Overexpression of adenosine deaminase acting on RNA 1 in chordoma tissues is associated with chordoma pathogenesis by reducing miR-125a and miR-10a expression

Chordoma is a rare, slow-growing primary malignant neoplasm of the axial skeleton, which arises from the remnants of the notochord. Emerging evidence suggests that microRNAs (miRs) are dysregulated in chordoma tissues and crucially involved in chordoma pathogenesis. In the present study, the expression of 11 candidate miRs were analyzed in chordoma tissues and miR-10a and miR-125a were found to be significantly downregulated compared with controls. Notably, the expression of the primary transcripts, pri-miR-125a and pri-miR-10a was unaltered, suggesting that disturbed microRNA expression may be induced by altered pri-miRNA processing. Previous studies have indicated that disturbed adenosine deaminase acting on RNA (ADAR) expression is able to alter mRNA and miRNA adenosine to inosine (A-to-I) levels associated with cancer pathogenesis. Therefore, the expression of ADAR1 and ADAR2 was analyzed in chordoma tissues. It was found that ADAR1 was significantly overexpressed, which was accompanied by enhanced pre-miR-10a and pri-miR-125a A-to-I editing. These findings suggest that ADAR2 overexpression causes enhanced pre-miR-10a and pri-miR-125a A-to-I editing, which alters mature miR-10a and miR-125a expression and may contribute to chordoma pathogenesis.


Introduction
Chordoma is a rare tumor of the bone, known to arise from notochord remnants and is the most common primary malignant tumor of the sacrum and the mobile spine. The clinical management of chordoma is extremely challenging. Surgical resection is the primary curative strategy for chordoma, however, it is often not possible to perform adequately due to the anatomical location of the tumor (1)(2)(3). At present, there are no effective drug treatments for patients with chordoma, although radiation has been used as an adjuvant if complete resection is not possible, as well as when non-contaminated surgical margins are achieved (4,5).
A microRNA (miRNA) is a type of short, non-coding RNA that suppresses the expression of protein coding genes by partial complementary binding, particularly to the 3'-untranslated regions (3'-UTRs) of messenger RNAs (mRNAs). Alterations in the expression of miRNAs are associated with the initiation, progression and metastasis of human cancer and it is hypothesized that miRNAs function as tumor suppressors and oncogenes in cancer development (6,7). Previous studies have revealed that disturbed expression of microRNA (miR)-1 and miR-31 may be involved in chordoma pathogenesis, however, the mechanisms remain to be elucidated (8)(9)(10).
RNA editing is a widespread post-transcriptional process contributing to cellular transcriptome diversity in eukaryotes. The most well-characterized type of RNA editing identified in mammals converts cytosine to uracil and adenosine to inosine (A-to-I). In humans, the most frequent type of editing is the conversion of A-to-I, which is catalyzed by the double-stranded RNA specific adenosine deaminase acting on RNA (ADAR) family of proteins. Accumulating evidence has indicated that disturbed RNA editing may have a role in the pathogenesis of numerous types of cancers, including prostate (11), lung (12), liver (13) and malignant gliomas (14,15). Several studies have demonstrated that altered miRNA precursor editing is able to modulate miRNA processing and expression and is associated with cancer pathogenesis (15,16).
In the present study, in order to investigate the association between miRNA editing and chordoma, the sequences of miRNA precursors were compared with those of their coding regions in chordoma tissues. In addition, the expression levels of ADAR1 and ADAR1 were determined. Therefore, the present study aimed to reveal a novel association between A-to-I RNA editing and chordoma. miRNA reverse transcription-quantitative polymerase chain reaction (RT-qPCR). RT-qPCR analysis was used to determine the relative expression level of candidate miRNAs. The expression level of miRNAs was detected by TaqMan miRNA RT-qPCR. Single-stranded cDNA was synthesized using a TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) and then amplified using TaqMan Universal PCR Master mix (Applied Biosystems) together with miRNA-specific TaqMan MGB probes (Applied Biosystems). miR-RNU48, one of the most highly abundant and stably expressed miRs in human tissues, was used as an endogenous control. Each sample in each group was measured in triplicate and the experiment was repeated at least three times for the detection of miRNAs.

Samples
DNA collection and genotyping. DNA from tissue samples was isolated using a TIANamp Genomic DNA kit (Tiangen Biotech (Beijing) Co., Ltd., Beijing, China). DNA specimens were amplified using standard PCR protocols. A total of 322 bp of the pri-miR-125a coding region and 315 bp of the pri-miR-10a coding region were obtained. The PCR products were sequenced in the forward direction using the ABI 3730xl sequencing platform (Applied Biosystems

Differential expression of miRNAs in chordoma samples.
To determine whether there are differences in the miRNA expression between chordoma and nucleus pulposus tissues, the expression levels of 11 miRNAs that are associated with cancer initiation were analyzed using RT-qPCR. The present results demonstrated that the expression of miR-10a, miR-31 and miR-125a was significantly downregulated in chordoma tissues (Fig. 1). Furthermore, only the expression of miR-10a and miR-125a was downregulated in all the sample pairs (Fig. 2). To elucidate whether the altered miRNA expression was caused at the transcriptional level or post-transcriptional level, the expression of pri-miR-10a and pri-miR-125a was also analyzed. As shown in Fig. 2C, the expression levels of pri-miR-125a and pri-miR-10a were not altered in chordoma tissues, suggesting that an altered post-transcriptional processing step was performed.

Adenosine to guanine (A-to-G) nucleotide variations identified in sequencing results
. Accumulating evidence has indicated that nucleotide mutation may disturb miRNA processing and lead to altered miRNA expression (17,18). Several studies have also reported that RNA editing is common in mammals and altered RNA editing may be associated with cancer initiation and processing (15,16).
To understand the reason for the reduction in miR-10a and miR-125a expression, the DNA and cDNA sequences of these two miRNA precursors were compared. Notably, an A-to-G variation in the miR-10a and miR-125a cDNA sequence obtained from chordoma tissues was identified, which was not observed in genomic DNA and cDNA from the nucleus pulposus tissues (Fig. 3A). These results suggested that A-to-I RNA editing and overexpression of ADARs may be associated with chordoma.
ADAR1 and ADAR2 are overexpressed in chordoma tissues. To confirm the above hypothesis, the expression of ADAR1 and ADAR2 was analyzed in chordoma and control tissues. The results of western blot analysis and immunohistochemistry indicated that the expression of ADAR1 was overexpressed in almost all the chordoma tissues with upregulated ADAR2 in a number of the cancer samples ( Fig. 3B and C).

Overexpression of ADAR1 reduces miR-10a and miR-125a expression and upregulates expression of their target genes.
To confirm the association between overexpressed ADARs and downregulated miRNA expression, a FLAG-ADAR1 (110 kDa isoform) expression vector was constructed. In HEK293T cells, miR-10a and miR-125a were downregulated following overexpression of ADAR1 using FLAG-ADAR1 expression vector transfection (Fig. 4A). miRNAs function as important regulators in the initiation, progression and metastasis of multiple types of cancer through suppressing gene expression (19)(20)(21)(22). In order to determine the biological function of downregulated miRNAs, ERBB2 and HOXA1 3'-UTR reporter vectors were constructed, which were confirmed to be target genes of miR-10a or miR-125a. 3'-UTR reporter vectors and FLAG-ADAR1 expression vectors were co-transfected into HEK293T cells. After 48 h, total RNAs were extracted and miR-10a and miR-125a were detected using RT-qPCR. As shown in Fig. 4B, the relative luciferase activities were significantly increased in the ADAR1 overexpression groups.

Discussion
Chordoma, a rare type of bone tumor originating from notochord remnants, is a slow growing malignant form of cancer. Since it is relatively rare, few molecular studies have been performed, however, further studies are required in order to enable the development of novel strategies to treat chordoma. Previous studies have demonstrated that disturbed expression of miR-1 and miR-31 may be involved in chordoma pathogenesis, however, the underlying mechanisms remain to be elucidated (8-10). In the present study, the expression of 11 miRNAs were analyzed using RT-qPCR and miR-125a, miR-10a and miR-31 were found to be significantly downregulated. By comparing the sequences of cDNA and genomic DNA, it was identified that A-to-I RNA editing was present in the pre-miR-10a and pri-miR-125a. In addition, the expression of ADAR1 and ADAR2 was upregulated in chordoma tissues, which is collateral evidence for RNA editing.
In the present study, the expression levels of several miRNAs were detected, which have been reported tobe dysregulated in chordoma, including miR-181a, miR-31, miR-1 and miR-146b (8,9). Among them, only the reduction of miR-31 expression was in accordance with the results reported in a previous study (9). This difference may be caused by differences in ancestries and the small sample size.
It has been reported that miR-125a and miR-10a act as tumor suppressors in numerous types of cancer, including   (24), lung cancer (25,26) and breast cancer (27,28). In the present study, ERBB2 (a target gene of miR-125a) and HOXA1 (a target gene of miR-10a) were selected to represent the biological function of ADAR1 overexpression. It was revealed that ADAR1 overexpression upregulated luciferase activity by reducing the expression of miR-125a and miR-10a, suggesting that ADAR1 is associated with cancer pathogenesis by reducing antitumor miRNAs.
In conclusion, to the best of our knowledge, the present study is the first to demonstrate disturbed ADAR1 expression in chordoma tissues. Overexpression of ADAR1 is associated with chordoma pathogenesis by reducing tumor suppressor miRNA expression, including miR-125a and miR-10a expression.