<p>MiR-486-5p Act as a Biomarker in Endometrial Carcinoma: Promotes Cell Proliferation, Migration, Invasion by Targeting MARK1</p>
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Abstract:
miRNA expression acts as a potential biomarker in many diseases including endometrial carcinoma (EC). miR-486-5p dysregulation is observed in several tumor types, but the roles of miR-486-5p in EC are hardly ever studied.This study aimed to analyze the expression profile of miR-486-5p in tumor tissues and serum samples of patients with EC and explore the target prediction, function analysis and validation in immortal cell lines.A total of 42 freshly paired EC tissues, the corresponding adjacent non-neoplastic tissues and serum samples were also collected from patients with EC, and 42 matched normal serum samples were included as control group. The level of miR-486-5p expression was tested by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was determined by colony formation assay and CCK-8 assay. Furthermore, functional evaluation of miR-486-5p on migration was performed by wound-healing assay and invasion was estimated by transwell invasion assay. qRT-PCR, luciferase reporter assay and Western blotting (WB) were performed to verify the targeting of MARK1 by miR-486-5p.miR-486-5p was significantly up-regulated in EC tissues and serum samples, promoting the proliferation, migration and invasive activities of EC cells by targeting MARK1.These data indicated miR-486-5p as a novel molecular biomarker for diagnosing and treating EC, and MARK1 might act as a critical and functional target of miR-486-5p with the implications on cell proliferation, migration and invasiveness of EC tumor cells.Keywords:
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The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration and invasion abilities to understand how cancer cells disseminate, with the aim of developing new treatment strategies. Analysis of the cellular and molecular basis of these abilities has led to the characterization of cell mobility and the physicochemical properties of the cytoskeleton and cellular microenvironment. For many years, the Boyden chamber assay and the scratch wound assay have been the standard techniques to study cell invasion and migration. However, these two techniques have limitations. The Boyden chamber assay is difficult and time consuming, and the scratch wound assay has low reproducibility. Development of modern technologies, especially in microscopy, has increased the reproducibility of the scratch wound assay. Using powerful analysis systems, an "in-incubator" video microscope can be used to provide automatic and real-time analysis of cell migration and invasion. The aim of this paper is to report and compare the two assays used to study cell invasion and migration: the Boyden chamber assay and an optimized in vitro video microscope-based scratch wound assay.
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Alterations in cell migration are a hallmark of cancer cell invasion and metastasis. In vitro assays commonly used to study cell migration, including the scratch wound healing assay, Boyden chamber assay, and newly developed advanced systems with microfluidics, each have several disadvantages.Here we describe an easy and cost-effective in vitro assay for cell migration employing cloning rings to create gaps in the cell monolayer ("ring cell migration assay"). The assay was used to quantitate innate differences in cell motility and the effect of various extracellular matrix proteins on migration of five cancer cell lines: U87 and U251N glioma cells, MDA-MB-231 and MCF-7 breast cancer cells, and HeLa cervical cancer cells. Interestingly, collagen was a general promoter of cell migration for all five cancer cell lines, without affecting cell proliferation.Taken together, the ring cell migration assay is an easy, convenient and cost-effective assay to study cell migration in vitro.
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Objective:To investigate the effect of NS-398,a selective cyclooxygenase-2(COX-2) inhibitor,on the proliferation and invasion of human colon carcinoma cells in vitro,so as to determine the possibility of COX-2 as a new target for treatment of colon carcinoma.Methods: The expression of COX-2 in colorectal cancer cells(CW-2,COLO-320) was detected by RT-PCR and Western blotting.COLO-320 cell proliferation was measured by MTT after treatment with NS-398.Cell invasion ability was measured using migration and invasion chamber systems.Western blotting assay was used to examine the influence of NS-398 on MMP-2 expression.Results: Our results showed that CW-2,COLO-320 cells expressed COX-2 mRNA and protein.NS-398 inhibited the proliferation of COLO-320 cells in a time-and concentration-dependent manner.Invasion test showed that NS-398 inhibited the migration and invasion of COLO-320 cells.Western blotting revealed that NS-398 inhibited the expression of MMP-2 in COLO-320 cells.Conclusion: The selective COX-2 inhibitor NS-398 can inhibit COLO-320 cell proliferation and invasion,indicating COX-2 may serve as a new target for colon carcinoma treatment.
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Objective: To investigate the effect of NS-398, a selective cyclooxygenase-2 (COX-2) inhibitor, on the proliferation and invasion of human colon carcinoma cells in vitro, so as to determine the possibility of COX-2 as a new target for treatment of colon carcinoma. Methods: The expression of COX-2 in colorectal cancer cells (CW-2, COLO-320) was detected by RT-PCR and Western blotting. COLO-320 cell proliferation was measured by MTT after treatment with NS-398. Cell invasion ability was measured using migration and invasion chamber systems. Western blotting assay was used to examine the influence of NS-398 on MMP-2 expression. Results: Our results showed that CW-2, COLO-320 cells expressed COX-2 mRNA and protein. NS-398 inhibited the proliferation of COLO-320 cells in a time- and concentration-dependent manner. Invasion teat showed that NS-398 inhibited the migration and invasion of COLO-320 cells, Western blotting revealed that NS-398 inhibited the expression of MMP-2 in COLO-320 cells. Conclusion: The selective COX-2 inhibitor NS 398 can inhibit COLO-320 cell proliferation and invasion, indicating COX-2 may serve as a new target for colon carcinoma treatment.
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Aim To investigate the inhibitory effect of heparanase inhibitor OGT2115 on the invasion and migration in KB cells,in order to provide a new target points for the treatment of cancer metastasis.Methods MTT assay was used to detect the cell viability by different concentrations of OGT2115(0.4,0.8,1.6,3.2,6.4 μmol·L-1) and OGT2115(0.4 μmol·L-1)combine with ADM(2 μmol·L-1)in KB.Transwell assay was applied to detect the invasion and migration by OGT2115 and ADM.Wound healing assay was employed to observe cell migration,and heparanase activity was detected by ELISA.Results MTT results showed that OGT2115 could inhibit the proliferation of KB;IC50 was 8.59 μmol·L-1;the inhibitory activity increased with the role of time and concentration(P0.05).OGT2115 could inhibit the invasion and migration of KB;using OGT2115(0.4 μmol·L-1)significantly enhanced anti-invasion and anti-proliferation of ADM(P0.05).Conclusion OGT2115 can inhibit the invasion and migration of KB,and OGT2115 enhances anti-invasion and anti-migration of ADM,which is closely related to heparanase.
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Objectives: The purpose of this study is to investigate the expression pattern of lncRNA H19 in OC tissues and to detect the ability of H19 to influence OC cell migration and invasion in vitro. Material and methods: We quantified the levels of H19 within the obtained cancerous and adjacent noncancerous tissues from 258 OC patients. H19 association with patient progression-free survival (PFS) was analyzed by a Kaplan-Meier plot. Expression levels of H19 were reduced by small interfering RNA transfection against H19 or restored by a H19 overexpression plasmid transfection in OC cells. H19 effects on OC cell migration and invasion in vitro were evaluated using wound-healing assay and transwell invasion assay. Wound healing assay and transwell invasion assay were used to evaluate the effects of H19 on OC cell migration and invasion in vitro. Results: H19 is upregulated remarkably in primary OC tissues and human OC cell lines (OVCAR3, SKOV3, A2780, and Caov-3). We found that the median PFS was longer in patients with lower levels of H19 than in those with high levels, suggesting that overexpres- sion of H19 was linked to poor prognosis in OC patients. Intriguingly, the depletion of H19 expression induced by small interfering RNA inhibited the capability of migration and invasion of OC cell lines. Restoration of H19 in OC cell lines significantly increased cell migration and invasion. Conclusions: The key finding of the present study suggests that overexpression of H19 may be associated with an unfavorable prognosis for OC and is likely to be a possible contributory force involved in OC cell migration and invasion. H19 may provide a new and attractive target for future prognostic and therapeutic intervention of OC patients.
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Objective: The actin binding protein Vasodilator‐stimulated phosphoprotein(VASP) has complex effects on cytoskeletal organization and cell motility, which play a positive role on tumour cells migration and invasion. Our research aim to investigate the effect of epigallocatechin gallate (EGCG), an extractent from green tea, on SGC‐7901 cells migration and invasion. Methods: The expression level of VASP in SGC‐7901 cells was detected by RT‐PCR and Western Blotting. Down regulation of VASP expression was performed with VASP siRNA transfection, while the activation of Rac1 pathway was enhanced by fMLP. Wound‐healing assay (2D assay) and transwell migration assay (3D assay) were employed to analyze invasive migration ability of SGC‐7901 cells. Results: After treated with EGCG, the expression level of VASP in SGC‐7901 cells decreased significantly at mRNA and protein level compared with control group (P<0.05). In 2D assay, the cell migration velocity was reduced by EGCG (P<0.05), accordingly the EGCG group showed lower invasion capacity than the control group in 3D assay (P<0.05).The effect of EGCG on SGC‐7901 cells migration and invasion was enhanced by VASP siRNA transfection, on the contrary decreased by fMLP. Conclusion: EGCG has a inhibitive effect on SGC‐7901 cells migration and invasion by decreasing the expression level of VASP via Rac1 pathway. (This work was supported by National Natural Sciences Foundation of China under Grant No.30770966)
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