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.     

miR-133 is a key negative regulator of CDC42–PAK pathway in gastric cancer

Cell division cycle 42 (CDC42), an important member of the Ras homolog (Rho) family, plays a key role in regulating multiple cellular processes such as cell cycle progression, migration, cell cytoskeleton organization, cell fate determination and differentiation. Among the downstream effectors of CDC42, P21-activated kinases (PAKs) obtain the most attention. Although a large body of evidence indicates that CDC42/PAKs pathway plays important role in tumor growth, invasion and metastasis, the mechanism of their negative regulation remains unclear. Here, we identified CDC42, a PAKs activating factor, was a target of miR-133. Ectopic overexpression of miRNAs not only downregulated CDC42 expression and PAKs activation, but also inhibited cancer cell proliferation and migration. We also found that miR-133 was down-regulated in 180 pairs gastric cancer tissues. miR-133 expression was negatively associated with tumor size, invasion depth and peripheral organ metastasis. Besides, dysfunction of miR-133 was an independent prognosis factor for overall survival. Our findings could provide new insights into the molecular mechanisms of gastric carcinogenesis, and may help facilitating development of CDC42/PAK-based therapies for human cancer.     

RNF7 promotes glioma growth via the PI3K/AKT signalling axis

RNF7 has been reported to play critical roles in various cancers. However, the underlying mechanisms of RNF7 in glioma development remain largely unknown. Herein, the expression level of RNF7 was examined in tissues by quantitative real-time PCR, Western blotting and immunohistochemistry. The effect of RNF7 on glioma progression was measured by performing CCK-8 and apoptosis assays, cell cycle-related experiments and animal experiments. The effect of RNF7 on PI3K/AKT signalling pathway was tested by Western blotting. First, we found that RNF7 was upregulated in tumour tissue compared with normal brain tissue, especially in high-grade glioma, and the high expression of RNF7 was significantly related to tumour size, Karnofsky Performance Scale score and a poor prognosis. Second, RNF7 overexpression facilitated tumour cell cycle progression and cell proliferation and suppressed apoptosis. Conversely, RNF7 knockdown suppressed tumour cell cycle progression and cell proliferation and facilitated apoptosis. Furthermore, follow-up mechanistic studies indicated that RNF7 could facilitate glioma cell proliferation and cell cycle progression and inhibit apoptosis by activating the PI3K/AKT signalling pathway. This study shows that RNF7 can clearly promote glioma cell proliferation by facilitating cell cycle progression and inhibiting apoptosis by activating the PI3K/AKT signalling pathway. Targeting the RNF7/PI3K/AKT axis may provide a new perspective on the prevention or treatment of glioma.     

Prefoldin 6 promotes glioma progression via the AKT signalling pathway

Gliomas are one of the most aggressive primary tumours, accounting for 81% of malignant brain tumours, and are associated with a significant mortality. Therefore, the elucidation of the molecular mechanism underlying glioma progression and identification of promising treatment targets are necessary. Here, the expression of prefoldin (PFDN) 6 in human glioma tissues and cell lines was evaluated using immunohistochemistry and quantitative polymerase chain reaction. Celigo and CCK-8 assays were performed for assessing cell viability. Flow cytometry was used to analyse apoptosis and cell cycle distribution. Wound-healing and transwell assays were performed to observe cell migration. Lastly, xenograft models were developed for the in vivo validation of the results, and a human phospho-kinase array was used to explore the downstream signalling pathways. PFDN6 was upregulated in gliomas, and PFDN6 overexpression was significantly correlated with a low survival rate, estimated glomerular filtration rate (EGFR) expression, and tumour grade and recurrence. Moreover, PFDN6 knockdown significantly attenuated cell proliferation and migration, induced apoptosis, and blocked cell cycle progression in the G2 phase, which was further confirmed in the in vivo experiments. Mechanistically, the effects of PFDN6 may be mediated via the AKT signalling pathway. In conclusion, we showed that PFDN6 promotes glioma development by activating AKT signalling and emphasised the potential of PFDN6 as a crucial target in glioma therapy.     

Retracted: Long noncoding RNA MEG3 inhibits proliferation and migration but induces autophagy by regulation of Sirt7 and PI3K/AKT/mTOR pathway in glioma cells

Glioma is a common primary brain tumor with high mortality rate and poor prognosis. Long noncoding RNA maternally expressed gene 3 (MEG3) is a tumor suppressor in diverse cancer types. However, the role of MEG3 in glioma remains unclear. We aimed to explore the effects of MEG3 on U251 cells as well as the underlying mechanisms. U251 cells were stably transfected with different recombined plasmids to overexpress or silence MEG3. Effects of aberrantly expressed MEG3 on cell viability, migration, apoptosis, expressions of apoptosis-associated and autophagy-associated proteins, and phosphorylated levels of key kinases in the PI3K/AKT/mTOR pathway were all evaluated. Then, messenger RNA (mRNA) and protein expression of Sirt7 in cells abnormally expressing MEG3 were estimated. In addition, effects of abnormally expressed MEG3 and Sirt7 on U251 cells were determined to reveal the underlying mechanism of MEG3-associated modulation. Cell viability and migration were significantly reduced by MEG3 overexpression whereas cell apoptosis as well as Bax and cleaved caspase-3/-9 proteins were obviously induced. Beclin-1 and LC3-II/LC3-I were upregulated and p62 was downregulated in MEG3 overexpressed cells. In addition, the autophagy pharmacological inhibitor (3-methyladenine, 3-MA) affected the effect of MEG3 overexpression on cell proliferation. Furthermore, the phosphorylated levels of key kinases in the PI3K/AKT/mTOR pathway were all reduced by MEG3 overexpression. Sirt7 was positively regulated by MEG3 expression, and effects of MEG3 overexpression on U251 cells were ameliorated by Sirt7 silence. MEG3 suppressed cell proliferation and migration but promoted autophagy in U251 cells through positively regulating Sirt7, involving in the inhibition of the PI3K/AKT/mTOR pathway.     

miR-18a Inhibits CDC42 and Plays a Tumour Suppressor Role in Colorectal Cancer Cells

The miR-17-92 cluster of microRNAs is elevated in colorectal cancer, and has a causative role in cancer development. Of the six miR-17-92 cluster members, miR-19a and b in particular are key promoters of cancer development and cell proliferation, while preliminary evidence suggests that miR-18a may act in opposition to other cluster members to decrease cell proliferation. It was hypothesised that miR-18a may have a homeostatic function in helping to contain the oncogenic effect of the entire miR-17-92 cluster, and that elevated miR-17-92 cluster activity without a corresponding increase in miR-18a may promote colorectal tumour progression. In colorectal cancer samples and corresponding normal colorectal mucosa, miR-18a displayed lower overall expression than other miR-17-92 cluster members. miR-18a was shown to have an opposing role to other miR-17-92 cluster members, in particular the key oncogenic miRNAs, miR-19a and b. Transfection of HCT116 and LIM1215 colorectal cancer cell lines with miR-18a mimics decreased proliferation, while a miR-18a inhibitor increased proliferation. miR-18a was also responsible for decreasing cell migration, altering cell morphology, inducing G1/S phase cell cycle arrest, increasing apoptosis, and enhancing the action of a pro-apoptotic agent. CDC42, a mediator of the PI3K pathway, was identified as a novel miR-18a target. Overexpression of miR-18a reduced CDC42 expression, and a luciferase assay confirmed that miR-18a directly targets the 3′UTR of CDC42. miR-18a mimics had a similar effect on proliferation as a small molecule inhibitor of CDC42. Inhibition of CDC42 expression is likely to be a key mechanism by which miR-18a impairs cancer cell growth, with a target protector experiment revealing miR-18a influences proliferation via direct inhibition of CDC42. Inhibition of CCND1 by miR-18a may also assist in this growth-suppression effect. The homeostatic function of miR-18a within the miR-17-92 cluster in colorectal cancer cells may be achieved through suppression of CDC42 and the PI3K pathway.     

LncRNA PTCSC3 inhibits triple-negative breast cancer cell proliferation by downregulating lncRNA H19

Long noncoding RNA (lncRNA) PTCSC3 (hereafter PTCSC3 is used to represent lncRNA PTCSC3) inhibits glioma and thyroid cancer, indicating its potential tumor suppression function in other types of cancers. We explored the potential involvement of PTCSC3 in triple-negative breast cancer (TNBC). In the current study, we found that PTCSC3 was downregulated in tumor tissues of patients with TNBC. PTCSC3 expression was positively correlated with plasma levels of PTCSC3. LncRNA H19 was upregulated and was inversely correlated with PTCSC3 in tumor tissues. PTCSC3 overexpression led to downregulated H19 in TNBC cells, while H19 overexpression did not affect PTCSC3 expression. PTCSC3 inhibited and H19 promoted proliferation of TNBC cells. H19 overexpression attenuated the effects of PTCSC3 overexpression. Cancer cell migration and invasion were not significantly affected by PTCSC3 overexpression. Therefore, lncRNA PTCSC3 inhibits TNBC cell proliferation by downregulating lncRNA H19.     

LncRNA-135528 inhibits tumor progression by up-regulating CXCL10 through the JAK/STAT pathway

Spontaneous tumor regression can be observed in many tumors, however, studies related to the altered expression of lncRNA in spontaneous glioma regression are limited, and the potential contributions of lncRNAs to spontaneous glioma regression remain unknown. To investigate the biological roles of lncRNA-135528 in spontaneous glioma regression. The cDNA fragment of lncRNA-135528 was obtained by rapid-amplification of cDNA ends (RACE) technology and cloned into the plvx-mcmv-zsgreen-puro vector. Additionally, we stably silenced or overexpressed lncRNA-135528 in G422 cells by transfecting with siRNA against lncRNA-135528 or lncRNA-135528 overexpression plasmid. Then, we examined lncRNA-135528 overexpressing and lncRNA-135528 silencing on glioma cells and its effects on CXCL10 and JAK/STAT pathways. The main findings indicated that lncRNA-135528 promoted glioma cell apoptosis, inhibited cell proliferation and arrested cell cycle progression; the up-regulation of lncRNA135528 led to significantly increased CXCL10 levels and the differential expression of mRNA associated with JAK/STAT pathway in glioma cells. lncRNA-135528 can inhibit tumor progression by up-regulating CXCL10 through the JAK/STAT pathway.     

TROY (TNFRSF19) is overexpressed in advanced glial tumors and promotes glioblastoma cell invasion via Pyk2-Rac1 signaling

A critical problem in the treatment of malignant gliomas is the extensive infiltration of individual tumor cells into adjacent brain tissues. This invasive phenotype severely limits all current therapies, and to date, no treatment is available to control the spread of this disease. Members of the tumor necrosis factor (TNF) ligand superfamily and their cognate receptors regulate various cellular responses including proliferation, migration, differentiation, and apoptosis. Specifically, the TNFRSF19/TROY gene encodes a type I cell surface receptor that is expressed on migrating or proliferating progenitor cells of the hippocampus, thalamus, and cerebral cortex. Here, we show that levels of TROY mRNA expression directly correlate with increasing glial tumor grade. Among malignant gliomas, TROY expression correlates inversely with overall patient survival. In addition, we show that TROY overexpression in glioma cells activates Rac1 signaling in a Pyk2-dependent manner to drive glioma cell invasion and migration. Pyk2 coimmunoprecipitates with the TROY receptor, and depletion of Pyk2 expression by short hairpin RNA interference oligonucleotides inhibits TROY-induced Rac1 activation and subsequent cellular migration. These findings position aberrant expression and/or signaling by TROY as a contributor, and possibly as a driver, of the malignant dispersion of glioma cells.     

miR-34a attenuates glioma cells progression and chemoresistance via targeting PD-L1

Objective

To investigate the roles of miR-34a in progression and chemoresistance of glioma cells.

Results

Quantitative real-time PCR analysis showed that miR-34a level was lower, but PD-L1 expression level was higher in glioma tissue specimens compared with normal brain tissues and their expression levels were negatively correlated. Ectopic expression of miR-34a inhibited glioma cell proliferation, promoted cell cycle arrest in G1/S phase and cell apoptosis. Additionally, miR-34a/PD-L1 axis was again confirmed and co-expression of PD-L1 with miR-34a mimics attenuated the effects of miR-34a on cell proliferation and apoptosis in glioma cells. Importantly, PD-L1 overexpression resulted in chemoresistance in glioma cells, this effect was attenuated by miR-34a overexpression.

Conclusions

miR-34a inhibits glioma cells progression and chemoresistance via targeting PD-L1.

     

miR-3940-5p Functions as a Tumor Suppressor in Non–Small Cell Lung Cancer Cells by Targeting Cyclin D1 and Ubiquitin Specific Peptidase-28

miR-3940-5p level was lower in non–small cell lung cancer (NSCLC) tumor tissues than that in the matched tumor-adjacent tissues and correlated with clinicopathological features. Cyclin D1 (CCND1), a key driver of malignant transformation in NSCLC, was overexpressed in many cancers, including NSCLC. The ubiquitin specific peptidase-28 (USP28) was also overexpressed in NSCLC and associated with poor prognosis of NSCLC patients. We searched for miR-3940-5p targets by using TargetScan and miRanda online tools and found that CCND1 and USP28 were potential targets of miR-3940-5p. Based on these findings, we speculated that miR-3940-5p might target CCND1 and USP28 to inhibit NSCLC growth. We determined the expression of miR-3940-5p, CCND1, and USP28 by quantitative real-time polymerase chain reaction and Western blot assays, respectively, and found downregulation of miR-3940-5p and upregulation of CCND1 and USP28 in NSCLC tissues and cell lines. Cell proliferation and apoptosis assays showed that miR-3940-5p suppressed proliferation and promoted apoptosis in NSCLC cells, and silencing CCND1 and USP28 both recapitulated the effects of miR-3940-5p on NSCLC cells. Furthermore, we verified that CCND1 and USP28 were direct targets of miR-3940-5p and also found that the effects of NSCLC cell proliferation and apoptosis by miR-3940-5p were attenuated by overexpression of CCND1 or USP28. The animal experiments also showed that overexpression of miR-3940-5p inhibited the growth of NSCLC tumors in vivo. These results confirmed our speculation that miR-3940-5p inhibits proliferation and induces apoptosis in NSCLC cells by targeting CCND1 and USP28. These findings facilitate a better understanding of the molecular mechanisms underlying NSCLC initiation and progression and provide promising diagnostic markers and therapeutic targets for NSCLC.     

The neural stem cell fate determinant TLX promotes tumorigenesis and genesis of cells resembling glioma stem cells

A growing body of evidence indicates that deregulation of stem cell fate determinants is a hallmark of many types of malignancies. The neural stem cell fate determinant TLX plays a pivotal role in neurogenesis in the adult brain by maintaining neural stem cells. Here, we report a tumorigenic role of TLX in brain tumor initiation and progression. Increased TLX expression was observed in a number of glioma cells and glioma stem cells, and correlated with poor survival of patients with gliomas. Ectopic expression of TLX in the U87MG glioma cell line and Ink4a/Arf-deficient mouse astrocytes (Ink4a/Arf^-/- astrocytes) induced cell proliferation with a concomitant increase in cyclin D expression, and accelerated foci formation in soft agar and tumor formation in in vivo transplantation assays. Furthermore, overexpression of TLX in Ink4a/Arf^-/- astrocytes inhibited cell migration and invasion and promoted neurosphere formation and Nestin expression, which are hallmark characteristics of glioma stem cells, under stem cell culture conditions. Our results indicate that TLX is involved in glioma stem cell genesis and represents a potential therapeutic target for this type of malignancy.     

MicroRNA-383 inhibits anchorage-independent growth and induces cell cycle arrest of glioma cells by targeting CCND1

In recent years, microRNAs (miRNAs) have been proved to be closely related to the tumorigenesis and progression. An increasing number of researches have shown that microRNAs function as oncogenes or tumor suppressor genes in human malignant tumors. This study aims to explore the effects of microRNA-383 (miR-383) on malignant biological function of human gliomas. We detected the expression of miR-383 in glioma tissues and normal brain tissues by quantitative real-time PCR. Anchorage-independent growth assays, and flow cytometry were used to evaluate the functions of miR-383 that involves in cell growth and cell cycle. Western blotting assay was used to examine protein expression levels of Cyclin D1 (CCND1), a cell cycle-associated oncogene which has a predicted binding site of miR-383 within its 3′-untranslated region (3′-UTR), and luciferase activity assay was used to evaluate the 3′-UTR activity of CCND1. In this study, we found that miR-383 expression level was lower in gliomas than normal brain tissues. Overexpression of miR-383 in U251 and U87 cells showed a significant inhibitory effect on cell growth, which accompanied with cell cycle G0/G1 arrest as well as downregulation of CCND1 expression. Moreover, CCND1 was verified to be one of the direct targets of miR-383. In summary, this study suggested that miR-383 plays the role of tumor suppressor by targeting CCND1 in glioma cells, and may be useful for developing a new therapeutic strategy for gliomas.     

Retracted: Long noncoding RNA BCAR4 promotes glioma cell proliferation via EGFR/PI3K/AKT signaling pathway

Long noncoding RNA Breast Cancer Antiestrogen Resistance 4 (BCAR4) has been identified to be oncogenic in several cancers. In our study, we demonstrated that BCAR4 expression was significantly upregulated in glioma tissues compared with paired nontumor tissues. In addition, higher BCAR4 level was associated with poor overall survival in patients with glioma. Besides, we also discovered that knockdown of BCAR4 inhibited cell proliferation, whereas BCAR4 overexpression promoted this process. Intriguingly, we proved a cellular transformation of normal human astrocyte cells (NHAs) in response to enforced expression of BCAR4. In addition, we revealed that BCAR4 affected cell proliferation in glioma cells by promoting cell cycle progression and inhibiting cell apoptosis. Mechanistically, we uncovered that BCAR4 activated PI3K/AKT signaling pathway in glioma through upregulating EGFR and interacting with it. Moreover, activating PI3K/AKT pathway could reverse the repressive effects caused by BCAR4 silence on the biological behaviors of glioma cells, whereas inhibition of this pathway rescued the impact of BACR4 upregulation in NHAs. These findings disclosed that BCAR4 contributes to glioma progression by enhancing cell growth via activating EGFR/PI3K/AKT pathway, providing potent evidence that BCAR4 could be an effective new target for treatment and prognosis of glioma patients.     

Hirudin inhibits glioma growth through mTOR-regulated autophagy

Glioma is the most common primary malignant brain tumour, and survival is poor. Hirudin has anticancer pharmacological effects through suppression of glioma cell progression, but the molecular target and mechanism are poorly understood. In this study, we observed that hirudin dose- and time-dependently inhibited glioma invasion, migration and proliferation. Mechanistically, hirudin activated LC3-II but not Caspase-3 to induce the autophagic death of glioma cells by decreasing the phosphorylation of mTOR and its downstream substrates ULK1, P70S6K and 4EBP1. Furthermore, hirudin inhibited glioma growth and induced changes in autophagy in cell-derived xenograft (CDX) nude mice, with a decrease in mTOR activity and activation of LC3-II. Collectively, our results highlight a new anticancer mechanism of hirudin in which hirudin-induced inhibition of glioma progression through autophagy activation is likely achieved by inhibition of the mTOR signalling pathway, thus providing a molecular basis for hirudin as a potential and effective clinical drug for glioma therapy.     

miR‐150‐5p suppresses the stem cell‐like characteristics of glioma cells by targeting the Wnt/β‐catenin signaling pathway

Glioma is the most common brain tumor malignancy with high mortality and poor prognosis. Emerging evidence suggests that cancer stem cells are the key culprit in the development of cancer. MicroRNAs have been reported to be dysregulated in many cancers, while the mechanism underlying miR‐150‐5p in glioma progression and proportion of stem cells is unclear. The expression levels of miR‐150‐5p and catenin beta 1 (CTNNB1, which encodes β‐catenin) were measured by quantitative real‐time polymerase chain reaction (qRT‐PCR) and western blot. The expression levels of downstream genes of the Wnt/β‐catenin pathway and stem cell markers were detected by qRT‐PCR. Tumorigenesis was investigated by cell viability, colony formation, and tumor growth in vitro and in vivo. The interaction between miR‐150‐5p and β‐catenin was explored via bioinformatics analysis and luciferase activity assay. We found that miR‐150‐5p was downregulated in glioma and its overexpression inhibited cell proliferation, colony formation, and tumor growth. Moreover, miR‐150‐5p directly suppressed CTNNB1 and negatively regulated the abundances of downstream genes of the Wnt/β‐catenin pathway and stem cell markers. Furthermore, miR‐150‐5p expression was decreased and β‐catenin level was enhanced in CD133+ glioma stem cells. Knockdown of miR‐150‐5p contributed to CD133? cells with stem cell‐like phenotype, whereas overexpression of miR‐150‐5p suppressed CD133+ glioma stem cell‐like characteristics. In conclusion, miR‐150‐5p inhibited the progression of glioma by controlling stem cell‐like characteristics via regulating the Wnt/β‐catenin pathway, providing a novel target for glioma treatment.     

MiR-125a-3p Regulates Glioma Apoptosis and Invasion by Regulating Nrg1

The current study was designed to examine the functional role and mechanism of miR-125a-3p in glioma development. Quantitative RT-PCR was used to evaluate miR-125a-3p expression in 60 glioma cases of different malignant grades. Then, the clinic pathologic significance of miR-125a-3p expression was determined in combination with the prognosis of the patients. In addition, the effects and mechanisms of miR-125a-3p on the proliferation, apoptosis and invasion of glioma cells were further investigated. The results showed that the expression of miR-125a-3p was decreased significantly in most malignant glioma samples relative to normal brain tissues and glioma tissues of low-malignant degree. Further kaplan-meier survival analysis showed that the lower expression of miR-125a-3p was associated with a poor prognosis of GBM patients. Functional analysis showed that the reintroduction of miR-125a-3p into glioblastoma cell lines induces markedly the apoptosis and suppresses the proliferation and migration of glioblastoma cells in vitro and in vivo. Luciferase assay and Western blot analysis revealed that Nrg1 is a direct target of miR-125a-3p. Furthermore, an increased expression of Nrg1 could reverse the effects of overexpression of miR-125a-3p on the proliferation, apoptosis and migration of glioblastoma cells. These findings suggest that miR-125a-3p performed an important role in glioma development mediated by directly regulating the expression of Nrg1. This study also provides a potential target for diagnosis and treatment of malignant glioma.