MicroRNAs play a pivotal part in multiple cellular functions such as proliferation, apoptosis, differentiation and development. However, aberrant miRNAs occurs in carcinogenesis and metastasis during cancer progression [8]. Mounting evidence highlights a crucial role of miRNAs in OC progression. For instance, alterations in miR-101, miR-206, miR-200a and miR-203 are associated with OC cell proliferation and invasion [9–11]. In recent years, increasing numbers of studies elucidated that miRNAs participated in the invasive and metastatic processes of tumors. One miRNA can regulate nearly 100 different mRNAs; in addition, a single mRNA can bind several different miRNAs, thus forming a miRNA–target gene network regulatory relationship [12, 13]. Different types of cancer samples show varying gene expression patterns; even the same miRNAs often regulate different target genes in different types of cancer. In studies on OC, the application of a microRNA chip technique is helpful in identifying differentially expressed miRNAs affecting proliferation, apoptosis, invasion and metastasis in OC in a more rapid and comprehensive manner. Using a microRNA chip technique, Fu et al. [14] screened and identified eight differentially expressed miRNAs, including miR-93 from cisplatin-resistant OC cell strains and OC parental cell strains. With the application of a miRCURY LNA™ microRNA Array chip technique, Liu et al. [15] screened and identified a total of 31 differentially expressed miRNAs from another two pairs of cisplatin-resistant OC cell lines, of which the expression of 21 miRNAs was up-regulated and that of 10 miRNAs was down-regulated. Cheng et al.[16] found by using an Affymetrix miRNA 3.0 Array chip that 37 miRNAs were differentially expressed in adult and juvenile granulosa cell tumors (p < 0.05, fold change ≥ 2), and that these could be used as markers for the diagnosis and recurrence of ovarian granulosa cell tumors.
In studies on the mechanism of metastasis in OC, it is critical to screen differentially expressed miRNAs influencing the invasion and metastasis of OC. Conventional miRNA detection platforms only support the detection of miRNA expression but fail to allow the parallel detection of miRNA and target genes; as a result, it is impossible to comprehensively understand microRNA–target gene regulatory networks. In our study, we analyzed the miRNA expression profiles of primary OC tissues and their respective metastases from six patients using a miRStar™ Human Cancer Focus miRNA and Target mRNA PCR Arraychip. This chip contained 184 cancer-related miRNAs and 178 target mRNAs. All 184 miRNAs were carefully screened and identified from the latest literature, including miRNAs relating to such common cancers as gastric, liver, lung, breast, colorectal, and prostate. Numerous studies provided support for 178 target mRNAs, and their expression correlated to the functional activity of miRNAs in tumors. With an innovative experimental design, the miRStar™ Human Cancer Focus miRNA and Target mRNA PCR Array chip allows the synchronous detection of miRNA and target mRNA. It can be used not only to rapidly detect expression changes of cancer-related miRNAs in test samples, but can also be used to evaluate the activity of these miRNAs against target mRNAs, thus facilitating the screening of core functional miRNAs in diseases and the rapid comprehension of their regulatory mechanisms and functions.
It was found that 15 miRNAs and 10 mRNAs were dysregulated in both primary and metastatic OC tissues. Meanwhile, miRNAs and mRNAs with a possible targeting relationship were obtained by microRNA-mRNA conjoint analysis. By using our chip and combining the experimental results obtained from microRNA and mRNA chips, and the famous target gene prediction websites, targetscan and Miranda, possible target genes were predicted as follows: TGFβ2, MTSS1, HOXB5 and TNC (for miR-200a-3p); TGFβ2, MTSS1 and HOXB5 (for miR-141-3p); TGFβ2, CD34 and HOXB5 (for miR-7-5p); and HOXB5 (for miR-187-5p). Furthermore, GO and Pathway analysis was performed on mRNAs with a substantial difference in microRNA–mRNA conjoint analysis. GO analysis consisted of three parts: Biological Process, Cellular Component and Molecular Function. For Biological Process, we identified a total of 668 molecular function nodes showing a prominent difference (p < 0.05); differential genes were mainly involved in anatomical structure morphogenesis, embryo development, cell differentiation and cellular developmental processes. For Cellular Component, we identified a total of 52 molecular function nodes showing a remarkable difference (p < 0.05); differential genes primarily participated in ruffles, adherens junctions, anchoring junctions and cell junctions. For Molecular Function, we identified a total of 39 molecular function nodes showing a noteworthy difference (p < 0.05). Differential genes dominantly joined in RNA polymerase II distal enhancer sequence-specific DNA binding transcription factor activity involved in the positive regulation of transcription. In this study, we listed Enrichment Score Top 10 nodes in Biological Process, Cellular Component and Molecular Function. Using Pathway analysis, we found 21 strikingly different pathways (p < 0.05) and listed the top 10 appreciably different pathways, of which the pathway of MicroRNAs in Cancer was associated with TGFβ2, TNC and ITGB3. Based on these study results, we found several differentially expressed miRNAs and mRNAs, as well as miRNAs and mRNAs with a possible targeted regulatory relationship in metastatic and primary OC tissues that may play a critical part in invasion and metastasis links of OC.
As is well known, miRNAs can post-transcriptionally mediate a number of genes through a combination of specific sequences in target mRNA molecules [17]. MicroRNAs were differently expressed in OC [18]. Furthermore, miR-187 has been found to exert a dual role in OC by regulating the disabled homolog-2 gene [19]. Gao et al. also found that miR-141 acted as a potential diagnostic and prognostic biomarker for OC [20], which was concurred with our results. Zhu et al. demonstrated that miR-7-5p suppresses cell migration and invasion by targeting SOX18 in pancreatic ductal adenocarcinoma [21]. Another study also claimed that miR-7-5p under-expression was associated with recurrence in glioblastoma patients, and that its overexpression decreased glioblastoma cell stemness [22]. Furthermore, it was also elucidated that ectopic expression of miR-7 functioned as an anti-oncogene in OC by repressing cell invasion and proliferation [7].
MiR-7-5p has been found to target and regulate the genes as SATB1 and PARP1 in some cancer diseases as we have mentioned before [23, 24]. Furthermore, in this study, we found that TGFβ2 was targeted by hsa-miR-7-5p. The results of miRNA-mRNA conjoint analysis with our chip showed that hsa-miR-7-5p and TGFβ2 were differentially expressed in metastatic and primary OC tissues at a striking level, and may have a targeted regulatory relationship. To confirm this, we further validated the expression of four miRNAs (including hsa-miR-7-5p), TGFβ2 and TNC in 25 pairs of metastatic and primary OC tissues, a larger sample size. We found that TGFβ2 was negatively targeted by miR-7-5p using a dual luciferase reporter assay. MiR-7-5p may have several target genes similar to miR-137; such target genes are critical oncogenic factors, including TGFβ2, that could further regulate brain tumorigenesis [25]. In addition, we found that OC metastases expressed higher levels of TGFβ2 and TNC compared with primary OC tissues. This suggested that TGFβ2 was closely associated with OC metastasis. In studying potential mechanisms, we identified TGFβ2 as an mRNA target using bioinformatics analysis, as well as functional and binding assays. Taken together, our findings suggested that hsa-miR-7-5p and TGFβ2 were inversely correlated with regard to mRNA and protein expression; they demonstrated a targeted regulatory relationship that could influence the invasive and metastatic processes of OC. The TGFβ pathway takes part in many cellular processes, including cell proliferation, differentiation, extracellular matrix accumulation, tissue repair, immune and inflammatory responses. TGFβ2 is an isoform of TGFβ in mammals [26] and is abnormally expressed in various cancers such as human melanoma and hepatocellular carcinoma [27, 28]. In addition, many researchers have also explored the role of TGFβ in OC. Cao and his colleagues reported that TGFβ-induced transglutaminase gave rise to EMT and a cancer stem cell phenotype that consequently enhanced ovarian tumor metastasis [29]. TGFβ induced EMT and a more invasive phenotype in epithelial OC cells in collaboration with the EGF pathway, indicating TGFβ may be a promising target candidate for the treatment of metastatic OC in future [30]. TNC is an extracellular matrix glycoprotein that shows forced expression in cell proliferation and migration, and in EMT during organogenesis [31]. Moreover, a recent study highlighted that serum TNC levels were much higher in patients with epithelial OC than in normal controls. Such high serum TNC levels were related to poorer overall survival, which was consistent with our findings [32].