MiR-30a-5p Inhibits Epithelial-to-Mesenchymal Transition and Upregulates Expression of Tight Junction Protein Claudin-5 in Human Upper Tract Urothelial Carcinoma Cells
"> Figure 1
<p>Expression profiles of miRNAs in human UTUC tumor and non-tumor samples. Three tumor (T1–T3; light blue to navy blue) and three adjacent normal tissues (N1–N3; orange-yellow-green) were subjected to RNA extraction and NGS-miRNA sequencing through the Illumina MiSeq platform. (<b>A</b>) Heat-map clustering analysis was conducted to examine the overall miRNA expression profiles among samples. A line scatterplot was used to present miRNA expression profiles; (<b>B</b>) Downregulation of miR-30a-5p in UTUC tumor tissues (<span class="html-italic">n</span> = 22) compared with adjacent normal controls (<span class="html-italic">n</span> = 14) by RT-qPCR analysis. The asterisk denotes <span class="html-italic">p</span> < 0.001 using an unpaired <span class="html-italic">t</span>-test.</p> "> Figure 2
<p>Comparative transcriptome analysis in human UTUC tumor and non-tumor samples. The total RNA isolated from human UTUC tumors (<span class="html-italic">n</span> = 9) and adjacent normal tissues (<span class="html-italic">n</span> = 3) was subjected to transcriptome analysis on Illumina HT12 microarray chips. The expression data was used for heat-map clustering analysis.</p> "> Figure 3
<p>KEGG pathway enrichment analysis on the tight junction pathway. The differentially-expressed genes in human UTUC tumor tissues compared to adjacent normal tissues were subjected to KEGG pathway enrichment analysis using Partek Genomics software. Note that a total of six genes (highlighted in green boxes) in the tight junction pathway, including claudin-5, were significantly downregulated in UTUC tumor tissues.</p> "> Figure 4
<p>The suppressive effect of miR-30a-5p on the proliferation and migration of UTUC cells. BFTC-909 cells were transfected with miR-30a-5p mimetic agent, followed by WST-1 and trans-well assays. NTC denotes non-transfection controls with scrambled mimetic treatment. (<b>A</b>) After mimetic transfection for 48 h, the WST-1 assay was used to evaluate cell numbers. The WST-1 value is directly proportional to cell number; (<b>B</b>) After mimetic transfection for 72 h, trans-well migration assay was performed to evaluate the cell migration ability; (<b>C</b>) The crystal violet-stained cells are those penetrating the trans-well membrane. All data are shown as means ± SEM from three independent experiments. * indicates that <span class="html-italic">p</span> < 0.001 between the indicated groups.</p> "> Figure 5
<p>miR-30a-5p overexpression inhibited epithelial-to-mesenchymal transition in UTUC cells. The BFTC-909 cells transfected with miR-30a-5p were subjected to immunofluorescent staining for cellular distributions of (<b>A</b>) E-cadherin (red), vimentin (green), and nuclei (blue); (<b>C</b>) Alternatively, α-SMA (red), fibronectin (green), and nuclei (blue) were immunofluorescently visualized. The fluorescence intensities of E-cadherin and vimentin (<b>B</b>), as well as EMT markers, α-SMA, and fibronectin (<b>D</b>), were quantified by counting 5–10 different fields per sample. NTC denotes non-transfection controls with scrambled mimetic treatment. Data are expressed as means ± SEM (<span class="html-italic">n</span> = 3). * indicates that <span class="html-italic">p</span> < 0.05 between the indicated groups. Original magnification: 200×.</p> "> Figure 5 Cont.
<p>miR-30a-5p overexpression inhibited epithelial-to-mesenchymal transition in UTUC cells. The BFTC-909 cells transfected with miR-30a-5p were subjected to immunofluorescent staining for cellular distributions of (<b>A</b>) E-cadherin (red), vimentin (green), and nuclei (blue); (<b>C</b>) Alternatively, α-SMA (red), fibronectin (green), and nuclei (blue) were immunofluorescently visualized. The fluorescence intensities of E-cadherin and vimentin (<b>B</b>), as well as EMT markers, α-SMA, and fibronectin (<b>D</b>), were quantified by counting 5–10 different fields per sample. NTC denotes non-transfection controls with scrambled mimetic treatment. Data are expressed as means ± SEM (<span class="html-italic">n</span> = 3). * indicates that <span class="html-italic">p</span> < 0.05 between the indicated groups. Original magnification: 200×.</p> "> Figure 6
<p>miR-30a-5p overexpression inhibits epithelial-to-mesenchymal transition marker expression in UTUC cells. Western blot analysis of expression levels of epithelial marker E-cadherin (<b>A</b>), and mesenchymal markers, including vimentin (<b>B</b>), fibronectin (<b>C</b>), and α-SMA (<b>D</b>) in cultured BFTC-909 cells transfected with miR-30a-5p or miR-NC. GAPDH was used as the loading control. Data are expressed as means ± SEM (<span class="html-italic">n</span> = 3). * indicates that <span class="html-italic">p</span> < 0.05 between the indicated groups.</p> "> Figure 7
<p>Claudin-5 expression is enhanced by the miR-30a-5p. The qPCR (<b>A</b>) and Western blot (<b>B</b>) analysis of <span class="html-italic">CLDN-5</span> expression levels in cultured BFTC-909 cells transfected with miR-30a-5p or miR-NC. GAPDH was used as the loading control. Data are expressed as means ± SEM (<span class="html-italic">n</span> = 3). * indicates that <span class="html-italic">p</span> < 0.05 between the indicated groups.</p> "> Figure 8
<p>Proposed effects of miR-30a-5p on UTUC. The miR-30a-5p-enhanced <span class="html-italic">CLDN-5</span> expression may trigger tight junction pathway signal transduction. Moreover, miR-30a-5p suppresses the EMT process via increasing E-cadherin and suppressing vimentin, fibronectin, and α-SMA expression, thereby inhibiting the migration activity of UTUC cells.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Profiling the miRNA Expression Signature of UTUC by NGS and qPCR
2.2. Transcriptome Profiles in UTUC Tissues on Illumina HT12 Microarray Chips
2.3. Pathway Analysis on the Involvement of miRNAs in the Tumorigenesis of UTUC
2.4. miR-30a-5p Overexpression Reduced Proliferation and Migration of BFTC-909 Cells
2.5. miR-30a-5p Overexpression Inhibits Epithelial-to-Mesenchymal Transition (EMT) in BFTC-909 Cells
2.6. miR-30a-5p Overexpression Increased TJ Protein Claudin-5 Expression in BFTC-909 Cells
3. Discussion
4. Materials and Methods
4.1. Clinical Specimens
4.2. RNA Isolation
4.3. Library Preparation and Sequencing
4.4. Pathway Enrichment Analysis
4.5. Reverse Transcription (RT) and Real-Time PCR
4.6. Cell Culture and miRNA Treatment
4.7. Cell Proliferation Assay
4.8. Trans-Well Cell Migration Assay
4.9. Western Blot Analysis
4.10. Immunofluorescent Staining
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Pathway Name | p-Value | Pathway ID |
---|---|---|
MAPK signaling pathway | 0.00274259 | kegg_pathway_261 |
PI3K-Akt signaling pathway | 0.00308062 | kegg_pathway_262 |
Tight junction | 0.00451891 | kegg_pathway_257 |
Protein digestion and absorption | 0.00458404 | kegg_pathway_279 |
Notch signaling pathway | 0.00534759 | kegg_pathway_55 |
Cytokine-cytokine receptor interaction | 0.00924043 | kegg_pathway_79 |
Endocytosis | 0.0151682 | kegg_pathway_232 |
Focal adhesion | 0.0158629 | kegg_pathway_188 |
Regulation of actin cytoskeleton | 0.0164765 | kegg_pathway_139 |
Morphine addiction | 0.0191389 | kegg_pathway_101 |
Calcium signaling pathway | 0.022008 | kegg_pathway_237 |
Amoebiasis | 0.0234208 | kegg_pathway_214 |
Amino sugar and nucleotide sugar metabolism | 0.0254128 | kegg_pathway_203 |
GABAergic synapse | 0.0258019 | kegg_pathway_235 |
Ras signaling pathway | 0.0265302 | kegg_pathway_265 |
Endocrine and other factor-regulated calcium reabsorption | 0.0266089 | kegg_pathway_174 |
HTLV-I infection | 0.0280212 | kegg_pathway_190 |
ECM-receptor interaction | 0.0314539 | kegg_pathway_242 |
Jak-STAT signaling pathway | 0.0400457 | kegg_pathway_40 |
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Chung, Y.-H.; Li, S.-C.; Kao, Y.-H.; Luo, H.-L.; Cheng, Y.-T.; Lin, P.-R.; Tai, M.-H.; Chiang, P.-H. MiR-30a-5p Inhibits Epithelial-to-Mesenchymal Transition and Upregulates Expression of Tight Junction Protein Claudin-5 in Human Upper Tract Urothelial Carcinoma Cells. Int. J. Mol. Sci. 2017, 18, 1826. https://doi.org/10.3390/ijms18081826
Chung Y-H, Li S-C, Kao Y-H, Luo H-L, Cheng Y-T, Lin P-R, Tai M-H, Chiang P-H. MiR-30a-5p Inhibits Epithelial-to-Mesenchymal Transition and Upregulates Expression of Tight Junction Protein Claudin-5 in Human Upper Tract Urothelial Carcinoma Cells. International Journal of Molecular Sciences. 2017; 18(8):1826. https://doi.org/10.3390/ijms18081826
Chicago/Turabian StyleChung, Yueh-Hua, Sung-Chou Li, Ying-Hsien Kao, Hao-Lun Luo, Yuan-Tso Cheng, Pey-Ru Lin, Ming-Hong Tai, and Po-Hui Chiang. 2017. "MiR-30a-5p Inhibits Epithelial-to-Mesenchymal Transition and Upregulates Expression of Tight Junction Protein Claudin-5 in Human Upper Tract Urothelial Carcinoma Cells" International Journal of Molecular Sciences 18, no. 8: 1826. https://doi.org/10.3390/ijms18081826