LncRNA DLX6-AS1 promotes tumor proliferation and metastasis in osteosarcoma through modulating miR-641/HOXA9 signaling pathway

Osteosarcoma (OS) is the most common primary malignant bone tumor. Recently, increasing evidence has shown that the long noncoding RNA (lncRNA) DLX6-AS1 (distal-less homeobox 6 antisense 1) plays significant roles in various types of cancers. However, the functions and underlying mechanisms of DLX6-AS1 have not been explored in OS yet. In this study, we assessed the expression of DLX6-AS1 in OS tissues and cell lines and explored the underlying molecular mechanisms. DLX6-AS1 was found to be significantly upregulated in OS tissues and OS cell lines. High expression of DLX6-AS1 was significantly correlated with advanced TNM stage, high tumor grade, and distant metastasis of patients with OS. Knockdown of DLX6-AS1 suppressed OS cell proliferation, invasion, and migration, and induced cell apoptosis. Knockdown of DLX6-AS1 also suppressed in vivo tumor growth. Bioinformatics and luciferase assay analysis showed that DLX6-AS1 functioned as a competing endogenous RNA (ceRNA) to negatively regulate miR-641 expression. Furthermore, miR-641 was found to target the 3′ untranslated region of homeobox protein Hox-A9 (HOXA9) and suppressed the expression of HOXA9. Mechanistic studies showed that DLX6-AS1 regulated OS cell proliferation, invasion, and migration via regulating HOXA9 by acting as a ceRNA for miR-641. Our results suggested that DLX6-AS1 functions as a ceRNA by targeting miR-641/HOXA9 signal pathway to suppress OS cell proliferation and metastasis. Our study may provide novel insights into understanding pathogenesis and development of OS.     

LncRNA CRNDE promotes the epithelial-mesenchymal transition of hepatocellular carcinoma cells via enhancing the Wnt/β-catenin signaling pathway

Colorectal neoplasia differentially expressed (CRNDE) is a significantly upregulated long noncoding RNA in hepatocellular carcinoma (HCC). CRNDE could promote cell proliferation, migration, and invasion, while its molecular mechanisms were still largely unclear. In this study, we investigated the expression and function of CRNDE. CRNDE was significantly upregulated in tumor tissues compared with adjacent normal tissues. In vitro, we revealed that knockdown of CRNDE inhibited cell proliferation, migration, and cell invasion capacities in HCC. Animal studies indicated that CRNDE knockdown represses both growth and metastasis of HCC tumors in vivo. Moreover, knockdown of CRNDE suppressed the cell epithelial-mesenchymal transition (EMT) process by increasing the expression of E-cadherin and ZO-1, whereas, decreasing the expression of N-cadherin, slug, twist, and vimentin in HCC cells. We also revealed that knockdown of CRNDE suppressed the Wnt/β-catenin signaling in HCC. Thus, CRNDE could modulate EMT of HCC cells and knockdown of CRNDE impaired the mesenchymal properties. CRNDE increased invasion of HCC cells might be through activating the Wnt/β-catenin signaling pathway.     

lncRNA OSER1-AS1 acts as a ceRNA to promote tumorigenesis in hepatocellular carcinoma by regulating miR-372-3p/Rab23 axis

Long non-coding RNAs (lncRNAs) are crucial regulators of tumorigenesis and progression in human cancer, including hepatocellular carcinoma (HCC). However, the role of most lncRNAs that are dysregulated in HCC remains to be elucidated. Here, we investigated the role of OSER1-AS1 in the progression of HCC. The results of database and qRT-PCR analysis demonstrated that OSER1-AS1 was highly expressed in HCC tissues and the high expression of OSER1-AS1 was closely associated with larger tumor size, advanced tumor stages, lower disease free survival and overall survival of HCC patients. OSER1-AS1 knockdown significantly inhibited the proliferation, invasion and migration of HCC cells, and induced the apoptosis. In addition, the dual luciferase reporter assay directly demonstrated that OSER1-AS1 functioned as a molecular sponge for miR-372-3p to promote Rab23 expression. Moreover, the results of immunohistochemistry and western blot analysis showed that Rab23 was highly expressed in HCC tissues, and the high expression of Rab23 was closely associated with the poor overall survival of HCC patients. Immunofluorescence assay also found the subcellular localization of Rab23 in HCC cells. Rab23 was obviously downregulated in cells that were transfected with miR-372-3p mimics. MiR-372-3p mimics significantly inhibited the proliferation and invasion of HCC cells). Rab23 restoration partially reversed miR-372-3p-induced tumor suppressive effects on HCC cells. In conclusion, we found that OSER1-AS1 acted as a ceRNA to sponge miR-372-3p, thereby positively regulating the Rab23 expression and ultimately acting as a tumor suppressor gene in HCC progression.     

LncRNA FGD5-AS1 functions as an oncogene to upregulate GTPBP4 expression by sponging miR-873-5p in hepatocellular carcinoma

The long non-coding FGD5-AS1 (LncFGD5-AS1) has been reported to be a novel carcinogenic gene and participant in regulating tumor progression by sponging microRNAs (miRNAs). However, the pattern of expression and the biological role of FGD5-AS1 in hepatocellular carcinoma (HCC) remains largely unknown. The expression level of FGD5-AS1 in tumor tissues and cell lines was measured by RT-qPCR. CCK-8, EdU, flow cytometry, wound healing and transwell chamber assays were performed to investigate the role of FGD5-AS1 in cell proliferation, apoptosis, migration, and invasion in HCC. Dual luciferase reporter, and RNA pull-down assays were performed to identify the regulatory interactions among FGD5-AS1, miR-873-5p and GTP-binding protein 4 (GTPBP4). We found that the expression of FGD5-AS1 was upregulated in HCC tissues and cell lines. Moreover, the knockdown of FGD5-AS1 suppressed cell proliferation, migration and invasion, and induced apoptosis in HCC cells. Further studies demonstrated that FGD5-AS1 could function as a competitive RNA by sponging miR-873-5p in HCC cells. Moreover, GTPBP4 was identified as direct downstream target of miR-873-5p in HCC cells and FGD5-AS1mediated the effects of GTPBP4 by competitively binding with miR-873-5p. Taken together, this study demonstrated the regulatory role of FGD5-AS1 in the progression of HCC and identified the miR-873-5p/GTPBP4 axis as the direct downstream pathway. It represents a promising novel therapeutic strategy for HCC patients.Key words: Hepatocellular carcinoma, FGD5-AS1, miR-873-5p, GTPBP4     

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。     

lncRNA FLVCR1-AS1 silencing inhibits lung cancer cell proliferation,migration, and invasion by inhibiting the activity of the Wnt/β-catenin signaling pathway

Long noncoding RNAs have been reported to be essential regulators in several human diseases, including tumorigenesis. A recent report revealed that FLVCR1-AS1 promotes the progression of hepatocellular carcinoma. However, whether FLVCR1-AS1 is involved in lung cancer remains unclear. In this study, we found that the expression of FLVCR1-AS1 was increased in lung cancer tissues according to The Cancer Genome Atlas database. Similarly, FLVCR1-AS1 was significantly upregulated in lung cancer cell lines. Knockdown of FLVCR1-AS1 dramatically reduced the cell proliferation, migration, and invasion of SPCA1 and A549. Mechanistically, we found that the expression levels of CTNNB1, SOX4, CCND1, CCND2, c-MYC, as well as nucleus β-catenin were decreased in lung cancer cells after FLVCR1-AS1 silencing. Thus, FLVCR1-AS1 positively regulates the activation of the Wnt/β-catenin pathway. Overexpression of CTNNB1 reversed the effect of FLVCR1-AS1 knockdown on A549 cells. In sum, FLVCR1-AS1 silencing inhibited the proliferation, migration, and invasion of lung cancer cells by inhibiting the activity of the Wnt/β-catenin signaling pathway.     

Long noncoding RNA DLX6-AS1 promotes liver cancer by increasing the expression of WEE1 via targeting miR-424-5p

Long noncoding RNAs (lncRNAs) played an important role in tumorigenesis and development of hepatocellular carcinoma (HCC). In this study, we first demonstrated that lncRNA DLX6 antisense RNA 1 (DLX6-AS1) was upregulated in cancer tissues and cells lines compared with normal adjacent and cell line. Knock-down DLX6-AS1 by transfection with small interfering RNA (siRNA) suppressed cell proliferation, migration, and invasion of HCC cells. Cell cycle analysis showed that cells transfected with siRNA were arrested in G0/G1 phase. Then, we performed dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay to show that DLX6-AS1 could bind with miR-424-5p. And cotransfection inhibitor of miR-424-5p with siRNA of DLX6-AS1 could abolish the inhibitory effect of siRNA of DLX6-AS1 on cell proliferation, migration, and invasion. Moreover, we further demonstrated that the oncogene WEE1 G2 checkpoint kinase (WEE1) was the target of miR-424-5p and expression levels of WEE1 were positive correlation with that of DLX6-AS1. Taken together, these results suggested that upregulated DLX6-AS1 promoted cell proliferation, migration, and invasion of HCC through increasing expression of WEE1 via targeting miR-424-5p.     

Retracted: In vivo and in vitro effects of microRNA-221 on hepatocellular carcinoma development and progression through the JAK–STAT3 signaling pathway by targeting SOCS3

Hepatocellular carcinoma (HCC), as the third leading cancer-caused deaths, prevails with high mortality, and affects more than half a million individuals per year worldwide. A former study revealed that microRNA-221 (miR-221) was involved in cell proliferation of liver cancer and HCC development. The current study aims to evaluate whether miR-221 targeting SOCS3 affects HCC through JAK–STAT3 signaling pathway. A series of miR-221 mimic, miR-221 inhibitor, siRNA against SOCS3, and SOCS3 plasmids were introduced to SMMC7721 cells with the highest miR-221 expression assessed. The expression of JAK–STAT3 signaling pathway–related genes and proteins was determined by Western blot analysis. Cell apoptosis, viability, migration, and invasion were evaluated by means of flow cytometry, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide, and transwell assays, respectively. HCC xenograft in nude mice was performed to measure HCC tumor growth. miR-221 was found to be highly expressed but SOCS3 was poorly expressed in HCC tissues. miR-221 expression was correlated with lymph node metastasis (LNM) and tumor node metastasis (TNM) of HCC, and SOCS3 expression was correlated with LNM, differentiation and TNM of HCC. SOCS3 is a target gene of miR-221. MiR-221 mimic or si-SOCS3 exposure was found to induce cell viability, migration, and invasion, and reduce apoptosis. MiR-221 inhibitor was observed to have inhibitory effects on HCC cell proliferation, migration, and invasion. Moreover, the expression of JAK–STAT3 signaling pathway was suppressed by miR-221 inhibitor. Downregulated miR-221 expression could promote its target gene SOCS3 to inhibit the proliferation, invasion and migration of HCC cells by repressing JAK–STAT3 signaling pathway.     

沉默脂肪细胞增强子结合蛋白2通过PI3K/Akt途径抑制肝癌细胞增殖和迁移

脂肪细胞增强子结合蛋白2(AEBP2)作为多梳抑制复合物2(PRC2)的组成蛋白质,参与多种肿瘤细胞的增殖和迁移,然而其在肝癌中的作用尚不清楚。本研究基于UALCAN和Kaplan-Meier Plotter数据库分析发现,AEBP2在肝癌组织中高表达,并且与患者的不良预后呈正相关。实时荧光定量PCR和蛋白质印迹结果证实,AEBP2在肝癌细胞中的表达高于正常肝细胞。在HepG2和Huh-7细胞中转染AEBP2 siRNA,平板克隆、CCK-8、流式细胞术、划痕愈合和Transwell结果显示,沉默AEBP2可以抑制肝癌细胞增殖、迁移和侵袭,并促进细胞凋亡(P＜0.05)。免疫荧光检测和蛋白质印迹结果显示,沉默AEBP2能够抑制肝癌细胞上皮-间质转化(EMT)(P＜0.05)。生物信息学分析结果表明,AEBP2参与调控PI3K/Akt信号通路。蛋白质印迹结果证实,沉默AEBP2能下调PI3K、p-AKT (S473)、mTOR、MMP-2和MMP-9的蛋白质表达水平(P＜0.05)。此外,沉默AEBP2对HepG2细胞迁移和侵袭的影响可被PI3K/Akt通路激动剂胰岛素样生长因子1(IGF-1)部分逆转(P＜0.01)。综上所述,AEBP2可能通过调节PI3K/Akt途径促进肝癌细胞增殖和迁移。本研究为AEBP2在肝癌中的作用提供理论依据。     

Long non-coding RNA HOMER3-AS1 drives hepatocellular carcinoma progression via modulating the behaviors of both tumor cells and macrophages

The crosstalk between cancer cells and tumor microenvironment plays critical roles in hepatocellular carcinoma (HCC). The identification of long non-coding RNAs (lncRNAs) mediating the crosstalk might promote the development of new therapeutic strategies against HCC. Here, we identified a lncRNA, HOMER3-AS1, which is over-expressed in HCC and correlated with poor survival of HCC patients. HOMER3-AS1 promoted HCC cellular proliferation, migration, and invasion, and reduced HCC cellular apoptosis. Furthermore, HOMER3-AS1 promoted macrophages recruitment and M2-like polarization. In vivo, HOMER3-AS1 significantly facilitated HCC progression. Mechanism investigations revealed that HOMER3-AS1 activated Wnt/β-catenin signaling via upregulating HOMER3. Functional rescue experiments revealed that HOMER3/Wnt/β-catenin axis mediated the roles of HOMER3-AS1 in promoting HCC cellular malignant phenotypes. Furthermore, colony stimulating factor-1 (CSF-1) was also identified as a critical downstream target of HOMER3-AS1. HOMER3-AS1 increased CSF-1 expression and secretion. Blocking CSF-1 reversed the roles of HOMER3-AS1 in inducing macrophages recruitment and M2 polarization. Furthermore, positive correlations between HOMER3-AS1 and HOMER3 expression, HOMER3-AS1 and CSF-1 expression, and HOMER3-AS1 expression and M2-like macrophages infiltration were found in human HCC tissues. In summary, our findings demonstrated that HOMER3-AS1 drives HCC progression via modulating the behaviors of both tumor cells and macrophages, which are dependent on the activation of HOMER3/Wnt/β-catenin axis and CSF-1, respectively. HOMER3-AS1 might be a promising prognostic and therapeutic target for HCC.Subject terms: Liver cancer, Oncogenes, Long non-coding RNAs, Translational research     

Long noncoding RNA STXBP5-AS1 inhibits cell proliferation,migration, and invasion via preventing the PI3K/AKT against STXBP5 expression in non–small-cell lung carcinoma

Long noncoding RNAs participate in carcinogenesis and tumor progression in non–small-cell lung carcinoma (NSCLC), but the mechanisms underlying NSCLC tumorigenesis remain to be fully elucidated. Here, we reported the functional role and potential mechanism of long noncoding RNA syntaxin-binding protein 5-antisense RNA 1 (STXBP5-AS1) in NSCLC. First, our data revealed that the expression levels of STXBP5-AS1 in 31 NSCLC tissues were lower than in adjacent tissues using quantitative polymerase chain reaction (qPCR) and its expression was significantly associated with tumor metastasis of NSCLC patients. Moreover, CCK-8, scratch wound healing and transwell assay suggested that upregulation of STXBP5-AS1 repressed the proliferation, migration, and invasion in A549, NCI-H292, and NCI-H460 cells. To explore the potential mechanism of STXBP5-AS1 in NSCLC, we first investigated the relationship among STXBP5-AS1, STXBP5, and AKT1 in A549 cells. Results indicated that STXBP5-AS1 was negatively related with STXBP5 and AKT1 at messenger RNA expression level using qPCR. In addition, we observed that STXBP5-AS1 had reverse effects on the protein levels of STXBP5 and phosphorylated AKT1 (p-AKT1) in A549 cells via Western blot assay, despite no significant effects on AKT1. Subsequently, LY294002, as the phosphatidylinositol 3 kinase/protein kinase B (PI3K/AKT) pathway inhibitor, was used to further confirm the regulatory mechanism of STXBP5-AS1, which showed that knockdown of STXBP5-AS1 could rescue the expression of STXBP5 and p-AKT1 protein expression levels in A549 cells. Taken together, our results suggested that STXBP5-AS1, as a tumor suppressor, inhibits cell proliferation, migration, and invasion by preventing the PI3K/AKT against STXBP5 expression in NSCLC.     

RANKL Promotes Migration and Invasion of Hepatocellular Carcinoma Cells via NF-κB-Mediated Epithelial-Mesenchymal Transition

Background

Metastasis accounts for the most deaths in patients with hepatocellular carcinoma (HCC). Receptor activator of nuclear factor kappa B ligand (RANKL) is associated with cancer metastasis, while its role in HCC remains largely unknown.

Methods

Immunohistochemistry was performed to determine the expression of RANK in HCC tissue (n = 398). Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to examine the expression of RANK, E-cadherin, N-cadherin, vimentin, Snail, Slug, Twist and MMPs in HCC cells. Wound healing and Transwell assays were used to evaluate cell migration and invasion ability.

Results

We found that expression of RANK, the receptor of RANKL, was significantly higher in HCC tumor tissues than in peritumor liver tissues (p<0.001). Constitutive expression of RANK was detected in HCC cell lines, which can be up-regulated when HCC cells were stimulated with RANKL. Notably, in vitro experiments showed that activation of RANKL-RANK axis significantly promoted migration and invasion ability of HCC cells. In addition, RANKL stimulation increased the expression levels of N-cadherin, Snail, and Twist, while decreased the expression of E-cadherin, with concomitant activation of NF-κB signaling pathway. Moreover, administration of the NF-κB inhibitor attenuated RANKL-induced migration, invasion and epithelial-mesenchymal transition of HCC cells.

Conclusions

RANKL could potentiate migration and invasion ability of RANK-positive HCC cells through NF-κB pathway-mediated epithelial-mesenchymal transition, which means that RANKL-RANK axis could be a potential target for HCC therapy.     

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.