Figure 1.
Quantitative analysis of miR-200c expression in human pancreatic cancer cell lines and ectopic expression of miR-200c in S2.028 and T3M-4 cells.
(A) The expression of miR-200c in seven pancreatic cancer cells were determined by Real-time PCR. Each sample was run in quadruplicate and error bars represent SD. S2.028 (B) and T3M-4 cells (C) stably expressing the primary transcript of miR-200c were evaluated for miR-200c expression by Real-time PCR. Each measurement was carried out in triplicate. These values were normalized with internal control U6 rRNA. The fold increase in transcript levels over vector control is expressed as Mean ± S.D. The p value was determined by using the Student’s t-test. Differences with a p value < 0.05 were considered statistically significant.
Figure 2.
Lysates from (A) S2.028 and (B) T3M-4-miR-200c and respective vector control transfected cells were separated on SDS-Agarose gel electrophoresis, subjected to western blot using an anti-MUC4 antibody (left panels) and signals were quantified by densitometry and analyzed using the ImageJ program (right panels). The miR-200c expressing cells showed a significant reduction of MUC4 protein compared to control cells in (A) S2.028 and (B) T3M-4 cells. Detection of alpha tubulin was used as a loading control.
Figure 3.
(A) S2.028 and (B) T3M-4-miR200c and respective vector control transfected cell lysates were separated by SDS-Agarose gel electrophoresis and subjected to western blot using an anti-MUC16 antibody (left panels). Band intensity was quantified by densitometry and analyzed using the imageJ program (right panels). Significant reductions of both high and low molecular weight MUC16 protein isoforms were observed in miR-200c expressing S2.028 (A) and T3M-4 cells (B) than compared to vector control cells. α-tubulin was used as a loading control.
Figure 4.
miR-200c targets transcript levels of MUC4 and MUC16 in S2.028 and T3M-4 cells.
qRT-PCR analysis of MUC4 (A and B) and MUC16 (C and D) were carried out in vector control and miR-200c transfected S2.028 (A and C) and T3M-4 cells (B and D). The relative expression of MUC4 and MUC16 was evaluated by the 2-ΔΔCt method using GAPDH as an internal control. The miR-200c expressing cells (S2.028 and T3M-4) showed significantly reduced levels of MUC4 mRNAs compared to vector control cells (A and B). Expression of MUC16 transcript was reduced in miR-200c expressing S2.028 and T3M-4 cells (C and D). All measurements were carried out in triplicate. The fold increase in transcript levels over vector control were expressed as Mean ± S.D. A p-value less than 0.05 was considered to be statistically significant.
Figure 5.
Prediction of miR-200c interaction sites in MUC4 and MUC16 genes.
Possible miR-200c targeting regions in MUC4 and MUC16 were identified by using the RegRNA MicroRNA target prediction web server (http://regrna.mbc.nctu.edu.tw/index1.php). A, RegRNA miRNA target prediction shows that miR-200c binds between base pairs 820-842 in the first exon of MUC4. B, In MUC16 mRNA, the miR-200c is predicted to bind nine different exons including E1, E3, E19, E39, E44, E49, E54, E64 and E73. The numbers indicate the region of mRNAs that interact with miR-200c.
Figure 6.
Luciferase assay showing that miR-200c regulates MUC4 and MUC16 expression in S2.028 and T3M-4 cells.
(A) Wild type and mutated sequences of MUC4 and MUC16 were cloned into the pMIR-Luciferase vector (pMIR-MUC4 wt and MUC16 wt) and (pMIR-MUC4 mt and MUC16 mt) respectively. Vectors expressing pMIR-Luc, pMIR-MUC4 wt, pMIR-MUC16 wt, pMIR-MUC4 mt and pMIR-MUC16 mt were transfected into S2.028miR-200c (B and D) and T3M-4miR-200c cells (C and E) and luciferase activity was quantified 48 h after transfection. The results were expressed as relative luciferase activity (Mean ± SEM of three independent determinations). The p value was determined using Student’s t-test (