A human telomerase reverse transcriptase promoter mutant and its application

Abstract

The present invention belongs to the field of biomedicine and molecular biology technology, and specifically relates to a human telomerase reverse transcriptase promoter mutant and its application. The present invention has found through research that the KLF binding site CACCC on the hTERT core promoter mutates to CAATC, which can significantly inhibit the hTERT promoter activity and can inhibit the most common activating mutation ‑124G>A in the hTERT promoter region. The hTERT promoter region base mutation proposed in the present invention is a tumor suppressor factor, plays a certain role in regulating the hTERT function, and provides an experimental material basis for the targeted hTERT gene therapy of HCC, so it has good practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and molecular biology, and specifically relates to a human telomerase reverse transcriptase promoter mutant and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Telomeres are simple repeating structures containing TTAGGG at the ends of eukaryotic chromosomes. As cells age and die, their length gradually shortens, so they are vividly called "cellular biological clocks." Human telomerase is a ribonucleoprotein complex that can reverse transcribe telomere DNA sequences using its own RNA as a template to compensate for the shortening of chromosome ends during cell division, extend telomeres, and restore telomere function. Human telomerase reverse transcriptase (hTERT) is the rate-limiting factor of telomerase activity, and hTERT gene transcription regulation is a major mechanism for regulating human telomerase activity.

[0004] The core sequence of the hTERT gene promoter region is the 283 bases in front of the gene transcription start point, containing 2 E boxes (CACGTG sequence, E-box) and 5 GC boxes (GGGGCGGGG sequence, GC-box), and almost no TATA box or CAAT box. E-box can bind to transcription factors such as c-myc, Mad1, USF1 / 2, and transcription factors Sp1 and Sp3 can bind to GC-box. The inventor's research group found in the previous study that the HBX protein encoded by hepatitis B virus (HBV) upregulates hTERT expression and telomerase activity by enhancing the binding of Sp1 to DNA. The preS2 protein encoded by HBV activates the hTERT promoter by acting on the target sequence preS2-Resiponsible Region (PRR), enhances the expression of hTERT and telomerase activity, promotes the growth and proliferation of liver cancer cells, and then participates in the occurrence and development of hepatocellular carcinoma (HCC).

[0005] Activating mutations often occur in the hTERT promoter region in HCC, melanoma, malignant glioma, bladder cancer, and thyroid cancer. The mutation rate of the hTERT promoter region in HCC is about 60%, and the two independent mutation hotspots are respectively at -124bp upstream of the translation start point ATG (G>A mutation, with an incidence of 93%; G>T mutation, with an incidence of 1%) and -146bp (G>A mutation, with an incidence of 6%). After the mutation, new ETS- / TCF-binding sites are generated, which affect the occurrence and development of tumors such as HCC. Therefore, it is necessary to conduct further in-depth research on the hTERT promoter. Summary of the invention

[0006] In view of the above-mentioned prior art, the present invention provides a human telomerase reverse transcriptase promoter mutant and its application. Specifically, the present invention unexpectedly discovered through research that the Krüppel like factor (KLF) binding site CACCC on the hTERT core promoter mutated to CAATC, which can significantly inhibit the hTERT promoter activity, and can inhibit the most common activating mutation -124G>A in the hTERT promoter region, providing basic experimental materials and experimental basis for further research on gene targeted therapy of tumors such as HCC. Based on the above research results, the present invention is completed.

[0007] Specifically, the present invention relates to the following technical solutions:

[0008] The first aspect of the present invention provides a human telomerase reverse transcriptase promoter mutant, wherein the nucleotide sequence of the KLF binding site of the human telomerase reverse transcriptase promoter mutant is CAATC, and the nucleotide sequence is located at -324 to -320 upstream of the transcription start point of the human telomerase reverse transcriptase promoter;

[0009] Specifically, the human telomerase reverse transcriptase promoter mutant has a nucleotide sequence as shown in SEQ ID NO.1.

[0010] The second aspect of the present invention provides a recombinant expression vector, which contains the nucleotide sequence of the human telomerase reverse transcriptase promoter mutant.

[0011] Specifically, the recombinant expression vector is obtained by effectively connecting the above-mentioned nucleotide sequence to an expression vector, and the expression vector is any one or more of a viral vector, a plasmid, a phage, a phagemid, a cosmid or an artificial chromosome; the viral vector may include an adenoviral vector, a retroviral vector or an adeno-associated viral vector, and the artificial chromosome includes a bacterial artificial chromosome, a phage P1-derived vector, a yeast artificial chromosome or a mammalian artificial chromosome; further, the expression vector is a plasmid.

[0012] The third aspect of the present invention provides a host cell, wherein the host cell contains the above-mentioned recombinant expression vector or has the above-mentioned human telomerase reverse transcriptase promoter mutant integrated into its chromosome.

[0013] The host cell may be a prokaryotic cell or a eukaryotic cell, which is not specifically limited herein.

[0014] The fourth aspect of the present invention provides the use of the human telomerase reverse transcriptase promoter mutant, recombinant expression vector or host cell in any one or more of the following:

[0015] (a) inhibiting the activity of human telomerase reverse transcriptase promoter or preparing a product that inhibits the activity of human telomerase reverse transcriptase promoter;

[0016] (b) inhibiting the activity of human telomerase reverse transcriptase or preparing a product that inhibits the activity of human telomerase reverse transcriptase;

[0017] (c) Preparation of tumor gene targeted therapeutic drugs.

[0018] Wherein, in the application (a), the human telomerase reverse transcriptase promoter includes a human telomerase reverse transcriptase promoter activating mutant, and the human telomerase reverse transcriptase promoter activating mutant includes a human telomerase reverse transcriptase promoter activating mutant (-124G>A) and a human telomerase reverse transcriptase promoter activating mutant (-124G>T).

[0019] In the applications (a)-(b), the product may be a drug or a test reagent. The test reagent may be used for basic research, thereby facilitating research on the mechanisms of human telomerase reverse transcriptase and related diseases (such as tumors).

[0020] When the product is a drug, the drug also includes pharmaceutically necessary carriers, and the pharmaceutically acceptable carrier dosage should be harmless to the subject, specifically including but not limited to buffers (such as acetate, Tris, phosphate, citrate and other organic acids), antioxidants (such as ascorbic acid and methionine), preservatives (such as octadecyldimethylbenzyl ammonium chloride, phenol, butyl alcohol or benzyl alcohol, methyl paraben or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol), bactericides (such as hexamethonium chloride, benzalkonium chloride, benzethonium chloride), Proteins (such as serum albumin, gelatin or immunoglobulins), hydrophilic polymers (such as polyvinyl pyrrolidone), amino acids (such as glycine, glutamine, asparagine, histidine, arginine or lysine), monosaccharides, disaccharides and other carbohydrates (including glucose, mannose or dextrin), chelating agents (such as EDTA), tonicity regulators (such as trehalose and sodium chloride), sugars (such as sucrose, mannitol, trehalose or sorbitol), surfactants (such as polysorbates), salt-forming counterions (such as sodium), metal complexes (such as Zn-protein complexes) and / or non-ionic surfactants.

[0021] In the application (c), the tumor is a solid tumor, including but not limited to hepatocellular carcinoma, melanoma, malignant glioma, bladder cancer and thyroid cancer; further, hepatocellular carcinoma. The gene is the hTERT gene.

[0022] Beneficial technical effects of one or more of the above technical solutions:

[0023] The above technical scheme constructs a series of promoter reporter plasmids with truncated and mutated hTERT promoters, and uses the dual luciferase method to analyze the specific inhibitory effect of CC>AT mutation on hTERT promoter activity and the interaction with the activating mutation in the promoter region of the hTERT gene. The results show that the reporter vector promoter activity detection shows that the CC>AT specific mutation at the CACCC site significantly inhibits the hTERT promoter activity (inhibition efficiency is 82.68%). The inhibitory effect on the activating mutation site -124G>A (mutation incidence accounts for 93%) is 99.45%, the inhibitory effect on the activating mutation site -124G>T (mutation incidence accounts for 1%) is 21.05%, and there is almost no inhibitory effect on the activating mutation site -146G>A (mutation incidence accounts for 6%). The regulatory sequence CAATC can also inhibit the activity of the SV40 promoter.

[0024] In summary, the base mutation in the hTERT promoter region proposed in the above technical scheme is a tumor suppressor factor, which plays a certain role in regulating the function of hTERT and provides an experimental material basis for targeted hTERT gene therapy of HCC, and therefore has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0026] Figure 1 It is the restriction enzyme digestion, PCR and sequencing identification of the reporter vectors pGL3B-329 (A), pGL3B-MUT329 (B) and pGL3B-MUT329-2nd (C) in the examples of the present invention.

[0027] Figure 2 It is the restriction enzyme digestion and sequencing identification of the reporter vectors pGL3B-MUT329-124A (A) and pGL3B-329-124A (B) in the examples of the present invention.

[0028] Figure 3 This is the sequencing identification of the reporter vectors pGL3B-MUT329-124T (A) and pGL3B-329-124T (B) in the examples of the present invention.

[0029] Figure 4 This is the sequencing identification of the reporter vectors pGL3B-MUT329-146A (A) and pGL3B-329-146A (B) in the examples of the present invention.

[0030] Figure 5 It is a comparison of the activities of the full-length, truncated and CACCC site mutated hTERT promoter reporter plasmids in the examples of the present invention in the liver cancer cell HepG2.

[0031] Figure 6 This is the effect of CC>AT mutation on the activity of hTERT promoter containing activating mutation in the embodiment of the present invention. DETAILED DESCRIPTION

[0032] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0033] It should be noted that the terms used here are only for describing specific embodiments, and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should also be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof. If the experimental methods of specific conditions are not indicated in the following specific embodiments, the conventional methods and conditions of molecular biology in the art are usually followed, and such techniques and conditions are fully explained in the literature. See, for example, Sambrook et al., the techniques and conditions described in "Molecular Cloning: Laboratory Manual", or according to the conditions recommended by the manufacturer.

[0034] The present invention is further described in conjunction with specific examples. The following examples are only for explaining the present invention and are not intended to limit the content thereof. If the specific experimental conditions are not specified in the examples, they are usually carried out under conventional conditions or under conditions recommended by the sales company; the materials and reagents used in the examples, unless otherwise specified, can be purchased through commercial channels.

[0035] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0036] Example

[0037] Materials and methods

[0038] Plasmid construction: hTERT full-length promoter reporter plasmid pGL3B-TRTP was kindly provided by Dr. Horikawa (Laboratory of Biosystems and Cancer, Center for Cancer Research, National Cancer Institute, NIH, USA). Plasmids pGL3-Basic, pGL3-promoter and pRL-TK were purchased from Promega. According to the human hTERT genome sequence published by GenBank [gi: 4226057], specific PCR primers were designed, and high GC Taq enzyme was used to amplify a series of truncated and mutated hTERT promoter fragments. The reporter vectors were named pGL3B-329, pGL3B-MUT329, pGL3B-MUT329-2nd, pGL3B-329-124A, pGL3B-329-124T, pGL3B-329-146A, pGL3B-MUT329-124A, pGL3B-MUT329-124T and pGL3B-MUT329-146A (124 and 146 indicate the position relative to the translation start site, and other numbers indicate the position relative to the transcription start site). All ampicillin-resistant plasmids were transformed into Escherichia coli JM109 for amplification and prokaryotic expression. (Primers are shown in Table 1)

[0039] Table 1

[0040]

[0041]

[0042] Note: F1 is the common upstream primer of pGL3B-329, pGL3B-329-124A, pGL3B-329-124T, and pGL3B-329-146A; F2 is the common downstream primer of pGL3B-MUT329, pGL3B-MUT329-124A, pGL3B-MUT329-124T, and pGL3B-MUT329-146A.

[0043] Cell experiment: HepG2 (ATCC HB-8065) ​​hepatoma cell line was purchased from the Institute of Cell Biology, Chinese Academy of Sciences, Shanghai, China, and cultured in Minimum Essential Medium containing 10% fetal bovine serum plus 1mM sodium pyruvate (EMEM culture medium) at 37°C and 5% CO2. 1μg of truncated and mutant hTERT promoter reporter plasmids expressing firefly luciferase and 25ng of internal reference plasmid pRL-TK expressing Renilla luciferase were added with 2μl lipofectamine TM2000The encapsulated cells were co-transfected into HepG2 cells. Each transfection experiment was repeated three times and the cells were lysed after 48 hours.

[0044] Dual fluorescence detection: Dual luciferase reporter gene test system (PerRinElmer, USA) was used to measure luciferase activity. The firefly fluorescence intensity M1 and the Renilla fluorescence intensity expression intensity M2 were used. The ratio of M1 to M2 represented the luciferase expression intensity.

[0045] Statistical methods: GraphPad Prism 9.0 was used for statistical analysis and plotting. The quantitative data between the two groups were compared using Student-t test. All p values ​​are two-tailed 95% confidence intervals. p < 0.05 was considered statistically significant.

[0046] result

[0047] 1. Construction of a series of truncated and mutated hTERT promoter reporter vectors: The full-length hTERT promoter reporter plasmid (-1665 to +135) was selected as a PCR amplification template, and 9 truncated and mutated hTERT promoter reporter vectors were successfully constructed through restriction digestion, PCR amplification or DNA sequencing identification ( Figure 1-4 ).

[0048] 2. The CC>AT mutation at the CACCC site significantly inhibited the hTERT promoter activity: The results of the dual luciferase activity assay showed that the constructed truncated hTERT promoter reporter plasmid pGL3B-329 had comparable activity to the full-length promoter. When the KLF binding site CACCC mutated to CAATC, the promoter activity was significantly inhibited (inhibition efficiency was 82.68%). When the KLF binding site CACCC was randomly mutated to CAGAC again, the promoter activity remained essentially unchanged. Figure 5 )

[0049] 3. The mutual influence between inhibitory mutations and known activating mutations in the promoter region of hTERT gene: The results of dual luciferase activity detection showed that CC>AT mutation (CACCC site) in the promoter region of hTERT could significantly inhibit the activating mutation -124G>A (inhibition efficiency was 99.45%), and still had some inhibitory effect on the activating mutation -124G>T (inhibition efficiency was 21.05%), but had no inhibitory effect on the activating mutation -146G>A. ( Figure 6 )

[0050] Mutations in the hTERT promoter region are the most common genetic alterations in HCC, with an overall frequency of approximately 60%. Almost all hTERT promoter mutations occur in the first hotspot - 124G>A. Mutations are associated with the activation and expression of telomerase, and play a key role in the maintenance of telomeres during the development of HCC. In this study, the CC>AT mutation at the CACCC site in the hTERT promoter region significantly inhibited the activity of the hTERT promoter by constructing a reporter vector and performing dual luciferase activity assays. First, luciferase analysis of reporter vectors for different mutations at the CACCC site showed that specific CC>AT mutations inhibited hTERT promoter activity. Second, after cloning the regulatory sequence CAATC, which strongly inhibited hTERT promoter activity, into the pGL3-promoter reporter plasmid containing the SV40 promoter, it was found that the CAATC sequence could inhibit the activity of the SV40 promoter, indicating that the negative regulatory sequence CAATC has nonspecific inhibition and silencing functions on the promoter. Most importantly, when studying the interaction between activating mutations and CC>AT mutations, we found that CC>AT mutations could almost completely suppress the most common activating mutation in the hTERT promoter - 124G>A.

[0051] The promoter binding site software showed that the CACCC sequence in the hTERT promoter region is a binding site for KLF. KLF is a subfamily of zinc finger protein transcription factors, which is highly homologous to SP1. There are currently 17 members in the family, which can compete with each other to bind to the CACCC site and participate in cell proliferation, apoptosis, differentiation and individual development. Studies have reported that KLF-2 can directly bind to the hTERT promoter in T cells and inhibit its activity. Therefore, the CACCC site plays an active regulatory role in the development of HCC.

[0052] The most likely explanation for the loss of promoter activity caused by the CC>AT mutation at the CACCC site in the hTERT promoter region is the binding of transcriptional repressors. This study also found that the CAATC sequence can inhibit the activity of the SV40 promoter. Because the 283 bases upstream of the transcription start site are the core sequence of the hTERT promoter, another possible explanation for the loss of promoter activity caused by the CC>AT mutation at the CACCC site in the hTERT promoter region is that the mutated CAATC sequence may become a silencing regulatory region of the hTERT core promoter. In addition, the CC>AT mutation may also interfere with the methylation of the hTERT promoter region, but the specific mechanism needs further confirmation.

[0053] Continuous activation of telomerase can lead to cell immortalization, which is the premise and basis for cell carcinogenesis and malignant proliferation. hTERT is considered to be the rate-limiting enzyme of telomerase activity. Studies have found that hTERT expression is upregulated in more than 90% of tumors, including HCC, and it is a potential therapeutic target for HCC. Immunotherapy, small molecule inhibitors, gene therapy or anti-hTERT vaccines for tumors with high telomerase activity are currently under development. Since CC>AT mutation can significantly inhibit the most common activating mutation in the hTERT promoter -124G>A, it can be used as a potential target for HCC gene therapy by inhibiting hTERT transcription and thus reducing telomerase activity.

[0054] In 2013, with the emergence of a new artificial nuclease Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / CRISPR-associated (Cas) 9, tumor gene mutation therapy was pushed to the forefront of tumor gene therapy. It has been reported that CRISPR / Cas technology can be applied to personalized molecular surgical treatment of EGFR mutation gene-mutated lung cancer, and scientists have found that the improved SRIRACCHA (ie, a stable, but reversible, integrated reporter for assaying CRISPR / Cas-stimulated HDR activity) can maintain the integrity of the genome during cell editing therapy. The latest study found that small molecule-assisted shut-off (SMASh) tags can improve the accuracy, safety and versatility of the CRISPR-cas9 gene editing system. Therefore, the next step is to conduct more in vivo and in vitro studies to further clarify whether editing the hTERT promoter can be used for anti-hTERT treatment of a portion of HCC patients.

[0055] It is well known that tumor growth depends on the proliferation of tumor cells on the one hand and on the generation of blood vessels on the other. Cultured tumor cells cannot exceed a certain size due to hypoxia and lack of necessary nutrients. Angiogenesis is a necessary condition for the transformation of tumor cell clusters into malignant tumors, and the exponential growth of tumors mainly depends on angiogenesis. Therefore, when applying the hTERT promoter CC>AT mutation as a potential target for gene therapy in the future, it is best to apply it locally to the tumor during the avascular stage of the tumor.

[0056] In summary, this study constructed truncated and mutated hTERT promoter reporter vectors and performed dual luciferase activity assays and found that the CC>AT mutation at the CACCC site could inhibit hTERT promoter activity and the most common activating mutation in the hTERT promoter region -124G>A, providing basic experimental materials and experimental basis for further research on gene targeted therapy for HCC.

[0057] The nucleotide sequence of the human telomerase reverse transcriptase promoter mutant in the embodiment is:

[0058]

[0059] Among them, italic bold letters represents the KLF binding site, the capital letter G represents the transcription start site (-1), and the lowercase bold letters Represents the translation start site.

[0060] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of a human telomerase reverse transcriptase promoter mutant, a recombinant expression vector or a host cell in any one or more of the following: (a) preparing a product for inhibiting hTERT promoter activity; (b) Preparation of tumor gene-targeted therapeutic drugs; In the application (a), the hTERT promoter activity is human telomerase reverse transcriptase promoter activation type -124G>A and -124G>T; In the application (b), the tumor is hepatocellular carcinoma; the gene is hTERT gene; The nucleotide sequence of the human telomerase reverse transcriptase promoter mutant is shown in SEQ ID NO.1; The recombinant expression vector contains the nucleotide sequence of the human telomerase reverse transcriptase promoter mutant; The host cell contains the recombinant expression vector or has the human telomerase reverse transcriptase promoter mutant integrated into its chromosome.

2. The use according to claim 1, characterized in that: The recombinant expression vector is obtained by connecting the nucleotide of the human telomerase reverse transcriptase promoter mutant to an expression vector, and the expression vector is any one or more of a viral vector, a plasmid, a phage or an artificial chromosome; The nucleotide sequence of the human telomerase reverse transcriptase promoter mutant is shown in SEQ ID NO.

1.

3. The use according to claim 2, characterized in that: The viral vector includes an adenoviral vector, a retroviral vector or an adeno-associated viral vector.

4. The use according to claim 2, characterized in that: The artificial chromosome includes a bacterial artificial chromosome, a yeast artificial chromosome or a mammalian artificial chromosome.

5. The use according to claim 2, characterized in that: The expression vector is a plasmid.

6. The use according to claim 1, characterized in that: The host cell is a prokaryotic cell or a eukaryotic cell.

7. The use according to claim 1, characterized in that: In the application (a), the product is a drug or a test reagent, and the test reagent is used for basic research.

8. The use according to claim 1, characterized in that When the product is a medicine, the medicine further includes a pharmaceutically necessary carrier.

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