Ese-3 Inhibits the Proliferation, Migration, and Invasion of HaCaT Cells by Downregulating PSIP1 and NUCKS1.
Chao ZhangJunqiang LiYongdong GuoRonglin WangLei HuaDongxue GanLiaoliao ZhuPeixiang MaJing YangHong LiShanshan LiYang SongHaichuan Su
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OBJECTIVE Epithelium-specific ETS 3 (Ese-3) is a member of the ETS family that is associated with tumor progression. However, there is little knowledge about Ese-3 in skin cancer. This study was conducted to explore the effects of Ese-3 on clinical prognosis in skin cancer and the functions of HaCaT cells. MATERIALS AND METHODS Gene expression and clinical data were collected from The Cancer Genome Atlas (TCGA), The Genotype-Tissue Expression (GTEx), and three GSE datasets (GSE15605, GSE46517, and GSE114445). Comparison of data between groups was performed by Student's t-test and chi square test. Survival analysis was performed using log-rank test. Univariate and multivariate analyses were performed using Cox proportional hazards models. Enrichment analysis was used to predict Ese-3 related functions. Cell proliferation assays, colony formation assays, and flow cytometry were used to assess cell proliferation, while Transwell assays analyzed cell migration and invasion. RESULTS Compared with normal tissues, the Ese-3 mRNA in cutaneous malignant melanoma (CMM) patients was downregulated (P<0.0001). Ese-3 mRNA was associated with the T stage (χ2=10.015, P=0.018), clinical stage (χ2=4.122, P=0.042), and prognosis in CMM patients (P=0.0219) and was an independent prognostic predictor in CMM (HR=1.878, P=0.048). Enrichment analysis showed that differentially expressed proteins were associated with protein kinase B (AKT) binding. CONCLUSION Ese-3 inhibited the proliferation, migration, and invasion of HaCaT cells by downregulating PSIP1 and NUCKS1 expression levels to inactivate the phosphorylation of AKT.Keywords:
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Glioblastoma multiforme (GBM) was one of the first cancer types systematically studied at a genomic and transcriptomic level due to its high incidence and aggressivity; however, the detailed mechanism remains unclear, even though it is known that numerous cytokines are involved in the occurrence and development of GBM. The present study aimed to determine whether the SET gene has a role in human glioblastoma carcinogenesis. A total of 32 samples, including 18 cases of glioma, 2 cases of meningioma and 12 normal brain tissue samples, were detected using the streptavidin-peroxidase method through immunohistochemistry. To reduce SET gene expression in U251 and U87MG cell lines, the RNA interference technique was used and transfection with small interfering (si)RNA of the SET gene was performed. Cell apoptosis was detected by flow cytometry, cell migration was examined by Transwell migration assay and cell proliferation was determined by Cell Counting Kit-8. SET, Bcl-2, Bax and caspase-3 mRNA and protein expression levels were detected by reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. Positive protein expression of SET was observed in the cell nucleus, with the expression level of SET significantly higher in glioma tissues compared with normal brain tissue (P=0.001). Elevated expression of SET was significantly associated with gender (P=0.002), tumors classified as World Health Organization grade II (P=0.031), III (P=0.003) or IV (P=0.001), and moderately (P=0.031) or poorly differentiated (P=0.001) tumors. Compared with the negative and non-treatment (blank) control cells, SET gene expression was significantly inhibited (P=0.006 and P<0.001), cell apoptosis was significantly increased (P=0.001 and P<0.001), cell proliferation was significantly inhibited (P=0.002 and P=0.015), and cell migration was significantly decreased (P=0.001 and P=0.001) in siRNA-transfected U87MG-SET and U251-SET cells, respectively. In addition, mRNA and protein expression levels of Bcl-2 were significantly inhibited in U87MG-SET and U251-SET cells, while mRNA and protein expression levels of Bax and caspase-3 were significantly increased, compared with the two control groups. Thus, the current data suggests that SET may regulate the proliferation and apoptosis of glioblastoma cells by upregulating Bcl-2, and downregulating Bax and caspase-3.
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The functions of miR-126-mediated signal transducers and activators of the transcription 3 (STAT3) signal pathway were investigated in regulating the behavior of cells in non-small cell lung cancer (NSCLC). Cultured NSCLC A549 cells were transfected with empty, miR-126 overexpression or miR-126 knocked-down expression plasmids. After transfection efficiency verification by reverse transcription polymerase chain reaction (RT-PCR) and culture for 24 h, methyl thiazolyl tetrazolium (MTT) was applied to detect cell proliferation rate, migration distance was measured in scratch assays, cell cycle was determined through flow cytometry, the mRNA expression level of caspase-3 in cells was detected using RT-PCR and protein expression levels of STAT3 were detected using western blotting. Our results showed the cell proliferation rate was significantly higher in cells of the overexpression group than that in those of the control group (p<0.05) and the rate in the cells of the low-expression group was the lowest among the three groups (p<0.05). The migration distance of the overexpression group cells was significantly longer than that in the control group cells and the shortest migration distance was found in the low-expression group cells (p<0.05). The amount of cells in mitotic phase in the overexpression group was significantly higher than that in the control group and the same amount in the low-expression group was the lowest (p<0.05). The mRNA expression level of caspase-3 of cells in the overexpression group was significantly lower than that of cells in the control group and the highest expression level was found in the low-expression group (p<0.05). Finally, the protein expression levels of STAT3 in cells in the overexpression group were significantly lower than those in the control group and the highest expression levels were identified in the low-expression group (p<0.05). Based on our findings, the cancer-promoting miR-126 can mediate the activation of the STAT3 signal pathway to regulate the malignant biological behavior of NSCLC cells affecting their proliferation, migration, cycle and apoptosis susceptibility.
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Ovarian cancer (OC) is one of the most lethal gynecological malignancies in the world. The aim of the present study was to examine the role of microRNA (miR)-134-3p in OC. Reverse transcription-quantitative PCR was used to measure the expression levels of miR-134-3p. Cell Counting Kit-8, TUNEL, flow cytometric and colony formation assays were performed to examine the effects of miR-134-3p on OC cell proliferation. Moreover, wound healing and Transwell assays were performed to examine the effects on migration and invasion. In addition, western blot analyses were used to assess protein expression. Finally, the target genes of miR-134-3p were analyzed by bioinformatics analysis and dual-luciferase reporter assay. The results revealed that miR-134-3p expression was low in OC cells compared with in normal ovarian cells. The overexpression of miR-134-3p decreased cell viability, facilitated cell apoptosis, inhibited cell proliferation and arrested the cell cycle in SKOV-3 and OVCAR-3 cells. Furthermore, transfection using a miR-134-3p mimic inhibited the migration and invasion of SKOV-3 and OVCAR-3 cells, and decreased the protein expression levels of cyclooxygenase-2, matrix metalloproteinase (MMP)2 and MMP9. Bioinformatics analysis indicated that one of the potential target genes of miR-134-3p was flap structure-specific endonuclease 1 (FEN1), which was confirmed by dual-luciferase reporter assay. Moreover, overexpression of miR-134-3p decreased the expression levels of FEN1 in SKOV-3 and OVCAR-3 cells. Additionally, overexpression of FEN1 reversed the effects of the miR-134-3p mimic on the proliferation, migration and invasion of SKOV-3 and OVCAR-3 cells. Overall, the findings of the present study demonstrated that miR-134-3p may inhibit OC cell proliferation, migration and invasion by directly targeting FEN1.
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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.
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BACKGROUND:This study aimed to investigate the role of miRNA-339-5p in pancreatic cancer cell invasion and migration. MATERIAL AND METHODS:The differences between exosomal miRNAs of PANC02 and PANC02-H7 were studied by microarray analysis. We measured miRNA-339-5p expression in different groups; differences in cell invasion and migration were evaluated using the Transwell and wound healing assays and expression of relative proteins (E-cadherin, vimentin and ZNF689) was measured by WB assay. The correlation between miRNA-339-5p and ZNF689 expression was evaluated by luciferase reporter gene assay. RESULTS:Compared with PANC02 exosome, microarray analysis indicated that miRNA-339-5p mRNA expression was significantly suppressed (P<0.001) in the PANC02-H7 exosome. Supplementation with miR-339-5p mimics led to a significant decrease in the invasion cell number and wound healing rate (P<0.001), with significantly enhanced E-cadherin expression and suppressed vimentin expression (P<0.001). However, transfection of a miR-339-5p inhibitor led to a significant increase in the invasion cell number and wound healing rate (P<0.001), with significantly suppressed E-cadherin expression and increased vimentin expression (P<0.001). Luciferase reporter gene assay demonstrated ZNF689 gene to be the target of miR-339-5p in the PANC02-H7 cell. With miR-339-5p and ZNF689 transfection, the invasion cell number and wound healing rate were significantly increased compared with those in the miR-339-5p group (P<0.001), with significantly increased expression of ZNF689 and vimentin and suppressed E-cadherin expression (P<0.001). CONCLUSIONS:miR-339-5p suppresses the invasion and migration of pancreatic cancer cells via direct regulation of ZNF689 in vitro.
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This investigation examined the effects of the microRNA miR-34c-5p on the proliferation, migration, and invasion of oral squamous cell carcinoma (OSCC) and the mechanisms involved.The Gene Expression Omnibus (GEO) database was used to filter the chips, and the GEO2R software (https://www.ncbi.nlm.nih.gov/geo/geo2r/) was used to analyze the microarray data (GSE28100 and GSE45238). Gene set enrichment analysis (GSEA) was used to study the relationship between the expression of miR-34c-5p and the distant metastasis and pathological grade of OSCC. The correlation between TRIM29 (tripartite motif containing 29) expression and the malignant clinical phenotype of OSCC was also examined. The mRNA and protein expression levels of miR-34c-5p and TRIM29 were measured by real time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot analysis. The proliferation, migration, invasion and apoptosis of the human oral squamous carcinoma cell lines CAL-27 and Tca8113 was assessed by performing cell-counting kit-8 (CCK-8) assays, colony formation assays, transwell tests, wound scratch tests and flow cytometry. Luciferase reporter assays were used to predict the relationship between miR-34c-5p and TRIM29. A xenograft nude model was established and used to evaluate the effect of miR-34c-5p on tumor growth in female BALB/c mice.The expression of miR-34c-5p was significantly correlated with the proliferation, migration, and metastasis of OSCC. Overexpression of miR-34c-5p promoted the proliferation, migration, and invasion of CAL-27 and Tca8113 cells, and suppressed their apoptosis. Inversely, low expression of miR-34c-5p suppressed the proliferation, migration, and invasion of CAL-27 and Tca8113 cells, and promoted their apoptosis. Overexpression of miR-34c-5p promoted tumor growth in the xenograft nude mice model. The expression of TRIM29 was related to malignant clinical phenotype of OSCC. Overexpression of TRIM29 inhibited the proliferation, migration and invasion of CAL-27 and Tca8113 cell, and induced their apoptosis. TRIM29 knockout had just the opposite effect. Importantly, miR-34c-5p binds to TRIM29 and inhibited TRIM29 expression.MiR-34c-5p regulates the proliferation, migration, invasion, and apoptosis of OSCC through targeted binding of TRIM29. This may represent a novel therapeutic target for the treatment of patients with OSCC.
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Esophageal cancer (EC) is a common cancer worldwide. Sine oculis homeobox homolog (SIX3) is a human transcription factor that regulates the progression of vertebrate eye and fetal forebrain. However, studies on the function of SIX3 in human tumorigenesis remain rare. In this study, we aim to evaluate the role and the significance of SIX3 in EC. The TCGA database and clinical samples were used to assess the expression of SIX3 in EC patients. The Kaplan-Meier method and Cox's proportional hazards model were performed to analyze the correlations between SIX3 expression and EC clinical outcomes. The expressions of SIX3 in EC cells were measured by quantitative reverse transcription PCR analysis. The cell proliferation was detected using cell counting kit-8 and colony formation assay. The migration and invasion capacity of EC cells were evaluated using wound healing and Transwell methods. Western blot assay was used to measure the alterations in some important protein expression levels in the PI3K/Akt signaling pathway. We found that SIX3 was highly expressed in the EC tissues and cells. In addition, high expression of SIX3 was related to poor survival. The knockdown of SIX3 significantly inhibited the proliferation, migration, and invasion of ECA109 cells. A similar pattern was also found in the proliferation and migration of SKGT-4 cell line. The expression levels of some key proteins in the PI3K/Akt signaling pathway were obviously decreased after cells were transfected with si-SIX3, possibly resulting in PI3K/AKT signaling inactivation. In addition, E-cadherin and N-cadherin showed some change. Collectively, the results shed light on a potentially promoting role of SIX3 in human EC. Thus, SIX3 might be considered a novel prognostic biomarker and therapeutic target for EC patients.
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This study aims to investigate the expression of microRNA (miR)-16 in non-small cell lung carcinoma (NSCLC) and to identify its potential mechanism.A total of 45 NSCLC patients were included in the present work. NSCLC tissues and adjacent normal tissues were resected and collected. The Reverse Transcription-quantitative Polymerase Chain Reaction was used to determine miR-16 expression. Regulatory effects of miR-16 on proliferation, migration and invasion, and cell cycle of A549 cells were determined by Cell-Counting Kit 8 assay, transwell assay, and flow cytometry, respectively. Western blotting was performed to measure the protein expression of matrix metalloproteinase (MMP)-19 in cells overexpressing miR-16. Dual-luciferase reporter gene assay was conducted to identify the interaction between miR-16 and MMP-19.MiR-16 expression in NSCLC significantly decreased compared with that in healthy tissue (p<0.05). The expression level of miR-16 was negatively correlated to the clinical staging of NSCLC. In addition, the expression of miR-16 in NSCLC patients with lymph node metastasis was significantly lower than that in patients without lymph node metastasis (p<0.05). In vitro studies demonstrated that miR-16 inhibited the proliferation, migration, and invasion of A549 cells. Western blotting analyses indicated that overexpression of miR-16 down-regulated the expression of MMP-19. Additionally, the dual-luciferase reporter gene assay determined that miR-16 directly regulated the expression of MMP-16.The present study demonstrates that miR-16 acts as a tumor-suppressor gene by inhibiting the proliferation, migration, and invasion of NSCLC cells via downregulating MMP-19 expression.
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Objective
To investigate the effect of miR-491-5p on proliferation, migration and invasion of esophageal squamous cell carcinoma (ESCC) cells and its mechanism.
Methods
The immortalized esophageal epithelial cell line HET-1A and human esophageal cancer cell lines EC109, EC9706, KYSE510 were cultured, and the levels of miR-491-5p and SHOC2 mRNA were detected by qRT- PCR. EC109 cells were divided into control group, miR-491-5p group, miR-NC group, miR-491-5p+pcDNA-SHOC2 group and miR-491-5p+pcDNA group. MTT was used to detect cell proliferation, Transwell was used to detect cell migration and invasion, and Western Blot was used to detect the expression levels of CyclinD1, Vimentin, E-cadherin and MAPK/ERK signaling pathway-related protein. The dual luciferase reporter gene assay was used to verify the regulatory relationship between miR-491-5p and SHOC2. The comparison between the two groups was performed using independent sample t tests. One-way analysis of variance was used for comparison between multiple groups, and SNK-q test was used for further comparisons.
Results
The expression levels of miR- 491-5p in esophageal squamous cells EC109, EC9706 and KYSE510 were lower than those in HET-1A cells (0.32±0.06, 0.62±0.10, 0.61±0.08 vs 1.00±0.08, F = 106.340, P < 0.001) ; The mRNA expression level of SHOC2 was higher than that of HET-1A cells (2.85±0.16, 1.73±0.10, 1.45±0.06 vs 1.02±0.09, F = 464.949, P < 0.001) . The OD value, cell migration number, invasion number, and CyclinD1, Vimentin, p-MEK, and p-ERK protein levels in miR-491- 5p group of EC109 cells after 72 h of culture were lower than those in miR-NC group (0.70±0.06 vs 1.42±0.08, 65.01±10.36 vs 150.01±12.48, 70.03±10.26 vs 140.02±11.85, 0.30±0.03 vs 0.93±0.16, 0.41±0.05 vs 0.86±0.08, 0.32±0.06 vs 0.95±0.11, 0.40±0.06 vs 0.92±0.13, F = 236.565, 159.440, 120.706, 101.071, 98.619, 130.766, 77.046, P < 0.001) , E-cadherin protein levels were higher than miR-NC group (0.89±0.13 vs 0.48±0.08, F = 816.432, P < 0.001) . miR-491-5p negatively regulated SHOC2 expression in EC109 cells. SHOC2 overexpression reversed the effect of miR-491-5p overexpression on proliferation, migration and invasion of EC109 cells and MAPK/ERK signaling pathway.
Conclusion
miR-491-5p can inhibit the proliferation, migration and invasion of ESCC cells, and its mechanism may be related to the down-regulation of SHOC2 expression and inhibition of MAPK/ERK signaling pathway activity.
Key words:
Esophageal squamous cell carcinoma; miR-491-5p; SHOC2; Cell proliferation; Cell migration; Cell invasion
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Previous studies have shown that miR-100-5p expression is abnormal in prostate cancer. However, the role and regulatory mechanism of miR-100-5p requires further investigation. Thus, the aim of this study was to observe the effects of miR-100-5p on the proliferation, migration and invasion of prostate cancer (PCa) cells and to explore the potential related regulatory mechanism.Differential miRNA expression analysis was performed using next-generation sequencing (NGS) in the patients with PCa and benign prostatic hyperplasia (BPH). The expression levels of miR-100-5p were detected using real-time fluorescence quantitative PCR (qRT-PCR). PCa cells were transfected with NC-mimics or miR-100-5p mimics, inhibitor by using liposome transfection. Moreover, the CCK-8 proliferation assay, colony formation assay, cell scratch assay and Transwell assay were used to detect the effects of miR-100-5p on cell proliferation, migration, and invasion. In addition, the target gene of miR-100-5p was verified by luciferase reporter gene assay, and the influence of miR-100-5p on the expression of mTOR mRNA by qRT-PCR and the expression of mammalian target of rapamycin (mTOR) protein was detected by western blot and immunohistochemical staining.Differential expression analysis of high-throughput sequencing data showed low expression of miR-100-5p in the patients of PCa. It was further confirmed by qRT-PCR that the expression of miR-100-5p in PCa cells was significantly lower than that in RWPE-1 cells (P<0.01). miR-100-5p expression in lymph node carcinoma of prostate(LNCaP) cells was markedly upregulated after transfection with miR-100-5p mimics (P<0.01), while cell proliferation, migration and invasion capacities were clearly reduced (P<0.01). mTOR mRNA and protein expression was also substantially lowered (P<0.01) and mTOR adjusted the expression of NOX4. Finally, we further confirmed by immunohistochemical staining that miR-100-5p regulated the expression of mTOR and NOX4.miR-100-5p is expressed at low levels in PCa cells, and it can suppress PCa cell proliferation, migration and invasion, the mechanism of which is related to downregulating the expression of mTOR.
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