Effect of LINC00320 Regulating the Expression of PLEKHA1 through the Transcription Factor MYC in Glioma Cell Proliferation, Migration, Invasion and Apoptosis In vivo and In vitro

Aim: This study was carried out to explore the mechanism and function of LINC00320 in the development of glioma by regulating PLEKHA1 expression through transcription factor MYC. Methods: By searching LINCDISEASE database and through difference analysis of glioma chip, glioma related lncRNAs were screened, and lncRNA-transcription factor-mRNA triplet was predicted through lncMAP database. The expressions of LINC00320 and PLEKHA1 were detected in glioma and normal controls, followed by the detection of the proliferation, invasion, migration, and apoptosis of glioma cells by using CCK-8 method, Transwell assay, and ow cytometry, respectively. In addition, the expression patterns of MMP9 and cleaved-Caspase 3 were detected with Western Blot. Furthermore, the possible mechanism of LINC00320 was predicted in gliomas by LncMAP. RIP assay was performed to verify the interaction between LINC00320 and MYC, and ChIP assay was applied to validate the binding of MYC and PLEKHA1 promoter. The existence of binding site between MYC and PLEKHA1 promoter were determined by dual luciferase reporter gene assay. Lastly, in vivo test was conducted by using nude mice as the objects of study for verication of the results obtained by in vitro tests. Results: LINC00320 was found to be signicantly down-expressed in glioma, and patients with low expression levels of LINC00320 exhibited an even worse prognostic outcome. Over-expression of LINC00320 in glioma cells brought about a signicant reduction in cell proliferation, migration, invasion, and promoted apoptosis. There was a signicant decrease in the protein expression of MMP9 but remarkable increase in that of cleaved-Caspase 3 after LINC00320 over-expression. LncRNA-transcription factor-mRNA triplet prediction showed that LINC00320 regulated the expression of PLEKHA1 through MYC. RIP assay demonstrated that MYC could signicantly enrich LINC00320, Chip assay showed that MYC bound with the PLEKHA1 promoter, and dual luciferase


Background
Gliomas are the most common malignancy of the brain, accounting for about 50% of primary brain tumors [1,2]. Unfortunately, the median survival of patients with gliomas is extremely low despite the advent of surgical regimens and postoperative adjuvant radiotherapy and chemotherapy [3,4]. At present, it is imperative to improve the therapeutic effect of gliomas and reduce the recurrence considering the limited clinical use of various therapeutic approaches for gliomas [5]. In recent decades, rapid development in the eld of tumor cell research has been greatly attributed to the gradual elucidation of functions of transcription factors. Transcription factors are proteins that possess the ability to bind to speci c DNA sequences and regulate gene transcription [6][7][8]. Several transcription factors can also contribute to tissue-or cell-type-speci c gene expressions, and are thus involved in the development of malignancies. Moreover, some transcription factors, such as Octamer-binding transcription factor (Oct4), B-lymphoma Moloney murine leukemia virus insertion region-1 (Bmi1) and Nanog, have been found to be implicated in the early development of embryo, which not only regulate the maintenance of normal cell attributes, but also play a vital role in the maintenance of tumor cell biological characteristics [9][10][11]. Also, the gene product of MYC, especially c-MYC, also plays an important role in the induction of apoptosis [12]. MYC is one of the well-known transcription factors that serves as an oncogene to promote the genesis of various human tumors [13], and recent studies on its expression and function have further highlighted its therapeutic opportunities [14][15][16]. Furthermore, studies have uncovered that MYC can mediate the expressions of both protein and non-coding RNA to regulate cell growth, proliferation, differentiation and drug resistance, etc. [17,18]. However, it is still unclear with respect to the molecular and genetic mechanisms of MYC in patients with glioma.
Long noncoding RNAs (lncRNAs) are a class of transcribed RNA molecules of >200 nucleotides in length [19][20][21][22]. LncRNAs are capable of modulating the expressions of target genes at transcriptional, posttranscriptional, and epigenetic levels, not encoding proteins [23,24]. More importantly, a large number of studies have demonstrated that lncRNAs confer signi cant functions in various biological steps such as cell cycle processes, cell differentiation, etc. [25][26][27]. Numerous lncRNAs such as lncRNA LOC441204 and PVT1 are known to play multi-roles in glioma cells acting as oncogenes or tumor suppressors. Furthermore, previous studies also support the important roles of numerous oncogenic and antioncogenic lncRNAs in the development of gliomas [28][29][30][31]. Another such lncRNA, LINC00320, was previously indicated to be human brain-speci c and highly-expressed in the cortical white matter [32].
Moreover, a recent study showed that LINC00320 as a tumor suppressive lncRNA, was down-regulated in glioma tissue and suppressed the proliferation of glioma cells by inhibiting the activation of Wnt/βcatenin signaling pathway, and further proposed a novel axis of HMGB1/LINC00320/β-catenin in glioma [33]. However, there are no such reports about LINC00320 involvement in glioma, and was thus chosen as the prime focus lncRNA for a more comprehensive and systematic investigation. Meanwhile, PH domaincontaining family A member 1 (PLEKHA1), also known as TAPP1, is known as a member of the superfamily of proteins containing the PH domain [34]. Previous evidence has documented that PLEKHA1 can bind with PtdIns(3,4)P2 speci cally through its C-terminal PH domain [35]. So far, there is a dearth of studies relating its role in the regulation of cell migration. More pressingly, there is a lack of evidence investigating the relationship between LINC00320, PLEKHA1 and MYC, which was hypothesized to be correlated interactively in our study and would be explored subsequently. In view of the above interpretations, the present study was carried out to explore the mechanism and function of LINC00320, the roles of PLEKHA1 and MYC as well as their relationship in the development of glioma.

qRT-PCR Test
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 uorescent quantitative PCR to perform uorescent quantitative PCR according to the instruction of SYBR®Premix ExTaqTM kit (RR820A, TaKaRa). The reaction system was 50 μL in amount, including 25 μL of SYBR®Premix Ex TaqTM (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 ddH 2 O. Real-time uorescence quantitative PCR system (ABI 7500, ABI, Foster City, CA, USA) was then carried out for uorescence 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 nally. 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).

Western Blot
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 βactin (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 uorescence detection kit, mixed in dark conditions, and dropped onto the lm 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.
Immuno uorescence Subcellular localization of LINC00320 and MYC in the SHG-44 cell line was identi ed using immuno uorescence 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 xation 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, ve different elds of vision were observed and photographed under a uorescence microscope (Olympus, Japan) after anti-uorescence quenching agent for sealing.

CHIP Assay
SHG-44 cells from each group was treated with formaldehyde for 10 min to produce DNA protein crosslinking. 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 speci c 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-speci c complex, the cross-linking was removed overnight at 65℃, and DNA fragments were extracted and puri ed with phenol/chloroform. Finally, the binding of MYC to the promoter region of PLEKHA1 was detected by means of PCR with PLEKHA1 using speci c primers.

RIP Assay
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 coprecipitation. As for the speci c 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 Fire y 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 1×10 5 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.

Transwell Assay
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 solidi cation 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.

Flow Cytometry
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 ow 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 1×10 6 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 uorescence were detected by 525 nm and 620 nm band-pass lters 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 1.0×10 6 /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×b 2 . 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.

Immunohistochemistry
Para n 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% H 2 O 2 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 elds (×200) were randomly selected for each section, with 200 cells in each eld, with the purpose of analyzing the ratio of positive cells [36].

Statistical Analysis
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 posthoc 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 signi cant.

LINC00320 Involved in the Development of Glioma
The GSE15824 dataset with glioma expression patterns was obtained by retrieving the GEO database. Subsequent difference analysis of the gene expression between normal samples and glioma samples in the dataset reared a total of 657 signi cantly different expressed genes ( Figure 1A). Additionally, 142 glioma related lncRNAs were obtained from LNCDISEASE database to explore the mechanism of lncRNA regulating disease through transcription factors in glioma. Moreover, the lncMAP database was searched to obtain the triplet data of lncRNA-transcription factor-mRNA in lncMAP database. Microarray analysis was then performed to obtain the intersection of lncRNA in the LNCDISEASE database and that in the triplet within lncMAP database ( Figure 1B), with one lncRNA of LINC00320 obtained nally. In addition, microarray analysis indicated that the expression of LINC00320 was signi cantly downregulated in glioma ( Figure 1C). Meanwhile, the expression patterns of LINC00320 in glioma in TCGA and GTEX databases were analyzed by GEPIA database (Figure 1D), with a similar down-regulated expression trend observed in the tumor. In order to further determine whether LINC00320 was involved in the development of glioma, qRT-PCR was applied to detect the expression levels of LINC00320 in the collected glioma tissues and controls. It was found that the expression levels of LINC00320 in human gliomas were signi cantly lower than those in non-tumor brain tissues (p<0.05) ( Figure 1E). According to the average expression level of LINC00320 in gliomas (0.414), the high expression group (n =31) and low expression group (n =29) were established to analyze the relation between high and low expressions of LINC00320 and the clinicopathological characteristics of patients. LINC00320 expression was found to be not signi cantly correlated with age, gender and recurrence, while showing correlation with tumor size, stage, WHO grade and KPS score (p<0.05) ( Table 2). According to the results of Kaplan-Meier analysis, the mean survival time of patients with high expression of LINC00320 was obviously prolonged than those with relatively low LINC00320 expressions (51.10 months vs. 38.77 months; t=2.874, p<0.05). Besides, the recurrence-free survival of patients with high expression of LINC00320 was found to signi cantly longer than those with poorly-expressed LINC00320 (47.93 months vs. 32.35 months; t=3.346, p<0.05) ( Figure 1F-G). In addition, the expression levels of LINC00320 were detected in glioma cells, and it was observed that LINC00320 expressions in glioma cells were signi cantly decreased, with a more signi cant expression of LINC00320 in SHG-44 cell line. Hence, SHG-44 cells were selected for subsequent experimentation ( Figure 1H).

Effect of LINC00320 on Proliferation, Migration, Invasion and Apoptosis of Glioma Cells
In view of the low expressions of LINC00320 in gliomas, LINC00320 over-expression lentivirus was constructed rstly and transfected into the glioma cell line SHG-44 to determine how it speci cally affects the proliferation, migration and invasion of gliomas. A signi cant increase in LINC00320 expression levels was detected by qRT-PCR (Figure 2A). In addition, CCK8 assay results showed that cell proliferation activity decreased signi cantly after LINC00320 over-expression ( Figure 2B); Similarly, Transwell assay revealed that an obvious decrease in the number of migrated and invaded cells after LINC00320 overexpression ( Figure 2CD). Meanwhile, ow cytometry with Annexin-V/PI double staining showed that the apoptosis rate increased signi cantly when LINC00320 was over-expressed ( Figure 2E). Western Blot indicated that compared with Vector group, the protein expression of MMP9 decreased obviously, while that of cleaved-Caspase-3 increased signi cantly in LINC00320 group ( Figure 2F). These ndings indicated that over-expression of LINC00320 decreased glioma cell proliferation, migration and invasion, while promoted cell apoptosis.

LINC00320 Regulated PLEKHA1 through Transcription Factor MYC
In order to elucidate the mechanism of LINC00320 involvement in the development of glioma, transcription factors in the downstream of LINC00320 predicted by lncMAP were further intersected with the differentially expressed genes in GSE15824 ( Figure 3A). MYC was not only present in the triplet, but also expressed in the GSE15824 dataset. Furthermore, microarray analysis showed that MYC expression levels were signi cantly up-regulated in glioma ( Figure 3B). Analysis of the expression patterns of MYC in TCGA and GTEx ( Figure 3C) also indicated that MYC was highly-expressed in glioma. Next, the differentially expressed genes between the downstream gene of transcription factor MYC in the triplet and the result of microarray analysis were intersected (Figure 3D), and a downstream target gene with differential expression in glioma was nally obtained, namely, PLEKHA1. Subsequently, the expression of PLEKHA1 in glioma was analyzed in GSE15824, and the results revealed that the expression of PLEKHA1 was evidently decreased in microarray analysis of GSE15824 ( Figure 3E); besides, our experiment also analyzed the differential expression of PLEKHA1 in TCGA glioma database, with an obviously low expression in glioma as well ( Figure 3F). The study continued to verify the expressions of MYC and PLEKHA1 in glioma samples and control tissues we collected clinically, and qRT-PCR results indicated that compared with control tissues, the mRNA expression level of MYC was up-regulated signi cantly, while that of PLEKHA1 was down-regulated obviously in glioma tissues (p<0.05) ( Figure 3G-H). Further Pearson correlation analysis showed that LINC00320 was negatively-correlated with MYC in glioma tissue (r 2 =-0.748, p=0.001), but positively-correlated with PLEKHA1 (r 2 =0.795, p=0.001) ( Figure 3I). These results suggested that LINC00320 was correlated with the expression of transcription factor MYC and PLEKHA1.

LINC00320 Regulated the Expression of LEKHA1 by Recruiting Transcription Factor MYC
Cell veri cation was performed to determine the mechanism of the aforementioned LINC00320/MYC/PLEKHA1 signal axis. The location of LINC00320 and MYC in cells was rstly determined using immuno uorescence staining, and LINC00320 and MYC were found to be primarily located in the nucleus, and a few scattered in the cytoplasm ( Figure 4A). Using the lncMAP database (http://bio-bigdata.hrbmu.edu.cn/LncMAP/survival.jsp), the triplet of downstream transcription factors and regulatory genes of LINC0032 in low-grade glioma were predicted ( Figure 4B). Subsequent RIP experiment indicated that when LINC00320 was mutated, compared with IgG group, LINC00320-MUT group showed no MYC enrichment, while LINC00320-WT group had signi cantly enriched MYC compared with that in LINC00320-MUT group (p<0.05), suggesting that LINC00320 could be pulled-down by combining with MYC ( Figure 4C). Meanwhile, based on the results of CHIP assay, analysis after precipitation showed that the primer of PLEKHA1 promoter could signi cantly detect the enrichment of PLEKHA1 mRNA, suggesting that MYC could bind to the promoter of PLEKHA1 ( Figure 4D, p<0.05); besides, over-expression of LINC00320 inhibited the binding of MYC to the PLEKHA1 promoter ( Figure 4E, p<0.05). Furthermore, dual luciferase reporter gene assay indicated that compared with the vector group, uorescence intensity of MYC group decreased signi cantly in WT (p<0.05), and when the predicted binding site (MUT) was mutated, the uorescence intensity did not shown signi cant changes (p>0.05) ( Figure 4F). Finally, after over-expression of LINC00320 in SHG-44 cells, qRT-PCR results demonstrated that compared with the vector group, the expression levels of LINC00320 and PLEKHA1 were increased signi cantly in LINC00320 group. In the MYC group, MYC expression was signi cantly increased, while that of PLEKHA1 was signi cantly decreased; besides, compared with MYC group, the expressions of LINC00320 and PLEKHA1 were signi cantly increased in MYC+LINC00320 group ( Figure 4G, p<0.05). These results suggested that over-expression of LINC00320 could inhibit the binding of MYC and PLEKHA.

Effects of LINC00320/MYC/PLEKHA1 Axis on Proliferation, Migration, Invasion and Apoptosis of Glioma Cells
Our experiment further observed the effect of LINC00320/MYC/PLEKHA1 axis on the proliferation, migration, invasion and apoptosis of glioma cells. According to the results of qRT-PCR and Western blot, LINC0032 over-expression signi cantly promoted the expressions of LINC0032 and PLEKHA1, while the simultaneous over-expression of LINC0032 and MYC signi cantly promoted the expression of MYC and inhibited that of PLEKHA1. Moreover, LINC0032 over-expression combined with PLEKHA1 expression interference signi cantly inhibited the expression of PLEKHA1. The results of CCK8 assay demonstrated that although LINC00320 signi cantly reduced the proliferation and migration of cells, over-expression of MYC or interference of PLEKHA1 resulted in the reversed effect of over-expression of LINC00320, accompanied by upregulated proliferation ability of glioma cells ( Figure 5A). Transwell assay further illustrated that when LINC00320 was transfected alone, the number of cells migrating and invading were signi cantly reduced. However, when MYC or si-PLEKHA1 were transfected simultaneously, the effect of LINC00320 could be blocked, while the number of cells migrating and invading increased, which promoted the migration and invasion of cells ( Figure 5BC). Meanwhile, the results of ow cytometry with Annexin-V/PI double staining showed that LINC00320 transfection increased the proportion of apoptosis.
However, co-transfection of MYC over-expression or si-PLEKHA1 with LINC00320 over-expression reversed the effect of LINC00320 and reduced the proportion of apoptosis ( Figure 5D). Similarly, Western blot showed that LINC00320 reduced the expression of MMP9 and promoted that of cleaved-Caspase 3, which could be reversed after co-transfection of MYC over-expression or si-PLEKHA1 ( Figure 5E). These results suggested that LINC00320 can regulate PLEKHA1 through the role of MYC and participated in the proliferation, migration and invasion of glioma cells.

LINC00320 Regulated in vivo Tumorigenesis in Glioma
Mouse models of xenotransplantation were established to con rm the anti-tumor effect of LINC00320 in vivo, and stably expressed LINC00320 in SHG-44 cells were inoculated into mice. Tumor growth and weight of mice treated with LINC00320 were found to be lower relative to the vector group ( Figure 6A-C). Western blot results demonstrated that PLEKHA1 protein expression levels were signi cantly up-regulated after LINC00320 treatment ( Figure 6D). Immunohistochemistry showed that the positive expression of GFAP and Ki67 were signi cantly reduced after LINC00320 transfection ( Figure 6E). These results indicated that LINC00320 can inhibit tumor growth in vivo.

Discussion
To data, microRNAs or lncRNAs are hot-spots with the emergence of genetic research. A small proportion of lncRNAs have been characterized in depth, however, lncRNAs accepted and recognized to exhibit extensive roles in cellular and development processes of human disorders [37][38][39]. According to prior investigation, lncRNAs are also known to serve as prevalent regulators of gene expression, however, it still remains unclear with regard to the consequences of lncRNA inactivation or over-expression in vivo and in vitro generally in the development of tumors or non-tumor diseases [40,41]. Moreover, the human brain with its heterogeneous cellular structure is a rich source of lncRNAs; yet, the functions of the majority of lncRNAs remain to be discovered. In lieu of this, the current study performed a series of experiments to elucidate the mechanism and function of LINC00320 in the development of glioma by regulating the expression of PLEKHA1 through the transcription factor MYC.
Firstly, our study was initiated with bioinformatic prediction, which indicated that LINC00320 was implicated in the development of glioma. It has been reported that there was aberrant expression of LINC00320 in human malignancies, especially in glioma [33,42]. Our subsequent microarray analyses identi ed low expression levels of LINC00320 in glioma, while the screened patient data also indicated that LINC00320 was poorly-expressed in glioma patients. Moreover, prognoses were found to be worse in glioma patients presenting with low expressions of LINC00320. After uncovering the aberrant expression of LINC00320 in gliomas, we further explored the effect of LINC00320 on proliferation, migration, invasion and apoptosis of glioma cells, with the clari cation of intrinsic mechanism of action. To our surprise, cell proliferation activity and the number of cell migration and invasion were signi cantly decreased, while the apoptosis rate exhibited a marked increase after over-expression of LINC00320.Together, these ndings suggested that over-expression of LINC00320 may function positively on the progression of glioma.
Furthermore, our ndings demonstrated that LINC00320 regulated PLEKHA1 through the transcription factor MYC, which indicated that LINC00320 could play a role in the development of glioma by regulating the expression of PLEKHA1 through MYC. Additional in vivo cell experimentation in our study revealed that LINC00320 and MYC were primarily located in the nucleus, LINC00320 could be pulled-down by combining with MYC, and MYC could bind to the target gene PLEKHA1. The hard-done work of our peers has further indicated that MYC is a canonical stem cell factor, and is involved in the development and progression of multiple solid tumors [43][44][45], especially in the development of glioma [46], which can be explained by its function in modulating the role of several lncRNAs, miRNAs and proteins, etc. [47,48]. Moreover, one particular study documented that inhibition of Myc can inhibit cell proliferation and induce apoptosis of glioma stem cells [46,49]. Besides, our study further veri ed that over-expression of LINC00320 brought about an inhibitory effect on the binding of MYC to PLEKHA1.
After uncovering the potential mechanism, our study nally explored the regulation of PLEKHA1 by LINC00320 through the transcription factor MYC and its consequent effects on the proliferation, migration and invasion of glioma cells. Our ndings present with their own basis and advantages. Previous studies have shown that lncRNA APTR represses the CDKN1A/p21 promoter independent of p53 to promote cell proliferation by recruiting polycomb proteins [50]. Meanwhile, knock-down of lncRNA MALAT1 was previously demonstrated to promote cell proliferation in gastric cancer cell line by recruiting the splicing factor SF2/ASF [51]. Returning to the focus of our study, down-regulated expressions levels LINC00320 have also been reported to serve as a tumor suppressor in glioma tissues; over-expression of LINC00320 could inhibit glioma cell proliferation by restraining the Wnt/β-catenin signaling pathway via the segregation of β-catenin and TCF4, which reiterates the signi cance of LINC00320 and relevant targeting in the progression of glioma [33]. More importantly, the above-mentioned evidence are in complete accordance with the current study, wherein step by step experimentation highlighted that overexpression of LINC00320 could suppress the abnormal biological process of glioma cells by recruiting the transcription factor MYC to weaken the binding of MYC and PLEKHA1. The strength of our study lies in that in vitro and in vivo experiments were combined jointly on the basis of bioinformatic prediction. Furthermore, lncRNAs are known to function as key regulatory molecules in various fundamental cellular processes, and their deregulation is often assumed to contribute to carcinogenesis. Accordingly, intervention with LINC00320 in our study exactly provides potential research approach and clinical reference value in target therapy of human tumors on the basis of mechanism research. However, our study does present with limitations. The molecular mechanism of transcription regulation of transcription factor MYC for PLEKHA1 requires more exploration. Moreover, the sample size e is small in the literature. Besides, there is only one GSE dataset. While the strength of this article lies in that in vitro and in vivo experiments were combined jointly on the basis of bioinformatic prediction. Intervention with LINC00320 in this study exactly provides potential research approach and clinical reference value in target therapy of human tumors on the basis of mechanism research. Signi cantly, the innovation of this research lies in elaborating the effect of LINC00320/MYC/PLEKHA1 signaling axis on glioma.

Conclusion
In conclusion, ndings obtained in our study indicate that over-expression of LINC00320 can weaken the binding of MYC with PLEKHA1 by recruiting the transcription factor MYC, and ultimately inhibit the proliferation, migration and invasion, and promote the apoptosis of glioma. Moreover, interfering PLEKHA1 could reverse the effects of LINC00320 over-expression on glioma cells. These ndings shed a new light on the potential of LINC00320 to as molecular biomarker for the clinicopathology and prognosis of glioma, which offers a novel molecular approach for the target therapy of glioma.    Effects of LINC00320 on proliferation, migration, invasion and apoptosis of glioma cells Note: A: qRT-PCR analysis showed LINC00320 was signi cantly highly expressed in glioma following over-expression of LINC00320. B: CCK8 assay showed that LINC00320 inhibited the proliferation of glioma cells. C: Transwell assay indicated that LINC00320 over-expression reduced cell migration. D: Transwell assay indicated that LINC00320 over-expression reduced cell invasion. E: Annexin-V/PI ow cytometry showed that the apoptotic rate of overexpressed LINC00320 cells was signi cantly up-regulated. F: Western blot detection of protein expression. The data were expressed as mean ± standard deviation, and t-est test was used to analyze the data between groups, *compared with vector group, p<0.05, Figure 3 The presence of correlation in the LINC00320/MYC/PLEKHA1 axis in glioma Note: A: The predicted results of transcription factors in the downstream of lncRNA and the intersection of differential expressed Over-expression of LINC00320 inhibited the binding of MYC to PLEKHA1 Note: A: Immuno uorescence assay showed that LINC00320 and MYC were mainly located in the nucleus. B: The binding site between transcription factor and regulatory gene. C: RIP test showed that LINC00320 could bind with MYC. D:

List Of Abbreviations
CHIP test indicated that PLEKHA1 could be enriched signi cantly after the complex was precipitated with MYC antibody. E: CHIP test revealed that over-expression of LINC00320 inhibited the binding of MYC with PLEKHA1. F: Dual luciferase reporter assay showed the existence of binding site between MYC and PLEKHA1. G: qRT-PCR detection of the regulation of LINC00320 and MYC on PLEKHA1 expression. The data were expressed as mean ± standard deviation, t-est test was used to analyze the data between groups, one-way ANOVA was used for statistical analysis among multiple groups, and the experiment was repeated three times. *p<0.05. Transwell assay to evaluate the ability of MYC/PLEKHA1 to reverse the inhibition of LINC00320 on glioma cell invasion. F: Annexin-V/PI double staining to detect the effect of LINC00320/MYC/PLEKHA1 axis on apoptosis of glioma cells. G: Western blot detection of MMP9 and cleaved-Caspase 3 expression in cells. The data were expressed as mean ± standard deviation, one-way ANOVA was used for statistical analysis among multiple groups, and the experiment was repeated three times. *p<0.05. Ki67 expression. The data were expressed as mean ± standard deviation, t-est test was used to analyze the data between groups, N=5, *p<0.05.