CN105803056B - Application of human IARS2 gene and related medicine thereof - Google Patents
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Abstract
The invention discloses application of a human IARS2 gene and related medicines thereof. The invention discloses an application of a human IARS2 gene in tumor treatment, tumor diagnosis and medicine preparation. The invention further constructs human IARS2 gene small interfering RNA, human IARS2 gene interfering nucleic acid construct, human IARS2 gene interfering slow virus and discloses the application thereof. The siRNA or the nucleic acid construct containing the siRNA sequence and the lentivirus provided by the invention can specifically inhibit the expression of human IARS2 gene, especially the lentivirus can efficiently infect target cells, efficiently inhibit the expression of IARS2 gene in the target cells, further inhibit the growth of tumor cells and promote the apoptosis of the tumor cells, and have important significance in tumor treatment.
Description
Technical Field
The invention relates to the technical field of biology, in particular to application of a human IARS2 gene and a related medicine thereof.
Background
RNA interference (RNAi) is the post-transcriptional gene silencing by short double-stranded RNA (dsrna) composed of nucleotides. It can block the expression of specific gene in body efficiently and specially to result in its degradation, so that it can cause the silencing of specific gene in the body and make the cell show the deletion of some gene phenotype. Studies have shown that double-stranded RNA of 21-23nt in length is capable of specifically causing RNAi at the transcriptional and post-transcriptional levels (Tuschl T, Zamore PD, Sharp PA, Bartel DP. RNAi: double-stranded RNA direct the ATP-dependent cleavage of mRNA at 21to23nucleotide intervals. cell 2000; 101: 25-33.). The five-year survival rate of tumor patients is very low although chemotherapy, radiotherapy and comprehensive treatment are carried out on the tumor patients, and if genes related to the tumor morbidity and progression can be intervened, a new way for treating tumors can be developed. In recent years, RNAi has become an effective strategy for gene therapy of tumors. RNAi technology can be used to inhibit the expression of proto-oncogenes, mutated anti-oncogenes, cell cycle-related genes, anti-apoptosis-related genes, etc., to inhibit tumor progression (Uplichard, Susan L. the therapeutic potential of RNA interference. FEBS Letters 2005; 579: 5996-.
IARS2 is a mitochondrial protein (Cotter D, Guda P, Fahy E, Subramaniam S.2004.MitoProteome: mitochondrial protein sequence database and a nanotationsystem. nucleic Acids Res32: D463-D467.), IARS2 gene has a highly conserved evolutionary sequence, and studies have shown that in many species, the IARS2 gene was aligned, showing a conserved site in the map, and that p.P90L 9is a functional protein that is effective when queried by SIFT, but is likely to be disrupted by PolyPhon 2HumDiv and PolyPhon 2 HumVar. Further studies have shown that IARS2 protein in patient skin cells directly affects the p.P909L mutation [ Yao and Shoubabr idge,1999 ]. The IARS2 antibody is detected to be more in the mutant cells than in the wild-type fiber cells by the binding method of the immunohistochemical polyclonal antibody. The presence of the p.P909L mutation in the IARS2 gene did not affect the transcriptional activity of mitochondrial enzymes (Sasarman F, Shoubridge EA.2012.radio active labeling of mitochondral transformation products in pooled cells. methods Mol Biol 837: 207-217).
Clinical studies have shown that a reduction in the levels of IARS2 protein is found in cells in some patients, and it can be concluded that the alteration in IARS2 is a pathological mutation that is the initial cause of the pathological phenotype in some later-stage patients. Several functional experiments also demonstrated that a reduction in IARS2 protein leads to aberrant translation of the OXPHHO respiratory system (Konovalova S, Tyynisma H.2013.Mitochondrial a minoacyl-tRNA synthenases in hu-mandiscease. mol Genet Metab108: 206-.
However, the current experimental report of the IARS2 gene in tumor-related fields is still a blank, especially in the field of human glioma research.
Disclosure of Invention
The invention aims to disclose a treatment method and a medicament related to a human IARS2 gene, and research the action of the IARS2 gene in the survival and apoptosis process of tumor cells by taking RNA interference (RNAi) as a means.
In the first aspect of the invention, the role of IARS2 gene in tumorigenesis and development is studied by means of RNA interference, and a method for inhibiting or reducing tumor cell growth, proliferation, differentiation and/or survival is disclosed, which comprises the following steps: administering to the tumor cell a molecule that specifically inhibits transcription or translation of the IARS2 gene, or that specifically inhibits expression or activity of the IARS2 gene, thereby inhibiting growth, proliferation, differentiation, and/or survival of the tumor cell.
The tumor cell is selected from tumor cells whose growth is associated with expression or activity of IARS2 gene. Preferably, the tumor cell is selected from the group consisting of gliomas.
In the method of inhibiting or reducing growth, proliferation, differentiation and/or survival of a tumor cell, the molecule is administered in an amount sufficient to reduce transcription or translation of the IARS2 gene, or to reduce expression or activity of the IARS2 gene. Further, the expression of the IARS2 gene is reduced by at least 50%, 80%, 90%, 95%, or 99%.
The molecule may be selected from, but is not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemical drugs, antibody drugs, polypeptides, proteins, or interfering lentiviruses.
Such nucleic acids include, but are not limited to: antisense oligonucleotides, double-stranded RNA (dsRNA), ribozymes, small interfering RNA (esiRNA) produced by endoribonuclease III, or short hairpin RNA (shRNA).
The double-stranded RNA, ribozyme, esiRNA or shRNA contains a promoter sequence of IARS2 gene or an information sequence of IARS2 gene.
Further, the double-stranded RNA is small interfering RNA (siRNA). The small interfering RNA comprises a first strand and a second strand, the first strand and the second strand are complementary to form an RNA dimer, and the sequence of the first strand is substantially identical to a sequence of 15-27 contiguous nucleotides in the IARS2 gene. The small interfering RNA can specifically bind to an mRNA fragment encoded by a target sequence and specifically silence the expression of the human IARS2 gene.
Further, the first strand sequence of the small interfering RNA is substantially identical to the target sequence in the IARS2 gene. Preferably, the target sequence in the IARS2 gene comprises SEQ ID NO. 1.
The target sequence in the IARS2 gene is a fragment in the IARS2 gene corresponding to an mRNA fragment complementarily combined with the small interfering RNA when the small interfering RNA specifically silences the expression of the IARS2 gene.
Preferably, the IARS2 gene is of human origin.
The invention also discloses the application of the separated human IARS2 gene in preparing or screening tumor treatment medicines or preparing tumor diagnosis medicines.
Further, the tumor is selected from glioma.
The application of the isolated IARS2 gene in preparing or screening the tumor treatment medicine comprises two aspects: firstly, the IARS2 gene is used as a drug or a preparation to be applied to the preparation of tumor treatment drugs or preparations aiming at the action target of tumor cells; secondly, the IARS2 gene is used as a drug or a preparation to be applied to screening tumor treatment drugs or preparations aiming at the action target of tumor cells.
The application of the IARS2 gene as a drug or preparation aiming at the action target of tumor cells in preparing tumor treatment drugs or preparations specifically comprises the following steps: the IARS2 gene is used as a target of RNA interference effect to develop a medicament or a preparation aiming at tumor cells, so that the expression level of the IARS2 gene in the tumor cells can be reduced.
The application of the IARS2 gene as an action target of a medicine or a preparation aiming at tumor cells in screening tumor treatment medicines or preparations specifically comprises the following steps: the IARS2 gene is used as an action object, and the medicine or the preparation is screened to find the medicine which can inhibit or promote the expression of the human IARS2 gene and is used as a candidate medicine for treating the tumor. The IARS2 gene small interfering RNA (siRNA) is obtained by screening human IARS2 gene serving as an action object and can be used as a medicament with the effect of inhibiting tumor cell proliferation. In addition, IARS2 gene and its protein can be used as target for antibody drug, small molecule drug, etc.
The IARS2 gene is used for preparing the tumor diagnosis medicament, and the IARS2 gene expression product is used as a tumor diagnosis index for preparing the tumor diagnosis medicament.
The expression level of IARS2 gene in 4 glioma cells was detected by Real-time Quantitative PCR. The research finds that: IARS2 was expressed in high abundance in 4 glioma cells. The IARS2 is suggested to play an important role in the generation and development of tumors as an oncogene; the expression level of IARS2 gene can be a marker for tumor diagnosis.
The tumor treatment drug is a molecule which can specifically inhibit the transcription or translation of IARS2 gene, or can specifically inhibit the expression or activity of IARS2 protein, thereby reducing the expression level of IARS2 gene in tumor cells and achieving the purpose of inhibiting the proliferation, growth, differentiation and/or survival of the tumor cells.
The tumor therapeutic drug or tumor diagnostic drug prepared or screened by the isolated IARS2 gene includes but is not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemical drugs, antibody drugs, polypeptides, proteins, or interfering lentiviruses.
Such nucleic acids include, but are not limited to: antisense oligonucleotides, double-stranded RNA (dsRNA), ribozymes, small interfering RNA (esiRNA) produced by endoribonuclease III, or short hairpin RNA (shRNA).
The amount of the tumor treatment agent administered is a dose sufficient to reduce transcription or translation of the human IARS2 gene, or to reduce expression or activity of the human IARS2 gene. Such that the expression of the human IARS2 gene is reduced by at least 50%, 80%, 90%, 95%, or 99%.
The method for treating the tumor by adopting the tumor treatment medicine mainly achieves the aim of treating by reducing the expression level of human IARS2 gene and inhibiting the proliferation of tumor cells. In particular, in therapy, a substance effective to reduce the expression level of human IARS2 gene is administered to a patient.
In a second aspect, the present invention discloses an isolated nucleic acid molecule for reducing expression of the IARS2 gene in a tumor cell, said nucleic acid molecule comprising:
a) a double-stranded RNA comprising a nucleotide sequence capable of hybridizing to the IARS2 gene under stringent conditions;
or
b) An shRNA comprising a nucleotide sequence capable of hybridizing to the IARS2 gene under stringent conditions.
Further, the double-stranded RNA comprises a first strand and a second strand, the first strand and the second strand are complementary to form an RNA dimer, and the sequence of the first strand is substantially identical to 15-27 consecutive nucleotide sequences in the IARS2 gene. Preferably, the sequence of the first strand is substantially identical to a 19-23 contiguous nucleotide sequence in the IARS2 gene; more preferably, the sequence of the first strand is substantially identical to a 19, 20 or 21 contiguous nucleotide sequence of the IARS2 gene.
Further, the double-stranded RNA comprises a first strand and a second strand, the first strand and the second strand are complementary to form an RNA dimer, and the sequence of the first strand is substantially identical to a target sequence in the IARS2 gene.
The length of the first strand and the second strand of the double-stranded RNA are both 15-27 nucleotides; preferably, the length is 19-23 nucleotides; most preferably, the length is 19, 20 or 21 nucleotides.
Further, the double-stranded RNA is small interfering RNA (siRNA). Further, the sequence of the first strand of the small interfering RNA is shown in SEQ ID NO: shown at 9, specifically 5'-GUACUUGCAGUCAUCCAUUAA-3'.
SEQ ID NO: the siRNA shown in the 9 is designed by taking the sequence shown in SEQ ID NO. 1 as an RNA interference target sequence and aiming at one strand of small interfering RNA of human IARS2 gene, the sequence of the other strand, namely the second strand, is complementary with the sequence of the first strand, and the siRNA can play a role in specifically silencing the expression of endogenous IARS2 gene in tumor cells.
Further, the shRNA comprises a sense strand segment and an antisense strand segment, and a stem-loop structure connecting the sense strand segment and the antisense strand segment, wherein the sequences of the sense strand segment and the antisense strand segment are complementary, and the sequence of the sense strand segment is substantially identical to 15-27 consecutive nucleotide sequences in the IARS2 gene. The shRNA can become small interfering RNA (siRNA) after being processed, and further plays a role in specifically silencing the expression of endogenous IARS2 genes in tumor cells.
Further, the shRNA comprises a sense strand segment and an antisense strand segment, and a stem-loop structure connecting the sense strand segment and the antisense strand segment, wherein the sequences of the sense strand segment and the antisense strand segment are complementary, and the sequence of the sense strand segment is substantially identical to a target sequence in the IARS2 gene.
Preferably, the sense strand fragment is substantially identical to 19-23 contiguous nucleotide sequences in the IARS2 gene; more preferably, the sense strand fragment is substantially identical to a 19, 20 or 21 contiguous nucleotide sequence of the IARS2 gene.
Further, the sequence of the stem-loop structure of the shRNA can be selected from any one of the following sequences: UUCAAGAGA, AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU, and CCACACC.
Furthermore, the sequence of the shRNA is shown as SEQ ID NO:14, and specifically comprises the following steps: 5'-GUACUUGCAGUCAUCCAUUAAUUCAAGAGAUUAAUGGAUGACUGCAAGUAC-3' are provided.
The shRNA can become siRNA after enzyme digestion processing, and further plays a role in specifically silencing the expression of endogenous human IARS2 genes in tumor cells.
The interfering slow virus vector of the gene segment for encoding the shRNA contains SEQ ID NO 1 and a complementary sequence thereof.
The first strand of the double-stranded RNA or the sense strand segment of the shRNA is basically the same as a target sequence in an IARS2 gene, and the target sequence of the IARS2 gene is a segment in an IARS2 gene corresponding to an mRNA segment which is identified and silenced by the siRNA when the siRNA is used for specifically silencing expression of the IARS2 gene.
Preferably, the target sequence in the IARS2 gene comprises SEQ ID NO. 1.
Further, the IARS2 gene is derived from a human.
In a third aspect of the invention, an IARS2 gene interfering nucleic acid construct is disclosed, which comprises a gene segment for encoding shRNA in the isolated nucleic acid molecule of the invention and can express the shRNA.
The human IARS2 gene interfering nucleic acid construct can be obtained by cloning a gene segment for coding the human IARS2 gene shRNA into a known vector. Further, the IARS2 gene interference nucleic acid construct is an IARS2 gene interference lentiviral vector.
The IARS2 gene interference lentiviral vector is obtained by cloning a DNA fragment for coding the IARS2 gene shRNA into a known vector, wherein the known vector is mostly a lentiviral vector, the IARS2 gene interference lentiviral vector is packaged into infectious viral particles by virus, then infects tumor cells, further transcribes the shRNA, and finally obtains the siRNA through the steps of enzyme digestion processing and the like, so that the siRNA is used for specifically silencing the expression of the IARS2 gene.
Further, the IARS2 gene interference lentiviral vector also contains a promoter sequence and/or a nucleotide sequence encoding a marker which can be detected in tumor cells; preferably, the detectable label is Green Fluorescent Protein (GFP).
Further, the lentiviral vector may be selected from the group consisting of: pLKO.1-puro, pLKO.1-CMV-tGFP, pLKO.1-puro-CMV-tGFP, pLKO.1-CMV-Neo, pLKO.1-Neo-CMV-tGFP, pLKO.1-puro-CMV-TagCFP, pLKO.1-puro-CMV-TagYFP, pLKO.1-puro-CMV-TagFP635, pLKO.1-puro-UbC-TurboGFP, pLKO.1-puro-UbC-TagFP635, pLKO-puro-IPTG-1xLacO, pLKO-puro-IPTG-3xLacO, pLP1, pLP2, pLP/VSV-G, pENTR/U6, pLenti6/BLOCK-iT-DEST, pLenti 6-GW/U6-laminsham, pcDNA1.2/V5-GW/lacZ, pLenti6.2/N-Lumio/V5-DEST, pGCSIL-GFP or pLenti 6.2/N-Lumio/V5-GW/lacZ.
The embodiment of the invention specifically discloses a human IARS2 gene interference lentiviral vector constructed by taking pGCSIL-GFP as a vector, which is named as pGCSIL-GFP-IARS 2-siRNA.
The isolated nucleic acid molecule of the invention can be used for preparing a medicament for preventing or treating tumors, wherein the tumors are gliomas.
The IARS2 gene siRNA can be used for inhibiting the proliferation of tumor cells, and further can be used as a medicament or a preparation for treating tumors. The IARS2 gene interference lentiviral vector can be used for preparing the IARS2 gene siRNA. When used as a medicament or formulation for treating tumors, a safe and effective amount of the nucleic acid molecule is administered to a mammal. The particular dosage will also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.
The invention discloses an IARS2 gene interference lentivirus in the fourth aspect, which is prepared by virus packaging of the IARS2 gene interference lentivirus vector under the assistance of lentivirus packaging plasmid and cell line. The lentivirus can infect tumor cells and generate small interfering RNA aiming at IARS2 gene, thereby inhibiting the proliferation of glioma tumor cells. The IARS2 gene interference lentivirus can be used for preparing medicaments for preventing or treating tumors.
In the fifth aspect of the invention, a pharmaceutical composition for preventing or treating tumor is disclosed, the effective substance of which comprises one or more combinations of the isolated nucleic acid molecule, IARS2 gene interference nucleic acid construct or IARS2 gene interference lentivirus.
Further, the pharmaceutical composition contains 1-99 wt% of the double-stranded RNA, shRNA, IARS2 gene interference nucleic acid construct or IARS2 gene interference lentivirus, and a pharmaceutically acceptable carrier, diluent or excipient.
In preparing these compositions, the active ingredient is typically mixed with, or diluted with, excipients or enclosed within a carrier which may be in the form of a capsule or sachet. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material that acts as a vehicle, carrier, or medium for the active ingredient. Thus, the composition may be in the form of tablets, pills, powders, solutions, syrups, sterile injectable solutions and the like. Examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starch, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, and the like. The preparation may further comprise a humectant, an emulsifier, a preservative (such as methyl and propyl hydroxybenzoate), a sweetener, etc.
The invention also discloses application of the pharmaceutical composition in preparing a tumor treatment medicament for treating glioma.
The application of the pharmaceutical composition provides a method for treating tumors, in particular to a method for preventing or treating tumors in a subject, which comprises the step of administering an effective dose of the pharmaceutical composition to the subject. Further, the tumor is glioma.
When the pharmaceutical composition is used for preventing or treating tumors in a subject, an effective dose of the pharmaceutical composition needs to be administered to the subject. Using this method, the growth, proliferation, recurrence and/or metastasis of the tumor is inhibited. Further, at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% fraction of the growth, proliferation, recurrence and/or metastasis of the tumor is inhibited.
The subject of the method may be a human.
In a sixth aspect of the invention, a kit for reducing expression of IARS2 gene in a tumor cell is disclosed, the kit comprising: the isolated nucleic acid molecule, the IARS2 gene interfering nucleic acid construct, and/or the IARS2 gene interfering lentivirus present in the container.
In conclusion, the invention designs 1 RNAi target sequence of human IARS2 gene, constructs corresponding IARS2 gene RNAi vector, wherein the coding sequence SEQ ID NO: the RNAi vector pGCSIL-GFP-IARS2-siRNA of 1 can obviously reduce the expression of IARS2 gene at mRNA level and protein level. Lentivirus (lentivirus, abbreviated as Lv) is used as a gene operation tool to carry an RNAi vector pGCSIL-GFP-IARS2-siRNA, so that the RNAi sequence aiming at the IARS2 gene can be efficiently introduced into glioma U251 cells in a targeted manner, the expression level of the IARS2 gene is reduced, and the proliferation capacity of the tumor cells is remarkably inhibited. Lentivirus-mediated IARS2 gene silencing is therefore a potential clinical non-surgical treatment modality for malignancies.
The siRNA or the nucleic acid construct containing the siRNA sequence and the lentivirus provided by the invention can specifically inhibit the expression of human IARS2 gene, especially the lentivirus can efficiently infect target cells, efficiently inhibit the expression of IARS2 gene in the target cells, promote apoptosis, reduce the invasion and transfer capacity of tumor cells and the like, further inhibit the growth of the tumor cells, promote the apoptosis of the tumor cells and have important significance in tumor treatment.
Drawings
FIG. 1: pGCSIL-GFP plasmid DNA map
FIG. 2: IARS 2-after 5 days when human glioma U251 cells are infected by RNAi lentivirus, the expression level of IARS2mRNA is remarkably reduced
FIG. 3: IARS 2-inhibition of cell proliferation caused by 5 days after human glioma U251 cells are infected by RNAi lentivirus
Detailed Description
The invention relates to a group of small interfering RNA (siRNA) sequences, RNA interference vectors and RNA interference lentiviruses aiming at human IARS2 genes. The coding region sequence of human IARS2mRNA is selected as the target site of siRNA, and the siRNA target site sequence is designed according to the continuous 10-30 (preferably 15-27, more preferably 19-23) base sequences in the target site. Constructing a nucleic acid construct expressing the siRNA through gene cloning, and packaging the lentivirus expressing the siRNA. Cell experiments prove that the siRNA sequence can specifically silence the expression of an endogenous IARS2 gene in human tumor cells.
The inventor finds that after the expression of the human IARS2 gene is down-regulated by an RNAi method, the proliferation of tumor cells and the like can be effectively inhibited, the growth process of tumors can be effectively controlled, and the research result shows that the IARS2 gene is a protooncogene and can be used as a target for tumor treatment. The inventor further synthesizes and tests a plurality of siRNAs aiming at IARS2 gene, screens out the siRNA which can effectively inhibit IARS2 expression and further inhibit the proliferation and growth of human glioma U521 cell, and completes the invention on the basis.
The invention provides a series of small interfering RNA (siRNA) sequences interfering human IARS2 gene, and constructs a lentivirus capable of specifically silencing IARS2 gene expression. The research of the invention finds that the small interfering RNA and RNAi lentivirus designed aiming at the human IARS2 gene can stably and specifically down-regulate the expression of the IARS2 gene and effectively inhibit the proliferation of human tumor cells. The invention shows that the IARS2 gene can promote the growth of tumor cells and is expected to become a target for early diagnosis and treatment of tumors. In addition, silencing the expression of IARS2 gene by RNAi method can be used as an effective means for inhibiting tumor development.
The design idea of the invention is as follows:
the invention obtains a human IARS2 gene RNAi lentivirus by screening through the following method: the human IARS2 gene sequence is adjusted from Genbank; predicting the siRNA site; synthesizing an effective siRNA sequence aiming at IARS2 gene and a double-stranded DNA Oligo with two ends containing sticky ends of enzyme cutting sites; after double enzyme digestion, the lentiviral vector is connected with a double-stranded DNA Oligo to construct an RNAi plasmid for expressing an IARS2 gene siRNA sequence; the RNAi plasmid and a helper vector (packaging Mix, Sigma-aldrich company) required by lentivirus packaging are cotransfected with a human embryonic kidney cell 293T to generate recombinant lentivirus particles, and the lentivirus capable of efficiently silencing IARS2 gene can be prepared.
Based on the method, the invention provides 1 effective target (shown as SEQ ID NO: 1) for interfering IARS2 gene, and constructs the lentivirus of specific interference human IARS2 gene.
Meanwhile, the invention also discloses an RNAi lentivirus (IARS2-RNAi) of the human IARS2 gene and preparation and application thereof.
The research discovers that the proliferation of the tumor cells can be effectively inhibited after the expression of IARS2 gene in the tumor cells is reduced by using a lentivirus-mediated RNAi method. The research shows that the IARS2 gene is a protooncogene, can promote the proliferation of tumor cells, has important biological functions in the occurrence and development of tumors, the IARS2 gene can be a target for tumor treatment, and the lentivirus-mediated IARS2 gene specific silencing can be used as a new means for tumor treatment.
The invention is further illustrated by the following examples. It should be understood that the examples are for illustrative purposes only and are not intended to limit the scope of the present invention. In the examples, the experimental methods and reagents having no specific conditions described therein were performed under conventional conditions, such as those described in [ U.S. Sambrook.J.; huang Beitang, etc. Molecular cloning test guidelines, third edition. Beijing: the conditions described in scientific press 2002 or conditions suggested by the manufacturer.
Example 1 preparation of RNAi lentivirus against human IARS2 Gene
1. Screening effective siRNA target point aiming at human IARS2 gene
The IARS2 (NM-018060) gene information is called from Genbank; designing effective siRNA target point aiming at IARS2 gene. Table 1 lists 1 of the effective siRNA target sequences against IARS2 gene.
TABLE 1 siRNA target sequences targeting the human IARS2 gene
| SEQ ID NO | TargetSeq |
| 1 | GTACTTGCAGTCATCCATTAA |
2. Preparation of Lentiviral vectors
Synthesizing double-stranded DNA Oligo sequences (Table 2) containing Age I and EcoR I enzyme cutting sites at two ends aiming at siRNA targets (taking SEQ ID NO:1 as an example); the restriction enzymes Age I and EcoR I were used to act on pGCSIL-GFP vector (supplied by Shanghai Jikai Gene chemistry, Ltd., FIG. 1), which was linearized, and the cleaved fragments were identified by agarose gel electrophoresis.
TABLE 2 double-stranded DNA Oligo with Age I and EcoR I cleavage sites at both ends
The vector DNA linearized by double digestion (digestion system shown in Table 4, 37 ℃, reaction 1h) was ligated to the purified double-stranded DNA Oligo by T4DNA ligase, ligated overnight at 16 ℃ in an appropriate buffer system (ligation system shown in Table 5), and the ligation product was recovered. The ligation product was transformed into calcium chloride prepared fresh E.coli competent cells (transformation protocol reference: molecular cloning protocols second edition, pages 55-56). Dipping the surface of the clone of the strain growing from the connected transformation product, dissolving the surface in 10 mu lLB culture medium, uniformly mixing, and taking 1 mu l as a template; designing universal PCR primers at the upstream and downstream of RNAi sequence in the lentiviral vector, wherein the upstream primer sequence: 5'-CCTATTTCCCATGATTCCTTCATA-3' (SEQ ID NO: 6); the sequence of the downstream primer is as follows: 5'-GTAATACGGTTATCCACGCG-3' (SEQ ID NO: 7), and a PCR identification experiment was performed (PCR reaction system shown in Table 6-1, reaction conditions shown in Table 6-2). Sequencing and comparing the clones which are identified to be positive by the PCR, wherein the correctly compared clones are the clones which are successfully constructed and are directed at the nucleotide sequence shown in SEQ ID NO:1, named pGCSIL-GFP-IARS 2-siRNA.
pGCSIL-GFP-Scr-siRNA negative control plasmid was constructed with negative control siRNA target sequence 5'-TTCTCCGAACGTGTCACGT-3' (SEQ ID NO: 8). When pGCSIL-GFP-Scr-siRNA negative control plasmids are constructed, double-stranded DNA Oligo sequences (table 3) containing adhesive ends of Age I and EcoR I enzyme cutting sites at two ends are synthesized aiming at the Scr siRNA target spots, and the rest construction methods, identification methods and conditions are the same as pGCSIL-GFP-IARS 2-siRNA.
TABLE 3 double-stranded DNA Oligo with Age I and EcoR I cleavage sites at both ends
The vector was linearized by T4DNA ligase (digestion system shown in Table 4, 37 ℃ C., reaction time 1h)
TABLE 4 pGCSIL-GFP plasmid digestion reaction System
| Reagent | Volume (μ l) |
| pGCSIL-GFP plasmid (1. mu.g/. mu.l) | 2.0 |
| 10×buffer | 5.0 |
| 100×BSA | 0.5 |
| Age I(10U/μl) | 1.0 |
| EcoR I(10U/μl) | 1.0 |
| dd H2O | 40.5 |
| Total | 50.0 |
TABLE 5 ligation reaction System of vector DNA and double-stranded DNA Oligo
| Reagent | Positive control (μ l) | Self-contained control (μ l) | Connecting group (mu l) |
| Linearized vector DNA (100 ng/. mu.l) | 1.0 | 1.0 | 1.0 |
| Annealed double stranded DNA Oligo (100 ng/. mu.l) | 1.0 | - | 1.0 |
| 10 × T4 phage DNA ligase buffer | 1.0 | 1.0 | 1.0 |
| T4 phage DNA ligase | 1.0 | 1.0 | 1.0 |
| dd H2O | 16.0 | 17.0 | 16.0 |
| Total | 20.0 | 20.0 | 20.0 |
TABLE 6-1 PCR reaction System
| Reagent | Volume (μ l) |
| 10×buffer | 2.0 |
| dNTPs(2.5mM) | 0.8 |
| Upstream primer | 0.4 |
| Downstream primer | 0.4 |
| Taq polymerase | 0.2 |
| Form panel | 1.0 |
| ddH2O | 15.2 |
| Total | 20.0 |
TABLE 6-2 PCR reaction System Programming
3. Packaging IARS2-siRNA lentivirus
The DNA of RNAi plasmid pGCSIL-GFP-IARS2-siRNA was extracted with a plasmid extraction kit from Qiagen corporation to prepare 100 ng/. mu.l stock solution.
24h before transfection, human embryonic kidney cell 293T cells in logarithmic growth phase were trypsinized and cell density was adjusted to 1.5 × 10 in DMEM complete medium containing 10% fetal bovine serum5Cells/ml, seeded in 6-well plates at 37 ℃ with 5% CO2Culturing in an incubator. The cell density can reach 70-80% to be used for transfection. 2h before transfection, the original medium was aspirated and 1.5ml of fresh complete medium was added. Mu.l of Packing Mix (PVM), 12. mu.l of PEI, and 400. mu.l of serum-free DMEM medium were added to a sterilized centrifuge tube according to the instructions of the MISSION Lentiviral Packing Mix kit from Sigma-aldrich, and 20. mu.l of the above-mentioned extracted plasmid DNA was added to the above-mentioned PVM/PEI/DMEM mixture.
The transfection mixture was incubated at room temperature for 15min, transferred to medium of human embryonic kidney 293T cells at 37 ℃ with 5% CO2Culturing for 16h in an incubator. The medium containing the transfection mixture was discarded, washed with PBS solution, 2ml of complete medium was added and incubation continued for 48 h. The cell supernatant was collected, and the lentivirus was purified and concentrated by a Centricon Plus-20 centrifugal ultrafiltration device (Millipore) according to the following steps: (1) centrifuging at 4 deg.C and 4000g for 10min to remove cell debris; (2) filtering the supernatant with a 0.45 μm filter in a 40ml ultracentrifuge tube; (3) centrifuging at 4000g for 10-15min to obtain the required virus concentration volume; (4) after the centrifugation is finished, separating the filter cup from the lower filtrate collecting cup, reversely buckling the filter cup on the sample collecting cup, and centrifuging for 2min until the centrifugal force is not more than 1000 g; (5) the centrifuge cup is removed from the sample collection cup, and the virus concentrate is obtained. Subpackaging the virus concentrated solution and storing at-80 ℃. The sequence of the first strand of siRNA contained in the virus concentrated solution is shown in SEQ ID NO. 9. Packaging procedure for control lentivirusLike IARS2-siRNA lentivirus, pGCSIL-GFP-IARS2-siRNA vector was replaced by pGCSIL-GFP-Scr-siRNA vector alone.
Example 2 detection of silencing efficiency of IARS2 Gene by real-time fluorescent quantitative RT-PCR
Glioma U251 cells in logarithmic growth phase were trypsinized to prepare a cell suspension (cell number about 5 × 10)4/ml) were inoculated in 6-well plates and cultured until the degree of cell confluence reached about 30%. According to the value of the multiplicity of infection (MOI, U251:5), an appropriate amount of the virus prepared in example 1 was added, the medium was changed after 24 hours of culture, and after the infection time reached 5 days, the cells were collected. Total RNA was extracted according to the Trizol protocol of Invitrogen corporation. The RNA was reverse-transcribed to obtain cDNA according to the M-MLV protocol of Promega (reverse transcription reaction system shown in Table 7, reaction at 42 ℃ for 1 hour, followed by inactivation of the reverse transcriptase by water bath in a 70 ℃ water bath for 10 min).
Real-time quantitative detection was carried out using a TP800 Real time PCR instrument (TAKARA). Primers for the IARS2 gene were as follows: an upstream primer 5'-TGGACCTCCTTATGCAAACGG-3' (SEQ ID NO: 10) and a downstream primer 5'-GGCAACCCATGACAATCCCA-3' (SEQ ID NO: 11). The housekeeping gene GAPDH is used as an internal reference, and the primer sequences are as follows: an upstream primer 5'-TGACTTCAACAGCGACACCCA-3' (SEQ ID NO: 12) and a downstream primer 5'-CACCCTGTTGCTGTAGCCAAA-3' (SEQ ID NO: 13). The reaction system was prepared in the proportions shown in Table 8.
TABLE 7 reverse transcription reaction System
| Reagent | Volume (μ l) |
| 5×RT buffer | 4.0 |
| 10mM dNTPs | 2.0 |
| RNasin | 0.5 |
| M-MLV-RTase | 1.0 |
| DEPC H2O | 3.5 |
| Total | 11.0 |
TABLE 8 Real-time PCR reaction System
| Reagent | Volume (μ l) |
| SYBR premix ex taq: | 10.0 |
| Upstream primer (2.5 μ M): | 0.5 |
| downstream primer (2.5 μ M): | 0.5 |
| cDNA | 1.0 |
| ddH2O | 8.0 |
| Total | 20.0 |
the program was a two-step Real-time PCR: pre-denaturation at 95 ℃ for 15 s; then, denaturation is carried out at 95 ℃ for 5s in each step; annealing and extending for 30s at 60 ℃; a total of 45 cycles were performed. Each time reading the absorbance value during the extension phase. After the PCR was completed, the DNA was denatured at 95 ℃ for 1min, and then cooled to 55 ℃ to allow the DNA double strands to be sufficiently bound. Melting curves were prepared by increasing the temperature from 55 ℃ to 95 ℃ by 0.5 ℃ for 4 seconds and reading the absorbance. By adopting 2-ΔΔCtThe analysis method calculates the expression abundance of IARS2 infected mRNA. Cells infected with a control virus (Lv-Scr-siRNA) served as controls. The results of the experiment (fig. 2) show that the expression level of IARS2mRNA in glioma U251 cells was down-regulated by 87.3%.
Example 3 examination of the proliferative Capacity of IARS 2-siRNA-infected tumor cells
Glioma U251 cells in logarithmic growth phase were trypsinized to prepare a cell suspension (cell number about 5 × 10)4Perml) was inoculated into 6-well plates and cultured until the degree of cell confluence reached about 30%. according to the multiplicity of infection (MOI, U251:5), an appropriate amount of virus was added, the medium was changed after 24h of culture, and the experimental cells in the logarithmic growth phase were collected after the infection time reached 5 days-complete medium was resuspended to a cell suspension (2 × 10)4Per ml) at a cell density of about 2000 per well, 96-well plates were seeded. Each set of 5 duplicate wells, 100. mu.l per well. After the plate is laid, the plate is placed at 37 ℃ and 5% CO2Culturing in an incubator. The plate readings were performed once a day with Cellomics apparatus (Thermo Fisher) for 5 consecutive days starting the second day after plating. The number of green fluorescent cells in the well plate for each scan was accurately calculated by adjusting the input parameters of the Cellomics arrayscan, and the data were statistically plotted to generate a cell proliferation curve (the results are shown in FIG. 3). The results show that after the tumor cells of the lentivirus infection group are cultured in vitro for 5 days, the proliferation speed is obviously slowed down and is far lower than that of the tumor cells of the control group, and the number of the active cells is reduced80.2% indicates that IARS2 gene silencing results in inhibition of tumor cell proliferation capacity.
While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.
Claims (8)
1. Use of a molecule capable of specifically inhibiting the transcription or translation of the IARS2 gene, or capable of specifically inhibiting the expression or activity of the IARS2 gene, in the manufacture of a medicament for the treatment of a tumor selected from a glioma, said molecule being selected from a nucleic acid molecule comprising:
a) a double-stranded RNA comprising a nucleotide sequence capable of hybridizing under stringent conditions to the IARS2 gene, the double-stranded RNA comprising a first strand and a second strand, the first strand and the second strand being complementary to each other to form an RNA dimer, and the sequence of the first strand being substantially identical to a target sequence in the IARS2 gene; or
b) An shRNA which contains a nucleotide sequence capable of hybridizing with an IARS2 gene under stringent conditions, wherein the shRNA comprises a sense strand segment and an antisense strand segment, and a stem-loop structure connecting the sense strand segment and the antisense strand segment, the sequences of the sense strand segment and the antisense strand segment are complementary, and the sequence of the sense strand segment is basically identical to a target sequence in the IARS2 gene;
the target sequence of the IARS2 gene contains SEQ ID NO. 1.
2. The use of claim 1, wherein the double-stranded RNA is a small interfering RNA having a first strand with a sequence as set forth in SEQ ID NO: shown at 9.
3. The use of claim 1, wherein the sequence of the shRNA is as set forth in SEQ ID NO: as shown at 14.
4. Use of an IARS2 gene interfering nucleic acid construct in the manufacture of a medicament for the treatment of a tumour selected from a glioma, the IARS2 gene interfering nucleic acid construct comprising a gene segment encoding an shRNA capable of expressing the shRNA, the shRNA comprising a nucleotide sequence capable of hybridising under stringent conditions to an IARS2 gene, the shRNA comprising a sense strand segment and an antisense strand segment, and a stem-loop structure linking the sense strand segment and the antisense strand segment, the sense strand segment and the antisense strand segment having complementary sequences and the sense strand segment having substantially the same sequence as a target sequence in the IARS2 gene;
the target sequence of the IARS2 gene contains SEQ ID NO. 1.
5. The use of claim 4, wherein the IARS2 gene interfering nucleic acid construct is an interfering lentiviral vector.
6. The use of claim 5, wherein the interfering lentiviral vector is obtained by cloning a gene segment encoding the shRNA into a lentiviral vector selected from the group consisting of: pLKO.1-puro,
pLKO.1-CMV-tGFP, pLKO.1-puro-CMV-tGFP, pLKO.1-CMV-Neo, pLKO.1-Neo-CMV-tGFP, pLKO.1-puro-CMV-TagCFP, pLKO.1-puro-CMV-TagYFP, pLKO.1-puro-CMV-TagFP635, pLKO.1-puro-UbC-TurboGFP, pLKO.1-puro-UbC-TagFP635, pLKO-puro-IPTG-1xLacO, pLKO-puro-IPTG-3xLacO, pLP1, pLP2, pLP/VSV-G, pENTR/U6, pLenti6/BLOCK-iT-DEST, pLenti 6-GW/U6-laminsham, pcDNA1.2/V5-GW/lacZ, pLenti6.2/N-Lumio/V5-DEST, pGCSIL-GFP or pLenti 6.2/N-Lumio/V5-GW/lacZ.
7. The use of an IARS2 gene interfering lentivirus in the preparation of a medicament for treating tumors, wherein the tumors are selected from gliomas, the IARS2 gene interfering lentivirus is formed by viral packaging of an interfering lentivirus vector under the assistance of a lentivirus packaging plasmid and a cell line, the interfering lentivirus vector contains a gene segment for coding shRNA (short hairpin ribonucleic acid), the shRNA can be expressed, the shRNA contains a nucleotide sequence capable of hybridizing with an IARS2 gene under a strict condition, the shRNA comprises a sense strand segment and an antisense strand segment, and a stem-loop structure for connecting the sense strand segment and the antisense strand segment, the sequences of the sense strand segment and the antisense strand segment are complementary, and the sequence of the sense strand segment is basically identical to a target sequence in an IARS2 gene;
the target sequence of the IARS2 gene contains SEQ ID NO. 1.
8. The use of a pharmaceutical composition for the prevention or treatment of a tumor, the active agent of which comprises an isolated nucleic acid molecule that reduces the expression of the IARS2 gene in tumor cells, for the preparation of a medicament for the treatment of a tumor for the treatment of glioma;
or the effective substance of the pharmaceutical composition contains an IARS2 gene interfering nucleic acid construct, the IARS2 gene interfering nucleic acid construct contains a gene segment for coding shRNA and can express the shRNA;
or the effective substance of the pharmaceutical composition contains an IARS2 gene interference lentivirus, the IARS2 gene interference lentivirus is formed by virus packaging of an interference lentivirus vector under the assistance of a lentivirus packaging plasmid and a cell line, and the interference lentivirus vector contains a gene segment for coding shRNA and can express the shRNA;
the shRNA contains a nucleotide sequence capable of hybridizing with an IARS2 gene under stringent conditions, and comprises a sense strand segment and an antisense strand segment, and a stem-loop structure connecting the sense strand segment and the antisense strand segment, wherein the sequences of the sense strand segment and the antisense strand segment are complementary, and the sequence of the sense strand segment is basically identical to a target sequence in the IARS2 gene;
the target sequence of IARS2 gene contains SEQ ID NO. 1;
the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or excipient.
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| CN102186987A (en) * | 2008-04-24 | 2011-09-14 | 阿肯色大学托管委员会 | Gene expression profiling based identification of genomic signature of high-risk multiple myeloma and uses thereof |
| WO2015144929A1 (en) * | 2014-03-28 | 2015-10-01 | Erasmus University Medical Center Rotterdam | Method for diagnosing and treating multiple myeloma. |
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| CN102186987A (en) * | 2008-04-24 | 2011-09-14 | 阿肯色大学托管委员会 | Gene expression profiling based identification of genomic signature of high-risk multiple myeloma and uses thereof |
| WO2015144929A1 (en) * | 2014-03-28 | 2015-10-01 | Erasmus University Medical Center Rotterdam | Method for diagnosing and treating multiple myeloma. |
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