Biosynthetic circ-PLEKHA5 stabilized by SRSF7 promotes the vasculogenic mimicry of glioblastoma cells by encoding a novel protein 622aa

Background: Increasing studies have been demonstrated that circRNAs play vital regulatory roles in the biological behaviors of glioblastoma cells, and increasing circRNAs are found to have the capacity of encoding small peptides, which are involved in the regulatory process. Methods: Western blot and qRT-PCR were conducted to conrm the expression of SRSF7 and circ-PLEKHA5 respectively. RNase R digestion assay and uorescence in situ hybridization assays were conducted to evaluate the existence and cellular location of circ-PLEKHA5. RIP assay was used to access the relationship between SRSF7 and circ-PLEKHA5. Dual-luciferase assay and FLAG tag assays were performed to test the coding capability of circ-PLEKHA5. Immunouorescence assay was conducted to evaluate the location of circ-PLEKHA5-622aa. CCK-8, vasculogenic mimicry (VM) formation and transwell assays were used to evaluate the roles of SRSF7, circ-PLEKHA5, circ-PLEKHA5-622aa on proliferation, VM formation, migration and invasion. Nude mice xenograft studys with PAS-CD34 staining were used to clarify the functional roles of SRSF7 and circ-PLEKHA5 on VM formation in vivo. Results: SRSF7 was up-regulated in glioma, and promoted the proliferation, migration, invasion, VM formation and the expression of VM-associated proteins of glioma cells by increasing the expression of circ-PLEKHA5. Circ-PLEKHA5 was mainly localized in cytoplasm and promoted the proliferation, migration, invasion, VM formation and the expression of VM-associated proteins of glioma cells by encoding a novel protein circ-PLEKHA5-622aa. The application of SRSF7 and circ-PLEKHA5 inhibitor signicantly suppressed the tumor growth and VM formation in vivo. Conclusions: This study rst demonstrated the coding ability of circ-PLEKHA5, and identied the regulatory roles of SRSF7/circ-PLEKHA5/circ-PLEKHA5-622aa pathway in VM formation of glioblastoma cells. Our ndings might provide a novel strategy for glioma treatment. encoded by circ-β-catenin protects β-catenin by prevent it from binding GSK3β and ubiquitination, thereby promoting tumor growth [38] . Based on the prediction on database circRNADb, we investigated the coding ability of circ-PLEKHA5 and conrmed that circ-PLEKHA5 could encode a 622 amino acid (aa) peptide circ-PLEKHA5-622aa. Moreover, the expression level of 622aa was signicantly elevated in glioma. We next investigated the potential function of circ-PLEKHA5-622aa, and the results showed that the elevated 622aa, independent of circ-PLEKHA5, signicantly increased the proliferation, migration, invasion and VM formation abilities of glioma cells and also reversed the inhibitory effects caused by circ-PLEKHA5-depletion. These results indicated that circ-PLEKHA5 regulated the malignant behaviors of glioma cells by encoding circ-PLEKHA5-622aa.

RNA-binding protein serine/arginine-rich splicing factor 7 (SRSF7) belongs to the SR splicing factor family, which is known for its regulatory roles in alternative precursor mRNA (pre-mRNA) splicing [4] . Besides pre-mRNA splicing, SRSFs also have been considered to regulate mRNA stability, translation and export, thereby affecting a series of cell behaviors and disease developments [5] . In a pathway of DNA damage response, SRSF7 binds to the precursor mRNA of p53 and promotes its alternative splicing into p53β, which affect the senescence and aging process of mammalian cells [6] . Furthermore, SRSF7 is indicated to be up-regulated and act as an oncogene in various cancer types, such as renal cancer, gastric cancer, colon cancer and lung cancer [7] . In glioblastoma, SRSFs can act as target of the m6A methyltransferase METTL3 caused nonsense-mediated mRNA decay, which lead to a disorder of alternative spliceosome expressions [8] . SRSF3 is up-regulated and promotes the proliferation, self-renewal and tumorigenic processes of glioma-stem like cells [5] . Nevertheless, the effects of SRSF7 on glioma genesis remain unclear.
Circular RNAs (circRNAs) are a class of non-canonical RNA transcripts that rst discovered in the 1990s and identi ed to be widely expressed in eukaryotes. Unlike the linear RNA transcripts, most circRNAs are basically generated through pre-mRNA alternative back-splicing of exons, which forming a covalently circular structure with no 5' and 3' ends [9] . As RNA deep-sequencing technologies develops and comprehensive studies of circRNAs continues, increasing circRNAs have been identi ed along with their participation in biological processes, such as innate immune responses and tumorigenesis [10] . Endogenous circRNAs have previously been considered as a member of non-coding RNAs until Chen CY et al. rst discovered the translation ability of them [11] . In recent years, more and more endogenous circRNAs are found to encode functional polypeptides or proteins through the open read frames (ORFs) contained, which could further play speci c roles in the tumorigenesis and development of various tumors including glioma [12][13][14] . Despite the development of ribosome pro ling technique and the increasing of computational databases, there are still few studies on the functional peptides that encoded by circRNAs.
In this study, we identi ed circ-PLEKHA5 (hsa_circ_0098068) which was up-regulated in glioma tissues and cells, and promoted by SRSF7. Furthermore, a 622 amino acid (aa) peptide encoded by circ-PLEKHA5 ORF was up-regulated in glioma tissues and cells. We also explored the molecular mechanisms among them and their effects on the VM formation of glioma cells. Our results provided a new sight for discovering the VM formation in glioma and a new strategy for the treatment of glioma.

Materials And Methods
Cell lines and human tissue samples All cells that used in this study, including human astrocyte (HA) cell, human glioma cells (U373, U251) and human embryonic kidney (HEK) 293T cell were purchased from Shanghai Institutes for Biological Sciences Cell Resource Center. HA cell was cultured in RPMI-1640 culture medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA). U373, U251 and 293T cells were cultured in Dulbecco's modi ed Eagle medium (DMEM)/high glucose with 10% FBS. All cells were maintained at 37℃ with 5% CO 2 . All human glioma tissues and normal brain tissues (NBTs) were collected from the Department of Neurosurgery of Shengjing Hospital of China Medical University with informed consent, and the study was approved by the Ethics Committee of Shengjing Hospital of China Medical University.

Plasmid Construction and Cell transfection
The silencing plasmids of SRSF7 and circ-PLEKHA5 gene with their nontargeting sequences were ligated into pGPU6/GFP/Neo vectors. Human SRSF7 mRNA sequence was ligated into the pcDNA3.1 vector (GenePharma, Shanghai, China). Human circ-PLEKHA5 overexpressed vector and the control plasmid were obtained from GenePharma Company. Full length circ-PLEKHA5-622aa gene and respective nontargeting sequence were ligated into pIRES2-EGFP.
Transfection was performed using Lipofectamine 3000 and Opti-MEM (Life Technologies, Carlsbad, USA), and the stable transfected cells were selected by Geneticin (G418) and Puromycin (Sigma-Aldrich, St Louis, USA). The transfection e ciency was evaluated by qRT-PCR and western blot.

CircRNAs microarray
The circRNAs analysis and microarray hybridization were performed by Kangchen Biotechnology Corporation (Shanghai, China).

RNA uorescence in situ hybridization (FISH)
To identify the distribution of circ-PLEKHA5, the probe of circ-PLEKHA5 (5'-TTATGGCTGATGATCCAAATA -3', green-labeled, Biosense, Guangzhou, China) were designed and used. Brie y, U373 and U251 were rst seed in 6-well plates and climbed to the carry sheet glass subsequently. Cells were next washed by cold PBS solution, xed by 4% RNase-free paraformaldehyde (PFA) solution for 15min and permeabilized by 0.5% Triton X-100 at room temperature for 5min. After dehydrated by graded ethanol, cells were then added prepared hybridization mix with circ-PLEKHA5 probe and hybridized at 50℃ overnight. Next day, the carry glass was washed using 50% formamide with 0.1% saline sodium citrate (SSC) solution for 10min and stained with 4',6-diamidino-2-phenylindole (DAPI, Beyotime, China) for 5min at time temperature. Finally, cells were covered with cover glass and visualized using the confocal microscopy.
Immuno uorescence (IF) assay Cells were seeded on chamber slides, xed with 4% paraformaldehyde and permeabilized in PBS containing 0.2% Triton X-100 for 10 min at -20℃, and then blocked with 5% BSA for 2 hours at room temperature. The immunostaining was carried out with primary antibody against GFP fusion proteins (1:100; Abcam, MA, USA) (overnight at 4℃) and anti-rabbit IgG secondary antibody (1:500; Beyotime Institute of Biotechnology, Jiangsu, China) (2h at room temperature). Then the nuclei were counterstained with DAPI (Beyotime Institute of Biotechnology, Jiangsu, China) for 5min, and the images were captured using confocal microscope.

Cell proliferation assay
Proliferation assay of glioma cells were performed using Cell Counting Kit-8 (CCK-8, Beyotime Institute of Biotechnology, Jiangsu, China) as previously described [15] . Brie y, cells were seeded at a density of 2500 cells per eld, 20μl CCK-8 was added subsequently, then the absorbance was analyzed at 450nm.

Transwell assay
Cell migration and invasion abilities were evaluated by transwell assays as previously described [15] . Brie y, cells were resuspended and seeded onto the upper transwell chamber (Corning, UY, USA), and the medium with 10% FBS was added into the lower chamber. Cells on the lower surface of membrane were then xed and captured after 48h under a light microscope.
In Vitro VM formation assay The U373 and U251 cells were resuspended in 100μl FBS-free medium at a number of 6 × 10 4 cells and seeded into 96-well culture plates, which was covered by 60μl Matrigel Basement Membrane Matrix (BD Bioscience, MA, USA) before. Next, cells were incubated at 37℃ for 8h, then visualized and captured under an inverted microscope (Olympus, Tokyo, Japan).

RNA-protein immunoprecipitation (RIP) assay
RIP assay was performed using EZ-Magna RIP Kit (Millipore, MA, USA) according to the manufacturer's instruction. Brie y, cells were lysed by RNA lysis buffer with protease inhibitor, and incubated with RIP buffer that contains magnetic beads conjugated with anti-SRSF7 antibody or with negative control anti-IgG respectively. Next, wash buffer was added into RIP reactions and vortex gently. The fold enrichments of immunoprecipitated RNAs was next isolated and puri ed, and then analyzed followed qRT-PCR.

Nude mice xenograft model
For the in vivo study, four weeks old female BALB/C nude mice were purchased from the Cancer Institute of the Chinese Academy of Medical Science and subcutaneously injected with U373 or U251 cells with stable knockdown of SRSF7 and circ-PLEKHA5 (3 × 10 5 cells each mouse) in the right ank area. Mice were divided into ve groups: Control, SRSF7(-)NC+circ-PLEKHA5(-)NC, SRSF7(-), Circ-PLEKHA5(-) and SRSF7(-)+circ-PLEKHA5(-) groups (n=6 each group), and the volume of transplanted tumors were observed and recorded every 5 days after the inoculation following length × width 2 /2 mm 3 formula. Tumors were isolated out of the mice after sacri cing them. The experiment was complied with the Administrate Panel on Laboratory Animal Care of China Medical University.
Immunohistochemical dual-staining of CD34 and Periodic Acid-Schiff (PAS) The tumor slices were dewaxed under 60℃ for 30min, hydrated by xylene and ethanol solution in turn, and then soaked in citric acid buffer at 90℃ for antigen retrieval. Next, slices were blocked using BSA (Thermo Scienti c, MA, USA), incubated with the speci c rabbit anti-human antibody of CD34 (Beijing Zhongshan Golden bridge, China) and left in the fridge at 4℃ overnight. Next day, the goat anti-rabbit secondary antibody and the color reagent DAB (Fuzhou MaiXin Biotech, China) were subsequently added onto the slides. For the staining of PAS, slices were stained with periodic for 10min, Schiff solution for 10min, and counterstained with hematoxylin (Baso, Guangdong, China) for 1min, and PBS solution was used for washing at each interval. Finally, by using the microscope, CD34 and PAS signals were captured evaluated.

Statistical analysis
In this work, all data were described as mean ± standard deviation (SD) from at least three times independent experiments. All statistical analysis was conducted using GraphPad Prism 7 software with student's t test (between two groups) or one-way ANOVA analysis (three or more) of variance. P value < 0.05 was considered to be statistically signi cant.

Results
SRSF7 was up-regulated in glioma, knockdown of SRSF7 inhibited the VM formation of glioma cells The elevated expression level of SRSF7 in glioma samples was rst found by using The Cancer Genome Atlas (TCGA) database (Figure S1A), and western blot was further used to verify the prediction. As shown in Figure 1A-B, compared with in normal brain tissues (NBTs) and human astrocyte (HA) cell, the expression level of SRSF7 was signi cantly up-regulated in glioma tissues and cells respectively, and the expression level was positively correlated with the histopathological grade. To further elucidate the role of SRSF7 in glioma, we constructed SRSF7 silenced U373 and U251 cell lines, and SRSF7 re-expressed cell lines respectively, and evaluated the cell proliferation, VM formation, migration and invasion ability. The results of CCK-8 assay, in vitro VM formation assay and transwell assay indicated that knockdown SRSF7 signi cantly inhibited the cell proliferation, VM formation, migration and invasion abilities of glioma cells and re-expression of SRSF7 signi cantly restored the inhibitory effect ( Figure 1C-E). Matrix metallopeptidase 2 (MMP-2) and vascular endothelial cadherin (VE-cadherin) are critical VM-associated proteins [16,17] . As shown in Figure 1F, knockdown of SRSF7 expressly restrained the expression levels of MMP-2 and VE-cadherin in glioma cells, while re-expressed SRSF7 restored their expression signi cantly.
By using circRNA microarray analysis, we obtained the effect of SRSF7 on the expression levels of dysregulated circRNAs in glioma, and we found that has_circ_0098068 was up-regulated in glioma cells and signi cantly down-regulated by transient knockdown of SRSF7 ( Figure S1B). Through matching the circRNA database circRNADb, we obtained the generation of hsa_circ_0098068, also regarded as circ-PLEKHA5, was formed by the circularization of exon4 to exon15 of the pre-mRNA of PLEKHA5 ( Figure  2A). In order to con rm the existence of circ-PLEKHA5, we used convergent and divergent primers to detect the existence of linear and circular PLEKHA5, and RNase R was used to eliminate the interference of linear PLEKHA5. As shown in Figure 2B, circ-PLEKHA5 was detected in DNA samples with or without RNase R, while linear PLEKHA5 was only detected in RNase R -group. Furthermore, no circ-PLEKHA5 was found in genomic DNA (gDNA), which con rmed the existence of endogenous circ-PLEKHA5. Next, qRT-PCR was carried out to evaluate the expression of circ-PLEKHA5 in glioma tissues and cells, as shown in Figure 2C, circ-PLEKHA5 was signi cantly up-regulated in glioma tissues and cells, and positively correlated with the pathological grades of glioma tissues. In the meantime, as shown in Figure S1C, there was no dysregulated expression of linear PLEKHA5 was found in glioma tissues or cells. Moreover, circ-PLEKHA5 was almost located in the cytoplasm of HA, U373 and U251 cells, and was visually elevated in glioma cells ( Figure 2D). To evaluate the effect of circ-PLEKHA5 in glioma, stable circ-PLEKHA5 knockdown glioma cells were established. As shown in Figure 2E-H, knockdown of circ-PLEKHA5 signi cantly inhibited the proliferation, VM formation, migration and invasion abilities of glioma cells, and also inhibited the expression of MMP-2 and VE-cadherin, while re-expressed circ-PLEKHA5 restored these inhibitory effects.

SRSF7 promoted VM formation of glioma cells by increasing the expression of circ-PLEKHA5
As previously mentioned, the expression of circ-PLEKHA5 was signi cantly decreased in SRSF7 silenced cells ( Figure S1B), and several studies have found that RNA-binding proteins (RBPs) could increase the generation of circRNAs by binding to the upstream and downstream of their circRNA-forming pre-mRNAs that near the junction sites [18] . By using the RNA-binding protein database RBPDb, the putative binding sites between SRSF7 and PLEKHA5 pre-mRNA (300 bases upstream of exon4; 300 bases downstream of exon 15) was found ( Figure S1D), therefore, we proposed that circ-PLEKHA5 might be involved in the regulation of SRSF7 on glioma cells. We rstly checked the effect of SRSF7 on circ-PLEKHA5 via qRT-PCR and results showed the expression of circ-PLEKHA5 was reduced in stable SRSF7 silenced glioma cells while re-expressed SRSF7 restored circ-PLEKHA5( Figure 3A). We next carried out the RIP assay and the result showed that PLEKHA5 pre-mRNA was signi cantly enriched in Anti-SRSF7 groups compared with Anti-IgG group, which con rmed our prediction ( Figure 3B). Moreover, as shown in Figure 3C-F, overexpression or knockdown of circ-PLEKHA5 signi cantly counteracted the inhibitory or promoting role of SRSF7 on the proliferation, VM, migration, invasion and the expression of VM-associated proteins in glioma cells, implied that circ-PLEKHA5 is associated with the effect of SRSF7 on glioma cells.
Circ-PLEKHA5 encoded a novel peptide through its ORF By matching circ-PLEKHA5 in circRNADb, a predicted ORF sequence and an IRES sequence were found and circ-PLEKHA5 may encode a 622aa peptide protein by initiation-termination codon "ATG-TGA" ( Figure  2A). To investigate the existence of circ-PLEKHA5-622aa, a speci c antibody was designed and western blot was carried out. As shown in Figure 4B, compared with circ-PLEKHA5(-)NC group, knockdown of circ-PLEKHA5 signi cantly reduced the expression of circ-PLEKHA5-622aa, while re-expressed circ-PLEKHA5 restored the expression of circ-PLEKHA5-622aa. Furthermore, we analyzed the activity of predicted IRES in circ-PLEKHA5 by performing dual-luciferase assay. The full-length and mutated IRES sequence of circ-PLEKHA5 were cloned and constructed between Rluc and Luc reporters along with their own initiation and termination codons, and the relative luciferase (Luc/Rluc) were analyzed. As shown in Figure 4C, the mutation signi cantly reduced the luciferase activity, which con rmed the activity of IRES in circ-PLEKHA5. Moreover, to further verify that endogenous 622aa is translated from circ-PLEKHA5 ORF, we added FLAG tag sequence separately at each end of circ-PLEKHA5 ORF along with anking sequences for helping cyclization (2#FLAG-circ-PLEKHA5). For a negative control, one anking sequence was deleted (3#AG-circ-PLEKHA5-FL). For a positive control, the integrated sequence that encodes 622aa was constructed with FLAG tag forward termination codon (4#FLAG-circ-PLEKHA5-ORF). All plasmids above were transfected into U373 and U251 subsequently, and the results followed western blot showed that FLAG tag was found in FLAG-circ-PLEKHA5 group and FLAG-circ-PLEKHA5-ORF group, and the expression of 622aa was visually elevated in these two groups compared with in control and negative groups ( Figure 4D). Furthermore, the expression level of circ-PLEKHA5 only up-regulated in FLAG-circ-PLEKHA5 group but not in FLAG-circ-PLEKHA5-ORF group ( Figure S1E), and these results further suggested that 622aa was encoded by circ-PLEKHA5 ORF.
622aa was up-regulated in glioma and promoted VM formation of glioma cells We further evaluated the expression level of circ-PLEKHA5-622aa, and as shown in Figure 5A-B, circ-PLEKHA5-622aa was signi cantly up-regulated in glioma tissues and cells, and the expression level was positively correlated with the histopathological grade. Moreover, compared with in glioma cells that transfected with GFP vector alone, the localization of GFP was visually more concentrated in the cytoplasm of cells that transfected with 622aa-GFP ( Figure 5C). By using bioinformatic software SMART, a pleckstrin homology (PH) domain was found in the amino acid sequence of 622aa ( Figure S1F). PH domain was known to be shared with key mediators of cellular biological behaviors due to its phospholipid-binding speci cities [19] . To further investigate the role of circ-PLEKHA5-622aa but not circ-PLEKHA5 in glioma, we constructed 622aa overexpressed glioma cells by using FLAG-circ-PLEKHA5-ORF vector, and analyzed the changes in biological behaviors of these cells. As shown in Figure 5D-G, U373 and U251 cells with overexpressed 622aa exhibited increased proliferation, VM formation, migration and invasion abilities, and increased expression of MMP-2 and VE-cadherin. Moreover, overexpressed 622aa also counteracted the inhibitory effect that caused by circ-PLEKHA5 knockdown. These results indicated that circ-PLEKHA5 regulated the malignant biological behaviors by encoding circ-PLEKHA5-622aa. It has been demonstrated that PH domain-containing proteins are important components that involved in the regulation of signal transduction pathways on cell behaviors [20] . In order to comprehend the potential regulatory mechanism of circ-PLEKHA5-622aa in glioma cells VM formation, we evaluated the effect of PH domain in circ-PLEKHA5-622aa on the expression of p-Akt as well. As shown in Figure S1G, the expression level of p-Akt was signi cantly increased in circ-PLEKHA5-622aa(+) group, while remained static in circ-PLEKHA5-622aa (+) with mutated PH domain group.

The combination using of SRSF7 and circ-PLEKHA5 inhibitors suppressed tumor growth and VM formation in nude mice
In order to further determine the roles of SRSF7 and circ-PLEKHA5 in vivo, the nude mice model was used. As shown in Figure 6A-B, the volume of transplanted tumor in SRSF7(-) group and circ-PLEKHA5(-) group was signi cantly reduced compared with control and negative control groups. In addition, the combined use of SRSF7 and circ-PLEKHA5 inhibitors produced the smallest tumor volume than single use of them respectively. Moreover, the result followed CD34-PAS showed that knockdown of SRSF7 and circ-PLEKHA5 respectively reduced the VM number of the transplanted tumors and the VM number in SRSF7(-)+circ-PLEKHA5(-) group was the least ( Figure 6C).

Discussion
As critical regulators of various cellular biological processes, RBPs play roles in gene expression, RNA splicing, localization, translation and degradation at post-transcriptional level. Based on this, RBPs can also participated in the development of tumors by in uencing speci c RNA expression [21,22] . SRSF7 is a member of SR protein family which was rst found by Cavaloc Y et al. at 1994. SRSF7 contains a motif with function of recognizing speci c RNAs at the N-terminus and a structure with function of facilitating spliceosome assembly, and based on this, SRSF7 participates in alternative splicing processes of multiple pre-mRNAs [23] . So far SRSF7 is known to express abnormally and play regulatory roles in multiple cancers including lung cancer, kidney cancer and colon cancer [7,23,24] . In this study, based on the TCGA database, we found for the rst time that SRSF7 was highly expressed in glioma tissues and cells, especially in high grade. Knockdown of SRSF7 signi cantly inhibited the proliferation, VM formation, migration and invasion abilities of glioma cells, and also decreased the expression level of VM-related proteins MMP-2 and VE-cadherin. Furthermore, the re-expression of SRSF7 eliminated the effect of SRSF7 inhibition on glioma cells, suggesting that SRSF7 plays a role as an oncogene in glioma. Similar to our research, SRSF7 was found to highly express in lung cancer and colon cancer, knockdown of SRSF7 inhibited the proliferation and promoted the apoptosis of tumor cells. In addition, SRSF7 is also upregulated in gastric and prostate cancers, and the expression level is positively correlated with poor prognosis of patients [25] .
PLEKHA5 (Pleckstrin homology domain-containing protein family A member 5) is a member of the PLEKHA family which is known to contain Trp-Trp (WW) and pleckstrin homology (PH) domains. Yamada et al. found that two forms of PLEKHA5 mRNA are widely expressed in human tissues and play a role in brain development due to contain PH domains and their ability to associate with PI3P, PI(3.5)P2 and other speci c phosphoinositides compounds [26] . In addition, PLEKHA5 is also been found to abundantly expressed in the cytoplasm and cell membrane, and closely related to the process of cell migration and vascular formation [27] . In the cell model of brain metastasis of melanoma, knockdown of PLEKHA5 inhibits the proliferation of tumor cells, blocks cell cycle at G1-S progression, thereby inhibiting the tumorigenesis of tumor cells in vivo [28] . CircRNAs is a class of endogenous RNA with an atypical structure of covalently binding of 5'-and 3'-ends. As the forefront of biological researches, a growing number of circRNAs have been found to be involved in various tumor progression and viewed as potential therapeutic targets [29] . Many circRNAs including circ-U2AF1, circ-SMARCA5 and circ-DENND2A are abnormally expressed in gliomas and involved in regulating glioma cell behaviors [30][31][32] . Yang P et al. found that knockdown of circ-ZNF292 could down-regulate the proliferation and angiogenesis of glioma cells by inhibiting the activity of Wnt/β pathway [33] . Circ-PLEKHA5, by matching in database CircBase (hsa_circ_0098067) and CircRNADb (hsa_circ_00571), is composed of 12 exons of PLEKHA5 pre-mRNA with 1810 nucleotides long. In our study, we rst identi ed the existence of circ-PLEKHA5 and found that circ-PLEKHA5 was up-regulated in glioma tissues and cells and mainly located in cytoplasm. Knockdown of circ-PLEKHA5 signi cantly inhibited the proliferation, VM formation, migration and invasion abilities of glioma cells, and also decreased the expression level of VM-related proteins MMP-2 and VE-cadherin. Furthermore, the re-expression of circ-PLEKHA5 eliminated the effect of circ-PLEKHA5 inhibition on glioma cells, indicating circ-PLEKHA5 as an oncogene in glioma.
Several studies have found that RBPs could bind to multiple pre-mRNAs at the upstream or downstream of circRNAs forming exon adjacent sites, especially anking sequences or intronic repeated sequences, thereby promoting the back-splicing of pre-mRNA along with the covalent binding of adjacent exons, and the generation of circRNAs [18] . Based on UCSC and RBPmap database, two potential binding sites of SRSF7 and PLEKHA5 pre-mRNA were found at the upstream and downstream of the circ-PLEKHA5 sequence respectively. After a series of experiments, we con rmed the molecular mechanism that SRSF7 promoted the formation and expression level of circ-PLEKHA5 through binding to PLEKHA5 pre-mRNA. Similar to our nding, RBP-Sam68 has been reported to promote the circularization of SMN exons along with the generation of circ-SMN by binding to SMN pre-mRNA [34] . RBP-QKI binds to the upstream and downstream of SMARCA5 pre-mRNA anking sequence and promotes the cyclization of adjacent exons to form circ-SMARCA5 [31] .
With the technical evolution of deep RNA sequencing and ribosomal pro ling technologies, circRNAs that previously considered as non-coding RNAs, have been identi ed to encode small peptides through its own open reading frames (ORFs) and driven by internal ribosome entry site (IRES) [35] . In addition, the peptide proteins, instead of their initial circRNAs, could regulate the development of various cancers including glioma according to their amino acid sequence and functional structure [36] . For instance, circ-PPP1R12A-73aa that encoded by circ-PPP1R12A ORF is highly expressed in colon cancer and promotes colon cancer cell proliferation, migration and invasion abilities by activating Hippo-YAP signaling pathway [37] . In liver cancer, β-catenin-370aa that encoded by circ-β-catenin protects β-catenin by prevent it from binding GSK3β and ubiquitination, thereby promoting tumor growth [38] . Based on the prediction on database circRNADb, we investigated the coding ability of circ-PLEKHA5 and con rmed that circ-PLEKHA5 could encode a 622 amino acid (aa) peptide circ-PLEKHA5-622aa. Moreover, the expression level of 622aa was signi cantly elevated in glioma. We next investigated the potential function of circ-PLEKHA5-622aa, and the results showed that the elevated 622aa, independent of circ-PLEKHA5, signi cantly increased the proliferation, migration, invasion and VM formation abilities of glioma cells and also reversed the inhibitory effects caused by circ-PLEKHA5-depletion. These results indicated that circ-PLEKHA5 regulated the malignant behaviors of glioma cells by encoding circ-PLEKHA5-622aa.
Pleckstrin homology (PH) domain is a region of approximately 100 to 120 amino acids that mostly occurs in a wide range of proteins which are involved in intracellular signaling or as constituents of the cytoskeleton [39] . It has been reported that the PH domain is essential for PH-domain-containing proteins, especially cytosolic signaling proteins, to interact with phosphatidylinositol-(3, 4, 5)-triphosphate (PIP3) and recruit proteins to participated in multiple signal pathways [40] . For instance, the PH domain in Akt pathway regulator PDK1 is recognized and binds to PIP3, which activates Akt signaling and promotes cell angiogenesis subsequently [41] . As a critical modulating molecule, the phosphorylation of Akt has been found to promote the proliferation, migration and VM formation of glioma cells by regulating the downstream effector MMP-2 [42,43] . Due to the PH domain that PLEKHA5 contains, PLEKHA5 was supposedly to be associated with multiple phosphoinositide binding properties and PI3K-AKT pathway, thereby participating in intracellular functions [44] . It was reported that the expression of phosphorylated AKT (p-Akt) was decreased in PLEKHA5-depleted melanoma cells [28] . By scanning the amino acid sequence, PH domain was also found in circ-PLEKHA5-622aa. In our study, we found that the PH domain in circ-PLEKHA5-622aa signi cantly increased the expression level of p-Akt, indicating that circ-PLEKHA5-622aa may play the roles by activating p-Akt. Similarly, circ-SHPRH and circ-SHPRH-146aa were downregulated and functioned as tumor suppressors in glioma [13] , circ-PINT suppressed the proliferation and migration of glioma cells via encoding circ-PINT-87aa [14] .
Finally, by performing the in vivo study, we demonstrated that the alone or combined use of SRSF7 inhibitor and circ-PLEKHA5 inhibitor observably inhibited the tumor growth and prolonged the survival times of nude mice, and the combined use resulted in the smallest tumors and the highest survival times.
Moreover, the CD34-PAS that performed in xenografted tumors showed that knockdown SRSF7 or circ-PLEKHA5 signi cantly inhibited the VM formation in vivo, and the combined use resulted in the least VMs in vivo. These suggested that the combination use of SRSF7 inhibitor and circ-PLEKHA5 inhibitor might has potential clinical application value in the treatment of glioma.

Conclusion
Collectively, our study found the elevated expression levels of SRSF7, circ-PLEKHA5 in glioma tissues and cells, and knockdown of SRSF7 or circ-PLEKHA5 inhibited VM formation of glioma cells in vitro and in vivo. SRSF7 promoted the generation of circ-PLEKHA5 through binding to PLEKHA5 pre-mRNA and a novel peptide protein 622aa that encoded by circ-PLEKHA5 ORF was identi ed. Moreover, we found that circ-PLEKHA5-622aa was elevated in glioma and promoted the VM formation of glioma cells itself. In summary, our nding rst reveled that SRSF7/circ-PLEKHA5/circ-PLEKHA5-622aa module regulated the biological behaviors and VM formation of glioma cells, as well as provided a novel insight for the treatment of glioma.

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In this article, the material original research and has not been published elsewhere in whole or in part. All