Supplementary Figures 1-11 from Genome-Wide miRNA Analysis Identifies miR-188-3p as a Novel Prognostic Marker and Molecular Factor Involved in Colorectal Carcinogenesis

Abstract

<p>Supplementary Figure S1 In situ hybridization of U6 in normal mucosae (n = 3, upper panel) and corresponding tumor tissue (n = 3, lower panel). Supplementary Figure S2 (A) MiR-188-3p is significantly up-regulated in matched cancer tissue compared to the adjacent non-cancerous mucosa (p<0.001, paired studentÂ's t-test). (B) MiR-188-3p is significantly expressed at higher levels in tumors of higher tumor stage (p<0.001, ANOVA). (C) MiR-188-3p is significantly expressed at higher levels in tumors of microsatellite stable background. Supplementary Figure S3. Kaplan-Meier curve for overall survival in the validation set of 332 colorectal cancer patients stratified according to the quartile of expression levels (p = 0.001, log-rank test). Supplementary Figure S4 (A) MiR-188-3p is expressed in all tested colorectal cancer cell lines at varying levels. (B-D) The WST-1 cellular growth assay shows no significant differences or trend in three different colorectal cancer cell lines transfected by control, mir-188-3p mimetic or inhibitor. Supplementary Figure S5 (A-C) No effects of miR-188-3p expression levels on drug sensitivity against commonly used colorectal cancer drugs in HCT116 cells could be observed. Supplementary Figure S6 (A-C) No effects of miR-188-3p expression levels on drug sensitivity against commonly used colorectal cancer drugs in HRT18 cells could be observed. Supplementary Figure S7 (A-C) No effects of miR-188-3p expression levels on drug sensitivity against commonly used colorectal cancer drugs in RKO cells could be observed. Supplementary Figure S8 (A) Forced expression of miR-188-3p led to a higher migration rate in HCT116 cells or (B) even significantly earlier and higher migration rate in RKO colorectal cancer cells (p<0.05, n=3). Supplementary Figure S9 (A) Forced expression of miR-188-3p led to early and higher rate of migration in HRT18 colorectal cancer cells (p<0.05, n = 3). Supplementary Figure S10 (A-B) Transfection with a miR-188-3p inhibitor led to decreased cell migration in HCT116 cell line (C-D), in HRT18 cell line (E-F) and in the RKO cell line (p<0.05, unpaired studentÂ's t-test, n=4) Supplementary Figure S11 (A) Treatment with miR-188-3p inhibitor led to about 30% increase in MLLT4 expression after 48 hours in two independent cell lines (*p<0.05, unpaired studentÂ's t-test). (B-C) MLLT4 mRNA expression after 48 hours of MLLT4-directed siRNA transfection as measured by RT-qPCR in HCT116 and HRT18 cell line. Data are presented as mean {plus minus} SD of three independent transfection experiments in HCT116 and HRT18 cells. (D) MLLT4 protein expression after 48 hours of siRNA transfection as quantified by Western blot analysis in both cell lines.</p>