Altered editing level of microRNAs is a potential biomarker in lung adenocarcinoma

Adenosine‐to‐inosine (A‐to‐I) microRNA editing is associated with tumor phenotypes in various cancer types. Recent analyses of The Cancer Genome Atlas (TCGA) dataset have shown several microRNAs that undergo A‐to‐I editing in human cancers, some of which have been reported to be associated with prognosis. Herein, we examined published small RNA deep sequencing data of 74 cases of lung adenocarcinoma (AD) and the corresponding normal counterpart (NC) specimen in silico in order to identify A‐to‐I microRNA editing events. Editing levels of miR‐379‐5p, miR‐99a‐5p, and miR‐497‐5p were lower in AD than in NC and, in a large number of cases, the editing level of miR‐200b‐3p was higher in AD than in NC. Difference in the editing level between AD and NC was largest for miR‐99a‐5p. Then, we examined the editing level of miR‐99a‐5p in 50 surgically resected lung adenocarcinoma cases at our institution by a conventional sequence‐based method, and its association with clinical outcomes. The editing level of miR‐99a‐5p was significantly lower in 19 cases of AD (38%) than in corresponding NC. These cases showed a shorter overall survival as assessed using the log‐rank test (P = .047). This trend was consistent with previous analyses of TCGA dataset. The altered editing level of microRNAs in lung adenocarcinoma could serve as a potential biomarker.

most frequently edited. 10,11 Since 2004, when primary microRNAs, precursors of microRNAs were first demonstrated to undergo A-to-I editing, 12 some studies have shown the role of microRNA editing in normal organs 13,14 and various types of cancers. [15][16][17] MicroRNAs are single-stranded RNA molecules that are approximately 22 nucleotides in length and function as posttranscriptional gene regulators.
They bind to the 3′ UTR of mRNAs which contain a sequence complementary to the seed sequence (positions 2-8) on the microRNAs, resulting in degradation or downregulation of the target mRNAs. 18 As a consequence of microRNA editing, altered base complementation of a primary microRNA molecule affects the cleavage activity of microRNA processing proteins, DROSHA and DICER. 19,20 Moreover, when the seed sequence of a microRNA undergoes RNA editing, the edited microRNA targets another set of mRNAs. Therefore, the alteration in the editing levels of microRNAs affects the phenotype of a cancer through the alterations of the gene expression profile. 13 Recently, in silico analyses of small RNA deep sequencing data of The Cancer Genome Atlas (TCGA) dataset showed the comprehensive microRNA editing profile of 32 types of cancer. 21,22 In the cases of lung adenocarcinoma, significant differences in the editing levels of 5 microRNAs (miR-200b-3p, miR-379-5p, miR-411-5p, miR-497-5p, and miR-99a-5p) were found between adenocarcinoma (AD) samples and the corresponding normal counterpart (NC) samples, and correlations were found between the editing levels of 4 of these microRNAs (miR-376c-3p, miR-379-5p, miR-381-3p, and miR-99a-5p) in AD and the survival of the patients.
In the present study, we analyzed the published small RNA deep sequencing data of 74 cases of lung adenocarcinoma, including the data of both AD and NC in silico, and detected 3 micro-RNAs showing loss of editing in AD (the editing level was significantly lower in AD than in NC) and 1 microRNA showing gain of editing in AD (the editing level was significantly higher in AD than in NC). Regarding miR-99a-5p, which showed the largest difference of the editing level between AD and NC, we set up a conventional sequence-based method to quantify the editing levels. We examined surgically resected specimens from 50 cases of lung adenocarcinoma at our institution to detect differences of the editing level of miR-99a-5p between AD and NC. In our dataset, loss of editing of miR-99a-5p was significantly associated with shorter overall survival (OS), which was consistent with the analyses of TCGA dataset.

| In silico analysis of deep sequencing data
We searched the Sequence Read Archives (SRA) at the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/ sra) using a search phrase, "miRNA lung adenocarcinoma." We included paired data of lung adenocarcinomas and the corresponding normal tissues published before March 2015.
The downloaded SRA files were analyzed using perl scripts as previously described. 23,24 In short, 5′ and 3′ adaptors were removed by Process_reads.pl. The reads were mapped to the human reference genome (hg38) using Bowtie. 25,26 Here, we trimmed 2 bases at the 3′ end that undergo massive modifications. 27 We allowed a maximum of 1 mismatch per read with a quality score of at least 30, best unique alignment, and no cross-mapping. Then, the reads were remapped to the pre-microRNA sequence database by Analyze_mutation.pl, to count the read depth and the number of reads with mismatch. The original script used release 18 from miRBase (http:// www.mirbase.org) as reference. Instead, we used the latest version, release 21. The data format differs slightly according to versions; revised scripts are shown in Docs S1 and S2, and reference files are shown in Docs S3-S5. Finally, sequencing errors were removed for each position of each pre-microRNA by Binomial_analysis.pl. Output data included name of the pre-microRNA, mismatch position, mismatch type (such as C-to-U), number of mismatched reads, total number of reads in the position, and P-value with Benjamini-Hochberg correction with a false discovery rate (FDR) of 0.05. Known singlenucleotide polymorphisms (SNP) were discarded by reference to miR-NASNP Release 2.0 (http://bioinfo.life.hust.edu.cn/miRNASNP2/).

| Patient population and collection of samples
The study protocol was approved by the Institutional Ethical Review Board at the University of Tokyo Hospital (Tokyo, Japan), and written informed consent was obtained from each patient.   ligase (New England Biolabs) was added, followed by incubation at room temperature for 30 minutes to concatenate the amplicons. 29 Formation of a ladder with intervals of 73 base pairs was confirmed by electrophoresis. Taq DNA Polymerase (New England Biolabs), dATP, and dTTP were added, followed by incubation at 70°C for 20 minutes. The solution was loaded onto 1.2% Low Melting Agar (PH Japan, Hiroshima, Japan) with 1% Gel Indicator (BioDynamics Laboratory, Tokyo, Japan), and electrophoresed for 30 minutes. The ladder between approximately 300-700 base pairs was cut out, and melted at 65°C. β-Agarase (New England Biolabs) was added, followed by incubation at 42°C for 60 minutes. The solution was salted out, precipitated with ethanol, and eluted with nuclease-free water.

The concatenated amplicons were ligated into the pGEM-T Easy
Vector System (Promega), and clones with inserts of the expected length were sequenced using BigDye Terminator v3.1 (Thermo Fisher Scientific) with a primer on the vector, 5′-GCC AAG CTA TTT AGG TGA C-3′. We repeated the process of cloning and sequencing until we obtained in excess of 100 amplicons for each sample. The editing level in each sample was calculated as the number of edited reads divided by total reads.
To confirm that the proportion of edited reads reflected the editing level of miR-99a-5p, the unedited amplicon which consists of the 5′ adaptor, unedited miR-99a-5p and polyA was ligated into the pGEM-T Easy Vector System (Promega). The edited amplicon was ligated similarly. Edited and unedited plasmids were mixed, so that the proportion of the copy number of the edited plasmids was 5%, 15%, and 25%. These 3 templates were amplified, concatenated, and sequenced by the same method described above until amplicons in excess of 100 were obtained for each template.

| Statistical analysis
Student's t test (age) and Fisher's exact test (the others) were used to compare patient characteristics between the two groups. Statistical significance of the difference in the editing level between AD and NC was evaluated using the chi-squared test with Benjamini-

| In silico analysis of deep sequencing data
We downloaded the SRA files of 94 cases (188 samples) as candidates for analysis. They were from a single study which examined the relationship between smoking history and alteration of micro-RNA expression level in lung adenocarcinoma. 31 Table 2.
The perl scripts output, including all mismatch types, included a total of 4518 significant mismatch events in 148 samples. The events were detected in 186 mismatch spots. We detected 64 mismatch hotspots, in which 1-base mismatch events were detected in F I G U R E 1 Schema of the method for quantifying the editing level of miR-99a-5p. Position 1 of miR-99a-5p is "A" in the absence of editing, and "I" with editing. First-strand cDNA carries "T" and "C", respectively. The proportion of edited miR-99a-5p is assumed to be constant before and after PCR and ligation. "GAATTC" in the PCR primer is the EcoRI site  ber of total mapped reads) and were detected in fewer samples than the A-to-G mismatch hotspots ( Figure 2). Therefore, we regarded the non-A-to-G mismatch hotspots as noises, random sequencing errors which could not be eliminated by statistical procedure in the perl scripts. In regard to the 10 A-to-G mismatch hotspots, all of them were also detected in the previous study of TCGA dataset. 22 These mismatch hotspots can be regarded as true editing sites, because they were firmly reproducible; we call them "editing hotspots" from here.
For the 10 A-to-I editing hotspots, we compared the editing levels  F I G U R E 4 A, Editing levels of miR-99a-5p in the 50 cases at our institution. The format of the scatter plot is the same as that in Figure 2

| Quantification of the editing level of miR-99a-5p
Our improved method to quantify the editing level of miR-99a-5p shown in Figure 1 is based on two assumptions: (i) PCR efficiency of edited and unedited miR-99a-5p is equivalent; and (ii) the proportion of edited and unedited amplicons in concatemers is equivalent to that before ligation. To confirm that the proportion of edited reads reflected the editing level of the sample, we examined the standard template which contains 5%, 15%, and 25% edited amplicons (5′ adaptor, miR-99a-5p, and polyA). As shown in Table 3, each template showed results consistent with its mixture fractions.
Next, we extracted total RNA from 100 resected tissue specimens obtained from a cohort of 50 cases of lung adenocarcinoma and examined the editing level of miR-99a-5p in these specimens. In 43 cases (86%), the editing level in AD was lower than that in NC.
Loss of editing was not significantly associated with any unique clini-    Kaplan-Meier curves are shown in Figure 5B.

| DISCUSSION
In the present study, in silico analysis identified 8 microRNAs with paired A-to-I editing hotspots. Furthermore, we demonstrated an improved method to quantify the editing level of miR-99a-5p. In our patient cohort, loss of editing was associated with poor prognosis after complete resection of lung adenocarcinoma. This tendency was consistent with the results of previous studies of TCGA dataset; 21,22 reproducibility was confirmed by a well-validated conventional RT-PCR method. Along with these studies, we believe that the alteration of microRNA editing in lung adenocarcinoma could serve as a potential biomarker for predicting patient outcomes.
Before this biomarker can be applied practically in clinical medicine, the method for quantifying the editing level of microRNA needs to be further refined. In the first study to report microRNA editing, the authors amplified the precursor of miR-22, pri-miR-22, cloned the PCR product and sequenced at least 100 amplicons. 12  showed that the difference in the editing level between a primary microRNA and mature microRNA is >10% in approximately half of the microRNAs. 34 An improved method to amplify mature microRNA was published in 2012. 28  Previous studies have shown that miR-99a-5p itself functions as a tumor-suppressing microRNA through repressing invasion, migration, 36 and stemness. 37 However, the functional impact of editing miR-99a-5p is not clear, because it undergoes RNA editing at position 1. The seed sequence of the microRNAs, spanning positions 2-8, must be completely complementary to the 3′ UTR part of the target mRNA. 18 A previous study reported that miR-17-5p and miR-106a-5p, whose sequences differ only in position 1, are regulated and function in parallel. 38 Although the previous study of TCGA dataset showed that the editing level of miR-99a-5p was correlated with the mutation frequency of 14 important genes, including TP53, HRAS, NRAS and PIK3CA in 6 cancer types, 21 this cannot be explained by the known function of microRNA editing. Therefore, we assume that the difference in the editing level of miR-99a-5p being the largest among the paired editing hotspots caused a "passenger change" in lung adenocarcinoma, rather than alteration of a specific function of miR-99a-5p.
In a previous study of TCGA dataset, expression level of ADAR1 in lung adenocarcinoma was reported to be upregulated as compared with that in the corresponding normal tissue. 39 Moreover, amplification of the ADAR1 region 40 and higher expression level of ADAR1 in cancerous tissue 21 were associated with a tendency toward a poorer prognosis, although the association was not statistically significant for either. Our dataset showed an inverse tendency. We did not identify any clinical features (age, gender, smoking history, stage, EGFR mutation status) that were significantly associated with the expression levels of ADAR1 in our dataset. The largest difference between our cohort and TCGA cohort was in the ethnicity of the patients (100% East Asian vs 67.2% White and 1.4% Asian), which may have affected tumor biogenesis. We consider that study of more cases is needed to verify the function of ADAR1 in lung adenocarcinoma. Regarding ADAR2, our findings were consistent with previous studies; ADAR2 was downregulated in lung adenocarcinoma tissues, 39 and a lower ADAR2 expression level was significantly associated with a poorer prognosis. 41 In summary, we analyzed the alterations of microRNA editing in lung adenocarcinoma both in silico and by a conventional RT-PCR method. Loss of editing of miR-99a-5p in cancerous tissue was associated with poor survival in surgically resected lung adenocarcinoma specimens. Further investigations are required to elucidate the cause and effect of this association and to refine the method for quantification of the editing level.

CONFLI CTS OF INTEREST
Authors declare no conflicts of interest for this article.