N6-methyladenosine-modified HOTAIRM1 promotes vasculogenic mimicry formation in glioma.

Vasculogenic mimicry (VM) has been reported to accelerate angiogenesis in malignant tumors, yet the mechanism underlying VM has not been fully elucidated. N6-methyladenosine (m6A) mainly modulates mRNA fate and affects multiple tumorigenesis. Here, we aimed to investigate m6A-modified HOXA transcript antisense RNA myeloid-specific 1 (HOTAIRM1) in the regulation of glioma-associated VM formation. Gene expression was analyzed by quantitative RT-PCR. Cell viability, metastases, and VM formation capacity were determined by CCK-8, migration and invasion, as well as tube formation assays, respectively. The function and mechanisms of m6A-modified HOTAIRM1 were defined through liquid chromatography-tandem mass spectrometry m6A quantification, methylated RNA immunoprecipitation sequencing, RNA stability assays, and RNA pull-down experiments. A glioma xenograft mouse model was further established for VM evaluation in vivo. The results showed that HOTAIRM1, methyltransferase-like 3 (METTL3), and insulin-like growth factor binding protein 2 (IGFBP2) were upregulated in glioma tissues and cell lines. HOTAIRM1 functions as an oncogene in glioma progression; however, knockdown of HOTAIRM1 significantly reduced cell viability, migration, invasion, and VM formation. Notably, METTL3-dependent m6A modification enhanced HOTAIRM1 mRNA stability, whereas knockdown of METTL3 deficiency significantly suppressed VM in glioma. Moreover, HOTAIRM1 was found to bind IGFBP2, and HOTAIRM1 deficiency blocked glioma progression and VM formation in vivo. Our results indicated that METTL3-dependent m6A-modified HOTAIRM1 promoted VM formation in glioma.

GLI1 is involved in HIF-1α-induced migration, invasion, and epithelial-mesenchymal transition in glioma cells.

INTRODUCTION: Glioma is characterized by hypoxia that activates the hypoxia inducible factor (HIF) pathway and controls a myriad of genes that drive cancer progression. HIF-1α promotes GLI1 transferring to the nucleus by activating the hedgehog pathway under hypoxic conditions. However, their mechanisms in glioma cells under hypoxia remain unknown. MATERIAL AND METHODS: Human glioma cell lines (LN229 and LN18) were transfected with HIF-1α or GLI1-specific short hairpin RNAs (shRNAs) and cultured under normoxic or hypoxic conditions. The protein levels of HIF-1α, GLI1, and epithelial-mesenchymal transition (EMT) markers including E-cadherin and vimentin were measured by Western blot analysis. RT-qPCR analysis was performed for the detection of HIF-1α and GLI1 mRNA expression. Cell migratory and invasive capacities were evaluated by wound healing and Transwell assays, respectively. RESULTS: Hypoxia blocked the breakdown of the HIF-1α protein and upregulated GLI1 expression in glioma cells. Downregulation of HIF-1α expression inhibited hypoxia-induced cell migration and invasion, as well as reversed the effects of hypoxia on GLI1, E-cadherin, and vimentin expression in LN229 and LN18 cells. Depletion of GLI1 inhibited glioma cell migration and invasion induced by hypoxia. Silenced GLI1 did not affect HIF-1α expression but completely offset hypoxia-regulated expression of E-cadherin and vimentin in glioma cells. CONCLUSIONS: GLI1 is involved in HIF-1α-induced migration, invasion, and EMT in glioma cells, thus revealing a novel molecular mechanism for glioma research.

Long non-coding RNA HOTAIRM1 promotes proliferation and inhibits apoptosis of glioma cells by regulating the miR-873-5p/ZEB2 axis.

BACKGROUND: Glioblastoma is one of the most common malignant brain tumors. Conventional clinical treatment of glioblastoma is not sufficient, and the molecular mechanism underlying the initiation and development of this disease remains unclear. Our study aimed to explore the expression and function of miR-873a-5p in glioblastoma and related molecular mechanism. METHODS: We analyzed the most dysregulated microRNAs from the Gene Expression Omnibus (GEO) database and examined the expression of miR-873-5p in 20 glioblastoma tissues compared with ten normal brain tissues collected in the Zhejiang Tongde Hospital. We then overexpressed or inhibited miR-873-5p expression in U87 glioblastoma cell lines and analyzed the phenotype using the cell counting kit-8 assay, wound healing assay, and apoptosis. In addition, we predicted upstream and downstream genes of miR-873-5p in glioblastoma using bioinformatics analysis and tested our hypothesis in U87 cells using the luciferase reporter gene assay and Western blotting assay. The differences between two groups were analyzed by Student's t test. The Kruskal-Wallis test was used for the comparison of multiple groups. A P < 0.05 was considered to be significant. RESULTS: The miR-873-5p was downregulated in glioblastoma tissues compared with that in normal brain tissues (normal vs. tumor, 0.762 ±â€Š0.231 vs. 0.378 ±â€Š0.114, t = 4.540, P < 0.01). Overexpression of miR-873-5p inhibited cell growth (t = 6.095, P < 0.01) and migration (t = 3.142, P < 0.01) and promoted cell apoptosis (t = 4.861, P < 0.01), while inhibition of miR-873-5p had the opposite effect. Mechanistically, the long non-coding RNA HOTAIRM1 was found to act as a sponge of miR-873-5p to activate ZEB2 expression in U87 cells. CONCLUSIONS: We uncovered a novel HOTAIRM1/miR-873-5p/ZEB2 axis in glioblastoma cells, providing new insight into glioblastoma progression and a theoretical basis for the treatment of glioblastoma.

Long noncoding RNA LINC00515 promotes cell proliferation and inhibits apoptosis by sponging miR-16 and activating PRMT5 expression in human glioma.

Introduction: In recent years, an increasing amount of literature has demonstrated the functional role of long non-coding RNA (lncRNA) in human diseases. LINC00515 is a newly defined lncRNA and is reported to act as an oncogene in multiple myeloma. However, the function of LINC00515 in glioma is still uncertain. Materials and methods: We examined the expression levels of LINC00515 in human glioma tissues and cell lines using real-time PCR analysis. In addition, we confirmed the distribution of LINC00515 in glioma cells and suppressed LINC00515 expression with siRNAs. CCK-8, colony formation assay and apoptosis analysis were used to study the function of LINC00515 in glioma progression. Then, we used bioinformatics prediction and subsequent experiments to reveal the underlying molecular mechanism. Results: We found that LINC00515 was up-regulated in glioma tissues and cell lines. LINC00515 was mainly located in the cytoplasm in glioma cells. Knockdown of LINC00515 led to decreased proliferation and increased apoptosis of glioma cells. Mechanistically, our data indicated that there was a LINC00515/miR-16/PRMT5 regulatory axis in glioma. LINC00515 could activate PRMT5 expression and promote glioma progression by acting as a sponge of miR-16. Conclusion: LINC00515 expression is elevated in human glioma and promotes growth and inhibits apoptosis of glioma cells. The regulatory cascade LINC00515/miR-16/PRMT5 plays a critical role in glioma progression.

Hsa-mir-127 impairs survival of patients with glioma and promotes proliferation, migration and invasion of cancerous cells by modulating replication initiator 1.

This work aimed to investigate the inter-regulatory functions of hsa-mir-127 and replication initiator 1 (REPIN1) on the proliferation and metastasis of glioma cells. The in-silico data on the implication of hsa-mir-127 and REPIN1 in glioma were retrieved from The Cancer Genome Atlas (TCGA). The expression levels of hsa-mir-127 and REPIN1 mRNA were determined by qRT-PCR, whereas Western blot was used to detect REPIN1 protein expression in glioma cell lines. The proliferation of glioma cells was determined by means of the MTT assay, whereas the transwell assay was employed for assessing the extent of cell migration and invasion. The interaction among REPIN1 and hsa-mir-127 was checked using the luciferase reporter assay. The expression of hsa-mir-127 was markedly increased in clinical data obtained from TCGA and in glioma cells compared with normal tissues and control cells, respectively. Increased expression of hsa-mir-127 and decreased expression of REPIN1 were both associated with poor overall survival. Moreover, hsa-mir-127 overexpression noticeably promoted proliferation, inhibited apoptosis and increased the invasive and migratory capacities of glioma cells. Inverse effects were found with hsa-mir-127 antisense inhibitor. Interestingly, overexpression of hsa-mir-127 downregulated REPIN1 expression, and luciferase reporter assay showed that the tumorigenesis effect of hsa-mir-127 requires, in part, its direct targeting of REPIN1. In conclusion, the hsa-mir-127/REPIN1 pathway is involved in gliomas and could be a potential therapeutic target.

MicroRNA-128 inhibits proliferation and invasion of glioma cells by targeting COX-2.

MicroRNAs (miRNA), a class of small noncoding RNAs, regulates message RNA (mRNA) by targeting the 3'-untranslated region (3'-UTR) resulting in suppression of gene expression. In this study, we identified the expression and function of miR-128, which was found to be downregulated in glioma tissues and glioma cells by real time PCR. Overexpression of miR-128 mimics into LN229 and U251 cells could inhibit proliferation and invasion of glioma cells. However, the inhibitory effects of miR-128 mimics on the invasion and proliferation of glioma cells were reversed by overexpression of cyclooxygenase-2 (COX-2). Our data showed that COX-2 was a candidate target of miR-128. Luciferase activity of 3'-UTR of COX-2 was reduced in the presence of miR-128. Additionally, miR-128 obviously decreased COX-2 mRNA stability determined by real time PCR. Contrarily, we found that miR-128 inhibitor significantly increased the COX-2 mRNA expression, and elevated the protein expression of MMP9 and ki67, and promoted the proliferation of glioma cells. Furthermore, luciferase activity of the 3'-UTR was upregulated by miR-128 inhibitor. All of these results supported that miR-128 was a direct regulator of COX-2. Further studies proved that COX-2 was elevated in glioma tissues and its expression was negatively correlated with the levels of miR-128. These findings may establish miR-128 as a new potential target for the treatment of patients with gliomas.

Use of Pneumoperitoneal Puncture for Peritoneal Catheter Placement in Lumboperitoneal Shunt Surgery: Technical Note.

BACKGROUND: In hydrocephalus shunt surgery, a peritoneal catheter is traditionally inserted with laparotomy incision. The abdominal incision length will not be shorter than 3 cm in most cases. A longer incision has to be made in obese patients. The lateral position in lumboperitoneal shunt (LPS) surgery also increases the difficulty of laparotomy. This report introduces a simple technique of pneumoperitoneal puncture for peritoneal catheter placement in LPS surgery. METHODS: Twenty-eight communicating hydrocephalus cases underwent pneumoperitoneal puncture in an LPS operation. Abdominal incision length, time for peritoneal catheter placement, and postoperative complications were recorded. RESULTS: The length of the abdominal incision was 1 cm, and the average time for peritoneal catheter placement was 3.5 minutes. No patient suffered from infection and obstruction. Two cases of subdural hematoma because of cerebrospinal fluid overdrainage occurred. CONCLUSIONS: The pneumoperitoneal puncture technique has proven, in our experience, to be a minimally invasive, simple, and reliable method in a peritoneal catheter placement procedure. This technique, which needs to be assessed further by larger case series, may be considered a new method of choice for peritoneal catheter placement in LPS surgery.