ITRM20100256A1 - A NEW BIOMARCER FOR EARLY DIAGNOSIS AND CANCER IMMUNOTHERAPY - Google Patents
A NEW BIOMARCER FOR EARLY DIAGNOSIS AND CANCER IMMUNOTHERAPY Download PDFInfo
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- ITRM20100256A1 ITRM20100256A1 IT000256A ITRM20100256A ITRM20100256A1 IT RM20100256 A1 ITRM20100256 A1 IT RM20100256A1 IT 000256 A IT000256 A IT 000256A IT RM20100256 A ITRM20100256 A IT RM20100256A IT RM20100256 A1 ITRM20100256 A1 IT RM20100256A1
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Classifications
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57423—Specifically defined cancers of lung
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Description
DESCRIZIONE DESCRIPTION
Il tumore al polmone rappresenta senza dubbio la più frequente neoplasia in uomini e donne. Si stimano circa 200.000 morti dovute al tumore al polmone ogni anno nei soli Stati Uniti. Tra tutti gli stadi e le tipologie di tumore al polmone, la sopravvivenza a 5 anni è solo del 15%. La prognosi peggiore è quella del carcinoma polmonare non a cellule piccole ( Non-small celi lung cancer, NSCLC) che rappresenta approssimativamente l 80% di tutti i casi di tumore al polmone. Per questi motivi, c’è assoluta necessità di sviluppare nuovi biomarcatori del tumore al polmone e identificare nuovi bersagli specifici per nuove terapie. Lung cancer is undoubtedly the most frequent malignancy in men and women. There are an estimated 200,000 deaths from lung cancer each year in the United States alone. Among all stages and types of lung cancer, 5-year survival is only 15%. The worst prognosis is non-small cell lung cancer (NSCLC) which accounts for approximately 80% of all lung cancer cases. For these reasons, there is an absolute need to develop new lung cancer biomarkers and identify new specific targets for new therapies.
Una delle caratteristiche peculiari di molti tumori è che spesso esprimono proteine diverse da quelle dei tessuti normali. L’origine di tali proteine è attribuibile in larga parte ad uno o più dei seguenti meccanismi: instabilità e cambiamenti del genoma in seguito a delezioni, inversioni, traslocazioni; fenomeni epigenetici che portano a cambiamenti dei livelli di trascrizione genica; o a meccanismi di splìcing alternativo diversi da quelli che avvengono nelle cellule normali. Tutti questi processi possono dare origine a nuove proteine normalmente inesistenti o espresse a livelli inferiori in cellule normali, e che hanno la potenzialità di essere utilizzate come biomarcatori specifici del tumore e/o come nuovi bersagli di terapie oncologiche. Infatti, in seguito ad una delezione cromosomica, un’inversione o una traslocazione, porzioni di geni contenenti elementi regolatori dell’espressione come promotori della trascrizione e/o enhancer possono creare nuovi geni ibridi costituiti da regioni cromosomiche normalmente non adiacenti e generare fusioni geniche specifiche nelle cellule tumorali. In un numero crescente di scoperte, infatti, vengono individuate aberrazioni cromosomiche in tumori ematologici ed epiteliali, che generano fusioni geniche. Tra gli ultimi, fusioni ricorrenti caratterizzano, tra gli altri, sottogruppi di tumori alla prostata, al seno, al polmone e al rene {Curr Opin Genet & Develop, 2009, 19: 82-91). Nella prostata, ad esempio, sono state descritte fusioni ricorrenti tra geni che codificano per i fattori di trascrizione della famiglia Ets, ERG ed ETV1 e l androgen-regulated transmembrane serine protease, TMPRSS2. Tali fusioni, sono successivamente emerse come li maggiore fattore causale nella tumorigenesi della prostata. Recentemente, inoltre, chimerismi indotti dai meccanismi di trascrizione o di post-trascrizione sono stati osservati nei geni dei mammiferi. In particolare, una serie di condizioni ambientali e di fattori cellulari possono influenzare lo splicing. Nel cancro, infatti, il meccanismo di splicing è significativamente alterato e presenta un repertorio di varianti molto diverso da quello dei tessuti normali. Molte di queste derivano da geni che hanno un ruolo riconosciuto nella carcinogenesi e nella progressione tumorale e per questo si pensa che lo splicing alternativo possa avere un ruolo patogenico nel cancro. La figura 1 mostra i possibili chimerismi nel trascrittoma di cellule tumorali. Nel pannello A viene mostrato il cis- e trans -splicing intergenico. Nel pannello B sono indicate fusioni oncogeniche associate a mutazioni. Da una recente analisi della trascrizione genica con la tecnica del 5 '-RACE e dei microarrays, è emerso che circa il 50 per cento degli RNA messaggeri è costituito da esoni al 5' che possono formare giunzioni con altri esoni presenti in geni distanti fino a 200 kb di distanza sullo stesso cromosoma ( cis-splicing ) ovvero anche su cromosomi diversi ( trans -splicing). Ad esempio il cis-splicing intergenico tra i geni MDS1 e EVI sul cromosoma 3q è stato frequentemente osservato nei casi di leucemia acuta {Proc Nati Acad Sci USA 1996;93:1642-7). Inoltre, un RNA chimerico derivato dalla fusione tra il gene JAZF1 presente sul cromosoma 7pl5 ed il gene JJAZ1 sito sul cromosoma 17ql 1, teoricamente attribuibile a un potenziale riarrangiamento t(7; 17) e che tradotto dà origine alla proteina JAZF1/JJAZ1 con funzioni antiapoptotiche, è stato recentemente isolato in campioni di tumore stromale dell’endometrio con cromosomi 7 e 17 intatti per citogenetica, dimostrando l’evento del meccanismo di trans-splicing in tali cellule tumorali ( Science . 2008 Sep 5;321: 1357-61). Anche nei tumori al polmone questa è la direzione della ricerca scientifica: 1) è stata dimostrata l’espressione anormale dei regolatori dello splìcing come ASF/SF2 ed altre ribonucleoproteine (J Thorac Oncol. 2009 Jun;4:674-8)\ 2) ben 2369 su 17 800 geni noti generano forme alternative di splicing; e 3) circa il 30% di queste ha funzioni oncogeniche (Nucleic Acids Res. One of the unique characteristics of many tumors is that they often express proteins that are different from those of normal tissues. The origin of these proteins is largely attributable to one or more of the following mechanisms: instability and changes in the genome following deletions, inversions, translocations; epigenetic phenomena that lead to changes in the levels of gene transcription; or to alternative splitting mechanisms other than those occurring in normal cells. All of these processes can give rise to new proteins that are normally non-existent or expressed at lower levels in normal cells, and which have the potential to be used as tumor-specific biomarkers and / or as novel targets for cancer therapies. In fact, following a chromosomal deletion, an inversion or a translocation, portions of genes containing expression regulatory elements such as transcription promoters and / or enhancers can create new hybrid genes consisting of normally non-adjacent chromosomal regions and generate specific gene fusions. in cancer cells. In fact, in an increasing number of discoveries, chromosomal aberrations are identified in hematological and epithelial tumors, which generate gene fusions. Among the latter, recurrent fusions characterize, among others, subgroups of prostate, breast, lung and kidney cancers (Curr Opin Genet & Develop, 2009, 19: 82-91). In the prostate, for example, recurrent fusions have been described between genes encoding transcription factors of the Ets family, ERG and ETV1 and the androgen-regulated transmembrane serine protease, TMPRSS2. These fusions subsequently emerged as the major causative factor in prostate tumorigenesis. Furthermore, chimerisms induced by transcription or post-transcription mechanisms have recently been observed in mammalian genes. In particular, a number of environmental conditions and cellular factors can influence splicing. In fact, in cancer, the splicing mechanism is significantly altered and presents a repertoire of variants very different from that of normal tissues. Many of these derive from genes that have a recognized role in carcinogenesis and tumor progression and therefore it is thought that alternative splicing may have a pathogenic role in cancer. Figure 1 shows the possible chimerisms in the transcriptome of tumor cells. Panel A shows intergenic cis- and trans-splicing. Panel B shows oncogenic fusions associated with mutations. From a recent analysis of gene transcription with the 5 '-RACE technique and microarrays, it emerged that about 50 percent of messenger RNAs are made up of 5' exons that can form junctions with other exons present in genes up to 200 kb of distance on the same chromosome (cis-splicing) or also on different chromosomes (trans-splicing). For example, intergenic cis-splicing between the MDS1 and EVI genes on chromosome 3q has been frequently observed in cases of acute leukemia (Proc Nati Acad Sci USA 1996; 93: 1642-7). Furthermore, a chimeric RNA derived from the fusion between the JAZF1 gene present on chromosome 7pl5 and the JJAZ1 gene located on chromosome 17ql 1, theoretically attributable to a potential rearrangement t (7; 17) and which translated gives rise to the JAZF1 / JJAZ1 protein with functions antiapoptotic, was recently isolated in endometrial stromal tumor specimens with intact chromosomes 7 and 17 by cytogenetics, demonstrating the event of the trans-splicing mechanism in these tumor cells (Science. 2008 Sep 5; 321: 1357-61) . Also in lung tumors this is the direction of scientific research: 1) the abnormal expression of splitting regulators such as ASF / SF2 and other ribonucleoproteins has been demonstrated (J Thorac Oncol. 2009 Jun; 4: 674-8) \ 2) 2369 out of 17 800 known genes generate alternative forms of splicing; and 3) about 30% of these have oncogenic functions (Nucleic Acids Res.
2008 November; 36: 6535-6547 ). 2008 November; 36: 6535-6547).
La sequenza del peptide ottamerico RTNKEASI (qui denominato EASI-PRO) isolato alcuni decenni fa {J Celi Physiol. 2009 Oct; 22 1:26-30) non corrisponde ad alcuna proteina presente in banca dati. Inoltre, un cDNA parziale di 300 bp (SEQ ID: 1) è stato ottenuto per Polymerase Chain Reaction (PCR) utilizzando oligonucleotidi degenerati codificanti per Γ ottamero. Tale cDNA presenta una cornice di lettura aperta {open reading frame - ORF) di 93 aa che, anche in questo caso, non è stata ritrovata nell’attuale banca dati delle proteine. Questo risultato non permette l’isolamento di sequenze geniche e proteiche più lunghe assolutamente indispensabili per l’ottenimento di reagenti necessari allo sviluppo di diagnostici e di terapeutici che pertanto permettano la definizione di un nuovo biomarcatore. Da tenere presente che varie preparazioni di anticorpi generati contro il peptide EASI-PRO, hanno generato dati positivi preliminari di immunoistochimica (IHC) e di microarray di tessuti tumorali (TMA) in lesioni di polmone e di colon, evidenziando il concetto che la proteina riconosciuta potrebbe essere espressa in modo selettivo in un'alta percentuale di tumori in confronto a tessuti normali. La mancanza di un allineamento con una sequenza proteica nota è ancora più sorprendente alla luce del fatto che negli anni recenti è stata determinata la sequenza dell’intero genoma umano e molte più proteine ed ORF siano state caratterizzate. The sequence of the octameric peptide RTNKEASI (here called EASI-PRO) isolated some decades ago {J Celi Physiol. 2009 Oct; 22 1: 26-30) does not match any protein in the database. Furthermore, a partial cDNA of 300 bp (SEQ ID: 1) was obtained for Polymerase Chain Reaction (PCR) using degenerated oligonucleotides encoding for Γ octamer. This cDNA has an open reading frame (ORF) of 93 aa which, also in this case, has not been found in the current protein database. This result does not allow the isolation of longer gene and protein sequences that are absolutely essential for obtaining the reagents necessary for the development of diagnostics and therapeutics that therefore allow the definition of a new biomarker. It should be noted that various antibody preparations generated against the EASI-PRO peptide generated preliminary positive immunohistochemical (IHC) and tumor tissue microarray (TMA) data in lung and colon lesions, highlighting the concept that the recognized protein it could be selectively expressed in a high percentage of tumors compared to normal tissues. The lack of alignment with a known protein sequence is even more surprising in light of the fact that in recent years the sequence of the entire human genome has been determined and many more proteins and ORFs have been characterized.
La presente invenzione consiste in nuovi metodi per la rivelazione di un RNA messaggero (SEQ ID: 2) non canonico e specifico delle cellule tumorali rispetto alle cellule normali dello stesso tessuto, che unisce due regioni geniche distinte di cui una è localizzata nel cromosoma 1 umano, l’altra è localizzata preferenzialmente ma non limitatamente nel cromosoma 1 umano. In una applicazione specifica, questo RNA messaggero dà origine ad una nuova proteina di fusione (SEQ ID: 3), precedentemente ignota, contenente al suo interno il peptide RTNKEASI, dove i primi cinque residui aminoacidici (RTNKE) risiedono preferibilmente, ma non limitatamente, al interno del gene hmm 1820564 e gli altri tre (ASI) sono localizzati in una ORF localizzata all’interno di una regione intergenica, e pertanto in precedenza considerata come una regione di DNA non codificante per nessuna proteina. The present invention consists of novel methods for the detection of a non-canonical and tumor cell specific messenger RNA (SEQ ID: 2) compared to normal cells of the same tissue, which unites two distinct gene regions, one of which is located on human chromosome 1 , the other is preferentially but not limited to human chromosome 1. In a specific application, this messenger RNA gives rise to a new fusion protein (SEQ ID: 3), previously unknown, containing the RTNKEASI peptide, where the first five amino acid residues (RTNKE) preferably, but not limitedly, reside. within the hmm 1820564 gene and the other three (ASI) are located in an ORF located within an intergenic region, and therefore previously considered as a DNA region not coding for any protein.
La rilevazione di questo RNA atipico e/o della sua corrispondente sequenza proteica consentirà di fornire nuovi marcatori diagnostici e prognostici che possano contribuire alla diagnosi precoce del cancro, al monitoraggio della risposta alla terapia e all'individuazione della malattia residua minima. Inoltre, da tale scoperta si possono sviluppare terapie mirate ad inibire la funzione della proteina e vaccini antitumorali. The detection of this atypical RNA and / or its corresponding protein sequence will allow to provide new diagnostic and prognostic markers that can contribute to the early diagnosis of cancer, the monitoring of response to therapy and the identification of minimal residual disease. Furthermore, from this discovery, therapies aimed at inhibiting the function of the protein and anticancer vaccines can be developed.
Gli esempi che seguono illustrano l’invenzione in maggiore dettaglio: ESEMPIO 1 The following examples illustrate the invention in greater detail: EXAMPLE 1
Analisi Bioinformatica Bioinformatics Analysis
Da una consultazione in banca dati utilizzando il motore di ricerca BLASTn (http://blast.ncbi.nlm.nih.gov/Blast.cgi) abbiamo constatato per la prima volta in letteratura che la maggior parte della sequenza del cDNA codificante per EASI-PRO è identica ad una porzione del cromosoma 1 umano. In particolare, la sovrapposizione dei nucleotidi 16-300 del cDNA aviene sul contig genomico GRCh37 del cromosoma 1 di Homo sapiens Allineamento della sequenza nucleotidica del cDNA codificante per EASI-PRO è mostrato in Fig. 2. Il cDNA (query) è stato analizzato tramite il motore di ricerca BLASTn. In particolare, l’allineamento del contig avviene tra la posizione 36,909K e la 37,009K bp all’interno della banda citogenica lp34.2 (Fig. 3). In tale regione, non sono predette dal modello Gnomon sequenze geniche complete o parziali (Fig. 4, Model). I geni noti più vicini alla sequenza sono il colony stimulating factor 3 receptor isoform b che si trova 14253 bp al 5' e il glutamate receptor, ionotropic, kainate 3 posizionato 307818 bp al 3' (Fig. 2 e 4). La figura 4 mostra la posizione dei loci associati con fenotipi (JPheno) descritti nelle banche dati Online Mendelian Inheritance in Man (OMIM) e quantitative trait loci (QTLs). E’ interessante notare che modifiche a carico di questa regione sono state osservate per l’Inflammatory Bowel Disease-7 (IBD-7). Tra le numerose mutazioni osservate in questa regione (si veda Morbid, Fig. 4), alcune sono associate a poliposi multiple del colon e del retto con ereditarietà autosomica recessiva causata da mutazioni in omozigosi del gene MYH, che codifica per una DNA glicosilasi responsabile del riparo del danno ossidativo del DNA. From a consultation in the database using the BLASTn search engine (http://blast.ncbi.nlm.nih.gov/Blast.cgi) we found for the first time in the literature that most of the sequence of the cDNA encoding for EASI -PRO is identical to a portion of the human chromosome 1. In particular, the overlap of the 16-300 nucleotides of the cDNA occurs on the genomic contig GRCh37 of chromosome 1 of Homo sapiens Alignment of the nucleotide sequence of the cDNA coding for EASI-PRO is shown in Fig. 2. The cDNA (query) was analyzed via the search engine BLASTn. In particular, the alignment of the contig occurs between the position 36.909K and 37.009K bp within the cytogenic band lp34.2 (Fig. 3). In this region, complete or partial gene sequences are not predicted by the Gnomon model (Fig. 4, Model). The closest known genes to the sequence are the colony stimulating factor 3 receptor isoform b located 14253 bp at 5 'and the glutamate receptor, ionotropic, kainate 3 positioned 307818 bp at 3' (Fig. 2 and 4). Figure 4 shows the location of the loci associated with phenotypes (JPheno) described in the Online Mendelian Inheritance in Man (OMIM) and quantitative trait loci (QTLs) databases. It is interesting to note that changes in this region have been observed for Inflammatory Bowel Disease-7 (IBD-7). Among the numerous mutations observed in this region (see Morbid, Fig. 4), some are associated with multiple polyposis of the colon and rectum with autosomal recessive inheritance caused by homozygous mutations of the MYH gene, which encodes a DNA glycosylase responsible for the repair of oxidative DNA damage.
Tale osservazione dimostra che la SEQ ID: 1 è in larga parte presente in una regione intergenica del cromosoma 1, coinvolta in talune patologie. Da questa porzione di cromosoma, tuttavia, viene trascritto un RNA messaggero specifico in campioni tumorali. This observation demonstrates that SEQ ID: 1 is largely present in an intergenic region of chromosome 1, involved in certain pathologies. From this portion of the chromosome, however, a specific messenger RNA is transcribed in tumor samples.
ESEMPIO 2 EXAMPLE 2
Analisi Bioinformatica Bioinformatics Analysis
In seguito ad una ricerca nella banca dati descritta sopra, si osserva che i primi 15 nucleotidi del cDNA danno una sovrapposizione con centinaia di sequenze contenute nel genoma umano, di cui un sottoinsieme è presente all’ interno del cromosoma 1. Tra queste ultime, le sovrapposizioni fisicamente più vicine alla regione contenente i nucleotidi 16-300 sono state selezionate (Fig. 5). Tali regioni si trovano a distanze variabili dai geni del colony stimulating factor 3 receptor isoform b e del glutamate receptor, ionotropic, kainate. In particolare, 3 di esse (si vedano le sequenze 6, 7 e 8 della Fig. 5) mappano al interno dell’ultimo gene. La figura 6 mostra la posizione di alcune tra queste sovrapposizioni in formato grafico. Risulta evidente che 2 di esse (Blast Hits), in particolare le sovrapposizioni 3 e 4, mappano all’intemo del gene hmml820564, mai identificato sperimentalmente ma predetto dall’ algoritmo Gnomon e del quale non è stato ipotizzato sinora alcun ruolo o funzione. Following a search in the database described above, it is observed that the first 15 nucleotides of the cDNA overlap with hundreds of sequences contained in the human genome, of which a subset is present within chromosome 1. Among these, the overlaps physically closest to the region containing 16-300 nucleotides were selected (Fig. 5). These regions are located at variable distances from the colony stimulating factor 3 receptor isoform b and glutamate receptor, ionotropic, kainate genes. In particular, 3 of them (see sequences 6, 7 and 8 of Fig. 5) map within the last gene. Figure 6 shows the location of some of these overlays in graphic format. It is clear that 2 of them (Blast Hits), in particular the overlaps 3 and 4, map within the hmml820564 gene, never experimentally identified but predicted by the Gnomon algorithm and of which no role or function has been hypothesized so far.
Tale analisi bioinformatica suggerisce che eventi di riarrangiamento molecolare tra una di queste sequenze e la regione intergenica del cromosoma 1 descritta nell’esempio 1, possano dare origine ad un mRNA che nel suo interno contiene una sequenza che codifica per il peptide EASI-PRO specifico delle cellule tumorali. This bioinformatic analysis suggests that molecular rearrangement events between one of these sequences and the intergenic region of chromosome 1 described in example 1, may give rise to an mRNA which contains within it a sequence that codes for the EASI-PRO peptide specific to cancer cells.
ESEMPIO 3 EXAMPLE 3
RT-PCR RT-PCR
Dall’analisi del cDNA clonato è possibile osservare che un potenziale sito di splicing è presente tra i codoni codificanti il peptide ottamerico RTNKEASI e in particolare tra i codoni per E ed A (Fig. 7). Da questa osservazione nasce l’ipotesi che uno splicing alternativo (cis -splicing o trans-splicing) che avviene solo nel contesto delle cellule tumorali, possa generare una proteina unica e specifica. From the analysis of the cloned cDNA it is possible to observe that a potential splicing site is present between the codons encoding the octameric peptide RTNKEASI and in particular between the codons for E and A (Fig. 7). From this observation, the hypothesis arises that an alternative splicing (cis-splicing or trans-splicing) that occurs only in the context of cancer cells, can generate a unique and specific protein.
A tale scopo, abbiamo selezionato le sequenze delle regioni cromosomiche al fianco delle sovrapposizioni presenti nel gene presunto hmml820564 (SEQ ID: 4 e 6, sequenze invertite e complementari) e abbiamo disegnato degli oligonucleotidi per effettuare una RT-PCR e verificare sperimentalmente se avviene un evento di splicing alternativo che includa tali sequenze. In particolare, la SEQ ID: 5 e la SEQ ID: 7, derivate dalle SEQ ID: 4 e 6, rispettivamente, sono state utilizzate in coppia con un oligonucleotide disegnato all’intemo del cDNA contenente EASI-PRO (SEQ ID: 8). In particolare, l’RNA totale è stato estratto da 3 campioni di tessuto tumorale di NSCLC e 3 biopsie di polmone normale con il sistema Qiagen RNeasy columns (QIAGEN) e retrotrascritto con il Reverse Transcription System della PROMEGA. Una reazione di PCR è stata poi eseguita per 35 cicli (1 min a 95°C, 2 min a 50°C e 1 min a 72°C) utilizzando le combinazioni degli oligonucleotidi SEQ ID: 5 e 8 e SEQ ID: 7 e 8 . I prodotti di PCR sono stati analizzati per elettroforesi con un gel d’agarosio all’ 1%, contenente etidio bromuro. Una banda di circa 400 coppie di basi, corrispondente a SEQ ID: 2 è stata amplificata con la combinazione SEQ ID: 7 e 8, ma solo da RNA estratto da 2 su 3 campioni tumorali e non dai tessuti normali. Nessuna amplificazione è stata ottenuta con la combinazione SEQ ID: 5 e 8. Tale osservazione dimostra che è avvenuto un fenomeno di splicing tra due regioni trascritte situate ambedue nel cromosoma 1 ma localizzate a distanza: la prima situata all’ interno del gene hmml820564, la seconda situata in una regione comunemente considerata intergenica tra la posizione 36,909K e la 37,009K bp all’intemo della banda citogenica lp34. Tale meccanismo può avvenire all’intemo dello stesso pre-mRNA, ipotizzando una sequenza intronica di circa 167kb (cis -splicing) ovvero uno splicing tra due diversi pre-mRNA (trans-splicing). For this purpose, we have selected the sequences of the chromosomal regions alongside the overlaps present in the presumed gene hmml820564 (SEQ ID: 4 and 6, inverted and complementary sequences) and we have designed oligonucleotides to perform an RT-PCR and experimentally verify if a alternative splice event that includes such sequences. In particular, SEQ ID: 5 and SEQ ID: 7, derived from SEQ ID: 4 and 6, respectively, were used in tandem with an oligonucleotide drawn inside the cDNA containing EASI-PRO (SEQ ID: 8) . In particular, total RNA was extracted from 3 NSCLC tumor tissue samples and 3 normal lung biopsies with the Qiagen RNeasy columns (QIAGEN) system and back-transcribed with the PROMEGA Reverse Transcription System. A PCR reaction was then performed for 35 cycles (1 min at 95 ° C, 2 min at 50 ° C and 1 min at 72 ° C) using the combinations of the oligonucleotides SEQ ID: 5 and 8 and SEQ ID: 7 and 8. The PCR products were analyzed by electrophoresis with a 1% agarose gel containing ethidium bromide. A band of approximately 400 base pairs, corresponding to SEQ ID: 2 was amplified with the combination SEQ ID: 7 and 8, but only by RNA extracted from 2 of 3 tumor samples and not from normal tissues. No amplification was obtained with the combination SEQ ID: 5 and 8. This observation demonstrates that a splicing phenomenon occurred between two transcribed regions located both on chromosome 1 but located at a distance: the first located inside the hmml820564 gene, the second located in a region commonly considered intergenic between the 36.909K and 37.009K bp position within the cytogenic band lp34. This mechanism can occur within the same pre-mRNA, assuming an intronic sequence of about 167kb (cis-splicing) or a splicing between two different pre-mRNAs (trans-splicing).
Lista delle sequenze List of sequences
SEQ ID: 1 SEQ ID: 1
ACCAACAAAG AAGCCTCCAT CTGTCCATCC GTATATCTAT CCACCCTACC ATCCATCCAT TCACCCACTA ATTCATCCAT TTATTATCCA TGCATCCATC TGTCCATAAG TCTATCCGTC CACCCACCAC TTATCCATCC ATCCATTTAC CCATCATACT CATCCATTCA TTCATCCAGC CACCACCCAT GCACTCACCT ATCCACCCAT TCAGTCATTA ATCCAGTAAA AAATTTTGAG CACCTACTAC CAATCAGGCC CTGCACTTGG ACCTTAGGGT AGTGTGTAAA TAAAACC<'>CCA ACCAACAAAG AAGCCTCCAT CTGTCCATCC GTATATCTAT CCACCCTACC ATCCATCCAT TCACCCACTA ATTCATCCAT TTATTATCCA TGCATCCATC TGTCCATAAG TCTATCCGTC CACCCACCAC TTATCCATCC ATCCATTTAC CCATCATACT CATCCATTCA TTCATCCAGC CACCACCCAT GCACTCACCT ATCCACCCAT TCAGTCATTA ATCCAGTAAA AAATTTTGAG CACCTACTAC CAATCAGGCC CTGCACTTGG ACCTTAGGGT AGTGTGTAAA TAAAACC<'>CCA
SEQ ID: 2 SEQ ID: 2
1 ACACTCTCAG ATCTTGCAGA GAATCTTCCA CACACTCACC TGCACACTAA GTGGTTGGAG ACTCACTCCC CAAGGTGCCA ATTTTCTTAC CATGGTAACA 1 ACACTCTCAG ATCTTGCAGA GAATCTTCCA CACACTCACC TGCACACTAA GTGGTTGGAG ACTCACTCCC CAAGGTGCCA ATTTTCTTAC CATGGTAACA
101 AAACCAACAG AACCAACAAA GAAGCCTCCA TCTGTCCATC CGTATATCTA TCCACCCTAC CATCCATCCA TTCACCCACT AATTCATCCA TTTATTATCC 101 AAACCAACAG AACCAACAAA GAAGCCTCCA TCTGTCCATC CGTATATCTA TCCACCCTAC CATCCATCCA TTCACCCACT AATTCATCCA TTTATTATCC
201 ATGCATCCAT CTGTCCATAA GTCTATCCGT CCACCCACCA CTTATCCATC _ CATCCATTTA CCCATCATAC TCATCCATTC ATTCATCCAG CCACCACCCA 201 ATGCATCCAT CTGTCCATAA GTCTATCCGT CCACCCACCA CTTATCCATC _ CATCCATTTA CCCATCATAC TCATCCATTC ATTCATCCAG CCACCACCCA
301 TGCACTCACC TATCCACCCA TTCAGTCATT AATCCAGTAA AAAATTTTGA GCACCTACTA CCAATCAGGC CCTGCACTTG GACCTTAGGG TAGTGTGTAA 301 TGCACTCACC TATCCACCCA TTCAGTCATT AATCCAGTAA AAAATTTTGA GCACCTACTA CCAATCAGGC CCTGCACTTG GACCTTAGGG TAGTGTGTAA
401 ATAAAACCCC A 401 ATAAAACCCC A
In nero: sequenza del gene hmml820564 In black: gene sequence hmml820564
In rosso: sequenza del cDNA in SEQ ID: 1 In red: cDNA sequence in SEQ ID: 1
SEQ ID : 3 SEQ ID: 3
1 TLSDLAENLP HTHLHTKWLE THSPRCQFSY HGNKTNRTNK EASICPSVYL 1 TLSDLAENLP HTHLHTKWLE THSPRCQFSY HGNKTNRTNK EASICPSVYL
51 STLPSIHSPT NSSIYYPCIH LSISLSVHPP LIHPSIYPSY SSIHSSSHHP 101 CTHLSTHSVI NPVKNFEHLL PIRPCTWTLG 51 STLPSIHSPT NSSIYYPCIH LSISLSVHPP LIHPSIYPSY SSIHSSSHHP 101 CTHLSTHSVI NPVKNFEHLL PIRPCTWTLG
In nero: sequenza del polipeptide derivata dal gene hmml820564 In black: polypeptide sequence derived from the hmml820564 gene
In rosso: sequenza del polipeptide derivata dal cDNA in SEQ ID:1 In red: cDNA-derived polypeptide sequence in SEQ ID: 1
SEQ ID: 4 SEQ ID: 4
Posizione 7104088-7104277 (reverse complemen&) Position 7104088-7104277 (reverse complement)
CCTGAAAGACT AGAAC AGCTTCT AATGGGGGAGGT GGGCAT GAAAAGACAGAGGAAAAGGGAAAGGCA CTGATCCCGGGCTGGAGGGGAGCATGAGGTAGTCATCAGGGATGATGCTTCATGGTTGGTTGTGGAGG GTTCTTGGCGGCCAAAGAGTTTCCAGTGACTACGAGGGATCAGAGGCAAAGAAGCC CCTGAAAGACT AGAAC AGCTTCT AATGGGGGAGGT GGGCAT GAAAAGACAGAGGAAAAGGGAAAGGCA CTGATCCCGGGCTGGGGGGGAGCATGAGGTAGTCATCAGGGATGATGCTTCATGGTTGCCGTAGTGTGTAGCAGGTCAGGTAGTAGTAGCAGCAGGTAGGTAGTAGTAGC
SEQ ID: 5 SEQ ID: 5
CCTG AAAGACTAGAAC AGC CCTG AAAGACTAGAAC AGC
SEQ ID: 6 SEQ ID: 6
Posizione 7098577-7098700 (reverse complement) ACACTCTCAGATCTTGCAGAGAATCTTCCACACACTCACCTGCACACTAAGTGGTTGGAGACTCACTC CCCAAGGTGCCAATTTTCTTACCATGGTAACAAAACCAACAGAACCAACAAAGAA Position 7098577-7098700 (reverse complement) ACACTCTCAGATCTTGCAGAGAATCTTCCACACACTCACCTGCACACTAAGTGGTTGGAGACTCACTC CCCAAGGTGCCAATTTTCTTACCATGGTAACAAAACCAACAGAACCAACAAAGAA
SEQ ID: 7 SEQ ID: 7
ACACTCTCAGATCTTGC ACACTCTCAGATCTTGC
SEQ ID: 8 SEQ ID: 8
GGGGTTTTATTTACACACTACCC GGGGTTTTATTTACACACTACCC
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WO2001062786A2 (en) * | 2000-02-25 | 2001-08-30 | Unihart Corporation | Tlp peptides and dna sequences coding the same |
WO2003002591A2 (en) * | 2001-06-29 | 2003-01-09 | Unihart Corporation | Tlp antigen extract, its preparation and diagnostic applications thereof |
WO2003045997A2 (en) * | 2001-11-30 | 2003-06-05 | Unihart Corporation | Fusion proteins containing tlp peptides |
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WO2011145125A1 (en) | 2011-11-24 |
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