Application of CPT2 protein and mutant thereof in preparation of anti-kidney cancer drugs

Abstract

The invention discloses application of CPT2 protein and a mutant thereof in preparation of anti-kidney cancer drugs, and belongs to the technical field of biology. The application provided by the invention specifically refers to the application of the CPT2 protein and the mutants CPT-G600E and CPT-G600R of the CPT2 protein in inhibition of proliferation and metastasis of the kidney cancer. Experiments prove that the overexpression of the CPT2 protein and the mutants of the CPT2 protein, namely CPT-G600E and CPT2-G600R, has a remarkable inhibition effect on the proliferation, migration and invasion ability of kidney cancer 786-O and Caki-1 cells. The invention is of great significance to treatment and research of kidney cancer.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to the application of CPT2 protein and its mutants in the preparation of anti-kidney cancer drugs. Background Art

[0002] Kidney cancer is a common malignant tumor of the urinary system. According to data released in 2022, the annual number of new cases of kidney cancer in the world ranks sixth among men and ninth among women. Renal clear cell carcinoma (ccRCC) is the most common pathological type of kidney cancer, accounting for about 70% of all renal malignancies. Early ccRCC patients can obtain a good prognosis with a 5-year survival rate of more than 90% after partial nephrectomy or radical resection. However, due to the lack of typical clinical symptoms in the early stage of ccRCC, one-quarter of patients are found to have distant metastases at the time of initial diagnosis. Metastatic ccRCC is insensitive to chemotherapy and radiotherapy, and its 5-year overall survival rate is only 12%. Therefore, drug development for metastatic renal cancer is currently a hot research direction.

[0003] CPT2 protein is a key enzyme for β-oxidation of fatty acids. CPT2 is responsible for catalyzing the release of carnitine and acyl CoA from acylcarnitine, which can be oxidized and decomposed by the lipid oxidase system in mitochondria. In recent years, reports have shown that CPT2 is abnormally expressed in many tumors: the expression level of CPT2 is significantly reduced in hepatocellular carcinoma, ovarian cancer, and renal cancer, while CPT2 is significantly highly expressed in prostate cancer and breast cancer. Among them, downregulation of CPT2 in hepatocellular carcinoma significantly improves the stemness, metastatic ability and tolerance to cisplatin of liver cancer cells. Downregulation of CPT2 in breast cancer with high CPT2 expression can inhibit the proliferation and migration of breast cancer, and increase the sensitivity of breast cancer to radiotherapy and glutaminase inhibitors.

[0004] It can be seen that CPT2 may play a completely different role in different tumors, and the specific role of CPT2 in the progression of renal cancer has not been reported. Our latest study found that the proliferation, migration and invasion of renal cancer cells were significantly inhibited after overexpression of CPT2, while the proliferation, migration and invasion of renal cancer cells were significantly enhanced after knocking down CPT2. This shows that CPT2 protein has a potential therapeutic effect on renal cancer. Summary of the invention

[0005] One object of the present invention is to provide a use of a CPT2 protein and its mutants CPT-G600E and CPT2-G600R in the preparation of anti-renal cancer drugs.

[0006] The CPT2 protein and its mutants CPT-G600E and CPT2-G600R are specifically the proteins shown in sequences 1, 2 and 3 in the sequence table; the specific application is to prepare a product that inhibits the proliferation, migration and invasion of renal cancer cells.

[0007] The use of genes encoding proteins shown in sequences 1, 2, and 3 in the sequence table in the preparation of products that inhibit the proliferation, migration, and invasion of renal cancer cells also falls within the scope of protection of the present invention. The gene can be a DNA molecule as shown in sequence 4, 5, or 6, or any other DNA sequence that can encode a protein as shown in sequence 1, 2, or 3.

[0008] The use of expression cassettes, recombinant vectors, transgenic cell lines or recombinant bacteria containing the gene in the preparation of products for inhibiting the proliferation, migration and invasion of renal cancer cells also falls within the protection scope of the present invention.

[0009] In the present invention, the recombinant vector is a recombinant expression vector. The recombinant expression vector is specifically a recombinant plasmid obtained by inserting the gene (sequence 4, 5, 6) at the multiple cloning site of the lentiviral vector GV341. The multiple cloning site is specifically located at XmnI and NotI. The vector is packaged by lentivirus and used as a drug to reduce the proliferation, migration and invasion ability of renal cancer cells.

[0010] In the present invention, the renal cancer cells are human renal cancer cells, specifically 786-O and Caki-1 cells.

[0011] Beneficial effects: The present invention utilizes a lentivirus-packaged overexpression vector to achieve stable and high expression of CPT2 protein and mutants CPT2-G600E and CPT2-G600R in renal cancer cells, and detects the proliferation, migration and invasion abilities of the cells. Experiments have shown that overexpression of CPT2 protein and mutants CPT2-G600E and CPT2-G600R can significantly inhibit the proliferation, migration and invasion abilities of renal cancer cells. The present invention is of great significance for the treatment of renal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is the overexpression detection of human CPT2 gene and mutants CPT2-G600E and CPT2-G600R in 786-O and Caki-1 cells;

[0013] Figure 2 For CCK8 detection, the effects of overexpression of protein CPT2 and mutants CPT2-G600E and CPT2-G600R on cell proliferation;

[0014] Figure 3 To detect the effect of overexpression of protein CPT2 and mutants CPT2-G600E and CPT2-G600R on cell proliferation for clone formation experiments;

[0015] Figure 4The effects of overexpression of protein CPT2 and its mutants CPT2-G600E and CPT2-G600R on cell invasion and migration;

[0016] Figure 5 This is the effect of protein CPT2 overexpression on the proliferation of 786-O cell subcutaneous transplanted tumors. DETAILED DESCRIPTION

[0017] The present invention is further described below in conjunction with specific examples. Experimental methods without specific conditions in the examples are usually carried out under conventional conditions or conditions recommended by the manufacturer.

[0018] 786-O and Caki-1 cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0019] Lentiviral vector GV341: 10393 bp in length, with puromycin as the selection marker. Provided by Shanghai Genetech Biotechnology Co., Ltd.

[0020] RMPI 1640, Trypsin-EDTA, PBS were purchased from Gibco; FBS was purchased from Cellmax; CPT2 antibody was purchased from abcam. CCK8 and crystal violet solution were purchased from Beyotime Biotechnology Co., Ltd.

[0021] Example 1. Construction of overexpression plasmids for wild-type, CPT2-G600E, and CPT2-G600R mutant human CPT2 genes

[0022] The present invention relates to human CPT2 gene wild type (Genebank: NM_000098.2), CPT2-G600E (G mutates to A at 1799bp, causing glycine codon GGG to mutate to glutamate codon GAG), and CPT2-G600R (G mutates to C at 1798bp, causing glycine codon GGG to mutate to arginine codon CGG) mutant sequences. The nucleotide sequence of the CPT2 gene wild type is specifically shown in sequence 4 in the sequence table, encoding the wild type CPT2 protein shown in sequence 1 in the sequence table. The nucleotide sequence of CPT2-G600E is specifically shown in sequence 5 in the sequence table, encoding the CPT2-G600E mutant shown in sequence 2 in the sequence table. The nucleotide sequence of CPT2-G600R is specifically shown in sequence 6 in the sequence table, encoding the CPT2-G600R mutant shown in sequence 3 in the sequence table.

[0023] In this example, the wild type of CPT2 gene (sequence 4), CPT2-G600E (sequence 5), and CPT2-G600R (sequence 6) were respectively inserted between the multiple cloning sites XmnI and NotI of the lentiviral vector GV341 to construct recombinant expression vectors GV341-CPT2, GV341-CPT2-G600E, and GV341-CPT2-G600R. The construction of the recombinant expression vector was completed by Shanghai Jikai Biotechnology Co., Ltd., and the sequencing verification of the recombinant plasmid was performed.

[0024] Example 2: Verification of expression of wild-type human CPT2 gene, CPT2-G600E, and CPT2-G600R mutants in renal cancer cells

[0025] To verify the working efficiency of the overexpression vector, the recombinant expression vectors GV341-CPT2, GV341-CPT2-G600E and GV341-CPT2-G600R constructed in Example 1 were mixed with the lentiviral packaging plasmids PAX2 and PMD2.G at a gene:PAX2:PMD2.G mass ratio of 2:2:1 and transfected into 293T cells. At the same time, the empty vector GV341-vector was set as a blank control. The virus particles were collected 48 and 72 hours after infection, respectively, and directly infected 786-O and Caki-1 cells after filtration through a 0.22 μM filter. After 48 hours, puromycin was added for screening and positive cells were obtained after one week.

[0026] 1. Detection of CPT2 mRNA levels in cells using qPCR

[0027] 1) RNA extraction: The puromycin-resistant positive cells screened above were added to 500 μL RNA lysis buffer (Yishan Biotechnology), and then RNA was extracted according to the Yishan RNA Rapid Extraction Kit. The purity and concentration of the extracted RNA were quantified using a NanoDrop ND-1000 nucleic acid quantifier, and agarose quality inspection was performed to ensure the integrity of the extracted RNA.

[0028] 2) cDNA synthesis: The extracted total RNA was reverse transcribed and synthesized into cDNA using the HiFiScript gDNA Removal RTMasterMix reverse transcription kit.

[0029] 3) Real-time quantitative PCR: Specific primers were designed based on the nucleic acid sequences of CPT2 and β-actin, and the PCR reaction was performed using UltraSYBR Mixture from Kangwei Century. The primer sequences for CPT2 were: upstream 5'-; downstream 5'-. The primer sequences for β-actin were: upstream; downstream

[0030] The reaction system is as follows:

[0031] Table 1 PCR reaction system

[0032] Reagents Usage amount (μL) 2×UltraSYBR Mixture 5 Forward primer, 10 μM 1 Reverse primer, 10 μM 1 Template DNA 2 <![CDATA[ddH2O]]> 1 Total 10

[0033] After mixing the above components evenly, follow the following procedure: pre-denaturation at 95°C for 10 min, 40 cycles: 95°C for 15 s, 60°C for 30 s. The specificity of the reaction was determined based on the melting curve, and the formula 2 was used to determine the specificity of the reaction. -ΔΔCt The relative expression of CPT2 was calculated and the results are shown in Figure 1 .Depend on Figure 1 It can be seen that after infection with CPT2, CPT2-G600E, and CPT2-G600R, the mRNA level of CPT2 in renal cancer cells was significantly increased.

[0034] 2. Detect the expression of CPT2 protein in cells by western blot

[0035] 1) Total protein was extracted from the above-mentioned puromycin-positive cells and protein quantification was performed. 10 μg of protein was heat-denatured at 95°C for 5 minutes in protein loading buffer, and the proteins were separated by 10% SDS-PAGE electrophoresis and then transferred to a PVDF membrane (Biorad).

[0036] 2) After the transfer, wash with 20 ml TBST on a decolorizing shaker for 5 minutes, and then block with 5% skimmed milk powder for 1 hour.

[0037] 3) After blocking, the primary antibody was incubated as follows: rabbit anti-human CPT2 monoclonal antibody (abcam, ab231162) and mouse anti-human β-actin (ab8226) were used for incubation at 4°C overnight, and then washed with 20 ml TBST for 5 minutes, three times.

[0038] 4) Then incubate with rabbit secondary antibody and mouse secondary antibody (1:5000) for 1 hour, wash with 20 ml TBST for 5 minutes, and wash three times.

[0039] 5) The target band is developed with ECL developer and photographed. Figure 1 .

[0040] Example 3 Human CPT2 gene wild type, CPT2-G600E, and CPT2-G600R mutants inhibit the proliferation of renal cancer cells

[0041] 1. CCK8 method to detect cell proliferation ability

[0042] 1) Digest the GV341-vector, GV341-CPT2, GV341-CPT2-G600E and GV341-CPT2-G600R (787-O, Caki-1) in the logarithmic growth phase into a single cell suspension one day in advance, count the cells, adjust the cell concentration to 10,000 cells / ml, and inoculate them into a 96-well plate with 100 μL per well, i.e., 1,000 cells per well.

[0043] 2) After the cells adhered, CCK8 reagent was added at different time points (1, 2, 3, 4, and 5 days) at a ratio of 1:10, that is, 10 μL of detection solution was added to 100 μL of culture medium.

[0044] 3) After incubation at 37°C for 2 h, the absorbance at 450 nm was measured using an ELISA reader.

[0045] 4) Figure 2 is a schematic diagram of the growth curve of renal cancer cells. Figure 2 It can be seen that overexpression of CPT2 and its mutants can significantly inhibit the proliferation of renal cancer cell lines.

[0046] 2. Cell clone formation experiment to detect cell proliferation ability

[0047] 1) GV341-vector, GV341-CPT2, GV341-CPT2-G600E and GV341-CPT2-G600R (787-O, Caki-1) in the logarithmic growth phase were digested into single cell suspensions, inoculated into 6-well plates, 500 cells per well, and cultured statically for 1-2 weeks.

[0048] 2) Observe frequently. When visible clones appear in the culture dish, terminate the culture. Discard the supernatant and carefully rinse twice with PBS. Stain with 0.3% crystal violet staining solution for 10-30 minutes, then slowly wash away the staining solution with running water and air dry.

[0049] 3) Take a photo of the six-well plate in an imaging system to observe the colony formation.

[0050] 4) Figure 3 Image the colony formation of renal cancer cells by Figure 3 It can be seen that overexpression of CPT2 and its mutants can significantly inhibit the clone formation of renal cancer cells.

[0051] Based on the results of 1)-2) above, it can be seen that overexpression of CPT2 and its mutants can inhibit the proliferation of renal cancer cells.

[0052] Example 4 Human CPT2 gene wild type, CPT2-G600E, and CPT2-G600R mutants inhibit migration and invasion of renal cancer cells

[0053] 1. Transwell assay to detect migration and invasion ability of renal cancer cells

[0054] 1) GV341-vector, GV341-CPT2, GV341-CPT2-G600E and GV341-CPT2-G600R (787-O, Caki-1) in the logarithmic growth phase were digested and centrifuged, then resuspended in serum-free RIPM1640 culture medium into a single cell suspension, counted, and the 786-O cell concentration was adjusted to 1×10 5 / ml, Caki-1 cell concentration was 3×10 5 / ml, take 100μl and add it to the upper chamber, add 600μl RIMP1640 complete medium to the lower chamber, and culture in a 37℃ CO2 incubator for 18-24 hours.

[0055] 2) Discard the liquid in the upper chamber, wipe off the cells on the upper chamber with a cotton swab, fix with paraformaldehyde, stain with crystal violet, and count the cells under a 200x light microscope.

[0056] 3) The cell invasion experiment is basically the same as the above process, except that a layer of Matrigel needs to be laid in the upper chamber.

[0057] 4) Transwell results such as Figure 4 As shown, compared with the cells in the blank control group, the migration and invasion speed of the cells in the overexpression CPT2 and mutant groups were significantly reduced.

[0058] Example 5: Observation of the effect of CPT2 on tumor proliferation in a subcutaneous transplant model

[0059] The constructed stable overexpression GV341-CPT2 cells and blank control GV341-vector cells were cultured at 10 7 The nude mice were inoculated subcutaneously with 100 μg / ml of cytokines. The nude mice were killed 5 to 6 weeks later, the tumors were collected, and the tumor volume was measured. The results are shown in Figure 5 .like Figure 5 As shown, overexpression of CPT2 significantly inhibited the growth of transplanted tumors.

[0060] Sequence Listing

[0061] Sequence 1

[0062] >CPT2 protein sequence

[0063] MVPRLLLRAWPRGPAVGPGAPSRPLSAGSGPGQYLQRSIVPTMHYQDSLPRLPIPKLEDTIRRYLSAQKPLLNDGQFRKTEQFCKSFENGIGKELHEQLVALDKQNKHTSYISGPWFDMYLSARDSVVLNFNPFMAFNPDPKSEYNDQLTRATNMTVSAIRFLK TLRAGLLEPEVFHLNPAKSDTITFKRLIRFVPSSSLSWYGAYLVNAYPLDMSQYFRLFNSTRLPKPSRDELFTDDKARHLLVLRKGNFYIFDVLDQDGNIVSPSEIQAHLKYILSDSSPAPEFPLAYLTSENRDIWAELRQKLMSSSGNEESLRKVDSAVFCLCLDD FPIKDLVHLSHNMLHGDGTNRWFDKSFNLIIAKDGSTAVHFEHSWGDGVAVLRFFNEVFKDSTQTPAVTPQSQPATTDSTVTVQKLNFELTDALKTGITAAKEKFDATMKTLTIDCVQFQRGGKEFLKKQKLSPDAVAQLAFQMAFLRQYGQTVATYESCSTAA FKHGRTETIRPASVYTKRCSEAFVREPSRHSAGELQQMMVECSKYHGQLTKEAAMGQGFDRHLFALRHLAAAKGIILPELYLDPAYGQINHNVLSTSTLSSPAVNLGGFAPVVSDGFGVGYAVHDNWIGCNVSSYPGRNAREFLQCVEKALEDMFDALEGKSIKS

[0064] Sequence 2

[0065] >CPT2-G600E protein sequence

[0066] MVPRLLLRAWPRGPAVGPGAPSRPLSAGSGPGQYLQRSIVPTMHYQDSLPRLPIPKLEDTIRRYLSAQKPLLNDGQFRKTEQFCKSFENGIGKELHEQLVALDKQNKHTSYISGPWFDMYLSARDSVVLNFNPFMAFNPDPKSEYNDQLTRATNMTVSAIRFLK TLRAGLLEPEVFHLNPAKSDTITFKRLIRFVPSSSLSWYGAYLVNAYPLDMSQYFRLFNSTRLPKPSRDELFTDDKARHLLVLRKGNFYIFDVLDQDGNIVSPSEIQAHLKYILSDSSPAPEFPLAYLTSENRDIWAELRQKLMSSSGNEESLRKVDSAVFCLCLDD FPIKDLVHLSHNMLHGDGTNRWFDKSFNLIIAKDGSTAVHFEHSWGDGVAVLRFFNEVFKDSTQTPAVTPQSQPATTDSTVTVQKLNFELTDALKTGITAAKEKFDATMKTLTIDCVQFQRGGKEFLKKQKLSPDAVAQLAFQMAFLRQYGQTVATYESCSTAA FKHGRTETIRPASVYTKRCSEAFVREPSRHSAGELQQMMVECSKYHGQLTKEAAMGQGFDRHLFALRHLAAAKGIILPELYLDPAYGQINHNVLSTSTLSSPAVNLEGFAPVVSDGFGVGYAVHDNWIGCNVSSYPGRNAREFLQCVEKALEDMFDALEGKSIKS

[0067] Sequence 3

[0068] >CPT2-G600R protein sequence

[0069] MVPRLLLRAWPRGPAVGPGAPSRPLSAGSGPGQYLQRSIVPTMHYQDSLPRLPIPKLEDTIRRYLSAQKPLLNDGQFRKTEQFCKSFENGIGKELHEQLVALDKQNKHTSYISGPWFDMYLSARDSVVLNFNPFMAFNPDPKSEYNDQLTRATNMTVSAIRFLKTLRAGLLEPEVFHLNPAKSDTITFKRLIRFVPSSLSWYGAYLVNAYPLDMSQYFRLFNSTRLPKPSRDELFTDDKARHLLVLRKGNFYIFDVLDQDGNIVSPSEIQAHLKYILSDSSPAPEFPLAYLTSENRDIWAELRQKLMSSGNEESLRKVDSAVFCLCLDDFPIKDLVHLSHNMLHGDGTNRWFDKSFNLIIAKDGSTAVHFEHSWGDGVAVLRFFNEVFKDSTQTPAVTPQSQPATTDSTVTVQKLNFELTDALKTGITAAKEKFDATMKTLTIDCVQFQRGGKEFLKKQKLSPDAVAQLAFQMAFLRQYGQTVATYESCSTAAFKHGRTETIRPASVYTKRCSEAFVREPSRHSAGELQQMMVECSKYHGQLTKEAAMGQGFDRHLFALRHLAAAKGIILPELYLDPAYGQINHNVLSTSTLSSPAVNLRGFAPVVSDGFGVGYAVHDNWIGCNVSSYPGRNAREFLQCVEKALEDMFDALEGKSIKS

[0070] Sequence 4

[0071] >CPT2 DNA sequence (1977 nt):

[0072]

[0073] Sequence 5

[0074] >CPT2-G600E DNA sequence (1977 nt):

[0075]

[0076] Sequence 6

[0077] >CPT2-G600R DNA sequence (1977 nt):

[0078]

Claims

1. The use of CPT2 protein in the preparation of anti-renal cancer drugs, characterized in that: The CPT2 protein sequence is shown in Sequence 1.

2. The use of CPT2 mutant CPT2-G600E in the preparation of anti-renal cancer drugs, characterized in that: The CPT2-G600E protein sequence is shown in Sequence 2.

3. The use of CPT2 mutant CPT2-G600R in the preparation of anti-renal cancer drugs, characterized in that: The CPT2-G600R protein sequence is shown in Sequence 3.

4. Use of a gene encoding CPT2 protein or its mutant in the preparation of an anti-renal cancer drug, wherein the mutant is CPT2-G600E or CPT2-G600R.

5. The use according to claim 4, characterized in that The gene is a DNA molecule as shown in sequence 4, sequence 5, or sequence 6.

6. Use of an expression cassette, a recombinant vector, a transgenic cell line or a recombinant bacterium containing the gene as claimed in claim 4 in the preparation of an anti-renal cancer drug.

7. The use according to any one of claims 1 to 6, characterized in that: The drug has at least one of the following functions: 1) Inhibit the proliferation of renal cancer cells; 2) Inhibit renal cancer cell migration; 3) Inhibit the invasive ability of renal cancer cells.

8. The use according to claim 7, characterized in that The renal cancer cells are at least one of 786-O and Caki-1.

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