Printer Friendly

Copy number and expression alterations of miRNAs in the ovarian cancer cell line OVCAR-3: impact on kallikrein 6 protein expression.

Cancer arises through the dysregulated interplay and regulatory networks between DNA and the expression of proteins and pathways encoded within it. Elucidating these regulatory mechanisms provides insight into the observed changes seen at the DNA, RNA, and protein levels, which are frequently used as cancer biomarkers. The kallikrein (KLK) [4] family of serine proteases is a putative source of such cancer biomarkers (1, 2). KLK6, the protein encoded by the Kallikrein-related peptidase 6 (KLK6) [5] gene, is a promising biomarker that is frequently overexpressed at the mRNA and protein levels in tissues and fluids derived from patients with ovarian cancer (OCa) and implicated in a variety of normal physiological processes (3). This observed overexpression been associated with poor prognosis and enhanced malignancy. Several prognostic studies have shown that the combination of KLK6 with CA-125 enhances their diagnostic power (4,5). Increases in the KLK6 gene copy number have been previously reported in OCa (6), and the KLK locus (19q13.3/4) is subject to copy number changes in OCa (7, 8). Early cytogenetic studies (8) revealed that the entire locus was involved in the copy number change, rather than individual members, a result that was recently confirmed by array studies (9). The highly aneuploid and structurally abnormal OCa karyotypes also showed that the locus was subject to copy number heterogeneity (10, 11), reflecting chromosomal instability often not revealed by the averaging algorithms of genome-wide methods (12). Although copy number gains of KLK6 were associated with increased KLK6 protein expression, a number of cancers with either 2 copies and/or a subpopulation containing 1 copyof the locus exhibited protein levels comparable to cancers with extra copies or amplification of the locus (approximately 28.0%). Conversely, some cancers with populations of copy number gains or amplifications exhibited KLK6 protein expression deemed as moderate/low (approximately 15.0%). Therefore, these data suggest that in addition to copy number, other mechanisms influence the observed overexpression, particularly posttranscriptionally (13-15). Although a number of mechanisms have been investigated, more study is needed (3). A growing body of evidence shows that miRNAs are key players in regulating and fine-tuning protein expression (16). miRNAs comprise a class of noncoding RNAs, in which perfect complementarity between the miRNA and the target gene mRNA 3' untranslated region (UTR) results in the cleavage and degradation of the target mRNA, whereas less than perfect pairing represses the translation process. In this fashion, miRNAs can target different mRNAs, increasing the diversity of gene regulation. Alterations in miRNAs have been analyzed in many cancers, including OCa (17, 18), and are believed to be affected by copy number (17, 19,20). Recent bioinfomatic and experimental findings (21-23) suggest that the KLK genes are also subject to regulation by miRNAs. In this study, we examined the contribution of copy number, of both the KLK locus and of miRNAs predicted to target KLK6, to the observed protein expression of KLK6 in a representative KLK6-overexpressing OCa cell line, OVCAR-3.

Materials and Methods

MOLECULAR CYTOGENETICS

The OVCAR-3 cell line was obtained from the ATCC and maintained according the product insert. For fluorescence in situ hybridization (FISH) analysis, differentially labeled bacterial artificial chromosome clones mapping to 3 regions of chromosome 19q (19q12, 19q13.2, the KLK locus at 19q13.3/4) were used as described previously (7) and hybridized to OVCAR-3 metaphase chromosomes (24). For spectral karyotyping (SKY) analysis, metaphase preparations were pretreated and hybridized with SKY Paints (Applied Spectral Imaging) and processed as described (25). The images were collected using a Zeiss Axioplan microscope (Carl Zeiss) and processed using the ASI image capturing and analysis system (ASI). For array comparative genomic hybridization (aCGH) analysis, the Agilent human genome 244K microarray platform was used (Agilent Technologies). The array design included a total of 5045 features and internal controls based on the UCSChg17 Build [National Center for Biotechnology Information (NCBI) build 35, May 2004]. The aCGH assay was performed as previously described (8). The normalized ratios for all features on the array are provided in Table 1 of the Data Supplement accompanying the online version of this article at http://www.clinchem.org/content/vol59/issue1.

miRNA PROFILING

We extracted miRNAs using the mirVana[TM] miRNA isolation kit (Ambion Life Technologies) and stored at -80[degrees]C until ready for use. Quantification of miRNA expression was performed by reverse-transcription (RT)-PCR using the TaqMan[R] MicroRNAv1.0 system [Applied Biosystems Inc. (ABI), Life Technologies] and compared to normal miRNAs derived from total ovarian tissues (Ambion). The TaqMan low-density array (TLDA) human miRNA panel and multiplex RT pools of 8 48-miRNA sets (ABI) were used for global miRNA expression. Briefly, 10 ng of miRNAs were converted into specific cDNAs and subsequently-quantified using the TaqMan MicroRNA TLDA card, containing 365 lyophilized human TaqMan miRNA sequences plus 3 small nucleolar RNAs (RU6B, RNU4, and RNU44) as endogenous controls. Data were quantified and analyzed using Sequence Detection System (version 2.3) (ABI). miRNA relative expression was normalized against endogenous controls and normal ovarian miRNAs according to the following threshold cycle (CT) calculation: 2-ACT, where ACT = (CTmiRNA - [CT.sub.snoRNAs]) (26). The final log10-transformed and normalized expression values for the miRNAs on the array can be found in online Supplemental Table 2.

IDENTIFICATION OF PREDICTED miRNAS TARGETING KLK6

To identify putative miRNAs predicted to target the 3'UTR of KLK6, 3 public databases were accessed: the Sanger Institute miRNA Registry (27), Memorial Sloan-Kettering Cancer Center's miRNA database (28), and TargetScan (29). Predicted miRNAs common to all 3 databases were considered candidates for further study.

TRANSIENT TRANSFECTION OF miRNAS TO OVCAR-3

For transient transfections, the PremiR[TM] miRNA Precursor Kit (Ambion) was used. The hsa-let-7a-5p precursor miRNA (PM10050, Ambion, Life Technologies), corresponding to identical mature miRNAs of hsa-let-7a-1, hsa-let-7a-2, and hsa-let-7a-3 loci, plus a scrambled miRNA negative control (Life Technologies) were prepared to a final concentration of 6.25 [micro]mol/L. OVCAR-3 cells were trypsinized and adjusted to a final concentration of 1 X [10.sup.5] cells/mL. In 6-well culture plates, 2.4 mL of OVCAR-3 cells (performed in duplicate) were combined with the transfection mixture comprised of the Ambion siPORT NeoFX transfection agent and Opti-Mem I medium, as instructed. The cultures were incubated at 37[degrees]C in a CO2 incubator for 24 h, after which time the medium was replaced with fresh normal growth medium. Biological replicates for each condition were prepared. After 72 h, the supernatant was collected and stored at -80[degrees]C, the cells were counted by trypan blue staining, and the cell pellet was stored for later protein and RNA extraction.

QUANTITATIVE RT-PCR

Quantification of mature hsa-let-7a, using the TaqMan miRNA Assays (ABI, ID RT000377, and TM000377), including a control small nuclear RNA, RNU44 (ABI, ID RT001094, and TM001094), were performed according to the manufacturer's instructions and analyzed as described above. The assays were performed in triplicate for each sample.

MEASUREMENT OF KLK6 AND KLK10 BY ELISA AND WESTERN BLOTTING

The assays were based on sandwich-type ELISA principles, with one antibody used for capture and one for detection. We used a monoclonal--monoclonal ELISA configuration for the immunodetection of KLKs 6 and 10 (8), and for standard Western analysis we used the same antibody clones for KLK6 (26 kDa) and 10 (30 kDa) with [beta]-actin (41 kDa) as a control (Abcam).

Results

IDENTIFICATION OF PREDICTED miRNAS REGULATING KLK6 AND miRNA PROFILING

The most commonly predicted miRNAs for the 3' UTR of KLK6 were from the members of the hsa-let-7 family of miRNAs, namely Hsa-let-7a, Hsa-let-7b, Hsa-let-7c, Hsa-let-7d, and Hsa-let-7e (Fig. 1A). With the use of the TLDA arrays, miRNA profiling of the KLK6overexpressing OVCAR-3 showed the dysregulation of these miRNAs compared to normal ovarian-derived miRNA, with the decrease in expression of hsa-let-7 family members. Table 1 summarizes the expression of miRNAs that were present on the TLDA arrayand predicted to target KLK6. Moreover, Table 1 indicates only those miRNAs that were identified in 2 or more databases. A comprehensive list detailing the log10 ratios of these and other miRNAs predicted to target KLK6 is provided in online Supplemental Table 3. On the basis of the miRNAs present on the TLDA array, and those previously predicted (23), 10 of the 13 miRNAs predicted to target KLK6 showed decreased expression (see online Supplemental Table 3). Expression profiling of the other predicted miRNAs showed a similar trend, with 13 of 24 miRNAs showing decreased expression (see online Supplemental Table 3). Because hsa-let-7a has been identified as an important member of the OCa miRNA signature (17), it was chosen as a candidate modulator of KLK6 protein expression. Validation of the miRNA findings by mature hsa-let-7a-specific qPCR confirmed the relative low-level expression of hsa-let-7a in OVCAR-3 compared to the normal ovarian miRNA control (Fig. 1B).

To test whether these candidate miRNAs could affect protein expression, hsa-let-7a-5p was transiently transfected into OVCAR-3. After a 72-h transfection with either hsa-let-7a-5p or a scrambled miRNA control, the amount of secreted KLK6 in the supernatants was determined by ELISA. After adjustment for the final cell number, a mean decrease of 25%-35% in the concentration of KLK6 protein in the hsa-let-7a-5-p-treated cells was detected compared to the controls. Western blotting confirmed the results observed with ELISA, with a modest decrease in KLK6 protein from the celllysate (Fig. 1C). On the basis of previous studies showing the modulation of KLK6 and 10-protein expression by hsa-let-7f (21, 23), we surveyed the databases from miRNAs predicted to target KLK10 and their expressions. Table 2 summarizes the relative miRNA expression of those miRNAs predicted to target KLK10 in 2 or more databases. Similarly, a detailed list of the relative log10 expression ratios of these miRNAs and others is provided in online Supplemental Table 4. Of the miRNAs identified in all 3 databases, 3 of 4 miRNAs showed decreases in expression, including hsa-let-7b, hsa-miR-214, and hsa-miR-485-5p (Table 2; also see online Supplemental Table 4). Of the 20 miRNAs identified in 2 databases, 11 miRNAs also showed a relative decrease in expression (see online Supplemental Table 4). On the basis of these database algorithms, among the hsa-let-7 family members hsa-let-7b is more strongly predicted to target KLK10 than hsa-let-7a, hsa-let-7c, hsa-let-7d, or hsa-let-7e, with hsa-let-7a identified in 2 databases as a putative regulating miRNA. Nevertheless, we tested whether hsalet-7a could also affect KLK10 protein expression. ELISA and Western blotting for KLK10 in the hsa-let7a-5p transfected line showed an 8%-15% decrease in secreted KLK10 protein compared to the controls (Fig. 1C).

OVCAR-3 CYTOGENOMICS

SKY analysis of OVCAR-3 showed a hypertriploid cell line (59-70 chromosomes) with numerous simple and complex structural rearrangements consistent with previous reports from the NCBI SKY/M-FISH and CGH Database. Structural rearrangements involving chromosome 19 were identified in addition to 2 apparently normal copies of chromosome 19. A complex chromosome described as a der(22)(16;19;22) was shown to contain a large homogeneously staining region indicative of amplification (Fig. 2A). aCGH analysis of the cell line revealed a high-level amplification spanning 19q11 to 19q13.2 and focal amplification at 19q13.4 (Fig. 2B). These copy number changes were consistent with other aCGH findings for this cell line (9). Using a multicolor 19q probe set (7), we confirmed the copy number findings identified by aCGH. Amplification of 19q11 (green) and 19q13.2 (blue) were identified on the der(22), as evidenced by the colocalization of FISH signals. Single hybridization signals, representing the KLK locus, were observed only on the 2 normal versions of chromosome 19 (red) (Fig. 2C). aCGH ratios for the KLK locus suggested its net loss, reflecting the limitations of CGH to account for the ploidy status of the cell line and the possibility of nonclonal losses and gains of chromosome 19. Thus, at most, there are 2 copies of the KLK locus in this triploid cell line.

[FIGURE 1 OMITTED]

Because copy number has been shown to be a contributing mechanism to changes in miRNA expression (17, 19, 20), we assessed the copy number status of the 3 genes encoding for an identical mature hsa-let7a miRNA (30), Hsa-let-7a-1 (9q22.32), Hsa-let-7a-2 (11q24.1), and Hsa-let-7a-3 (22q.13.3), using the aCGH data (Fig. 3). Both Hsa-let-7a-2 and Hsa-let7a-3 mapped to loci generating ratios indicative of an overall net loss, whereas Hsa-let-7a-1 indicated ratios indicative of 2 copies. Because OVCAR-3 possesses a triploid karyotype, with both clonal and nonclonal changes of these chromosomes, there is the presence of at least 2 copies of Hsa-let-7a-1, and at least 1, but not more than 2 copies each of Hsa-let-7a-2 and Hsa-let7a-3, consistent with the SKY findings. When we analyzed the structural rearrangements of those chromosomes to which these miRNAs mapped, we found that although the majority of metaphases possessed these structural rearrangements, which affect these loci, for some only 50% (5/10) of metaphases possessed these derivative chromosomes (Fig. 3). For Hsa-let-7a-1 (and Hsa-let-7f-1) mapping to 9q22.3, 2 normal versions of chromosome 9 were detected in all cells (10/10); however, the der(9)t(8;9) was detected in only 60% of metaphases, with the loss of material telomeric to 9q12. For Hsa-let-7a-2 mapping at 11q24.1, no normal versions of chromosome 11 were identified, however, 8/10 of metaphases possessed the der(11)t(11;14) rearrangement, accounting for copies of Hsa-let-7a-2 at the telomeric end of the chromosome. However, the der(11)t(11;16;18) rearrangement, present in only 60% (6/10) of metaphases, possesses a deletion telomeric to 11q23. Together, these account for the net deletion of the locus. Finally, Hsa-let-7a-3, which maps to 22q13.2, showed a net loss and contributed primarily by the combination of losses of the locus in the following rearrangements: i(22)(p10) (8/10 metaphases) and der(22)t(11;8;16) (5/10 metaphases), and the pres ence of the locus in the der(22)t(16;22) (7/10 metaphases) and der(22)t(16;19;22) (9/10 metaphases). Table 1 summarizes the net copy number changes for the miRNAs predicted to target KLK6 in at least 2 of the databases and for those previously identified (23) by combined aCGH and cytogenetic analysis, and accounting for the ploidy. For each miRNA, there is a general concordance in the association between overexpression of the mature miRNAs and net copy number gains of their genomic mapping locations, or decreased expression associated with net copy number losses. The exceptions to these findings are hsa-let-7c, hsa-let-7f-2, and hsa-miR-639. Table 2 summarizes the net copy number changes by combined aCGH and cytogenetic analysis for the miRNAs predicted to target KLK10 in at least 2 of the databases and for those previously identified (23). These results show a similar trend of positive association between the copy number status of the miRNAs and their expression, with the exception of 7 that showed an inverse association.

[FIGURE 2 OMITTED]

Discussion

In this study, we investigated the role of miRNAs in the regulation of KLK6 protein expression in a representative OCa cell line, OVCAR-3, and whether copy number may contribute to this mode of regulation. Mining of the publically available miRNA databases identified the hsa-let-7 family of miRNAs as likely regulators of KLK6. Together with aCGH and molecular cytogenetic validation, the copy number status of these and other miRNAs was shown to be subject to copy number losses, in keeping with the observed decrease in their expression, as determined by miRNA profiling. Indeed, the transient transfection of hsa-let-7a into OVCAR-3 resulted in the decrease of KLK6 protein expression.

[FIGURE 3 OMITTED]

Our TLDA profiling of this KLK6-overexpressing OCa cell line showed that the majority of miRNAs predicted to target KLK6 had decreased expression (Table 1; also see online Supplemental Table 3). All hsa-let-7 family members present on the array showed decreased expression of their mature miRNAs, compared to miRNAs derived from normal ovary, consistent with previous findings of decreased hsa-let-7 expression in OCas (17).

The KLK locus is subject to copy number heterogeneity, and cancers deemed to have net losses of the locus often possess minor subpopulations of cells with 2 or more copies (7, 8). Moreover, cytogenetic studies support the observation that chromosome 19 is typically involved in both clonal and nonclonal numerical and structural aberrations (8, 31), and that the vast majority of OCas are polyploid (8, 10, 11, 32). Therefore, the averaging algorithms of aCGH used in studies resulting in the frequent loss of the locus (33, 34) failed to take into account ploidy and copy number heterogeneity (12). FISH showed 2 copies of the KLK locus at the normal mapping location (19q13.3/4). Moreover, SKY analysis showed OVCAR-3 to be structurally and numerically abnormal, consistent with the reported karyotypic changes. Complementing the karyotypic analyses, aCGH confirmed the shift in ratio indicating the net loss of the KLK locus, including KLK6 (Fig. 2B), in keeping with identification of 2 copies within a triploid background. Therefore, OVCAR-3 represents the KLK6-overexpressing and diploid/net loss copy number scenario, observed in our primary tumor study (7).

Because copy number has been suggested to play a role in miRNA expression (17, 19, 20), we assessed the copy number status of the Hsa-let-7 family members predicted to target KLK6 (Table 1). There was concordance between the copy number status and the expression of the miRNA (Table 1, Table 2). The Hsa-let-7 members map to regions susceptible to frequent copy number changes (9, 33), and the net copy number changes exhibited in this cell line are representative of the genomic profiles seen in most OCas. With the exception of hsa-let-7f-2 and hsa-let-7c, we found that expression of miRNAs was associated with their copy number status. The combined aCGH and karyotypic analyses of Hsa-let-7a, which is encoded by 3 distinct genes and was processed to result in an identical mature hsa-let-7a, showed the overall net loss of genomic material (Fig. 3). Because hsa-let-7a has been identified in many OCa miRNA profiling and functional studies to be an important member of the OCa miRNA signature (17), and because the mature form results from 3 distinct copies within the normal human genome, hsalet-7a was chosen as a candidate modulator of KLK6 protein expression. When hsa-let-7a miRNA was transiently transfected into OVCAR-3, there was a decrease in KLK6 protein in the hsa-let-7a-transfected cell line supernatant and cell lysate, compared to the scrambled and untransfected controls (Fig. 1C). These findings were consistent with those of other investigators (2123 ), who showed the decrease in KLK6 expression by hsa-let-7f, which possesses the identical seed sequence to hsa-let-7a (30) and binding site on the KLK6 3' UTR (21-23). Additionally, previous bioinfomatic analyses (23), as well as our own (Table 2; also see online Sup plemental Table 4), led to the identification of hsa-let-7a as a possible modulator of KLK10. ELISA for KLK10 showed that hsa-let-7a was able to decrease KLK10 protein expression but to a lower extent than KLK6. Like KLK6, KLK10 overexpression has also been reported in OCa and associated with unfavorable prognosis and late-stage disease (35).

These findings add to the growing number of mRNA targets for the hsa-let-7 members, including MYC [v-myc myelocytomatosis viral oncogene homolog (avian)] (36), KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) (37), and HMGA2 (high mobility group AT-hook) (38), whose increased expression contributes to cancer progression. Indeed, although the increase of KLK6 mRNA and protein expression has been demonstrated to enhance malignant potential (3), the increased KLK6 transcript may also act to sequester such miRNAs with tumor-suppressive qualities from their endogenous targets. Interestingly, the bioinfomatic predictions performed to date suggest that members of the hsa-let-7 familymaypreferentially target KLK6 (21-23), and to a lesser extent, KLK10 (Table 1; also see online Supplemental Table 3). Although this theory is speculative and requires further investigation, we envision that the preferential targeting of KLK6 by the members of the let-7 family acts to simultaneously decrease KLK6-associated aggressive phenotypes while permitting the expression of the recently elucidate tumor suppressive properties of KLK10 (39). The downregulation of hsa-let-7a has been consistently reported among OCas (17), and its relative expression has been shown to have an impact on survival outcomes of patients treated with chemotherapy (40). The dysregulation of hsa-let-7a expression could have profound biological consequences linked to changes in the concentrations of target proteins, suggesting that such proteins could be putative biomarkers. Identifying and verifying the putative targets of this and other miRNAs provides the opportunityto discover relevant biomarkers and protein-based signatures that could enhance genomically and transcriptomically-derived signatures and define important clinical subgroups.

In summary, we demonstrated that the hsa-let-7 family member, hsa-let-7a, is a modulator of KLK6 protein expression that is independent of the KLK6 copy number status. Additionally, we demonstrated that hsa-let-7a can also weakly affect the protein expression of KLK10. Our cytogenomic analyses showed the strong contribution of copy number and miRNA expression in this representative OCa cell line. With the continued elucidation of other hsa-let-7a targets, it is possible that a clinically significant proteomic signature (including KLK6) can be developed to improve the diagnostic and predictive needs in OCa.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:

Employment or Leadership: E.P. Diamandis, Clinical Chemistry, AACC.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: None declared.

Expert Testimony: None declared.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.

References

(1.) Borgono CA, Diamandis EP. The emerging roles of human tissue kallikreins in cancer. Nat Rev Cancer 2004;4:876-90.

(2.) Lawrence MG, Lai J, Clements JA. Kallikreins on steroids: structure, function, and hormonal regulation of prostate-specific antigen and the extended kallikrein locus. Endo Rev 2010;31:407-46.

(3.) Bayani J, Diamandis EP. The physiology and pathobiology of human kallikrein-related peptidase 6 (KLK6). Clin Chem Lab Med 2011;50:211 33.

(4.) Shan SJ, Scorilas A, Katsaros D, Diamandis EP. Transcriptional upregulation of human tissue kallikrein 6 in ovarian cancer: clinical and mechanistic aspects. Br J Cancer 2007;96:362-72.

(5.) White NM, Mathews M, Yousef GM, Prizada A, Fontaine D, Ghatage P, et al. Human kallikrein related peptidases 6 and 13 in combination with CA125 is a more sensitive test for ovarian cancer than CA125 alone. Cancer Biomark 2009;5:279 87.

(6.) Ni X, Zhang W, Huang KC, Wang Y, Ng SK, Mok SC, et al. Characterisation of human kallikrein 6/protease M expression in ovarian cancer. Br J Cancer 2004;91:725-31.

(7.) Bayani J, Marrano P, Graham C, Zheng Y, Li L, Katsaros D, et al. Genomic instability and copy number heterogeneity of chromosome 19q, including the kallikrein locus, in ovarian carcinomas. Mol Oncol 2011;5:48-60.

(8.) Bayani J, Paliouras M, Planque C, Shan SJ, Graham C, Squire JA, et al. Impact of cytogenetic and genomic aberrations of the kallikrein locus in ovarian cancer. Mol Onc 2008;2:250-60.

(9.) Beroukhim R, Mermel CH, Porter D, Wei G, Raychaudhuri S, Donovan J, et al. The landscape of somatic copy-number alteration across human cancers. Nature 2010;463:899-905.

(10.) Bayani J, Brenton JD, Macgregor PF, Beheshti B, Albert M, Nallainathan D, et al. Parallel analysis of sporadic primary ovarian carcinomas by spectral karyotyping, comparative genomic hybridization, and expression microarrays. Cancer Res 2002;62:3466-76.

(11.) Bayani J, Paderova J, Murphy J, Rosen B, Zielenska M, Squire JA. Distinct patterns of structural and numerical chromosomal instability characterize sporadic ovarian cancer. Neoplasia 2008;10: 1057-65.

(12.) Albertson DG, Collins C, McCormick F, Gray JW. Chromosome aberrations in solid tumors. Nat Genet 2003;34:369-76.

(13.) Pampalakis G, Diamandis EP, Sotiropoulou G. The epigenetic basis for the aberrant expression of kallikreins in human cancers. Biol Chem 2006; 387:795-9.

(14.) Pampalakis G, Prosnikli E, Agalioti T, Vlahou A, Zoumpourlis V, Sotiropoulou G. A tumorprotective role for human kallikrein-related peptidase 6 in breast cancer mediated by inhibition of epithelial-to-mesenchymal transition. Cancer Res 2009;69:3779-87.

(15.) Pampalakis G, Sotiropoulou G. Multiple mechanisms underlie the aberrant expression of the human kallikrein 6 gene in breast cancer. Biol Chem 2006;387:773-82.

(16.) Schickel R, Boyerinas B, Park SM, Peter ME. MicroRNAs: key players in the immune system, differentiation, tumorigenesis and cell death. On cogene 2008;27:5959-74.

(17.) Dahiya N, Morin PJ. MicroRNAs in ovarian carcinomas. Endocr Relat Cancer 2010;17:F77-89.

(18.) Iorio MV, Visone R, Di Leva G, Donati V, Petrocca F, Casalini P, et al. MicroRNA signatures in human ovarian cancer. Cancer Res 2007;67:8699-707.

(19.) Croce CM. Causes and consequences of microRNA dysregulation in cancer. Nat Rev Genet 2009;10: 704-14.

(20.) Zhang L, Volinia S, Bonome T, Calin GA, Greshock J, Yang N, et al. Genomic and epigenetic alterations deregulate microRNA expression in human epithelial ovarian cancer. Proc Natl Acad Sci USA 2008;105:7004-9.

(21.) Chow TF, Crow M, Earle T, El-Said H, Diamandis EP, Yousef GM. Kallikreins as microRNA targets: an in silico and experimental-based analysis. Biol Chem 2008;389:731-8.

(22.) White NM, Bui A, Mejia-Guerrero S, Chao J, Soosaipillai A, Youssef Y, et al. Dysregulation of kallikrein-related peptidases in renal cell carcinoma: potential targets of miRNAs. Biol Chem 2010;391:411-23.

(23.) White NM, Chow TF, Mejia-Guerrero S, Diamandis M, Rofael Y, Faragalla H, et al. Three dysregulated miRNAs control kallikrein 10 expression and cell proliferation in ovarian cancer. Br J Cancer 2010;102:1244-53.

(24.) Bayani J, Squire JA. Preparation of cytogenetic specimens from tissue samples. Curr Protoc Cell Biol 2004;Chapter 22:Unit 22.2.

(25.) Bayani J, Squire JA. Spectral karyotyping. Methods Mol Biol 2002;204:85-104.

(26.) Schmittgen TD, Jiang J, Liu Q, Yang L. A high-throughput method to monitor the expression of microRNA precursors. Nucleic Acids Res 2004;32: e43.

(27.) Griffiths-Jones S, Saini HK, van Dongen S, Enright AJ. miRBase: tools for microRNA genomics. Nucleic Acids Res 2008;36:D154-8.

(28.) Betel D, Koppal A, Agius P, Sander C, Leslie C. Comprehensive modeling of microRNA targets predicts functional non-conserved and noncanonical sites. Genome Biol 2010;11:R90.

(29.) Grimson A, Farh KK, Johnston WK, Garrett-Engele P, Lim LP, Bartel DP. MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol Cell 2007;27:91-105.

(30.) Boyerinas B, Park SM, Hau A, Murmann AE, Peter ME. The role of let-7 in cell differentiation and cancer. Endocr Relat Cancer 2010;17:F19-36.

(31.) Micci F, Weimer J, Haugom L, Skotheim RI, Grunewald R, Abeler VM, et al. Reverse painting of microdissected chromosome 19 markers in ovarian carcinoma identifies a complex rearrangement map. Genes Chromosomes Cancer 2009;48:184-93.

(32.) Taetle R, Aickin M, Yang JM, Panda L, Emerson J, Roe D, et al. Chromosome abnormalities in ovarian adenocarcinoma: I. Nonrandom chromosome abnormalities from 244 cases. Genes Chromo Cancer 1999;25:290-300.

(33.) Cancer Genome Atlas Research Network Integrated genomic analyses of ovarian carcinoma. Nature 2011;474:609-15.

(34.) Gorringe KL, Campbell IG. Large-scale genomic analysis of ovarian carcinomas. Mol Oncol 2009; 3:157-64.

(35.) Koh SC, Razvi K, Chan YH, Narasimhan K, Ilanch eran A, Low JJ, et al. The association with age, human tissue kallikreins 6 and 10 and hemostatic markers for survival outcome from epithelial ovarian cancer. Arch Gynecol Obstet 2011;284: 183-90.

(36.) Nadiminty N, Tummala R, Lou W, Zhu Y, Zhang J, Chen X, et al. MicroRNA let-7c suppresses androgen receptor expression and activity via regulation of Myc expression in prostate cancer cells. J Biol Chem 2011;287:1527-37.

(37.) Johnson SM, Grosshans H, Shingara J, Byrom M, Jarvis R, Cheng A, et al. RAS is regulated by the let-7 microRNA family. Cell 2005;120:635-47.

(38.) Shell S, Park SM, Radjabi AR, Schickel R, Kistner EO, Jewell DA, et al. Let-7 expression defines two differentiation stages of cancer. Proc Natl Acad SciUSA 2007;104:11400-5.

(39.) Pepin D, Shao ZQ, Huppe G, Wakefield A, Chu CW, Sharif Z, et al. Kallikreins 5, 6 and 10 differentially alter pathophysiology and overall survival in an ovarian cancer xenograft model. PLoS One 2011;6:e26075.

(40.) Lu L, Schwartz P, Scarampi L, Rutherford T, Canuto EM, Yu H, et al. MicroRNA let-7a: a potential marker for selection of paclitaxel in ovarian cancer management. Gynecol Oncol 2011;122: 366-71.

Jane Bayani, [1,2] Uros Kuzmanov, [1,2] Punit Saraon, [1,2] William A. Fung, [1] Antoninus Soosaipillai, [2] Jeremy A. Squire, [3] and Eleftherios P. Diamandis [1,2] *

[1] Department of Laboratory Medicine and Pathobiology, University of Toronto, and [2] Mount Sinai Hospital Joseph and Wolf Lebovic Health Complex, Mount Sinai Hospital, Toronto, Ontario, Canada; [3] Department of Laboratory Medicine and Pathobiology, Queen's University, Kingston, Ontario, Canada. Kingston General Hospital, Translational Laboratory Research NCIC Clinical Trials Group, Queen's University, Kingston, Ontario, Canada.

[4] Nonstandard abbreviations: KLK, kallikrein; OCa, ovarian carcinoma; UTR, untranslated region; FISH, fluorescence in situ hybridization; SKY, spectral karyotyping; aCGH, array comparative genomic hybridization; NCBI, National Center for Biotechnology Information; RT-PCR, reverse-transcription PCR; ABI, Applied Biosystems; TLDA, TaqMan low-density array; CT, threshold cycle.

[5] Human genes: KLK6, kallikrein-related peptidase 6; KLK10, Kallikrein-related peptidase 10; MYC, v-myc myelocytomatosis viral oncogene homolog (avian); KRAS, v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog; HMGA2, high mobility group AT-hook.

* Address correspondence to this author at: Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, 6th Floor Rm. 6-201, Box 32, 60 Murray St., Toronto, Ontario, Canada M5T 3L9. Fax 416-619-5521; e-mail ediamandis@mtsinai.on.ca.

Received July 20, 2012; accepted October 19, 2012.

Previously published online at DOI: 10.1373/clinchem.2012.193060
Table 1. Summary of miRNA expression and copy number changes of
miRNAs predicted to target KLK6. (a)

                                                      Net copy
    Database             miRNA         aCGH locus      number

Target Scan#       hsa-let-7a-1 (b)#   9q22.32#     Balanced (c)#
Sloan Kettering#   hsa-let-7a-2 (b)#   11q24.1#     Loss#
Sanger#            hsa-let-7a-3 (b)#   22q13.3#     Loss#
                   hsa-let-7b (b)#     22q13.3#     Loss#
                   hsa-let-7c#         21q21.21#    Gain#
                   hsa-let-7d (b)#     9q22.32#     Balanced#
                   hsa-let-7e (b)#     19q13.3#     Loss#
                   hsa-miR-98 (b)#     Xp11.22#     Gain#
Target Scan        hsa-let-7f-1 (b)    9q22.32      Balanced
Sloan Kettering    hsa-let-7f-2 (b)    Xp11.22      Gain
                   hsa-let-7g          3p21.1       Balanced
Target Scan        hsa-miR-296         20q13.32     Gain
Sanger             hsa-miR-575         4q21.22      Balanced
                   hsa-miR-639         19p13.2      Loss
                   hsa-miR-659         22q13.1      Loss
                   hsa-miR-140 (b)     16q22.1      Loss

                                          miRNA
                                        expression
    Database             miRNA         mature miRNA

Target Scan#       hsa-let-7a-1 (b)#   Decreased#
Sloan Kettering#   hsa-let-7a-2 (b)#
Sanger#            hsa-let-7a-3 (b)#
                   hsa-let-7b (b)#     Decreased#
                   hsa-let-7c#         Decreased#
                   hsa-let-7d (b)#     Decreased#
                   hsa-let-7e (b)#     Decreased#
                   hsa-miR-98 (b)#     Increased#
Target Scan        hsa-let-7f-1 (b)    Decreased
Sloan Kettering    hsa-let-7f-2 (b)
                   hsa-let-7g          Decreased
Target Scan        hsa-miR-296         Increased
Sanger             hsa-miR-575         Decreased
                   hsa-miR-639         Increased
                   hsa-miR-659         Decreased
                   hsa-miR-140 (b)     Decreased

(a) Shown are the combined expression and cytogenomic analyses of
miRNAs predicted to target KLK6, which were identified in 2 or more
databases. miRNAs in bold are those miRNAs identified in all 3
publically accessible databases as described in the Materials and
Methods.

(b) miRNAs previously reported by White et al. (23) to be predicted
to target KLK6.

(c) Balanced, net copy number of 2/no change in copy number; loss,
net decrease in copy number; gain, net increase in copy number.

Note: Those miRNAs identified in all 3 publically accessible
databases as described in the Materials and Methods are indicated
with #.

Table 2. Summary of miRNA expression and copy number changes of
miRNAs predicted to target KLK10. (a)

                                               aCGH

                                                        Net copy
     Database                miRNA            Locus      number

Target Scan          hsa-let-7b (b)#         22q13.3#   Balanced#
Sloan Kettering      hsa-miR-214 (b)         1q24.3     Gain
Sanger               hsa-miR-224 (b)         Xq28       Balanced
                     hsa-miR-485-5p          14q32.3    Balanced
Sanger               hsa-let-7a-1 (b)#       9q22.32#   Balanced#
Target Scan          hsa-let-7a-2 (b)#       11q2.1#    Loss#
                     hsa-let-7a-3 (b)#       22q13.3#   Loss#
                     hsa-let-7c#             21q21.21#  Gain#
                     hsa-let-7d#             9q22.32#   Balanced#
                     hsa-let-7e#             19q13.3#   Loss#
                     hsa-miR-148 (b)         12q13.13   Gain
                     hsa-miR-152             7q21.32    Gain
                     hsa-miR-197             1p13.3     Loss
                     hsa-miR-326             11q13.4    Amplified
                     hsa-miR-98 (b)          Xp11.22    Gain
Target Scan          hsa-miR-1-1             20q13.33   Gain
Sloan Kettering      hsa-miR-1-2             18q11.2    Loss
                     hsa-miR-143 (b)         5q32       Loss
                     hsa-miR-18a             13q31.1    Loss
                     hsa-miR-192             11q13.1    Gain
                     hsa-miR-193 (b)         16p13.2    Loss
                     hsa-miR-206 (b)         6p12.2     Balanced
                     hsa-miR-215             1q41       Gain
                     hsa-miR-510             Xq27.3     Loss
                     hsa-miR-515-3p          19q13.42   Amplified
                     hsa-miR-613             12p13.1    Gain
                     hsa-miR-646             20q13.3    Gain
White et al. (23)    hsa-miR-125 (b)-1 (b)   11q24.1    Loss
                     hsa-miR-125 (b)-2 (b)   21q21.1    Gain
                     hsa-miR-140 (b)         16q22.1    Loss
                     hsa-miR-149 (b)         2q37.3     Gain
                     hsa-miR-432 (b)         14q32.2    Balanced

                                             miRNA expression
     Database                miRNA             mature miRNA

Target Scan          hsa-let-7b (b)#         Decreased#
Sloan Kettering      hsa-miR-214 (b)         Decreased
Sanger               hsa-miR-224 (b)         Increased
                     hsa-miR-485-5p          Decreased
Sanger               hsa-let-7a-1 (b)#       Decreased#
Target Scan          hsa-let-7a-2 (b)#       Decreased#
                     hsa-let-7a-3 (b)#       Decreased#
                     hsa-let-7c#             Decreased#
                     hsa-let-7d#             Decreased#
                     hsa-let-7e#             Decreased#
                     hsa-miR-148 (b)         Increased
                     hsa-miR-152             Decreased
                     hsa-miR-197             Decreased
                     hsa-miR-326             Increased
                     hsa-miR-98 (b)          Increased
Target Scan          hsa-miR-1-1             Decreased
Sloan Kettering      hsa-miR-1-2             Decreased
                     hsa-miR-143 (b)         Decreased
                     hsa-miR-18a             Increased
                     hsa-miR-192             Decreased
                     hsa-miR-193 (b)         Decreased
                     hsa-miR-206 (b)         Increased
                     hsa-miR-215             Increased
                     hsa-miR-510             Decreased
                     hsa-miR-515-3p          Decreased
                     hsa-miR-613             Increased
                     hsa-miR-646             Increased
White et al. (23)    hsa-miR-125 (b)-1 (b)   Decreased
                     hsa-miR-125 (b)-2 (b)   Decreased
                     hsa-miR-140 (b)         Decreased
                     hsa-miR-149 (b)         Increased
                     hsa-miR-432 (b)         Decreased

(a) Shown are the combined expression and cytogenomic analyses of
miRNAs predicted to target KLK10 in two or more databases. miRNAs in
bold represent those miRNAs also predicted to target KLK6.

(b) miRNAs previously reported by White et al. (23) to be predicted
to target KLK10.

(c) Balanced, net copy number of 2/no change in copy number; gain,
net increase in copy number; loss, net decrease in copy number.

Note: Those miRNAs also predicted to target KLK6 are indicated with
#.
COPYRIGHT 2013 American Association for Clinical Chemistry, Inc.
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2013 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Title Annotation:Cancer Diagnostics
Author:Bayani, Jane; Kuzmanov, Uros; Saraon, Punit; Fung, William A.; Soosaipillai, Antoninus; Squire, Jere
Publication:Clinical Chemistry
Article Type:Report
Geographic Code:1USA
Date:Jan 1, 2013
Words:5608
Previous Article:Oral cancer diagnosis via a ferrocenylnaphthalene diimide-based electrochemical telomerase assay.
Next Article:Multicenter evaluation of [-2]proprostate-specific antigen and the prostate health index for detecting prostate cancer.
Topics:

Terms of use | Privacy policy | Copyright © 2019 Farlex, Inc. | Feedback | For webmasters