A shRNA transgenic recombinant plasmid promoting tumor cell cavitation and its application

Abstract

The present invention provides a shRNA transgenic recombinant plasmid that promotes tumor cell cavitation and its application. The transgenic recombinant plasmid is a shRNA expression gene inserted into an AAV2 plasmid, and the nucleotide sequence of the shRNA expression gene is SEQ ID NO.1; the application of the shRNA transgenic recombinant plasmid in the preparation of anti-tumor drugs; the shRNA transgenic recombinant plasmid provided by the present invention can promote tumor cell cavitation, and the prognosis of cavitary RB is better. Therefore, the present invention provides a new treatment method and therapeutic drug for the treatment of cancer, especially retinoblastoma, improves the effectiveness of treatment, reduces the rate of enucleation, and improves the visual prognosis of patients.

Description

Technical Field

[0001] The invention relates to a shRNA transgenic recombinant plasmid promoting tumor cell cavitation and application thereof, belonging to the technical field of biomedicine. Background Art

[0002] Retinoblastoma (RB) is the most common ocular malignancy in children. The "secondary mutation hypothesis" holds that the two alleles of the RB1 tumor suppressor gene are successively inactivated during retinal development, leading to RB1 － / － However, the latest research found that about 2% of RB patients have highly amplified MYCN oncogene without RB1 gene mutation, so RB1 + / + MYCN A As a new pathogenic genotype of RB, this type of RB is more common than the classic RB1 － / － RB type has early onset, strong invasiveness and low eye preservation rate.

[0003] Tumor cell cavitation is due to the incomplete development of tumor blood vessels. When the central part of the tumor lacks nutrition, necrosis will occur, and when the necrotic material is absorbed, a cavity will be formed. Tumor cavitation is often seen in malignant tumors such as retinoblastoma and liver cancer, but the existing technology has not yet analyzed the prognosis of cavitary RB.

[0004] Viral vectors are the main gene delivery tools for gene therapy. Lentivirus vectors can effectively deliver genes, but lentiviruses have a high host genome integration rate and high safety risks in clinical applications. In recent years, studies have found that adeno-associated viruses (AAVs) can widely infect tissue cells, have low immunogenicity and integration ability, and have high delivery efficiency and safety.

[0005] Adenovirus is a linear double-stranded DNA non-enveloped virus that can infect both dividing and non-dividing cells and has epithelial cell tropism. Recombinant adenovirus vector is a tool virus vector developed based on adenovirus, which has the characteristics of wide host range, strong immunogenicity, large gene capacity, no integration with the genome, and transient expression. Summary of the invention

[0006] The present invention provides a shRNA transgenic recombinant plasmid that promotes tumor cell cavitation and its application. The shRNA transgenic recombinant plasmid provided by the present invention can promote tumor cell cavitation, and the prognosis of cavitary RB is better. Therefore, the present invention provides a new treatment method and therapeutic drug for the treatment of cancer, especially retinoblastoma, improves the effectiveness of treatment, reduces the rate of enucleation, and improves the visual prognosis of patients.

[0007] To achieve this object, the present invention provides the following technical solutions:

[0008] The first aspect of the present invention provides a shRNA transgenic recombinant plasmid that promotes tumor cell cavitation. The transgenic recombinant plasmid is a shRNA expression gene inserted into an AAV2 plasmid, and the nucleotide sequence of the shRNA expression gene is SEQ ID NO.1.

[0009] SEQ ID NO. 1 5'-GCCAGTATTAGACTGGAAGTT-3'.

[0010] Preferably, the shRNA expression gene is inserted at the end of the U6 promoter, and the front end of the U6 promoter and the end of the shRNA expression gene are connected to the ubiquitin ligase E3A.

[0011] Preferably, the tumor includes retinoblastoma, neuroblastoma, and hepatoma.

[0012] The present application has proved through experiments that RB PDX has more cavities after using shRNA transgenic recombinant plasmid. And shRNA transgenic recombinant plasmid inhibits MYCN expression of RB PDX. In the prior art, MYCN expression is related to tumors such as retinoblastoma, neuroblastoma, and hepatoma. Therefore, it is speculated that the shRNA transgenic recombinant plasmid of the present application can be used to treat retinoblastoma, neuroblastoma, hepatoma, etc.

[0013] Neuroblastoma (NB) originates from the postganglionic sympathetic nerves and is the most common extracranial malignant solid tumor in children. According to the 2014 US childhood cancer statistics, the incidence of NB in ​​children under 15 years old is 10.54 / million. The biggest feature of NB is that it is prone to distant metastasis in the early stage of the disease and has a high degree of malignancy. Age, International Neuroblastoma Staging System (INSS), tumor differentiation, MYCN amplification, histological type, etc. are all important factors affecting the prognosis of children. Among them, MYCN gene amplification is an independent factor for poor prognosis.

[0014] Preferably, the tumor is RB1 + / + MYCN A Retinoblastoma.

[0015] The second aspect of the present invention provides the use of the shRNA transgenic recombinant plasmid of the present invention in the preparation of anti-tumor drugs.

[0016] Preferably, the anti-tumor drug can promote tumor cell cavitation and / or inhibit the expression level of tumor MYCN protein.

[0017] Preferably, the anti-tumor drug is an adenovirus packaging plasmid.

[0018] Preferably, the administration method of the anti-tumor drug includes injection.

[0019] Preferably, the shRNA transgenic recombinant plasmid comprises a shRNA expression gene inserted into an AAV2 plasmid, and the nucleotide sequence of the shRNA expression gene is SEQ ID NO.1.

[0020] SEQ ID NO. 1 5'-GCCAGTATTAGACTGGAAGTT-3'.

[0021] Preferably, the shRNA expression gene is inserted at the end of the U6 promoter, and the front end of the U6 promoter and the end of the shRNA expression gene are connected to the ubiquitin ligase E3A.

[0022] Preferably, the tumor includes retinoblastoma, neuroblastoma, and hepatoma.

[0023] Preferably, the tumor is RB1 + / + MYCN A Retinoblastoma.

[0024] In summary, for RB1 + / + MYCN A This type of retinoblastoma has an early onset, strong invasiveness, and low eye preservation rate. The present invention successfully constructed an adeno-associated virus (AAV2-shRNA) that efficiently and accurately targets RB cells, and AAV2-shRNA can inhibit the expression of MYCN. On this basis, the inventors conducted intervention experiments using the PDX animal model of RB to verify the therapeutic effect and evaluate the therapeutic effect of AAV2-shRNA on RB, in order to establish a reliable solution for targeting MYCN gene therapy for RB.

[0025] Compared with the prior art, the beneficial effects and significant improvements of the technical solution of the present invention are:

[0026] 1. The shRNA transgenic recombinant plasmid of the present invention can promote cancer cell cavitation, and cancer cell cavitation is beneficial to the prognosis of cancer patients;

[0027] 2. The shRNA transgenic recombinant plasmid of the present invention can inhibit the expression of MYCN and has an impact on cancers related to MYCN expression, such as retinoblastoma, neuroblastoma, hepatoma, etc.;

[0028] 3. The shRNA transgenic recombinant plasmid of the present invention has good biosafety and is non-toxic to important organs such as the heart, liver, spleen, lungs, and kidneys;

[0029] 4. The present invention provides a new treatment method and therapeutic drug for the treatment of retinoblastoma, thereby improving the effectiveness of treatment, reducing the rate of enucleation, and improving the visual prognosis of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solution of the present invention, the drawings used in the embodiments of the present invention are briefly introduced below.

[0031] Figure 1 is the plasmid map of the shRNA transgenic recombinant plasmid of Example 1;

[0032] Figure 2 is the plasmid sequencing result of the shRNA transgenic recombinant plasmid of Example 1;

[0033] Figure 3 is the restriction enzyme electrophoresis diagram of the plasmid of the shRNA transgenic recombinant plasmid of Example 1;

[0034] Figure 4 The results of the therapeutic effect of the shRNA transgenic recombinant plasmid in Example 2 on RB are shown in Figure A, wherein Figure A is a microscopic image of cancer cells in the experimental group and the control group; Figure B is a microscopic image and statistical result of RB PDX tumor after AAV-shRNA gene therapy; Figure C is an OCT image of the anterior segment of the eye;

[0035] Figure 5 This is the result of the effect of the shRNA transgenic recombinant plasmid of Example 2 on the expression level of MYCN protein;

[0036] Figure 6 It is a graph of the prognostic analysis results of cavitary RB in Example 3, wherein Figure A is a statistical graph of metastasis risk, and Figure B is a statistical graph of survival rate. DETAILED DESCRIPTION

[0037] To make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific examples. 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. The experimental methods in the following examples that do not specify specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0038] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the technical field to which the application belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of the present application.

[0039] In order to more fully understand the present invention, the professional terms in the present invention are explained below.

[0040] The transgenic recombinant plasmid contains the target gene and the plasmid, and then the two are connected using enzymes to create the recombinant plasmid.

[0041] shRNA is the abbreviation of the English word short hairpin RNA. It is translated as "short hairpin RNA". shRNA includes two short inverted repeat sequences. The shRNA cloned into the shRNA expression vector includes two short inverted repeat sequences separated by a stem-loop sequence in the middle, forming a hairpin structure controlled by the polⅢ promoter. Then 5-6 Ts are connected as the transcription terminator of RNA polymeraseⅢ.

[0042] Injection refers to the use of medical devices such as syringes to inject liquid or gas into the human body for the purpose of diagnosing, treating, and preventing diseases. It is different from taking medicine. After injection, the medicine can quickly reach the blood and take effect.

[0043] A promoter is a DNA sequence that RNA polymerase recognizes, binds to, and starts transcription. It contains conserved sequences required for RNA polymerase specific binding and transcription initiation. Most of them are located upstream of the transcription start point of the structural gene, and the promoter itself is not transcribed. However, some promoters (such as tRNA promoters) are located downstream of the transcription start point, and these DNA sequences can be transcribed.

[0044] Ligase, also known as synthetase, is an enzyme that can catalyze the connection of two molecules into one molecule or connect the head and tail of a molecule. This reaction is coupled with the decomposition reaction of ATP. When connecting two molecules, the high-energy phosphate bond of adenosine triphosphate (ATP) is broken, such as DNA ligase.

[0045] Ubiquitin ligase, also known as E3 ubiquitin ligase, is an enzyme that can attach ubiquitin molecules to a lysine on a target protein.

[0046] A tumor refers to a new growth formed by the proliferation of local tissue cells under the action of various tumorigenic factors. Because this new growth often appears as a space-occupying mass-like protrusion, it is also called a neoplasm.

[0047] The experimental equipment used in the following examples includes:

[0048] TRIZOL was purchased from Invitrogen (USA); protein lysis buffer was purchased from Thermo Fisher (USA); fluorescent secondary antibody was purchased from Sigma-Aldrich (USA); DAPI dye was purchased from Sangon (China); and optical microscope was purchased from OLYMPUS (Japan).

[0049] Example 1 Preparation of shRNA transgenic recombinant plasmid

[0050] The shRNA transgenic recombinant plasmid includes a target gene, shRNA: 5'-GCCAGTATTAGACTGGA AGTT-3'. AAV2 (RC plasmid, Guangzhou Paizhen Biotechnology Co., Ltd.) was used to prepare AAV. The plasmid map is as follows Figure 1 As shown, the insertion position of the shRNA expression gene is the end of the U6 promoter, and the front end of the U6 promoter and the end of the shRNA expression gene are connected to the ubiquitin ligase E3A.

[0051] like Figure 2-3 As shown, Figure 3 The enzyme digestion result bands of the sample from left to right are: Lane 1: DL10,000DNA, Marker, Lane 2: C-12978 (SmaI), Lane 3: C-12978 (AhdI), Lane 4: C-12978 (NheI + EcoRI), Lane 5: C-12978. The sequencing results and enzyme electrophoresis patterns were compared, which were consistent with the patterns and simulated patterns, proving that the plasmid vector construction test was qualified.

[0052] Example 2 Evaluation of the efficacy of shRNA transgenic recombinant plasmid in the RB PDX animal model cohort

[0053] Experimental methods

[0054] 2.1. This example uses the RB second-generation PDX model (P2) after serial passage and histological stability evaluation as the experimental mice for this part of the experiment. After the PDX P2 transplanted tumor grows for 1 week and white pupils can be seen with the naked eye, the shRNA transgenic recombinant plasmid (named AAV2-shRNA) prepared in Example 1 is injected into the vitreous cavity for treatment, and the control group is injected with AAV2-shCtrl for treatment;

[0055] 2.2. After administration, the morphology of mouse eye tumors was measured and recorded every week for relevant safety assessment;

[0056] 2.3. After 3 weeks of treatment, the experiment was terminated, the nude mice were treated by cervical dislocation, the tumors were removed by dissection, the size of the tumors was measured, and photos were taken. The dissected tumors were retained and HE staining was performed on the tumors.

[0057] Experimental Results

[0058] The relevant indexes will be compared with the control group RB PDX model mice treated with AAV2-shCtrl. Figure 4 As shown in Figure 4A, after AAV2-shRNA was injected into the RB tumor in vivo, the formation of RB cavities increased significantly; Figure 4B showed that the RB PDX tumor showed more rosette structures after AAV-shRNA gene therapy; Figure 4C showed that the anterior segment OCT showed that the RB PDX had more cavities after AAV2-shRNA gene therapy.

[0059] Furthermore, the RNA expression level of MYCN in mouse intraocular tumors was analyzed, and the results were as follows Figure 5 The expression level of MYCN protein in AAV2-shRNA was significantly inhibited. Therefore, it was analyzed that the promotion of tumor cavitation by the shRNA transgenic recombinant plasmid of the present invention may be related to the inhibition of MYCN expression by the shRNA transgenic recombinant plasmid.

[0060] Example 3 Prognostic analysis of cavitary RB

[0061] Patients with retinoblastoma who visited the applicant's hospital between 2018 and 2024 were enrolled, and their prognostic data were collected and statistically analyzed.

[0062] The results are as follows Figure 6 As shown in Figure 6A, the risk of metastasis in patients with cavitary RB is significantly lower than that in patients with non-cavitary RB; the overall survival rate in patients with cavitary RB is significantly better than that in patients with non-cavitary RB. It can be seen that cavitary RB has a better prognosis.

[0063] The applicant declares that, in the description of the above specification:

[0064] The descriptions of the terms "this embodiment", "an embodiment of the present invention", "as shown in...", "further", "a further improved technical sub-scheme", etc., mean that the specific features, structures, materials or characteristics described in the embodiment or example are included in at least one embodiment or example of the present invention; in this specification, the schematic expressions of the above terms are not necessarily for the same embodiment or example, and the specific features, structures, materials or characteristics described may be combined or combined in a suitable manner in any one or more embodiments or examples; in addition, a person of ordinary skill in the art may combine or combine the different embodiments or examples and the features of the different embodiments or examples described in this specification without causing any contradiction.

[0065] Finally, it should be noted that:

[0066] The above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit them.

[0067] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some or all of the technical features therein, and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention. The non-essential improvements, adjustments or replacements made by those skilled in the art based on the contents of this specification are all within the scope of protection required by the present invention.

Claims

1. A shRNA transgenic recombinant plasmid that promotes cavitation of retinoblastoma cells, characterized in that: The transgenic recombinant plasmid is obtained by inserting the shRNA expression gene into the AAV2 plasmid, and the nucleotide sequence of the shRNA expression gene is SEQ ID NO.

1.

2. The shRNA transgenic recombinant plasmid for promoting retinoblastoma cell cavitation according to claim 1, characterized in that: The insertion position of the shRNA expression gene is the end of the U6 promoter, and the front end of the U6 promoter and the end of the shRNA expression gene are connected to the ubiquitin ligase E3A.

3. The shRNA transgenic recombinant plasmid for promoting cavitation of retinoblastoma cells according to claim 2, characterized in that: The retinoblastoma is RB1 ＋ / ＋ MYCN A Retinoblastoma.

4. Use of the shRNA transgenic recombinant plasmid according to any one of claims 1 to 3 in the preparation of an anti-retinoblastoma drug.

5. The use according to claim 4, characterized in that The anti-retinoblastoma drug can promote retinoblastoma cell cavitation and / or inhibit the expression level of retinoblastoma MYCN protein.

6. The use according to claim 4, characterized in that The anti-retinoblastoma drug is an adenovirus packaging plasmid.

7. The use according to claim 4 or 6, characterized in that The administration method of the anti-retinoblastoma drug includes injection.

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