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@# Objective: To explore the roles and mechanisms of long non-coding RNA (lncRNA) small nucleolar RNA host gene 6 (SNHG6) in promoting invasion and metastasis of esophageal squamous carcinoma (ESCC). Methods: Real time quantitative polymerase chain reaction (qPCR) was used to detect the expression of SNHG6 in ESCC and matched para-carcinoma tissues. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect the expression of SNHG6 in ESCC cell lines (TE1, Yes-2, Eca9706 and Kyse150). Then, TE1 cell line which harbored highest expression of SNHG6 was used in following experiments. siRNAs were used to knock down the expression of SNHG6. Clone formation, wound-healing and transwell assay were used to detect the abilities of proliferation, migration andinvasionofTE1cells,respectively.Westernblottingwasusedtodetecttheexpressions of MMP-2, MMP-9andZEB1 protein before and after knockdownofSNHG6inTE1cells.Results:SNHG6washighlyexpressedinESCC tissues, compared to para-carcinoma tissues (P<0.01). The expression of SNHG6 was significantly decreased after transfection of SNHG6siRNA (all P<0.01). The abilities of proliferation, migration and invasion of TE1 cells in si-SNHG6-1 and si-SNHG6-2 group were significantly lower than those in the control group (all P<0.01). The expressions of ZEB1, MMP-2and MMP-9 in si-SNHG6-1 and si-SNHG6-2 group were significantly lower than those in the control group (all P<0.05). Conclusion: SNHG6 is highly expressed in ESCC tissues and promotes the malignant biological behavior of ESCC cells. Its mechanism of promoting the occurrence and development of ESCC may be related to the upregulation of ZEB1 expression.
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@#[Abstract] Objective：To explore the regulatory effect of miR-9 on biological behaviors of small cell lung cancer (SCLC) cells by targeting zinc finger E-box binding homeobox 2 (ZEB2), and to analyze the role of miR-9 in SCLC and its possible mechanism. Methods: qPCR, WB and immunohistochemistry methods were used to detect the mRNA and protein expressions of ZEB2 in cancer tissues and corresponding adjacent tissues of 67 SCLC patients who received surgical treatment at the Department of Oncology, Fourth Hospital of Hebei Medical University from February 2018 to November 2019. TargetScan was used to predict the potential target gene of miR-9, which was later verified by Dual luciferase reporter gene assay, qPCR and WB methods. CCK-8 method, Flow cytometry and Transwell experiment were used to detect the effect of miR-9 and ZEB2 over-expression on the biological behaviors of NCI-H446 cells, and WB was used to detect the protein expressions of E-cadherin, N-cadherin and Vimentin in cells. NCI-H446 cells overexpressing miR-9 were used to construct SCLC nude mouse xenograft model, and the effect of miR-9 on the growth of xenografts was observed. Results: The mRNA and protein expression levels of ZEB2 in SCLC tissues were significantly higher than those in adjacent tissues (P<0.01). There is a potential binding site on the 3' UTR of ZEB2 to bind with miR-9. Compared with the control group, the mRNA and protein expression levels of ZEB2 in NCI-H446 cells of the miR-9 over-expression group were significantly reduced (P<0.01); the proliferation, migration and invasion abilities of NCI-H446 cells were significantly suppressed (P<0.05 or P<0.01), and the expression of EMT protein was reduced; However, simultaneous over-expression of ZEB2 could reverse above effects. In in vivo experiments, the size and weight of transplanted tumors in the miR-9 over-expression group were significantly lower than those in the control group (P<0.05 or P<0.01). The expression of ZEB2 protein in the tumor tissues of nude mice in the miR-9 overexpression group was significantly lower than that in the control group (P<0.01). Conclusion: miR-9 can inhibit the biological behaviors of SCLC cells and the growth of NCI-H446 transplanted tumors in nude mice by targeting and regulating ZEB2.
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Objective: To investigate the effects of silencing zinc finger E-box binding homeoboxl (ZEB1) gene on the expressions of mesenchymal markers and cell migration in the glioma U87 cells, and to clarify the effect of ZEB1 on the epithelial to mesenchymal transition (EMT) in the glioma cells. Methods: The constructed ZEB1 shRNA interfering plasmid and control plasmid (shCtrl) were transfected into the glioma U87 cells and the interfering effects were detected by Western blotting method. The glioma U87 cells were divided into control group (the glioma U87 cells were transfected with shCtrl), EMT group (EMT was induced by TGF-fil in the glioma U87 cells transfected with shCtrl) and ZEB1 silence group (EMT was induced by TGF-fil in the glioma U87 cells transfected with ZEB1 shRNAs plasmid). The protein expression levels of mesenchymal markers (N-cadherin, Vimentin), and matrix metalloproteinase-9 (MMP-9) in the glioma U87 cells were measured by Western blotting method. The scratch-healing assay was performed to examine the migration ability of glioma cells. Results: The Western blotting results showed that the expression levels of ZEB1 in the glioma U87 cells transfected with shZEBl # 1 and shZEBl # 2 were significantly lower than that in the cells transfected with shCtrl (P<0. 05 or P< 0. 01), and the inhibitory effect of shZEBl #2 on the ZEB1 expression was more obvious, indicating that ZEB1 was stably transfected into the U87 cells. Compared with control group, the expression levels of mesenchymal markers N-cadherin, Vimentin, and MMP-9 in EMT group were significantly increased (P<0. 05 or P<0. 01). Compared with EMT group, the expression levels of the above proteins in ZEB1 silencing group were markedly reduced (P< 0. 05 or P<0. 01). The cell migration rate in EMT group was obviously elevated compared with control group (P< 0. 01), and the cell migration rate of the glioma U87 cells in ZEB1 silence group was significantly lower than that in EMT group (P<0. 01). Conclusion: Silencing ZEB1 gene expression can inhibit the EMT in the glioma U87 cells and reduce the cell migration abilities, suggesting ZEB1 as an important therapeutic target of invasive glioma.
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@#Objective: To explore the effect of glycyrrhizin (GA) on the proliferation, invasion and migration of non-small cell lung cancer HCC827 andA549 cells via regulating miR-142/ZEB1 (Zinc finger E-box-binding homeobox 1) axis. Methods:After being cultured and transfected, HCC827 andA549 cells were divided into 4 groups: NC group (untransfected+3 mmol/L GA), miR-142 inhibitor group (miR-142 knockdown+3 mmol/L GA), pcDNA3.1-ZEB1 group (ZEB1 over-expression+3 mmol/L GA) and pcDNA3.1-ZEB1+ miR-142 mimic group (ZEB1 over-expression+miR-142+3 mmol/L GA). qPCR was used to detect the expression level of miR-142 in HCC827 andA549 cells treated with different concentrations of GA. MTT and Transwell assays were used to examine the proliferation, invasion and migration of HCC827 and A549 cells. WB was used to detect the expression level of ZEB1 protein in HCC827 and A549 cells. Dual-luciferase reporter gene assay was used to explore the relationship between miR-142 and ZEB1. Results: GA significantly inhibited the proliferation, invasion and migration of HCC827 and A549 cells, and up-regulated the expression level of miR-142 ( P < 0.05 or P <0.01). Dual-luciferase reporter gene assay showed that miR-142 could targetedly combine with 3'-UTR of ZEB1 and downregulate the expression of ZEB1 ( P <0.05 or P <0.01). Further experiment validated that GAinhibited ZEB1 expression via up-regulating miR-142, thus suppressed proliferation, invasion and migration of HCC827 and A549 cells ( P <0.05 or P <0.01). Conclusion: GA inhibits the proliferation, invasion and migration of NSCLC HCC827 and A549 cells, the mechanism of which is that GA inhibits the malignant biological behavior of NSCLC HCC827 andA549 cells via up-regulating the inhibition effect of miR-142 on ZEB1.
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@#To investigate the molecular mechanism of lncRNA-HCG11 promoting progression and metastasis of colorectal cancer (CRC) via up-regulating zinc finger E box binding homeobox 1 (ZEB1) by regulating miR-144-3p expression in CRC. Methods:Atotal of 78 pairs of CRC tissues and corresponding adjacent tissues were obtained from patients in Department of Colorectal Surgery, Cancer Hospital of Yunnan Province during January 2013 and January 2018. HCG11 expression level in CRC cell lines and tissues was determined by qPCR; HCG11-knockdown vector, miR-144-3p mimic and miR-144-3p inhibitor were constructed and transfected into CRC cells lines (SW480 and SW620); and then, cell viability was detected by using CCK-8 assay and colony formation assay, while cell migration and invasion was assessed by using transwell assay; the expression levels of ZEB1 and epithelial mesenchymal markers (E-cadherin, Vimentin, ɑ-catenin, Sox2, Nestin, Oct4 and Nanog) were detected by Wb and immunofluorescence assay; and the relationship between HCG11, miR-144-3p and ZEB1 was validated by dual-luciferase reporter gene assay. Nude mice xenograft model was constructed and the effect of HCG11 knock-down on the growth of xenograft was evaluated. Results: The expression of HCG11 was significantly higher in CRC cell lines (all P<0.05) and tissues (P<0.01) compared with that in normal colon epithelial cells and para-cancerous tissues; HCG11 expression was closely related with cancer metastasis, clinical staging and prognosis of CRC patients (all P<0.05). Knockdown of HCG11 significantly inhibited cells proliferation, migration, invasion, epithelial-mesenchymal transition and CRC stem cell formation (all P<0.05). Moreover, knockdown of HCG11 significantly up-regulated miR-144-3p expression (P<0.05), while over-expression of miR-144-3p significantly inhibited ZEB1 expression (P<0.05) and reduced dual-luciferase activity (P＜0.05). Conclusion: HCG11 regulates miR-144-3p to up-regulate ZEB1 expression, and further promotes CRC progression and metastasis; therefore, HCG11 could be used as a target for clinical diagnosis and treatment for CRC.
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Objective: To investigate the effects of miR-192 on the expression of zinc-finger E-box binding homeobox 2 (ZEB2) and the abilities of migration and invasion of colorectal cancer (CRC) SW480 and SW620 cells. Methods: After transfection of miR-192 mimics into SW480 and SW620 cells, the expression levels of miR-192 and ZEB2 mRNA and ZEB2 protein were detected by real-time fluorescence quantitative RT-PCR and Western blotting, respectively. The abilities of migration and invasion of SW480 and SW620 cells after transfection with miR-192 mimics were determined by wound healing and Transwell assays, respectively. The recombinant vector pmiR-ZEB2-wt and miR-192 mimics were co-transfected into SW480 cells. The binding site of 3′-untranslated region (3′-UTR) of ZEB2 gene with miR-192 was verified by Dual Luciferase™ Reporter Gene Assay. Results: As compared with the negative control (NC) group (transfection with miR-192 mimics-NC), the expression levels of miR-192 in SW480 and SW620 cells after transfection with miR-192 mimics were increased (P < 0.05), and the expression levels of ZEB2 mRNA and protein were decreased (P < 0.05), as well as the abilities of migration and invasion of SW480 and SW620 cells were inhibited (P < 0.05). The Dual Luciferase™ Reporter Gene Assay revealed that the luciferase activity in SW480 cells after co-transfection with recombinant vector pmiR-ZEB2-wt and miR-192 mimics was inhibited (P < 0.05), which indicated that the 3′-UTR of ZEB2 harbored a binding site for miR-192. Conclusion: ZEB2 may be one of the target genes for miR-192. Overexpression of miR-192 may down-regulate the ZEB2 expression and inhibit the migration and invasion of SW480 and SW620 cells. Copyright© 2014 by TUMOR.
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Objective To explore the effects of zinc finger E-box binding protein (ZEB)2 3′UTR gene transfection on proliferation, invasion and migration in human gastric epithelial cell line GES-1. Methods The synthetic ZEB2 3′UTR and miR-200b micmics were transfected into GES-1 cell line by lipofectamine 2000. We set up control grop, the mutation group and ZEB2 3′UTR group. Real-time quantitative PCR was performed to evaluate the expression levels of miR-200a/b/c and ZEB1/ZEB2 mRNAs after transfection.And then we set up control group, ZEB2 3′UTR group, ZEB2 3′UTR+negative control group and ZEB2 3′UTR+miR-200b micmics group. The protein expression levels of ZEB1, ZEB2, matrix metallopro-teinases (MMP) 2/9 and proliferating cell nuclear antigen (PCNA) were detected by Western blot assay. The invasion and mi-gration capability were analyzed by transwell assay and wound healing test. MTT assay was used to detect the proliferation ability. Results Compared with control group and mutation group, the expressions of miR-200a/b/c were significantly de-creased, especially for miR-200b. And the expressions of ZEB1/ZEB2 were significantly increased at both mRNA and pro-tein levels after transfected with the ZEB2 3′UTR, enhancing the capability of migration,invasion,and proliferation (P <0.05). Compared with ZEB2 3′UTR group, the capabilities of proliferation,invasion and migration were significantly lower in combined group. Conclusion ZEB2 3′UTR can increase the ability of cell proliferation, invasion and metastasis through regulating the levels of miR-200a/b/c, and then influence the regulation of transcription of the target gene, which could lead to malignant transformation of GES-1 cells.