CN105586391B - Application of human GTPBP4 gene and related medicine thereof - Google Patents
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Abstract
The invention discloses an application of a human GTPBP4 gene and a related medicament thereof. The invention discloses the use of human GTPBP4 gene in tumor treatment, tumor diagnosis and medicine preparation. The invention further constructs human GTPBP4 gene small interfering RNA, human GTPBP4 gene interfering nucleic acid construct, human GTPBP4 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 GTPBP4 gene, especially the lentivirus can efficiently infect target cells, efficiently inhibit the expression of GTPBP4 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 GTPBP4 gene and a related medicament 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-.
GTPBP4 is a GTP binding protein, originally discovered by differential display PCR with differential expression in renal disease, which functions as a gtpase that can act as a molecular switch to exist in two distinct states of inactivity; is inactive when bound to GTP and active when bound to GDP, at which time the molecular switch opens some downstream signaling pathway in the activated cell. When the exogenous receptor binds to a GTP-like receptor. The conformation of the receptor is changed, opening of the trimeric G protein switch releases GDP, thereby forming GTP, when the G protease hydrolyzes the high energy phosphate bond of GTP bound thereto, the switch is closed, and the conversion of GTP into GDP (Laning NJ1, Olson BA, Zhu Y. identification of a novel nuclear hormone expressed In amino acids such as Soc protein 2001 May; 12 (883-90.) is necessary that the complex formed by GTPBP4 and Nog1 can participate In the synthesis of 60S ribosomal subunit (Jensen BC, Wang Q, Safer CT, Parsons help 1GTP-binding protein expressed In biological genes for the synthesis of 60S ribosomal subunit of chemical tissue of molecular tissue of GTPaseNog1.mol Cell Biol 27: 7735-
Using the white gene DNA microarrays technique, tens of proteins, including the GTPBP4 protein, were detected in association with hypertensive rat (SHR) cells compared to normotensive rats. (Kinoshita K, Ashanar MS, Tabuchi M, Higashino H., white rate DNA array summary for reliable gene expression to hypertension in a kit of protein from the same porous water uptake peptides at two stages of and with a porous index used by hydrophilic reagent water chloride. exp. the. Med. 2011Mar; 2(2):201-212. Ep.) there are reports of using microarray analysis in terms of environmental contamination, analyzing the presence of GTPBP4 protein expression in a sample of wastewater that is capable of being upregulated by estrogen regulation. (Wang DY1, McKague B, Liss SN, Edwards EA. Gene expression profiles for detecting and distinguishing potential end rings-distinguishing complexes in environmental samples Environ technique 2004 Dec1; 38(23):6396-
There are few reports of GTPBP4 in tumor-related fields. In order to research the correlation between GTPBP4 and tumors, a colon cancer cell model is selected, and the role of GTPBP4 in the occurrence and development of colon cancer is researched by taking RNAi as a means.
Disclosure of Invention
The invention aims to disclose a treatment method and a medicament related to human GTPBP4 gene, and research the effect of GTPBP4 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 effect of the GTPBP4 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 capable of specifically inhibiting transcription or translation of the GTPBP4 gene, or capable of specifically inhibiting expression or activity of the GTPBP4 protein, thereby inhibiting growth, proliferation, differentiation and/or survival of the tumor cell.
The tumor cells are selected from tumor cells the growth of which is associated with the expression or activity of GTPBP4 protein. Preferably, the tumor cell is selected from colon cancer.
In the method of inhibiting or reducing tumor cell growth, proliferation, differentiation and/or survival, the molecule is administered in an amount sufficient to reduce transcription or translation of the GTPBP4 gene, or to reduce expression or activity of GTPBP4 protein. Further, the expression of the GTPBP4 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, the ribozyme, the esiRNA or the shRNA contains a promoter sequence of a GTPBP4 gene or an information sequence of a GTPBP4 gene.
Further, the double-stranded RNA is small interfering RNA (siRNA). The small interfering RNA comprises a first strand and a second strand, wherein the first strand and the second strand are complementary to form an RNA dimer, and the sequence of the first strand is basically identical to 15-27 continuous nucleotide sequences in a GTPBP4 gene. The small interfering RNA can specifically bind to an mRNA fragment coded by a target sequence and specifically silence the expression of a human GTPBP4 gene.
Further, the small interfering RNA first chain sequence and GTPBP4 gene target sequence basically the same. Preferably, the target sequence in the GTPBP4 gene contains any sequence in SEQ ID NO 1-5.
The target sequence in the GTPBP4 gene is the fragment in the GTPBP4 gene corresponding to the mRNA fragment complementarily combined with the small interfering RNA when the small interfering RNA specifically silences the expression of the GTPBP4 gene.
Preferably, the GTPBP4 gene is of human origin.
The invention also discloses the application of the separated human GTPBP4 gene in preparing or screening tumor treatment medicines or preparing tumor diagnosis medicines.
Further, the tumor is selected from colon cancer.
The application of the separated GTPBP4 gene in preparing or screening tumor treatment medicines comprises two aspects: firstly, the GTPBP4 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 GTPBP4 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 GTPBP4 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 GTPBP4 gene is used as a target of RNA interference effect to develop a medicine or a preparation aiming at tumor cells, so that the expression level of the GTPBP4 gene in the tumor cells can be reduced.
The application of the GTPBP4 gene as a drug or preparation for screening tumor treatment drugs or preparations aiming at the action target of tumor cells specifically comprises the following steps: the GTPBP4 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 GTPBP4 gene and is used as a candidate medicine for treating the tumor. The GTPBP4 gene small interfering RNA (siRNA) is obtained by screening human GTPBP4 gene as an action object and can be used as a medicament for inhibiting the proliferation of tumor cells. In addition, GTPBP4 gene and its protein can be used as target of action, such as antibody drug, small molecule drug, etc.
The GTPBP4 gene is used for preparing tumor diagnosis medicines, and the GTPBP4 gene expression product is used as a tumor diagnosis index for preparing the tumor diagnosis medicines.
The expression level of the GTPBP4 gene in tumor tissue, normal tissue and normal tissue around tumor was examined by immunohistochemical method. The research finds that: the expression level of the GTPBP4 gene in tumor tissues is obviously higher than that of normal tissues and normal tissues around the tumor. The GTPBP4 gene is suggested to be possibly used as an oncogene and play an important role in the occurrence and development of tumors; the expression level of GTPBP4 gene may be the marker for tumor diagnosis.
The tumor treatment drug is a molecule which can specifically inhibit the transcription or translation of the GTPBP4 gene, or can specifically inhibit the expression or activity of the GTPBP4 protein, so that the expression level of the GTPBP4 gene in tumor cells is reduced, and the purposes of inhibiting the proliferation, growth, differentiation and/or survival of the tumor cells are achieved.
The tumor therapeutic drug or tumor diagnostic drug prepared or screened by the isolated GTPBP4 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 tumor treatment drug is administered in an amount sufficient to reduce transcription or translation of the human GTPBP4 gene, or to reduce expression or activity of the human GTPBP4 protein. Such that the expression of the human GTPBP4 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 GTPBP4 gene to inhibit the proliferation of tumor cells. Specifically, in treatment, a substance effective to reduce the expression level of human GTPBP4 gene is administered to the patient.
In a second aspect, the present invention discloses an isolated nucleic acid molecule for reducing the expression of the GTPBP4 gene in a tumor cell, said nucleic acid molecule comprising:
a) a double-stranded RNA comprising a nucleotide sequence capable of hybridizing to the GTPBP4 gene under stringent conditions; or
b) shRNA containing a nucleotide sequence capable of hybridizing with the GTPBP4 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 GTPBP4 gene. Preferably, the sequence of said first strand is substantially identical to 19-23 contiguous nucleotide sequences in the GTPBP4 gene; more preferably, the sequence of the first strand is substantially identical to 19, 20 or 21 contiguous nucleotide sequences in the GTPBP4 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 GTPBP4 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 17, specifically 5'-GCGUAGUCUUGGUGUUGACAU-3'.
SEQ ID NO: the siRNA shown in 17 is designed by taking the sequence shown in SEQ ID NO. 1 as an RNA interference target sequence and is aimed at one strand of small interfering RNA of human GTPBP4 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 GTPBP4 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 continuous nucleotide sequences in the GTPBP4 gene. The shRNA can become small interfering RNA (siRNA) after being processed, and further plays a role in specifically silencing the expression of endogenous GTPBP4 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 a target sequence in the GTPBP4 gene.
Preferably, the sense strand fragment is substantially identical to 19-23 contiguous nucleotide sequences in the GTPBP4 gene; more preferably, the sense strand fragment is substantially identical to 19, 20 or 21 contiguous nucleotide sequences in the GTPBP4 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. 18, and specifically comprises the following steps: 5'-CCGGGCGUAGUCUUGGUGU CUCGAGAUGUCAACACCAAUUUU-3' are provided.
The shRNA can become siRNA after enzyme digestion processing, and further plays a role in specifically silencing endogenous human GTPBP4 gene expression in tumor cells.
The interfering slow virus vector of the gene segment for encoding shRNA contains any sequence in SEQ ID NO 1-5 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 a GTPBP4 gene, and the target sequence of the GTPBP4 gene is a segment in a GTPBP4 gene corresponding to an mRNA segment which is identified and silenced by siRNA when the siRNA is used for specifically silencing GTPBP4 gene expression.
Preferably, the target sequence in the GTPBP4 gene contains any sequence of SEQ ID NO 1-5.
Further, the GTPBP4 gene is derived from human.
In a third aspect of the invention, a GTPBP4 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 GTPBP4 gene interfering nucleic acid construct can be obtained by cloning a gene segment for coding the human GTPBP4 gene shRNA into a known vector. Further, the GTPBP4 gene interference nucleic acid construct is a GTPBP4 gene interference lentiviral vector.
The GTPBP4 gene interference lentiviral vector is obtained by cloning a DNA fragment for coding the GTPBP4 gene shRNA into a known vector, wherein the known vector is mostly a lentiviral vector, the GTPBP4 gene interference lentiviral vector is packaged into infectious viral particles by viruses, 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 GTPBP4 gene.
Further, the GTPBP4 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 provides a human GTPBP4 gene interference lentiviral vector constructed by taking pGCSIL-GFP as a vector, which is named as pGCSIL-GFP-GTPBP 4-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 colon cancers.
The GTPBP4 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 GTPBP4 gene interference lentiviral vector can be used for preparing the GTPBP4 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 also discloses a GTPBP4 gene interference lentivirus, which is prepared by virus packaging of the GTPBP4 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 the GTPBP4 gene, thereby inhibiting the proliferation of colon cancer. The GTPBP4 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 of the combination of the isolated nucleic acid molecule, the GTPBP4 gene interference nucleic acid construct or the GTPBP4 gene interference lentivirus.
Further, the pharmaceutical composition contains 1-99 wt% of the double-stranded RNA, shRNA, GTPBP4 gene interference nucleic acid construct or GTPBP4 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 colon cancer.
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 selected from colon cancer.
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 the expression of GTPBP4 gene in tumor cells is disclosed, the kit comprising: the isolated nucleic acid molecule, the GTPBP4 gene interfering nucleic acid construct, and/or the GTPBP4 gene interfering lentivirus present in the container.
In conclusion, the invention designs 5 RNAi target sequences aiming at human GTPBP4 gene, constructs corresponding GTPBP4RNAi vector, wherein the coding sequence SEQ ID NO: the RNAi vector pGCSIL-GFP-GTPBP4-siRNA of 1 can obviously down-regulate the expression of GTPBP4 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-GTPBP4-siRNA, so that the RNAi sequence aiming at the GTPBP4 gene can be efficiently introduced into the RKO cell of the colon cancer in a targeted manner, the expression level of the GTPBP4 gene is reduced, and the proliferation capacity of the tumor cell is obviously inhibited. Lentivirus-mediated GTPBP4 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 GTPBP4 gene, especially the lentivirus can efficiently infect target cells, efficiently inhibit the expression of GTPBP4 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: after 4 days of infection of colon cancer cells by GTPBP4-RNAi lentivirus, the expression level of GTPBP4mRNA is remarkably reduced
FIG. 3: 5 days after the GTPBP4-RNAi lentivirus infects intestinal cancer cells, cell proliferation inhibition is caused
Detailed Description
The invention relates to a group of small interfering RNA (siRNA) sequences, RNA interference vectors and RNA interference lentiviruses aiming at human GTPBP4 gene. The coding region sequence of human GTPBP4mRNA 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 GTPBP4 gene in human tumor cells.
The inventor of the invention has found through extensive and intensive research that the GTPBP4 gene is significantly highly expressed in tumor tissues; the inventor finds that after the expression of the human GTPBP4 gene is down-regulated by an RNAi method, the tumor cell proliferation can be effectively inhibited, the cell apoptosis can be promoted, the invasion and the metastasis capacity of the tumor cell can be reduced, and the growth process of the tumor can be effectively controlled, and the research result shows that the GTPBP4 gene is a protooncogene and can be used as a target point for tumor treatment. The inventor further synthesizes and tests a plurality of siRNAs aiming at the GTPBP4 gene, screens out the siRNA which can effectively inhibit the expression of GTPBP4 and further inhibit the proliferation and growth of RKO cells of human colon cancer, and completes the invention on the basis.
The invention provides a series of small interfering RNA (siRNA) sequences interfering human GTPBP4 gene, and constructs a lentivirus capable of specifically silencing GTPBP4 gene expression. The research of the invention finds that the small interfering RNA and RNAi lentivirus designed aiming at the human GTPBP4 gene can stably and specifically regulate the expression of the GTPBP4 gene and effectively inhibit the proliferation of human tumor cells. The invention shows that the GTPBP4 gene can promote the growth of tumor cells and is expected to become a target for early diagnosis and treatment of tumors. Further, silencing the expression of the GTPBP4 gene by RNAi means can be an effective means for inhibiting tumor development.
The design idea of the invention is as follows:
the invention obtains a human GTPBP4 gene RNAi lentivirus by screening through the following method: the sequence of human GTPBP4 gene is adjusted from Genbank; predicting the siRNA site; synthesizing an effective siRNA sequence aiming at the GTPBP4 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 a GTPBP4 gene siRNA sequence; the RNAi plasmid and a helper vector (Packingmix, Sigma-aldrich company) required by lentivirus packaging are cotransfected with the human embryonic kidney cell 293T to generate recombinant lentivirus particles, and the lentivirus capable of efficiently silencing the GTPBP4 gene can be prepared.
Based on the method, the invention provides 5 effective targets (shown as SEQ ID NO: 1-5) for interfering the GTPBP4 gene, and constructs the lentivirus specifically interfering the human GTPBP4 gene.
Meanwhile, the invention also discloses a human GTPBP4 gene RNAi lentivirus (GTPBP4-RNAi) and preparation and application thereof.
The research discovers that the slow virus mediated RNAi method can effectively inhibit the proliferation of the tumor cells after the expression of the GTPBP4 gene in the tumor cells is reduced. The research shows that the GTPBP4 gene is a protooncogene, can promote the proliferation of tumor cells, has important biological functions in the occurrence and development of tumors, the GTPBP4 gene can be a target for tumor treatment, and the lentivirus-mediated GTPBP4 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 GTPBP4 Gene
1. Screening effective siRNA target point aiming at human GTPBP4 gene
Calling GTPBP4 (NM-012341) gene information from Genbank; designing effective siRNA target point aiming at GTPBP4 gene. Table 1 lists 5 effective siRNA target sequences against GTPBP4 gene.
TABLE 1 siRNA target sequences targeting the human GTPBP4 gene
| SEQ ID NO | TargetSeq |
| 1 | |
| 2 | GCTGGAGAGTATGACAGTGTA |
| 3 | |
| 4 | GCGTCAGCATTTATCCCGTTT |
| 5 | CCAACCGTTATTCATAAACAT |
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'-TCCCAGGAGTGTCTACAACCT-3' (SEQ ID NO: 10); the sequence of the downstream primer is as follows: 5'-CAGCCCTATGCGTTCACAAC-3' (SEQ ID NO: 11), 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-GTPBP 4-siRNA.
pGCSIL-GFP-Scr-siRNA negative control plasmid was constructed with negative control siRNA target sequence 5'-TTCTCCGAACGTGTCACGT-3' (SEQ ID NO: 12). When pGCSIL-GFP-Scr-siRNA negative control plasmids are constructed, double-stranded DNA Oligo sequences (table 3) containing Age I and EcoR I enzyme cutting sites at two ends are synthesized aiming at Scr siRNA targets, and the rest construction methods, identification methods and conditions are the same as pGCSIL-GFP-GTPBP 4-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 |
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 GTPBP4-siRNA lentivirus
The DNA of RNAi plasmid pGCSIL-GFP-GTPBP4-siRNA was extracted using a plasmid extraction kit from Qiagen, Inc., and 100 ng/. mu.l of stock solution was prepared.
24h before transfection, human embryonic kidney cell 293T cells in logarithmic growth phase were trypsinized and cell density was adjusted to 1.5X 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 and 2ml of complete medium was added. Because the lentivirus vector carries a reporter gene of green fluorescent protein, the lentivirus vector can be placed under a fluorescent microscope to observe the expression condition of GFP of two groups of cells after 24 hours, and whether the lentivirus plasmid is packaged by 293T cells is determined. After an additional 48h of culture, cell supernatants were collected and lentiviruses were purified and concentrated by a Centricon Plus-20 centrifugal ultrafiltration unit (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 0.45 μm filter in 40ml ultracentrifuge tube, adding virus crude extract sample into filter cup (up to 19ml), covering lid, inserting the filter cup into filtrate collecting tube; (3) after the virus is well mixed, well balanced, placed on a rotating head, centrifuged at 4000g for 10-15min until the required virus concentration volume is reached; (4) after the centrifugation is finished, taking out the centrifugal device, 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 concentrate is shown in SEQ ID NO 13.The packaging procedure for the control lentivirus was identical to that for the GTPBP4-siRNA lentivirus, except that the pGCSIL-GFP-Scr-siRNA vector was used in place of the pGCSIL-GFP-GTPBP4-siRNA vector.
Example 2 detection of silencing efficiency of GTPBP4 Gene by real-time fluorescent quantitative RT-PCR
Colon cancer RKO cells in logarithmic growth phase were trypsinized to prepare a cell suspension (cell number approximately 5X 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, RKO: 10), 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 4 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 (reverse transcription reaction system shown in Table 7, reaction at 42 ℃ C. for 1 hour) according to the M-MLV protocol of Promega, and then the reverse transcriptase was inactivated by a water bath at 70 ℃ for 10 min.
Real-time quantitative detection was carried out using a TP800 Real time PCR instrument (TAKARA). The primers for the GTPBP4 gene were as follows: an upstream primer 5'-TCCCAGGAGTGTCTACAACCT-3' (SEQ ID NO: 13) and a downstream primer 5'-CAGCCCTATGCGTTCACAAC-3' (SEQ ID NO: 14). 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: 15) and a downstream primer 5'-CACCCTGTTGCTGTAGCCAAA-3' (SEQ ID NO: 16). 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 assay calculated the expression abundance of GTPBP 4-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 GTPBP4mRNA was down-regulated by 79.2% in colon cancer RKO cells.
Example 3 examination of the proliferative Capacity of tumor cells infected with GTPBP4-siRNA lentivirus
Colon cancer RKO cells in logarithmic growth phase were trypsinized to prepare a cell suspension (cell number approximately 5X 10)4/ml) were inoculated in 6-well plates and cultured until the degree of cell confluence reached about 30%. According to the infection complex number (MOI, RKO: 10), adding a proper amount of virus, culturing for 24h, then replacing the culture medium, and collecting cells of each experimental group in the logarithmic growth phase after the infection time reaches 4 days. Complete medium resuspension into cell suspension (2X 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 reads were examined once daily for 5 consecutive days starting the second day after plating using a Cellomics ArrayScan VTI high content screening Analyzer (Thermo Fisher). 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 slow virus infected colon cancer RKO cells are cultured in vitro for 5 days, the proliferation speed is obviously reducedSlowly, the proliferation speed of the tumor cells is far lower than that of the control group, the number of the viable cells is respectively reduced by 83.1%, and the GTPBP4 gene silencing leads to the inhibition of the proliferation capacity of the tumor cells.
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 (11)
1. The application of an interference RNA aiming at a GTPBP4 gene in the preparation of a medicine for treating colon cancer, wherein the target sequence of the GTPBP4 gene is shown as SEQ ID NO:1 is shown.
2. An isolated nucleic acid molecule that reduces expression of the GTPBP4 gene in a colon cancer cell, the nucleic acid molecule comprising:
a) a double-stranded RNA containing a nucleotide sequence capable of hybridizing with the GTPBP4 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 identical to a target sequence in the GTPBP4 gene, the target sequence of the GTPBP4 gene being as set forth in SEQ ID NO:1 is shown in the specification; or
b) The shRNA contains a nucleotide sequence capable of hybridizing with a GTPBP4 gene, 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, the sequence of the sense strand segment is identical to a target sequence in a GTPBP4 gene, and the target sequence of the GTPBP4 gene is shown as SEQ ID NO:1 is shown.
3. The isolated nucleic acid molecule of claim 2, 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 17.
4. The isolated nucleic acid molecule of claim 2, wherein the sequence of said shRNA is as set forth in SEQ ID NO:18, respectively.
5. A GTPBP4 gene interfering nucleic acid construct containing a gene segment encoding shRNA in the isolated nucleic acid molecule of any one of claims 2-4, capable of expressing the shRNA.
6. The GTPBP4 gene interfering nucleic acid construct of claim 5, wherein the GTPBP4 gene interfering nucleic acid construct is an interfering lentiviral vector.
7. The GTPBP4 gene interfering nucleic acid construct according to claim 6, 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.
8. A GTPBP4 gene interference lentivirus, which is prepared by virus packaging of the GTPBP4 gene interference nucleic acid construct of any one of claims 5-7 with the aid of lentivirus packaging plasmid and cell line.
9. A pharmaceutical composition for treating tumor, wherein the main effective component comprises the isolated nucleic acid molecule of any one of claims 2-4, the GTPBP4 gene interference nucleic acid construct of any one of claims 5-7, or the GTPBP4 gene interference lentivirus of claim 8, and a pharmaceutically acceptable carrier, diluent or excipient.
10. Use of the isolated nucleic acid molecule of any one of claims 2-4, the GTPBP4 gene interfering nucleic acid construct of any one of claims 5-7, or the GTPBP4 gene interfering lentivirus of claim 8 for the preparation of a tumor therapeutic drug for the treatment of colon cancer.
11. A kit for reducing the expression of GTPBP4 gene in a tumor cell, the kit comprising: the isolated nucleic acid molecule of any one of claims 2-4, the GTPBP4 gene interfering nucleic acid construct of any one of claims 5-7, or the GTPBP4 gene interfering lentivirus of claim 8, in a container.
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Denomination of invention: The use of human GTPBP4 gene and its related drugs Granted publication date: 20200612 Pledgee: Agricultural Bank of China Limited Shanghai Huangpu Sub branch Pledgor: Shanghai Jikai gene Medical Technology Co.,Ltd. Registration number: Y2024310000749 |