The effects of MicroRNA transfections on global patterns of gene expression in ovarian cancer cells are functionally coordinated

Background MicroRNAs (miRNAs) are a class of small RNAs that have been linked to a number of diseases including cancer. The potential application of miRNAs in the diagnostics and therapeutics of ovarian and other cancers is an area of intense interest. A current challenge is the inability to accurately predict the functional consequences of exogenous modulations in the levels of potentially therapeutic miRNAs. Methods In an initial effort to systematically address this issue, we conducted miRNA transfection experiments using two miRNAs (miR-7, miR-128). We monitored the consequent changes in global patterns of gene expression by microarray and quantitative (real-time) polymerase chain reaction. Network analysis of the expression data was used to predict the consequence of each transfection on cellular function and these predictions were experimentally tested. Results While ~20% of the changes in expression patterns of hundreds to thousands of genes could be attributed to direct miRNA-mRNA interactions, the majority of the changes are indirect, involving the downstream consequences of miRNA-mediated changes in regulatory gene expression. The changes in gene expression induced by individual miRNAs are functionally coordinated but distinct between the two miRNAs. MiR-7 transfection into ovarian cancer cells induces changes in cell adhesion and other developmental networks previously associated with epithelial-mesenchymal transitions (EMT) and other processes linked with metastasis. In contrast, miR-128 transfection induces changes in cell cycle control and other processes commonly linked with cellular replication. Conclusions The functionally coordinated patterns of gene expression displayed by different families of miRNAs have the potential to provide clinicians with a strategy to treat cancers from a systems rather than a single gene perspective.

Successful transfection of miR-128 was confirmed by assaying the relative down-regulation of its validated target BMI1. Here we confirmed successful transfection of the miRNA into HEY cells by measuring BMI1 RNA level using qPCR following transfection of either miR-NC or miR-128 into HEY cells. Transfection of miR-128 down-regulated BMI1 by ~60 % relative to miR-NC. *** p <0.005.

Supplementary Table 1. Differentially expressed genes in miR-7 transfected HEY cells.
Genes differentially expressed (fold change ≥1.4, FDR ≤5%) in HEY ovarian cancer cells transfected with miR-7 compared to HEY cells transfected with negative control miRNA (miR-NC). 'Probeset ID' refers to Affymetrix HG-U133 Plus 2.0 probeset identifier. 'Gene Symbol' shows the official gene symbol for the corresponding Probeset ID. 'miR7-miR-NC' refers to the difference between average log 2 signal values (i.e. the log 2 of the ratio of signal values) of the miR-7 transfected group and the miR-NC transfected group. 'q-value (%)' shows the false discovery rate calculated using the SAM algorithm. miRanda predicted targets of miR-7 are listed in the column titled 'miR7tgts_M', TargetScan predicted targets of miR-7 are listed under the heading miR-7tgts_TS, and predicted PicTar targets of miR-7 are listed under the heading miR-7tgts_PT. Genomica enrichment (FDR <0.05) of miRNA targets among significantly up-regulated genes following miR-128 transfection into HEY cells. Regulating miRNAs are predicted using miRanda (those with the prefix John04 or SloanKettering), TargetScan or PicTar.

Probeset ID
Only "up" genes, i.e. up-regulated in the transfection, are analyzed ('Set'). The other columns show the p-value of enrichment, the number of probesets in our dataset that are up-regulated and present in Genomica database ('Set Size'), compared to the number of probesets in each set in the background Affymetrix chip ('Total Size'). If a probeset is a predicted target of a miRNA then it is considered a 'hit' ('Set Hits' and 'Total Hits'). The percentages calculated from raw numbers of genes that belong in each set are also shown. Supplementary Table 6. miRNA target enrichment analysis among up-regulated genes following miR-7 transfection. Genomica enrichment (FDR <0.05) of miRNA targets among significantly up-regulated genes following miR-7 transfection into HEY cells. Regulating miRNAs are predicted using miRanda (those with the prefix John04 or SloanKettering), TargetScan or PicTar. Only "up" genes, i.e. up-regulated in the transfection, are analyzed ('Set').

Set Enriched Set
The other columns show the p-value of enrichment, the number of probesets in our dataset that are up-regulated and present in Genomica database ('Set Size'), compared to the number of probesets in each set in the background Affymetrix chip ('Total Size'). If a probeset is a predicted target of a miRNA then it is considered a 'hit' ('Set Hits' and 'Total Hits'). The percentages calculated from raw numbers of genes that belong in each set are also shown.

Supplementary Table 7. Hub genes and their targets affected by miR-7 in HEY cells.
GeneGo was used to analyze the most significant interactions within the set of differentially expressed genes following miR-7 transfection. The analysis gives a list of "hub" genes interacting with other differentially expressed genes (i.e. nodes) which suggests that miRNAs are regulating the node genes indirectly through regulating the hubs. "IDs in active dataset" refer to the Probeset IDs in the dataset that passed the GeneGo FDR threshold of 5% and are acting as hubs. Corresponding gene names/symbols are given in "Object name" column. "IDs of corresponding network object from GeneGo network" refer to probeset IDs of objects in the dataset that the hubs are interacting with in GeneGo networks. The names of these nodes are given in the last column. If a hub gene is found to regulate another gene which may also act as a hub then the row is shaded. Genes that are predicted to be miR-7 targets by miRanda or may act as transcription factors are marked with 'x'.

Supplementary Table 8. Hub genes and their targets affected by miR-128 in HEY cells.
GeneGo was used to analyze the most significant interactions within the set of differentially expressed genes following miR-128 transfection. The analysis gives a list of "hub" genes interacting with other differentially expressed genes (i.e. nodes) which suggests that miRNAs are regulating the node genes indirectly through regulating the hubs. "IDs in active dataset" refer to the probeset IDs in the dataset that passed the GeneGo FDR threshold of 5% and are acting as hubs. Corresponding gene names/symbols are given in "object name" column. "IDs of corresponding network object from GeneGo network" refer to probeset IDs of objects in the dataset that the hubs are interacting with in GeneGo networks. The names of these nodes are given in the last column. If a hub gene is found to regulate another gene which may also act as a hub then the row is shaded. Genes that are predicted to be miR-128 targets by miRanda or may act as transcription factors are marked with 'x'.  Beta. Differentially expressed genes following miR-7 transfection in HEY cells that are directly regulated by NF-κB, by IL-1 beta, or by both are listed. IL-1 beta (IL1B) is a transcriptional target of NF-κB and both are significantly down-regulated following miR-7 transfection. The genes that are only targeted by IL-1 beta may be differentially expressed because RELA/NF-κB is a target of miR-7 and in turn regulates IL-1 beta. ACO2  KCTD12 CASP1  ASS1  ADAM19 LIFR  DPP4  BID  ASPH  LITAF  IGFBP3  BMP2  ATXN3  MAT2A  IL1RAP  CXCL1  BACE1  MATR3  IL7  CXCL2  CALM1  NQO1  IRS1  DDIT3  CALM3  OGT  LAMC1  FN1  CBS  PAX6  ME1  HBEGF  CCND2  PCK2  MMP14  ICAM1  CCRL2  PDPN  NFKBIZ  IL8  CDK6  PHLDA1 NR1H3  MIR21  CDKN1B PMEPA1 NRP1  NUPR1  CSNK1A1 PSME3  NRP2  PTX3  DDIT4  RRM2  PAPPA  RUNX2  DDX21  S100A6  PRLR  SAA1  DLG1  SH2B3  SERPINH1 SAA2  DTNA  SLC11A2 SLC38A2  SERPINE2  EHD1  SLC44A1 SLC7A2  SOD2  EIF4A1  ST6GAL1 SOCS7  SPP1  ERAP1  ST8SIA1 THBD  TGFA  GAS5  TFRC  THBS1  TGFB2  GFPT2  TGM2  TIMP3  TGFBR2  GLO1  TNIP1  VEGFA  GREM1  TXNIP  VEGFC  HSPA9  VIM  XIAP  IL1B  ZBED4  IL7R  Supplementary Table 10. Direct downstream targets of Caveolin-1 and SMAD2. Caveolin-1 and SMAD2 are both identified as 'hub genes' among the genes differentially expressed after miR-128 transfection. CAV1 is a predicted target of miR-128 but SMAD2 is not. By targeting CAV1, miR-128 indirectly regulates SMAD2 and triggers the differential expression of genes regulated by SMAD2 as well. Differentially expressed genes that are direct targets of either Caveolin-1 or SMAD2 are listed. The relative fold change of these genes following miR-128 transfection into HEY cells are also provided.

Caveolin-1 and its direct targets SMAD2 and its direct targets Gene Symbol
Fold change Gene Symbol Fold change GeneGo and the ratio of number of genes actually present in the dataset to the number of genes in the Affymetrix array that are in each pathway. In this case only 1 pathway was found to be significant.