Figure 4. MiR-338-5p and miR-421 overexpression attenuates EMT and Stemness.

(A) Heatmap depicting change in the expression of EMT and pluripotency markers in 22RV1-miR-338-5p and 22RV1-miR-421 cells. Shades of blue represents log₂ fold-change in gene expression (n=3 biologically independent samples).

(B) QPCR analysis depicts expression of EMT markers in 22RV1-miR-338-5p, 22RV1-miR-421 and control cells. Expression for each gene was normalized to GAPDH.

(C) Immunostaining showing SLUG and SNAIL expression in the same cells as in (B).

(D) Same cells as in (B), except immunostained for E-cadherin and Vimentin.

(E) Same cells as in (B), except qPCR analysis for stem cell markers.

(F) Same cells as in (B), except immunostained for c-Kit and SOX-9.

(G) Hoechst 33342 staining for side population (SP) analysis using same cells as in (B). Percentages of SP were analyzed using the blue and far red filters, gated regions as indicated (red) in each panel.

(H) Phase contrast microscope images for the prostatospheres using same cells as in (B). Scale bar 100μm.

(I) Bar plot depicts percent sphere formation efficiency and mean area of the prostatosphere.

(J) Expression of TET1 by qPCR and Western blot using same cells as in (B).

(K) Schematic describing the role of miR-338-5p and miR-421 in regulating EMT, cancer stemness and drug resistance in SPINK1+ cancer.

For panels (C), (D) and (F), scale bar represents 20µm. In the panels (B), (E), (I) and (J) biologically independent samples were used (n=3); data represents mean ± SEM *P≤ 0.05 and **P≤ 0.005 using two-tailed unpaired Student's t test.