The primary aim of this study was to try to elucidate the effects of the RET proto-oncogene on gene expression and transcriptional regulation in a matrix of cells lines harbouring ret/PTC 1 rearrangement. This was performed using Applied Biosystems whole genome expression array technology and TaqMan® microRNA assays. The sequencing of the human genome and advances in microarray technology has significantly increased understanding of the molecular pathogenesis of thyroid tumour formation in recent years. On order to gain a better understanding of the genes and pathways involved in PTC and unveil potential biomarkers, several groups have applied DNA microarray technology to show the genetic profiles in PTC [23–26]. These profiles uncovered genes that were suggested to be involved in processes such cell adhesion and cell structure, inhibition and induction of apoptosis, cell proliferation and differentiation.
This study showed a group of differentially expressed genes in two cell lines contingent on a rearrangement of the RET oncogene when compared to normal thyroid. Among the genes found to be over-expressed, those involved in biological processes such as cell differentiation and proliferation were over-represented. Such genes included CEBPB, CCNG1, IFITM3 and HTRA1. CEBPB is a bZip transcription factor that cooperates with Stat 6 to activate transcription . It has been shown to have a critical role in Ras mediated tumourigenesis and cell survival and has been implicated as a target for tumour inhibition. Studies have shown that it is expressed in the cytoplasm of PTC and is implicated to play a role in the regulation of thyroid specific genes and processes and has altered function in thyroid carcinoma . It is involved in the positive regulation of genes involved in the immune and inflammatory response, a group which has been shown to be associated with ret/PTC 1 and tumour formation in this study and in previous thyroid literature . CEBPB also has been shown to be a regulator of Cox-2; Cox-2 has recently been implicated as a potential biomarker in thyroid neoplasm .
HTRA1  is a member of the trypsin family of serine proteases and is a regulator of cell growth. It has been implicated in the pathology of several diseases. Several papers have implicated matrix metalloproteases in PTC tumour progression; during tumour invasion they degrade protein components of the extracellular matrix aiding angiogenesis . HTRA has been proven to cause the induction of matrix metalloproteases 1 and 3.
Cell signalling also showed an increase in ret/PTC 1 cell lines, SEMA3F is a protein involved in signalling. Endogenous SEMA3F causes cells to round and loose extracellular contacts a process which may contribute to the classic appearance of PTC. Membralin (C19orf6) is a novel gene originally cloned from a human ovarian cancer cell line. It is suggested to be a tumour associated marker in ovarian and colorectal cancer [33, 34]. It was also shown to be over-expressed in ret/PTC 1 cell lines when compared to normal. It is interesting to hypothesize that there may be a link between membralin overexpression and the papillary architecture seen in PTC and in ovarian cancers.
Furthermore, genes involved in p53 pathway were overrepresented. P300/CBP- associated factor (PCAF) was one such gene and is a co- activator of the tumour suppressor p53. PCAF was found to be induced by p53 in breast cancer cell lines and indirectly regulated ERK1 effecting cellular proliferation .
Among the genes shown to be down-regulated in ret/PTC 1 cell lines when compared to normal, those involved in cell structure and motility were over-represented. Examples of such genes include DCTN5, TPM1, TPM3, CRP1 and Keratin type 1. Tumour cells are held together by direct cell – cell contact and by adhesion to the extracellular matrix. Loss of adhesion is thought to promote tumour invasiveness and increase the metastatic potential of carcinoma. Loss of these cell structure genes might also be involved in the papillary pattern of PTC. Tropomysoin (TM) has a role in regulating cellular functions associated with cytoskeletal remodelling. Suppression of TMs is a prominent feature of many transformed cells. TM1 expression is abolished in human breast cancer cell lines and also in the ret/PTC 1 cell lines in this study. This downregulation may destabilize microfilament architecture and facilitate survival of neoplastic cells and promote malignant growth . PSMD2 (TRAP2) participates in TNF signalling pathway and was found to be downregulated. This suggested a role for PSMD2 in cell survival. Rab32 inactivation may represent a component of the oncogenic pathway of microsatellite-unstable gastrointestinal adenocarcinoma. It encodes an A-kinase anchoring protein and is often hypermethylated in gastric and colon cancers and was found to also be downregulated in PTC cell lines. Rab32 has also been shown to induce apoptosis [37, 38].
Of particular interest in this study was the discovery of the underexpression of DROSHA in PTC cell lines. This is the core nuclease that executes the initiation step of miRNA processing in the nucleus. DROSHA collaborates with Dicer in stepwise processing of miRNAs and has a key role in miRNA mediated gene regulation processes such as development and differentiation . Components of the miRNA-machinery have been implicated in tumourigenesis and reduced levels have been shown to be associated with cancer . Its differential expression may effect the expression of certain miRNAs such as those in table 3 and consequently play a role in gene expression regulation.
This data corroborates the importance of these pathways in PTC. The genes involved may play a role in tumour initiation and in the morphological characteristic of classic PTC due to the ret/PTC 1 oncogene. Several reports have shown that ret/PTC 1 rearrangements are in general associated with classic papillary architecture in thyroid carcinoma. The profiles examined contained genes that were suggested to be involved in processes such as cell adhesion and cell structure, such as tropomysoins, and inhibition and induction of apoptosis. This aberrant expression has an impact on the molecular structure and the extracellular matrix which may facilitate destabilization of the microfilament architecture. While also adding to tumour spread this phenomenon may contribute to the classic papillary appearance associated with this rearrangement.
Gene expression has recently been shown to be regulated by small noncoding RNAs called microRNAs (miRNAs). It is believed that miRNAs are responsible for fine tuning gene expression and are thought to regulate approximately 30% of the human genome, making them one of the largest classes of gene regulator. Several clues have pointed to the potential role of miRNAs in cancer and suggest that aberrations in miRNA expression may be important in tumour progression by regulating genes and pathways involved in cancer associated processes. Studies have reported that reduced levels of the Let-7 family of miRNAs have an association with lung cancer tumourigenesis [41, 42]. The miRNA cluster of miR-17- miR-92 is highly expressed in many B-cell chronic lymphomas . Furthermore, Mir 15a-Mir 16 expression is upregulated in B-cell chronic lymphoma and leukaemia and falls within a region thought to harbour a tumour suppressor gene. Glioblastomas and breast tumours have also been reported to show over expression of miR-21 [44, 45].
This study examined, by real-time PCR, the expression of miRNA in papillary thyroid carcinoma cell lines with ret/PTC1 rearrangement.
Tables 3 shows a set of differentially expressed miRNAs in 2 cell lines with ret/PTC1 rearrangement when compared to normal thyroid cell line. 21 miRNAs were found to be overexpressed and 14 miRNAs were underexpressed in both cell lines when compared to Nthy-ori 3-1. These miRNAs are effected by direct ret/PTC 1 expression or by the expression of other genes that are induced by ret/PTC 1 such as transcription factors. Little is known about the function of these miRNAs and only a subset has been found to be differentially expressed in other cancers. Of the miRNAs in table 3, miR-181a expression was down-regulated and expression of miR-200b and miR-141 was up-regulated. Ciafre et al found miR-181a expression to be downregulated in glioblastoma . Studies have reported that miR-181 upregulated during differentiation and suggest that miR-181 downregulates the homeobox protein Hox-A11 (a repressor of the differentiation process), thus establishing a functional link between miR-181 and the complex process of differentiation. In fully differentiated tissue miR-181 was found to be downregulated, PTCs are well-differentiated carcinomas therefore this may explain downregulation of miR-181a in this study.
miR-200b and miR-141 have also been shown to be highly overexpressed in malignant cholangiocytes and in colon carcinoma [46, 47]. Studies showed that their inhibition increased sensitivity to the anticancer drug gemcitabine and also decreased cell growth suggesting a role for their involvement in tumour formation.
An immediate obstacle facing research groups at present is the lack of confirmed miRNA gene targets. Although multiple approaches have been suggested from bioinformatics including many prediction algorithms accurate target prediction and validation are still major obstacles facing miRNA researchers. Each miRNA found to be significantly up/down regulated from table 3 was imputed into the algorithms, miRBase, PICTAR and TARGETSCAN to search for potential miRNA:miRNA target pairings. The potential binding partners of 6 of these miRNAs are listed in table 4. It is interesting to note that these differentially expressed miRNAs potentially bind and regulate a number of genes that have implications in thyroid carcinoma progression such as genes involved in thyroid function, MAPKKK cascade, retinoic acid receptors, cell adhesion and cell structure associated genes, genes involved in cell signalling, G-protein mediated signalling, oncogenesis and cell cycle control. Examples of these genes include thyroid hormone interacting protein, protocadherin, MAP3K10, KIT, PDGFRA and exportin 4. Therefore, it seems plausible that this subset of miRNAs may be important in the progression of thyroid carcinoma by regulating genes involved in thyroid neoplasia such as those found in this study discussed above.
Analysis of DNA microarray data showed a group of genes that are differentially expressed in ret/PTC1 harbouring cell lines (Table 1 and 2). Comparing this list of genes with the list of putative miRNA targets yielded from analysis using PICTAR, TARGETSCAN and miRBase showed that 15 of the differentially expressed genes are targeted by several miRNAs that are significantly up/downregulated in cell lines with ret/PTC 1 rearrangement. This data is shown in Table 5. Again it is interesting to speculate that these genes are regulated by their corresponding miRNA thereby contributing to tumour development in thyroid. It remains difficult to identify the precise target mRNA of each miRNA species. Therefore without further experimental investigation any mRNA:miRNA partners are essentially speculative and selection involves a degree of subjectivity.
Recent studies on miRNA deregulation in PTC found an aberrant miRNA expression profile in PTCs compared to normal thyroid tissues. In particular, a significant increase in miR- 222, miR-221, miR-146 and miR-181b was seen [48, 49]. In this study miR-146 was upregulated 500 fold in TPC-1 cell line only. It is possible this miRNA may play a role in a late stages of tumour formation and progression; given the transfected cell line had only been exposed to ret/PTC1 for a short period (3 passages). Alternatively, the difference may reflect the accumulation of several genetic insults. The other miRNAs [miR-222, miR- 221, miR-181b], however, were not found to be upregulated significantly. This may be due to the differences in experimental approaches; de la Chapelle et al and Fusco A et al used miRNA microarrays. They also used different chemistry and fresh tissue samples; also the RET status of each PTC case was not disclosed.
This field of miRNA research is young and many important questions still remain. It is clear that miRNAs exert a regulatory role on protein coding genes in a variety of species and their deregulation may contribute to the development of human disease. However, the precise biological function of miRNAs has yet to be fully elucidated.
Even without more in-depth knowledge of their exact function or role in PTC, it seems reasonable that a distinct signature of miRNAs or miRNA expression profiles assigned for thyroid tumours might be used as an adjunct in diagnosis and prognostication of thyroid neoplasia. It has been suggested that miRNA signatures may be more effective than profiles of protein coding genes in distinguishing tumours from benign lesions. Fortunately miRNAs, unlike mRNA targets remain intact in routinely collected paraffin embedded samples, a fact which increases their potential use as clinical biomarkers. miRNAs also have the potential as possible treatment targets. One example of potential miRNA targeted therapy has been shown with miR-127 in bladder cancer. This suggests the possibility of direct targeting of miRNAs that are amplified or upregulated in patient tumours.