Martin inhibitor and application thereof in preparation of anti-tumor drugs

Abstract

The invention relates to the technical field of biomedical engineering. The invention provides an application of an oncolytic vaccinia virus carrying a stritin interference sequence in an anti-tumor drug, a stritin inhibitor recombinant vector, an application of the stritin inhibitor recombinant vector in preparation of the anti-tumor drug, and an anti-tumor drug composition taking a stritin inhibitor or the stritin inhibitor recombinant vector as an active component. According to the present invention, the inhibition on the Stritin can be used for preparing the anti-tumor virus drug, and particularly the recombinant vector is formed by recombining the Stritin inhibitor and the vaccinia virus, such that the advantages of the Stritin inhibitor and the vaccinia virus can be combined, the new target spot is provided for the tumor virus treatment, and the broad clinical application prospect is provided.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and specifically to the application of striatin as a target in the preparation of anti-tumor drugs, carriers and drugs containing the same. Background Art

[0002] Oncolytic virus refers to a virus with therapeutic value that can selectively infect and damage tumor tissues. Currently, 10 different species of oncolytic viruses have entered clinical trials, including Adenovirus, Coxackie virus, Herpes simplex virus (HSV), Measlesvirus, Newcastledisease virus, Parvovirus, Poliovirus, Reovirus, Vaccinia virus and Vesicular stomatitis virus. Since 2005, three oncolytic viruses have been on the market, namely H101, T-VEC and G47Δ. Generally, oncolytic viruses have shown considerable safety and effectiveness in clinical trials.

[0003] Vaccinia virus is a double-stranded DNA virus in the poxvirus family and has the following advantages compared with other oncolytic viruses: (1) It replicates only in the cytoplasm, reducing the risk of virus integration into the host genome; (2) Vaccinia virus was first used as a vaccine against smallpox virus, and its safety is well guaranteed, and human clinical research on it is relatively thorough; (3) Vaccinia virus can carry foreign genes with a relatively large length (25-40Kb); (4) The virus replication cycle is short, and mature progeny viruses can be produced in 6 hours.

[0004] Currently, most common oncolytic vaccinia viruses have been attenuated, including the deletion of thymidine kinase TK gene, double deletion of TK / vaccinia virus growth factor (VGF) gene, etc. The deletion of the TK gene makes the replication of vaccinia virus more dependent on the intracellular TK level, and cancer cells usually have a higher level of TK compared with normal cells; in addition, the replication of vaccinia virus also depends on the drive of the epidermal growth factor receptor EGFR / Ras pathway, making it highly selective for cancer cells. Oncolytic vaccinia virus has shown the ability to target cancer tissues in experimental animals and humans, and thus has become an ideal viral vector for cancer treatment.

[0005] Striatin is a part of the STRIPAK complex and is involved in many biological processes such as vesicle trafficking, cell migration, and regulation of the Hippo signaling transduction pathway (Hwang J and Pallas D C. STRIPAK complexes: structure, biological function, and involvement in human diseases. Internationla Journal of Biochemistry&Cell Biology. 201447:118-48; Chen R, Xie R, et al. STRIPAK integrates upstream signals to initiate the Hippo kinase cascade. Naturea Cell Biology. 2019, 21(12):1565-1577). The Striatin protein was initially isolated from rat brain synaptosomes and got its name because it was found most in the brain striatum. The Striatin protein family includes three members, namely Striatin (STRN), SG2NA (STRN3), and Zinedin (STRN4). These members are encoded by different genes but show high homology in amino acid sequences and domains. Striatin is a protein containing 780 amino acids and has four structural regions, from the amino terminus to the carboxyl terminus, which are: caveolin-binding region, coiled-coil binding region, calcium-calmodulin binding region, and a large WD repeat region (Benoist, M, Gaillard, S and Castets, F. The striatin family: a newsignaling platform in dendritic spines. Journal of Physiology-Paris. 2006, 99(2):146–153).

[0006] Striatin (STRN) is mainly present in the central and peripheral nervous systems, especially in the striatum and motor neurons. Expression of the Striatin gene has also been detected in other tissues, including but not limited to the lung, liver, kidney, skeletal muscle, myocardium, testis, B and T lymphocytes, and fibroblasts ( J, Przygodzka P, et al. Downregulation of striatin leads to hyperphosphorylation of MAP2, induces depolymerization of microtubules and inhibits proliferation of HEK293T cells. FEBS Letter. 2015, 589(2): 222-230). Another study showed that the Striatin gene can also be expressed at the epithelial cell junctions. Striatin is highly expressed in certain brain regions (such as the olfactory tubercle, spinal cord, especially the striatum), mainly expressed in neurons of the motor system: the fifth layer of the sensorimotor cortex, the motor nuclei of the brain, the dorsal part of the caudate nucleus and motor neurons, the basal ganglia and the brainstem motor cell nuclei of mammals, especially in dendritic spines, plays a role in the regulation of calcium-dependent neuronal signaling, is crucial for the maintenance and growth of dendrites, and controls motor function by participating in all aspects of motor control through the cortico-basal ganglia-thalamo-cortical loop (Salin P, Kachidian P, Bartoli M, et al. Distribution of Striatin, a newly identified calmodulin-binding protein in the rat brain: An in situ hybridization and immunocytochemical study. The Journal of Comparative Neurology, 1998, 397(1): 41-59). Since Striatin is expressed in the brain and brainstem motor nuclei and spinal motor neurons, and spinal motor neurons themselves are targets of severe neurodegenerative diseases, striatin affects neurodegenerative diseases such as Parkinson's disease and Huntington's disease (Moqrich A, Mattei M G, Bartoli M, et al. Cloning of human striatin cDNA (STRN), gene mapping to 2p22-p21, and preferential expression in brain. Genomics, 1998, 51(1): 136-139). Striatin has also been identified as a new fusion partner of platelet-derived growth factor receptor α (PDGFRA), leading to PDGFRA activation and the development of chronic eosinophilic leukemia.STRN is associated with the mechanism of arrhythmia and the development of unique myocardial fiber fat infiltration in the myocardium (Meurs K M, Mauceli E, Lahmers S, et al. Genome-wide association identifies a deletion in the 3′ untranslated region of Striatin in a canine model of arrhythmogenic right ventricular cardiomyopathy[J]. Human Genetics, 2010, 128(3):315). Striatin regulates the phosphorylation of microtubule-associated proteins by acting on the catalytic subunit of protein phosphatase 2A (PP2A), and in addition, regulates the assembly of microtubular proteins. Inhibiting the expression of striatin to promote microtubule instability can inhibit the proliferation of HEK293T cells. In addition, striatin interacts with estrogen receptors, thus contributing to nongenomic steroid signaling. By mediating the nongenomic effects of steroids, such as the interactions between aldosterone and mineralocorticoid receptors, estrogen and estrogen receptor-α, etc., it increases aldosterone levels and salt-sensitive hypertension. (Gupta T, Connors M, Tan J W, et al. Striatin gene polymorphic variants are associated with salt sensitive blood pressure in normotensives and hypertensives. American Journal of Hypertension. 2017, 31(1):124-131; Stone IB, Green JAEM, Koefoed AW, et al. Striatin genotype-based, mineralocorticoid receptor antagonist-driven clinical trial: study rationale and design. Pharmacogenet Genomics. 2021, 31(4):83-88).Mutations in the Striatin gene are also associated with the development of a few cancers. The Striatin gene fuses with the anaplastic lymphoma kinase (ALK) gene to form STRN-ALK, which is associated with the development of papillary thyroid carcinoma and lung cancer, providing a basis for radiotherapy and lymph node metastasis (Pérot G, Soubeyran I, Ribeiro A, et al. Identification of a recurrent STRN / ALK fusion in thyroid carcinomas. PLoS One. 2014, 9(1): e87170). The STRN-NTRK2 fusion occurs in pediatric soft tissue tumors (Knezevich S R, Mcfadden D E, Tao W, et al. A novel ETV6-NTRK3 gene fusion in congenital fibrosarcoma[J]. Nature Genetics, 1998, 18(2): 184-187).

[0007] In terms of viral infection, it has been found that overexpression of Striatin in MLE-15 cells or 293T cells leads to an increase in the titer of influenza virus SC35M. However, inhibitory expression of Striatin has no effect on the replication of SC35M. Meanwhile, co-inhibition of Striatin and STRN3 expression results in a decrease in virus titer. Downregulation of Striatin leads to an increase in the level of STRN3, while downregulation of STRN3 leads to an increase in the amount of STRN. It has also been found in cells infected with the human IAV subtype (H1N1pdm09) that STRN and STRN3 are associated with IAV replication. Inhibition of the expression of Striatin and STRN3 can reduce the virus titer and downregulate the expression of viral proteins. Knockdown of Striatin and STRN3 results in severe impairment of the multimerization of M1 in SC35M-infected cells (Liu L, Weber A, Linne U, et al. Phosphorylation of Influenza A Virus Matrix Protein 1 at Threonine 108 Controls Its Multimerization State and Functional Association with the STRIPAK Complex. mBio. 2023, 14(1): e0323122).

[0008] It can be seen that Striatin plays an important role in cells, but there are no reports on the relationship between Striatin and the growth of other tumors, and whether the inhibition of Striatin regulates tumors. In addition, there are no reports on whether the oncolytic vaccinia virus is used as a vector and Striatin is used to match the two, whether there is only a simple vector expression relationship between the two, or whether Striatin can also regulate the replication of oncolytic vaccinia virus in tumor cells.

[0009] This study regulated the replication level of oncolytic vaccinia virus in tumor cells by inhibiting Striatin, induced interferon expression, and significantly improved the tumor-lytic effect of oncolytic vaccinia virus, providing a new technical approach for the development of oncolytic virus anti-tumor drugs. Summary of the invention

[0010] The first purpose of the present invention is to study the relationship between Striatin and tumor cells and provide new uses of Striatin inhibitors; another purpose of the present invention is to study the regulatory relationship between Striatin and oncolytic vaccinia viruses in infected tumor cells, providing a new technical approach for the research and development of oncolytic virus anti-tumor drugs.

[0011] Through research, the present invention found that the inhibition of Striatin expression can significantly reduce the survival rate of liver cancer cells (PLC / PRF / 5, Hep3B, SK-HEP-1, Huh7), lung cancer cells H460, colorectal cancer cells HCT116, ovarian cancer cells HELA, ovarian cancer cells A2780 and glioma cells U87MG ( Figure 2 , Figure 3 and Figure 4 ), among which the effect of oncolytic vaccinia virus expressing Striatin shRNA was significantly better than that expressing GM-CSF (a similar virus to JX-594), and it had a significant inhibitory effect on tumor growth in the pancreatic cancer A2780 cell tumor model and the liver cancer cell Hep3B tumor model in mice ( Figure 8 ).

[0012] Further studies have shown that the inhibition of Striatin can significantly upregulate the replication level of oncolytic vaccinia virus ( Figure 5 ), upregulating the apoptosis rate of tumor cells ( Figure 6 ), induce cells to produce anti-tumor cytokines, namely type I interferons IFN-α and IFN-β (see Figure 7 ). By inhibiting Striatin, the replication ability of oncolytic viruses can be significantly promoted, and can be used to prepare anti-tumor viral drugs.

[0013] Based on the above research, the specific technical solutions of the present invention are as follows:

[0014] In a first aspect of the present invention, there is provided the use of a striatin inhibitor in the preparation of an anti-tumor drug.

[0015] The striatin inhibitor is any substance that can reduce the activity of Striatin, reduce the stability of Striatin, inhibit the expression of Striatin, reduce the effective action time of Striatin, or inhibit the transcriptional activity of Striatin, including but not limited to: small interfering molecules that specifically interfere with the expression and processing of the Striatin gene, such as shRNA molecules, siRNA molecules, antisense nucleotides, etc.; antagonists, downregulators, blockers, and inhibitors of Striatin. Preferably, small interfering RNA molecules, short hairpin RNA, or antisense nucleotides that specifically interfere with the expression of the Striatin gene are used, and more preferably, short hairpin RNA with a simple structure is used.

[0016] The sequence of the short hairpin RNA is shown in the following table:

[0017]

[0018] The coding DNA sequence of the short hairpin RNA is shown in the following table:

[0019]

[0020] In a second aspect of the present invention, there is provided a recombinant vector of a striatin inhibitor, including an expression vector and a striatin siRNA, a striatin shRNA, or a striatin antisense nucleotide inserted and disposed on the expression vector.

[0021] The vector includes a viral vector and a non-viral vector.

[0022] The "viral vector" includes adenovirus, adeno-associated virus, lentivirus, coxsackievirus, herpes simplex virus, measles virus, Newcastle disease virus, parvovirus, poliovirus, reovirus, vaccinia virus, and vesicular stomatitis virus, etc. Suitable viral vectors are well known to those of ordinary skill in the art.

[0023] The "non-viral vector" includes liposomes or lipid complexes, cationic polymers, chitosan polymers, and nanoparticle carriers, etc. Suitable non-viral vectors are well known to those of ordinary skill in the art.

[0024] In addition, the inventors found through research that the interference expression of shRNA of Striatin significantly enhances the replication level of oncolytic vaccinia virus in tumor cells (as Figure 5 shown).

[0025] Therefore, in the third aspect of the present invention, there is particularly provided an oncolytic vaccinia virus Striatin shRNA recombinant vector and its use in the preparation of anti-tumor drugs. Preferred vaccinia viruses are Vaccinia virus Western Reserve strain, Vaccinia virus Tian Tan strain, Vaccinia virus Wyeth strain, Vaccinia virus Copenhagen strain, Vaccinia virus Lister strain or Vaccinia virus NYCBH strain.

[0026] In the fourth aspect of the present invention, there is provided an anti-tumor drug composition, which includes an active ingredient and a pharmaceutically acceptable excipient, carrier or diluent. The active ingredient includes a striatin interacting protein inhibitor or a striatin interacting protein inhibitor recombinant vector.

[0027] The striatin inhibitor, striatin inhibitor recombinant vector, oncolytic vaccinia virus oncoVV-Striatin shRNA, etc. of the present invention can inhibit the growth of most tumor cells such as liver cancer, lung cancer, gastrointestinal carcinoid tumors and ovarian cancer.

[0028] The recombinant virus of the present invention and pharmaceutically acceptable excipients together form an anti-tumor drug composition, so as to exert the curative effect more stably. These preparations can ensure the conformational integrity of the amino acid core sequence of the bispecific antibody disclosed in the present invention, and at the same time protect the multiple functional groups of the protein and prevent its degradation (including but not limited to aggregation, deamination or oxidation).

[0029] Generally, liquid preparations can be stored stably for at least one year at 2°C - 8°C, and freeze-dried preparations are stable at 30°C for at least six months. The preparations can be commonly used suspensions, aqueous injections, freeze-dried preparations, etc. in the pharmaceutical field.

[0030] When the recombinant virus and its composition of the present invention are administered to animals including humans, the dosage varies depending on the age and weight of the patient, the characteristics and severity of the disease, and the administration route. The total dosage should not exceed a certain range with reference to the results of animal experiments and various circumstances.

[0031] Beneficial guarantees and effects of the present invention:

[0032] The present invention provides the use of Striatin inhibitors in the preparation of anti-tumor drugs. Through experiments, it is confirmed that Striatin inhibitors can kill tumor cells by inducing the expression of type I interferon and inducing apoptosis. At the same time, the replication level of oncolytic vaccinia virus oncoVV-Striatin shRNA in tumor cells is significantly higher than that of the control virus, indicating that Striatin inhibitors promote the replication of oncolytic viruses. Therefore, the inhibition of Striatin in the present invention can be used to prepare anti-tumor viral drugs. In particular, when it is recombined with vaccinia virus into a recombinant vector, it can combine the advantages of Striatin inhibitors and vaccinia virus, providing a new target for the viral treatment of tumors and having broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is the plasmid map of pCB-Striatin shRNA.

[0034] Figure 2 It is the growth inhibitory effect of oncolytic vaccinia virus oncoVV-Striatin shRNA#1 on ovarian cancer cells A2780 (A), liver cancer cells PLC / PRF / 5 (B), and Hep3B (C) in vitro detected by MTT method.

[0035] Figure 3 It is the comparison of the growth inhibitory effects of oncolytic vaccinia viruses oncoVV-Striatin shRNA#1, oncoVV-Striatin shRNA#2 and the control virus oncoVV on tumor cells Huh7 (A) and SK-Hep-1 (B).

[0036] Figure 4 It is the comparison of the killing effects of oncolytic vaccinia viruses oncoVV-Striatin shRNA#1, oncoVV-Striatin shRNA#2 and the control virus oncoVV-GM-CSF on cervical cancer cells HELA (A), lung cancer cells H460 (B), colorectal cancer cells HCT116 (C), and glioma cells U87MG (D).

[0037] Figure 5 It is that oncoVV-Striatin shRNA#1 promotes the replication of the virus in cells Huh7 (A), SK-Hep-1 (B), PLC / PRF / 5 (C), and A2780 (D).

[0038] Figure 6 It is the proportion of apoptotic cells of oncoVV-Striatin shRNA#1-induced tumor cells A2780 (A), Hep3B (B), and Huh7 (C).

[0039] Figure 7 oncoVV-Striatin shRNA#1 promoted the expression of type I interferon in Hep3B (A), PLC / PRF / 5 (B), and SK-Hep-1 (C) cells.

[0040] Figure 8 oncoVV-Striatin shRNA#1 significantly inhibited the growth of Hep3B (A) and A2780 (B) xenograft tumors in mice. Detailed implementation manners

[0041] The following examples and experimental examples further illustrate the present invention and should not be construed as limiting the present invention. The examples do not include detailed descriptions of traditional methods, such as PCR methods, those for constructing vectors and plasmids, methods for inserting genes encoding proteins into such vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those of ordinary skill in the art and are described in many publications, including Sambrook, J., Fritsch, E.F. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2 nd edition, Cold spring Harbor Laboratory Press.

[0042] Unless otherwise indicated, percentages and parts are by weight. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention, and the preferred implementation methods and materials described in the detailed implementation manners are for illustrative purposes only.

[0043] Example 1. Construction and identification of vaccinia viruses oncoVV-Striatin shRNA#1 and oncoVV-Striatin shRNA#2 expressing Striatin shRNA

[0044] 1. Construction of pCB-Striatin shRNA recombinant plasmid

[0045] Construct the pCB-Striatin shRNA recombinant plasmid according to the plasmid map ( Figure 1 ). Among them, the shRNA sequences specifically inhibiting Striatin expression are shown in Table 1, and their encoding DNA sequences are shown in Table 2 below. Of course, RNA sequences can also be used for recombinant plasmid construction.

[0046] Table 1 shRNA Sequences Specifically Inhibiting Striatin Expression

[0047]

[0048] Table 2 Summary of Encoding DNA Sequences of shRNAs Specifically Inhibiting Striatin Expression

[0049]

[0050] The encoding DNA sequences of the Striatin shRNA sequences shown in Table 2 above were inserted into the pCB plasmid through BglII and Xba I sites to obtain the pCB-Striatin shRNA plasmid. The vTK-L and vTK-R regions in the pCB plasmid were homologously recombined with the thymidine kinase (TK) region of the wild-type virus to insert the foreign gene sequence into the TK region, while causing TK deletion. The replication of vaccinia virus depends on TK, and the TK level in tumor cells is much higher than that in normal cells. Therefore, the vaccinia virus with TK deletion has the characteristic of specifically replicating in tumor cells.

[0051] In addition, this plasmid also carries the xanthine-guanine phoshporibosyl transferase (gpt) gene as a selection gene. The gpt gene comes from Escherichia coli. In the presence of mycophenolic acid (MPA), since MPA can block guanine synthesis, the nucleic acid synthesis of the virus or cells cannot proceed normally and they will die. However, in the presence of the gpt gene, the cells or viruses can utilize hypoxanthine and xanthine to synthesize guanine through an alternative pathway, making the nucleic acid synthesis unrestricted. The wild-type virus was removed by adding mycophenolic acid, hypoxanthine, and xanthine to the culture medium to obtain purified recombinant virus.

[0052] 2. Recombination of Western Reserve (WR) Strain Vaccinia Virus and pCB-Striatin shRNA Plasmid

[0053] (1) In a 6 cm 2 culture dish, an appropriate number of 293A cells were inoculated so that they could grow to 80 - 90% confluent the next day;

[0054] (2) The culture medium was discarded, and 1 mL of Western Reserve (WR) strain vaccinia virus (0.05 - 0.1 MOI, the virus solution was diluted with the medium containing 2% serum) was gently added along the side wall and cultured in an incubator at 37°C and 5% CO2 for 2 - 4 hours. During this period, it was shaken about every 15 minutes to prevent local cell death.

[0055] (3) Cell transfection was performed according to the instructions of the kit (Effectene) as follows:

[0056] Add buffer EC to 1 μg of pCB-Striatin shRNA to make up to 150 μL, then add 8 μL of Enhancebuffer respectively, shake for 1 s, and let stand at room temperature for 5 min; add 25 μL of Effectene buffer to each of the above three mixtures respectively, invert and mix 5 times, shake for 10 s, and let stand at room temperature for 5 - 10 min, then add 1 mL of fresh culture medium (which may contain serum and antibiotics) to the above mixtures respectively, and invert twice; at the same time, discard the culture medium in step 2, add 4 mL of fresh culture medium containing 10% FBS, and add the mixed transfection solution to it respectively; place the culture dish in an incubator at 37 °C and 5% CO2 for 6 - 18 hours, and then add 5 mL of fresh culture medium to continue the culture.

[0057] (4) After complete cytopathic effect, collect the virus solution in a biosafety cabinet, aliquot it into centrifuge tubes, label it, and freeze-thaw the centrifuge tubes at -80 °C and 37 °C three times repeatedly to completely lyse the cells and release the virus. Centrifuge at 2000 rmp for 5 min to collect the supernatant, and store it in a -80 °C ultra-low temperature freezer for later use.

[0058] 3. Screening of recombinant virus

[0059] (1) Inoculate 293A cells with good growth status into a culture dish, and the cell density can reach about 80% - 90% the next day.

[0060] (2) Prepare three screening drugs: xanthine, hypoxanthine, and mycophenolic acid.

[0061] (3) Carefully add 500 μL of the previously packaged virus solution along the side wall of each culture dish in (1), and place it in an incubator at 37 °C and 5% CO2 for 2 - 4 h. After about 2 - 4 h, aspirate and discard the suspended virus solution, and add 3 mL of fresh culture medium, which contains 7.5 μL of (1×) mycophenolic acid, 75 μL of (1×) xanthine, and 7.5 μL of (1×) hypoxanthine.

[0062] (4) Observe the cytopathic effect of the cells every day. After about two or three days, collect all the diseased cell fluids in a biosafety cabinet, freeze-thaw three times repeatedly, and store it in a -80 °C ultra-low temperature freezer for later use.

[0063] (5) Repeat the screening of the virus solution collected each time 3 - 4 times according to the above method.

[0064] 4. Virus plaque picking and identification

[0065] (1) Preparation of 5% low melting point glue: Weigh 0.25 g of low melting point glue and dissolve it in 5 mL of PBS. Autoclave it at 121 °C for 20 min, and then store it in a 4 °C refrigerator for later use.

[0066] (2) Seed 293A cells in good condition into a six-well plate. The next day, when the cell density reaches about 90%, serially dilute the virus solution in a gradient of 10 -4 ~10 -6 . Then discard the old culture medium in the six-well plate, add 1 mL of the diluted virus solution to each well to allow the virus to adsorb, and incubate it in an incubator for 2 - 4 h. Place the boiled low melting point glue in a 40 °C water bath to keep it warm, then put it in a laminar flow hood, add three volumes of DMEM culture medium to make its final concentration 1.25%, quickly mix it with a pipette, and quickly aspirate the suspended virus solution in the plate with a pipette gun. Carefully add 2 mL of the culture medium containing 1.25% low melting point glue along the side wall with a pipette, being careful not to blow up the cells, and then incubate it in a 37 °C, 5% CO2 cell incubator.

[0067] (3) Observe the cytopathic effect under an inverted microscope every day. If isolated virus plaques appear, pick them and place them in a 12-well plate pre-coated with 293A cells, label them, and incubate them in a 37 °C, 5% CO2 cell incubator. After they are fully diseased, collect the virus solution in a 1.5 mL centrifuge tube in a biosafety cabinet and store it in an -80 °C ultra-low temperature refrigerator for the next identification.

[0068] (4) Perform PCR identification using the characteristic that wild-type virus has a complete TK region while recombinant virus does not, and obtain purified vaccinia virus oncoVV-Striatin shRNA.

[0069] Example 2. Detection of the in vitro inhibitory effect of oncoVV-Striatin shRNA#1 and oncoVV-Striatin shRNA#2 on tumor cells by MTT method

[0070] In this experiment, liver cancer cells PLC / PRF / 5, Hep3B, SK-Hep-1, Huh7, ovarian cancer cells A2780, lung cancer cells H460, colorectal cancer cells HCT116, and ovarian cancer cells HELA were selected. They were seeded into a 96-well plate at a density of 5×10 3 / well, and 90 μL of cell culture medium was added to each well and cultured overnight. The cells were respectively added with a certain dose of oncoVV or oncoVV-GM-CSF, oncoVV-Striatin shRNA#1 or oncoVV-Striatin shRNA#2. Six replicate wells were set. The control group was cells without virus, and the blank group was cell-free culture medium.

[0071] The cells were cultured at 37 °C and 5% CO2, and four time gradients of 24 h, 48 h, 72 h, and 96 h were set. At the corresponding time points, 20 μL of MTT solution (5 mg / mL) was added to each well in the dark. After standing in the incubator for 4 h, the culture medium of each group was aspirated, and then 150 μL of dimethyl sulfoxide was added to each well and shaken on a shaker for 10 min to fully dissolve the crystals. The OD value was measured on an enzyme-linked detector at a detection wavelength of 490 nm.

[0072] Calculate the cell survival rate according to the measured OD value. The formula is:

[0073] Cell survival rate = (OD value of the treatment group - OD value of the zero-adjustment group) / (OD value of the control group - OD value of the zero-adjustment group) × 100%.

[0074] The analysis results are as Figure 2 、 Figure 3 and Figure 4 shown. oncoVV-Striatin shRNA#1 or oncoVV-Striatin shRNA#2 significantly inhibited the proliferation of tumor cells in vitro in a dose- and time-dependent manner.

[0075] Example 3. The replication level of oncolytic vaccinia virus oncoVV-Striatin shRNA#1 in tumor cells was significantly higher than that of the control virus

[0076] On the first night, hepatoma cells PLC / PRF / 5, SK-Hep-1, and Huh7 were seeded at 100,000 cells per well, and ovarian cancer cells A2780 were seeded at 50,000 cells per well in a 24-well plate and cultured overnight. Each group was set with 3 replicate wells. For hepatoma cells PLC / PRF / 5, SK-Hep-1, and Huh7, 5 MOI of oncoVV and oncoVV-Striatin shRNA#1 were added, and for ovarian cancer cells A2780, 1 MOI of oncoVV and oncoVV-Striatin shRNA#1 were added, and cultured at 37 °C. At five time points (0 h, 12 h, 24 h, 36 h, 48 h), the cells were collected, frozen and thawed three times in an -80 °C refrigerator, and then the replication efficiency of the virus in tumor cells was detected by the TCID 50 method (50% tissue culture infective dose). The method is as follows:

[0077] (1) Cell plating. HEK-293A cells were seeded in a 96-well plate at 3000 cells per well.

[0078] (2) Preparation of virus dilution: Virus sample dilution was performed aseptically in a laminar flow hood: The purified virus was diluted with serum-free DMEM to 10 -2 、10-3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , and 10 -8 and several different gradients, etc.

[0079] (3) Infected virus samples: Infect the 96-well plates with the diluted virus samples in sequence. Repeat 8 replicate wells for each concentration gradient, and add 100 μL of virus solution to each well. Incubate in a 37 °C, 5% CO2 incubator for 5 - 8 days under normal conditions.

[0080] (4) Result calculation

[0081] Virus titer calculation formula: For a 100 μL sample, titer T = 10 1+d(s-0.5) ;

[0082] d = log10 dilution factor = 1 (for a 10-fold dilution factor);

[0083] s = sum of positive ratios (starting from the first 10-fold dilution);

[0084] According to the following formula, convert TCID 50 / mL to PFU / mL:

[0085] T = a × 10 b TCID 50 / mL = a × 10 b-0.7 PFU / mL;

[0086] The detection results are as Figure 5 shown: The replication level of oncoVV-Striatin shRNA#1 in the above six types of tumor cells is significantly better than that of the control virus oncoVV.

[0087] Example 4. oncoVV-Striatin shRNA#1 induces apoptosis in cancer cells

[0088] 1. Detection of apoptosis by flow cytometry

[0089] (1) Seed the cells into six-well plates, 600,000 cells per well, with three replicate wells in each group, and incubate overnight at 37 °C.

[0090] (2) The next day, dilute oncoVV and oncoVV-Striatin shRNA#1 to 5 MOI with culture medium and add them to the corresponding wells, 50 μL / well; the PBS group is made up to volume with PBS. Incubate in an incubator for 24 h.

[0091] (3) Aspirate the supernatant, wash once with pre-cooled PBS, and add 1 mL of trypsin without EDTA to each well for digestion.

[0092] (4) Remove the trypsin, add 1 mL of the corresponding cell medium to terminate the digestion, then blow down the cells and collect them in a 1.5 mL centrifuge tube, centrifuge at 1500 rpm for 5 min.

[0093] (5) Remove the supernatant, and wash the cell pellet twice with PBS.

[0094] (6) Add 100 μL of 1× Binding Buffer (diluted with PBS) to each centrifuge tube and gently resuspend the cells.

[0095] (7) Add 5 μL of Annexin V-FITC and 5 μL of Propidium Iodide solution to each tube in sequence in the dark, flick to mix well, and incubate in the dark for 15 min.

[0096] (8) Add another 400 μL of 1× Binding Buffer to each tube and mix well.

[0097] (9) Filter the sample through a 300-mesh nylon mesh into a new tube.

[0098] (10) Detect the apoptosis of the sample with a flow cytometer.

[0099] The detection results are as Figure 6 shown. The ability of oncoVV-Striatin shRNA#1 to induce apoptosis in tumor cells is significantly better than that of the control virus oncoVV.

[0100] Example 5. oncoVV-Striatin shRNA#1 Induces the Expression of Type I Interferon in Cells

[0101] 1. RNA Extraction

[0102] (1) Inoculate plasma cells in a 24-well plate (60,000 cells / well), with 4 replicates in each group, and culture at 37 °C for 36 h.

[0103] (2) After 36 h, discard the supernatant and wash the cells once with PBS.

[0104] (3) Add 500 μL of Lysis Buffer in the RNA rapid extraction kit to each well to lyse the cells.

[0105] (4) Use the lysis solution to lyse the cells, then transfer to a centrifuge tube and vortex for 10 s.

[0106] (5) Add 500 μL of absolute ethanol to each tube, blow and suck to mix well, and transfer the liquid to a centrifugal column.

[0107] (6) Centrifuge at 4000g for 1 min.

[0108] (7) Add 500 μL of Wash Buffer in the kit to the centrifuge column and centrifuge at 12000g for 1 min.

[0109] (8) Discard the waste liquid, place the centrifuge column on a new RNase-free centrifuge tube, open the lid and air dry for 2 min.

[0110] (9) Add 30 μL of DEPC water to the middle part of each centrifuge column and centrifuge at 12000g for 1 min.

[0111] (10) Measure the concentration of the eluted RNA using an absorbance photometer.

[0112] 2. Reverse Transcription

[0113] (1) Place the RNA sample at 65 °C, pre-denature for 5 min, and immediately cool on ice.

[0114] (2) Perform reverse transcription using the TOYOBO reverse transcription kit.

[0115] The reaction system is as follows: 5×RT Buffer, 4 μL; RT Enzyme Mix, 0.5 μL; Primer Mix, 0.5 μL; RNA, 1 μg; Nuclease-free Water, Up to 20 μL.

[0116] The reaction time is: 37 °C, 15 min; 98 °C, 5 min; 4 °C, 10 min. The cDNA is stored in a -20 °C refrigerator for later use.

[0117] 3. Real-Time Fluorescent Quantitative PCR (qPCR)

[0118] (1) Dilute the cDNA from the previous step tenfold before use.

[0119] (2) Perform qPCR using the TOYOBO qPCR enzyme, and the primers are as follows:

[0120] GAPDH Primer 1: 5’-GACAG TCAGC CGCAT CTTCT-3’ (SEQ ID NO.5);

[0121] GAPDH Primer 2: 5’-GCGCC CAATACGACCAAATC-3’ (SEQ ID NO.6);

[0122] IFN-α Primer 1: 5’-GTGAG GAAATACTTC CAAAG AATCA C-3’ (SEQ ID NO.7);

[0123] IFN-α Primer 2: 5’-TCTCATGATT TCTGC TCTGACAA-3’ (SEQ ID NO.8);

[0124] IFN-β Primer 1: 5’-CAGCAATTTT CAGTG TCAGAAGC-3’ (SEQ ID NO.9);

[0125] IFN-β Primer 2: 5’-TCATC CTGTC CTTGAGGCAG T-3’ (SEQ ID NO.10).

[0126] The qPCR reaction system is: ddH2O, 6.4 μL; SYBR Green Realtime PCR Master Mix, 10 μL; Primer1, 0.8 μL; Primer 2, 0.8 μL; cDNA, 2 μL.

[0127] The cycling conditions are: Holding stage: 95 °C, 1 min. Cycling stage (50×): 95 °C, 15 s; 60 °C, 1 min; 95 °C, 15 s; Melting stage: 60 °C, 1 min; 95 °C, 30 min; 60 °C, 15 s.

[0128] (3) Process the data: Using the PBS group as the control and GAPDH as the internal reference, calculate the relative expression fold of interferon in the oncoVV group and the oncoVV-Striatin shRNA#1 group.

[0129] The detection results are as Figure 7 shown that oncoVV-Striatin shRNA#1 significantly induces the expression of interferon in tumor cells, and the level is significantly higher than that of the control virus oncoVV.

[0130] Example 6, oncoVV-Striatin shRNA#1 significantly eliminates A2780 and Hep3B mouse transplanted tumors

[0131] Establish subcutaneous transplanted tumors in nude mice using ovarian cancer cell line A2780 and hepatocellular carcinoma cell line Hep3B. Inject oncoVV, oncoVV-Striatin shRNA#1 or an equal volume of PBS intraperitoneally, and regularly measure the tumor volume. The animal experiment method is as follows:

[0132] All animal experiment operations in this study were strictly carried out in accordance with the NIH experimental animal guidelines. Female BALB / c nude mice at 4 weeks of age were selected and injected with 2.3x10 7Cells at a density of / 100 μL were subcutaneously injected with A2780 cells at the axillary end of the forelimb. After cell injection, the growth of tumor volume was observed daily and measured with a micrometer, and the tumor volume ((mm 3 ) = (length × width 2 ) / 2). When the tumor grew to a stable state, it was divided into 2 groups: PBS group, oncoVV group and oncoVV-Striatin shRNA#1 group, with 6 - 8 nude mice in each group. After grouping, each nude mouse was intraperitoneally injected once with 1×10 7 PFU virus or an equal volume of PBS, and then the tumor volume was measured regularly.

[0133] The detection results are as Figure 8 shown. oncoVV-Striatin shRNA#1 significantly inhibited the growth of transplanted tumors in mice, and the effect was significantly better than that of the control virus oncoVV.

[0134] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art can also make various equivalent variations or substitutions without departing from the spirit of the present invention, and these equivalent variations or substitutions are all included within the scope defined by the claims of this application.

Claims

1. Use of striatin inhibitor in the preparation of anti-tumor drugs.

2. Use of the fibrillin inhibitor according to claim 1 in the preparation of an anti-tumor drug, characterized in that: The tumors include liver cancer, lung cancer, colorectal cancer, ovarian cancer, glioma, and pancreatic cancer.

3. The use of the striatin inhibitor in the preparation of anti-tumor drugs according to claim 1, characterized in that: Among them, The striatin inhibitor is any substance that can reduce the activity or stability of striatin, inhibit the expression or processing of striatin gene, reduce the effective action time of striatin or inhibit the transcriptional activity of striatin gene.

4. The use of the striatin inhibitor in the preparation of anti-tumor drugs according to claim 3, characterized in that: Among them, The striatin inhibitor is small interfering RNA, short hairpin RNA or antisense nucleotide that specifically interferes with the expression of striatin gene, The sequences of the small interfering RNA or short hairpin RNA are as shown in SEQ ID NO.1 or SEQ ID NO.

2.

5. A recombinant vector of a fibrillin inhibitor, characterized in that, It includes an expression vector and striatin siRNA, striatin shRNA or striatin antisense nucleotide inserted and set on the expression vector. Among them, the expression vector is a plasmid vector, cosmid vector, phage vector or viral vector, and the viral vector is selected from adenovirus, adeno-associated virus, lentivirus, coxsackievirus, herpes simplex virus, measles virus, Newcastle disease virus, parvovirus, poliovirus, reovirus, vaccinia virus and vesicular stomatitis virus.

6. The recombinant vector of striatin inhibitor according to claim 5, characterized in that: Among them, The expression vector is vaccinia virus, and the vaccinia virus is Vaccinia virus Western Reserve strain, Vaccinia virus Tian Tan strain, Vaccinia virus Wyeth strain, Vaccinia virus Copenhagen strain, Vaccinia virus Lister strain or Vaccinia virus NYCBH strain.

7. Use of the recombinant vector of striatin inhibitor according to claim 5 in the preparation of anti-tumor drugs.

8. The use of the recombinant vector of striatin inhibitor in the preparation of anti-tumor drugs according to claim 7, characterized in that: Among them, The recombinant vector of striatin inhibitor is STRIP shRNA vaccinia virus.

9. An anti-tumor pharmaceutical composition, characterized in that, The anti-tumor drug composition includes an active ingredient and a pharmaceutically acceptable excipient, carrier or diluent, Among them, the active ingredient is the striatin inhibitor according to any one of claims 1 to 4 or the recombinant vector of striatin inhibitor according to any one of claims 5 to 8.

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