Shanghai Qincheng Biotechnology Co., Ltd. SHG-44, The obtained cells were cultured in RPMI 1640 (w/o Hepes) medium containing 10% fetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin in a humidified incubator with 5%CO2 at 37 ℃.
According to the experimental requirements, SHG-44 cells at the logarithmic phase of growth were transfected, among which the lentivirus over-expression vectors were constructed according to the sequence information of LINC00320 and MYC. Subsequently, LINC00320 stable over-expression sequence(F: 5'-AATTTTACACATCTGTCTATACACAT-3’; F: 5'-TCAGTTGTCACTAAAGTAAGCAATGT-3’) and MYC over-expression sequence (F: 5’-TTCCTGTTGGTGAAGCTAACGTTGAGGGGCAT-3’; F: 5’-TCAACTGTTCTCGTCGTTTCCGCAACAAGTCC-3’) were transfected to the PLV vector. Based on the information of PLEKHA1, the specific siRNA sequence (SS sequence: GGGTAAATGTGTTAAACAA, and AS sequence: TTGTTTAACACATTTACCC) and its control sequence (SS sequence: GGUGGUGUGUGUCUGUAGU , and AS sequence: ACUACAGACACACACCACC) targeting human PLEKHA1 gene were connected to the PLKO-Puro vector. After correct sequencing, the plasmid was co-transfected with psPAX2 and pMD2.G (Addgen, USA) into the HEK293T cells. Supernatant of the culture medium containing lentivirus particles was collected to infect SHG-44 cells for screening the stable cell lines.
Total RNA content was extracted using Trizol kits (Invitrogen, California, USA), and PrimeScript RT kits (rr037a, Takara, Japan) were employed to reverse transcribe the RNA into cDNA. The system was 10ul and was operated in accordance with the manufacturer’s instructions. The reaction conditions were as follows: 37℃, 15 min×3 times (reverse transcription reaction), followed by processing at 85℃ for 5 s (reverse transcriptase inactivation reaction). The reaction solution was selected for the fluorescent quantitative PCR to perform fluorescent quantitative PCR according to the instruction of SYBR®Premix ExTaqTMⅡ kit (RR820A, TaKaRa). The reaction system was 50 μL in amount, including 25 μL of SYBR®Premix Ex TaqTMⅡ(2×), 2 μL of PCR upstream primer, 2 μL of PCR downstream primer, 1 μL of ROX Reference Dye (50×), 4 μL of DNA template, and 16 μL of ddH2O. Real-time fluorescence quantitative PCR system (ABI 7500, ABI, Foster City, CA, USA) was then carried out for fluorescence quantitative PCR, and the reaction conditions were as follows: pre-denaturation at 95℃ for 10 min, denaturation at 95℃ for 15 s, annealing at 60℃ for 30 s (40 cycles), and extension at 72℃ for 1 min finally. With β-actin serving as the internal reference, the relative expression of each target gene was calculated using the 2-ΔΔCt method according to the formula of ΔΔCt=ΔCt experimental group-ΔCt control group and ΔCt=Ct target gene-Ct internal reference, and each experiment was repeated three times. Relevant primers were designed by Sangon Biotech (Shanghai) Co., Ltd. (Table 1).
Total protein content in tissues or cells was extracted from RIFA lysate of SF, incubated on ice for 30 min, and centrifuged at 4 ℃ for 10min (8,000g) to obtain the supernatant. BCA kits were employed to detect the total protein concentration. Next, 50 μg protein was dissolved in 2 × SDS sampling buffer and boiled at 100℃ for 5 min. The above samples were then subjected to SDS-PAGE gel electrophoresis, and the proteins were transferred to PVDF membrane using the wet-transfer method, followed by sealing with 5% skim milk powder at room temperature for 1 h. After that, PVDF membrane was incubated overnight at 4℃ with the rabbit anti-MMP9 antibody (dilution ratio of 1:1000, ab38898, abcam, Cambridge, UK) and rabbit anti-cleaved-Caspase-3 antibody (dilution ratio of 1:500, ab49822, abcam, Cambridge, UK), with β-actin (dilution ratio of 1:2000, ab227387, abcam, Cambridge, UK) as the internal reference. After TBST washing three times (10min each), PVDF membrane was incubated with the HRP labeled goat anti-rabbit IgG H&L (HRP) (dilution ratio of 1:2000, ab97051, abcam, Cambridge, UK) for 1 h, followed by TBST washing. Same amounts of solution A and solution B solution were taken from the ECL fluorescence detection kit, mixed in dark conditions, and dropped onto the film for imaging in an gel imager. Photography was carried out with the Bio-Rad image analysis system (Bio-Rad company, USA), and associated with analysis using the Quantity One v4.6.2 software. The gray value of corresponding protein bands/β-actin protein bands represented the relative protein content. The experiment was repeated three times to obtain the mean value.
Subcellular localization of LINC00320 and MYC in the SHG-44 cell line was identified using immunofluorescence techniques. A cover glass was placed on a 6-well culture plate, followed by inoculation of SHG-44 cells, and then cultured for 1 d to make the cell fusion rate about 80%. The slides were taken out and washed with PBS. Then, 1 mL of 4% paraformaldehyde was added for cell fixation at room temperature, followed by the addition of 250 μL of pre-hybridization solution containing LINC00320 and MYC probes to incubate at 42℃ for 1 h after treatment with protease K (2 μg/mL), glycine and acetylate reagent. Next, the pre-hybridization solution was absorbed and 250 μL of hybridization solution containing probe (300 ng/mL) was added for hybridization overnight at 42℃. After PBST rinsing (×3 times), DAPI (dilution ratio of 1:800) staining solution diluted with PBST was added to stain the nucleus, followed by transferring to the 24-well culture plate, staining for 5 min; washing with PBST (×3 times), 3 min each time. Finally, five different fields of vision were observed and photographed under a fluorescence microscope (Olympus, Japan) after anti-fluorescence quenching agent for sealing.
SHG-44 cells from each group was treated with formaldehyde for 10 min to produce DNA protein cross-linking. An ultrasonic crusher was then employed and set to break the chromatin into fragments by 10s in 15 cycles, with time interval of 10s. After centrifugation at 4℃ for 10 min, the supernatant was collected and divided into two tubes. Negative control antibody of IgG of normal mice and specific antibody anti-MYC (#18583S, dilution ratio of 1:100, Cell Signaling Technology Inc.) of the target protein were added and incubated at 4℃ overnight for full binding. Next, the DNA protein complex was precipitated with Protein Agarose/Sepharose, followed by centrifugation (12,000g) for 5 min, and the supernatant was discarded. After washing the non-specific complex, the cross-linking was removed overnight at 65℃, and DNA fragments were extracted and purified with phenol/chloroform. Finally, the binding of MYC to the promoter region of PLEKHA1 was detected by means of PCR with PLEKHA1 using specific primers.
The binding of LINC00320 to MYC was detected using RIP kits (millipore, USA). After rinsing the SHG-44 cells with pre-cooled PBS, cells were lysed with RIPA lysate (P0013B, Beyotime) of equal volume in an ice bath for 5 min after discarding the supernatant, followed by centrifugation at 4℃ for 10 min (12,000 g). A portion of the cell extract was taken out as input and a part of that was incubated with antibody for co-precipitation. As for the specific steps, each co-precipitation reaction system was washed with 50 μL magnetic beads and then re-suspended in 100 μL RIP Wash Buffer, and 5 μg antibody was added to incubate for binding. After cleaning, the bead antibody complex was re-suspended in 900 μL RIP Wash Buffer and incubated overnight with 100 μL cell extract at 4 ℃. Next, the sample was placed on the magnetic base to collect the bead-protein complex. After digestion with protease K for the sample and input, RNA content was extracted for subsequent PCR detection. The antibody used for RIP was anti-MYC, which was mixed at room temperature for 30 min, and IgG was used as negative control.
Dual Luciferase Reporter Assay
The binding sites of the promoter region of PLEKHA1 with MYC were predicted by website analysis through lncMAP website. PLEKHA1 promoter region was transfected into pGL3-Basic vector (Promega) to serve as the recombinant vector of PLEKHA1-WT. Meanwhile, the MYC binding site mutation of PLEKHA1 was constructed into the pGL3-Basic vector (Promega) to serve as the recombinant vector of PLEKHA1-MUT. The correctly sequenced luciferase reporter plasmid WT and MUT were then transfected into HEK-293T cells with MYC respectively. After 48 h of transfection, luciferase activity was detected by collecting and cleaving cells. In accordance with the instructions of dual luciferase reporter assay kit, the cells were rinsed with PBS and lysed in 200 μL lysate for 15 min. Luciferase activity was measured at 560 nm using Firefly Luciferase Reporter Gene Assay kits (RG005, Beyotime, China) and a microplate reader. Each experiment was repeated three times to obtain the mean value.
CCK-8 Detection of Cell Growth Curve
The transfected SHG-44 cells were digested and re-suspended. Next, the cell density was adjusted to 1×105 cells/mL and inoculated in 96-well plate with 100μL per well, and allowed to culture overnight. The following day, the cells were treated according to the instructions of CCK-8 kit (Beyotime, Shanghai, China). The cell viability was detected using the CCK-8 method at 24, 48, 72, 96 h after inoculation. An amount of 10μl CCK-8 detection solution was added to each test, and placed in the incubator for 4 h. The absorbance of CCK-8 at wavelength of 450 nm was detected using a microplate reader to plot the growth curve.
Matrigel preserved at - 80 ℃ was taken out and allowed to melt into the liquid state overnight at 4 ℃. Next, 200 μL of Matrigel was added to 200 μL of serum-free medium at 4℃ and mixed well to dilute the matrix gel. An amount of 50 μL was then added to the upper chamber of each Transwell plate, placed into the incubator, and incubated for 2-3 h until solidification of glue. After digesting and counting, the cell suspension was prepared with serum-free medium. Then, 200 μL of cell suspension was added to the upper chamber of each well, and 800 μL of medium containing 20% FBS was added to the lower chamber, which was placed in an incubator at 37 ℃ for 20~24 h. Afterwards, the Transwell plate was taken out, rinsed twice with PBS, soaked in formaldehyde for 10 min, and then washed thrice with water. After 0.1% crystal violet staining, the cells were placed at room temperature for 30 min, rinsed twice with PBS and wiped off with a cotton swab. Finally, the cells were observed, photographed and counted under an inverted microscope. Transwell migration experiment required no matrix glue, and the incubation time was 16 h. Cells from at least four microscope areas were randomly selected to count. Each experiment was repeated three times to obtain the mean value.
After 48 h of transfection, cells were digested with 0.25% trypsin (free of EDTA) (YB15050057, Yu Bo Biotech Co., Ltd., Shanghai, China) and collected in a flow tube, centrifuged, and the supernatant was discarded. Next, the cells were rinsed thrice with PBS and centrifuged to discard the supernatant. According to the instructions of Annexin-V-FITC cell apoptosis detection kit (K201-100, Biovision, USA), Annexin-V-FITC, PI, HEPES buffer solution were prepared into an Annexin-V-FITC/PI staining solution at a ratio of 1:2:50. Cells at the density of 1×106 cells/mL were re-suspended every 100 μL of staining solution, and were evenly mixed by shaking. After incubation at room temperature for 15 min, 1 ml of HEPES buffer solution (PB180325, Procell, China, China) was added, and the cells were again evenly mixed by shaking. FITC and PI fluorescence were detected by 525 nm and 620 nm band-pass filters excited at 488 nm for the detection of cell apoptosis. Each experiment was repeated three times to obtain the mean value.
Tumorigenesis Experiment in Nude Mice
A total of 10 BALB/c male nude mice (aged 4-5 weeks; weighing about 18-22 g) were obtained from the Shanghai SLAC Laboratory Animal Co.,Ltd for in vivo experimentation. The lentivirus vector was constructed by over-expression of LINC00320 to obtain SHG-44 cell line with stable expression of LINC00320 and its empty vector. After cell concentration adjustment to 1.0×106/mL, 20 μL of cell suspension was taken and inoculated into the subcutaneous tissue of the abdomen of nude mice, and tumorigenesis was observed every 5 days. The maximum diameter ‘a’ and minimum diameter ‘b’ of transplanted tumor were measured using Vernier calipers, followed by calculating the weight and volume of tumor (TV) according to the formula TV=1/2×a×b2. After 30 d, nude mice in each group were euthanized, the tumor tissue was extracted, and the tumor mass was weighed with a balance. The measurement was repeated three times in each group. All the above experimental animals were approved by the Animal Care and Use Committee (Ethics Committee Approval No.201306005). Processing of the animal experiment in this study conformed to the management and usage principles of local experimental animals.
Paraffin embedded sections of mice were dewaxed and hydrated for 10 min, and xylene I and II were used to dewax for 10 min respectively, followed by dehydration with gradient alcohol and two PBS rinses (5 min each time). After soaking with 3% H2O2 for 10 min and another two times of PBS rinsing (5 min each time), high-pressure antigen repair (Beyotime, China) was conducted for 90s, followed by cooling at room temperature and section rinsing with PBS. Next, 5% BSA blocking solution was added for incubation at 37℃ for 30 min, followed by the addition of 50μl of VEGF rabbit monoclonal antibody (dilution ratio of 1: 250, ab32152, abcam, Shanghai, China) and CD31 rabbit monoclonal antibody (dilution ratio of 1:50, ab28364, abcam, Shanghai, China) in a refrigerator at 4℃ overnight. After PBS rinsing for 2min, 50 μL HRP labeled goat anti-rabbit secondary antibody (dilution ratio of 1:10000, ab205718, abcam, Shanghai, China) was added for incubation at 37℃ for 30 min. With the addition of SAB working solution, DBA (Fuzhou Maixin Biotechnology Development Co., Ltd.) was used for development, followed by re-staining with hematoxylin for 5min, observation and photography under optical microscope (XSP-36, Bostar Optical Instruments Co., Ltd., Shenzhen, China). Five high power fields (×200) were randomly selected for each section, with 200 cells in each field, with the purpose of analyzing the ratio of positive cells .
Statistical analyses were performed using the SPSS 21.0 software (IBM SPSS statistics, Chicago, IL, USA). Measurement data were expressed by mean ± standard deviation. Normal tissues and cancer tissue data were evaluated by paired t-test; data between groups were compared by unpaired t test; and that among multiple groups were compared by one-way analysis of variance (ANOVA) and Tukey's post-hoc test. Two-way ANOVA was applied for cell activity at different time points, and repeated ANOVA was used for tumor volume at different time points. Bonferroni correction was made in post-hoc tests. Pearson correlation analysis was performed for correlation between the two indexes. Kaplan-Meier test was used to analyze the survival of patients with high and low expression, and Log-rank test adopted for difference comparison. A value of p < 0.05 was regarded statistically significant.