Dysregulated circRNA_100876 suppresses proliferation of osteosarcoma cancer cells by targeting microRNA-136

The aim of this study was to explore the regulatory mechanism of circRNA_100876/ microRNA-136 (miR-136) axis in the development and progression of osteosarcoma cancer. Quantitative real-time polymerase chain reaction (RT-qPCR) was used to evaluate the expression levels of circRNA_100876 and miR-136 in osteosarcoma cancer samples and the adjacent nontumor tissues. Then, cell proliferation, cell cycle, apoptosis, and migration of circRNA_100876-knocked down cells were analyzed by in vitro and in vivo experiments, using cell counting kit-8 (CCK-8), flow cytometry, and transwell and tumorigenesis assays. The expression of circRNA_100876 was found to be significantly upregulated in osteosarcoma, and was closely correlated with the tumor size and tumor differentiation degree. In addition, the knockdown of circRNA_100876 could significantly inhibit the tumor growth, both in vitro and in vivo. Flow cytometry and Western blot analysis results showed that the downregulation of circRNA_100876 inhibited osteosarcoma cells proliferation via promoting apoptosis and arresting more cells in the G2/M stage, as suggested by the expression of apoptosis and cell cycle pathway-related proteins, which changed consistently. Furthermore, the level of miR-136 was negatively correlated with the expression of circRNA_100876, and miR-136 inhibitors were able to reverse the suppression of cell proliferation induced by silencing circRNA_100876. Our study demonstrates that the dysregulation of circRNA_100876 could induce apoptosis and arrest the cell cycle at G2/M stage, followed by suppression of cell proliferation in osteosarcoma, while silencing miR-136 could restore the cell growth. Therefore, circRNA_100876 might serve as a promising biomarker and treatment target for osteosarcoma.     

ANXA2P2/miR-9/LDHA axis regulates Warburg effect and affects glioblastoma proliferation and apoptosis

BackgroundAerobic glycolysis is a unique tumor cell phenotype considered as one of the hallmarks of cancer. Aerobic glycolysis can accelerate tumor development by increasing glucose uptake and lactate production. In the present study, lactate dehydrogenase A (LDHA) is significantly increased within glioma tissue samples and cells, further confirming the oncogenic role of LDHA within glioma.MethodsHematoxylin and eosin (H&E) and immunohistochemical (IHC) staining were applied for histopathological examination. The protein levels of LDHA, transporter isoform 1 (GLUT1), hexokinase 2 (HK2), phosphofructokinase (PFK) in target cells were detected by Immunoblotting. The predicted miR-9 binding to lncRNA Annexin A2 Pseudogene 2 (ANXA2P2) or the 3′ untranslated region (UTR) of LDHA was verified using Luciferase reporter assay. Cell viability or apoptosis were examined by MTT assay or Flow cytometry. Intracellular glucose and Lactate levels were measured using glucose assay kit and lactate colorimetric assay kit.ResultsThe expression of ANXA2P2 showed to be dramatically upregulated within glioma tissue samples and cells. Knocking down ANXA2P2 within glioma cells significantly inhibited cell proliferation and aerobic glycolysis, as manifested as decreased lactate and increased glucose in culture medium, and downregulated protein levels of glycolysis markers, GLUT1, HK2, PFK, as well as LDHA. miR-9 was predicted to target both lncRNA ANXA2P2 and LDHA. The overexpression of miR-9 suppressed the cell proliferation and aerobic glycolysis of glioma cells. Notably, miR-9 could directly bind to LDHA 3′UTR to inhibit LDHA expression and decrease the protein levels of LDHA. ANXA2P2 competitively targeted miR-9, therefore counteracting miR-9-mediated repression on LDHA. Within tissues, miR-9 exhibited a negative correlation with ANXA2P2 and LDHA, respectively, whereas ANXA2P2 and LDHA exhibited a positive correlation with each other.ConclusionsIn conclusion, ANXA2P2/miR-9/LDHA axis modulates the aerobic glycolysis progression in glioma cells, therefore affecting glioma cell proliferation.     

The mmu_circRNA_37492/hsa_circ_0012138 function as potential ceRNA to attenuate obstructive renal fibrosis

Circular RNAs (circRNAs) are involved in the pathogenesis of certain renal diseases, however, the function and mechanism of them in renal fibrosis remains largely unknown. In the present study, RNA expression data in unilateral ureteral obstruction (UUO) kidneys was obtained from our previous circRNA Microarray and public Gene Expression Omnibus datasets to construct a ceRNA network. The effects of target circRNA as long as the homologous human circRNA on renal fibrosis was examined in vitro and in vivo. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was further performed among genes regulated by the human circRNA. We found that circRNA_37492, showing well connection degree in the ceRNA network, was abundant expression and high sequence conservation. We observed that the expression of circRNA_37492 was induced by the TGF-β1 or UUO in BUMPT cells and C57BL/6 mice, respectively. In vitro, cytoplasmic circRNA_37492 inhibited type I, III collagen and fibronectin deposition by sponging miR-7682-3p and then upregulated its downstream target Fgb. In vivo, overexpression of circRNA_37492 attenuated fibrotic lesions in the kidneys of UUO mice via targeting miR-7682-3p/Fgb axis. Furthermore, hsa_circ_0012138, homologous with circRNA_37492, may potentially target miR-651-5p/FGB axis in human renal fibrosis. Not only that, GO and KEGG enrichment revealed that hsa_circ_0012138-regulated genes were previously demonstrated to related to the fibrosis. In conclusion, we for the first time demonstrated that circRNA_37492 attenuated renal fibrosis via targeting miR-7682-3p/Fgb axis, and the homologous hsa_circRNA_0012138 was speculated as a possible ceRNA to regulate multiple gene expressions and involve in human renal fibrosis, suggesting that circRNA_37492/hsa_circ_0012138 may serve as potent therapy target for obstructive renal fibrosis disease. Subject terms: End-stage renal disease, miRNAs     

miR-181a shows tumor suppressive effect against oral squamous cell carcinoma cells by downregulating K-ras

MicroRNAs (miRNAs) are epigenetic regulators of gene expression, and their deregulation plays an important role in human cancer, including oral squamous cell carcinoma (OSCC). Recently, we found that miRNA-181a (miR-181a) was upregulated during replicative senescence of normal human oral keratinocytes. Since senescence is considered as a tumor suppressive mechanism, we thus investigated the expression and biological role of miR-181a in OSCC. We found that miR-181a was frequently downregulated in OSCC. Ectopic expression of miR-181a suppressed proliferation and anchorage independent growth ability of OSCC. Moreover, miR-181a dramatically reduces the growth of OSCC on three dimensional organotypic raft culture. We also identified K-ras as a novel target of miR-181a. miR-181a decreased K-ras protein level as well as the luciferase activity of reporter vectors containing the 3′-untranslated region of K-ras gene. Finally, we defined a minimal regulatory region of miR-181a and found a positive correlation between its promoter activity and the level of miR-181a expression. In conclusion, miR-181a may function as an OSCC suppressor by targeting on K-ras oncogene. Thus, miR-181a should be considered for therapeutic application for OSCC.     

LINC00152 promotes cell cycle progression in hepatocellular carcinoma via miR-193a/b-3p/CCND1 axis

Long intergenic non-coding RNA 00152 (LINC00152) is aberrantly expressed in various human malignancies and plays an important role in the pathogenesis. Here, we found that LINC00152 is upregulated in hepatocellular carcinoma (HCC) tissues as compared to adjacent non-neoplastic tissues; gain-and-loss-of-function analyses in vitro showed that LINC00152 facilitates HCC cell cycle progression through regulating the expression of CCND1. LINC00152 knockdown inhibits tumorigenesis in vivo. MS2-RIP analysis indicated that LINC00152 binds directly to miR-193a/b-3p, as confirmed by luciferase reporter assays. Furthermore, ectopic expression of LINC00152 partially halted the decrease in CCND1 expression and cell proliferation capacity induced by miR-193a/b-3p overexpression. Thus, LINC00152 acts as a competing endogenous RNA (ceRNA) by sponging miR-193a/b-3p to modulate its target gene, CCND1. Our findings establish a ceRNA mechanism regulating cell proliferation in HCC via the LINC00152/miR-193a/b-3p/CCND1 signalling axis, and identify LINC00152 as a potential therapeutic target for HCC.     

H3K27 acetylation-induced lncRNA EIF3J-AS1 improved proliferation and impeded apoptosis of colorectal cancer through miR-3163/YAP1 axis

Long noncoding RNAs (lncRNAs) are found to be aberrantly expressed and pose significant impacts in colorectal cancer (CRC), the most prevalent type malignancy in the gastrointestinal tract. This study aimed to find out the regulation of lncRNA EIF3J antisense RNA 1 (EIF3J-AS1) on CRC progression. Expressions of EIF3J-AS1, microRNA-3163 (miR-3163), and Yes-associated protein 1 (YAP1) in tissues and cells were evaluated by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis. Association of EIF3J-AS1 with CRC prognosis was analyzed through the online bioinformatics tool GEPIA. The biological function of EIF3J-AS1 in CRC was investigated by Cell Counting Kit-8, colony formation, caspase-3 activity, and TUNEL staining. Competitive endogenous RNA (ceRNA) network of EIF3J-AS1/miR-3163/YAP1 was determined by luciferase reporter and RNA immunoprecipitation assays. Results showed that EIF3J-AS1 was upregulated in CRC tissues and cell lines, indicating poor prognosis of CRC patients. The silence of EIF3J-AS1 led to reduced proliferation and facilitated apoptosis of CRC cells. Mechanistcally, EIF3J-AS1 was upregulated by cAMP-response element-binding protein-binding protein-mediated histone H3 on lysine 27 acetylation (H3K27ac) at the promoter region, and EIF3J-AS1 upregulated YAP1 expression through sponging miR-3163 in CRC cells. In conclusion, we first found that H3K27 acetylation-induced lncRNA EIF3J-AS1 improved proliferation and impeded apoptosis of colorectal cancer through the miR-3163/YAP1 axis, which might potentially provide a novel molecular-targeted strategy for CRC treatment.     

PER1 suppresses glycolysis and cell proliferation in oral squamous cell carcinoma via the PER1/RACK1/PI3K signaling complex

There is increasing evidence that the core clock gene Period 1 (PER1) plays important roles in the formation of various tumors. However, the biological functions and mechanism of PER1 in promoting tumor progression remain largely unknown. Here, we discovered that PER1 was markedly downregulated in oral squamous cell carcinoma (OSCC). Then, OSCC cell lines with stable overexpression, knockdown, and mutation of PER1 were established. We found that PER1 overexpression significantly inhibited glycolysis, glucose uptake, proliferation, and the PI3K/AKT pathway in OSCC cells. The opposite effects were observed in PER1-knockdown OSCC cells. After treatment of PER1-overexpressing OSCC cells with an AKT activator or treatment of PER1-knockdown OSCC cells with an AKT inhibitor, glycolysis, glucose uptake, and proliferation were markedly rescued. In addition, after treatment of PER1-knockdown OSCC cells with a glycolysis inhibitor, the increase in cell proliferation was significantly reversed. Further, coimmunoprecipitation (Co-IP) and cycloheximide (CHX) chase experiment demonstrated that PER1 can bind with RACK1 and PI3K to form the PER1/RACK1/PI3K complex in OSCC cells. In PER1-overexpressing OSCC cells, the abundance of the PER1/RACK1/PI3K complex was significantly increased, the half-life of PI3K was markedly decreased, and glycolysis, proliferation, and the PI3K/AKT pathway were significantly inhibited. However, these effects were markedly reversed in PER1-mutant OSCC cells. In vivo tumorigenicity assays confirmed that PER1 overexpression inhibited tumor growth while suppressing glycolysis, proliferation, and the PI3K/AKT pathway. Collectively, this study generated the novel findings that PER1 suppresses OSCC progression by inhibiting glycolysis-mediated cell proliferation via the formation of the PER1/RACK1/PI3K complex to regulate the stability of PI3K and the PI3K/AKT pathway-dependent manner and that PER1 could potentially be a valuable therapeutic target in OSCC.Subject terms: Oral cancer, Cell growth, RNAi     

CircGFRA1 facilitates the malignant progression of HER-2-positive breast cancer via acting as a sponge of miR-1228 and enhancing AIFM2 expression

CircRNAs (circular RNA) are reported to regulate onset and progress multiple cancers. Nonetheless, the function along with the underlying mechanisms of circRNAs in HER-2-positive breast cancer (BC) remains unclear. CircRNA microarrays were performed to elucidate expression profiles of HER-2-positive BC cells. circRNA levels were quantified using qRT-PCR assay. Various in vitro along with in vivo assays were employed to further explore the effects of circGFRA1 in the progress of HER-2-positive BC and interactions of circGFRA1, miR-1228 and AIFM2 in Her-2-positive BC. CircGFRA1 was remarkably upregulated in HER-2-positive BC. Knockdown of circGFRA1 could attenuate HER-2-positive BC progression by inhibiting the proliferation, infiltration and migratory ability of HER-2-positive BC cells. Through ceRNA mechanism, circGFRA1 could bind to miR-1228 and alleviate inhibitory activity of miR-1228 on targeted gene AIFM2. In summary, circGFRA1-miR-1228-AIFM2 axis regulates HER-2-positive BC. CircGFRA1 is a novel promising treatment option for HER-2-positive BC.     

microRNA-211 promotes proliferation,migration, and invasion ability of oral squamous cell carcinoma cells via targeting the bridging integrator 1 protein

Oral squamous cell carcinoma (OSCC), the most common pathological type of oral cancer, is still a frequent malignancy with unsatisfactory prognosis. Accumulating studies have proven some microRNAs (miRNAs) can function as oncogenes in OSCC by targeting tumor suppressors. In this study, we first investigated the expression and role of tumor suppressor bridging integrator-1 (BIN1) in OSCC tissues and cells. Our results indicated that BIN1 was low expressed in the OSCC tissues and cell lines (SCC6, SCC9, SCC25, HN4, and HN6) along with miR-211 was highly expressed in OSCC tissues and cell lines, and BIN1 overexpression could evidently inhibit their proliferation, migration, and invasion abilities. Next, we used bioinformation algorithms to predict the potential miRNA targeting BIN1 and chose miR-211 for further study. miR-211, a highly expressed miRNA in OSCC cells, could specifically bind with the 3′-untranslated region (3′-UTR) of BIN1 to trigger its degradation. Addition of miR-211 inhibitor could evidently suppress the malignant behaviors of OSCC cells by upregulating BIN1 expression and inhibit the activation of the EGFR/MAPK pathway. Taken together the findings of the study indicated that miR-211 mediated BIN1 downregulation had crucial significances in OSCC, suggesting the miR-211 might be a novel potential therapeutic target for the OSCC treatment.     

miR-30a attenuates drug sensitivity to 5-FU by modulating cell proliferation possibly by downregulating cyclin E2 in oral squamous cell carcinoma

We aimed to determine the functional role of the miRNA, which affects drug sensitivity to 5-FU in oral squamous cell carcinoma (OSCC), using two types of 5-FU-resistant and parental OSCC cell lines. MiRNA microarray data showed that miR-30a was significantly upregulated in two resistant cell lines. Therefore, we investigated the effects and molecular mechanism of miR-30a on 5-FU sensitivity. Stable overexpression of miR-30a in parental OSCC cells decreased cell proliferation and attenuated drug sensitivity to 5-FU. Cell cycle analysis indicated that miR-30a overexpression increased the proportion of G1 phase cells and decreased the proportion of S phase cells. MiR-30a knockdown using siRNA reversed the effects of miR-30a overexpression. DNA microarray analysis using miR-30a-overexpressing cell lines and a TargetScan database search showed that cyclin E2 (CCNE2) is a target of miR-30a. A luciferase reporter assay confirmed that a miR-30a mimic interacted with the specific binding site in the 3' UTR of CCNE2. CCNE2 knockdown with siRNA in OSCC cells yielded decreased drug sensitivity to 5-FU, similar to miR-30a overexpressing cells. These findings suggest that miR-30a in OSCC may be a novel biomarker of 5-FU-resistant tumors, as well as a therapeutic target for combating resistance.     

LncRNA EWSAT1 Regulates the Tumorigenesis of NSCLC as a ceRNA by Modulating miR-330-5p/ITGA5 Axis

The aim of the study is to investigate how lncRNA EWSAT1 regulates the tumorigenesis of non-small cell lung cancer (NSCLC) as a ceRNA by modulating miR-330-5p/ITGA5 axis. qRT-PCR was conducted to evaluate the expression of EWSAT1 in NSCLC tissue. Then, A549 cells were selected and divided into Blank shScramble, shEWSAT1, miR-330-5p inhibitor, shEWSAT1?+?miR-330-5p inhibitor, and siITGA5 and miR-330-5p inhibitor?+?siITGA5 groups. Besides, a series of in-vitro experiments were carried out to determine the changes in cell proliferation, apoptosis, invasion, and migration in each group. In addition, xenograft models were also constructed on nude mice to detect the tumor volume and weight, and the expression of Ki67 and apoptosis in xenograft tumor were evaluated. In NSCLC tissue and cell, EWSAT1 was upregulated significantly, demonstrating a correlation with tumor diameter, differentiation, lymph node metastasis, and TNM stage. Dual luciferase reporter gene assay confirmed targeting relationships among miR-330-5p, EWSAT1, and ITGA5. In comparison with the Blank group, the number of cell clones in the shEWSAT1 group and siITGA5 decreased, with declined invasion and migration but increased apoptotic rate. Meanwhile, ITGA5, MMP-2, and MMP-9 were downregulated with upregulated cleaved caspase-3. However, the changes above were totally reversed in the miR-330-5p inhibitor group, and miR-330-5p inhibitor transfection abolished the effect of shEWSAT1. In addition, subcutaneous xenotransplantation showed that the tumor growth in shEWSAT1 group retarded significantly, with downregulation of Ki67 and increase apoptotic rate. Silencing EWSAT1 could inhibit the expression of ITGA5 via upregulating miR-330-5p, thus, resulting in the inhibition of NSCLC cell growth.

     

E2F1-mediated MNX1-AS1-miR-218-5p-SEC61A1 feedback loop contributes to the progression of colon adenocarcinoma

The long noncoding RNA MNX1-AS1 has been reported to facilitate the progression of glioblastoma and ovarian cancer. Nevertheless, the biological roles and underlying mechanisms of MNX1-AS1 in colon adenocarcinoma have not been studied until now. In the current study, MNX1-AS1 was upregulated in colon adenocarcinoma. JASPAR prediction tool showed that E2F1 could bind to the promoter region of MNX1-AS1. The chromatin immunoprecipitation assay and luciferase reporter assay were used to verify the interactions between MNX1-AS1 and E2F1. Then functional assays revealed that downregulation of MNX1-AS1 decreased cell proliferation, migration, and invasion in colon adenocarcinoma, but upregulation of E2F1 reversed the effects. Moreover, subcellular fractionation assay manifested that MNX1-AS1 was enriched in the cytoplasm of colon adenocarcinoma cells, thus we speculated whether MNX1-AS1 could function as a competing endogenous RNA (ceRNA) to play roles in colon adenocarcinoma. Bioinformatics analysis and luciferase reporter assay indicated that MNX1-AS1 could sponge microRNA-218-5p (miR-218-5p). Furthermore, we discovered that SEC61A1 was downstream target of miR-218-5p, and MNX1-AS1 acted as a ceRNA to upregulate the expression of SEC61A1 through sponging miR-218-5p. Finally, rescue assays confirmed that MNX1-AS1 facilitated the progression of colon adenocarcinoma through regulating miR-218-5p/SEC61A1 axis. Taken together, we concluded that E2F1-mediated MNX1-AS1-miR-218-5p-SEC61A1 feedback loop contributed to the progression of colon adenocarcinoma.     

Monocarboxylate Transporter 4 Facilitates Cell Proliferation and Migration and Is Associated with Poor Prognosis in Oral Squamous Cell Carcinoma Patients

Monocarboxylate transporter 4 (MCT4) is a cell membrane transporter of lactate. Recent studies have shown that MCT4 is over-expressed in various cancers; however, its role in cancer maintenance and aggressiveness has not been fully demonstrated. This study investigated the role of MCT4 in oral squamous cell carcinoma (OSCC), and found that it is highly expressed in OSCC patients by using immunohistochemistry. Moreover, this over-expression of MCT4 was closely associated with tumor size, TNM classification, lymphatic metastasis, distant metastasis and tumor recurrence, and also poor prognosis. To further study mechanisms of MCT4 in vitro, we used small-interfering RNA to silence its expression in OSCC cell lines. The results showed that knock-down of MCT4 decreased cell proliferation, migration, and invasion. The inhibition of proliferation was associated with down-regulation of p-AKT and p-ERK1/2, while decreased cell migration and invasion may be caused by down-regulation of integrin β4-SRC-FAK and MEK-ERK signaling. Together, these findings provide new insight into the critical role of MCT4 in cell proliferation and metastasis in OSCC.     

Overexpressed MicroRNA-182 Promotes Proliferation and Invasion in Prostate Cancer PC-3 Cells by Down-Regulating N-myc Downstream Regulated Gene 1 (NDRG1)

MicroRNAs, non-coding 20–22 nucleotide single-stranded RNAs, result in translational repression or degradation and gene silencing of their target genes, and significantly contribute to the regulation of gene expression. In the current study, we report that miR-182 expression was significantly upregulated in prostate cancer tissues and four cell lines, compared to benign prostatic hyperplasia tissues and normal prostatic epithelial (RWPE-1) cells. Ectopic overexpression of miR-182 significantly promotes the proliferation, increases the invasion, promotes the G1/S cell cycle transition and reduces early apotosis of PC-3 cells, while suppression of miR-182 decreased the proliferation and invasion, inhibits the G1/S cell cycle transition and increase early apotosis of PC-3 cells. Additionally, we demonstrated that miR-182 could downregulate expression of NDRG1 by directly targeting the NDRG1 3′-untranslated region. In conclusion, our results suggest that miR-182 plays an important role in the proliferation of human prostate cancer cells by directly suppressing the tumor supressor gene NDRG1. We uncovered a new epigenetic regulation of NDRG1.