AGAP2-AS1 serves as an oncogenic lncRNA and prognostic biomarker in glioblastoma multiforme

Long noncoding RNA (lncRNA) AGAP2 antisense RNA 1 (AGAP2-AS1) has been suggested to function as an oncogenic lncRNA in lung cancer, breast cancer, and anaplastic glioma. However, the expression pattern and molecular mechanism of AGAP2-AS1 in glioblastoma multiforme (GBM) remains unknown. The purpose of this study is to present more evidence about the clinical and biological function of AGAP2-AS1 in GBM. In our results, we found AGAP2-AS1 expression was increased in GBM compared with adjacent normal brain tissues or low-grade glioma tissues, and there was no significantly different between low-grade glioma tissues and normal tissues. Kaplan-Meier survival analysis indicated patients with GBM having high-expression of AGAP2-AS1 had shorter overall survival time than those with low expression of AGAP2-AS1. The loss-of-function studies showed that downregulation of AGAP2-AS1 depressed cell proliferation, migration, and invasion, and promoted cell apoptosis in GBM. In summary, AGAP2-AS1 is a prognostic biomarker for patients with GBM, and functions as an oncogenic lncRNA to modulate GBM cell proliferation, apoptosis, migration, and invasion, which suggests that AGAP2-AS1 is potential therapeutic target for GBM.     

Long noncoding RNA FLVCR1-AS1 aggravates biological behaviors of glioma cells via targeting miR-4731-5p/E2F2 axis

Long noncoding RNAs (lncRNAs) display essential roles in cancer progression. FLVCR1-AS1 is a rarely investigated lncRNAs involved in various human cancers, such as hepatocellular carcinoma and lung cancer. However, its function in glioma has not been clarified. In our study, we found that FLVCR1-AS1 was highly expressed in glioma tissues and cell lines. And upregulation of FLVCR1-AS1 predicted poor prognosis in patients with glioma. Moreover, FLVCR1-AS1 knockdown inhibited proliferation, migration and invasion of glioma cells. Through bioinformatics analysis, we identified that FLVCR1-AS1 was a sponge for miR-4731-5p to upregulate E2F2 expression. Moreover, rescue assays indicated that FLVCR1-AS1 modulated E2F2 expression to participate in glioma progression. Altogether, our research demonstrates that the FLVCR1-AS1/miR-4731-5p/E2F2 axis is a novel signaling in glioma and may be a potential target for tumor therapy.     

Long noncoding RNA FOXD2-AS1 promotes glioma cell cycle progression and proliferation through the FOXD2-AS1/miR-31/CDK1 pathway

Long noncoding RNAs (lncRNAs) are vital mediators involved in cancer progression. Previous studies confirmed that FOXD2 adjacent opposite strand RNA 1 (FOXD2-AS1) is upregulated in tumor diseases. The potential influence of FOXD2-AS1 in glioma progression, however, remains unknown. In this paper, FOXD2-AS1 was found to be upregulated in glioma tissues. Its level was linked with glioma stage. Moreover, glioma patients expressing high level of FOXD2-AS1 suffered worse prognosis. Biological functions of FOXD2-AS1 in glioma cells were analyzed through integrative bioinformatics and TCGA RNA sequencing data analysis. Pathway enrichment analysis uncovered that FOXD2-AS1 was mainly linked with cell cycle regulation in both low-grade glioma and glioblastoma. Further experiments demonstrated that silence of FOXD2-AS1 inhibited proliferation, arrested cell cycle and downregulated cyclin-dependent kinase 1 (CDK1) in human glioma cells. Dual-luciferase reporter assay confirmed that FOXD2-AS1 upregulated CDK1 by sponging miR-31. Rescue assays were performed and confirmed the regulatory loop FOXD2-AS1/miR-31/CDK1 in glioma. Collectively, our results indicated that the FOXD2-AS1/miR-31/CDK1 axis influenced glioma progression, providing a potential new target for glioma patients.     

Long noncoding RNA FOXD2-AS1 promotes glioma malignancy and tumorigenesis via targeting miR-185-5p/CCND2 axis

Glioma is the most aggressive malignant tumor in the adult central nervous system. Abnormal long noncoding RNA (lncRNA) FOXD2-AS1 expression was associated with tumor development. However, the possible role of FOXD2-AS1 in the progression of glioma is not known. In the present study, we used in vitro and in vivo assays to investigate the effect of abnormal expression of FOXD2-AS1 on glioma progression and to explore the mechanisms. FOXD2-AS1 was upregulated in glioma tissue, cells, and sphere subpopulation. Upregulation of FOXD2-AS1 was correlated with poor prognosis of glioma. Downregulation of FOXD2-AS1 decreased cell proliferation, migration, invasion, stemness, and epithelial-mesenchymal transition (EMT) in glioma cells and inhibited tumor growth in transplanted tumor. We also revealed that FOXD2-AS1 was mainly located in cytoplasm and microRNA (miR)-185-5p both targeted FOXD2-AS1 and CCND2 messenger RNA (mRNA) 3′-untranslated region (3′-UTR). miR-185-5p was downregulated in glioma tissue, cells, and sphere subpopulation. Downregulation of miR-185-5p was closely correlated with poor prognosis of glioma patients. In addition, miR-185-5p mimics decreased cell proliferation, migration, invasion, stemness, and EMT in glioma cells. CCND2 was upregulated in glioma tissue, cells, and sphere subpopulation. Upregulation of CCND2 was closely correlated with poor prognosis of glioma patients. CCND2 knockdown decreased cell proliferation, migration, invasion, and EMT in glioma cells. In glioma tissues, CCND2 expression was negatively associated with miR-185-5p, but positively correlated with FOXD2-AS1. FOXD2-AS1 knockdown and miR-185-5p mimics decreased CCND2 expression. Inhibition of miR-185-5p suppressed FOXD2-AS1 knockdown-induced decrease of CCND2 expression. Overexpression of CCND2 suppressed FOXD2-AS1 knockdown-induced inhibition of glioma malignancy. Taken together, our findings highlight the FOXD2-AS1/miR-185-5p/CCND2 axis in the glioma development.     

长链非编码RNA HOXA11-AS在胶质瘤中的表达及其临床意义的研究

目的:探讨长链非编码RNA HOXA11-AS在胶质瘤组织中的表达以及与胶质瘤患者临床预后的相关性。方法:首先,应用RT-PCR法检测人胶质瘤组织以及正常脑组织中HOXA11-AS的表达情况;其次,分析HOXA11-AS的表达水平与胶质瘤患者临床病理学特征之间的关系;最后,探讨HOXA11-AS的表达水平与胶质瘤患者预后之间的相关性。结果:RT-PCR显示,较之于正常脑组织(1.00±0.17),HOXA11-AS在胶质瘤组织(3.89±0.34)中的表达水平显著升高(P0.001),且随着肿瘤学分级的增高,HOXA11-AS的表达水平也随着升高(Grade Ⅰ-Ⅱ, 2.96±0.21 vs. Grade Ⅲ-Ⅳ, 4.83±0.50, p=0.003)。x~2检验提示HOXA11-AS表达水平与胶质瘤患者的肿瘤学分级、KPS评分以及患者的复发情况具有显著相关性,而与患者的年龄、性别、肿瘤大小等无相关性。Kaplan-Meier分析患者生存率,结果显示,HOXA11-AS低表达组患者的生存率明显高于HOXA11-AS高表达组患者,差异具有统计学意义(P0.001)。最后,我们的研究结果发现,HOXA11-AS的高表达水平、肿瘤学分级的增高、KPS评分80分均为影响胶质瘤患者预后的独立危险因素(P0.05)。结论:HOXA11-AS与胶质瘤患者预后密切相关,且为预测患者预后的独立因素。     

Long non-coding RNA IGF2-AS represses breast cancer tumorigenesis by epigenetically regulating IGF2

Long non-coding RNAs are a kind of endogenous ncRNAs with a length of more than 200 bp. Accumulating evidence suggests that long non-coding RNAs function as pivotal regulators in tumorigenesis and progression. However, their biological roles in breast cancer remain largely unknown. Here, we found that IGF2 antisense RNA (IGF2-AS) was significantly decreased in breast cancer tissues, cell lines, and plasma. Patients with low IGF2-AS were more likely to develop larger tumor size and later clinical stage. Overexpression of IGF2-AS evidently inhibited the proliferation and induced apoptosis of MCF-7 and T47D cells in vitro, as well as retarded tumor growth in vivo. Further investigation revealed that IGF2-AS inhibited the expression of its sense-cognate gene IGF2 in an epigenetic DNMT1-dependent manner, resulting in the inactivation of downstream oncogenic PI3K/AKT/mTOR signaling pathway. Enforced expression of IGF2 could significantly block the tumor inhibitory effect of IGF2-AS. Importantly, we found that IGF2-AS could be used as an effective biomarker for breast cancer diagnosis and prognosis. Taken together, our study indicates that IGF2-AS is a tumor suppressor in breast cancer, restoration of IGF2-AS may be a promising treatment for this fatal disease.     

Serine/Threonine Kinase CHEK1-Dependent Transcriptional Regulation of RAD54L Promotes Proliferation and Radio Resistance in Glioblastoma

Accumulating evidence indicates that Checkpoint kinase 1 (CHEK1) plays an essential role in tumor cells and that it could induce cell proliferation and could be related to prognosis in multiple types of cancer. However, the biological role and molecular mechanism of CHEK1 in GBM still remain unclear. In this study, we identified that CHEK1 expression was enriched in glioblastoma (GBM) tumors and was functionally required for tumor proliferation and that its expression was associated to poor prognosis in GBM patients. Mechanically, CHEK1 induced radio resistance in GBM cells, and CHEK1 knockdown increased cell apoptosis when combined with radiotherapy via regulation of the DNA repair/recombination protein 54L (RAD54L) expression. Therapeutically, we found that CHEK1 inhibitor attenuated tumor growth both in vitro and in vivo. Collectively, CHEK1 promotes proliferation, induces radio resistance in GBM, and could become a potential therapeutic target for GBM.     

FOXO1 associated with sensitivity to chemotherapy drugs and glial-mesenchymal transition in glioma

Mesenchymal subtype of glioblastoma (GBM), identified as one of four clinically relevant molecular subtypes, has worst prognosis because of its close relation with the malignant biological properties induced by glial-mesenchymal transition (GMT). However, the molecular mechanism of GMT and its characterized molecule of GBM have not been studied. Forkhead box protein O1 (FOXO1) is at a convergence point of receptor tyrosine kinase signaling as one of the three core pathways implicated in GBM. Our previous study indicated that the inactivation of FOXO1 involved in the inhibition of GMT is an independent prognosis factor of GBM. In this study, we will further confirm the role of FOXO1 in GMT through cytological experiments to clarify how FOXO1 regulates GMT and its clinical significance. We established virus-infected FOXO1 overexpression and FOXO1 knockdown cells of U373 MG and U251 mediated by lentivirus, based on the effect of which FOXO1-correlated-GMT experiments were performed in vitro and in vivo. Our data suggested that FOXO1 played a crucial role in resistance to TMZ, BCNU, and CDDP; migration and invasion; and stem cell properties of glioma cells. FOXO1 may serve as a targeted biomarker for prediction of sensitivity to chemotherapy drugs, metastasis, and prognosis, which provides a new idea for mesenchymal GBM treatment.     

Identification of novel LncRNA targeting Smad2/PKCα signal pathway to negatively regulate malignant progression of glioblastoma

Glioblastoma multiforme (GBM) is a highly proliferative cancer with generally poor prognosis and accumulating evidence has highlighted the potential of long noncoding RNAs (lncRNAs) in the biological behaviors of glioma cells. This study focused on the identification of lncRNAs to identify targets for possible GBM prognosis. Microarray expression profiling found that 1,759 lncRNAs and 3,026 messenger RNAs (mRNAs) were upregulated, and 1932s lncRNA and 2,979 mRNAs were downregulated in GBM. Bioinformatics analysis and experimental verification identified TCONS_00020456 (TCON) for further analysis. In situ hybridization, along with immunohistochemical and receiver operating characteristic analysis determined TCON (truncation value = 3.5) as highly sensitive and specific in GBM. Grade IV patients with glioma life span with different lncRNA staining scores were analyzed. TCON staining scores below 3.5 indicated poor prognosis (life span ranging from 0.25 to 7 months), even if the glioma was surgically removed. TCON decreased significantly in GBM, and showed a coexpressional relationship with Smad2 and protein kinase C α (PKCα). Overexpression of TCON reduced the proliferation on one hand and migration, invasion on the other. TCON also inhibited epithelial–mesenchymal transformation and glioma progression in vivo, based on a nude mouse tumorigenicity assay. In addition, we predicted a potential binding site and intersection that microRNAs targeting Smad2, PKCα, and TCON through RACE pretest and bioinformatics analysis. Taken together, TCON, regarded as oncosuppressor, targeting the Smad2/PKCα axis plays a novel role in inhibiting the malignant progression of glioma. Moreover, it also demonstrates that the level of TCON can be used as a prognostic and diagnostic biomarker for GBM.     

Long noncoding RNA TPT1-AS1 downregulates the microRNA-770-5p expression to inhibit glioma cell autophagy and promote proliferation through STMN1 upregulation

Through the microarray analysis, long noncoding RNA TPT1-AS1 (TPT1-AS1) was identified in the development of glioma. However, the specific effect of TPT1-AS1 on glioma autophagy in the recent years has not fully been investigated. Therefore, the purpose of our present study is to investigate the function of TPT1-AS1 on affecting autophagy of glioma cells through regulation of microRNA-770-5p (miR-770-5p)-mediated stathmin 1 (STMN1). Initially, the expression of TPT1-AS1, miR-770-5p, and STMN1 were determined in glioma cell lines, followed by the prediction and validation of their interaction. After that, the effects of TPT1-AS1, miR-770-5p, and STMN1 on the in vitro glioma cell proliferation and autophagy were assessed using EdU assay and macrophage-derived chemokine (MDC) and on the in vivo tumor development and autophagy were evaluated using a nude mouse xenograft tumor assay and immunofluorescence assay. In comparison with the normal cells, the glioma cells displayed upregulated expression of TPT1-AS1 and STMN1, but a downregulated miR-770-5p expression. miR-770-5p, which directly targeted STMN1, could be downregulated by TPT1-AS1. Subsequently, in glioma cells, TPT1-AS1 can function to competitively bind to miR-770-5p, thus regulatEing STMN1 expression. Moreover, glioma cell proliferation and autophagy could be mediated through the TPT1-AS1/miR-770-5p/STMN1 axis. From our data we conclude an inhibitory function of TPT1-AS1 in glioma cell autophagy by downregulating miR-770-5p and upregulating STMN1, which may be instrumental for the therapeutic targeting and clinical management of glioma.     

Long noncoding RNA SOX21-AS1 promotes cervical cancer progression by competitively sponging miR-7/VDAC1

Growing evidence has shown that long noncoding RNAs (lncRNAs) play crucial roles in cervical cancer. Dy000sregulation of lncRNA SOX21 antisense RNA 1 (SOX21-AS1) has been reported in several tumors. However, its expression pattern and potential biological function in cervical cancer (CC) have not been investigated. In this study, we first reported that SOX21-AS1 expression was significantly upregulated in both CC tissues and cell lines. High expression of SOX21-AS1 was found to be significantly correlated with Federation of Gynecology and Obstetrics (FIGO) stage, lymph node metastasis and depth of cervical invasion. Further clinical assay confirmed that high SOX21-AS1 expression was associated with shorter overall survival and could be used as a potential prognostic biomarker for CC patients. Functional investigation showed that knockdown of SOX21-AS1 suppressed CC cells proliferation, migration, and invasion, as well as epithelial to mesenchymal transition progress. Furthermore, our data showed that microRNA-7 (miR-7) interacted with SOX21-AS1 by directly targeting the miRNA-binding site in the SOX21-AS1 sequence, and quantitative real-time polymerase chain reaction results showed overexpression of SOX21-AS1 decreased the levels of miR-7 in CC cells. Moreover, we confirmed that miR-7 directly targeted the 3′-untranslated region of voltage dependent anion channel 1 (VDAC1). Final in vitro assay suggested that in CC cells with SOX21-AS1, VDAC1 overexpression resulted in an increase of cell proliferation, migration, and invasion. Overall, our findings illuminate how SOX21-AS1 formed a regulatory network to confer an oncogenic function in CC and SOX21-AS1 could be regarded as an efficient therapeutic target and potential biomarker for CC patients.     

Long noncoding RNA CHL1-AS1 promotes cell proliferation and migration by sponging miR-6076 to regulate CHL1 expression in endometrial cancer

Endometrial cancer (EC) is deemed to be the most typical gynecologic malignant tumor. Despite the incidence of EC being lower in Asia than that in western countries, substantial increased incidence has been observed in the past few decades in Asia. Although various molecular testing methods and genomic science have developed, the overall prognosis is still disappointing. LncRNAs have been found to influence the progression of various cancers. CHL1-AS1 has been found to be upregulated in ovarian endometriosis, nevertheless, the molecular mechanism and biological function of CHL1-AS1 in EC have not been explored. In our exploration, both CHL1-AS1 and CHL1 were upregulated in EC cells. Knockdown of CHL1-AS1 or CHL1 inhibited cell proliferation and migration in EC. Furthermore, microRNA-6076 (miR-6076) could bind with CHL1-AS1 or CHL1, and regulate the expression of CHL1. Finally, absence of miR-6076 or overexpression of CHL1 can partially rescue the effect of CHL1-AS1 knockdown or miR-6076 upregulation on cell proliferation and migration, respectively. All in all, our research was the first endeavor to study the underlying mechanism of CHL1-AS1 in EC and confirmed that CHL1-AS1 regulated EC progression via targeting the miR-6076/CHL1 axis, offering new insight into treating EC.     

SUMOylation of IGF2BP2 promotes vasculogenic mimicry of glioma via regulating OIP5-AS1/miR-495-3p axis

Rationale: Glioma is the most common primary malignant tumor of human central nervous system, and its rich vascular characteristics make anti-angiogenic therapy become a therapeutic hotspot. However, the existence of glioma VM makes the anti-angiogenic therapy ineffective. SUMOylation is a post-translational modification that affects cell tumorigenicity by regulating the expression and activity of substrate proteins.Methods: The binding and modification of IGF2BP2 and SUMO1 were identified using Ni²⁺-NTA agarose bead pull-down assays, CO-IP and western blot; and in vitro SUMOylation assays combined with immunoprecipitation and immunofluorescence staining were performed to explore the detail affects and regulations of the SUMOylation on IGF2BP2. RT-PCR and western blot were used to detect the expression levels of IGF2BP2, OIP5-AS1, and miR-495-3p in glioma tissues and cell lines. CCK-8 assays, cell transwell assays, and three-dimensional cell culture methods were used for evaluating the function of IGF2BP2, OIP5-AS1, miR-495-3p, HIF1A and MMP14 in biological behaviors of glioma cells. Meantime, RIP and luciferase reporter assays were used for inquiring into the interactions among IGF2BP2, OIP5-AS1, miR-495-3p, HIF1A and MMP14. Eventually, the tumor xenografts in nude mice further as certained the effects of IGF2BP2 SUMOylation on glioma cells.Results: This study proved that IGF2BP2 mainly binds to SUMO1 and was SUMOylated at the lysine residues K497, K505 and K509 sites, which can be reduced by SENP1. SUMOylation increased IGF2BP2 protein expression and blocked its degradation through ubiquitin-proteasome pathway, thereby increasing its stability. The expressions of IGF2BP2 and OIP5-AS1 were up-regulated and the expression of miR-495-3p was down-regulated in both glioma tissues and cells. IGF2BP2 enhances the stability of OIP5-AS1, thereby increasing the binding of OIP5-AS1 to miR-495-3p, weakening the binding of miR-495-3p to the 3''UTR of HIF1A and MMP14 mRNA, and ultimately promoting the formation of VM in glioma.Conclusions: This study first revealed that SUMOylation of IGF2BP2 regulated OIP5-AS1/miR-495-3p axis to promote VM formation in glioma cells and xenografts growth in nude mice, providing a new idea for molecular targeted therapy of glioma.