Lentiviral CRISPRv2 / dCas9-VP64-sgRNAs transcriptional activation system based on tumor suppressor gene BRD7 and preparation and application thereof

Abstract

This invention discloses a LentiCRISPRv2 / dCas9-VP64-sgRNA transcriptional activation system based on the tumor suppressor gene BRD7, its preparation, and its application, belonging to the field of tumor molecular biology. Transcriptional silencing of BRD7 is a key mechanism leading to the downregulation of BRD7 expression in nasopharyngeal carcinoma cells. By using sgRNAs to specifically target the proximal promoter region of BRD7, the expression of the tumor suppressor gene BRD7 can be transcribed and activated, enabling personalized and precise treatment for cancer patients. This invention constructs a LentiCRISPRv2 / dCas9-VP64-sgRNA transcriptional activation system based on lentiviral delivery and dCas9-guided transcriptional regulation technology. This system, used to activate BRD7 expression, significantly inhibits the proliferation of nasopharyngeal carcinoma cells and tumor growth in vivo, thus exhibiting an anti-tumor effect and showing promise as an important molecular strategy for personalized cancer treatment.

Description

Technical Field

[0001] This invention belongs to the field of tumor molecular biology, specifically relating to the LentiCRISPRv2 / dCas9-VP64-sgRNAs transcriptional activation system that targets the proximal promoter region of BRD7 to transcribe and activate the expression of the tumor suppressor gene BRD7, and its construction method. It also relates to the application of this transcriptional activation system in the preparation of tumor therapeutic agents. Background Technology

[0002] With the continuous development of science and technology, our understanding of the occurrence and development of tumors has become more microscopic, gradually achieving a leap from the organ and tissue level to the molecular level. Gene therapy, as a revolutionary treatment method, treats diseases by introducing modified genes into the patient's body. Based on the molecular mechanisms of disease, it provides precise intervention and achieves personalized treatment, demonstrating enormous potential in the field of tumor treatment.

[0003] The BRD7 gene is a novel, full-length gene that is expressed at low levels in nasopharyngeal carcinoma cells. It was previously isolated and cloned independently using methods such as cDNA representativeness analysis and library screening. It has been shown to be downregulated in various malignant tumors, including nasopharyngeal carcinoma, breast cancer, and lung cancer, playing an important tumor-suppressive role and negatively correlated with clinical progression and poor prognosis. However, the mechanism by which BRD7 expression is downregulated in nasopharyngeal carcinoma and other tumors is not fully understood. Studies have shown that BRD7 expression can be regulated through transcription and epigenetics, therefore, it is inferred that BRD7 reactivation is feasible.

[0004] In this invention, a LentiCRISPRv2 / dCas9-VP64 plasmid was constructed based on the LentiCRISPRv2 plasmid, and sgRNAs specifically targeting the proximal promoter region of BRD7 were ligated into this plasmid system, thus successfully constructing the LentiCRISPRv2 / dCas9-VP64-sgRNAs transcriptional activation system. Therefore, the modified LentiCRISPRv2 / dCas9-VP64-sgRNAs vector system can constitutively express the sgRNA and dCas9-VP64 fusion protein, thereby achieving efficient and specific targeting and sustained transcriptional activation of the target gene.

[0005] In summary, the construction of the LentiCRISPRv2 / dCas9-VP64-sgRNAs transcriptional activation system and the preparation of tumor therapeutic agents can be used for clinical targeted therapy of patients with BRD7 low expression, and are expected to provide an important molecular strategy for personalized treatment of cancer patients. Summary of the Invention

[0006] The primary objective of this invention is to provide a method for preparing the LentiCRISPRv2 / dCas9-VP64-sgRNAs transcriptional activation system. The obtained LentiCRISPRv2 / dCas9-VP64-sgRNAs transcriptional activation system can effectively activate the expression of BRD7 in nasopharyngeal carcinoma cells and inhibit cell proliferation and tumor growth in vivo. It can be used for targeted or adjuvant therapy in patients with tumors that have low BRD7 expression.

[0007] Preparation method of LentiCRISPRv2 / dCas9-VP64-sgRNAs transcriptional activation system based on tumor suppressor gene BRD7

[0008] The Cas9 sequence of the LentiCRISPRv2 plasmid was replaced with dCas9-VP64-EGFP (sequence shown in SEQ NO: 1) to obtain the LentiCRISPRv2 / dCas9-VP64 vector; sgRNAs specifically targeting the proximal promoter region of BRD7 were ligated into the LentiCRISPRv2 / dCas9-VP64 vector.

[0009] The preparation method includes the following steps:

[0010] (1) First, the LentiCRISPRv2 vector was digested with enzymes to remove the Cas9 element, and the LentiCRISPRv2-ΔCas9 fragment of Cas9 was removed by gel recovery;

[0011] (2) PCR amplification of the dCas9-VP64-EGFP sequence;

[0012] (3) Ligate the LentiCRISPRv2-ΔCas9 fragment from step (1) with the dCas9-VP64-EGFP fragment amplified in step (2) to construct the LentiCRISPRv2 / dCas9-VP64 vector;

[0013] (4) Design sgRNAs targeting the proximal promoter region of BRD7; anneal single-stranded sgRNAs to form double-stranded sgRNAs;

[0014] (5) Enzymatic digestion of the LentiCRISPRv2 / dCas9-VP64 vector constructed in step (3);

[0015] (6) The long fragment product recovered after enzyme digestion in step (5) is ligated with the double-stranded sgRNA formed by annealing in step (4) to obtain LentiCRISPRv2 / dCas9-VP64-sgRNAs.

[0016] Furthermore,

[0017] Step (1) The LentiCRISPRv2 vector was digested with Xba I and BamH I to remove the Cas9 element, and the LentiCRISPRv2-ΔCas9 fragment was recovered by gel extraction.

[0018] Step (3) Use T4 DNA ligase to ligate the LentiCRISPRv2-ΔCas9 fragment with the dCas9-VP64-EGFP fragment to construct the LentiCRISPRv2 / dCas9-VP64 vector;

[0019] Step (5) Digest the constructed LentiCRISPRv2 / dCas9-VP64 plasmid with BsmBI enzyme;

[0020] Step (6) uses T4 DNA ligase to ligate the long fragment product recovered after digestion of LentiCRISPRv2 / dCas9-VP64 plasmid with double-stranded sgRNA formed by annealing.

[0021] Furthermore,

[0022] The PCR amplification primer sequences in step (2) are as follows:

[0023] Upstream primer sequence: 5'-GCTCTAGAATGAGCCCCAAGAAGAAG-3';

[0024] Downstream primer sequence: 5'-CGCGGATCCTTCTTACTTGTACAGCTCGTCCAT-3'.

[0025] The sgRNA sequence in step (4) is as follows:

[0026]

[0027] Preferably, the sgRNA in step (4) is at least one of sgRNA7-F / R and sgRNA10-F / R.

[0028] A second aspect of the present invention aims to provide a LentiCRISPRv2 / dCas9-VP64-sgRNA transcriptional activation system based on the tumor suppressor gene BRD7, which is prepared by the method described above.

[0029] A third aspect of the present invention aims to provide the application of the LentiCRISPRv2 / dCas9-VP64-sgRNA transcriptional activation system based on the tumor suppressor gene BRD7, for the preparation of formulations for treating tumors.

[0030] The tumors mentioned include nasopharyngeal carcinoma.

[0031] The formulation includes: a lentiviral system, specifically comprising: viral packaging plasmids psPAX2 and pMD2.G, transfection reagent Polyplus, and viral concentrate PEG8000.

[0032] The LentiCRISPRv2 plasmid of this invention was purchased from Addgene #52961.

[0033] In this invention, the constructed LentiCRISPRv2 / dCas9-VP64 vector was transformed into DH5α competent cells, and after resistance screening, single clones were selected. After plasmid extraction, double enzyme digestion was performed to detect whether the dCas9-VP64-EGFP fragment was successfully inserted into the LentiCRISPRv2-ΔCas9 vector.

[0034] The long fragment product recovered from gel digestion of LentiCRISPRv2 / dCas9-VP64 plasmid and the ligation product of double-stranded sgRNA formed by annealing were used for DH5α competent cell transformation, resistance screening, selection of single clones, and sequencing to determine whether the sgRNAs were successfully inserted into the LentiCRISPRv2 / dCas9-VP64 vector.

[0035] Other conventional reagents required for preparing lentiviral supernatant expressing the LentiCRISPRv2 / dCas9-VP64-sgRNA transcriptional activation system include: 5×PEG8000 viral concentrate, physiological saline, DH5α competent cells; LB liquid medium; tryptone; yeast extract; and Tiangen endotoxin-free plasmid small-volume extraction kit.

[0036] The preferred therapeutic formulation comprises a mixture of LentiCRISPRv2 / dCas9-VP64-sgRNA7 and LentiCRISPRv2 / dCas9-VP64-sgRNA10.

[0037] The present invention also provides the application of the described LentiCRISPRv2 / dCas9-VP64-sgRNAs transcriptional activation system in the preparation of formulations for activating the tumor suppressor gene BRD7.

[0038] The tumor therapeutic agents expressing the LentiCRISPRv2 / dCas9-VP64-sgRNAs transcriptional activation system are mainly administered via local tumor injection, but other administration routes to reach the tumor target site are not excluded.

[0039] This invention modifies the CRISPR / Cas9 system to construct a LentiCRISPRv2 / dCas9-VP64-sgRNA transcriptional activation system that specifically targets the proximal promoter region of BRD7. Results showed that this system exhibits excellent BRD7 activation efficacy. Furthermore, screening in various nasopharyngeal carcinoma cells revealed that among the 10 designed sgRNA pairs, sgRNA7 and sgRNA10 showed the most significant activation effects. Both in vitro and in vivo experiments confirmed that this system can inhibit tumor cell proliferation and colony formation, as well as tumor growth in vivo, indicating that it can specifically target the proximal promoter region of BRD7 and thereby activate BRD7 expression. This can compensate for the insufficient BRD7 expression in tumor cells and can be used to prepare formulations for tumor therapy. Therefore, the LentiCRISPRv2 / dCas9-VP64-sgRNA transcriptional activation system prepared in this invention helps to further elucidate the transcriptional repression mechanism of the BRD7 gene in tumor cells, providing a powerful molecular biology tool for personalized treatment of tumor patients with low BRD7 expression, and has significant clinical value and translational application prospects. Attached Figure Description

[0040] Figure 1 Construction of a LentiCRISPRv2 / dCas9-VP64-sgRNA transcriptional activation system targeting the proximal promoter region of BRD7;

[0041] Figure 1 A: Schematic diagram of the construction process of the transcriptional activation system;

[0042] Figure 1 B: The LentiCRISPRv2 vector was double-digested with Xba I and BamH I;

[0043] Figure 1 C: Agarose gel electrophoresis detection of the gel-recovered fragment LentiCRISPRv2-ΔCas9;

[0044] Figure 1 D: Detection of PCR products of the dCas9-VP64-EGFP fragment by agarose gel electrophoresis;

[0045] Figure 1 E: Detection of PCR purification products of the dCas9-VP64-EGFP fragment by agarose gel electrophoresis;

[0046] Figure 1 F: Results of agarose gel electrophoresis detection of the double-digested plasmid LentiCRISPRv2 / dCas9-VP64-EGFP;

[0047] Figure 1G: BsmBI restriction enzyme digestion plasmid LentiCRISPRv2 / dCas9-VP64-EGFP;

[0048] Figure 1 H: Gel recovery of long fragments of BsmBI digested plasmid LentiCRISPRv2 / dCas9-VP64-EGFP;

[0049] Figure 1 I: Sanger sequencing was used to detect whether sgRNAs were successfully ligated into the vector LentiCRISPRv2 / dCas9-VP64-EGFP.

[0050] Figure 2 The effect of the LentiCRISPRv2 / dCas9-VP64-sgRNAs transcriptional activation system on BRD7 expression;

[0051] Figure 2 A: Distribution of sgRNAs (sgRNA1-sgRNA10) in the proximal promoter region of BRD7;

[0052] Figure 2 B-2C: RT-qPCR ( Figure 2 B) and Western Blot ( Figure 2 C) Experimental detection of the effects of the LentiCRISPRv2 / dCas9-VP64-sgRNAs (sgRNA1-10) transcriptional activation system on the expression levels of BRD7 mRNA and protein in nasopharyngeal carcinoma cells 5-8F and CNE2;

[0053] Figure 2 D: Western blotting assays were performed to detect the effects of the LentiCRISPRv2 / dCas9-VP64-sgRNAs (sgRNA1, 3, 5, 7, 8, 10) transcriptional activation system on the expression level of BRD7 protein in nasopharyngeal carcinoma cells 5-8F, CNE2, HK1, and C666-1.

[0054] Figure 3 Effects of LentiCRISPRv2 / dCas9-VP64-sgRNA7 and LentiCRISPRv2 / dCas9-VP64-sgRNA10, alone or in combination, on BRD7 expression;

[0055] Figure 3 A-3B: RT-qPCR ( Figure 3 A) and Western Blot ( Figure 3 B) Experimentally examine the effects of delivery of sgRNA7 and sgRNA10 alone or in combination on the expression levels of BRD7 mRNA and protein.

[0056] Figure 4 The effects of the LentiCRISPRv2 / dCas9-VP64-sgRNA transcriptional activation system on nasopharyngeal carcinoma cell proliferation and colony formation;

[0057] Figure 4 A-4B: CCK-8 ( Figure 4 A) and clonogenesis experiments ( Figure 4 B) Examine the effect of overexpression of the LentiCRISPRv2 / dCas9-VP64-sgRNAs transcriptional activation system (stable expression of sgRNA7 and sgRNA10 alone or in combination) on the proliferation and clonogenic ability of nasopharyngeal carcinoma cells.

[0058] Figure 5 Nude mouse xenograft experiments confirmed that the LentiCRISPRv2 / dCas9-VP64-sgRNAs transcriptional activation system can inhibit the growth of nasopharyngeal carcinoma tumors in vivo;

[0059] Figure 5 A: Tumor growth curves of nude mice in each group, with data recorded every 2 days;

[0060] Figure 5 B: Nude mice were sacrificed, tumors were dissected, and the effects of the LentiCRISPRv2 / dCas9-VP64-sgRNAs transcriptional activation system on the growth of transplanted tumors were observed.

[0061] Figure 5 C: Weigh the transplanted tumors in the control group and each experimental group;

[0062] Figure 5 D: Western blotting was used to detect the protein expression of BRD7 in tumors of nude mice in each group.

[0063] Figure 6 Lentiviral delivery of the BRD7 transcriptional activation system exerts antitumor effects in vivo;

[0064] Figure 6 A: Growth curves of xenograft tumors in nude mice treated with lentivirus, with data recorded every 2 days;

[0065] Figure 6 B: Sacrifice nude mice, dissect tumors, and observe the effect of the lentivirus-mediated transcriptional activation system on tumor growth;

[0066] Figure 6 C: Weigh the tumors in the control group and each lentivirus-mediated treatment group;

[0067] Figure 6D: Western blotting was used to detect the protein expression of BRD7 in tumors of nude mice in each group. Detailed Implementation

[0068] The present invention will be further described below with reference to specific embodiments, but not in a way that limits the invention.

[0069] The tumor cell lines used in this invention were all preserved by the Molecular Genetics Laboratory of the Cancer Institute of Central South University (purchased from the Biological Cell Laboratory of the Advanced Research Center of Central South University). The cell culture conditions were: DMEM liquid medium containing 10% fetal bovine serum and 1% penicillin antibiotics, and adherent growth in a constant temperature incubator at 37°C and 5% CO2 concentration.

[0070] The primers used in this invention were designed on the NCBI website, and the final primer synthesis was commissioned to Sangon Biotech.

[0071] Example 1: Construction of a transcriptional activation system targeting the proximal promoter of BRD7, LentiCRISPRv2 / dCas9-VP64-sgRNAs.

[0072] 1. Materials and Methods

[0073] (1) Replace the Cas9 sequence in the LentiCRISPRv2 plasmid with dCas9-VP64-EGFP.

[0074] ① The specific construction process is as follows Figure 1 As shown in Figure A, the LentiCRISPRv2 vector (purchased from Addgene #52961) was first digested with Xba I and BamH I at 37°C to excise the Cas9 element. The LentiCRISPRv2-ΔCas9 fragment was then recovered using a gel extraction kit.

[0075] ② PCR amplification of the dCas9-VP64-EGFP sequence (see SEQ ID NO.1), Table 1 shows the PCR primer sequences.

[0076]

[0077] After PCR, agarose gel electrophoresis was performed, and the PCR products were then purified and recovered using a purification kit.

[0078] ③ The dCas9-VP64-EGFP fragment was digested with Xba I and BamHI, purified, and then subjected to agarose gel electrophoresis.

[0079] The lentiCRISPRv2-ΔCas9 fragment, which was double-digested with Xba I and BamH I, was ligated with the dCas9-VP64-EGFP fragment at 16°C overnight using T4 DNA ligase to obtain the LentiCRISPRv2 / dCas9-VP64 vector.

[0080] ⑤ Transform DH5α competent cells with LentiCRISPRv2 / dCas9-VP64 plasmid, and incubate the plates upside down in a 37°C incubator for 12-16 h. Then pick single clones and incubate them in a 37°C shaker at 250 rpm for 12-16 h.

[0081] ⑥ Extract the plasmid and perform double enzyme digestion for identification. Send the correctly digested single clones for sequencing. If the sequencing results are completely consistent with the dCas9-VP64-EGFP sequence, it indicates that the LentiCRISPRv2 / dCas9-VP64 plasmid has been successfully constructed.

[0082] (2) sgRNAs targeting the proximal promoter region of BRD7 were ligated into the vector LentiCRISPRv2 / dCas9-VP64

[0083] ① Ten pairs of sgRNAs were designed targeting the proximal promoter sequence of BRD7, and their sequences are shown in Figure 2:

[0084]

[0085] The synthesized sgRNA powder was centrifuged at 5000 rpm for 5 min and diluted with sterile water to 100 µM.

[0086] ② Anneal the above 10 pairs of single-stranded sgRNAs to form double-stranded sgRNAs.

[0087] ③ The constructed LentiCRISPRv2 / dCas9-VP64 plasmid was digested with BsmBI, followed by agarose gel electrophoresis to recover the long fragment.

[0088] The recovered products from step ③ were ligated with the double-stranded sgRNAs formed by annealing in step ② using T4 DNA ligase. The ligation products were transformed into E. coli DH5α competent cells, and single clones were selected for sequencing verification.

[0089] 2. Experimental Results and Conclusions

[0090] The Cas9 element was excised by double digestion of the LentiCRISPRv2 vector with Xba I and BamH I. Figure 1 B), then the long fragment was recovered using gel electrophoresis, which is the LentiCRISPRv2-ΔCas9 fragment ( Figure 1 C). Simultaneously, the PCR-amplified dCas9-VP64-EGFP sequence showed the correct target band location and was the expected size. Figure 1 D), the purified and recovered bands were also clear and specific ( Figure 1 E). The lentiCRISPRv2-ΔCas9 fragment, after double digestion with Xba I and BamHI, was ligated with the dCas9-VP64-EGFP fragment. After transformation into *E. coli*, single clones were picked from plates, and identification and sequencing confirmed that single clone 1 met expectations. Figure 1 The presence of F indicates successful construction of the LentiCRISPRv2 / dCas9-VP64 plasmid. Subsequently, the constructed LentiCRISPRv2 / dCas9-VP64 plasmid was digested with BsmBI, and two specific bands were observed (F). Figure 1 G), long fragments recovered from the gel, with correctly and specifically positioned bands (G), Figure 1 H). Finally, the gel-recovered product was ligated with 10 pairs of annealed double-stranded sgRNAs, transformed into E. coli, and single clones were picked from plates for sequencing verification. Figure 1 Sequencing results showed that sgRNAs (sgRNA1-10) were successfully inserted into the LentiCRISPRv2 / dCas9-VP64 vector. In summary, the LentiCRISPRv2 / dCas9-VP64-sgRNA transcriptional activation system targeting the proximal promoter of BRD7 was successfully constructed.

[0091] Example 2: Screening for sgRNAs that can effectively activate BRD7 expression in nasopharyngeal carcinoma cells.

[0092] 1. Materials and Methods

[0093] (1) RT-qPCR and Western Blot experiments were used to detect the effect of the sgRNA1-10-driven dCas9-VP64 transcriptional activation system on BRD7 expression in nasopharyngeal carcinoma cells.

[0094] ① Nasopharyngeal carcinoma cells 5-8F and CNE2 were evenly seeded into 6-well plates and cultured in an incubator at 37°C.

[0095] ② When the cells reach 60-80% confluence, take an enzyme-free, sterile 0.6 mL EP tube and add 200 μL of jetPRIME® Buffer, 2 μg of LentiCRISPRv2 / dCas9-VP64-sgRNAs (sgRNA1-10) transcription activation system, and 4 μL of Polyplus transfection reagent (Polyplus, France; model: 101000046) (plasmid DNA mass: transfection reagent volume = 1:2). After vortexing, incubate at room temperature for 15 min. Then, gently add the incubated transfection mixture dropwise to the cell culture medium, gently shake the 6-well plate to mix the medium and transfection mixture evenly, and then return the cells to an incubator at 37°C and 5% CO2 for further culture. Collect cells after 36-48 h for subsequent experiments.

[0096] ③ Wash the cells 2-3 times with 1×PBS pre-cooled at 4℃.

[0097] RNA and protein were extracted separately and subjected to RT-qPCR and Western Blot experiments.

[0098] ⑤ Based on the results of RT-qPCR and Western Blot experiments, multiple sgRNAs that significantly activated BRD7 in both nasopharyngeal carcinoma cells 5-8F and CNE2 were screened.

[0099] (2) Western blot analysis was conducted to detect the effect of the dCas9-VP64 transcriptional activation system, which was initially screened and had activating effects, on BRD7 expression in different types of nasopharyngeal carcinoma cells.

[0100] ① Different types of nasopharyngeal carcinoma cells (5-8F, CNE2, HK1 and C666-1) were evenly seeded into 6-well plates and cultured in an incubator at 37°C.

[0101] ② When the cells reach 60-80% confluence, take an enzyme-free, sterile 0.6 mL EP tube and add 200 μL of jetPRIME® Buffer, 2 μg of LentiCRISPRv2 / dCas9-VP64-sgRNAs (sgRNA1, 3, 5, 7, 8, 10) transcription activation system, and 4 μL of polyplus transfection reagent (plasmid DNA mass: transfection reagent volume = 1:2). After vortexing, incubate at room temperature for 15 min. Then, gently add the incubated transfection mixture dropwise to the cell culture medium, gently shake the 6-well plate to mix the medium and transfection mixture evenly, and then return the cells to an incubator at 37℃ and 5% CO2 for further culture. Collect cells for subsequent experiments after 36-48 h.

[0102] ③ Wash the cells 2-3 times with 1×PBS pre-cooled at 4℃.

[0103] Total cellular protein was extracted and subjected to Western Blot experiments. Based on the results of the Western Blot experiments, two sgRNAs that were most effective in activating BRD7 were screened in different types of nasopharyngeal carcinoma cells (5-8F, CNE2, HK1, and C666-1).

[0104] 2. Experimental Results and Conclusions

[0105] Transiently transfecting nasopharyngeal carcinoma cells 5-8F and CNE2 with the sgRNA1-10-guided dCas9-VP64 system, cellular RNA and total protein were collected. The effect of the LentiCRISPRv2 / dCas9-VP64-sgRNAs (sgRNA1-10) transcriptional activation system on BRD7 expression was detected by RT-qPCR and Western blotting. The results showed that, compared with the control group, the dCas9-VP64 system guided by all six pairs of sgRNAs (sgRNA1, 3, 5, 7, 8, and 10) could promote the expression of BRD7 mRNA and protein in nasopharyngeal carcinoma cells to varying degrees (Figure 2B, C). To further screen for sgRNAs with better activation effects, the dCas9-VP64 system guided by the above six pairs of sgRNAs was transiently transfected into four types of nasopharyngeal carcinoma cells: 5-8F, CNE2, HK1, and C666-1. Western blot experiments showed that both sgRNA7 and sgRNA10 could effectively transcribe and activate the expression of BRD7 in the above four types of nasopharyngeal carcinoma cells, with a wide range of applications, and the effect was more pronounced in 5-8F and HK1 (Figure 2D).

[0106] Example 3: The optimal combination of two sgRNAs, sgRNA7 and sgRNA10, to guide the dCas9-VP64 system can synergistically promote BRD7 transcriptional activity and expression.

[0107] 1. Materials and Methods

[0108] (1) Construct a nasopharyngeal carcinoma cell line that stably expresses the LentiCRISPRv2 / dCas9-VP64-sgRNA7 & sgRNA10 transcriptional activation system.

[0109] ① When the density of 293T cells is about 60%~80%, perform plasmid transfection.

[0110] ② Take a sterile 1.5 mL EP tube without enzymes, add 20 μg of plasmid and 40 μL of polyplus to the EP tube according to the ratio of target plasmid: psPAX2: pMD2.G (purchased from Addgene) = 4:3:1, vortex to mix, briefly incubate at room temperature for 15 min.

[0111] ③ Wash the cells 2-3 times with D-Hanks, add 7 mL of fresh complete culture medium, add the incubated mixture to the corresponding culture dish, and place it in an incubator to continue culturing.

[0112] After 12 h of culture, add 6-8 mL of fresh culture medium and continue culturing. Collect lentivirus supernatant at 48 h and 72 h after transfection, respectively.

[0113] ⑤ Virus concentration: Transfer the collected viral supernatant to a 15 mL centrifuge tube, centrifuge at 3000 rpm for 10 min at 4 °C, filter into a 50 mL centrifuge tube, add 5×PEG8000 to the viral solution, invert to mix, place on ice, invert 5 times every 30 min, and incubate overnight at 4 °C. Subsequently, centrifuge at 4000 g for 20 min at 4 °C, aspirate the supernatant, resuspend the viral pellet in 200-400 μL of pre-chilled PBS, and immediately aliquot the collected virus into 1.5 mL EP tubes.

[0114] ⑥ Infecting target cells: Nasopharyngeal carcinoma cells 5-8F and HK1 were seeded in 10 cm culture dishes. When the cells grew to a cell density of 30%~50%, polybrene (8 μg / mL) and virus solution were added to the culture medium. After infection, the cell status was carefully observed. After the cells reached confluence, they could be passaged. When the cells reached the logarithmic growth phase, secondary infection could be performed.

[0115] ⑦ Antibiotic screening: After secondary infection, the medium was changed to complete medium and cultured for 48 h. Antibiotic screening was then performed. The constructed plasmid contained the puro selection marker, so 1 mg / mL of puromycin was added to the infected cells for screening. The cells obtained were stable cell lines that stably expressed the LentiCRISPRv2 / dCas9-VP64-sgNC and LentiCRISPRv2 / dCas9-VP64-sgRNA7 & sgRNA10 transcriptional activation systems.

[0116] ⑧ Validation of stable cell lines: Since the constructed vector system is fluorescently labeled, it can be observed with a fluorescence microscope. After the fluorescence efficiency reaches 100%, the antibiotic maintenance concentration is reduced to continue screening and amplification of infected cells. At the same time, the expression of BRD7 is detected after the cells are collected.

[0117] (2) RT-qPCR and Western Blot experiments were used to detect the effects of single or combined delivery of LentiCRISPRv2 / dCas9-VP64-sgRNA7 & sgRNA10 on BRD7 expression in nasopharyngeal carcinoma cells.

[0118] ① Nasopharyngeal carcinoma cells 5-8F and HK1, which stably express the LentiCRISPRv2 / dCas9-VP64-sgRNAs system, were uniformly seeded in 6-well plates and cultured in an incubator at 37°C.

[0119] ② A total of four groups were set up, namely dCas9-VP64-sgNC, dCas9-VP64-sg7, dCas9-VP64-sg10, and dCas9-VP64-sg7+10. The co-expression group was achieved by transiently transfecting dCas9-VP64-sg10 into cells that stably expressed dCas9-VP64-sg7.

[0120] ③ Collect cells for subsequent experiments 36-48 h after transfection with the co-expression group.

[0121] Wash cells 2-3 times with 1×PBS pre-cooled to 4°C.

[0122] ⑤ Extract RNA and protein separately, and perform RT-qPCR and Western Blot experiments.

[0123] 2. Experimental Results and Conclusions

[0124] Nasopharyngeal carcinoma cells 5-8F and HK1 were infected with lentiviral supernatant to establish cell lines stably expressing dCas9-VP64-sgRNA7 and dCas9-VP64-sgRNA10, with cells stably expressing dCas9-VP64-sgNC serving as a control. RT-qPCR and Western Blot results showed that, compared with the control group, the mRNA and protein expression levels of BRD7 were significantly increased in nasopharyngeal carcinoma cells stably expressing dCas9-VP64-sgRNA7 or dCas9-VP64-sgRNA10. Figure 3 (A, B). Simultaneously, it was found that compared to cells stably expressing dCas9-VP64-sgRNA7 or dCas9-VP64-sgRNA10, the combined delivery of sgRNA7 and sgRNA10 exhibited a synergistic effect, resulting in a more significant activation of BRD7 (Figure 3A, B). In summary, the LentiCRISPRv2 / dCas9-VP64-sgRNAs transcriptional activation system (sgRNA7 & sgRNA10) can specifically target the proximal promoter region of BRD7 to activate BRD7 expression; and the combined delivery of sgRNA7 & sgRNA10 has a synergistic effect, resulting in a more significant activation of BRD7, providing a basis for precision tumor treatment.

[0125] Example 4: In vitro experiments confirmed that the LentiCRISPRv2 / dCas9-VP64-sgRNAs transcriptional activation system can inhibit the proliferation and colony formation of BRD7-low-expressing tumor cells.

[0126] 1. Materials and Methods

[0127] (1) CCK-8 cell proliferation experiment

[0128] ① The cell line stably expressing the LentiCRISPRv2 / dCas9-VP64-sgRNAs system (obtained according to the infection method in Example 3) was diluted at 1000 cells / 100 μL of culture medium per well and seeded in 96-well plates with 5 replicates per group (for 6 days). After seeding, the plates were incubated at 37°C.

[0129] ② After the cells adhere to the culture medium, add CCK-8 reagent to each well at a ratio of 1:10 (CCK-8:culture medium volume) and incubate in an incubator for 2 hours.

[0130] ③ The absorbance of each group was measured using an ELISA reader and recorded as the data for day 0. Thereafter, measurements were taken every 24 hours for a total of 6 days.

[0131] After all data were measured, the data from day 0 were standardized, and the cell proliferation rate was statistically analyzed.

[0132] (2) Cloning experiment

[0133] ① Nasopharyngeal carcinoma cell lines stably expressing the LentiCRISPRv2 / dCas9-VP64-sgRNAs system were diluted at 1000 cells / 2 mL of culture medium per well, with 3 replicates per group, and seeded in 6-well plates and cultured at 37°C for 7-14 days.

[0134] ②When the cells have multiplied to a clone of more than 50 cells, wash the cells three times with 1×PBS, each time for 5 minutes.

[0135] ③ Fixation: Cells were fixed with 4% paraformaldehyde at room temperature for 20 min.

[0136] Staining: Add 1 mL of crystal violet to each well, stain for 5-10 min, rinse with distilled water until no crystal violet residue remains, air dry and take a picture.

[0137] ⑤ Count the number of effective cell clones in each group and analyze the data.

[0138] 2. Experimental Results and Conclusions

[0139] CCK-8 assays and colony formation assays revealed that, compared with the control group, stable expression of dCas9-VP64-sgRNA7 or dCas9-VP64-sgRNA10 inhibited the proliferation and colony formation ability of nasopharyngeal carcinoma cells, and the inhibitory effect was more significant in the co-expression group of dCas9-VP64-sgRNA7 & sgRNA10. Figure 4 (A, B). In summary, the transcriptional activation system driven by sgRNA7 and sgRNA10 (LentiCRISPRv2 / dCas9-VP64-sgRNA7 & sgRNA10) can inhibit the proliferation and colony formation of nasopharyngeal carcinoma cells in vitro, and the inhibitory effect of co-expression of both is more significant, indicating that this gene editing system can successfully activate the expression of BRD7 and promote the anti-tumor effect of BRD7.

[0140] Example 5: In vivo experiments confirmed that the LentiCRISPRv2 / dCas9-VP64-sgRNAs transcriptional activation system can inhibit tumor growth in vivo.

[0141] 1. Materials and Methods

[0142] (1) Constructing a subcutaneous xenograft model in nude mice

[0143] ① 24 female BALB / C nude mice, 4 weeks old, were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd., and raised under specific pathogen-free (SPF) conditions at the Department of Zoology, Central South University.

[0144] ② The cells inoculated were nasopharyngeal carcinoma cells 5-8F. The control group stable cells (LentiCRISPR / dCas9-VP64-sgNC) and the experimental group stable cells constructed in the materials and methods section of Example 3 were used, namely: the group stably expressing LentiCRISPRv2 / dCas9-VP64-sgRNA7, the group stably expressing LentiCRISPRv2 / dCas9-VP64-sgRNA10, and the group co-expressing LentiCRISPRv2 / dCas9-VP64-sgRNA7 & sgRNA10.

[0145] ③Each group contains 6 nude mice, and each mouse is prepared with 4×10 6 150 μL of cell suspension was injected into the subcutaneous area of ​​the nude mouse's right forelimb, slightly above the axilla, when the nude mouse was about 5 weeks old.

[0146] ④ After the tumor grows out, record the length and width of the tumor every 2 days and calculate the growth volume of the tumor.

[0147] ⑤ After the tumors have grown to a certain size, the mice were anesthetized with isoflurane, photographed, and then the nude mice in each group were sacrificed. The tumors were removed, photographed, and the weight of all tumors was measured.

[0148] (2) Western blot experiment to detect the expression of BRD7 in tumors of nude mice in each group.

[0149] ① Take a small portion of the dissected tumor tissue, place it in a pre-cooled grinder, add lysis buffer (add protease inhibitor and phosphatase inhibitor), and grind it thoroughly on ice.

[0150] ② Transfer the homogenized tissue suspension to a 1.5 mL EP tube and incubate on ice for 30 minutes, vortexing every 10 minutes to ensure complete lysis.

[0151] ③ Centrifuge at 4°C and 13,000 rpm for 15 min, and collect the protein supernatant.

[0152] ④ Proteins of equal mass after denaturation following BCA method for protein concentration determination.

[0153] ⑤ Electrophoresis: Add protein marker and an equal amount of protein sample in the order of sample loading, electrophoresis at 80 V for 40 min, then at 120 V for 60 min.

[0154] ⑥ Transfer: Peel off the gel and immerse it in pre-cooled transfer buffer. Immerse the PVDF membrane in methanol until it becomes transparent, then transfer it to the transfer buffer for later use. Then, place filter paper, gel, PVDF membrane, and filter paper in sequence above the transfer clamp, avoiding the formation of air bubbles between the gel and PVDF membrane. Transfer at 120 V for 2 hours. Keep the entire process on ice.

[0155] ⑦ Blocking: Block with 5% skim milk at room temperature at low speed for 1 hour.

[0156] ⑧ Incubate with primary antibody: Place the membrane in an antibody incubation box, add the prepared primary antibody working solution, and incubate overnight at 4°C.

[0157] ⑨ Wash with primary antibody: Wash the membrane with 1×TBST at high speed for 15 min, and repeat 3 times.

[0158] ⑩ Secondary antibody incubation: Prepare the appropriate secondary antibody using 5% skim milk. Place the membrane face up in the antibody incubation chamber, add an appropriate amount of secondary antibody solution, and incubate at 37°C for 1 hour.

[0159] Wash the secondary antibody: Same as step 9.

[0160] Luminescence: Dry the surface of the PVDF membrane, evenly add luminescent liquid to the surface of the membrane, and save the image after luminescence.

[0161] 2. Experimental Results and Conclusions

[0162] By constructing a nasopharyngeal carcinoma xenograft model stably infected with the LentiCRISPRv2 / dCas9-VP64-sgRNAs transcriptional activation system, it was found that compared with the control group, stable expression of LentiCRISPRv2 / dCas9-VP64-sgRNA7 or LentiCRISPRv2 / dCas9-VP64-sgRNA10 significantly inhibited tumor growth rate and reduced tumor weight. Furthermore, the co-expression of dCas9-VP64-sgRNA7 and sgRNA10 exhibited a synergistic effect, resulting in a more significant inhibitory effect. Figure 5 AC). Further collection of xenograft tumor fragments and Western blot analysis revealed that, compared with the control group, stable expression of LentiCRISPRv2 / dCas9-VP64-sgRNA7 or LentiCRISPRv2 / dCas9-VP64-sgRNA10 significantly activated BRD7 expression, and the co-expression group of dCas9-VP64-sgRNA7 & sgRNA10 exhibited a synergistic effect, with a more significant activation effect. Figure 5D). In summary, dCas9-VP64-sgRNA7 and dCas9-VP64-sgRNA10 can significantly activate BRD7 expression and thus inhibit tumor growth in vivo, and the combined delivery of the two drugs has a more significant BRD7 activation effect and tumor inhibition effect in vivo.

[0163] Example 6: The BRD7 transcriptional activation system delivered by lentivirus exerts antitumor effects in vivo.

[0164] 1. Materials and Methods

[0165] (1) Constructing a subcutaneous xenograft model in nude mice

[0166] ① Thirty female BALB / c nude mice, 4 weeks old, were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd., and raised under specific pathogen-free (SPF) conditions at the Department of Zoology, Central South University.

[0167] ② The cells inoculated were nasopharyngeal carcinoma cells 5-8F, each containing 4×10⁻⁶ cells. 6 150 μL of cell suspension was injected into the subcutaneous area of ​​the nude mouse's right forelimb, slightly above the axilla, when the nude mouse was about 5 weeks old.

[0168] ③ When the tumor grows to a size of 40~50mm 3 At that time, tumor-bearing mice were randomly divided into 5 groups, with 6 mice in each group. In the treatment group, 100 µL of a 1×10⁻⁶ concentration was injected into the tumor. 8 Mice stably expressing dCas9-VP64-sgRNA7, dCas9-VP64-sgRNA10, and a lentivirus combining both (target plasmid: psPAX2:pMD2.G=4:3:1) at TU / mL were used as the negative control group, while nude mice stably expressing dCas9-VP64-sgRNANC served as the blank control group. Multiple intratumoral injections were administered every 4 days for a total of three injections.

[0169] ④ Record the length and width of the tumor every two days and calculate the growth volume of the tumor.

[0170] ⑤ After the tumors have grown to a certain size, take photos, euthanize each group of nude mice, remove the tumors, take photos, and measure the weight of all tumors.

[0171] (2) Western blot experiment to detect the expression of BRD7 in tumors of nude mice in each group.

[0172] The specific steps are the same as in Example 5, 1. Materials and Methods (2).

[0173] 2. Experimental Results and Conclusions

[0174] Lentivirals stably expressing dCas9-VP64-sgRNA7 & sgRNA10 were used to evaluate their antitumor effects in xenograft tumor models. Results are as follows: Figure 6 As shown in AC, compared with the lentiviral control group stably expressing dCas9-VP64-sgRNANC, the tumor growth rate and tumor weight of the lentiviral treatment group stably expressing dCas9-VP64-sgRNA7 & sgRNA10 were significantly reduced, and the tumor suppression effect was more significant in the combined treatment group. Subsequently, tumor tissues from four nude mice were randomly selected from each group and proteins were extracted. Western blot results are shown below. Figure 6 As shown in Figure D, compared with the negative control group LV-dCas9-VP64-sgRNANC, the BRD7 protein expression level was increased in the lentiviral treatment groups guided by sgRNA7 and sgRNA10, and the combined treatment group showed a synergistic effect in increasing BRD7 expression. Figure 6 (D). However, compared with the blank control group, no significant change was observed in the expression of BRD7 in the tumor tissue of the negative control group (LV-dCas9-VP64-sgRNANC), further confirming the targeting of this transcriptional activation system to BRD7. In summary, lentiviruses stably expressing the dCas9-VP64-sgRNA7 & sgRNA10 transcriptional activation system can effectively deliver this system and exert a durable and effective antitumor effect in nasopharyngeal carcinoma xenografts by activating BRD7 expression through transcription.

[0175] SEQ NO: 1 dCas9-VP64-EGFP sequence:

Claims

1. A method for preparing a LentiCRISPRv2 / dCas9-VP64-sgRNA transcriptional activation system based on the tumor suppressor gene BRD7, characterized in that, The Cas9 sequence of the LentiCRISPRv2 plasmid was replaced with dCas9-VP64-EGFP to obtain the LentiCRISPRv2 / dCas9-VP64 vector; sgRNAs specifically targeting the proximal promoter region of BRD7 were ligated into the LentiCRISPRv2 / dCas9-VP64 vector.

2. The preparation method according to claim 1, characterized in that, Includes the following steps: (1) First, the LentiCRISPRv2 vector was digested with enzymes to remove the Cas9 element, and the LentiCRISPRv2-ΔCas9 fragment of Cas9 was removed by gel recovery; (2) PCR amplification of the dCas9-VP64-EGFP sequence; (3) Ligate the LentiCRISPRv2-ΔCas9 fragment from step (1) with the dCas9-VP64-EGFP fragment amplified in step (2) to construct the LentiCRISPRv2 / dCas9-VP64 vector; (4) Design sgRNAs targeting the proximal promoter region of BRD7; anneal single-stranded sgRNAs to form double-stranded sgRNAs; (5) Enzymatic digestion of the LentiCRISPRv2 / dCas9-VP64 vector constructed in step (3); (6) The long fragment product recovered after enzyme digestion in step (5) is ligated with the double-stranded sgRNA formed by annealing in step (4) to obtain LentiCRISPRv2 / dCas9-VP64-sgRNAs.

3. The preparation method according to claim 2, characterized in that, Step (1) The LentiCRISPRv2 vector was digested with Xba I and BamH I to remove the Cas9 element, and the LentiCRISPRv2-ΔCas9 fragment was recovered by gel extraction. Step (3) Use T4 DNA ligase to ligate the LentiCRISPRv2-ΔCas9 fragment with the dCas9-VP64-EGFP fragment to construct the LentiCRISPRv2 / dCas9-VP64 vector; Step (5) Digest the constructed LentiCRISPRv2 / dCas9-VP64 plasmid with BsmBI enzyme; Step (6) uses T4 DNA ligase to ligate the long fragment product recovered after digestion of LentiCRISPRv2 / dCas9-VP64 plasmid with double-stranded sgRNA formed by annealing.

4. The preparation method according to claim 2, characterized in that, The PCR amplification primer sequences in step (2) are as follows: Upstream primer sequence: 5'-GCTCTAGAATGAGCCCCAAGAAGAAG-3'; Downstream primer sequence: 5'-CGCGGATCCTTCTTACTTGTACAGCTCGTCCAT-3'.

5. The preparation method according to claim 2, characterized in that, The sgRNA sequence in step (4) is as follows: sgRNA1-F 5'-CACCGGCACCACCTCCGGTCGCCAG-3'; sgRNA1-R 5'-AAACCTGGCGACCGGAGGTGGTGCC-3'; sgRNA2-F 5'-CACCGGCGCGCCGTCTTCTCGAGAG-3'; sgRNA2-R 5'-AAACCTCTCGAGAAGACGGCGCGCC-3'; sgRNA3-F 5'-CACCGCGGCCGGAGCCCGAGAGCGG-3'; sgRNA3-R 5'-AAACCCGCTCTCGGGCTCCGGCCGC-3'; sgRNA4-F 5'-CACCGGGTCCCGGGCCAGGCGAGCGG-3'; sgRNA4-R 5'-AAACCCGCTCGCCTGGCCCGGACCC-3'; sgRNA5-F 5'-CACCGAAAGACGAGAGTCTGAGCGG-3'; sgRNA5-R 5'-AAACCCGCTCAGACTCTCGTCTTTC-3'; sgRNA6-F 5'-CACCGCCGCGAGACCCCGCGCCGCG-3'; sgRNA6-R 5'-AAACCGCGGCGCGGGGTCTCGCGGC-3'; sgRNA7-F 5'-CACCGTCCGGTCCGGGCCAGGCGAG-3'; sgRNA7-R 5'-AAACCTCGCCTGGCCCGGACCGGAC-3'; sgRNA8-F 5'-CACCGCGCGCCGCCCCCCTGCCTCG-3'; sgRNA8-R 5'-AAACCGAGGCAGGGGGGCGGCGCGC-3'; sgRNA9-F 5'-CACCGCGCGCCAGGCCCAGGCCGTG-3'; sgRNA9-R 5'-AAACCACGGCCTGGGCCTGGCGCGC-3'; sgRNA10-F 5'-CACCGACCGGGGCCCGGTCGGACAT-3'; sgRNA10-R 5'-AAACATGTCCGACCGGGCCCCGGTC-3'.

6. The preparation method according to claim 2, characterized in that, In step (4), the sgRNA is at least one of sgRNA7-F / R and sgRNA10-F / R.

7. A LentiCRISPRv2 / dCas9-VP64-sgRNA transcriptional activation system based on the tumor suppressor gene BRD7, characterized in that, It is prepared by the method described in any one of claims 1-6.

8. The application of the LentiCRISPRv2 / dCas9-VP64-sgRNA transcriptional activation system based on the tumor suppressor gene BRD7 as described in claim 7, characterized in that, Used to prepare formulations for treating tumors.

9. The application according to claim 8, characterized in that, The tumors mentioned include nasopharyngeal carcinoma.

10. The application according to claim 8, characterized in that, The formulation includes: a lentiviral system, specifically including: viral packaging plasmids psPAX2 and pMD2.G, transfection reagent Polyplus, and viral concentrate PEG8000.

Images