Article Figures & Data
Figures
- Figure 1.
A, transformation of HUC1 cells with long-term arsenic exposure: a, morphogic changes: morphology differences were monitored over the entire treatment schedule, using a light microscope (×20). Mock-treated cells and arsenic-treated cells were plated on 6-cm dishes at a density of 200,000 per plate. At 6 months, arsenic-treated HUC1 started to become more rounded and had a tendency to pile on to one another. b, MTT assay: the MTT assay was performed for each month of treatment to determine whether changes in cell proliferation occurred because of arsenic treatment. This figure depicts 2, 4, 6, 8, and 10 months of treated and mock-treated cells for simplicity reasons. Mean and SD were calculated using sample replicate values. c, soft agar assay: anchorage-independent growth was evaluated using soft agar assay. Cell were embedded in agar in triplicates and allowed to grow for 3 weeks. Cell colonies were counted using a light microscope in four different fields and averaged. Cell colonies were then stained with 0.05% ethidium bromide for 24 hours. UV transilluminator was used to determine the number of colonies per well greater than 50 μm in size. Left, colonies in arsenic-treated cells. No colony was observed in arsenic untreated cells (picture not shown). Pictures were taken at random using a digital camera attached to a high-resolution light microscope; right, bar graphs of colonies in different time periods. Statistical differences between 6, 8, and 10 months treated samples and untreated samples were calculated using the Student t test. (*, P < 0.05). d, invasion assay: left, invaded cells after arsenic treatment for 8 and 10 months. Right, graphical representation for the invasion assays performed at 8 and 10 months (UT, untreated; M, month). Invaded cells were counted using a light microscope at 10 different fields and a ×20 objective. Pictures were taken at random. The number of colonies was significantly increased in arsenic-treated samples (*, P < 0.05, Student t test). B, phenotypic observation after withdrawal of arsenic for 2.5 months from 8 months and 10 months arsenic-treated HUC1 cells. a, as determined by the MTT assay, no significant difference of cell viability was observed in 8 and 10 months arsenic-treated and 2.5M untreated cells. b, although no colony was observed in arsenic untreated HUC cells, 8 and 10 months arsenic-treated HUC1 cells retained the properties of anchorage-independent growth even after 2.5 months untreated period. c, similarly, 8 and 10 months arsenic-treated HUC1 cells maintained invasive properties even after culturing these cell for 2.5 months without arsenic. N.S., not significant; As, arsenic.
- Figure 2.
A, Western blot analysis of AKT pathway and EMT-related proteins. Bar graphs showed the results of densitomatric analysis (mean ± SD; *, P < 0.05, Student t test). Phosphorylated and total protein for EGFR, AKT, ERK, and PI3K were increased in arsenic-treated HUC1 cells. Cyclin D3 and mTOR were also increased in arsenic-treated HUC1 cells. The molecules that showed significant alterations due to arsenic treatment were bolded. As, arsenic. B, expression levels of miR-200 family in arsenic-exposed HUC1cell culture model. The HUC1 cell line was cultured in the presence of 1 μmol/L As2O3 for 6, 8, and 10 months. To study the reversibility of the arsenic exposal effect to the cells, we withdraw the drug for 2.5 months. Using the
method, the data are presented as the fold change in each miRNA expression normalized to mir-222 and relative to the untreated control (HUC1 cell line cultured in the presence of PBS for the same time course). Statistical difference between arsenic-treated and untreated cells were calculated using the Student t test. *, P < 0.05. C, Western blot analysis of two EMT-associated proteins: E-cadherin and Vimentin expression was analyzed using arsenic-treated and untreated HUC1 cells for 6 and 10 months. E-cadherin was significantly decreased in arsenic-treated cells of both 6 and 10 months (P < 0.001 for both 6 and 10 months, Student t test); no changes of expression was observed for Vimentin at 6 months arsenic-treated cells; however, Vimentin expression dramatically increased at 10 months arsenic-treated cells (P < 0.001, Student t test). - Figure 3.
Average 2−ΔCt of each miRNA expression (A: miR-200a; B: miR-200b; C: miR-200c; and D: miR-205) in urine sample was calculated and plotted after normalization with miR-222. Six groups of samples were listed. All normal urines (1: n = 177) were compared with cancer urines (6: n = 32). As subgroup analyses in normal urines, arsenic unexposed normal urines (2: n = 67) were compared with arsenic-exposed urines (3: n = 110), and nonsmoker normal urines (4: n = 46) were compared with smoker normal urines (5: n = 11). The Mann–Whitney U test was performed and a P value of <0.05 was considered as statistically significant. As for normal versus cancer, miR-200c and miR-205 expressions were significantly downregulated in cancer (P = 0.005 and P < 0.001, respectively). As for arsenic unexposed versus exposed, miR-200a and miR-205 were significantly downregulated in cancer (P = 0.002 and P < 0.001, respectively). As for nonsmoker versus smoker, miR-200c and miR-205 expression was significantly downregulated in cancer (P = 0.044 and P < 0.018, respectively). Notably, the expression of miR-205 was consistently downregulated in arsenic-exposed, smoker and cancer urines.
- Figure 4.
ROC curves for individual miRNA expression in 177 normal and 28 NMIBC urine samples were shown. The y-axis and x-axis denote sensitivity and 1 − specificity, respectively. AUC and the optimal cut off point with maximal sensitivity and specificity on each ROC curve were calculated. The curve of miR-205 showed the high AUC value (0.844) and promising sensitivity (92.9%) and specificity (76.8%) at the optimal cut off point (2−ΔCt=551).
Tables
- Table 1.
Characteristics of human urine samples tested for miRNA expression
A. Arsenic-exposed and unexposed normal urine samples Arsenic unexposed Characteristics Arsenic exposed (n = 110) Smokers (n = 11) Nonsmokers (n = 46) Total (n = 67a) Age, y Mean 36.8 59.4 68.2 66.5 Range 20—65 50—73 39–94 39–94 Gender Male 54 8 37 45 Female 56 3 9 12 Smoking history Never Not available N/A N/A 46 0–20 pack years Not available 5 N/A 5 >20–35 pack years Not available 1 N/A 1 >35 pack years Not available 5 N/A 5 Unknown Not available N/A N/A 10 Water arsenic, μg/L <10 28 N/A N/A N/A 10–50 22 N/A N/A N/A >50 21 N/A N/A N/A Unknown 39 N/A N/A N/A Creatinine-adjusted urine arsenic, μg/g <100 35 N/A N/A N/A 100–200 38 N/A N/A N/A >200 37 N/A N/A N/A B. Urothelial carcinoma and arsenic unexposed normal urine samples Arsenic unexposed Characteristics Cancer (n = 32) Smokers (n = 11) Nonsmokers (n = 46) Total (n = 67a) Age, y Mean 65.4 59.4 68.2 66.5 Range 36–84 50–73 39–94 39–94 Gender Male 26 8 37 45 Female 6 3 9 12 Stage CIS 7 N/A N/A N/A pTa 16 N/A N/A N/A T1 5 N/A N/A N/A ≥pT2 4 N/A N/A N/A Abbreviation: N/A, not applicable.
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↵aIncluded 10 unexposed samples with unknown smoking history.
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Supplementary Data
Files in this Data Supplement:
- Supp Table 1, Supp Table 2 - Supp Table 1, Supp Table 2. Supp Table 1: A.QMSP probe and primers, B.RT-PCR assay. Supplemental Table 2: The summary of notch methylation array findings
- Supp Figure 1 - Supp Figure 1. Technical validation of Notch Signaling Pathway DNA Methylation PCR Array.
- Supp Figure 2A - Supp Figure 2A. Promoter methylation is inversely associated with gene expression
- Supp Figure 2B - Supp Figure 2B. Methylated genes after arsenic treatment
- Supp Figure 3 - Supp Figure 3. Association of miRNA expression with water arsenic concentration and creatinine adjusted urine arsenic concentration (Mann-Whitney U test).
- Supp Figure 3 continued - Supp Figure 3 continued
- Supp Figure 4 - Supp Figure 4. ROC curves for individual micro RNA expression in 177 normal and 32 bladder cancer urine samples were shown.
- Supp Figure legends - Supp Figure legends
Volume 8, Issue 3
