Figure S1-S21 from miR-196b-5p Regulates Colorectal Cancer Cell Migration and Metastases through Interaction with HOXB7 and GALNT5
Authors: Stiegelbauer Verena , Vychytilova-Faltejskova Petra , Karbiener Michael , Pehserl Anna-Maria , Reicher Andreas , Resel Margit , Heitzer Ellen , Ivan Cristina , Bullock Marc , Ling Hui , Deutsch Alexander , Wulf-Goldenberg Annika , Adiprasito Jan Basri , Stoeger Herbert , Haybaeck Johannes , Svoboda Marek , Stotz Michael , Hoefler Gerald , Slaby Ondrej , Calin George Adrian , Gerger Armin , Pichler Martin 
Published: October 2024

<p>Supplementary Figure S1 Expression levels of miR-196b-5p in different tumor stages and tumor grade. (A-B) In cohort 1, miR-196b-5p expression decreased with (A) higher tumor stage, whereas no difference could be observed for (B) tumor grade. (C-D) In cohort 2, (C) stage IV tumors and (D) high grade tumors showed the lowest levels of miR-196b-5p. Supplementary Figure S2 Kaplan-Meier curve for 5-year overall survival in tumor stage II/III patients in (A) cohort 1 (n=51) and (B) cohort 2 (n=122). In both cohorts, low miR-196b-5p expression levels were associated with poor clinical outcome (p <0.05, log-rank test). (C) Kaplan-Meier curve for overall survival in stage IV metastatic disease in cohort 1 (n=59, p<0.05, log-rank test). Supplementary Figure S3 (A-B) Relative quantitative RT-PCR and absolute digital PCR quantification of miR-196b-5p in eight colorectal cancer cell lines. Different colors indicate each cell line, black vertical line separates the groups intoa low and high miR-196b-5p category. A degree of correlation (R=0.881, p=0.004) was found for both methods. (C-D) Relative and absolute quantification of miR-196b-5p in tumor tissue of ten selected colorectal cancer patients. Different colors indicate each patient (numbered with #1-10), black vertical line separates the groups into low and high miR-196b-5p category. Again, a high degree of correlation (R=0.794, p=0.006) was found for both methods. Supplementary Figure S4 (A-B) Comparison of miR-196b-5p mean expression levels in cell lines and patient samples by relative and absolute quantification methods. The cell lines and patient samples were categorized into low and high according to the threshold in previous Supplementary Figure. The cells belonging to the high category showed a mean 4 fold higher expression level of miR-196b-5p than the group of low expressing cell lines, independently which quantification method were used. Interestingly, the tissue levels of miR196b-5p were higher in the group of patients with high levels (ranging from 16 fold to 1000 fold, depending on the method, in comparison to the cells with low expression). In contrast, the group of patients belonging to the low category showed significantly less levels (ranging from 0.9 to 16 fold, depending on the method used) in comparison with cell lines of the with low expression category. (C-D) Quantitative RT-PCR confirmed a statistically significant overexpression (between 80 to 100 fold) and silencing (between 22 to 50%) of miR-196b-5p after 48 hours of transient transfection in all three CRC cell lines. Transfection of inhibitor led to a 55% reduction of miR-196b expression compared to control in HCT116 cells. In RKO and HRT18 transfection of miR-196b-5p inhibitor resulted in 23% reduction of miR-196b-5p expression compared to the control. (E-F) Successful transfection was validated by detecting well-known target genes GATA6 und HOXA9 mRNA expression levels 48 hours after miR-196b-5p mimic exposure in HCT116 and HRT18 cell lines (* = significant, p < 0.05; **, p < 0.01; ***, p < 0.001 Supplementary Figure S5 (A-C) The WST-1 cellular growth assay showed no significant differences in HCT116 and HRT18 cell lines transfected with control, miR-196b-5p mimic or inhibitor. In contrast, RKO cells showed a slight but significant (p=0.0028) growth inhibition after 96 hours in miR196b-5p overexpressing cells. (p-value <0.05 considered as significant). Supplementary Figure S6: (A-C) No significant effects of miR-196b-5p expression levels on drug sensitivity against commonly used colorectal cancer drugs were observed in HCT116 cells after transfection with miR-196b-5p mimic, inhibitor or the respective control as measured by WST-1 assay. Supplementary Figure S7: (A-C) No significant effects of miR-196b-5p expression levels on drug sensitivity against commonly used colorectal cancer drugs were observed in HRT18 cells after transfection with miR-196b-5p mimic, inhibitor or the respective control as measured by WST-1 assay. Supplementary Figure S8: (A-C) No significant effects of miR-196b-5p expression levels on drug sensitivity against commonly used colorectal cancer drugs were observed in RKO cells after transfection with miR-196b-5p mimic, inhibitor or the respective control as measured by WST-1 assay. Supplementary Figure S9 (A-C): Decreased expression of miR-196b-5p led to a higher migration rate in (A) HCT116 cells and (B) HRT18 cells as measured by the xCELLigence Real Time system (p<0.05, n=3). (C).Forced miR196b-5p expression resulted in decreased migration rates in RKO cells Supplementary Figure S10 (A-B) Scratch assays in SW48 (with low endogenous miR196b-5p expression) and DLD1 cells after transient miR-196b-5p overexpression or inhibition. Representative examples of scratch assays in SW48 and DLD1 after transfection of respective control, miR-196b-5p mimic (for SW480) or inhibitor (for DLD1). 0h = scratch at beginning, 16h = scratch after 16 hours, 36h = scratch after 36 hours. Bar charts graphs demonstrating the results of measurement of scratch closure after 36h and 16 hours for the miR-196-5p overexpression in SW48 cells or miR-196-5p inhibition in DLD1 cells. The miR-196b-5p inhibited DLD1 cells closed the scratch significantly earlier than the inhibitor control cells, whereas the miR-196b-5p overexpression in SW480 resulted in decreased scratch closure (* = significant, p < 0.05; **, p < 0.01; ***, p < 0.001; studentÂ's t-test). Supplementary Figure S11 (A) Decreased miR-196b-5p expression resulted in increased invasion in HCT116 cells as measured by transwell invasion assay. (B) Bar charts graph showing the results of the transwell invasion assay in HCT116 cells (p value <0.05 considered as significant). (C) Low expression of miR-196b-5p led to a higher rate of invasion in HCT116 colorectal cancer cells as measured by the xCELLigence Real Time Invasion Assay (n=3). (* = significant, p < 0.05; **, p < 0.01; ***, p < 0.001). Supplementary Figure S12 (A) quantitative RT-PCR confirmed a statistically significant overexpression (OE) of miR-196-5p in the lentiviral transfected CRC cells (p value <0.05 considered as significant). (B) Silencing (Inhibitor) of miR-196b-5p silencing was measured by qRT PCR. Supplementary Figure S13: (A) Bar chart of photon intensities in mice injected with control HCT116 cells and miR-196b-5p silenced cells (p=0.09). (B) Macroscopic assessment of all livers from seven controls and eight miR196b-5p silenced cells. White arrow heads indicate visible metastases (metastases were found in 7 out of 8 mice of the miR-196b-5p silenced cells versus 2 out of 7 control cells, p<0.05). (C) Hematoxylin-eosin staining of representative slides of livers from mice xenografts that had intra-spleen injection of stable HCT116 miR-196b-5p inhibitor cells (index mice I1-I8) or control cells (index mice C1-C7). Stars (*) indicate metastases in some areas of mice (metastases were found in 7 out of 8 mice of the miR-196b-5p silenced cells versus 2 out of 7 control cells, p<0.05). Note that for Inhibitor mouse 8 (I8) the whole liver was filled up with metastases as indicated by the stars, which corresponded to the macroscopic picture (D) Number of detectable metastases on hematoxylin -eosin stained slides for three different liver sections explored. Supplementary Figure S14: Relative mRNA quantification of markers for epithelial-mesenchymal transition was performed after stable miR196b-5p overexpression (A) or silencing (B) of three different CRC cells lines. E-Cadherin and N-Cadherin could not be detected in RKO cells. HCT116 cells did not show an N-Cadherin expression. There was no clear trend or opposite effects with regard to differential expressed EMT markers (* = significant, p < 0.05; **, p < 0.01; ***, p < 0.001). Supplementary Figure S15: (A) Confirmation of Array data in HCT116 cells. Downregulation of genes related to cell migration after miR-196b-5p overexpression (OE) in HCT116 were selected and validated by qRT-PCR. A downregulation of all seven selected genes could be observed in stably transduced HCT116 cells. (B) Validation of the candidate genes in HRT18 cell line. In this cell line we could only identify a downregulation of HOXB7 and GALNT5. CPA4 and DKK1 genes could not be detected in HRT18 cells. (C-E) qRT-PCR and Western blot confirmed a statistically significant upregulation of HOXB7 and GALNT5 mRNA expression after transient miR196b-5p inhibition in HCT116 and HRT18 cells. (F-G) In stable HCT116 miR-196b-5p silenced cells a statistically significant upregulation of HOXB7 and GALNT5 expression was detected by qRT PCR and western blot analysis. (* = significant, p < 0.05; **, p < 0.01; ***, p < 0.001; studentÂ's t-test) Supplementary Figure S16: (A) Human specific HOXB7 mRNA expression in liver tissue of mice xenografts injected intra-splenic with stable HCT116 miR-196b-5p inhibitor cells or control cells measured by qRT PCR. (B) Human specific GALNT5 mRNA expression in liver tissue of mice xenografts injected intra-splenic with stable HCT116 miR-196b-5p inhibitor cells or control cells measured by qRT PCR. (C-D) Luciferase activity after co-transfection of the HOXB7 and GALNT5 wild-type (WT) or mutated (MT) constructs and miR-196b-5p inhibitor/control in HCT116 stable miR196b-5p overexpression cells. Three independent biological experiments were performed and the means and standard deviations are shown (* = significant, p < 0.05; **, p < 0.01; ***, p < 0.001). Supplementary Figure S17 Spearman correlation analysis showed a negative and significant correlation between miR-196b-5p and its target genes HOXB7 and GALNT5. Supplementary Figure S18 Kaplan Meier curve analysis of 439 colorectal cancer patients showed a significant poor prognosis for patients with high HOXB7 expression levels (p<0.05, log-rank test). Data was generated by using the online tool http://www.oncolnc.org/ Supplementary Figure S19 Kaplan Meier curve analysis of 62 colorectal cancer patients showed a significant poor prognosis for patients with high HOXB7 expression levels (p<0.05, log-rank test). Data derived from GSE12945 (Staub et al. J Mol Med (Berl) 2009 Jun;87(6):633-44). Supplementary Figure S20 Kaplan Meier curve analysis of 290 colorectal cancer patients showed a significant poor prognosis for patients with high GALNT5 expression levels (p<0.05, log-rank test). R2: Genomics analysis and visualization platform (https://hgserver1.amc.nl/cgi-bin/r2/main.cgi?&species=hs). Supplementary Figure S21 (A,B) GALNT5 and HOXB7 mRNA expression after 48 hours of siRNA transfection measured by qRT-PCR in HCT116 und HRT18 cells. A statistically significant knockdown of HOXB7 and GALNT5 compared to control was confirmed in both cell lines. (C,D) Expression levels of well-known HOXB7 effector downstream genes CCND1 and CDKN1B were measured after 72h hours of transient miR-196b-5p mimic transfection in (C) HCT116 and (D) HRT18 cells (*= significant, *p < 0.05; **, p < 0.01; ***, p < 0.001).</p>