- Long non-coding RNA PSMA3-AS1. - promotes glioma progression through modulating the miR-411-3p/HOXA10 pathway. - However, the role of PSMA3-AS1 in glioma remains unknown. - Therefore, we concentrated on researching the regulatory mechanism of PSMA3-AS1 in glioma.. - Methods: PSMA3-AS1 expression was detected using RT-qPCR. - Functional assays were performed to measure the effects of PSMA3-AS1 on glioma progression. - After that, ENCORI (http://starbase.sysu.edu.cn/) database was used to predict potential genes that could bind to PSMA3-AS1, and miR-411-3p was chosen for further studies. - The interaction among PSMA3-AS1, miR-411-3p and homeobox A10 (HOXA10) were confirmed through mechanism assays.. - Results: PSMA3-AS1 was verified to be up-regulated in glioma cells and promote glioma progression. - Furthermore, PSMA3-AS1 could act as a competitive endogenous RNA (ceRNA) for miR-411-3p to regulate HOXA10 and thus affecting glioma progression.. - Conclusion: PSMA3-AS1 stimulated glioma progression via the miR-411-3p/HOXA10 pathway, which might offer a novel insight for the therapy and treatment of glioma.. - Keywords: PSMA3-AS1, miR-411-3p, HOXA10, Glioma. - has been published recently and disclosed that PSMA3-AS1 is actively involved in the progression of esophageal cancer and serves as a tumor-promoter [11].. - However, the role and molecular function of PSMA3- AS1 in glioma remain blurry. - In this study, we also designed related assays with the aim of exploring the potential ceRNA pattern of PSMA3- AS1 and its effects on glioma progression.. - In a word, we focused on the exploration of PSMA3- AS1 expression in glioma cells and how its abnormal ex- pression affects the biological behaviors of glioma cells such as cell proliferation and apoptosis. - In addition, we further probed into the regulatory mechanism of PSMA3-AS1 through the ceRNA approach, hoping to provide novel insight into the therapeutic options and treatment for glioma.. - The specific shRNAs tar- geting PSMA3-AS1 (sh-PSMA3-AS1#1/2) and HOXA10 (sh-HOXA10#1/2) were designed and constructed by GenePharma (Shanghai, China), using the nonspecific shRNAs as negative control (NC). - The knock- down and overexpression of miR-411-3p were separately achieved by transfecting cells with miR-411-3p inhibitor and miR-411-3p mimics, as well as NC inhibitor and NC mimics (all. - The contents of GAPDH, PSMA3-AS1 and U6 were separately analyzed in two cell fractions with GAPDH and U6 treated as the internal control of cytoplasm and nucleus, respectively.. - FISH assay was implemented in presence of the specific RNA probe to PSMA3-AS1 (Ribobio), following the established protocol. - Cell protein extracts were incubated with the PSMA3-AS1 biotin probe or control probe. - The antibodies used in the study were antibodies against IgG, AGO2, PSMA3-AS1, miR-411-3p and HOXA10. - Cells were co- transfected with miR-411-3p mimics or NC-mimics and the pmirGLO vectors containing the fragments of PSMA3-AS1 or HOXA10. - PSMA3-AS1 accelerates cell proliferation and suppresses apoptosis in glioma. - To figure out whether PSMA3-AS1 was involved in glioma progression, we applied RT-qPCR to assess the expression of PSMA3-AS1. - 1a, the expression of PSMA3-AS1 was higher in glioma cell lines (LN-229, T98G and SHG-44) than that in normal human brain as- trocytes HEB cell. - AS1 in glioma, sh-PSMA3-AS1#1 and sh-PSMA3-AS1#2 were used to induce PSMA3-AS1 down-regulation (Fig.. - After that, EdU and colony formation assays were adopted to evaluate glioma cell proliferation ability, which showed that the proliferative ability of LN-229 and T98G cells was effectively suppressed upon PSMA3-AS1 silencing (Fig. - Furthermore, JC-1 assay was conducted to de- tect cell apoptosis after PSMA3-AS1 was knocked down in glioma cells, and result showed that the JC-1 ratio was de- clined by PSMA3-AS1 silencing, which meant that the down-regulation of PSMA3-AS1 enhanced cell apoptosis in glioma (Fig. - Experimental data from flow cytometry and TUNEL assays further showed that PSMA3-AS1 silen- cing promoted glioma cell apoptosis (Fig. - Collectively, PSMA3-AS1 is up-regulated in glioma cells and it pro- motes glioma progression.. - PSMA3-AS1 functions AS a ceRNA for miR-411-3p. - Based on this, we first confirmed the location of lncRNA PSMA3-AS1 with the assistance of subcellular fractionation and FISH assays. - The results showed that PSMA3-AS1 was. - 1 PSMA3-AS1 accelerates cell proliferation and suppresses apoptosis in glioma. - A The expression level of PSMA3-AS1 was measured by RT- qPCR in glioma cell lines (LN-229, T98G and SHG-44) and normal HEB cell, using one-way ANOVA and Dunnett. - B RT-qPCR with t -test analysis was conducted to assess the interference efficiency of sh-PSMA3-AS1 in glioma cells. - C-D EdU and colony formation assays were adopted to test cell proliferation in glioma upon PSMA3-AS1 silencing. - E-G JC-1, flow cytometry and TUNEL assays were performed to evaluate cell apoptosis after PSMA3-AS1 was inhibited in glioma cells. - To further deter- mine the target gene, RNA pull down assay was carried out, and the finding showed that miR-411-3p was obvi- ously enriched with PSMA3-AS1 biotin compared with the control probe group in glioma cells, while no obvious changes were found in other miRNAs (Fig. - Figure 2e also exhibited the underlying binding sites between pre- dicted PSMA3-AS1 and miR-411-3p via ENCORI. - Fur- thermore, it was observed from RIP assay that PSMA3- AS1 and miR-411-3p were both abundant in Anti-AGO2 complex rather than in Anti-IgG group, reflecting the dir- ect interplay between them (Fig. - Besides, RT-qPCR was used to examine the overexpression efficiency of miR- 411-3p (Fig. - Finally, it was observed from luciferase reporter assay that PSMA3-AS1 wild type (PSMA3-AS1-. - WT) expressed lower luciferase activity with miR-411-3p overexpression, while the mutant type of PSMA3-AS1 expressed normally, suggesting the binding ability between PSMA3-AS1 and miR-411-3p (Fig. - Based on the above data, we conclude that PSMA3-AS1 functions as a ceRNA for miR-411-3p in glioma cells.. - MiR-411-3p is able to target HOXA10. - To explore the target gene of miR-411-3p, we applied miRmap (https://mirmap.ezlab.org/) and PicTar (https://. - Afterwards, we per- formed RT-qPCR assay under the circumstance of co- transfected cells with sh-PSMA3-AS1 and miR-411-3p mimics to further determine the target. - 2 PSMA3-AS1 functions as a ceRNA for miR-411-3p. - A-B Subcellular fractionation and FISH assays were used to confirm the location of PSMA3-AS1 in glioma cells. - C The possible target genes of PSMA3-AS1 were predicted via ENCORI database. - D RNA pull down assay was carried out to confirm the interaction between PSMA3-AS1 and potential miRNAs. - F RIP assay was used to test the correlation between PSMA3-AS1 and miR-411-3p. - G RT-qPCR was implemented to assess the overexpression efficiency of miR-411-3p. - H Luciferase reporter assay was conducted to test the interaction between PSMA3-AS1 and miR-411-3p using two-way ANOVA and Tukey methods. - that HOXA10 was highly expressed in glioma cell lines compared with the other two mRNAs (Fig. - The binding sites between miR-411-3p and HOXA10 were hypothe- sized through ENCORI database (Fig. - To prove the association and interaction between miR-411-3p and HOXA10, luciferase reporter and RNA pull down assays were utilized. - 3e, the luciferase activity of HOXA10-WT was obviously reduced by miR-411-3p mimics compared with NC mimics, while that of. - HOXA10-Mut was not affected in glioma cells. - Moreover, it was confirmed from RIP assay that PSMA3-AS1, miR- 411-3p and HOXA10 were co-existed in RNA induce- silencing complex (RISC) (Fig. - Taken together, miR- 411-3p is able to target HOXA10.. - 3 A MiR-411-3p is able to target HOXA10. - B The expression of possible mRNAs was examined upon PSMA3-AS1 knockdown and miR-411-3p overexpression. - C The expression of three mRNAs was evaluated via RT-qPCR in glioma cell lines with the help of two-way ANOVA and Tukey analysis. - D The binding sites of miR-411-3p and HOXA10 were presented by ENCORI database. - E Luciferase reporter assay was performed to confirm the binding situation between miR-411-3p and HOXA10 using two-way ANOVA and Tukey analysis. - F The connection among PSMA3-AS1, miR-411-3p and HOXA10 was testified by RIP assay. - of glioma cells, LN-229 and T98G cells was transfected with sh-HOXA10#1 and sh-HOXA10#2 and RT-qPCR was used to test the inhibitory efficiency of HOXA10 in glioma cells (Fig. - Besides, through JC-1, flow cytometry and TUNEL assays, we discovered that down-regulation of HOXA10 could en- hance cell apoptosis in glioma cells (Fig. - PSMA3-AS1 promotes glioma progression through modulating miR-411-3p/HOXA10 axis. - In the last step, a series of rescue assays were designed to confirm the interaction among PSMA3-AS1, miR- 411-3p and HOXA10 in glioma. - Firstly, the inhibitory ef- ficiency of miR-411-3p in glioma cells was examined via RT-qPCR (Fig. - Then, we divided experimental groups into sh-NC, sh-PSMA3-AS1#1 and sh-PSMA3- AS1#1 + miR-411-3p inhibitor and carried out the. - 5b that HOXA10 was primarily cut down by PSMA3-AS1 down-regulation but later rescued by miR-411-3p silen- cing, which suggested that PSMA3-AS1 could positively regulate HOXA10 via miR-411-3p, and thus the PSMA3-AS1/miR-411-3p/HOXA10 regulatory axis was successfully constructed. - 5d and e that the proliferative abil- ity of glioma cells was initially restrained due to the si- lencing of PSMA3-AS1 while recovered by miR-411-3p silencing or the overexpression of HOXA10. - Inversely, silencing PSMA3-AS1 accelerated glioma cell apoptosis, while this effect could be countervailed by miR-411-3p knockdown or HOXA10 up-regulation (Fig. - To conclude, PSMA3-AS1 promotes glioma progression through modulating the miR-411-3p/HOXA10 axis.. - PSMA3-AS1 has been disclosed to be actively involved in the progression of esophageal cancer in which it plays tumor-promoting function, while its detailed function and mechanism in glioma re- mains to be explored. - In this study, PSMA3-AS1 was testified to be up-regulated in glioma cells, and then loss-of-function assays were carried out to verify the ef- fects of PSMA3-AS1 on glioma cell proliferation and apoptosis. - Results showed that PSMA3-AS1 down- regulation had anti-proliferative and pro-apoptotic ef- fects on glioma cells. - On the whole, PSMA3-AS1 was regarded as a carcinogenic gene in glioma.. - LncRNAs have been illustrated to exert oncogenic roles as a ceRNA in glioma [17, 18]. - In this study, miR- 411-3p was selected and verified to be the target gene of PSMA3-AS1 and HOXA10 was identified as the down- stream target of miR-411-3p, thus forming the PSMA3- AS1/miR-411-3p/HOXA10 axis. - As for miR-411-3p, Halvorsen AR1 et al. - show that miR-411-3p is closely as- sociated with the overall survival rate of patient with lung cancer [19] as well as ovarian cancer [20].. - In conclusion, this study initially uncovered that PSMA3-AS1 could serve as an oncogene in glioma pro- gression by sponging miR-411-3p to regulate HOXA10.. - It is also the first time that we discovered PSMA3-AS1 may potentially act as a novel biomarker and therapeutic target for glioma treatment.. - PSMA3- AS1: Proteasome 20S subunit alpha 3 antisense RNA 1. - 5 PSMA3-AS1 promotes glioma progression through modulating the miR-411-3p/HOXA10 axis. - A The interference efficiency of miR-411-3p inhibitor in glioma cells was assessed through RT-qPCR. - B RT-qPCR was applied to test HOXA10 expression change in response to PSMA3-AS1 silencing and miR-411-3p down-regulation with the help of one-way ANOVA and Tukey analysis. - C The overexpression efficiency of pcDNA3.1- HOXA10 in glioma cells was tested via RT-qPCR. - D-E Cell proliferation in glioma under different transfection conditions was evaluated by EdU and colony formation assays. - New insights into long noncoding RNAs and their roles in glioma. - Long non-coding RNA PSMA3-AS1 promotes malignant phenotypes of esophageal cancer by modulating the miR-101/EZH2 axis AS a ceRNA. - Long noncoding RNA CDKN2B-AS1 interacts with miR-411-3p to regulate ovarian cancer in vitro and in vivo through HIF-1a/VEGF/P38 pathway. - HOXA10-AS: a novel oncogenic long non-coding RNA in glioma
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