Application of composition of DCLRE1C inhibitor and methotrexate in preparation of medicine for treating osteosarcoma

Abstract

The invention relates to the technical field of tumor drugs, in particular to application of a composition of a DCLRE1C inhibitor and methotrexate in preparation of drugs for treating osteosarcoma. According to the application, by down-regulating the expression of the non-homologous end joint repair core endonuclease DCLRE1C, the repair efficiency of NHEJ in osteosarcoma cells on DNA damage can be weakened, and the DNA damage condition caused by methotrexate is increased, so that the function of increasing the sensitivity of the osteosarcoma cells on methotrexate is realized, and therefore, the application of the methotrexate in the osteosarcoma cells has the advantages that the sensitivity of the osteosarcoma cells on methotrexate is improved; the DCLRE1C inhibitor and methotrexate can be combined for preparing the medicine for treating osteosarcoma.

Description

Technical Field

[0001] The present application belongs to the technical field of tumor drugs, and in particular, relates to the use of a combination of a DCLRE1C inhibitor and methotrexate in the preparation of a drug for treating osteosarcoma. Background Art

[0002] Osteosarcoma (OS) is one of the common primary malignant bone tumors, mainly occurring in children and adolescents, especially in the rapid growth period of long bones, such as the distal femur, proximal tibia and proximal humerus. Its main clinical manifestations include local pain, swelling and dysfunction, which seriously affect the patient's quality of life and social function. Currently, the standard treatment for osteosarcoma includes surgical resection combined with preoperative neoadjuvant chemotherapy and postoperative adjuvant chemotherapy, among which methotrexate (MTX) is the most commonly used chemotherapy drug in osteosarcoma chemotherapy.

[0003] Although high-dose methotrexate can greatly improve the therapeutic effect of osteosarcoma, its efficacy still faces the following major challenges: (1) Drug resistance: In osteosarcoma patients receiving MTX treatment, some tumor cells will gradually develop resistance to MTX by enhancing DNA damage repair ability or changing drug metabolic pathways. This drug resistance directly leads to a decrease in the effectiveness of chemotherapy, which in turn affects the patient's survival rate and disease control rate. (2) Limitations of mechanism research: At present, the molecular mechanism research on MTX resistance is relatively scattered, lacking systematicity and in-depthness. In particular, the key molecules in the DNA damage repair-related pathway have not been fully clarified, which limits the possibility of developing targeted treatments in clinical practice. (3) Lack of targeted treatment strategies: Existing studies are mostly focused on general chemotherapy strategies for osteosarcoma, while targeted treatment methods for resistance mechanisms are still in the exploratory stage, and there is no breakthrough treatment plan that can improve the prognosis of resistant patients. Summary of the invention

[0004] The purpose of the present application is to provide a use of a combination of a DCLRE1C inhibitor and methotrexate in the preparation of a drug for treating osteosarcoma, aiming to solve the technical problem of how to better treat osteosarcoma.

[0005] In order to achieve the above application purpose, the technical solution adopted in this application is as follows:

[0006] In a first aspect, the present application provides an application, namely, use of a combination of a DCLRE1C inhibitor and methotrexate in the preparation of a drug for treating osteosarcoma.

[0007] In a second aspect, the present application provides a pharmaceutical composition for treating osteosarcoma, comprising a DCLRE1C inhibitor and methotrexate.

[0008] The application provided in the first aspect of the present application is based on the research findings that the sensitivity of osteosarcoma cells to methotrexate can be enhanced by inhibiting the non-homologous end joining (NHEJ) repair mechanism. Specifically, by down-regulating the expression of the non-homologous end joining repair core endonuclease, DNA cross-link repair protein 1C (DNA cross-linkrepair 1C, DCLRE1C), the repair efficiency of NHEJ for DNA damage in osteosarcoma cells can be weakened, and the DNA damage caused by MTX can be increased, thereby achieving the function of increasing the sensitivity of osteosarcoma cells to MTX. Therefore, DCLRE1C inhibitors and methotrexate can be used in combination to prepare drugs for treating osteosarcoma.

[0009] The second aspect of the present application provides a pharmaceutical composition for treating osteosarcoma, including a DCLRE1C inhibitor and methotrexate. The combined treatment regimen is based on targeting the ZFP36L1-DCLRE1C-NHEJ signaling axis. The synergistic use of the DCLRE1C inhibitor and MTX can significantly improve the chemotherapy effect of osteosarcoma and provide a scientific basis for the early detection and precise treatment of osteosarcoma resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0011] Figure 1 It is a graph showing the detection results of ZFP36L1 overexpression and knockdown efficiency in the examples of the present application;

[0012] Figure 2 This is a graph showing the correlation between ZFP36L1 and DCLRE1C expression in the examples of this application;

[0013] Figure 3 This is a schematic diagram of the process of constructing a nude mouse subcutaneous tumor model in the embodiments of the present application;

[0014] Figure 4 is a diagram showing the growth of subcutaneous tumors in nude mice in each treatment group in the examples of the present application;

[0015] Figure 5 This is a schematic diagram of the process of constructing a nude mouse osteosarcoma lung metastasis model in the embodiments of the present application;

[0016] Figure 6 This is a diagram showing the growth of osteosarcoma cells in the lung metastases of nude mice in each treatment group in the examples of the present application. DETAILED DESCRIPTION

[0017] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0018] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0019] In the present application, "at least one" means one or more, "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0020] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0021] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0022] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass described in the embodiment description of the present application can be a mass unit known in the chemical industry such as μg, mg, g, kg, etc.

[0023] The terms "first" and "second" are used only for descriptive purposes to distinguish objects such as substances from each other, and should not be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX may also be referred to as the second XX, and similarly, the second XX may also be referred to as the first XX. Thus, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

[0024] Since there are still many limitations in the molecular mechanism of osteosarcoma resistance to methotrexate (MTX), especially the role of key regulatory factors in the DNA damage repair pathway has not been clarified, which greatly limits the clinical development of effective targeted treatment strategies. In response to this problem, this application discovered the close relationship between the ZFP36L1-DCLRE1C-NHEJ signaling axis and osteosarcoma resistance to MTX, and revealed its key role in enhancing the non-homologous end joining repair mechanism.

[0025] Specifically, the study found that in osteosarcoma MTX-resistant cells, the expression of RNA-binding protein ZFP36L1 was significantly downregulated. Further studies have confirmed that ZFP36L1 can increase DNA damage caused by MTX, thereby enhancing the sensitivity of osteosarcoma cells to MTX. Through in-depth research on the mechanism, it was found that ZFP36L1 mainly downregulated the expression of non-homologous recombination repair (NHEJ) core endonuclease DCLRE1C, weakened the repair efficiency of NHEJ for DNA damage in osteosarcoma cells, and increased the DNA damage caused by MTX, thereby achieving the function of increasing the sensitivity of osteosarcoma cells to MTX. Subsequently, by constructing a nude mouse subcutaneous tumor model and a lung metastasis model, it was found that overexpression of ZFP36L1 or knockdown of DCLRE1C targeting the ZFP36L1-DCLRE1C-NHEJ signaling axis can effectively increase the sensitivity of osteosarcoma cells in nude mice to MTX.

[0026] Therefore, the combined use of DCLRE1C inhibitor and methotrexate in the present embodiment can well overcome the MTX resistance problem of osteosarcoma and provide a new solution for improving the chemotherapy sensitivity and treatment effect of osteosarcoma patients. The specific technical solution is as follows:

[0027] In a first aspect, the present application provides an application. Specifically, the present application provides an application of a combination of a DCLRE1C inhibitor and methotrexate in the preparation of a drug for treating osteosarcoma.

[0028] The DCLRE1C inhibitor mentioned in the embodiments of the present application may specifically be a substance that reduces the activity or content of the DCLRE1C protein. For example, the DCLRE1C inhibitor has the effect of reducing the expression of the DCLRE1C protein in osteosarcoma patients, thereby effectively increasing the sensitivity of osteosarcoma cells to MTX-induced DNA damage and overcoming the limitations of existing chemotherapy methods.

[0029] In some embodiments, the DCLRE1C inhibitor includes at least one of a substance that inhibits the synthesis of DCLRE1C protein, a substance that promotes the degradation of DCLRE1C protein, a substance that knocks down the DCLRE1C gene, a substance that knocks out the DCLRE1C gene, and the like.

[0030] In some embodiments, the substance that knocks down the DCLRE1C gene can be any substance that can make the gene encoding the DCLRE1C protein difficult to express or reduce the expression level of its gene, such as a substance that silences the DCLRE1C gene. For example, it includes at least one of miRNA, siRNA, and shRNA that silence the DCLRE1C gene. Among them, miRNA (microRNA) is a type of non-coding single-stranded RNA molecule with a length of about 22 nucleotides encoded by endogenous genes, which can participate in post-transcriptional gene expression regulation. siRNA (Small interfering RNA) is a small infectious RNA, also known as short interfering RNA (short interfering RNA) or silencing RNA (silencing RNA), which is a double-stranded RNA of about 20 to 25 nucleotides in length, which regulates gene expression in a specific manner. shRNA (short hairpin RNA) is a small hairpin RNA, which is a section of RNA sequence with a tight hairpin loop, which is used to inhibit the expression of specific genes. The above types of RNA can effectively inhibit the expression of target genes.

[0031] In some embodiments, the substance that knocks out the DCLRE1C gene can be a substance that achieves in any way that osteosarcoma cells do not produce the functional protein product of the DCLRE1C gene, such as removing all or part of the coding gene sequence, introducing a frameshift mutation so that no functional protein is produced, removing or changing the regulatory component (e.g., promoter editing) so that the coding gene sequence is not transcribed, and preventing translation by binding to mRNA. Usually, the knockout is performed at the genomic DNA level so that the offspring of the cell also permanently carry the knockout. Specifically, the substance that knocks out the DCLRE1C gene can be any substance that can cause the DCLRE1C gene to mutate (the mutation form can be a deletion mutation and / or an insertion mutation and / or a base substitution) and thus lose its activity, including at least one of the zinc finger protein ZFN gene editing system, the TALENs gene editing system, the CRISPR / Cas9 gene editing system, and the like.

[0032] In some embodiments, the DCLRE1C inhibitor includes a recombinant plasmid that knocks down the expression of DCLRE1C. Exemplarily, the recombinant plasmid that knocks down the expression of DCLRE1C contains a shRNA target sequence, and the shRNA target sequence is shown in SEQ ID No. 1 or SEQ ID No. 2. For example, the above recombinant plasmid can be a vector recombinantly constructed by the pLKO.1 plasmid vector and the above shRNA target sequence.

[0033] In some embodiments, the DCLRE1C inhibitor includes a ZFP36L1 expression promoter. Specifically, the ZFP36L1 expression promoter is a recombinant plasmid that overexpresses ZFP36L1. For example, it can be a recombinant plasmid that overexpresses ZFP36L1 constructed based on a pLVX lentiviral vector.

[0034] The examples of this application discovered the ZFP36L1-DCLRE1C-NHEJ signaling axis. On the one hand, by targeted regulation of ZFP36L1: that is, downregulation of ZFP36L1 expression will enhance the tolerance of osteosarcoma cells to MTX-induced DNA damage. By restoring or promoting the expression of ZFP36L1, the NHEJ repair ability of osteosarcoma cells can be effectively reduced, DNA damage accumulation can be increased, and sensitivity to MTX can be restored. Therefore, by combining ZFP36L1 regulation and MTX chemotherapy, the responsiveness of osteosarcoma cells to MTX can be significantly enhanced, tumor cell proliferation can be inhibited, and the therapeutic effect can be improved. On the other hand, the specific role of ZFP36L1 in regulating the downstream molecule DCLRE1C is clarified, that is, overexpression of ZFP36L1 can inhibit the expression of DCLRE1C, which provides a theoretical basis and potential targets for the clinical development of new therapeutic drugs for the core protein DCLRE1C of the NHEJ pathway.

[0035] In a second aspect, the present invention provides a pharmaceutical composition for treating osteosarcoma. Specifically, the pharmaceutical composition provided in the present invention comprises: a DCLRE1C inhibitor and methotrexate.

[0036] The pharmaceutical composition of the embodiment of the present application is centered on the ZFP36L1-DCLRE1C-NHEJ signaling axis, and reduces drug resistance in MTX treatment and improves efficacy by regulating the NHEJ DNA damage repair mechanism in osteosarcoma cells.

[0037] In some embodiments, the DCLRE1C inhibitor includes a recombinant plasmid that knocks down the expression of DCLRE1C. For example, the recombinant plasmid that knocks down the expression of DCLRE1C contains a shRNA target sequence, and the shRNA target sequence is shown in SEQ ID No. 1 or SEQ ID No. 2.

[0038] In some embodiments, the DCLRE1C inhibitor includes a ZFP36L1 expression promoter. For example, the ZFP36L1 expression promoter is a recombinant plasmid that overexpresses ZFP36L1.

[0039] The embodiments of the present application significantly reduce the methotrexate resistance of osteosarcoma by regulating the ZFP36L1-DCLRE1C-NHEJ axis and its related pathways, providing a new solution for improving the chemotherapy sensitivity and treatment effect of osteosarcoma patients. Because the efficacy of methotrexate (MTX) chemotherapy is greatly reduced due to the drug resistance of osteosarcoma cells, the pharmaceutical composition of the embodiments of the present application significantly inhibits the non-homologous end joining (NHEJ) DNA repair pathway by regulating the expression of ZFP36L1 to inhibit DCLRE1C, thereby increasing the sensitivity of osteosarcoma cells to MTX-induced DNA damage.

[0040] In summary, the pharmaceutical composition of the embodiment of the present application can be targeted and precisely regulated to improve the therapeutic effect: inhibiting DCLRE1C through targeted regulation directly affects the DNA repair ability and survival ability of osteosarcoma cells, and effectively blocks the development of drug resistance at the molecular level. This precision treatment method significantly improves the safety and effectiveness of the treatment. The treatment strategy of combining inhibitors that inhibit DCLRE1C with methotrexate can inhibit tumor cell proliferation to a greater extent and improve the therapeutic effect compared to a single chemotherapy method. This innovative solution provides new possibilities for the personalized treatment of osteosarcoma in the clinic. At the same time, the embodiment of the present application also clarifies the core role of ZFP36L1 in the mechanism of osteosarcoma chemotherapy resistance, and further reveals its downstream molecular regulatory network, which provides a theoretical basis and technical support for the subsequent development of targeted drugs for the NHEJ repair pathway.

[0041] In some embodiments, the pharmaceutical composition can be administered orally, systemically or parenterally. The provided drugs can be prepared into various dosage forms, such as tablets, injections, capsules, and the like.

[0042] In some embodiments, the pharmaceutical composition contains an effective amount of a DCLRE1C inhibitor and methotrexate. Any of the aforementioned substances that inhibit the expression of DCLRE1C can be used to prepare the composition.

[0043] In some embodiments, provided pharmaceutical compositions further include a pharmaceutically acceptable carrier.

[0044] "Effective amount" refers to an amount that can produce functions or activities on humans and / or animals and can be accepted by humans and / or animals. "Pharmaceutically acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including (but not limited to) liposome nanoparticles, diluents, excipients, adhesives, disintegrants, surfactants, adsorption carriers, buffers, stabilizers, chelating agents, pH control agents, etc. This term refers to such pharmaceutical carriers: they are not necessary active ingredients themselves and are not overly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. In the composition, the pharmaceutically acceptable carrier may contain a liquid, such as water, saline, and buffer. In addition, these carriers may also contain auxiliary substances, such as fillers, lubricants, glidants, wetting agents or emulsifiers, pH buffer substances, etc. The carrier may also contain a cell transfection reagent. Specifically, a variety of methods well known in the art can be used to administer the inhibitor or its transcription gene, or its pharmaceutical composition to a mammal. Including but not limited to: subcutaneous injection, intramuscular injection, transdermal administration, local administration, implantation, sustained release administration, etc.

[0045] The following describes the invention in conjunction with specific embodiments.

[0046] Example 1 Construction of osteosarcoma cell line

[0047] The pLKO.1-shZFP36L1 recombinant plasmid, pLKO.1-shDCLRE1C recombinant plasmid and pLVX-ZFP36L1-FLAG recombinant plasmid were constructed by Suzhou Fubaao Biopharmaceutical Technology Co., Ltd. The target plasmids constructed above were co-transfected with psPAX2 and pMD2.G plasmids into 293T cells, and the cell supernatants were collected at 24h and 48h for culturing osteosarcoma cell line 143B. After 48h of lentiviral culture, puromycin or hygromycin was used for cell selection, and finally Mock, oeZFP36L1, shCTRL, shZFP36L1, shDCLRE1C, and shZFP36L1+shDCLRE1C stable cell lines were obtained.

[0048] pLKO.1-shZFP36L1 recombinant plasmid: a plasmid constructed by the pLKO.1 plasmid vector and shRNA targeting the ZFP36L1 gene.

[0049] pLKO.1-shDCLRE1C recombinant plasmid: a plasmid constructed by the pLKO.1 plasmid vector and shRNA targeting the DCLRE1C gene.

[0050] pLVX-ZFP36L1-FLAG recombinant plasmid: a plasmid overexpressing ZFP36L1 constructed based on the lentiviral vector pLVX.

[0051] Mock: blank control, i.e., 143B cell line successfully transfected with pLVX empty vector plasmid.

[0052] oeZFP36L1: 143B cell line successfully transfected with pLVX-ZFP36L1-FLAG plasmid.

[0053] shCTRL: irrelevant RNA sequence control group (control shRNA), namely the 143B cell line successfully transfected with pLKO.1-scrambleshRNA plasmid.

[0054] shZFP36L1: 143B cell line successfully transfected with pLKO.1-shZFP36L1 plasmid.

[0055] shDCLRE1C: 143B cell line successfully transfected with pLKO.1-shDCLRE1C plasmid.

[0056] shZFP36L1+shDCLRE1C: a mixture of shZFP36L1 and shDCLRE1C.

[0057] Among them, the plasmids used above, such as pLKO.1, pLVX, psPAX2, pMD2.G, etc., can be purchased from the market, and the sequence information of the ZFP36L1 gene and the DCLRE1C gene can be obtained from the database of the National Center for Biotechnology Information (NCBI). There are two shRNA designs targeting the ZFP36L1 gene (SEQ ID No. 3 is used if there is no special mark in the subsequent embodiments), and there are two shRNA designs targeting the DCLRE1C gene (SEQ ID No. 1 is used if there is no special mark in the subsequent embodiments). The specific shRNA target sequences are as follows:

[0058] shZFP36L1#1:5'-GCTCGCGAGACAGCCCGCTTCC-3'(SEQ ID No.3);

[0059] shZFP36L1#2:5'-GCTTCCGAGACCGCTCCTTCT-3'(SEQ ID No.4);

[0060] shDCLRE1C#1:5'-GATCCTCTGCCAATACCTTTA-3' (SEQ ID No. 1);

[0061] shDCLRE1C#2:5'-TATGGATAAAGTTGTCGAAAT-3' (SEQ ID No. 2).

[0062] Example 2 Correlation experiment between ZFP36L1 and DCLRE1C expression

[0063] The mRNA degradation experiment was used to detect the half-life of DCLRE1C mRNA in 143B cells overexpressing or knocking down ZFP36L1. The immunohistochemistry (IHC) staining method was used to detect the correlation between the expression levels of ZFP36L1, DCLRE1C and the DNA damage marker γ-H2AX in tumor tissues of osteosarcoma patients in clinical practice.

[0064] like Figure 1 As shown: A is the relative expression level of ZFP36L1 mRNA in different transfected cell lines, and B is the protein level expression detected by Western blot. Figure 2 As shown: A is the half-life of DCLRE1C mRNA in different transfected cell lines, B is the relative change in the amount of DCLRE1C mRNA in different transfected cell lines over time, C is the IHC staining test results of serial sections of tumor tissue wax blocks from different osteosarcoma patients (where case1 / 2 / 3 represent serial sections of different patients, respectively), and D is the expression correlation of ZFP36L1 and DCLRE1C.

[0065] The experimental results showed that overexpression of ZFP36L1 can effectively reduce the half-life of DCLRE1C mRNA in 143B cells. Correspondingly, knocking down ZFP36L1 can increase the half-life of DCLRE1C mRNA in 143B cells. This result suggests that ZFP36L1 can accelerate the degradation rate of DCLRE1C mRNA, thereby reducing the expression level of DCLRE1C. The results of IHC staining of osteosarcoma patient tissues showed that ZFP36L1 was negatively correlated with the expression level of DCLRE1C, and DCLRE1C was negatively correlated with the expression of γ-H2AX. This result suggests that ZFP36L1 in osteosarcoma patients can reduce the expression level of DCLRE1C, thereby increasing the sensitivity of osteosarcoma tissue to chemotherapy, more effectively causing cell DNA damage and inducing tumor cell apoptosis.

[0066] Example 3 Subcutaneous tumor animal model experiment

[0067] like Figure 3 As shown: The gene knockdown plasmid of Example 1 was used to transfer osteosarcoma cells and then injected into nude mice subcutaneously to construct a subcutaneous tumor model, and then the volume, mass and protein of the subcutaneous tumor were detected. The specific steps are as follows:

[0068] 1. Selection and grouping of subcutaneous tumor animal models

[0069] Eight-week-old female BALB / c nude mice were selected and divided into four groups, with five mice in each group.

[0070] a 1 Group: 143B cells inoculated subcutaneously with shCTRL of Example 1;

[0071] b 1 Group: 143B cells implanted subcutaneously with shZFP36L1 of Example 1;

[0072] c 1 Group: 143B cells expressing shDCLRE1C of Example 1 were implanted subcutaneously;

[0073] d 1 Group: 143B cells containing shZFP36L1+shDCLRE1C of Example 1 were subcutaneously implanted.

[0074] After subcutaneous implantation of tumor cells, the tumor cells were allowed to grow naturally subcutaneously in nude mice for 2 weeks, and then the nude mice were treated with MTX (5 mg / kg / week) by intraperitoneal injection for 3 weeks.

[0075] 2. Detection of subcutaneous tumor growth in mice

[0076] (1) Two weeks after subcutaneous injection of the above-mentioned 143B cells, the growth of subcutaneous tumors in nude mice was regularly monitored; (2) Three weeks after intraperitoneal injection of MTX, the mice were euthanized, and the subcutaneous tumors were removed and the mass of the subcutaneous tumors was detected; (3) Part of the subcutaneous tumor tissue was taken and protein was extracted to detect the protein expression in the subcutaneous tumor tissues of different groups.

[0077] The experimental results are as follows Figure 4 As shown: A is the expression of ZFP36L1 and DCLRE1C proteins in the cell samples used for subcutaneous injection detected by Western blot, B is the appearance growth of subcutaneous tumors in nude mice: C is the data of subcutaneous tumor weight in nude mice, D is the data of subcutaneous tumor volume in nude mice, and E is the protein expression of subcutaneous tumor lysis samples grown in mice 5 weeks after subcutaneous injection of 143B cells detected by Western blot.

[0078] The experimental results show that: compared with a 1 Group B 1 The group found that knocking down ZFP36L1 could enhance the resistance of osteosarcoma cells to MTX; 1 Group and c 1 The group found that knocking down DCLRE1C could reduce the resistance of osteosarcoma cells to MTX; compared with a1 Group b 1 Group, c 1 Group and d 1 The group found that knocking down DCLRE1C on the basis of knocking down ZFP36L1 could effectively attenuate the enhanced MTX resistance of osteosarcoma cells caused by knocking down ZFP36L1.

[0079] Example 4 Osteosarcoma lung metastasis animal model experiment

[0080] like Figure 5 As shown: The osteosarcoma lung metastasis model of nude mice was constructed by transferring the gene knockdown plasmid of Example 1 into osteosarcoma cells and then injecting them into the tail vein of nude mice, and then detected by in vivo visible light, HE staining of lung tumor tissue and IHC staining. The specific steps are as follows:

[0081] 1. Selection and grouping of animal models for osteosarcoma lung metastasis

[0082] Eight-week-old female BALB / c nude mice were selected and divided into four groups, with five mice in each group.

[0083] a 2 Group: Mock 143B cells of Example 1 were injected into the tail vein without MTX treatment;

[0084] b 2 Group: Mock 143B cells of Example 1 were injected into the tail vein and treated with MTX;

[0085] c 2 Group: 143B cells injected with oeZFP36L1 of Example 1 through tail vein without MTX treatment;

[0086] d 2 Group: oeZFP36L1 143B cells of Example 1 were injected into the tail vein and treated with MTX.

[0087] Two weeks after tail vein injection, the nude mice in the MTX treatment group were treated with MTX (5 mg / kg / week) intraperitoneally for 3 weeks; the nude mice in the non-MTX treatment group were intraperitoneally injected with an equal amount of DMSO for 3 weeks as an experimental control.

[0088] 2. Detection of osteosarcoma lung metastasis in mice

[0089] Five weeks after tail vein injection of 143B cells, (1) the in vivo visible light detection method was used to compare the pulmonary metastasis of osteosarcoma cells in different groups of mice; (2) the mice were killed by overdose anesthesia, and the lungs of the mice were removed and fixed with 4% paraformaldehyde, embedded in paraffin and sectioned (5-μm thick). H&E staining and IHC staining were performed to compare the apoptosis of osteosarcoma growth in the lungs of different groups of mice.

[0090] The experimental results are as follows Figure 6 As shown: A is the visible light detection result in the lungs of different treatment groups, B is the quantitative statistics of the bioluminescence intensity corresponding to A: C is the H&E staining result of different treatment groups, and D is the IHC staining result of different treatment groups.

[0091] The experimental results show that: compared with a 2 Group and c 2 The group found that overexpression of ZFP36L1 could reduce the ability of osteosarcoma cells to metastasize to the lungs; further comparison of b 2 Group and d 2 The group found that after MTX treatment, the group overexpressing ZFP36L1 had fewer lung metastases, and the experimental results showed that in the group overexpressing ZFP36L1, the death of osteosarcoma cells in the lung metastases of mice in the MTX treatment group was more obvious than that in the group without MTX treatment. This result suggests that overexpression of ZFP36L1 can increase the sensitivity of osteosarcoma cells in lung metastases to MTX.

[0092] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. Use of a combination of a DCLRE1C inhibitor and methotrexate in the preparation of a drug for treating osteosarcoma.

2. The use according to claim 1, characterized in that: The DCLRE1C inhibitor includes a recombinant plasmid for knocking down the expression of DCLRE1C.

3. The use according to claim 2, characterized in that: The recombinant plasmid for knocking down the expression of DCLRE1C contains a shRNA target sequence, and the shRNA target sequence is shown as SEQ ID No.1 or SEQ ID No.

2.

4. The use according to claim 1, characterized in that: The DCLRE1C inhibitors include ZFP36L1 expression promoters.

5. The use according to claim 4, characterized in that: The ZFP36L1 expression promoter is a recombinant plasmid that overexpresses ZFP36L1.

6. A pharmaceutical composition for treating osteosarcoma, characterized in that: include: DCLRE1C inhibitors and methotrexate.

7. The pharmaceutical composition for treating osteosarcoma according to claim 6, characterized in that: The DCLRE1C inhibitor includes a recombinant plasmid for knocking down the expression of DCLRE1C.

8. The pharmaceutical composition for treating osteosarcoma according to claim 7, characterized in that: The recombinant plasmid for knocking down the expression of DCLRE1C contains a shRNA target sequence, and the shRNA target sequence is shown as SEQ ID No.1 or SEQ ID No.

2.

9. The pharmaceutical composition for treating osteosarcoma according to claim 8, characterized in that: The DCLRE1C inhibitors include ZFP36L1 expression promoters.

10. The pharmaceutical composition for treating osteosarcoma according to claim 9, characterized in that: The ZFP36L1 expression promoter is a recombinant plasmid that overexpresses ZFP36L1.

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