TUMOR BIOLOGY

Over-expression of miR-31 or loss of KCNMA1 leads to increased cisplatin-resistance in ovarian cancer cells.

AUTHORS AND AFFILIATION

Priya Samuel a†, Ryan Charles Pink a†, Daniel Paul Caley a,b, James Michael Stevenson Currie a, Susan Ann Brooks a, David Raul Francisco Carter a.

a. Oxford Brookes University. Faculty of Health and Life Sciences. Department of Biological and Medical Sciences. Gipsy Lane, Oxford. OX3 0BP. UK.

b. Current address: Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, BC, V5Z 1L3, Canada.

†These authors contributed equally to this work

Corresponding author: DRF Carter () 01865484216

Supplementary File contains tables with top twenty deregulated genes from RNA-Seq data in CP70 and MCP1 cells compared to A2780 cells and the most enriched KEGG pathways in the deregulated genes and Supplementary figure with cisplatin dose response curves of A2780, MCP1 and CP70. The second supplementary table contains additional evidence for a role of KCNMA1 in regulating resistance and miR-31 targeting of KCNMA1.

Table S1 RNA-seq data showing top twenty up- and down-regulated genes in CP70 and MCP1 compared to A2780 cells. All genes indicated were concordantly de-regulated in both cell lines and all were significantly altered (p<0.05). “Infinite” fold change implies that either A2780, MCP1 or CP70 cells had undetectable levels of RNA as measured by RNA-seq.

Gene / Approved full name / CP70 fold / MCP1 fold / Fold direction
CRABP1 / cellular retinoic acid binding protein 1 / Infinite / 745.6 / down
MLH1 / mutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli) / Infinite / Infinite / down
FABP4 / fatty acid binding protein 4, adipocyte / 6953.9 / 940.1 / down
POU3F2 / POU class 3 homeobox 2 / 4993.8 / Infinite / down
PDGFD / platelet derived growth factor D / 2669.6 / 1082.7 / down
SV2A / synaptic vesicle glycoprotein 2A / 2310.8 / 3.4 / down
NRK / Nik related kinase / 2151.3 / 685.1 / down
POU3F4 / POU class 3 homeobox 4 / 2133.4 / Infinite / down
KCNMA1 / potassium large conductance calcium-activated channel, subfamily M, alpha member 1 / 1697.2 / 3.9 / down
ANXA1 / annexin A1 / 1554.6 / 15.0 / up
FREM1 / FRAS1 related extracellular matrix 1 / 936.8 / 104.3 / down
PTH1R / parathyroid hormone 1 receptor / 862.1 / 38.8 / down
ARMCX2 / armadillo repeat containing, X-linked 2 / 810.8 / Infinite / down
OLFML1 / olfactomedin-like 1 / 795.0 / 12.2 / down
LMX1B / LIM homeobox transcription factor 1, beta / 782.7 / 722.1 / up
NEFL / neurofilament, light polypeptide / 692.8 / 1319.1 / down
CYB5R2 / cytochrome b5 reductase 2 / 667.7 / 4.7 / down
STXBP5L / syntaxin binding protein 5-like / 609.4 / 185.4 / down
FAM50B / family with sequence similarity 50, member B / 603.6 / 247.4 / down
MUSK / muscle, skeletal, receptor tyrosine kinase / 575.4 / 153.9 / up
EBF2 / early B-cell factor 2 / 571.5 / Infinite / down
CARD16 / caspase recruitment domain family, member 16 / 465.2 / 253.5 / down
ZNF718 / zinc finger protein 718 / 428.5 / 8.4 / down
PRSS12 / protease, serine, 12 (neurotrypsin, motopsin) / 185.0 / 246.1 / up
MTMR3 / myotubularin related protein 3 / 151.7 / 114.9 / up
MYO6 / myosin VI / 137.4 / 23.9 / up
AP1M2 / adaptor-related protein complex 1, mu 2 subunit / 101.5 / 108.9 / up
SP100 / SP100 nuclear antigen / 90.2 / 11.0 / up
LPAR1 / lysophosphatidic acid receptor 1 / 86.6 / 37.5 / up
FOXE1 / forkhead box E1 (thyroid transcription factor 2) / 77.9 / 13.9 / up
ALX4 / ALX homeobox 4 / 54.3 / 125.6 / up
MARCKS / myristoylated alanine-rich protein kinase C substrate / 54.0 / 48.2 / up
SOX8 / SRY (sex determining region Y)-box 8 / 52.6 / 88.3 / up
MYEOV / myeloma overexpressed (in a subset of t(11;14) positive multiple myelomas) / 49.1 / 25.2 / up
SCNN1A / sodium channel, nonvoltage-gated 1 alpha / 45.5 / 22.2 / up
SLC27A6 / solute carrier family 27 (fatty acid transporter), member 6 / 45.1 / 72.1 / up
CEACAM1 / carcinoembryonic antigen-related cell adhesion molecule 1 (biliary glycoprotein) / 39.8 / 25.6 / up
ADRA2C / adrenergic, alpha-2C-, receptor / 37.1 / 63.0 / up
LAMA4 / laminin, alpha 4 / 33.8 / 217.1 / up

Table S2 Genes that were concordantly de-regulated in MCP1 and CP70 cells were analysed using the DAVID functional annotation tool. The most enriched KEGG pathways are shown with P Values.

KEGG Pathway / P Value
Focal adhesion / 0.005424
VEGF signaling pathway / 0.006648
Fc epsilon RI signaling pathway / 0.008029
Vascular smooth muscle contraction / 0.012941
Gap junction / 0.014878
Fc gamma R-mediated phagocytosis / 0.019963
Phosphatidylinositol signaling system / 0.025336
Calcium signaling pathway / 0.044906

Fig S1 Resistance to cisplatin was assessed in the three ovarian cell lines used in the study; A2780, MCP1 and CP70. Cells were seeded in 96 wells plate and treated after 48 hours. The indicated dose of cisplatin was added for 3 hours; cells were then grown for a further 48 hours in cisplatin-free media. MTT was performed and resistance was measured by normalising absorbance of each sample to the corresponding untreated cells. At least five biological replicates were used for each data point; error bars show standard error of the mean.

Fig S2 The effects of miR-31 and KCNMA1 on cisplatin-resistance in ovarian cancer cells. (A) miR-31 (50 μM) was transfected into OVCAR5 cells and the effect on cisplatin measured using the MTT assay. Error bars show SEM of 9 biological replicates; p = 0,002. (B) A2780 cells were transfected with control or KCNMA1 shRNAs and the effect on resistance measured. shRNAs 1, 2 and 3 induced an increase in resistance. Error bars represent SEM of 5 biological replicates. Stars indicate significance compared to control; p = 0.09, 0.002 and 0.001 respectively. (C) A pool of shRNAs against KCNMA1 was transfected into OVCAR5 cells, leading to a significant (p = 0.0006) increase in resistance compared to control. Error bars show SEM of 9 biological replicates. (D) OVCAR5 cells were treated with paxilline, leading to a significant (p = 0.03) increase in resistance. Error bars show SEM of 9 biological replicates. (E) Binding site for miR-31 on the KCNMA1 3’UTR using PITA (http://132.77.150.113/pubs/mir07/mir07_prediction.html).