CN113893337A - Application of DTX2 protein in preparation of preparation for regulating telomerase activity - Google Patents
Application of DTX2 protein in preparation of preparation for regulating telomerase activity Download PDFInfo
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Abstract
The invention discloses application of DTX2 protein in preparation of a preparation for regulating telomerase activity. The invention discovers that DTX2 is a novel telomerase activating factor, DTX2 protein participates in regulation and control of expression of telomerase catalytic subunit hTERT and regulation and control of telomere length, the expression level of the protein is in positive correlation with the expression level of hTERT and telomerase activity, and the interference with the expression of DTX2 protein or the knockout of DTX2 can obviously reduce the expression of telomerase catalytic subunit hTERT and the telomerase activity in cells. The DTX2 protein is a positive regulatory factor of telomerase, can be used for preparing a preparation for positively regulating the totipotency of stem cells, and the DTX2 protein inhibitor can also be used for preparing a telomerase positive tumor treatment medicine. The DTX2 protein has great significance for the clinical application of future stem cells and the treatment of malignant tumors.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to application of DTX2 protein in preparation of a preparation for regulating telomerase activity.
Background
Telomeres, which are the special structure of the end of eukaryotic chromosomes, are composed of highly repetitive DNA sequences (TTAGGG) and their interacting proteins. In normal somatic cells, with mitosis, the terminal sequences of chromosomes cannot be efficiently replicated, so that telomere DNA sequences of the cells are continuously shortened, and finally, senescence and apoptosis of the cells are triggered. Cells with strong proliferation capacity, such as stem cells, tumor cells and the like, have the activity of endogenous telomerase and can continuously extend telomeres, so that the telomeres of the cells cannot be shortened after multiple mitosis. Therefore, the activity of telomerase plays an important role in the proliferation of stem cells and the survival of tumor cells in vivo.
Telomerase is a ribonucleic acid protein complex consisting of an RNA subunit (hTR) complementary to telomeric DNA, a telomerase reverse transcriptase (hTERT) catalytic subunit with reverse transcriptase activity, and a telomerase associated protein (TEP), wherein hTR and hTERT constitute the most basic core structure of telomerase. hTR is highly expressed in all tissues, while hTERT is limited to cells with strong proliferation capacity, such as most tumor cells, e.g., gastric cancer, liver cancer, colorectal cancer, stem cells, germ cells, activated lymphocytes, and the like. A great deal of evidence indicates that the hTERT is a key component for regulating the telomerase activity, and the amount of the hTERT expression determines the telomerase activity of the cells in which the hTERT is expressed.
The activity regulation of telomerase takes place at multiple levels in cells, including processes of transcription, translation, post-translational modification, modification of hTR and hTERT, transport, subcellular localization, assembly of telomerase nucleoprotein on telomeres, and functional exertion. For example, chinese patent CN105582525A discloses the use of CIRP in the preparation of telomerase binding agents or telomerase activity modulators, where the CIRP acts directly on the RNA component of human telomerase and interacts with the telomerase protein component hTERT through the RNA component, and the CIRP participates in the regulation of the expression of the telomerase protein component and in the regulation of the length of telomeres. Therefore, more new factors capable of regulating the expression of telomerase catalytic subunit hTERT are discovered, and the method has great significance for regulating the activity of telomerase, and the clinical application of future stem cells and the treatment of malignant tumors.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, provides the application of the DTX2 protein in preparing a preparation for regulating and controlling telomerase protein subunit hTERT, and has important significance for biological research related to telomerase activity.
The invention also aims to provide application of the DTX2 protein in preparing a preparation for regulating telomerase activity.
The invention also aims to provide application of the DTX2 protein in preparing a preparation for regulating telomere length.
The invention also aims to provide application of the DTX2 protein inhibitor in preparing a telomerase positive tumor treatment medicine.
The invention also aims to provide application of the DTX2 protein in preparing a preparation for positively regulating totipotency of stem cells.
Another object of the present invention is to provide a telomerase positive cell growth regulator.
The invention also aims to provide a telomerase positive tumor treatment drug.
In order to achieve the purpose, the invention is realized by the following scheme:
the invention discloses a new factor-DTX 2 for activating telomerase, wherein the DTX2 Protein is named deltex E3 ubiquitin ligand 2, also named RING Finger Protein 58(RNF58), and is an E3 ubiquitin ligase. The full length comprises 622 amino acid residues, including two WWE domains and a RING domain (as shown in FIG. 1). The amino acid sequence of the DTX2 protein is shown as SEQ ID NO. 1. The inventor discovers that the DTX2 protein participates in regulation and control of the expression of telomerase catalytic subunit hTERT and the regulation and control of the length of telomere, the expression level of the protein is in positive correlation with the expression level of hTERT and the activity of telomerase, and the interference with the expression of DTX2 protein or the knockout of DTX2 can obviously reduce the expression of telomerase catalytic subunit hTERT and the activity of telomerase in cells; while overexpression of DTX2 resulted in an increase in the level of hTERT expression. Therefore, it was concluded that the DTX2 protein is a telomerase forward regulator.
Specifically, the inventors found through research that: (1) in HeLa cells, knocking down the expression of DTX2 using siRNA resulted in decreased hTERT expression levels and cellular telomerase activity; (2) conditional knock-out of DTX2 using CRISPR/Cas9 technology results in decreased hTERT expression levels and cellular telomerase activity in HeLa cells; (3) in an EGFP fluorescence report cell driven by an hTERT core promoter, the EGFP fluorescence intensity is weakened and the expression level is reduced by using siRNA to knock down the expression of DTX 2; (4) in HeLa cells, overexpression of DTX2 increased hTERT expression levels and cellular telomerase activity; (5) in HeLa cells with DTX2 knockout, complementation of DTX2 can increase the expression level of hTERT and the activity of telomerase.
Therefore, the invention requests to protect the application of the DTX2 protein in preparing a preparation for regulating the expression level of telomerase protein subunit hTERT.
Specifically, the DTX2 protein positively regulates the transcriptional expression of hTERT.
In addition, the inventor also researches and discovers that: in HeLa cells, interfering DTX2 by siRNA or conditionally knocking out DTX2 by CRISPR/Cas9 technology can lead to reduction of telomerase activity in cells, and overexpression of DTX2 can lead to increase of telomerase activity in cells.
Therefore, the invention also claims the application of the DTX2 protein in preparing a preparation for regulating telomerase activity.
Specifically, the DTX2 protein positively regulates telomerase activity.
Furthermore, the DTX2 protein simultaneously participates in the regulation of RNA level of telomerase protein subunit hTERT to regulate telomerase activity, and further participates in the regulation and maintenance of telomere length, so that the growth and proliferation of cells are regulated, and the protein expression level of the cells is positively correlated with the telomere length.
Therefore, the invention also claims the application of the DTX2 protein in preparing a preparation for regulating the length of telomeres. While the DTX2 protein inhibitor can be used as a negative regulator of telomere length.
In addition, the invention also requests to protect the application of the DTX2 protein inhibitor in the preparation of telomerase positive tumor treatment drugs, and the expression of DTX2 protein is inhibited, so that the activity of telomerase is inhibited, the growth, proliferation and differentiation of tumor cells are inhibited, the worsening condition of tumors is favorably alleviated, and the personalized effective treatment is facilitated.
In addition, the invention also requests to protect the application of the DTX2 protein or the expression promoter thereof in preparing a preparation for positively regulating the totipotency of the stem cells, and the DTX2 protein is overexpressed to further activate the activity of telomerase, so that the development and differentiation capacity of the stem cells in vivo is improved, and the totipotency and the pluripotency of the stem cells are maintained.
Preferably, the stem cell is a totipotent or pluripotent stem cell.
The invention also claims a telomerase positive cell growth regulator, which comprises DTX2 protein or an expression promoter thereof.
The invention also claims a telomerase positive tumor treatment drug which comprises a DTX2 protein inhibitor. The DTX2 protein inhibitor includes but is not limited to RNA interference agent, protein antibody, etc., and any agent that can inhibit the expression of DTX2 protein is within the scope of the present invention.
Since not all cancer cells are rendered immortal by telomerase, it has been shown by prior studies that about 85% of cancer cells are rendered immortal by telomerase; another 10% of tumor cells do not rely on telomerase for telomere length maintenance, and these cells have the ability to divide indefinitely, but no telomerase activity is detected. Therefore, according to the research result of the invention, a tumor treatment means for knocking down or inhibiting the DTX2 protein can have a further targeted treatment scheme, that is, when a DTX2 protein inhibitor is used for treating a tumor, whether the tumor is a telomerase positive tumor or not should be judged, for example, the activity of telomerase can be detected to judge the corresponding individual condition, and only in the case of excluding the telomerase negative tumor, the DTX2 protein inhibitor is used as a treatment means to reduce the time delay and drug abuse caused by ineffective medication and treatment.
Compared with the prior art, the invention has the following beneficial effects:
the invention discovers a novel telomerase activating factor-DTX 2, and the inventors find that DTX2 protein participates in the regulation of the expression of telomerase catalytic subunit hTERT and the regulation of telomere length, the expression level of the protein is in positive correlation with the expression level of hTERT and the telomerase activity, and the interference with the expression of DTX2 protein or the knockout of DTX2 can obviously reduce the expression of telomerase catalytic subunit hTERT and the telomerase activity in cells. Therefore, the DTX2 protein is a positive regulatory factor of telomerase, can be used for preparing a preparation for positively regulating the totipotency of stem cells, and the DTX2 protein inhibitor can also be used for preparing a telomerase positive tumor treatment medicine. The DTX2 protein has great significance for the clinical application of future stem cells and the treatment of malignant tumors.
Drawings
FIG. 1: schematic representation of the functional domains of the DTX2 protein.
FIG. 2: levels of endogenous DTX2 and hTERT mRNA were knocked down in HeLa cells after DTX 2.
FIG. 3: the telomerase activity at the opposite end of the cells after DTX2 was knocked down in HeLa cells.
FIG. 4: control group pMSCV-EF1 alpha-EGFP plasmid and pMSCV-hTERT-EGFP fluorescence report plasmid map
FIG. 5: the effect of knocking down DTX2 on EGFP fluorescence intensity, as well as the changes in DTX2 and EGFP mRNA levels in hTERT-EGFP reporter cell lines.
FIG. 6: changes in DTX2 and hTERT mRNA levels and in telomerase activity following conditional knockdown of DTX2 in HeLa cells.
FIG. 7: in monoclonal cells with DTX2 knocked out, the effect of DTX2 on hTERT mRNA levels and relative telomerase activity was complemented back.
FIG. 8: hTERT mRNA levels and relative telomerase activity after overexpression of DTX2 in HeLa cells.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Wherein, the siRNA is purchased from Shanghai Jima company,
RNAiMAX from Invitrogen (13778), RNAiso Plus from TAKARA (9109), and ChamQ qPCR mix from Vazyme (Q711-03); the real-time fluorescent quantitative PCR instrument used was Roche LightCycler 480II and the flow cytometer was BD Calibur.
1. RNA extraction and fluorescent quantitative RT-PCR experiment steps:
(1) RNA extraction method (taking 6-well plate as an example)
Add 1mL of RNAioso Plus per well and blow repeatedly with a micropipette until a clear, non-viscous liquid is obtained, transfer to a 1.5mL Eppendorf centrifuge tube (ep tube) and stand at room temperature for 5 minutes. 200 μ L of chloroform was added, followed by vigorous shaking for about 15 seconds to mix well, followed by centrifugation at 12,000g for 15 minutes at 4 ℃. Carefully transfer the supernatant to a new tube, add an equal volume of isopropanol, mix well and then stand at room temperature for 10 minutes. After centrifugation at 12,000g for 15 minutes at 4 ℃ and removal of the supernatant, 1mL of 75% ethanol was added and the precipitate was inverted several times to remove impurities. After centrifugation at 12,000g for 5 minutes at 4 ℃ and removal of the supernatant, the supernatant was air-dried, dissolved in DEPC-treated ultrapure water and stored at-80 ℃.
(2) Reverse transcription to synthesize cDNA
Using PrimeScriptTMRT reagent Kit (TAKARA, RR047A) reverse transcribes RNA to cDNA. First, 1. mu.g of RNA was subjected to a genomic DNA removal reaction (Table 1), followed by a reverse transcription reaction (Table 2), and the synthesized cDNA was stored at-20 ℃.
TABLE 1 reaction System for removing genomic DNA
42 ℃ for 2min (or room temperature for 5min)
TABLE 2 reverse transcription reaction System
37℃15min；85℃5sec
(3) Fluorescent quantitative PCR (qPCR)
After diluting cDNA at a ratio of 1:4 with ultrapure water free from nuclease contamination, 1. mu.L of the cDNA was used as a template in a 10. mu.L reaction system (Table 3), and the cDNA was detected by a Roche LightCycler 480II qPCR instrument. The relative expression abundance of the target gene is calculated by calculating the logarithmically transformed 2-ΔΔCtA value obtained.
TABLE 3 qPCR reaction System
Example 1 knock down of expression of DTX2 in HeLa cells
1. Method of producing a composite material
Cells were passaged the day before to a cell density of 60-80% at transfection. Using a 6-well plate as an example, 5. mu.L of 20. mu.M commercial siRNA was diluted with 250. mu.L of serum-free medium opti-MEM (Table 4), and then 5. mu.L of commercial siRNA was diluted with 250. mu.L of Lopti-MEM
RNAimax. Standing for 5min, adding the mixed solution containing RNAImax into the mixed solution containing siRNA, fully mixing uniformly, standing for 10-15min at room temperature, dropwise adding into the holes, and shaking uniformly. Cells can be harvested 48 hours after transfection for RNA extraction, cDNA synthesis by reverse transcription and qPCR detection of interference efficiency and other gene expression conditions (specific primer sequences are shown in Table 5).
TABLE 4 DTX2 siRNA sequences
TABLE 5 qPCR primer sequences
2. Results
The results are shown in fig. 2, and result in the reduction of hTERT expression level after knocking down the expression of DTX2 in HeLa cells by using siRNA interference technology.
Example 2 telomerase Activity detection (qTRAP) assay
1. Method of producing a composite material
Collecting 2X 105The individual cells were frozen in 1.5mL ep tubes using liquid nitrogen and stored in a freezer at-80 ℃. After a batch of samples was collected, 200. mu.L of pre-cooled NP-40 lysate (10mM Tris-Cl, pH 8.0; 1mM MgCl) was used for each sample2(ii) a 1mM EGTA; 1% NP-40; 0.25mM sodium deoxyholate; 10% glycol; 150mM NaCl; 1mM DTT and the protease inhibitor cocktail) were added prior to use and resuspended and incubated on ice for 30 minutes. Cell lysates were mixed as 1: after 4 dilutions, 1. mu.L of the DNA was used as template for qPCR-based telomere repeat amplification assay (qTRAP), and 0.05. mu.g of TS and ACX primers, 1mM EGTA and qPCR super mix (Table 6) were added to 10. mu.L of the reaction. Incubated at 30 ℃ for 30 minutes to allow telomerase to mimic TS primerAfter the plate is amplified, qPCR detection is carried out, and the strength of the signal reflects the strength of the telomerase activity. Quantification was performed using the internal reference protein GAPDH.
TABLE 6 qTRAP reaction System
2. Results
As shown in FIG. 3, the results indicate that in HeLa cells, interfering with DTX2 using siRNA results in a decrease in telomerase activity within the cells.
Example 3 Effect of siRNA interference with DTX2 on GFP expression in hTERT-GFP reporter
1. Construction of hTERT-GFP reporter gene cell line
To construct the hTERT-GFP fluorescence reporter system, we first constructed the pMSCV-EF1 alpha-EGFP expression vector. The encoded and optimized green fluorescent protein gene (EGFP) is amplified by PCR, cut by enzyme and connected to a pENTR cloning vector, and then a pMSCV-EF1 alpha-EGFP expression vector is constructed by a Gateway cloning method. According to the literature report, a fragment of 454bp upstream of the initiation codon of the hTERT gene is selected as a core promoter. Obtaining hTERT core promoter fragment by PCR amplification (cloning primer sequence is shown in table 7), replacing EF1 alpha promoter on pMSCV-EF1 alpha-EGFP vector with hTERT core promoter through AgeI and NotI enzyme digestion, connection and other steps, obtaining pMSCV-hTERT-EGFP expression vector (figure 4).
TABLE 7 primer sequences for cloning of hTERT core promoter
2. Flow cytometry analysis
The cells to be analyzed were digested with 0.25% trypsin and centrifuged to collect cell pellets, which were resuspended in 1 × PBS into single cells and added to the flow tube. GFP signals in the samples were detected using the FITC channel of a BD Calibur flow cytometer, and positive cell populations were selected using wild-type HeLa cells as negative controls. Cells that stably transfected hTERT-GFP shifted relative to the negative population, and were analyzed for GFP positivity and their signal intensity.
3. Results
The results are shown in fig. 5, and result in the decrease of the expression level of GFP in hTERT-GFP reporter gene after interfering with DTX2 by siRNA in HeLa cells, indicating that knocking down the expression level of DTX2 suppresses the promoter activity of hTERT.
Example 4 DTX2 knock-out test
1. Construction of DTX2 knockout cell line by CRISPR/Cas9 technology
First, stable transgenic lines were constructed that induced Cas9 expression by doxycline (dox). Cas 9-expressing plasmid was lentivirally infected with HeLa cells, screened 10 days with 250. mu.g/mL G418, and after Dox induction, Cas9 expressed stable monoclonals. Two sgrnas (table 8) were designed based on the gene sequence information of DTX2 and ligated to sgRNA expression vectors having Puromycin (Puromycin) resistance and Hygromycin (Hygromycin) resistance, respectively. And (3) stably transferring the two vectors into a cell line expressed by the Dox-induced Cas9 in a lentivirus infection mode, and sequentially screening by using two antibiotics, namely Puromycin and Hygromycin. And adding Dox to the surviving cells (namely the cells stably transferred with the two gRNAs) to induce the expression of Cas9, and detecting the expression conditions of DTX2 and hTERT by a fluorescent quantitative PCR method after 72 h.
TABLE 8 DTX2 sgRNA sequences
2. Results
The results are shown in fig. 6, in HeLa cells, the expression level of hTERT is reduced and the telomerase activity of the cells is reduced after knocking out DTX2 by using CRISPR/Cas9 technology. In addition, in HeLa cells with DTX2 knockout, complementation of DTX2 protein increased hTERT expression levels and telomerase activity back (fig. 7).
EXAMPLE 5 exogenous overexpression DTX2 experiment
1. Method of producing a composite material
Stable transgenic cell lines are constructed by integrating gene expression vectors into the cell genome by means of lentiviral infection. HEK293T cells were passaged the day before virus packaging to a cell density of 40-50% at transfection. Viral packaging plasmid (psPAX2), capsid plasmid (pMD2.G) and lentiviral expression plasmid were simultaneously transfected into HEK293T cells using PEI transfection reagent (Polysciences; 24765-1). The culture supernatant was collected 48 hours after packaging the virus and filtered through a 0.45 μm filter to infect cells (8 μ g/mL polybrene, Sigma; 107689 was added). Cells were infected with virus for 6-8 hours and replaced with fresh medium, and after 48 hours they were screened for 3 days with medium containing 2. mu.g/mL puromycin (InvivoGen; 58-58-2).
2. Results
The results are shown in fig. 8, and in HeLa cells, overexpression of DTX2 resulted in increased hTERT expression level and increased cell telomerase activity.
In conclusion, the expression level of the DTX2 protein is in positive correlation with the expression level of telomerase catalytic subunit hTERT and the activity of telomerase, and the interference of the expression of the DTX2 protein or the knockout of DTX2 can obviously reduce the expression of telomerase catalytic subunit hTERT and the activity of telomerase in cells. Therefore, the DTX2 protein is a positive regulatory factor of telomerase, can be used for preparing a preparation for positively regulating the totipotency of stem cells, and the DTX2 protein inhibitor can also be used for preparing telomerase positive tumor medicaments.
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Sequence listing
<110> Zhongshan university
Application of <120> DTX2 protein in preparation of preparation for regulating telomerase activity
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Claims (10)
- Application of DTX2 protein in preparation of a preparation for regulating the expression level of telomerase protein subunit hTERT.
- 2. The use of claim 1, wherein the DTX2 protein positively regulates the transcriptional expression of hTERT.
- Application of DTX2 protein in preparation of a preparation for regulating telomerase activity.
- 4. The use of claim 2, wherein the DTX2 protein positively regulates telomerase activity.
- Use of DTX2 protein in the preparation of a formulation for modulating telomere length.
- Application of DTX2 protein as a target point in preparation of telomerase positive tumor treatment drugs.
- Application of DTX2 protein expression inhibitor in preparation of telomerase positive tumor treatment drugs.
- The application of the DTX2 protein or the expression promoter thereof in preparing a preparation for positively regulating the totipotency of stem cells.
- 9. A telomerase positive cell growth regulator, comprising DTX2 protein or an expression promoter thereof.
- 10. A telomerase positive tumor treatment drug, which is characterized by comprising a DTX2 protein expression inhibitor.
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