CN110643705A - Application of human DGKZ gene and related medicine thereof - Google Patents

Abstract

The invention discloses application of human DGKZ gene and related medicines thereof. The invention discloses the use of human DGKZ gene in tumor treatment, tumor diagnosis and medicine preparation. The invention further constructs human DGKZ gene small interfering RNA, a human DGKZ gene interfering nucleic acid construct, a human DGKZ gene interfering lentivirus and discloses the application of the human DGKZ gene small interfering RNA and the human DGKZ gene interfering lentivirus. 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 DGKZ gene, especially the lentivirus can efficiently infect target cells and efficiently inhibit the expression of the DGKZ gene in the target cells, so that the growth of tumor cells is inhibited, the apoptosis of the tumor cells is promoted, and the siRNA or the nucleic acid construct containing the siRNA sequence and the lentivirus have important significance in tumor treatment.

Description

Application of human DGKZ gene and related medicine thereof

The application is a divisional application of the original application, and the application date of the original application is as follows: 2014-03-19; the application numbers are: 2014101030095, respectively; the invention provides the following: application of human DGKZ gene and related medicine.

Technical Field

The invention relates to the technical field of biology, in particular to application of a human DGKZ gene and a related medicament thereof.

Background

Ribonucleic acid interference (RNAi) phenomenon refers to the specific degradation of an endogenous mRNA coding region of a double-stranded RNA (dsrna) when the mRNA is introduced into a cell, resulting in the silencing of the expression of the gene. 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 (TuschlT, Zamore PD, Sharp PA, Bartel DP. RNAi: double-stranded RNA direct the ATP-dependent cleavage of mRNA at 21to 23nucleotide intervals. cell 2000; 101: 25-33.). Tumors are a major disease threatening the health of humans. 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 the development and progression of tumors (Uplicard, Susan L. the therapeutic potential of RNA interference. FEBS Letters 2005; 579: 5996-.

DGKZ, also known as DGK-ZETA, belongs to the family of eukaryotic Diacylglycerol Kinases (DGKs). DGKs comprise a series of enzymes, which are DGKalpha, DGKbeta, DGKgamma, DGKkdelta, DGKepsilon, DGKzeta, DGKeta, DGKtheta, DGKiota and DGKkappa. DGKs are widely distributed in mammals and regulate intracellular diglyceride content by metabolizing diglyceride to phosphatidic acid, regulate intracellular signaling, and respond to a variety of extracellular stimuli. "Wade D.Van horns, Charles R.Sanders.Prokaryotic diacylglycerol kinase and undecaprenol kinase. Annu Rev Biophys.2012; 41:81-101.Li D, Lyons JA, Pye VE, Vogeley L,D,Kenyon CP,Shah ST,Doherty C,Aherne M,Caffrey M.Crystal structure of the integral membrane diacylglycerolkinase.Nature.2013May23；497(7450):521-4.Miller DJ,Jerga A,Rock CO,WhiteSW.Analysis of the Staphylococcus aureus DgkB structure reveals a commoncatalytic mechanism for the soluble diacylglycerol kinases.Structure.2008Jul；16(7):1036-46.]

multiple members of the DGK family are involved in the development of cancer. DGKgamma and DGKbeta are target sites for phorbol esters with cancer-promoting effects [ Shindo M, Irie K, Masuda A, Ohigashi H, Shirai Y, MiyasakaK, Saito N.Synthesis and phorbol ester binding of the cysteine-rich domains of diacylglycerol kinase (DGK) isozymes. DGKgamma. and DGKbeta. are new targets of either-promoter phorbols esters. J Biol chem.2003May 16; 278(20):18448-54.]. An article reported in 2007 that DGKs play a key role in the invasion and growth process of HGF-induced thymus cancer cells [ Filigheddu N, Cutrupi S, Porporato PE, Riboni F, Baldanzi G, Chianale F, FortinaE, Piantandi P, De Bortoli M, Vacca G, Graziani A, Surico N.Diacryloyl kinase extract required for HGF-induced invasion and anchorage-independent growth of MDA-MB-231Breast cancer cells. 1489-92 ] of 27 (3B). Research in melanoma has shown that DGKalpha is a positive regulator of NF-kappaB and inhibits TNF-alpha induced melanoma apoptosis [ Yanagisawa K, Yasuda S, Kai M, Imai S, Yamada K, Yamashita T, JimbowK, Kanoh, Sakane F. Diacylglycerol kinase alpha supressases promoter regulator-alpha-induced apoptosis of human melanomas S triple drug pathway NF-kappaBactivation. Biochim Biophys Acta.2007Apr; 1771(4):462-74.]. In the study of endometrial cancer, DGKalpha is essential for the proliferation, migration and adhesion of cancer cells, suggesting that it is a potential target gene for cancer therapy [ Filigheddu N, Sampietro S, Chianale F, Porporo PE, Gagganensis M, Gregonin I, Rainero E, Ferrara M, Perego B, Riboni F, Baldanzi G, Graziani A, Suricon N.Diacryloyl glycerol kinase. alpha. indicators 17-beta-estradiol-induced promotion, motility, and anaerobic-index growth of Hec-1A endothelial cell ceramic membrane protein-linked protein receptor 30. signal.2011; 23(12):1988-96.].

There are few reports on DGKZ in the tumor-related field. In order to research the relevance of DGKZ and tumors, a tumor cell model is selected, and the role of DGKZ in the occurrence and development of the tumors 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 DGKZ gene, and researches the effect of the DGKZ 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 DGKZ gene in the occurrence and development of tumor is studied by means of RNA interference, and a method for inhibiting or reducing the growth, proliferation, differentiation and/or survival of tumor cells is disclosed, which comprises the following steps: administering to the tumor cell a molecule that specifically inhibits the transcription or translation of the DGKZ gene, or that specifically inhibits the expression or activity of the DGKZ protein, thereby inhibiting the growth, proliferation, differentiation and/or survival of the tumor cell.

The tumor is selected from any tumor of which the proliferation of tumor cells is related to the expression of human DGKZ gene, and preferably, the tumor is a malignant tumor: a glioma.

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 DGKZ gene, or to reduce expression or activity of the DGKZ protein. Further, the expression of the DGKZ gene is reduced by at least 50%, 80%, 90%, 95% or 99%.

Further, the molecule may be selected from, but 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 a DGKZ gene or an information sequence of the DGKZ 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 15-27 consecutive nucleotide sequences in a DGKZ 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 DGKZ gene.

Further, the small interfering RNA first strand sequence and DGKZ gene target sequence is basically the same. Preferably, the target sequence in the DGKZ gene comprises any one of SEQ ID NO 1-13.

The target sequence in the DGKZ gene is the segment in the DGKZ gene corresponding to the mRNA segment complementarily combined with the small interfering RNA when the small interfering RNA specifically silences the DGKZ gene expression.

Preferably, the DGKZ gene is derived from a human.

The invention also discloses the application of the separated human DGKZ gene in preparing or screening tumor treatment medicines or preparing tumor diagnosis medicines.

Further, the tumor is selected from any tumor whose tumor cell proliferation is associated with human DGKZ gene expression, preferably, the tumor is a malignant tumor: a glioma.

The application of the isolated human DGKZ gene in preparing or screening the tumor treatment medicine comprises two aspects: firstly, human DGKZ 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 human DGKZ gene is used as an action target of a medicine or a preparation aiming at tumor cells and is applied to screening of tumor treatment medicines or preparations.

The application of the human DGKZ gene as a drug or preparation aiming at the action target of tumor cells in preparing the tumor treatment drug or preparation specifically comprises the following steps: human DGKZ gene is used as the target of RNA interference to develop medicine or preparation for tumor cell, so that the expression level of human DGKZ gene in tumor cell can be reduced.

The application of the human DGKZ 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: human DGKZ gene is used as an action object, and the medicine or the preparation is screened to find out the medicine which can inhibit or promote the expression of the human DGKZ gene and is used as a candidate medicine for treating the tumor. The human DGKZ gene small interfering RNA is obtained by screening the human DGKZ gene serving as an action object and can be used as a medicament with the effect of inhibiting the proliferation of tumor cells. In addition, DGKZ gene and its protein can be used as target, such as antibody drug, small molecule drug, etc.

The application of the human DGKZ gene in preparing the tumor diagnosis medicament refers to the application of the human DGKZ gene expression product as a tumor diagnosis index in preparing the tumor diagnosis medicament.

The expression level of the DGKZ gene in tumor tissues, normal tissues and normal tissues around the tumor is detected by an immunohistochemical method. The research finds that: the expression level of the DGKZ gene in the tumor tissue is obviously higher than that of the normal tissue and the normal tissue around the tumor. The DGKZ 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 the DGKZ gene may be a marker for tumor diagnosis.

The tumor treatment drug is a molecule which can specifically inhibit the transcription or translation of DGKZ genes, or can specifically inhibit the expression or activity of DGKZ protein, so that the expression level of the DGKZ genes 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 drugs or tumor diagnostic drugs prepared or screened by the isolated DGKZ gene include, but are 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 agent is administered in an amount sufficient to reduce transcription or translation of the human DGKZ gene, or to reduce expression or activity of the human DGKZ protein. Such that the expression of the human DGKZ 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 DGKZ gene and inhibiting the proliferation of tumor cells. Specifically, in treatment, a substance effective in reducing the expression level of human DGKZ 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 DGKZ gene in a tumor cell, said nucleic acid molecule comprising:

a) a double-stranded RNA containing a nucleotide sequence capable of hybridizing with a DGKZ gene under stringent conditions; or

b) shRNA comprising a nucleotide sequence capable of hybridizing to a DGKZ 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 a 15-27 contiguous nucleotide sequence in a DGKZ gene. Preferably, the sequence of the first strand is substantially identical to a 19-23 contiguous nucleotide sequence in the DGKZ gene; more preferably, the sequence of the first strand is substantially identical to a sequence of 19, 20 or 21 contiguous nucleotides of the DGKZ 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 DGKZ 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 25, specifically 5'-CUCUGAAAGCAAGCAAGAA-3'.

SEQ ID NO:25 is designed by taking the sequence shown in SEQ ID NO.1 as an RNA interference target sequence, and aims at one strand of small interfering RNA of human DGKZ gene, and 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 DGKZ 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 basically identical to 15-27 continuous nucleotide sequences in the DGKZ gene. The shRNA can become small interfering RNA (siRNA) after being processed, and further plays a role in specifically silencing endogenous DGKZ gene expression 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 DGKZ gene.

Preferably, the sense strand fragment is substantially identical to a 19-23 contiguous nucleotide sequence in the DGKZ gene; more preferably, the sense strand fragment is substantially identical to a 19, 20 or 21 contiguous nucleotide sequence of the DGKZ gene.

Further, the sequence of the stem-loop structure of the shRNA is selected from any one of the following sequences: UUCAAGAGA, AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU, CCACACC. The invention specifically lists UUCAAGAGA as a stem-loop.

Furthermore, the sequence of the shRNA is shown as SEQ ID NO:26, and specifically comprises the following steps: 5'-CUCUGAAAGCAAGCAAGAA UUCAAGAGAUUCUUGCUUGCUUUCAGAG-3' are provided.

The shRNA can become siRNA after enzyme digestion processing, thereby playing a role in specifically silencing the expression of endogenous human DGKZ genes in tumor cells.

The interfering slow virus vector of the gene segment for encoding shRNA contains any sequence in SEQ ID NO 1-13 and a complementary sequence thereof.

And the sense strand segment of the first strand of the double-stranded RNA or the shRNA is basically the same as the target sequence in the DGKZ gene, and the target sequence of the DGKZ gene is the segment in the DGKZ gene corresponding to the mRNA segment which is identified and silenced by the siRNA when the siRNA is used for specifically silencing the expression of the DGKZ gene.

Preferably, the target sequence in the DGKZ gene comprises any one of SEQ ID NO 1-13.

Further, the DGKZ gene is derived from a human.

In the third aspect of the invention, a human DGKZ gene interference nucleic acid construct is disclosed, which comprises a gene segment for encoding the separated human DGKZ gene small interfering RNA, and can express the shRNA.

The human DGKZ gene interfering nucleic acid construct can be obtained by cloning a gene segment which codes the human DGKZ gene small interfering RNA into a known vector. Further, the human DGKZ gene interference nucleic acid construct is a human DGKZ gene interference lentiviral vector.

The DGKZ gene interference lentiviral vector is obtained by cloning a gene segment for coding the human DGKZ gene small interfering RNA into a known vector, wherein the known vector is mostly a lentiviral vector, the DGKZ gene interference lentiviral vector is packaged into infectious viral particles through viruses, then a tumor cell is infected, the shRNA is transcribed, and the siRNA is finally obtained through the steps of enzyme digestion processing and the like and is used for specifically silencing the expression of the DGKZ gene.

Further, the DGKZ 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).

The lentiviral vector may be selected from: 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 enumerates a human DGKZ gene interference lentiviral vector constructed by taking pGCSIL-GFP as a vector, and is named as pGCSIL-GFP-DGKZ-siRNA.

The isolated nucleic acid molecule of the invention can be used for preparing a medicament for preventing or treating tumors selected from any one of tumors in which the proliferation of tumor cells is related to the expression of human DGKZ gene, preferably a malignant tumor: a glioma.

The DGKZ gene siRNA can be used for inhibiting the proliferation of tumor cells, and further can be used as a medicament or preparation for treating tumors. DGKZ gene interference lentiviral vector can be used for preparing the DGKZ 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 fourth aspect of the invention discloses a human DGKZ gene interference lentivirus, which is formed by virus packaging of the human DGKZ gene interference nucleic acid construct under the assistance of lentivirus packaging plasmids and cell lines. The lentivirus can infect tumor cells and generate small interfering RNA aiming at human DGKZ gene, thereby inhibiting the proliferation of the tumor cells. The DGKZ gene interference lentivirus can be used for preparing medicaments for preventing or treating tumors. The tumor is selected from any tumor of which the proliferation of tumor cells is related to the expression of human DGKZ gene, and preferably, the tumor is a malignant tumor: a glioma.

In the fifth aspect of the invention, a pharmaceutical composition for preventing or treating tumor is also disclosed, the effective substance of which comprises one or more of the isolated nucleic acid molecule, the DGKZ gene interference nucleic acid construct or the DGKZ gene interference lentivirus.

Further, the pharmaceutical composition contains 1-99 wt% of the double-stranded RNA, shRNA, DGKZ gene interfering nucleic acid construct or DGKZ gene interfering slow virus, 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 any tumor treatment medicine for treating tumor cell proliferation and tumor cell expression related to human DGKZ gene expression.

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 any one of tumors in which the proliferation of tumor cells is associated with the expression of human DGKZ gene. Preferably, the tumor is a malignant tumor: a 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 the expression of the DGKZ gene in a tumor cell is disclosed, the kit comprising: the isolated nucleic acid molecule, DGKZ gene interfering nucleic acid construct, and/or the DGKZ gene interfering lentivirus present in the container.

In conclusion, the invention designs 13 RNAi target sequences aiming at human DGKZ genes, and constructs corresponding DGKZ RNAi vectors, wherein the coding sequence is SEQ ID NO: the RNAi vector pGCSIL-GFP-DGKZ-siRNA of 1 can obviously down-regulate the expression of DGKZ gene at mRNA level and protein level. Lentivirus (Lv) is used as a gene operation tool to carry an RNAi vector pGCSIL-GFP-DGKZ-siRNA, so that the RNAi sequence aiming at the DGKZ gene can be efficiently introduced into a human glioma U87 cell in a targeted manner, the expression level of the DGKZ gene is reduced, and the proliferation capacity of the glioma U87 cell is obviously inhibited. Lentivirus-mediated silencing of the DGKZ gene 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 DGKZ gene, especially the lentivirus can efficiently infect target cells, efficiently inhibit the expression of the DGKZ 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 shows the DNA map of pGCSIL-GFP plasmid

FIG. 2 shows that the expression level of DGKZ mRNA is significantly reduced after DGKZ-RNAi lentivirus infects human glioma U87 cells for 5 days

FIG. 3 shows that DGKZ-RNAi lentivirus infects human glioma U87 cells for 5 days and then causes inhibition of cell proliferation

FIG. 4 is a schematic diagram showing the results of detecting the clonogenic capacity of tumor cells infected with DGKZ-RNAi lentivirus in a clonogenic experiment

FIG. 5 is a schematic diagram of TTP detecting apoptosis of tumor cells after DGKZ-RNAi lentivirus infection

Detailed Description

Based on the research that zinc finger protein may be related to the proliferation, drug resistance, angiogenesis and the like of tumor cells, DGKZ is used as a newly discovered zinc finger protein and is presumed to be involved in the generation and development of malignant tumors.

The invention relates to a group of small interfering RNA (siRNA) sequences, RNA interference vectors and RNA interference lentiviruses aiming at human DGKZ genes. The coding region sequence of human DGKZ mRNA 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 endogenous DGKZ genes in human tumor cells.

The inventor finds that after the expression of the human DGKZ gene is down-regulated by adopting an RNAi method, the proliferation of tumor cells can be effectively inhibited, the apoptosis is promoted, the invasion and the transfer capacity of the tumor cells are reduced, and the growth process of the tumor can be effectively controlled. The research result shows that the DGKZ gene is protooncogene and can be used as a target point for tumor treatment. The inventors further synthesize and test various siRNAs aiming at the DGKZ gene, and screen out the siRNA which can effectively inhibit the expression of the DGKZ and further inhibit the proliferation and growth of human glioma U87 cells, thereby completing the invention.

The invention provides a series of small interfering RNA (siRNA) sequences interfering human DGKZ gene, and constructs a lentivirus capable of specifically silencing DGKZ gene expression. The research of the invention finds that the small interfering RNA and RNAi lentivirus designed aiming at the human DGKZ gene can stably and specifically down-regulate the expression of the DGKZ gene and effectively inhibit the proliferation of human tumor cells. The present invention shows that the DGKZ 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 DGKZ gene by RNAi can be an effective means for inhibiting tumor development.

The design idea of the invention is as follows:

the invention obtains a human DGKZ gene RNAi lentivirus by screening through the following method: a human DGKZ gene sequence is called from Genbank; predicting the siRNA site; synthesizing an effective siRNA sequence aiming at the DGKZ gene, wherein two ends of the effective siRNA sequence contain double-stranded DNA Oligo of the sticky ends of enzyme cutting sites; after double digestion, the lentivirus vector is connected with a double-stranded DNA Oligo to construct RNAi plasmid for expressing DGKZ gene siRNA sequence; the RNAi plasmid and a helper vector (packaging Mix, 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 DGKZ gene can be prepared.

Based on the method, the invention provides 13 effective targets (shown as SEQ ID NO 1-13) for interfering the DGKZ gene, and constructs the lentivirus specifically interfering the human DGKZ gene.

Meanwhile, the invention also discloses a human DGKZ gene RNAi lentivirus (DGKZ-RNAi) and preparation and application thereof.

The research discovers that the proliferation of the tumor cells can be effectively inhibited after the expression of the DGKZ gene in the tumor cells is reduced by using a lentivirus-mediated RNAi method. The research shows that the DGKZ gene is a proto-oncogene, can promote the proliferation of tumor cells, has important biological functions in the occurrence and development of tumors, can be a target for tumor treatment, and can be used as a new means for tumor treatment through lentivirus-mediated DGKZ gene specific silencing.

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 for human DGKZ gene

1. Screening of effective siRNA target against human DGKZ Gene

The DGKZ (NM-003646.3) gene information is called from Genbank; designing effective siRNA target points aiming at DGKZ genes. Table 1 lists 13 effective siRNA target sequences against DGKZ gene.

TABLE 1 siRNA target sequences targeting the human DGKZ gene

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 ℃ C., reaction time 1h) and the purified double-stranded DNA Oligo were ligated by T4 DNA ligase, and 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 out of the connected transformation product, dissolving the surface in 10 mul LB culture medium, uniformly mixing and taking 1 mul 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: 18); the sequence of the downstream primer is as follows: 5'-GTAATACGGTTATCCACGCG-3' (SEQ ID NO: 19), 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 PCR-identified positive clone, wherein the correctly compared clone is a successfully constructed RNAi vector containing SEQ ID NO.1 and is named as pGCSIL-GFP-DGKZ-siRNA.

pGCSIL-GFP-Scr-siRNA negative control plasmid was constructed with negative control siRNA target sequence 5'-TTCTCCGAACGTGTCACGT-3' (SEQ ID NO: 20). 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 the Scr siRNA target spots, and the rest construction methods, identification methods and conditions are the same as those of pGCSIL-GFP-DGKZ-siRNA.

TABLE 3 double-stranded DNA Oligo with Age I and EcoR I cleavage sites sticky ends at both ends

The vector was linearized by T4 DNA 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

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 DGKZ-siRNA lentivirus

The DNA of RNAi plasmid pGCSIL-GFP-DGKZ-siRNA was extracted with a plasmid extraction kit from Qiagen corporation to prepare 100 ng/. mu.l of stock solution.

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, 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. 25. The packaging process of the control lentivirus was the same as that of the DGKZ-siRNA lentivirus, and pGCSIL-GFP-DGKZ-siRNA vector was replaced with pGCSIL-GFP-Scr-siRNA vector alone.

Example 2: real-time fluorescence quantitative RT-PCR method for detecting silencing efficiency of DGKZ gene

Pancreatin digestion of human glioma U87 cells in logarithmic growth phaseCell suspension (cell number about 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, U87:10), 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 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 DGKZ gene were as follows: an upstream primer 5'-AGCAAGCAAGAAGAAGAAGAGG-3' (SEQ ID NO: 21) and a downstream primer 5'-GGATTGAGATACCAGAGGAAAGAC-3' (SEQ ID NO: 22). The housekeeping gene GAPDH is used as an internal reference, and the primer sequences are as follows: upstream primer 5'-TGACTTCAACAGCGACACCCA-3') (SEQ ID NO: 23) and a downstream primer 5'-CACCCTGTTGCTGTAGCCAAA-3' (SEQ ID NO: 24). The reaction system was prepared in the proportions shown in Table 8.

TABLE 7 reverse transcription reaction System

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 infected DGKZ 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 DGKZ mRNA in human glioma U87 cells is down-regulated by 76.5%.

Example 3: detecting the proliferation capacity of tumor cells infected with DGKZ-siRNA lentivirus

Human glioma U87 cells in logarithmic growth phase were trypsinized to prepare a cell suspension (about 5X 10 cells in number)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, U87:10), adding a proper amount of virus, culturing for 24h, then replacing the culture medium, and collecting the cells of each experimental group in the logarithmic growth phase after the infection time reaches 5 days. Complete medium resuspension into cell suspension (2 function10⁴Per 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 lentivirus infection group tumor is cultured in vitro for 5 days, the proliferation speed is obviously slowed down and is far lower than that of the contrast group tumor cells, the number of the viable cells is respectively reduced by 60.1 percent, and the DGKZ gene silencing leads to the inhibition of the proliferation capacity of the tumor cells.

Example 4 detection of the clonogenic Capacity of tumor cells infected with DGKZ-siRNA lentivirus

Human glioma U87 cells are trypsinized and then inoculated into a 12-well plate, and the cell density is 10-15%. The next day, the medium was changed to fresh medium containing 5ug/ml polybrene. DGKZ-siRNA lentiviruses were added to the plates at MOI (MOI, U87: 2) and the medium was replaced with fresh medium 12-24h after infection. After infection for 72h, fluorescence is observed under a fluorescence microscope, and the infection efficiency reaches 90%.

After the cells infected with the virus in the logarithmic growth phase are digested by pancreatin, the complete culture medium is re-suspended into cell suspension; after counting the cells, inoculating the cells into a 6-well plate (200 cells/well), continuously culturing the inoculated cells in an incubator until the number of the cells in 14 days or most of single clones is more than 50, changing the liquid at intervals of 3day, and observing the cell state; photographing the cell clone under a fluorescent microscope before the experiment is terminated; at the end of the experiment, cells were fixed with paraformaldehyde, washed with PBS, Giemsa stained, and photographed.

As shown in the result of fig. 4, the result of the colony forming ability of the tumor cell infected with DGKZ-siRNA is schematically shown, and taking U87 cell as an example, after the expression of DGKZ gene (KD group) is reduced by RNA interference, the number of colony spots formed by the tumor cell is significantly reduced, and the volume of the colony spots is significantly reduced; indicating that DGKZ silencing results in a reduction in the ability of tumor cells to form clones.

The plate clone formation assay detects that the clonogenic capacity of U87 cell tumor cells is reduced after reducing the expression of DGKZ.

Example 5 detection of apoptosis levels in tumor cells infected with DGKZ-siRNA lentivirus

Human glioma U87 cells are trypsinized and then inoculated into a 12-well plate, and the cell density is 10-15%. The next day, the medium was changed to fresh medium containing 5ug/ml polybrene. DGKZ-siRNA lentiviruses were added to the plates at MOI (MOI, U87: 2) and the medium was replaced with fresh medium 12-24h after infection. After infection for 72h, fluorescence is observed under a fluorescence microscope, and the infection efficiency reaches 90%.

After trypsinizing the cells in logarithmic growth phase, resuspending the complete medium into a cell suspension; inoculating to a 96-well plate with 100ul of each well; culturing in a 37 deg.C 5% CO2 incubator; after culturing for 36h, removing the culture solution, and fixing the cells by using 85% ethanol precooled at 4 ℃; washing the plate twice with PBS; after RNase treatment, cells were stained with Annexin-V for 15min in the dark. Scanning and analyzing the 96-well plate on a TTP instrument by using a preset template of analysis subG1 stage to obtain a result.

As shown in the schematic diagram of TTP detection of apoptosis of tumor cells infected with DGKZ-siRNA in FIG. 5, U87 cells are taken as an example. The Annexin-V staining-TTP method detects the change of the proportion of apoptotic bodies (sub G1 phase) of U87 cell tumor cells after the expression of DGKZ is reduced. It was found that the apoptosis rate of tumor cells increases after down-regulating the expression of the DGKZ gene. Following RNA interference to reduce expression of the DGKZ gene (KD group) compared to control interference (control), the number of apoptotic bodies produced by apoptotic tumor cells was significantly increased; indicating that DGKZ silencing results in tumor cell apoptosis. The Annexin-V staining-TTP method detects the change of the proportion of apoptotic bodies (sub G1 phase) of U87 cell tumor cells after the expression of DGKZ is reduced.

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.

Sequence listing

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Claims (15)

1. The use of an isolated human DGKZ gene in the preparation or screening of a medicament for the treatment of a tumor selected from any one of tumors in which the proliferation of tumor cells is associated with the expression of the human DGKZ gene, or in the preparation of a medicament for the diagnosis of a tumor.

2. The use according to claim 1, wherein the tumor is selected from the group consisting of: a glioma.

3. An isolated nucleic acid molecule that reduces the expression of a DGKZ gene in a tumor, the nucleic acid molecule comprising:

a) a double-stranded RNA containing a nucleotide sequence capable of hybridizing with a DGKZ gene under stringent conditions; or

b) shRNA comprising a nucleotide sequence capable of hybridizing to a DGKZ gene under stringent conditions.

4. The isolated nucleic acid molecule of claim 3, wherein said double stranded RNA comprises a first strand and a second strand, wherein said first strand and said second strand are complementary to form an RNA dimer, and wherein the sequence of said first strand is substantially identical to a target sequence in a DGKZ 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, 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 DGKZ gene.

5. The isolated nucleic acid molecule of claim 3 or 4, wherein the target sequence of the DGKZ gene comprises any one of SEQ ID NO 1-13.

6. The isolated nucleic acid molecule of claim 3 or 4, 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 25.

7. The isolated nucleic acid molecule of claim 3 or 4, wherein the sequence of said shRNA is as set forth in SEQ ID NO: shown at 26.

8. A human DGKZ gene interfering nucleic acid construct containing a gene fragment encoding shRNA in the isolated nucleic acid molecule of any one of claims 3-7, capable of expressing the shRNA.

9. The human DGKZ gene interfering nucleic acid construct of claim 8, which is a human DGKZ gene interfering lentiviral vector.

10. The human DGKZ gene interfering nucleic acid construct of claim 9, 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.

11. A human DGKZ gene interfering lentivirus, which is prepared by virus-packaging the human DGKZ gene interfering nucleic acid construct of any one of claims 8-10 with the aid of a lentivirus packaging plasmid or cell line.

12. A pharmaceutical composition for the prevention or treatment of a tumour, comprising a therapeutically effective amount of an isolated nucleic acid molecule according to any one of claims 3 to 7, a human DGKZ gene interfering nucleic acid construct according to any one of claims 8 to 10 and/or a human DGKZ gene interfering lentivirus according to claim 11, and a pharmaceutically acceptable carrier, diluent or excipient.

13. Use of a pharmaceutical composition according to claim 12 for the preparation of a medicament for the treatment of a tumor, wherein the tumor is any tumor whose proliferation of tumor cells is associated with expression of the human DGKZ gene.

14. Use of a pharmaceutical composition according to claim 13 for the manufacture of a medicament for the treatment of glioma.

15. A kit for reducing the expression of the human DGKZ gene in a tumor cell, comprising: human DGKZ gene small interfering RNA according to any one of claims 3 to 7, human DGKZ gene interfering nucleic acid construct according to any one of claims 8 to 10 and/or human DGKZ gene interfering lentivirus according to claim 11, present in a container.

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