A new cancer/testis long noncoding RNA, the OTP-AS1 RNA

Abstract

The orthopedia homeobox (OTP) gene encodes a homeodomain-containing transcription factor involved in brain development. OTP is mapped to human chromosome 5q14.1. Earlier we described transcription in the second intron of this gene in wide variety of tumors, but among normal tissues only in testis. In GeneBank these transcripts are represented by several 300-400 nucleotide long AI267901-like ESTs. We assumed that the AI267901-like ESTs belonged to the longer transcript(s). We used the Rapid Amplification of cDNA Ends (RACE) approach and other methods to find the full-length transcript. The transcript we found was a 2436 nucleotide polyadenylated sequence in antisense to OTP gene. The corresponding gene consisted of two exons separated by an intron of 2961 bp. The first exon was found to be 91 bp long and located in the third exon of OTP. The second exon was 2345 bp long and located in the second intron of OTP. We have shown the expression of this gene in many human tumors but as few as a single sample of normal testis. The transcript lacked significant ORFs suggesting that we discovered a new antisense cancer/testis (CT) sequence OTP-AS1 (OTP-antisense RNA 1), which belongs to the class of long noncoding RNAs (lncRNAs). According to our findings we assume that OTP-AS1 and OTP genes may be a CT-coding gene/CT-ncRNA pair, or sense-antisense gene pair involved in regulatory interactions.

Fig. 1

Two-round amplification of the studied gene 5’ and 3’ ends using gene specific and adaptor primers. (a) Two-round amplification of the studied gene 5’ end: 1—adaptor primer and rev(N), 2—negative control, first round of PCR, no template added, 3—negative control, second round of PCR, no template added. M1—GeneRuler™ 100 bp DNA ladder (Fermentas), M2—GeneRuler™ 1 kb DNA ladder (Fermentas). (b) Two-round amplification of the studied gene 3’ end: 1—adaptor primer and forv1(N), 2—negative control, first round of PCR, no template added, 3—adaptor primer and forv2(N), 4—negative control, second round of PCR, no template added, M—GeneRuler™ 1 kb DNA ladder (Fermentas).

Fig. 2

Exon/intron borders of the studied gene. *gt—donor splicing site. **ag—acceptor splicing sit.

Fig. 3

Genome localization of OTP gene, its regulatory region and sequenced fragments. Schematic alignment of the sequenced fragments (5’ end fragment—yellow, 3’ end fragment №1—green, 3’end fragment №3—cyan, assembled full-length sequence—red) with the 5th chromosome (black) and the OTP gene (blue), regulatory region (purple), core promoter (lilac).

Fig. 4

Two-round amplification of the full transcript on the cDNA from: 1—293 T cells, 2—uterus endothelium adenocarcinoma, 3—negative control, first round of PCR, no template added, 4—negative control, second round of PCR, no template added, M1—GeneRuler™ 100 bp DNA ladder (Fermentas), M2—GeneRuler™ 1 kb DNA ladder (Fermentas).

Fig. 5

Chromosome location of OTP and newly discovered gene with mapping of the known ESTs. The least conserved genomic DNA sequence is highlighted with blue. The most conserved genomic DNA sequence is highlighted with yellow. (This scheme was obtained with UCSC Genome Browser tool).

Fig. 6

The lack of expression of the newly discovered gene conservative region in normal human tissues. (a) 1—normal brain, 2—normal heart, 3—normal kidney, 4—normal liver, 5—normal lung, 6—normal pancreas, 7—normal placenta, 8—normal skeletal muscle, K−—PCR with no template, K + —PCR with human DNA (Full-length gel Fig. S16). (b) 1—normal colon, 2—normal ovary, 3—normal peripheral blood leukocytes, 4—normal prostate, 5—normal small intestine, 6—normal spleen, 7—normal testis, 8—normal thymus, K−—PCR with no template, K + —PCR with human DNA (Full-length gel Fig. S17). (c) 1—normal bone marrow, 2—fetal liver, 3—normal lymph node, 4—normal peripheral blood leukocyte, 5—normal spleen, 6—normal thymus, 7—normal tonsil, K−—PCR with no template, K + —PCR with human DNA (Full-length gel Fig. S18). (d) 1—fetal brain, 2—fetal heart, 3—fetal kidney, 4—fetal liver, 5—fetal lung, 6—fetal skeletal muscle, 7—fetal spleen, 8—fetal thymus, K−—PCR with no template, K + —PCR with human DNA (Full-length gel Fig. S19).

Fig. 7

Expression of the newly discovered gene conservative region in human tumors. (a) 1—brain astrocytoma, 2—breast invasive ductal carcinoma, 3—lung squamous cell carcinoma, 4—esophagus adenocarcinoma, 5—stomach adenocarcinoma, 6—small intestine adenocarcinoma, 7—colon adenocarcinoma, 8- hepatocellular carcinoma, 9—kidney clear cell carcinoma, 10—bladder transitional cell carcinoma, 11—uterus adenocarcinoma, 12—fallopian tube medullary carcinoma, 13—ovary mucinous adenocarcinoma, 14—testis seminoma, 15- ureter papillary transitional cell carcinoma, K−—PCR with no template, K + —PCR with human DNA. (Full-length gel Fig. S20). (b) 19—stage III mammary gland adenocarcinoma, 246, 250, 251, 252—stage II–III invasive duct mammary gland cancer; 2—squamous cell cervical carcinoma IV stage and its metastases into uterus (2a-1), left (2a-3) and right ovary (2a-4), 13- cervical myosarcoma, stage II-III, 6—ovary cancer, 156—stage II moderately differentiated endometrial adenocarcinoma, 270—stage III moderately differentiated endometrial adenocarcinoma with metastases, 7—seminoma, K−—PCR with no template, K + —PCR with human DNA (Full-length gel Fig. S21). 45, 63—meningiomas, 140—hypophyseal adenoma, 12,14—squamous cell lung cancer, 17—bronchus cancer III stage, 108 –stomach cancer, 30—stage IV chronic lymphacytic leukemia, 31—stage IV non-Hodgkin T-cell lymphoma, 67—lymphoadenpathy of unclear pathogenesis, 82—stage II non-Hodgkin lymphoma, stage II, 92—stage IV Hodgkin’s lymphoma, 94—hemolythic anaemia of unclear pathogenesis, 102—stage II non-Hodgkin lymphoma, 113T—stage IV non-Hodgkin lymphoma, K−—PCR with no template, K+—PCR with human DNA. (Full-length gel Fig. S22).

Fig. 8

Two-round amplification of the studied transcript on the cDNA from different clinical tumor subjects. 1—lymphadenopathy of unknown origin (67), 2—non-Hodgkin’s lymphoma at stage II (82), 3—non-Hodgkin’s lymphoma at stage IV (113), 4—invasive ductal breast cancer at stage II (246), 6—negative control, first round of PCR, no template added, 7—negative control, second round of PCR, no template added, 8—positive control, first round of PCR with plasmid containing full transcript of newly identified gene 9—positive control, second round of PCR with plasmid containing full transcript of newly identified gene M1—GeneRuler™ 1 kb DNA ladder (Fermentas).