A novel positive feedback loop of linc02042 and c-Myc mediated by YBX1 promotes tumorigenesis and metastasis in esophageal squamous cell carcinoma.

BACKGROUND: Long non-coding RNA (lncRNA) is a class of endogenous RNA with a length of more than 200 nucleotides, which is emerging as a pivotal player in cancer development and progression. However, the functional roles of many members in this class remain largely uncharacterized. In the present study, we explored the biological relevance of linc02042 in esophageal squamous cell carcinoma (ESCC). METHODS: qRT-PCR was used to detect the levels of linc02042 and c-Myc. Western blot was used to assess protein expression level. CCK-8 and Transwell assays were employed to test ESCC cell proliferation and invasion, respectively. The mice study including xenograft tumor and lung metastasis models was used to determine the role of linc02042 in vivo. RNA pull-down, ChIP and luciferase reporter assays were employed to test the relationship between linc02042, YBX1 and c-Myc. RESULTS: Linc02042 was found to be markedly upregulated in ESCC cell lines, tissues and plasma, and was closely correlated with malignant clinical features. Knockdown of linc02042 significantly inhibited ESCC cell viability and invasion in vitro as well as tumor growth and lung metastasis in vivo, whereas overexpression of linc02042 resulted in the opposite results. Mechanistically, linc02042 acted as a scaffold for YBX-1 binding to the 3'-UTR of c-Myc mRNA, leading to enhanced c-Myc mRNA stability, thereby facilitating ESCC growth and metastasis. Moreover, in turn, c-Myc was able to transcriptionally elevate linc02042 by directly binding to the E-box motif proximal to the transcription start site (TSS) of linc02042 promoter. Clinically, linc02042 was identified as an effective diagnostic and prognostic biomarker for ESCC patients, and its expression was strongly positively correlated with c-Myc expression in ESCC tissues. CONCLUSION: Our data suggest that linc02042 plays an important tumor-promoting role in ESCC, which lays a foundation for considering it as a potential target for ESCC patients.

The novel circular RNA circ-CAMK2A enhances lung adenocarcinoma metastasis by regulating the miR-615-5p/fibronectin 1 pathway.

BACKGROUND: Circular RNA (circRNA) has recently been considered as a key regulator in carcinogenesis. In this study, we investigated the functional significance and regulatory role of circ-CAMK2A (hsa_circ_0128332) in lung adenocarcinoma (LUAD). METHODS: GSE101586 was employed to screen differentially expressed circRNAs. = Relative expression levels of circ-CAMK2A, miR-615-5p, fibronectin 1 (FN1), MMP2, and MMP9 were tested by quantitative reverse transcription PCR (qRT-PCR) or western blotting. Functional experiments were performed by CCK-8, wound healing, and transwell assays. Luciferase reporter and biotin-labeled RNA pull-down assays were carried out to evaluate the interaction between circ-CAMK2A, miR-615-5p, and fibronectin 1. In addition, a lung metastasis model was constructed to determine the metastasis-promoting role of circ-CAMK2A in vivo. RESULTS: Circ-CAMK2A overexpression was observed in LUAD and was closely associated with lymph node metastasis, distant metastasis, advanced clinical stage, and poor prognosis. Circ-CAMK2A silencing evidently inhibited LUAD cell migration and invasion, whereas circ-CAMK2A overexpression had an opposite effect. Importantly, overexpression of circ-CAMK2A also enhanced LUAD metastasis in vivo. Mechanistically, miR-615-5p was identified as a direct target of circ-CAMK2A. Circ-CAMK2A up-regulates the expression level of fibronectin 1 by sponging miR-615-5p, thereby increasing MMP2 and MMP9 expression to promote the metastasis of LUAD. CONCLUSION: Circ-CAMK2A plays a crucial role in the metastasis of LUAD, at least partially, by regulating the miR-615-5p/fibronectin 1 axis.

LncRNA BLACAT1 Accelerates Non-small Cell Lung Cancer Through Up-Regulating the Activation of Sonic Hedgehog Pathway.

Recently, increasing evidence has displayed that lncRNAs can exhibit crucial function in cancer progression, including lung cancer. LncRNA bladder cancer-associated transcript 1 (BLACAT1) is reported to participate in various cancers. The aim of our current study was to investigate the function of BLACAT1 in non-small cell lung cancer progression and study the functional pathway. Here, we reported BLACAT1 was significantly up-regulated in lung cancer tissues in comparison to the adjacent normal tissues, which suggested BLACAT1 might act as an oncogene in lung cancer. Then, A549 and PC9 cells were infected with BLACAT1 overexpression plasmid and shRNA. As shown, we proved up-regulation of BLACAT1 greatly induced the growth of non-small cell lung cancer cells. Reversely, knockdown of BLACAT1 reduced A549 and PC9 cell proliferation, migration and invasion. Sonic hedgehog (shh) signaling is able to exert a significant role in carcinogenesis, including lung cancer. Currently, we proved that up-regulation of BLACAT1 activated shh signaling pathway, via inducing shh, Gli-1 and Smo expression. shh pathway inhibitor GANT-61 reversed the effect of overexpression of BLACAT1 on non-small cell lung cancer. Moreover, we manifested that loss of BLACAT1 remarkably reduced the in vivo growth and metastasis of A549 cells via enhancing infiltrating CD3+ T cells. In conclusion, our research revealed a critical role of BLACAT1 in the modulation of non-small cell lung cancer via modulating shh pathway.

miR-519d-3p Overexpression Inhibits P38 and PI3K/AKT Pathway via Targeting VEGFA to Attenuate the Malignant Biological Behavior of Non-Small Cell Lung Cancer.

BACKGROUND: Non-small cell lung cancer (NSCLC) is a heterogeneous tumor that accounts for approximately 85% of all lung cancer cases worldwide. microRNAs (miRNAs) are believed to play an important role in regulating a variety of biological processes, including immunity and cancer. We investigated the effect of miR-519d-3p on the mitigation of NSCLC in vitro and in vivo. METHODS: RT-PCR or immunohistochemical assays were used to assess the expression of miR-519d-3p. Colony formation, flow cytometry, and transwell assay were respectively used to detect proliferation, apoptosis, and invasion of A549 and NCI-H661 cell lines. Luciferase reporter assay was used to verify targeting the relationship between mir-519d-3p and VEGFA. Western blot was used to examine the expression of Ki67, caspase-3, E-cadherin, N-cadherin, VEGF, P38, and PI3K/AKT. Animal models were established by BABL/c mice to research the effect of mir-519d-3p overexpression in vivo. RESULTS: In vitro, miR-519d-3p overexpression inhibited A549 and NCI-H661 cells proliferation, invasion, and also promoted apoptosis. In addition, miR-519d-3p overexpression downregulated VEGFA expression and decreased the P38 and PI3K/AKT phosphorylation level. In vivo, miR-519d-3p overexpression significantly restrained tumor volume (2087±265 mm3 vs 599±135 mm3, *P< 0.05) and tumor weight (0.45±0.08 g vs 0.13±0.06 g, *P<0.05) compared with the control group. Overexpression of miR-519d-3p downregulated levels of Ki67 and N-cadherin significantly. CONCLUSION: The data indicated that miR-519d-3p could be a novel therapy or adjuvant against NSCLC.

[Heparanase and vascular endothelial growth factor expression in non-small-cell lung cancer].

OBJECTIVE: To study the clinicopathological significance of heparanase and VEGF expression in NSCLC. METHODS: Eighty-five lung samples were studied. Each sample was divided into two parts, one used for heparanase mRNA detection by reverse transcription PCR and the other for VEGF detection by immunohistochemistry. RESULTS: (1) The expressions of heparanase mRNA and VEGF were higher in NSCLC than in benign pulmonary diseases (P < 0.05). (2) The expression of heparanase mRNA was higher in cases with lymph-node invasion, metastasis, stage III and IV diseases, low-differentiation, and adenocarcinoma, as compared to cases without lymph-node invasion and metastasis, with stage I and II diseases, higher and moderate differentiation, and squamous cell cancer (P < 0.05). Its expression was higher in tumors larger than 5 cm in size (P < 0.05). (3) The expression of VEGF was higher in cases with lymph-node invasion, metastasis, and stage III and IV disease, as compared to cases without lymph-node invasion and metastasis, and with stage I and II diseases (P < 0.05). (4) There was no significant correlation between heparanase and VEGF expression (P > 0.05). CONCLUSIONS: Heparanase and VEGF are associated with NSCLC invasion and metastasis, and may be used to evaluate NSCLC metastasis status. Heparanase and VEGF promote NSCLC invasion and metastasis by independent mechanisms. Detection of these two markers may improve the sensitivity and specificity of the measurement of NSCLC metastatic potential.

MicroRNA-2 suppresses Lewis lung cancer cells proliferation, invasion, and migration in tumor-bearing mice.

We sought to find the biological effects of MicroRNA-2 in suppressing Lewis lung cancer cells proliferation, invasion, and migration in tumor-bearing mice. MicroRNA-2 was transfected into Lewis lung cancer cells of tumor-bearing mice by gene transient transfection technique and these Lewis-microRNA-2 cells were taken as MicroRNA transfection group. At the same time, Lewis cells were taken as control group and Lewis-EGFP cells as empty plasmid group. The growth curves of cells in the three groups were drawn by manual counting method, while the invasiveness of cells in the three groups was compared by transmembrane cell invasion assay. The three kinds of cells were seeded into BALB/Nude SPF level nude mice to detect the formation of tumors and the number of metastases by Xenograft experiments. The result showed that the MicroRNA transfection group has the lowest vitality of cells proliferation, fewest cells passed through matrigel matrix protein layer, and lowest cells invasive rate. Mice with Lewis-microRNA-2 cells apparently had a longer time of tumor formation. The average tumor mass and the number of metastases were significantly lower than the other two groups. MicroRNA-2 significantly inhibited Lewis lung cancer cell proliferation, invasion and migration in tumor-bearing mice, which may be associated with the regulation of target genes PLK1 and TGF-ß.