IT201800005807A1 - MODULATORS OF THE EXPRESSION OF THE FOXC2 GENE - Google Patents
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Description
“MODULATORI DELL’ESPRESSIONE DEL GENE FOXC2” "FOXC2 GENE EXPRESSION MODULATORS"
DESCRIZIONE DESCRIPTION
Campo tecnico dell’invenzione Technical field of the invention
La presente invenzione si riferisce ad una sequenza nucleotidica complementare alla sequenza umana lncRNA FOXC2-AS1 (denominata FOXC2-SAS) ed al suo uso per ridurre l’espressione della proteina FOXC2. Sono oggetto della presente invenzione anche vettori di espressione contenenti FOXC2-SAS, cellule e composizioni farmaceutiche relative a tali sequenze nucleotidiche. The present invention refers to a nucleotide sequence complementary to the human lncRNA FOXC2-AS1 sequence (called FOXC2-SAS) and its use to reduce the expression of the FOXC2 protein. Also object of the present invention are expression vectors containing FOXC2-SAS, cells and pharmaceutical compositions relating to such nucleotide sequences.
Stato della tecnica anteriore State of the prior art
Le molecole di tipo “Long non-coding RNAs” (lncRNAs) sono un gruppo eterogeneo di trascritti, cioè molecole di RNA, in grado di esplicare diverse funzioni, legate per esempio a meccanismi di regolazione epigenetica e di controllo dell’espressione genica. Le molecole lncRNAs sono lunghe più di 200 nucleotidi e non vengono tradotte in proteine, quindi non hanno capacità codificante. Nell’uomo sono state identificate circa 20.000 lncRNAs. Molecules of the "Long non-coding RNAs" type (lncRNAs) are a heterogeneous group of transcripts, that is, RNA molecules, capable of performing various functions, related for example to mechanisms of epigenetic regulation and gene expression control. Molecules lncRNAs are more than 200 nucleotides long and are not translated into proteins, so they have no coding capacity. About 20,000 lncRNAs have been identified in humans.
Sono state identificate varie classi di lncRNAs, tra cui i NATs che sono trascritti antisenso naturali, complementari a regioni codificanti di geni umani. Various classes of lncRNAs have been identified, including NATs which are natural antisense transcripts, complementary to coding regions of human genes.
Tra i lncRNAs di tipo NATs è stato recentemente descritto lncRNA FOXC2-AS1 che è una molecola di RNA non codificante e antisenso naturale del gene FOXC2. Questa macromolecola di RNA è stata identificata anche nell’osteosarcoma umano (Zhang CL et al. Cancer Lett. 2017 Jun 28; 396:66-75). In uno studio condotto da un gruppo di ricercatori dell‘ Università di Shanghai (China) è emerso che lncRNA FOXC2-AS1 è principalmente localizzato nel citoplasma delle cellule tumorali e forma, legandosi alla parte complementare dell’mRNA di FOXC2, una struttura a doppio filamento che regola l’espressione di FOXC2, sia a livello trascrizionale che post trascrizionale (Zhang CL et al. Cancer Lett. 2017 Jun 28;396:66-75). Among the NATs-type lncRNAs, FOXC2-AS1 lncRNA has recently been described, which is a natural non-coding and antisense RNA molecule of the FOXC2 gene. This RNA macromolecule has also been identified in human osteosarcoma (Zhang CL et al. Cancer Lett. 2017 Jun 28; 396: 66-75). In a study conducted by a group of researchers from the University of Shanghai (China) it emerged that FOXC2-AS1 lncRNA is mainly localized in the cytoplasm of cancer cells and forms, by binding to the complementary part of FOXC2 mRNA, a double-stranded structure which regulates the expression of FOXC2, both at the transcriptional and post-transcriptional level (Zhang CL et al. Cancer Lett. 2017 Jun 28; 396: 66-75).
In pratica, lncRNA FOXC2-AS1 si lega a RNA FOXC2 determinando una maggiore stabilità ed una maggior produzione della proteina FOXC2. In practice, FOXC2-AS1 lncRNA binds to FOXC2 RNA resulting in greater stability and greater production of the FOXC2 protein.
La proteina FOXC2 (forkhead box C2) è un potentissimo fattore di trascrizione nucleare (codice in banca dati NCBI NM_005251.2 (RNA): → NP_005242.1 (proteina); Ensembl: ENSG00000176692; MIM:602402). La proteina FOXC2 è nota anche con altri nomi: The FOXC2 protein (forkhead box C2) is a very powerful nuclear transcription factor (code in the NCBI database NM_005251.2 (RNA): → NP_005242.1 (protein); Ensembl: ENSG00000176692; MIM: 602402). FOXC2 protein is also known by other names:
MFH-1, mesenchyme forkhead 1 MFH-1, mesenchyme forkhead 1
forkhead box C2 (MFH-1, mesenchyme forkhead 1) forkhead box C2 (MFH-1, mesenchyme forkhead 1)
forkhead, Drosophila, homolog-like 14 forkhead, Drosophila, homolog-like 14
forkhead-related protein FKHL14 forkhead-related protein FKHL14
mesenchyme fork head protein 1 mesenchyme fork head protein 1
mesenchyme forkhead 1 mesenchyme forkhead 1
transcription factor FKH-14 transcription factor FKH-14
La proteina FOXC2 è fondamentale per lo sviluppo embrionale e prenatale. È un importante modulatore dell’angiogenesi e dello sviluppo del sistema linfatico. Inoltre FOXC2 si trova al crocevia del fenomeno di transizione delle proprietà delle cellule tumorali dall‘epitelio al mesenchima (EMT: epitelial-mesenchimal transition). Mutazioni nel gene FOXC2 determinano lo sviluppo del linfedema primario. Il linfedema si associa ad una distensione cronica degli arti, secondaria all'accumulo della linfa negli spazi interstiziali, in particolare nei tessuti sottocutanei e ad una malformazione sistemica o parziale dell'apparato linfatico. Sono stati sviluppati saggi funzionali per la caratterizzazione patogenetica delle diverse mutazioni del gene FOXC2, identificate nei pazienti con linfedema distichiasi (LD). In particolare, è stata verificata la stabilità, la corretta localizzazione intracellulare delle proteine FOXC2 mutate e la loro capacità di attivare un gene reporter. I risultati ottenuti hanno messo in evidenza che alcune di esse causano la perdita o la diminuzione dell’attività di FOXC2, mentre altre portano ad una iperattivazione del fattore di trascrizione (Tavian D et al. Oncotarget 7(34):54228-54239.). Pertanto (nei casi di iperattivazione) potrebbe essere utile ridurre la quantità di proteina FOXC2 espressa. Recentemente è stata messa in evidenza da diversi gruppi di ricerca nazionali ed internazionali una correlazione tra una maggiore quantità di proteina FOXC2 espressa ed il cancro. Questa correlazione è stata dimostrata in diversi tumori umani, per esempio il cancro alla mammella, allo stomaco, al fegato, alle ossa, alla cervice uterina e all’ovaio. Anche in questi casi, potrebbe rivelarsi utile un intervento molecolare che miri ad abbassare il livello della proteina FOXC2 presente nei tessuti tumorali. Allo stato attuale sono state proposte diverse strategie per modulare l’espressione del gene FOXC2; alcuni ricercatori hanno riportato diversi studi in cui sono stati utilizzati dei siRNA per diminuire l’espressione di FOXC2 (Zheng CH et al. Asian Pac J Cancer Prev, 2014;15). FOXC2 protein is critical for embryonic and prenatal development. It is an important modulator of angiogenesis and the development of the lymphatic system. Furthermore, FOXC2 is at the crossroads of the transition phenomenon of the properties of tumor cells from the epithelium to the mesenchyme (EMT: epithelial-mesenchimal transition). Mutations in the FOXC2 gene lead to the development of primary lymphedema. Lymphedema is associated with a chronic distension of the limbs, secondary to the accumulation of lymph in the interstitial spaces, particularly in the subcutaneous tissues and to a systemic or partial malformation of the lymphatic system. Functional assays have been developed for the pathogenetic characterization of the various mutations of the FOXC2 gene, identified in patients with distichiasis (LD) lymphedema. In particular, the stability, the correct intracellular localization of the mutated FOXC2 proteins and their ability to activate a reporter gene were verified. The results obtained have highlighted that some of them cause the loss or decrease of FOXC2 activity, while others lead to a hyperactivation of the transcription factor (Tavian D et al. Oncotarget 7 (34): 54228-54239.) . Therefore (in cases of hyperactivation) it might be useful to reduce the amount of FOXC2 protein expressed. A correlation between a greater quantity of FOXC2 protein expressed and cancer has recently been highlighted by various national and international research groups. This correlation has been demonstrated in various human cancers, for example breast, stomach, liver, bone, cervical and ovarian cancers. Also in these cases, a molecular intervention that aims to lower the level of the FOXC2 protein present in tumor tissues could prove useful. At present, various strategies have been proposed to modulate the expression of the FOXC2 gene; some researchers have reported several studies in which siRNAs were used to decrease the expression of FOXC2 (Zheng CH et al. Asian Pac J Cancer Prev, 2014; 15).
Recentemente è stato dimostrato che il miRNA let-7g regola negativamente l’espressione del gene FOXC2 (Wang L et al. Open Med. 2017; 12: 157-162). Nello studio sopra citato, si dimostra che l’espressione del miRNA definito “let-7g” è inversamente correlata all’espressione di FOXC2. Let-7g è un miRNA che appartiene ad una famiglia di molecole (let-7 family), coinvolte nello sviluppo del carcinoma mammario ed anche epatocellulare. È stato dimostrato che let-7g regola negativamente l’espressione anche di altri geni, come Bcl-xL e i collageni di tipo I e α2. Pertanto l’utilizzo del miRNA let-7g non può escludere ricadute nella modulazione d’espressione anche di altri geni, oltre che di FOXC2. It has recently been shown that the let-7g miRNA negatively regulates the expression of the FOXC2 gene (Wang L et al. Open Med. 2017; 12: 157-162). In the aforementioned study, it is shown that the expression of the miRNA defined as "let-7g" is inversely related to the expression of FOXC2. Let-7g is a miRNA that belongs to a family of molecules (let-7 family), involved in the development of breast and also hepatocellular cancer. It has been shown that let-7g negatively regulates the expression of other genes as well, such as Bcl-xL and type I and α2 collagens. Therefore, the use of let-7g miRNA cannot exclude relapses in the modulation of expression of other genes as well as FOXC2.
Sommario dell’invenzione Summary of the invention
Gli autori della presente invenzione hanno prodotto un vettore in grado di esprimere, in linee cellulari umane, una sequenza di RNA complementare alla sequenza umana di lncRNA FOXC2-AS1. Tale sequenza complementare rappresenta quindi una sequenza senso di un antisenso, che nella presente descrizione è anche definita FOXC2-SAS (Sense of Antisense). Gli inventori hanno scoperto che la sequenza FOXC2-SAS, interagendo con lncRNA FOXC2-AS1, riduce significativamente l’espressione del gene FOXC2 e quindi della proteina FOXC2. L’uso della sequenza FOXC2-SAS non determina la degradazione dell’mRNA di FOXC2, come accade utilizzando strategie di RNA interference. Inoltre è una macromolecola assolutamente specifica per il gene che si vuole silenziare (FOXC2), contrariamente a quanto accade nel caso dei miRNA, per i quali la specificità non è assicurata. Un miRNA può infatti avere effetto su diversi mRNA contemporaneamente. L’utilizzo del vettore di espressione di FOXC2-SAS in cellule HeLa, come riportato in dettaglio nella parte sperimentale della presente descrizione, ha evidenziato una netta riduzione dell’espressione della proteina FOXC2, del tutto paragonabile, se non superiore, alla diminuzione ottenuta con la strategia dei siRNA FOXC2. La molecola FOXC2-SAS ha una sequenza perfettamente complementare a FOXC2-AS1, quindi ha un livello di specificità molto elevato. Inoltre, come si può evincere dai risultati di comparazione di sequenza riportati nella sezione sperimentale, questa molecola non presenta complementarietà con altre molecole di mRNA. Pertanto un primo oggetto della presente invenzione è una sequenza nucleotidica per ridurre l’espressione della proteina FOXC2, in cui detta sequenza nucleotidica comprende o consiste in una sequenza complementare per almeno il 98% alla sequenza lncRNA FOXC2-AS1. Un ulteriore oggetto della presente invenzione è una sequenza nucleotidica come sopra definita per uso in un metodo di trattamento di patologie associate ad un’elevata espressione della proteina FOXC2. The authors of the present invention have produced a vector capable of expressing, in human cell lines, an RNA sequence complementary to the human FOXC2-AS1 lncRNA sequence. This complementary sequence therefore represents a sense sequence of an antisense, which in the present description is also defined as FOXC2-SAS (Sense of Antisense). The inventors have discovered that the FOXC2-SAS sequence, interacting with FOXC2-AS1 lncRNA, significantly reduces the expression of the FOXC2 gene and therefore of the FOXC2 protein. The use of the FOXC2-SAS sequence does not lead to the degradation of FOXC2 mRNA, as occurs using RNA interference strategies. Furthermore, it is an absolutely specific macromolecule for the gene to be silenced (FOXC2), contrary to what happens in the case of miRNAs, for which specificity is not guaranteed. A miRNA can in fact affect several mRNAs at the same time. The use of the FOXC2-SAS expression vector in HeLa cells, as reported in detail in the experimental part of the present description, showed a net reduction of the FOXC2 protein expression, fully comparable, if not greater, to the decrease obtained with the FOXC2 siRNA strategy. The FOXC2-SAS molecule has a perfectly complementary sequence to FOXC2-AS1, so it has a very high level of specificity. Furthermore, as can be seen from the sequence comparison results reported in the experimental section, this molecule does not show complementarity with other mRNA molecules. Therefore, a first object of the present invention is a nucleotide sequence to reduce the expression of the FOXC2 protein, in which said nucleotide sequence comprises or consists of a complementary sequence for at least 98% to the FOXC2-AS1 lncRNA sequence. A further object of the present invention is a nucleotide sequence as defined above for use in a method of treating pathologies associated with a high expression of the FOXC2 protein.
Un ulteriore oggetto della presente invenzione è un vettore per l’espressione in cellule eucariotiche di una sequenza nucleotidica complementare per almeno il 98% alla sequenza lncRNA FOXC2-AS1. Un ulteriore oggetto della presente invenzione è una cellula eucariotica trasformata con un vettore di espressione come sopra definito. A further object of the present invention is a vector for the expression in eukaryotic cells of a nucleotide sequence complementary for at least 98% to the FOXC2-AS1 lncRNA sequence. A further object of the present invention is a eukaryotic cell transformed with an expression vector as defined above.
Altri vantaggi e caratteristiche della presente invenzione risulteranno evidenti dalla seguente descrizione dettagliata. Other advantages and features of the present invention will become apparent from the following detailed description.
Descrizione delle figure Description of the figures
Figura 1: Mappa del plasmide pcDNA3.3-TOPO (Life Technologies), un vettore ad alta espressione per cellule eucariotiche, nel quale è stata clonata la sequenza di FOXC2-SAS (319 nucleotidi). La sequenza FOXC2-SAS è stata inserita tra il promotore Pcmv e la sequenza TK pA. Trasfettato in cellule umane, questo vettore permette l’espressione della molecola FOXC2-SAS. Figure 1: Map of the pcDNA3.3-TOPO plasmid (Life Technologies), a high-expression vector for eukaryotic cells, in which the FOXC2-SAS sequence (319 nucleotides) has been cloned. The FOXC2-SAS sequence was inserted between the Pcmv promoter and the TK pA sequence. Transfected in human cells, this vector allows the expression of the FOXC2-SAS molecule.
Figura 2. Figura 2: (A) Schematizzazione di FOXC2-AS1 e FOXC2-SAS. (B) Meccanismi di interazione tra FOXC2-AS1 e mRNA di FOXC2 e tra FOXC2-AS1 e FOXC2-SAS. Figure 2. Figure 2: (A) Schematic of FOXC2-AS1 and FOXC2-SAS. (B) Mechanisms of interaction between FOXC2-AS1 and FOXC2 mRNA and between FOXC2-AS1 and FOXC2-SAS.
1. la molecola FOXC2-AS1 (319 nt) lega l’mRNA di FOXC2 (1506 nt) nella regione compresa tra i nucleotidi 280 e 426, stabilizzando l’RNA e determinando un maggiore livello di produzione della proteina FOXC2; 2. la molecola FOXC2-SAS (che è una molecola non esistente in natura ed è stata prodotta artificialmente) lega FOXC2-AS1, ostacolando la sua interazione con FOXC2; questo causa una diminuzione della stabilità del trascritto FOXC2 e una diminuzione del livello di proteina FOXC2. 1. the FOXC2-AS1 molecule (319 nt) binds the FOXC2 mRNA (1506 nt) in the region between nucleotides 280 and 426, stabilizing the RNA and resulting in a higher production level of the FOXC2 protein; 2. the FOXC2-SAS molecule (which is a non-existent molecule in nature and has been artificially produced) binds FOXC2-AS1, hindering its interaction with FOXC2; this causes a decrease in the stability of the FOXC2 transcript and a decrease in the FOXC2 protein level.
Figura 3. Espressione di FOXC2 (1) e FOXC2-AS1 (2) in cellule HeLa e espressione di FOXC2-SAS (3) in cellule HeLa trasfettate con il vettore pcDNA-FOXC2-SAS. Il marker utilizzato è il GeneRuler 100 bp DNA Ladder Plus (Fermentas). Figure 3. FOXC2 (1) and FOXC2-AS1 (2) expression in HeLa cells and FOXC2-SAS (3) expression in HeLa cells transfected with the pcDNA-FOXC2-SAS vector. The marker used is the GeneRuler 100 bp DNA Ladder Plus (Fermentas).
Figura 4. Analisi di western blotting dei livelli di proteina FOXC2 e GAPDH in cellule HeLa non trasfettate e trasfettate con il vettore pcDNA-FOXC2-SAS. Figure 4. Western blotting analysis of FOXC2 and GAPDH protein levels in HeLa cells not transfected and transfected with the pcDNA-FOXC2-SAS vector.
Descrizione dettagliata dell’invenzione Detailed description of the invention
È qui descritta una sequenza nucleotidica per ridurre l’espressione della proteina FOXC2. Tale sequenza nucleotidica comprende o consiste in una sequenza complementare per almeno il 98% alla sequenza lncRNA FOXC2-AS1 (SEQ ID NO:4). La complementarità in biologia molecolare si riferisce a molecole di DNA o RNA in grado di formare un doppio filamento o ibridi temporanei tra le due sequenze. Queste infatti consistono di due filamenti complementari di nucleotidi, che non sono legati covalentemente, bensì connessi via due o tre legami idrogeno. Per ogni nucleotide esiste un nucleotide complementare specifico. L’Adenina complementa con la Timina (nel DNA) e l’Uracile (nell’RNA), mentre la Guanina complementa con la Citosina sia nel DNA che nell’RNA. A nucleotide sequence is described here to reduce the expression of the FOXC2 protein. This nucleotide sequence comprises or consists of a sequence complementary for at least 98% to the lncRNA FOXC2-AS1 sequence (SEQ ID NO: 4). Complementarity in molecular biology refers to DNA or RNA molecules capable of forming a double-stranded or temporary hybrids between the two sequences. These in fact consist of two complementary strands of nucleotides, which are not covalently linked, but connected via two or three hydrogen bonds. For each nucleotide there is a specific complementary nucleotide. Adenine complements with Thymine (in DNA) and Uracil (in RNA), while Guanine complements with Cytosine in both DNA and RNA.
Il termine "sequenza di acido nucleico" o "sequenza nucleotidica", secondo la presente invenzione, include DNA, come cDNA o, in una forma di realizzazione preferita, DNA genomico e RNA. Resta inteso che il termine "RNA" come qui utilizzato comprende tutte le forme di RNA. Il termine "sequenza di acido nucleico" è usato in modo intercambiabile secondo l'invenzione con il termine "polinucleotide". Con il termine complementare per almeno il 98% alla sequenza lncRNA FOXC2-AS1, s’intende che almeno il 98%, preferibilmente almeno il 99%, ancora più preferibilmente il 100% della sequenza è perfettamente complementare alla sequenza lncRNA FOXC2-AS1 (SEQ ID NO:4). Le sequenze di acido nucleico secondo una forma di realizzazione comprendono o consistono nella SEQ ID NO.3, o la corrispondente sequenza di RNA SEQ ID NO.13, in cui le T (Timina) sono sostituite dalle U (Uracile). Fanno parte della presente invenzione anche sequenze con almeno il 98% d’identità con le sequenze SEQ ID NO.3 e SEQ ID NO.13. The term "nucleic acid sequence" or "nucleotide sequence", according to the present invention, includes DNA, such as cDNA or, in a preferred embodiment, genomic DNA and RNA. It is understood that the term "RNA" as used herein includes all forms of RNA. The term "nucleic acid sequence" is used interchangeably according to the invention with the term "polynucleotide". With the term complementary for at least 98% to the FOXC2-AS1 lncRNA sequence, it is meant that at least 98%, preferably at least 99%, even more preferably 100% of the sequence is perfectly complementary to the FOXC2-AS1 lncRNA sequence (SEQ ID NO: 4). The nucleic acid sequences according to one embodiment comprise or consist of the SEQ ID NO.3, or the corresponding RNA sequence SEQ ID NO.13, in which the T (Thymine) are replaced by the U (Uracil). Also part of the present invention are sequences with at least 98% identity with the sequences SEQ ID NO.3 and SEQ ID NO.13.
Sono qui descritti anche vettori per l’espressione in cellule eucariotiche, in particolare in cellule eucariotiche umane, di una sequenza complementare per almeno per il 98% alla sequenza umana di lncRNA FOXC2-AS1. Un qualsiasi tipo di vettore ad espressione eucariotico che permetta la produzione di molecole di RNA può essere utilizzato per costruire vettori che esprimano le sequenze FOXC2-SAS. Un vettore di espressione eucariotico può essere definito come una sequenza di DNA circolare contenente tutti quei segnali capaci di ottimizzare la corretta trascrizione e traduzione dei geni eterologhi nell’ospite in cui avviene l’espressione. Esempi di vettori che possono essere usati per l’espressione di FOXC2-SAS sono il vettore Invitrogen (Life Technologies): pcDNA™3.3 TOPO vector, PROMEGA: pCI Mammalian expression vectors, Stratagene: pcMV-Script vectors, InvivoGen: pMONO expression plasmid p-SELECT, SIGMA-Aldrich: PSF-CMV-Selection plasmids, IBA-lifesciences: pDSG vector series, StarGateAcceptorVector, GenScript: pGen2.1. Un tipico vettore di espressione eucariotico contiene l'elemento promotore, che media l'inizio della trascrizione dell'mRNA, la sequenza codificante della proteina e i segnali richiesti per la terminazione della trascrizione e della poliadenilazione del trascritto. Inoltre, possono essere inclusi anche elementi come l'origine della replicazione, il gene di resistenza ai farmaci, i regolatori (ad esempio come parte di un promotore inducibile). Also described here are vectors for the expression in eukaryotic cells, in particular in human eukaryotic cells, of a sequence complementary for at least 98% to the human FOXC2-AS1 lncRNA sequence. Any type of eukaryotic expression vector that allows the production of RNA molecules can be used to construct vectors expressing FOXC2-SAS sequences. A eukaryotic expression vector can be defined as a circular DNA sequence containing all those signals capable of optimizing the correct transcription and translation of heterologous genes in the host in which the expression takes place. Examples of vectors that can be used for FOXC2-SAS expression are the Invitrogen vector (Life Technologies): pcDNA ™ 3.3 TOPO vector, PROMEGA: pCI Mammalian expression vectors, Stratagene: pcMV-Script vectors, InvivoGen: pMONO expression plasmid p -SELECT, SIGMA-Aldrich: PSF-CMV-Selection plasmids, IBA-lifesciences: pDSG vector series, StarGateAcceptorVector, GenScript: pGen2.1. A typical eukaryotic expression vector contains the promoter element, which mediates the initiation of mRNA transcription, the coding sequence of the protein, and the signals required for transcription and polyadenylation termination of the transcript. In addition, elements such as the origin of replication, drug resistance gene, regulators (e.g. as part of an inducible promoter) can also be included.
Sono anche oggetto della presente invenzione le cellule eucariotiche trasformate con i vettori qui descritti che esprimono le molecole FOXC2SAS. Tali cellule sono ad esempio linee cellulari umane tumorali. Preferibilmente la cellula trasformata sarà una cellula di mammifero come una cellula umana. Le cellule ospiti dei mammiferi che possono essere utilizzate includono cellule umane HeLa, 293, H9 e Jurkat, cellule NIH3T3 e C127, Cos 1, Cos 7 e CV1, cellule QC1-3 di quaglia, cellule L di topo e cellule ovariche di criceto cinese (CHO). La cellula può essere una parte di una linea cellulare, preferibilmente una linea cellulare umana. Sono noti nella tecnica mezzi di coltura appropriati e condizioni per le cellule ospiti sopra descritte. The eukaryotic cells transformed with the vectors described here that express the FOXC2SAS molecules are also object of the present invention. Such cells are for example human tumor cell lines. Preferably the transformed cell will be a mammalian cell such as a human cell. Mammalian host cells that can be used include human HeLa, 293, H9 and Jurkat cells, NIH3T3 and C127, Cos 1, Cos 7 and CV1 cells, quail QC1-3 cells, mouse L cells, and Chinese hamster ovary cells (CHO). The cell may be a part of a cell line, preferably a human cell line. Suitable culture media and conditions for the host cells described above are known in the art.
Sono anche oggetto composizioni farmaceutiche comprendenti le sequenze nucleotidiche o i vettori qui descritti. Le composizioni per uso secondo la presente invenzione potranno essere somministrate mediante qualsiasi mezzo convenzionale disponibile per uso unitamente a farmaci, o come agenti terapeutici individuali o in una combinazione di agenti terapeutici. Essi possono essere somministrati da soli, ma generalmente somministrati con un veicolante farmaceutico scelto sulla base della via di somministrazione scelta e della pratica farmaceutica standard. Il dosaggio somministrato, ovviamente, varierà dipendentemente da fattori noti, come le caratteristiche farmacodinamiche del particolare agente e dalla sua modalità e via di somministrazione; dall'età, salute e peso del ricevente; dalla natura e grado dei sintomi, dal tipo di trattamento concomitante; dalla frequenza di trattamento; e dall'effetto desiderato. L'ingrediente attivo sarà normalmente presente in una quantità di circa 0,1-95% in peso in base al peso totale della composizione Also subject matter are pharmaceutical compositions comprising the nucleotide sequences or vectors described herein. The compositions for use according to the present invention may be administered by any conventional means available for use in conjunction with drugs, or as individual therapeutic agents or in a combination of therapeutic agents. They can be administered alone, but generally administered with a pharmaceutical carrier selected based on the chosen route of administration and standard pharmaceutical practice. The dosage administered will obviously vary depending on known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the age, health and weight of the recipient; the nature and degree of symptoms, the type of concomitant treatment; the frequency of treatment; and the desired effect. The active ingredient will normally be present in an amount of about 0.1-95% by weight based on the total weight of the composition
Le sequenze ed i vettori FOXC2-SAS qui descritti e le composizioni che le comprendono possono trovare utilizzo in tutte le condizioni cellulari in cui si rende necessaria una riduzione dell’espressione della proteina FOXC2. In ambito oncologico è noto che due tipi di tumore contemporaneamente over esprimono FOXC2 e FOXC2-AS1: il tumore alla mammella e l’osteosarcoma (Zhang CL et al. Cancer Lett.2017 Jun 28;396:66-75; Yang H et al. Oncol Res. 2018). Per questi due tipi di tumore è indicato l’utilizzo di vettori FOXC2-SAS. The FOXC2-SAS sequences and vectors described here and the compositions that comprise them can be used in all cellular conditions in which a reduction in the expression of the FOXC2 protein is required. In oncology it is known that two types of tumor simultaneously over express FOXC2 and FOXC2-AS1: breast cancer and osteosarcoma (Zhang CL et al. Cancer Lett. 2017 Jun 28; 396: 66-75; Yang H et al. . Oncol Res. 2018). For these two types of cancer, the use of FOXC2-SAS vectors is indicated.
Inoltre, sono stati pubblicati diversi studi in cui l’over espressione di FOXC2 nelle cellule tumorali è correlata ad una aumentata capacità di proliferazione, di angiogenesi, di invasività tumorale e, in taluni casi, maggiore resistenza ai trattamenti chemioterapici. Recentemente questi studi sono stati sintetizzati in una review: Emerging roles and mechanisms of FOXC2 in cancer. Wang T, Zheng L, Wang Q, Hu YW. Clin Chim Acta. In addition, several studies have been published in which the over expression of FOXC2 in cancer cells is related to an increased capacity for proliferation, angiogenesis, tumor invasiveness and, in some cases, greater resistance to chemotherapy treatments. Recently these studies were summarized in a review: Emerging roles and mechanisms of FOXC2 in cancer. Wang T, Zheng L, Wang Q, Hu YW. Clin Chim Acta.
2018 Apr; 479:84-93. doi: 10.1016/j.cca.2018.01.019. Epub 2018 Jan 16. Review. In sintesi, i tumori umani in cui la proteina FOXC2 è stata trovata over espressa sono riportati di seguito: 2018 Apr; 479: 84-93. doi: 10.1016 / j.cca.2018.01.019. Epub 2018 Jan 16. Review. In summary, human cancers in which FOXC2 protein has been found to be over expressed are shown below:
- cancro al seno - breast cancer
- osteosarcoma - osteosarcoma
- carcinoma epatocellulare - HCC
- cancro alla prostata - prostate cancer
- cancro ovarico - ovarian cancer
- carcinoma a cellule renali - renal cell carcinoma
- carcinoma polmonare non a piccole cellule - non-small cell lung cancer
- cancro alla testa e al collo - head and neck cancer
- adenocarcinoma duttale pancreatico - pancreatic ductal adenocarcinoma
- carcinoma dell'endometrio - carcinoma of the endometrium
- glioma - glioma
- carcinoma a cellule squamose della lingua orale - squamous cell carcinoma of the oral tongue
- carcinoma cervicale - cervical cancer
- carcinoma gastrico - gastric cancer
- cancro del colon-retto - colorectal cancer
- adenocarcinoma del colon - colon adenocarcinoma
- colangiocarcinoma extraepatico - extrahepatic cholangiocarcinoma
- carcinoma squamoso esofageo. - esophageal squamous carcinoma.
In tutte queste tipologie di tumore umano, dove è stata dimostrata l’aumentata espressione di FOXC2, non è escludibile che sia presente anche la overespressione di molecole FOXC2-AS1. Se fosse vera quest’ ultima ipotesi, potrebbe trovare, ancora una volta, utile applicazione l’uso di molecole FOXC2-SAS. Un secondo ambito di applicazione dei vettori FOXC2-SAS è il linfedema primario. Il linfedema si associa ad una distensione cronica degli arti, secondaria all'accumulo della linfa negli spazi interstiziali, in particolare nei tessuti sottocutanei. È correlato ad una malformazione sistemica o parziale dell'apparato linfatico. Nelle forme familiari, la malattia si trasmette in modo autosomico dominante, con mutazioni in eterozigosi di geni coinvolti in processi di linfangiogenesi. In particolare, la sindrome “linfedema-distichiasi” è causata da mutazioni nel gene FOXC2 (OMIM 602402). Recentemente è stato dimostrato che alcune mutazioni del gene FOXC2 causano la perdita o la diminuzione dell’attività della proteina FOXC2, mentre altre portano ad una iperattivazione del fattore di trascrizione (Tavian et al, Oncotarget, 2016). In questi pazienti potrebbe quindi essere utile il trattamento con FOXC2-SAS per diminuire la quantità di proteina FOXC2 intracellulare. In all these types of human cancer, where the increased expression of FOXC2 has been demonstrated, it cannot be excluded that the overexpression of FOXC2-AS1 molecules is also present. If this last hypothesis were true, the use of FOXC2-SAS molecules could find, once again, a useful application. A second area of application of FOXC2-SAS vectors is primary lymphedema. Lymphedema is associated with chronic distension of the limbs, secondary to the accumulation of lymph in the interstitial spaces, particularly in the subcutaneous tissues. It is related to a systemic or partial malformation of the lymphatic system. In familial forms, the disease is transmitted in an autosomal dominant way, with heterozygous mutations of genes involved in lymphangiogenesis processes. In particular, the “lymphedema-distichiasis” syndrome is caused by mutations in the FOXC2 gene (OMIM 602402). It has recently been shown that some mutations in the FOXC2 gene cause the loss or decrease in the activity of the FOXC2 protein, while others lead to an overactivation of the transcription factor (Tavian et al, Oncotarget, 2016). Treatment with FOXC2-SAS may therefore be useful in these patients to decrease the amount of intracellular FOXC2 protein.
ESEMPI EXAMPLES
1. Realizzazione del plasmide di espressione pcDNA-FOXC2-SAS 1. Realization of the pcDNA-FOXC2-SAS expression plasmid
a) sono stati disegnati i primer (primer A, primer B) necessari ad amplificare la sequenza lncRNA FOXC2-AS1 a partire da cDNA di cellule HeLa. a) the primers (primer A, primer B) necessary to amplify the FOXC2-AS1 lncRNA sequence starting from HeLa cell cDNA were designed.
Primer A 5’-CTTGCCGGGCTTCTTGTCGT-3‘ (SEQ ID NO:1) Primer A 5'-CTTGCCGGGCTTCTTGTCGT-3 '(SEQ ID NO: 1)
Primer B 5‘-TACATTTTCGTCTTCTGTTCTTTTATTGG-3‘ (SEQ ID NO:2) Mediante RT-PCR si è ottenuto un frammento di 319 nucleotidi corrispondente alla sequenza lncRNA FOXC2-AS1. Primer B 5'-TACATTTTCGTCTTCTGTTCTTTTATTGG-3 '(SEQ ID NO: 2) A fragment of 319 nucleotides corresponding to the lncRNA FOXC2-AS1 sequence was obtained by RT-PCR.
b) Il prodotto di RT-PCR (frammento lncRNA FOXC2-AS1) è stato fatto correre in gel di agarosio al 2% e purificato. I frammenti purificati sono stati quantificati allo spettrofotometro e sono stati clonati nel plasmide pcDNA™3.3 TOPO. A seguito della trasformazione batterica, eseguita secondo protocollo standard (Life Technologies) sono stati ottenuti decine di cloni. Alcuni di questi cloni sono stati testati per verificare la correttezza di sequenza e l‘ orientamento del frammento inserito. b) The RT-PCR product (FOXC2-AS1 lncRNA fragment) was run in 2% agarose gel and purified. The purified fragments were quantified on the spectrophotometer and were cloned into the pcDNA ™ 3.3 TOPO plasmid. Following the bacterial transformation, performed according to standard protocol (Life Technologies), dozens of clones were obtained. Some of these clones have been tested to verify the sequence correctness and the orientation of the inserted fragment.
c) Sono stati selezionati dei cloni con la sequenza lncRNA FOXC2-AS1 inserita in orientamento opposto, cioè quello che viene definito orientamento antisenso. Poiché la sequenza utilizzata per il clonaggio è lncRNA FOXC2-AS1, tali plasmidi porteranno all’espressione di una sequenza senso di un antisenso, che in questa descrizione è stata definita FOXC2-SAS (Sense of Antisense). c) Clones were selected with the FOXC2-AS1 lncRNA sequence inserted in the opposite orientation, ie what is called antisense orientation. Since the sequence used for cloning is lncRNA FOXC2-AS1, these plasmids will lead to the expression of a sense sequence of an antisense, which in this description has been defined FOXC2-SAS (Sense of Antisense).
Il plasmide pcDNA-FOXC2-SAS è stato sequenziato interamente per verificare il corretto inserimento della sequenza SAS, nonché la correttezza della sequenza stessa. Il plasmide pcDNA-FOXC2-SAS è stato utilizzato per eseguire esperimenti di trasfezione in cellule HeLa. Dopo 24 o 48 ore di trasfezione, le cellule HeLa sono state lisate ed è stato estratto l’RNA totale. Mediante esperimenti di RT-PCR (Reverse Transcriptase and Polymerase Chain Reaction) è stata rilevata la presenza di molecole di RNA FOXC2-SAS nelle cellule trasfettate. The pcDNA-FOXC2-SAS plasmid was sequenced entirely to verify the correct insertion of the SAS sequence, as well as the correctness of the sequence itself. The pcDNA-FOXC2-SAS plasmid was used to perform transfection experiments in HeLa cells. After 24 or 48 hours of transfection, the HeLa cells were lysed and the total RNA was extracted. The presence of FOXC2-SAS RNA molecules in transfected cells was detected by means of RT-PCR (Reverse Transcriptase and Polymerase Chain Reaction) experiments.
Sequenza FOXC2-SAS (SEQ ID NO:3): FOXC2-SAS Sequence (SEQ ID NO: 3):
5’-TACATTTTCGTCTTCTGTTCTTTTATTGGAAATAAGTGGCACGCCCC ATTGCCTTCTAGTCGCCTCCCCGAAGCGAAGAGGCCGAAGCGAAGAG GCCTGGTGGGTTGTCTCAACATCCTTTTGCTGAGAATCGAATACGCAG CCGATGAACAGCCAGGAAGGGTGCAAGGAAACCTTGAACGGCATCTA CCAGTTCATCATGGACCGCTTCCCCTTCTACCGGGAGAACAAGCAGG GCTGGCAGAACAGCATCCGCCACAACCTCTCGCTCAACGAGTGCTTC GTCAAGGTGCCCCGCGACGACAAGAAGCCCGGCAAG-3‘ 5’-TACATTTTCGTCTTCTGTTCTTTTATTGGAAATAAGTGGCACGCCCC ATTGCCTTCTAGTCGCCTCCCCGAAGCGAAGAGGCCGAAGCGAAGAG GCCTGGTGGGTTGTCTCAACATCCTTTTGCTGAGAATCGAATACGCAG CCGATGAACAGCCAGGAAGGGTGCAAGGAAACCTTGAACGGCATCTA CCAGTTCATCATGGACCGCTTCCCCTTCTACCGGGAGAACAAGCAGG GCTGGCAGAACAGCATCCGCCACAACCTCTCGCTCAACGAGTGCTTC GTCAAGGTGCCCCGCGACGACAAGAAGCCCGGCAAG-3‘
La sequenza FOXC2-SAS è complementare alla sequenza lncRNA FOXC2-AS1 (FOXC2-AS1-201 ENST00000563280.1), di seguito riportata (SEQ ID NO:4): The FOXC2-SAS sequence is complementary to the FOXC2-AS1 lncRNA sequence (FOXC2-AS1-201 ENST00000563280.1), shown below (SEQ ID NO: 4):
Homo sapiens FOXC2 antisense RNA 1 (FOXC2-AS1), long non-coding RNA 886 NCBI Reference Sequence: NR_125795.1 Homo sapiens FOXC2 antisense RNA 1 (FOXC2-AS1), long non-coding RNA 886 NCBI Reference Sequence: NR_125795.1
>NR_125795.1 Homo sapiens FOXC2 antisense RNA 1 (FOXC2-AS1), long non-coding RNA > NR_125795.1 Homo sapiens FOXC2 antisense RNA 1 (FOXC2-AS1), long non-coding RNA
5‘-CTTGCCGGGCTTCTTGTCGTCGCGGGGCACCTTGACGAAGCACTC GTTGAGCGAGAGGTTGTGGCGGATGCTGTTCTGCCAGCCCTGCTTGT 5'-CTTGCCGGGCTTCTTGTCGTCGCGGGGCACCTTGACGAAGCACTC GTTGAGCGAGAGGTTGTGGCGGATGCTGTTCTGCCAGCCCTGCTTGT
TCTCCCGGTAGAAGGGGAAGCGGTCCATGATGAACTGGTAGATGCCG TCTCCCGGTAGAAGGGGAAGCGGTCCATGATGAACTGGTAGATGCCG
TTCAAGGTTTCCTTGCACCCTTCCTGGCTGTTCATCGGCTGCGTATTC TTCAAGGTTTCCTTGCACCCTTCCTGGCTGTTCATCGGCTGCGTATTC
GATTCTCAGCAAAAGGATGTTGAGACAACCCACCAGGCCTCTTCGCTT GATTCTCAGCAAAAGGATGTTGAGACAACCCACCAGGCCTCTTCGCTT
CGGCCTCTTCGCTTCGGGGAGGCGACTAGAAGGCAATGGGGCGTGC CGGCCTCTTCGCTTCGGGGAGGCGACTAGAAGGCAATGGGGCGTGC
CACTTATTTCCAATAAAAGAACAGAAGACGAAAATGTA-3‘ CACTTATTTCCAATAAAAGAACAGAAGACGAAAATGTA-3 '
È infine riportata la sequenza dell’RNA FOXC2, con il piccolo tratto di Finally, the FOXC2 RNA sequence is shown, with the small section of
sequenza (evidenziata in giallo) complementare ad una parte della sequence (highlighted in yellow) complementary to a part of the
molecola lncRNA FOXC2-AS1 (SEQ ID NO:5): FOXC2-AS1 lncRNA molecule (SEQ ID NO: 5):
Homo sapiens forkhead box C2 (FOXC2), mRNA Homo sapiens forkhead box C2 (FOXC2), mRNA
NCBI Reference Sequence: NM_005251.2 NCBI Reference Sequence: NM_005251.2
>NM_005251.2 Homo sapiens forkhead box C2 (FOXC2), mRNA > NM_005251.2 Homo sapiens forkhead box C2 (FOXC2), mRNA
5’-CGCGCTCTCTCGCTCTCAGGGCCCCCCTCGCTCCCCCGGCCGCAG TCCGTGCGCGAGGGCGCCGGCGAGCCGTCTCGGAAGCAGCATGCAG GCGCGCTACTCCGTGTCCGACCCCAACGCCCTGGGAGTGGTGCCCTA CCTGAGCGAGCAGAATTACTACCGGGCTGCGGGCAGCTACGGCGGC ATGGCCAGCCCCATGGGCGTCTATTCCGGCCACCCGGAGCAGTACAG CGCGGGGATGGGCCGCTCCTACGCGCCCTACCACCACCACCAGCCC GCGGCGCCTAAGGACCTGGTGAAGCCGCCCTACAGCTACATCGCGCT CATCACCATGGCCATCCAGAACGCGCCCGAGAAGAAGATCACCTTGA ACGGCATCTACCAGTTCATCATGGACCGCTTCCCCTTCTACCGGGAGA ACAAGCAGGGCTGGCAGAACAGCATCCGCCACAACCTCTCGCTCAAC GAGTGCTTCGTCAAGGTGCCCCGCGACGACAAGAAGCCCGGCAAGG GCAGTTACTGGACCCTGGACCCGGACTCCTACAACATGTTCGAGAAC GGCAGCTTCCTGCGGCGCCGGCGGCGCTTCAAAAAGAAGGACGTGT CCAAGGAGAAGGAGGAGCGGGCCCACCTCAAGGAGCCGCCCCCGGC GGCGTCCAAGGGCGCCCCGGCCACCCCCCACCTAGCGGACGCCCCC AAGGAGGCCGAGAAGAAGGTGGTGATCAAGAGCGAGGCGGCGTCCC CGGCGCTGCCGGTCATCACCAAGGTGGAGACGCTGAGCCCCGAGAG CGCGCTGCAGGGCAGCCCGCGCAGCGCGGCCTCCACGCCCGCCGG CTCCCCCGACGGCTCGCTGCCGGAGCACCACGCCGCGGCGCCCAAC GGGCTGCCTGGCTTCAGCGTGGAGAACATCATGACCCTGCGAACGTC GCCGCCGGGCGGAGAGCTGAGCCCGGGGGCCGGACGCGCGGGCCT GGTGGTGCCGCCGCTGGCGCTGCCCTACGCCGCCGCGCCGCCCGC CGCCTACGGCCAGCCGTGCGCTCAGGGCCTGGAGGCCGGGGCCGC CGGGGGCTACCAGTGCAGCATGCGAGCGATGAGCCTGTACACCGGG GCCGAGCGGCCGGCGCACATGTGCGTCCCGCCCGCCCTGGACGAG GCCCTCTCGGACCACCCGAGCGGCCCCACGTCGCCCCTGAGCGCTC TCAACCTCGCCGCCGGCCAGGAGGGCGCGCTCGCCGCCACGGGCCA CCACCACCAGCACCACGGCCACCACCACCCGCAGGCGCCGCCGCCC CCGCCGGCTCCCCAGCCCCAGCCGACGCCGCAGCCCGGGGCCGCC GCGGCGCAGGCGGCCTCCTGGTATCTCAACCACAGCGGGGACCTGA ACCACCTCCCCGGCCACACGTTCGCGGCCCAGCAGCAAACTTTCCCC AACGTGCGGGAGATGTTCAACTCCCACCGGCTGGGGATTGAGAACTC GACCCTCGGGGAGTCCCAGGTGAGTGGCAATGCCAGCTGCCAGCTG CCCTACAGATCCACGCCGCCTCTCTATCGCCACGCAGCCCCCTACTC CTACGACTGCACGAAATACTGACGTGTCCCGGGACCTCCCCTCCCCG GCCCGCTCCGGCTTCGCTTCCCAGCCCCGACCCAACCAGACAATTAA GGGGCTGCAGAGACGCAAAA -3’ 5’-CGCGCTCTCTCGCTCTCAGGGCCCCCCTCGCTCCCCCGGCCGCAG TCCGTGCGCGAGGGCGCCGGCGAGCCGTCTCGGAAGCAGCATGCAG GCGCGCTACTCCGTGTCCGACCCCAACGCCCTGGGAGTGGTGCCCTA CCTGAGCGAGCAGAATTACTACCGGGCTGCGGGCAGCTACGGCGGC ATGGCCAGCCCCATGGGCGTCTATTCCGGCCACCCGGAGCAGTACAG CGCGGGGATGGGCCGCTCCTACGCGCCCTACCACCACCACCAGCCC GCGGCGCCTAAGGACCTGGTGAAGCCGCCCTACAGCTACATCGCGCT CATCACCATGGCCATCCAGAACGCGCCCGAGAAGAAGATCACCTTGA ACGGCATCTACCAGTTCATCATGGACCGCTTCCCCTTCTACCGGGAGA ACAAGCAGGGCTGGCAGAACAGCATCCGCCACAACCTCTCGCTCAAC GAGTGCTTCGTCAAGGTGCCCCGCGACGACAAGAAGCCCGGCAAGG GCAGTTACTGGACCCTGGACCCGGACTCCTACAACATGTTCGAGAAC GGCAGCTTCCTGCGGCGCCGGCGGCGCTTCAAAAAGAAGGACGTGT CCAAGGAGAAGGAGGAGCGGGCCCACCTCAAGGAGCCGCCCCCGGC GGCGTCCAAGGGCGCCCCGGCCACCCCCCACCTAGCGGACGCCCCC AAGGAGGCCGAGAAGAAGGTGGTGATCAAGAGCGAGGCGGCGTCCC CGGCGCTGCCGGTCATCACCAAGGTGGAGACGCTGAGCCCCGAGAG CGCGCTGCAGGGCAGCCCGCGCAGCGCGGCCTCCACGCCCGCCGG CTCCCCCGACGGCTCGCTGCCGGAGCACCACGCCGCGGCGCCCAAC GGGCTGCCTGGCTTCAGCGTGGAGAACATCATGACCCTGCGAACGTC GCCGCCGGGCGGAGAGCTGAGCCCGGGGGCCGGACGCGCGGGCCT GGTG GTGCCGCCGCTGGCGCTGCCCTACGCCGCCGCGCCGCCCGC CGCCTACGGCCAGCCGTGCGCTCAGGGCCTGGAGGCCGGGGCCGC CGGGGGCTACCAGTGCAGCATGCGAGCGATGAGCCTGTACACCGGG GCCGAGCGGCCGGCGCACATGTGCGTCCCGCCCGCCCTGGACGAG GCCCTCTCGGACCACCCGAGCGGCCCCACGTCGCCCCTGAGCGCTC TCAACCTCGCCGCCGGCCAGGAGGGCGCGCTCGCCGCCACGGGCCA CCACCACCAGCACCACGGCCACCACCACCCGCAGGCGCCGCCGCCC CCGCCGGCTCCCCAGCCCCAGCCGACGCCGCAGCCCGGGGCCGCC GCGGCGCAGGCGGCCTCCTGGTATCTCAACCACAGCGGGGACCTGA ACCACCTCCCCGGCCACACGTTCGCGGCCCAGCAGCAAACTTTCCCC AACGTGCGGGAGATGTTCAACTCCCACCGGCTGGGGATTGAGAACTC GACCCTCGGGGAGTCCCAGGTGAGTGGCAATGCCAGCTGCCAGCTG CCCTACAGATCCACGCCGCCTCTCTATCGCCACGCAGCCCCCTACTC CTACGACTGCACGAAATACTGACGTGTCCCGGGACCTCCCCTCCCCG GCCCGCTCCGGCTTCGCTTCCCAGCCCCGACCCAACCAGACAATTAA GGGGCTGCAGAGACGCAAAA -3’
2. Analisi d’espressione di FOXC2 e FOXC2-AS1 in cellule HeLa Inizialmente è stato verificato se le cellule HeLa esprimessero l’mRNA di FOXC2 e le molecole di antisenso FOXC2-AS1. Da cellule HeLa coltivate in terreno DMEM con 10% di FBS è stato estratto l’RNA totale, secondo il protocollo Trizol (Life Technologies). L’RNA ottenuto è stato dosato spettrofotometricamente. Inoltre, 0,5 µg di RNA sono stati caricati in gel di agarosio all’1% per verificare la precisione della misurazione effettuata e l’integrità delle molecole di RNA. 2. FOXC2 and FOXC2-AS1 expression analysis in HeLa cells It was initially verified whether HeLa cells expressed FOXC2 mRNA and FOXC2-AS1 antisense molecules. Total RNA was extracted from HeLa cells grown in DMEM medium with 10% FBS, according to the Trizol protocol (Life Technologies). The RNA obtained was measured spectrophotometrically. In addition, 0.5 µg of RNA were loaded into 1% agarose gel to verify the accuracy of the measurement performed and the integrity of the RNA molecules.
Dopo aver trattato 2 µg di RNA con DNase I (LifeTechnologies) per eliminare eventuali contaminazioni di DNA, il campione è stato diviso in due parti: a) una parte (campione a), pari a 1 µg, è stata addizionata ad una mix di retrotrascrizione contenente esameri random e la trascrittasi inversa M-MLV (Life Technologies); b) la restante parte (campione b), pari ad 1 µg, è stata aggiunta ad una mix di reazione di retrotrascrizione con esameri random, non contenente M-MLV. Dato che in assenza dell’enzima M-MLV, la retrotrascrizione di RNA non avviene, utilizzando il campione b in saggi di RT-PCR semiquantitativa, non si dovrebbero ottenere prodotti di amplificazione, confermando la rimozione totale di DNA dal campione. Per valutare l’espressione di FOXC2 e FOXC2-AS1, 50 ng del cDNA ottenuto precedentemente (campione a) è stato aggiunto ad una mix di reazione contenente i primers FOXC2-F AGTGCAGCATGCGAGCGATG (SEQ ID NO:6) e FOXC2-R CGCTCGGGTGGTCCGAGAGGG (SEQ ID NO:7) mentre per FOXC2-AS1 sono stati utilizzati 100 ng di ciascun cDNA e i primers FOXC2-AS1F CTTGCCGGGCTTCTTGTCGT (SEQ ID NO:8) e FOXC2-AS1R TACATTTTCGTCTTCTGTTCTTTTATTGG (SEQ ID NO:9). I prodotti di amplificazione sono stati caricati su un gel di agarosio al 2%. L’analisi del gel ha mostrato la presenza di una banda da 102 nt per FOXC2 e una banda da 319 nt per FOXC2-AS1 (figura 3). Eseguendo le stesse reazioni utilizzando come templato il prodotto ottenuto dalla mix di retrotrascrizione a cui non è stata aggiunta la trascrittasi inversa (campione b) non si sono ottenuti prodotti di amplificazione. After treating 2 µg of RNA with DNase I (LifeTechnologies) to eliminate any DNA contamination, the sample was divided into two parts: a) one part (sample a), equal to 1 µg, was added to a mix of retro-transcription containing random hexamers and the reverse transcriptase M-MLV (Life Technologies); b) the remainder (sample b), equal to 1 µg, was added to a retro-transcription reaction mix with random hexamers, not containing M-MLV. Since in the absence of the M-MLV enzyme, the reverse transcription of RNA does not occur, using sample b in semi-quantitative RT-PCR assays, amplification products should not be obtained, confirming the total removal of DNA from the sample. To evaluate the expression of FOXC2 and FOXC2-AS1, 50 ng of the previously obtained cDNA (sample a) was added to a reaction mix containing the primers FOXC2-F AGTGCAGCATGCGAGCGATG (SEQ ID NO: 6) and FOXC2-R CGCTCGGGTGGTCCGAGAGGG ( SEQ ID NO: 7) while for FOXC2-AS1 100 ng of each cDNA and the primers FOXC2-AS1F CTTGCCGGGCTTCTTGTCGT (SEQ ID NO: 8) and FOXC2-AS1R TACATTTTCGTCTTCTGTTCTTTTATTGG (SEQ ID NO) were used. The amplification products were loaded onto a 2% agarose gel. The analysis of the gel showed the presence of a 102 nt band for FOXC2 and a 319 nt band for FOXC2-AS1 (Figure 3). By carrying out the same reactions using as template the product obtained from the reverse transcription mix to which the reverse transcriptase was not added (sample b), no amplification products were obtained.
3. Valutazione dell’inibizione dell’espressione della proteina FOXC2 a seguito della trasfezione di FOXC2-SAS in cellule HeLa 3. Evaluation of the inhibition of FOXC2 protein expression following transfection of FOXC2-SAS into HeLa cells
Per determinare l’effetto della molecola SAS sulla quantità di proteina FOXC2 prodotta, cellule HeLa coltivate in terreno DMEM con 10% di FBS sono state trasfettate con il vettore pcDNA-FOXC2-SAS. Dopo 48 ore dalla trasfezione, sono stati ottenuti gli estratti proteici dalle cellule trasfettate e non (FractionPREP Cell Fractionation Kit, BioVision). Gli estratti proteici sono stati quantificati mediante il metodo BCA Protein Assay Kit (Pierce).5 µg totali di estratto proteico per ciascun campione sono stati analizzati mediante western blotting. Per identificare la proteina FOXC2 è stato usato un anticorpo monoclonale anti-FOXC2 (Santa Cruz Biotechnology). Come mostrato in figura 4, la trasfezione di FOXC2-SAS causa una diminuzione della quantità di proteina FOXC2 presente nelle cellule HeLa di circa il 90%. L’espressione della proteina GAPDH è stata inoltre valutata in tutti i campioni (Fig 4). To determine the effect of the SAS molecule on the amount of FOXC2 protein produced, HeLa cells grown in DMEM medium with 10% FBS were transfected with the pcDNA-FOXC2-SAS vector. 48 hours after transfection, protein extracts from transfected and non-transfected cells (FractionPREP Cell Fractionation Kit, BioVision) were obtained. Protein extracts were quantified by the BCA Protein Assay Kit (Pierce) method. 5 µg total of protein extract for each sample were analyzed by western blotting. An anti-FOXC2 monoclonal antibody (Santa Cruz Biotechnology) was used to identify the FOXC2 protein. As shown in Figure 4, transfection of FOXC2-SAS causes a decrease in the amount of FOXC2 protein present in HeLa cells by approximately 90%. The expression of the GAPDH protein was also evaluated in all samples (Fig 4).
In parallelo al saggio di western appena descritto, da capsule di cellule HeLa trattate con il medesimo protocollo sperimentale specificato sopra (cioè trasfettate con pcDNA-FOXC2-SAS e non trasfettate) è stato estratto l’RNA totale e si è proceduto a verificare che le molecole del vettore pcDNA-FOXC2-SAS trasfettate esprimessero in vivo molecole di mRNA FOXC2-SAS. Dopo trattamento dell’RNA totale con DNase I è stata eseguita la retrotrascrizione utilizzando un primer reverse specifico per la molecola SAS (FOXC2-SASR CTTGCCGGGCTTCTTGTCGT (SEQ ID NO:10). Amplificando 100 ng di cDNA con i primers FOXC2-SASF TACATTTTCGTCTTCTGTTCTTTTATTGG (SEQ ID NO:11) e FOXC2-SASR si è ottenuto un prodotto di 319 nt corrispondente alla sequenza SAS (figura 3, corsia 3) nelle cellule HeLa trasfettate, mentre le cellule non trasfettate sono negative per FOXC2-SAS. In parallel to the western assay just described, total RNA was extracted from HeLa cell capsules treated with the same experimental protocol specified above (i.e. transfected with pcDNA-FOXC2-SAS and not transfected) and transfected pcDNA-FOXC2-SAS vector molecules expressed FOXC2-SAS mRNA molecules in vivo. After treatment of the total RNA with DNase I, reverse transcription was performed using a reverse primer specific for the SAS molecule (FOXC2-SASR CTTGCCGGGCTTCTTGTCGT (SEQ ID NO: 10). Amplifying 100 ng of cDNA with the primers FOXC2-SASF TACATTTTCGTCTTCTGTTCT ID NO: 11) and FOXC2-SASR a 319 nt product corresponding to the SAS sequence (Figure 3, lane 3) was obtained in transfected HeLa cells, while non-transfected cells are negative for FOXC2-SAS.
4. Omologia di sequenza FOXC2-SAS con molecole di RNA e complementarietà con lncRNA umani 4. FOXC2-SAS sequence homology with RNA molecules and complementarity with human lncRNA
Per investigare possibili omologie di FOXC2-SAS con altri RNA umani è stata fatta una ricerca tramite programmi di predizione. Da questa analisi è risultata una omologia del 95% tra 122 nt di FOXC2-SAS e l’mRNA di FOXL1. Di seguito sono riportate le sequenze dell’mRNA FOXL1 e di FOXC2-SAS, con i tratti di sequenza (evidenziata in rosso) omologhi. To investigate possible homologies of FOXC2-SAS with other human RNAs, a search was made through prediction programs. This analysis resulted in a 95% homology between 122 nt of FOXC2-SAS and the mRNA of FOXL1. Below are the sequences of FOXL1 and FOXC2-SAS mRNA, with the homologous sequences (highlighted in red).
>NM_005250.2 Homo sapiens forkhead box L1 (FOXL1), mRNA (SEQ ID NO:12) GCTAAGTCAACATATAACAGCAAACTTACAACAATTATTTAACATTTTTA AAGCCATGAAGAAGGGACAGAGCACGGAGCGGCCGGGGAGAGCGG CAGAGCCAGAGGCAGAGGCACGGCTGGCTCCCCGGGAGGGCCCTTG CGGCGCGGGGCGCAGTGCCTAGGCCGCCCGGGTCTCCTGCGTTGCG GGGAGCGCAGCGCAGGCTCTCGCTTGCCATGAGTCACCTCTTCGATC CCCGGCTGCCTGCCCTGGCCGCCTCGCCCATGCTGTATCTGTACGGT CCCGAGAGACCCGGCCTCCCTCTGGCCTTCGCCCCCGCGGCTGCTC TAGCTGCCTCGGGCCGGGCCGAGACCCCGCAGAAGCCTCCCTACAG CTACATCGCGCTCATCGCCATGGCGATCCAGGACGCGCCCGAGCAGA GGGTCACGCTCAACGGCATCTACCAGTTCATCATGGACCGCTTCCCCT TCTACCACGACAACCGGCAGGGCTGGCAGAACAGCATCCGCCACAAC CTCTCGCTCAACGACTGCTTCGTCAAGGTGCCCCGCGAGAAAGGGCG GCCGGGCAAGGGCAGCTACTGGACGCTGGACCCCCGCTGCCTGGAC ATGTTTGAGAACGGCAACTACCGGCGCCGGAAGAGGAAGCCCAAGCC GGGCCCCGGGGCCCCGGAGGCCAAGAGGCCCCGCGCCGAGACGCA CCAGCGCAGCGCGGAGGCGCAGCCGGAGGCGGGGAGCGGGGCAG GGGGCTCGGGCCCCGCAATCTCCCGCCTGCAGGCAGCGCCCGCGG GCCCCTCGCCCCTCCTGGACGGCCCCTCTCCGCCGGCGCCCCTCCA CTGGCCGGGGACCGCGTCCCCGAACGAGGACGCTGGTGACGCTGCC CAGGGCGCAGCGGCCGTGGCGGTCGGCCAGGCAGCGCGCACAGGG GACGGCCCGGGGTCCCCTCTGCGCCCCGCCTCCCGCAGCTCTCCGA AGAGCTCCGACAAGTCCAAGAGCTTCAGCATAGACAGCATCCTGGCG GGAAAGCAGGGCCAGAAGCCGCCTTCAGGGGACGAACTCCTAGGGG GTGCCAAGCCTGGGCCCGGCGGCCGTCTGGGTGCCTCGCTCCTGGC CGCCTCCTCCAGCCTCCGTCCGCCTTTCAACGCTTCCCTGATGCTCGA CCCGCATGTCCAGGGCGGCTTTTACCAGCTCGGGATCCCCTTCCTCT CTTATTTCCCCCTGCAGGTTCCCGACACGGTACTCCACTTCCAGTAAA GCAAACAATGGCACGGTTCTTCTCCCGGCCCAGCCTGAGCCTCCGCT GAGCGAAAGGCCACAGCTCCCACCGGCGGAGGATTTTAAAATGATCT TTGCCTGGGTCGGCCTGTGGGTTCAGGGAAGTGTTACCAACCATTGC GCGCAGGTGGGCGCGCTCGCCTGCCTTCTCCGAAGCAAACTTTTCCC AGCAACTGGGAGCAGCTACGGAGACTTAAAAGATCCCCGCGGGGTCG TGGGCACCGCACGTGGGCCCTGCAGGGACCTCCACCGCGGGAGATT CCTTGACGTTTGACCTGTCTAATGGAGTGTGGTCTTCAGCCGCCCACC GCAGGTCCTGCGAGTCCCGGGCATGCAAGGGCCCGCAGACCACCCA AGGCAGGCCTTACAGGTGCAGTTGAGGTCCTTTCGACCGGGGACCCC CGTATGACCGTCGCCTGCTTCGCTGTTGCCGTGTGGCTCAGGCCTTG GTTTTGAGCCCATCTGGGTGGTGGGTGGTCACAGCTGGGTTTGTTCC TGACTTGAAGTTGTGGAGGTGCCGGGGGTGACCCCGGGTTTTCAGGT TTTCTGAGAGATGTAGGGCCCGATGAGGATTTATGACCAATGTTCAGC TATAGAAGCTGATAACCACCCAGAGCAATAACACTTTTATTTTAAACAG AGACTTTTTCTCTTAATTTCTTTCTGGGTAGTAAAAAGAACATGTTTCAT TCTTTGCATAAATACTTGACTCTAAGCATTGACCTTTGAAAACGCTTGT ATTAACAATTTTTATTAAGAAAGTGCACTCTATATAACATCTTCTTGCAT TACGATAGCTCATTAGCCAATACACATGCAGCTATGTAAGCCACAACA GCAGACGTCCTATCCTTTTGCTTTTGTTTTTAAGGGATCAAAATATTTC AAGGGATACCATGAGGAAGGGTGTGAGGGGAAGGGGATATGCCTTG GACAATGTCAGAGTTTTGATTTCCATGTTTTTTCAAGATCCAGGCTTGG TGGTCGCTATATTTTTCAGAAACAGATAATGAAAGACTACATTTAATTTA AAAAGTTGGGTGAAACAACCAAAAGCAGATGCAGAGAGAGAGAGGGA TGAATGCGGGGGAAGAATAAGGGGACGTGCTGGTGTCCGCTGGAAG GGGCCTCTCTGGGCCCCAGAGGACCTGAGGTTTGATGGCAGGAATCT CCCAGACACTCCTGGCCCCCGCACCCCCTGCAGCCGCCCGCTCTTCC CTCCCAGCCCTGCCAGGCAGGCCTGGAGGCTGATACTAAATCAAAAC CTTAAAGAGGTGACTGGCAGTCTGGGGAGGGGAGAAGAGGTCTGCAA CGAAAATCTAATTAAGAACATTGAGATGTTCACAACCAAGTTCAGCTTG CAGCTTCCGGGGAAATGCAACCGACCTCTATGCCATCTCTAGCTGGG TAATCTTCGAGGCCCTGAAATGCAATTTTCTGAACCTCTAAGTCACTTT TATTTTATGTCATGTGATAAAGACACAAGTAGTCACGCACTATTGGAAA TAGGTTGGGAGGGAGGTCTTTAAGATCGCTTTTAAAATAGTTTCCAGG ACTTGTCTAAAAATGATGACAAACTTTGAATCCAGAGGGCCTGTTGGT CAATGTGTTGGAAAGGACATTTCAGGCCGGCAGAGTAATTGGATAATT CACAGGCATAGAGGTGACTTTTGGAAAATTCATTCTCCTATCCATGCT GAACCAGGCTTCCACGACTGGGTTCTACCCAGGCCAGTGGGAAGACT CTGGGAAACTTCAGGCCAGCCTTGATTGGAAGAACTTCCAGGAGGAG CCATTGCTGAGATCCAGGCTCACTGTGATTCACTGAAAATGTAGCGAA AGGGCTGTCACTGGATATTTTGCTATCCCAAACTCTCAGCCTCTGTGA ATAAAGTTGTTTTTTCATTAACCTCTCAAAAAAAAAAAAAAAAAA >NM_005250.2 Homo sapiens forkhead box L1 (FOXL1), mRNA (SEQ ID NO:12) GCTAAGTCAACATATAACAGCAAACTTACAACAATTATTTAACATTTTTA AAGCCATGAAGAAGGGACAGAGCACGGAGCGGCCGGGGAGAGCGG CAGAGCCAGAGGCAGAGGCACGGCTGGCTCCCCGGGAGGGCCCTTG CGGCGCGGGGCGCAGTGCCTAGGCCGCCCGGGTCTCCTGCGTTGCG GGGAGCGCAGCGCAGGCTCTCGCTTGCCATGAGTCACCTCTTCGATC CCCGGCTGCCTGCCCTGGCCGCCTCGCCCATGCTGTATCTGTACGGT CCCGAGAGACCCGGCCTCCCTCTGGCCTTCGCCCCCGCGGCTGCTC TAGCTGCCTCGGGCCGGGCCGAGACCCCGCAGAAGCCTCCCTACAG CTACATCGCGCTCATCGCCATGGCGATCCAGGACGCGCCCGAGCAGA GGGTCACGCTCAACGGCATCTACCAGTTCATCATGGACCGCTTCCCCT TCTACCACGACAACCGGCAGGGCTGGCAGAACAGCATCCGCCACAAC CTCTCGCTCAACGACTGCTTCGTCAAGGTGCCCCGCGAGAAAGGGCG GCCGGGCAAGGGCAGCTACTGGACGCTGGACCCCCGCTGCCTGGAC ATGTTTGAGAACGGCAACTACCGGCGCCGGAAGAGGAAGCCCAAGCC GGGCCCCGGGGCCCCGGAGGCCAAGAGGCCCCGCGCCGAGACGCA CCAGCGCAGCGCGGAGGCGCAGCCGGAGGCGGGGAGCGGGGCAG GGGGCTCGGGCCCCGCAATCTCCCGCCTGCAGGCAGCGCCCGCGG GCCCCTCGCCCCTCCTGGACGGCCCCTCTCCGCCGGCGCCCCTCCA CTGGCCGGGGACCGCGTCCCCGAACGAGGACGCTGGTGACGCTGCC CAGGGCGCAGCGGCCGTGGCGGTCGGCCA GGCAGCGCGCACAGGG GACGGCCCGGGGTCCCCTCTGCGCCCCGCCTCCCGCAGCTCTCCGA AGAGCTCCGACAAGTCCAAGAGCTTCAGCATAGACAGCATCCTGGCG GGAAAGCAGGGCCAGAAGCCGCCTTCAGGGGACGAACTCCTAGGGG GTGCCAAGCCTGGGCCCGGCGGCCGTCTGGGTGCCTCGCTCCTGGC CGCCTCCTCCAGCCTCCGTCCGCCTTTCAACGCTTCCCTGATGCTCGA CCCGCATGTCCAGGGCGGCTTTTACCAGCTCGGGATCCCCTTCCTCT CTTATTTCCCCCTGCAGGTTCCCGACACGGTACTCCACTTCCAGTAAA GCAAACAATGGCACGGTTCTTCTCCCGGCCCAGCCTGAGCCTCCGCT GAGCGAAAGGCCACAGCTCCCACCGGCGGAGGATTTTAAAATGATCT TTGCCTGGGTCGGCCTGTGGGTTCAGGGAAGTGTTACCAACCATTGC GCGCAGGTGGGCGCGCTCGCCTGCCTTCTCCGAAGCAAACTTTTCCC AGCAACTGGGAGCAGCTACGGAGACTTAAAAGATCCCCGCGGGGTCG TGGGCACCGCACGTGGGCCCTGCAGGGACCTCCACCGCGGGAGATT CCTTGACGTTTGACCTGTCTAATGGAGTGTGGTCTTCAGCCGCCCACC GCAGGTCCTGCGAGTCCCGGGCATGCAAGGGCCCGCAGACCACCCA AGGCAGGCCTTACAGGTGCAGTTGAGGTCCTTTCGACCGGGGACCCC CGTATGACCGTCGCCTGCTTCGCTGTTGCCGTGTGGCTCAGGCCTTG GTTTTGAGCCCATCTGGGTGGTGGGTGGTCACAGCTGGGTTTGTTCC TGACTTGAAGTTGTGGAGGTGCCGGGGGTGACCCCGGGTTTTCAGGT TTTCTGAGAGATGTAGGGCCCGATGAGGATTTATGACCAATGTTCAGC TATAGAAGCTGATAACCACCCAGA GCAATAACACTTTTATTTTAAACAG AGACTTTTTCTCTTAATTTCTTTCTGGGTAGTAAAAAGAACATGTTTCAT TCTTTGCATAAATACTTGACTCTAAGCATTGACCTTTGAAAACGCTTGT ATTAACAATTTTTATTAAGAAAGTGCACTCTATATAACATCTTCTTGCAT TACGATAGCTCATTAGCCAATACACATGCAGCTATGTAAGCCACAACA GCAGACGTCCTATCCTTTTGCTTTTGTTTTTAAGGGATCAAAATATTTC AAGGGATACCATGAGGAAGGGTGTGAGGGGAAGGGGATATGCCTTG GACAATGTCAGAGTTTTGATTTCCATGTTTTTTCAAGATCCAGGCTTGG TGGTCGCTATATTTTTCAGAAACAGATAATGAAAGACTACATTTAATTTA AAAAGTTGGGTGAAACAACCAAAAGCAGATGCAGAGAGAGAGAGGGA TGAATGCGGGGGAAGAATAAGGGGACGTGCTGGTGTCCGCTGGAAG GGGCCTCTCTGGGCCCCAGAGGACCTGAGGTTTGATGGCAGGAATCT CCCAGACACTCCTGGCCCCCGCACCCCCTGCAGCCGCCCGCTCTTCC CTCCCAGCCCTGCCAGGCAGGCCTGGAGGCTGATACTAAATCAAAAC CTTAAAGAGGTGACTGGCAGTCTGGGGAGGGGAGAAGAGGTCTGCAA CGAAAATCTAATTAAGAACATTGAGATGTTCACAACCAAGTTCAGCTTG CAGCTTCCGGGGAAATGCAACCGACCTCTATGCCATCTCTAGCTGGG TAATCTTCGAGGCCCTGAAATGCAATTTTCTGAACCTCTAAGTCACTTT TATTTTATGTCATGTGATAAAGACACAAGTAGTCACGCACTATTGGAAA TAGGTTGGGAGGGAGGTCTTTAAGATCGCTTTTAAAATAGTTTCCAGG ACTTGTCTAAAAATGATGACAAACTTTGAATCCAGAGGGCC TGTTGGT CAATGTGTTGGAAAGGACATTTCAGGCCGGCAGAGTAATTGGATAATT CACAGGCATAGAGGTGACTTTTGGAAAATTCATTCTCCTATCCATGCT GAACCAGGCTTCCACGACTGGGTTCTACCCAGGCCAGTGGGAAGACT CTGGGAAACTTCAGGCCAGCCTTGATTGGAAGAACTTCCAGGAGGAG CCATTGCTGAGATCCAGGCTCACTGTGATTCACTGAAAATGTAGCGAA AGGGCTGTCACTGGATATTTTGCTATCCCAAACTCTCAGCCTCTGTGA ATAAAGTTGTTTTTTCATTAACCTCTCAAAAAAAAAAAAAAAAAA
FOXC2-SAS (SEQ ID NO:3) TACATTTTCGTCTTCTGTTCTTTTATTGGAAATAAGTGGCACGCCCCAT TGCCTTCTAGTCGCCTCCCCGAAGCGAAGAGGCCGAAGCGAAGAGG CCTGGTGGGTTGTCTCAACATCCTTTTGCTGAGAATCGAATACGCAGC CGATGAACAGCCAGGAAGGGTGCAAGGAAACCTTGAACGGCATCTAC CAGTTCATCATGGACCGCTTCCCCTTCTACCGGGAGAACAAGCAGGG CTGGCAGAACAGCATCCGCCACAACCTCTCGCTCAACGAGTGCTTCG TCAAGGTGCCCCGCGACGACAAGAAGCCCGGCAAG FOXC2-SAS (SEQ ID NO:3) TACATTTTCGTCTTCTGTTCTTTTATTGGAAATAAGTGGCACGCCCCAT TGCCTTCTAGTCGCCTCCCCGAAGCGAAGAGGCCGAAGCGAAGAGG CCTGGTGGGTTGTCTCAACATCCTTTTGCTGAGAATCGAATACGCAGC CGATGAACAGCCAGGAAGGGTGCAAGGAAACCTTGAACGGCATCTAC CAGTTCATCATGGACCGCTTCCCCTTCTACCGGGAGAACAAGCAGGG CTGGCAGAACAGCATCCGCCACAACCTCTCGCTCAACGAGTGCTTCG TCAAGGTGCCCCGCGACGACAAGAAGCCCGGCAAG
FOXC2-SAS RNA (SEQ ID NO:13) FOXC2-SAS RNA (SEQ ID NO: 13)
UACAUUUUCG UCUUCUGUUC UUUUAUUGGA AAUAAGUGGC ACGCCCCAUU GCCUUCUAGU CGCCUCCCCG AAGCGAAGAG GCCGAAGCGA AGAGGCCUGG UGGGUUGUCU CAACAUCCUU UUGCUGAGAA UCGAAUACGC AGCCGAUGAA CAGCCAGGAA GGGUGCAAGG AAACCUUGAA CGGCAUCUAC CAGUUCAUCA UGGACCGCUU CCCCUUCUAC CGGGAGAACA AGCAGGGCUG GCAGAACAGC AUCCGCCACA ACCUCUCGCU CAACGAGUGC UUCGUCAAGG UGCCCCGCGA CGACAAGAAG CCCGGCAAG UACAUUUUCG UCUUCUGUUC UUUUAUUGGA AAUAAGUGGC ACGCCCCAUU GCCUUCUAGU CGCCUCCCCG AAGCGAAGAG GCCGAAGCGA AGAGGCCUGG UGGGUUGUCU CAACAUCCUU UUGCUGAGAA UCGAAUACGC AGCCGAUGAA CAGCCAGGAA GGGUGCAAGG AAACCUUGAA CGGCAUCUAC CAGUUCAUCA UGGACCGCUU CCCCUUCUAC CGGGAGAACA AGCAGGGCUG GCAGAACAGC AUCCGCCACA ACCUCUCGCU CAACGAGUGC UUCGUCAAGG UGCCCCGCGA CGACAAGAAG CCCGGCAAG
L’analisi di omologia è stata fatta tramite il sito https://blast.ncbi.nlm.nih.gov/Blast.cgi e il risultato ottenuto è riportato di seguito (sequenza query: FOXL1; sequenza sbjct: FOXC2-SAS): The homology analysis was done via the site https://blast.ncbi.nlm.nih.gov/Blast.cgi and the result obtained is shown below (query sequence: FOXL1; sbjct sequence: FOXC2-SAS):
Per escludere l’esistenza di complementarietà tra la molecola FOXC2-SAS e altri lncRNA nell’uomo, è stata fatta un’analisi di comparazione con circa 16000 sequenze di lncRNA riportate nella banca dati GENCODE (release 28 GRCh38.p12). Dall’analisi nessuna delle sequenze riportate è risultata complementare alla molecola FOXC2-SAS confermando la sua specificità con FOXC2-AS1. To exclude the existence of complementarity between the FOXC2-SAS molecule and other lncRNAs in humans, a comparison analysis was performed with about 16,000 lncRNA sequences reported in the GENCODE database (release 28 GRCh38.p12). From the analysis none of the sequences reported was complementary to the FOXC2-SAS molecule, confirming its specificity with FOXC2-AS1.
Poiché il legame tra FOXC2-AS1 e la molecola FOXC2-SAS è altamente specifico, è stato utilizzato un programma di predizione sull’interazione tra 2 sequenze di RNA (http://rna.informatik.uni-freiburg.de/IntaRNA) per cercare di determinare gli effetti di possibili mutazioni che coinvolgono percentuali diverse della sequenza di FOXC2-SAS. L’analisi ha mostrato che mutazioni che causano cambiamenti di almeno il 3-5% della sequenza FOXC2-SAS potrebbero modificare l’interazione con FOXC2-AS1. Since the link between FOXC2-AS1 and the FOXC2-SAS molecule is highly specific, a prediction program on the interaction between 2 RNA sequences (http://rna.informatik.uni-freiburg.de/IntaRNA) was used to try to determine the effects of possible mutations involving different percentages of the FOXC2-SAS sequence. The analysis showed that mutations that cause changes of at least 3-5% of the FOXC2-SAS sequence could modify the interaction with FOXC2-AS1.
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