A biomarker for screening drug-resistant lung cancer

Abstract

The present invention relates to the field of molecular diagnosis technology, and in particular to a biomarker for screening drug-resistant lung cancer, the transcription factor myeloid zinc finger gene 1 splice variant 2 (MZF1 L ) and myeloid zinc finger gene 1 splice variant 3 (MZF1 s ) showed significant differences between sensitive and resistant cells, namely MZF1 L / MZF1 S Co-expression of MZF1 in drug-resistant lung cancer cells L Drop MZF1 S Up-regulation pattern; overexpression of MZF1 was also found L By regulating the localization of EGFR on the cell membrane, the sensitivity of cells to EGFR-TKIs can be increased. Therefore, this specific molecule can be used as a biomarker combination for drug-resistant lung cancer patients to screen for drug-resistant patients during lung cancer treatment, providing a new means of accurate diagnosis and treatment for drug-resistant patients in lung cancer treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular diagnosis, and in particular to a biomarker for screening drug-resistant lung cancer. Background Art

[0002] Lung cancer is a highly prevalent cancer worldwide and ranks first among malignant tumors in my country. Non-small cell lung cancer (NSCLC) is the most common histological type of lung cancer, accounting for approximately 85% of lung cancer patients (Siegel RL, Miller KD, Fuchs HE, Jemal A. Cancer statistics, 2022. CA Cancer J Clin 2022, 72(1):7-33). EGFR mutation is an important oncogenic driver in NSCLC, opening the door to biomarker-guided treatment for patients with advanced disease (Cooper AJ, Sequist LV, Lin JJ. Third-generation EGFR and ALK inhibitors: mechanisms of resistance and management. Nature reviews Clinical oncology 2022.). Many EGFR tyrosine kinase inhibitors (EGFR-TKIs) have been developed, including the commonly used first-generation TKIs gefitinib and the highly effective third-generation TKIs osimertinib. Despite the remarkable efficacy of EGFR-TKIs in treating patients, resistance to them remains a fundamental unresolved challenge.

[0003] There are currently two mainstream views on the development of acquired resistance to EGFR-TKIs, namely, changes in the targeted kinase (such as EGFR C797S mutation (Thress KS, Paweletz CP, Felip E, Cho BC, Stetson D, Dougherty B, Lai Z, Markovets A, Vivancos A, Kuang Y, Ercan D, Matthews SE, Cantarini M, Barrett JC, PA, Oxnard GR.Acquired EGFR C797S mutation mediates resistance to AZD9291in non-small cell lung cancer harboring EGFR T790M.Nature medicine 2015,21(6):560-562)) and changes in downstream signaling pathways of the designed targets (such as BRAF fusion, KRAS mutation, NRAS mutation, MAP2K1 mutation in the RAS-MAPK pathway (SchoenfeldAJ, Chan JM, Kubota D, Sato H, Rizvi H, DaneshbodY, Chang JC, Paik PK, Offin M, Arcila ME, Davare MA, Shinde U, Pe'er D, Rekhtman N, Kris MG, Somwar R, Riely GJ, Ladanyi M, Yu HA.TumorAnalyses Reveal SquamousTransformation and Off-TargetAlterations As Early Resistance Mechanisms to First-line Osimertinib in EGFR-Mutant Lung Cancer.Clinical cancer research:affiliate journal of the American Association for Cancer Research 2020,26(11):2654-2663.)). However, most of these studies focused on genetic changes in EGFR-related genes. Epigenetic changes often occur in patients with EGFR-TKIs resistance, and regulating their epigenetic changes can enhance the cytotoxic effect of anti-tumor therapy in patients (Chen Z,Chen Q,Cheng Z,Gu J,Feng W,Lei T,Huang J,Pu J,Chen X,Wang Z.Long non-coding RNA CASC9 promotes gefitinib resistance in NSCLC by epigenetic repression of DUSP1.Cell death&disease 2020,11(10):858.).Compared with permanent gene mutations, epigenetic changes that control gene expression can gradually regulate cell differentiation and malignant transformation after cell resistance, play an important role, and can serve as potential sensitive markers and drug targets for drug resistance biological monitoring.

[0004] Zinc finger proteins bind Zn 2+ It forms a stable self-folding "finger" structure and is closely related to a variety of pathological processes including cancer invasion and metastasis and anti-tumor drug resistance. Therefore, it is regarded as an important molecular target (Jen J, Liu CY, Chen YT, Wu LT, Shieh YC, Lai WW, Wang YC. Oncogenic zinc finger protein ZNF322A promotes stem cell-like properties in lung cancer through transcriptional suppression of c-Myc expression. Cell death and differentiation 2019, 26(7): 1283-1298. Lin S, Ruan H, Qin L, Zhao C, Gu M, Wang Z, Liu B, Wang H, Wang J. Acquired resistance to EGFR-TKIs in NSCLC mediatesepigenetic downregulation of MUC17 by facilitating NF-κB activity via UHRF1 / DNMT1complex. International journal of biological sciences 2023, 19(3):832-851.) Zinc activates EGFR by activating intracellular Src and metalloproteinases. Activated EGFR induces airway inflammation by upregulating the expression of inflammatory proteins. Members of the zinc finger protein family play an important role in regulating the expression and activity of EGFR.

[0005] Methyl-sensitive zinc finger protein myeloid zinc finger 1 (MZF1) belongs to the Krüppel-like family of transcription factors and is involved in cell proliferation and differentiation. MZF1 itself is known to have three splice variants (MZF1-V1, MZF1-V2, and MZF1-V3). Among them, MZF1-V1 and MZF1-V2 have the same coding region, that is, they encode the complete isoform 1 (Isoform 1, MZF1 L) has functional domains including acidic domain, SCAN domain, TAD domain, and DNA binding domain, which is composed of 13 zinc fingers. The core sequence recognized by the C2H2 type zinc domain is rich in G sites, which is a methyl-sensitive transcription factor. MZF1-V3 encodes a truncated isoform 2 (Isoform 2, MZF1) that lacks the carboxyl C-terminus. S ), thus resembling a truncated DNA binding domain of MZF1. In our work, we found that the DNA binding abilities of the zinc finger domains of the two protein isoforms are different, which may play an important role in the transcriptional regulation of genes. Since the zinc finger domains of MZF1 often cluster to bind divalent cations (such as Zn 2+ ) and is methylation-sensitive. Therefore, the expression of different splice variants of MZF1 has different effects on EGFR phosphorylation activation, which can provide more reliable molecular markers and drug targets for precise diagnosis and personalized treatment. Summary of the Invention

[0006] The present invention found in the study that the transcription factor myeloid zinc finger gene 1 splice variant 2 (MZF1 L ) and myeloid zinc finger gene 1 splice variant 3 (MZF1 s ) showed significant differences between sensitive and resistant cells, namely MZF1 L / MZF1 S Co-expression of MZF1 in drug-resistant lung cancer cells L Drop MZF1 S Up-regulation pattern; overexpression of MZF1 was also found L The sensitivity of cells to EGFR-TKIs can be increased by regulating the localization of EGFR on the cell membrane. Based on this, the present invention was completed.

[0007] In a first aspect, the present invention provides a biomarker composition for screening drug-resistant lung cancer patients, the composition comprising MZF1 L and MZF1 S , among which, when MZF1 was detected in lung cancer patients L Low expression, MZF1 s When the expression level is high and MZF1 s Expression level relative to MZF1 L When the expression level of α is high, the patient can be judged as a drug-resistant patient.

[0008] Furthermore, the drug-resistant lung cancer patient is a patient resistant to EGFR-TKI inhibitor drugs.

[0009] Furthermore, the EGFR-TKI inhibitors include but are not limited to gefitinib, osimertinib, erlotinib, icotinib and afatinib.

[0010] Furthermore, the MZF1 L The nucleotide sequence of MZF1 is shown in SEQ ID NO.1; L The amino acid sequence is shown in SEQ ID NO.2;

[0011] Furthermore, the MZF1 s The nucleotide sequence is shown in SEQ ID NO.3; MZF1 s The amino acid sequence is shown in SEQ ID NO.3.

[0012] In a second aspect, the present invention provides a kit for detecting drug-resistant lung cancer patients, wherein the kit contains reagents for detecting a biomarker composition, wherein when MZF1 is detected in a lung cancer patient, L Low expression, MZF1 s When the expression level is high and MZF1 s Expression level relative to MZF1 L When the expression level of MZF1 is high, the patient can be judged as a drug-resistant patient. The biomarker composition is as described in the first aspect. L and MZF1 S .

[0013] In a third aspect, the present invention provides a group of biomarker compositions for use in preparing a reagent for diagnosing drug-resistant lung cancer patients, wherein when MZF1 is detected in lung cancer patients, L Low expression, MZF1 s When the expression level is high and MZF1 s Expression level relative to MZF1 L When the expression level of MZF1 is high, the patient can be judged as a drug-resistant patient. The biomarker composition is as described in the first aspect. L and MZF1 S .

[0014] Furthermore, the drug-resistant lung cancer patient is a patient resistant to EGFR-TKI inhibitor drugs.

[0015] Furthermore, the EGFR-TKI inhibitors include but are not limited to gefitinib, osimertinib, erlotinib, icotinib and afatinib.

[0016] In a fourth aspect, the present invention provides a drug for improving or enhancing the sensitivity of drug-resistant lung cancer patients to EGFR-TKI inhibitors, wherein the drug contains MZF1 L protein.

[0017] Furthermore, the MZF1 L The amino acid sequence of the protein is shown in SEQ ID NO.2.

[0018] Furthermore, the MZF1 L The protein amino acid sequence also includes 80%-99% homologous sequences, preferably 80%-85%; preferably 85%-90%; preferably 90%-95%.

[0019] Furthermore, the MZF1 L The protein amino acid sequence also includes mutations, deletions, substitutions or additions of 1-20 amino acids in its sequence. L The number of amino acids mutated, deleted, substituted or added in the protein amino acid sequence is preferably 1-15; preferably 1-10, preferably 1-5; preferably 5-20, preferably 10-20.

[0020] Furthermore, the mutation, deletion, substitution or addition of amino acids includes mutation, deletion, substitution or addition of consecutive amino acids simultaneously or at different times.

[0021] Furthermore, the mutation, deletion, substitution or addition of amino acids also includes mutation, deletion, substitution or addition between discontinuous amino acids at the same time or at different times.

[0022] Furthermore, the MZF1 L The protein also includes its fusion protein, conjugate, nucleic acid encoding its fusion protein or conjugate, and vector expressing the aforementioned nucleic acid molecule.

[0023] Furthermore, the drug may further contain a pharmaceutically acceptable carrier.

[0024] Furthermore, the drug includes but is not limited to an injectable preparation, a gel, a suspension, an emulsion, a polymer, nanoparticles, microspheres, a rectal capsule, an enema, a paste, a drink, and an implant, and can optionally be controlled and / or sustained-released through a dosage form or device.

[0025] Furthermore, the drug-resistant lung cancer patient is a patient resistant to EGFR-TKI inhibitor drugs.

[0026] Furthermore, the EGFR-TKI inhibitors include but are not limited to gefitinib, osimertinib, erlotinib, icotinib and afatinib.

[0027] In a sixth aspect, the present invention provides a MZF1 L The protein is used in the preparation of a drug for improving or enhancing the sensitivity of drug-resistant lung cancer patients to EGFR-TKI inhibitors.

[0028] Furthermore, the MZF1 L The amino acid sequence of the protein is shown in SEQ ID NO.2.

[0029] Furthermore, the MZF1 L The protein amino acid sequence also includes 80%-99% homologous sequences, preferably 80%-85%; preferably 85%-90%; preferably 90%-95%.

[0030] Furthermore, the MZF1 L The protein amino acid sequence also includes mutations, deletions, substitutions or additions of 1-20 amino acids in its sequence. L The number of amino acids mutated, deleted, substituted or added in the protein amino acid sequence is preferably 1-15; preferably 1-10, preferably 1-5; preferably 5-20, preferably 10-20.

[0031] Furthermore, the mutation, deletion, substitution or addition of amino acids includes mutation, deletion, substitution or addition of consecutive amino acids simultaneously or at different times.

[0032] Furthermore, the mutation, deletion, substitution or addition of amino acids also includes mutation, deletion, substitution or addition between discontinuous amino acids at the same time or at different times.

[0033] Furthermore, the MZF1 L The protein also includes its fusion protein, conjugate, nucleic acid encoding its fusion protein or conjugate, and vector expressing the aforementioned nucleic acid molecule.

[0034] Beneficial effects

[0035] This study found that the transcription factor myeloid zinc finger gene 1 splice variant 2 (MZF1 L ) and myeloid zinc finger gene 1 splice variant 3 (MZF1 s ) showed significant differences between sensitive and resistant cells, namely MZF1 L / MZF1 S Co-expression of MZF1 in drug-resistant lung cancer cells L Drop MZF1 S Up-regulation pattern; overexpression of MZF1 was also found L By regulating the localization of EGFR on the cell membrane, the sensitivity of cells to EGFR-TKIs can be increased. Therefore, this specific molecule can be used as a biomarker combination for drug-resistant lung cancer patients to screen for drug-resistant patients during lung cancer treatment, providing a new means of accurate diagnosis and treatment for drug-resistant patients in lung cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1Effect of gefitinib / osimertinib resistance on MZF1 splicing variants. A. RT-qPCR and Western blot show the mRNA expression of MZF1 splicing variants in resistant cells; B. Schematic diagram of clinical case collection; C. RT-qPCR shows the mRNA expression of MZF1 splicing variants in clinical samples; DG. Multicolor immunofluorescence detection of MZF1 splicing variant expression in clinical tissues with inflammation (CP), before treatment (BT), gefitinib resistance (GR), and osimertinib resistance (OR), *p<0.05, **p<0.01.

[0037] Figure 2 Effects of overexpression of MZF1 splicing variants on the function of gefitinib- and osimertinib-resistant cells; A. RT-qPCR and Western blot were used to detect the expression of MZF1 splicing variants in resistant cells after overexpression of each splicing variant; B. EGFR-TKIs-sensitive and -resistant lung cancer cell lines were selected, and transfected cell lines expressing exogenous and stable high expression of MZF1 splicing variants were established; MTT assay (B), colony formation assay (C), and Transwell assay (D) were used to detect the effects of overexpression of each splicing variant of MZF1 on cell proliferation and invasion. Quantitative data are shown in the upper half of the figure, and representative images are shown in the lower half of the figure. *p<0.05, **p<0.01.

[0038] Figure 3 Effects of overexpressing MZF1 splice variants on the function of gefitinib- and osimertinib-resistant cells. AC. MTT, colony formation assays, and Transwell assays show the effects of gefitinib (1 μM) / osimertinib (0.1 μM)-treated cells overexpressing MZF1 splice variants on cell viability (A), colony formation ability (B), and invasion and migration (C). Quantitative data are shown in the upper half of the figure, and representative images are shown in the lower half. D. p-EGFR expression was measured in resistant cells treated with gefitinib, osimertinib, and / or transfected with MZF1 splice variants. E. Protein levels of EGFR subunits in resistant cells treated with gefitinib, osimertinib, and / or transfected with MZF1 splice variants. *p < 0.05, **p < 0.01. DETAILED DESCRIPTION

[0039] The following is a further description of specific embodiments of the present invention. It should be noted that the description of these embodiments is intended to facilitate understanding of the present invention and does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below may be combined with each other as long as they do not conflict with each other.

[0040] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0041] The term "nucleic acid" as used herein refers to any one or more nucleic acid segments present in a polynucleotide, such as DNA or RNA fragments. For the purposes of the present invention, the nucleotide sequence encoding the MZF1 splice variant protein contained in the vector may be isolated or may be the sequence of the MZF1 splice variant gene.

[0042] The term "fusion protein" as used herein refers to a protein that comprises amino acids in addition to the amino acid sequence encoding the original or native full-length protein or a subsequence thereof, an amino acid sequence that replaces the amino acid sequence encoding the original or native full-length protein or a subsequence thereof, an amino acid sequence that is less than the amino acid sequence encoding the original or native full-length protein or a subsequence thereof, and / or an amino acid sequence that is different from the amino acid sequence encoding the original or native full-length protein or a subsequence thereof.

[0043] The term "mutation" as used herein refers to a change in the nucleotide sequence of the genome of an organism, virus or extrachromosomal DNA, including sequence changes caused by base substitution, DNA insertion, DNA deletion or DNA duplication; the mutation described in the present invention can be an amino acid mutation in the amino acid sequence of the MZF1 splice variant protein, or a mutation in the sequence of its MZF1 splice variant gene.

[0044] The term "composition" or "pharmaceutical composition" as used herein may include a composition comprising the MZF1 splice variants described herein and, for example, a pharmaceutically acceptable carrier, excipient, or diluent, which is administered to a subject.

[0045] The term "pharmaceutically acceptable" refers to compositions that are suitable for use in contact with human and animal tissues without excessive toxicity or other complications, commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment. In some aspects, the MZF1 splice variant proteins, compositions, and vaccines described herein are pharmaceutically acceptable.

[0046] The MZF1 involved in the present invention L The nucleotide sequence is ( SEQ ID NO.1 )：atgaggcctgcggtgctgg gctccccaga ccgagcaccc ccagaagatg aggggcctgt catggtgaag ctagaggactctgaggagga gggtgaggct gccttatggg acccaggccc tgaagctgca cgcctgcgtt tccggtgcttccgctatgag gaggccacag ggccccaaga ggccctggcc cagctccgag agctgtgtcg ccagtggctgcgtccagagg tacgctccaa ggagcagatg ctggagctgt tggtgctgga gcagttcctg ggcgcactgccccctgagat ccaggcccgt gtgcaggggc agcggccagg cagccccgag gaggctgctg ccctagtagatgggctgcgc cgggagccgg gcggaccccg gagatgggtc acagtccagg tgcagggcca ggaggtcctatcagagaaga tggagccctc cagtttccag cccctacctg aaactgagcc tccaactcca gagcctgggcccaagacacc tcctaggact atgcaggaat caccactggg cctgcaggtg aaagaggagt cagaggttacagaggactca gatttcctgg agtctgggcc tctagctgcc acccaggagt ctgtacccac cctcctgcctgaggaggccc agagatgtgg gaccgtgctg gaccagatct ttccccacag caagactggg cctgagggtccctcatggag ggagcacccc agggccctgt ggcatgagga agctgggggc atcttctccc cagggttcgcgctgcagcta ggcagcatct ccgcaggtcc aggtagtgta agccctcacc tccacgtccc ctgggacctcggcatggctg gcctttctgg ccagatccaa tcaccctccc gcgaaggtgg ctttgcgcatgcgcttctgctccccagcga tctgaggagt gaacaggacc ccacggacga ggatccctgc cggggtgtgg gccctgctctgatcaccacc cgctggcgct cccccagggg ccggagccgg ggccgcccca gcactggggg cggggtggttaggggcggcc gttgcgatgt atgtggcaag gtgttcagcc aacgcagcaa cctgctgagg caccagaagatccacacggg tgagcgacca ttcgtgtgca gcgagtgcgg ccgcagcttc agccgcagct cgcacctgctgcgccaccag cttacgcaca ccgaggagcg gccgttcgtg tgcggcgact gtggccaggg cttcgtgcgcagcgcgcgcc tggaagagca tcggagagtg cacacgggcg aacagccttt ccgttgcgct gagtgcggccagagcttccg gcagcgctcc aatctgctgc agcaccagcg catccacggc gatcccccgg gccctggcgctaagcccccg gcccctcctg gtgcgcccga gcctcccggc ccctttccgt gcagcgagtg ccgcgagagcttcgcgcggc gcgccgtgct gctggagcac caggcggtac acacgggcga caagtccttt ggctgcgtcgagtgcggcga gcgcttcggc cgccgctcag tgctgctgca gcaccggcgc gtgcacagtg gcgagcggcccttcgcctgt gccgagtgcg gccagagctt ccggcagcgc tccaacctga cgcagcaccg gcgcatccacaccggggagc ggcccttcgc ctgcgccgag tgtggcaagg ccttccgcca gcggcctacg ctcacgcagcatctccgcgt acacacgggc gagaaaccct ttgcctgccc cgagtgtggc cagcgcttcagccagcgcctcaagctcacg cgtcatcaga ggacacacac cggcgaaaag ccctaccact gcggtgagtg cggcctgggcttcacgcagg tctcgcggct caccgagcac cagcgcatcc acacgggcga acggcccttc gcctgccccgagtgcggcca gagctttcgg cagcacgcca acctcaccca gcaccggcgc atccacacgg gtgaacggccctacgcatgc cctgagtgtg gcaaggcctt ccgccagcgg cccacgctca cgcagcatct gcgcacccaccgacgagaga agcccttcgc ctgccaggac tgtggccgcc gcttccacca gagcaccaag ctcattcagcaccagcgcgt ccacagcgcc gagtag。

[0047] MZF1 involved in the present invention L The amino acid sequence of SEQ ID NO.2 )：mrpavlgspd rappedegpvmvkledseee geaalwdpgp eaarlrfrcf ryeeatgpqe alaqlrelcr qwlrpevrsk eqmlellvleqflgalppei qarvqgqrpg speeaaalvd glrrepggpr rwvtvqvqgq evlsekmeps sfqplpetepptpepgpktp prtmqesplg lqvkeesevt edsdflesgp laatqesvpt llpeeaqrcg tvldqifphsktgpegpswrehpralwhee aggifspgfa lqlgsisagp gsvsphlhvp wdlgmaglsgqiqspsreggfahalllpsd lrseqdptde dpcrgvgpal ittrwrsprg rsrgrpstgggvvrggrcdvcgkvfsqrsn llrhqkihtg erpfvcsecg rsfsrsshll rhqlthteerpfvcgdcgqgfvrsarleeh rrvhtgeqpf rcaecgqsfr qrsnllqhqr ihgdppgpgakppappgapeppgpfpcsec resfarravl lehqavhtgd ksfgcvecge rfgrrsvllqhrrvhsgerpfacaecgqsf rqrsnltqhr rihtgerpfa caecgkafrq rptltqhlrvhtgekpfacpecgqrfsqrl kltrhqrtht gekpyhcgec glgftqvsrl tehqrihtge rpfacpecgqsfrqhanltq hrrihtgerp yacpecgkafrqrptltqhl rthrrekpfa cqdcgrrfhqstkliqhqrvhsae。

[0048] MZF1 involved in the present invention s The nucleotide sequence is ( SEQ ID NO. 3):atgaggcct gcggtgctgggctccccaga ccgagcaccc ccagaagatg aggggcctgt catggtgaag ctagaggact ctgaggaggagggtgaggct gccttatggg acccaggccc tgaagctgca cgcctgcgtt tccggtgctt ccgctatgaggccccccggc agctgtgtcg ccagtggctg cgtccagaggtacgctccaa ggagcagatg ctggagctgt tggtgctgga gcagttcctg ggcgcactgc cccctgagatccaggcccgt gtgcaggggc agcggccagg cagccccgag gaggctgctg ccctagtaga tgggccccgc gagatgggtc acagtccagg tgcagggcca ggaggtccta tcagagaagatggagccctc cagtttccag ccctacctg aaactgagcc tccaactcca gagcctgggc ccaagacacctcctaggact atgcaggaat caccactggg cctgcaggtg aagaggagt cagaggccctc tctagctgcc acccaggagt ctgtacccac cctcctgcct gaggaggcccagagatgtgg gaccgtgctg gaccagatct ttccccacag caagactggg cctgagggtc cctcatggaggggcacccc agggccctgt ggcatgagga agctgggggc atcttctccc caggggcc gtggttaggg gcggccgttg cgatgtatgt ggcaaggtgt tcagccaacgcagcaacctg ctga

[0049] MZF1 involved in the present invention s The amino acid sequence of SEQ ID NO.4)： mrpavlgspd rappedegpvmvkledseee geaalwdpgp eaarlrfrcf ryeeatgpqe alaqlrelcr qwlrpevrsk eqmlellvleqflgalppei qarvqgqrpg speeaaalvd glrrepggpr rwvtvqvqgq evlsekmeps sfqplpetepptpepgpktp prtmqesplg lqvkeesevt edsdflesgp laatqesvpt llpeeaqrcg tvldqifphsktgpegpswr ehpralwhee aggifspgag agaapalgag wlgaavamyv arcsanaatc.

[0050] Example 1 Detection of the relationship between the expression of MZF1 splicing variants in sensitive and resistant lung cancer cells and EGFR-TKI resistance

[0051] 1.1 Materials

[0052] The non-small cell lung cancer cells used for the test: HCC827 and PC9, were purchased from the National Biomedical Experimental Cell Resource Bank and cultured and passaged under normal conditions in the applicant's laboratory.

[0053] There were 57 bronchoalveolar lavage fluid samples for testing, including 10 patients with pneumonia, 22 patients with lung cancer before treatment, 13 patients with gefitinib resistance, and 12 patients with osimertinib resistance.

[0054] Paraffin-embedded tissues for testing: 180 tissues, including 61 pneumonia patients, 47 lung cancer patients before treatment, 38 gefitinib-resistant patients, and 33 osimertinib-resistant patients.

[0055] 1.2 Methods

[0056] 1.2.1 Cell RNA extraction and reverse transcription process

[0057] (1) The cell RNA extraction process is to select cells with good growth status and extract them at 1mL / 10 6 100 cells were added to Trizol reagent (Invitrogen, USA, Cat. No. 15596026). Tissue RNA extraction involved removing frozen tissue from liquid nitrogen, cutting approximately 200 mg, and grinding the tissue specimen in a cold mortar. 1 mL of Trizol reagent was added for every 100 mg of tissue specimen.

[0058] (2) Incubate at room temperature for 10 minutes. After complete lysis, extract with chloroform by adding 0.2 ml of chloroform per 1 ml of Trizol. Shake vigorously for 15 seconds, incubate at room temperature for 5 minutes, and centrifuge at 12,000 g at 4°C for 15 minutes.

[0059] (3) Transfer the colorless upper layer to a new centrifuge tube and precipitate with pre-cooled isopropanol (0.5 mL of isopropanol per 1 mL of Trizol). Place on ice for 20 min. Centrifuge at 12,000 g at 4°C for 10 min and discard the supernatant.

[0060] (4) Wash the precipitate with pre-cooled 75% ethanol. Add 1 mL of 75% ethanol to 1 mL of Trizol and centrifuge at 7500 g for 5 min at 4°C. Discard the supernatant. After drying, add an appropriate amount of DEPC-H2O to dissolve the precipitate. Confirm the RNA concentration by electrophoresis on a 0.8% agarose gel. Measure the RNA concentration by NanoDrop. Store at -80°C.

[0061] (5) 1.0 μg of RNA was reverse transcribed into cDNA using the TransScript II First-Strand cDNA Synthesis SuperMix Kit (Cat. No. AH301-02) produced by Beijing Quanshijin Biotechnology Co., Ltd.: 1 μL Anchored Oligo(dT)20, 10 μL 2×TS Reaction Mix, and 1 μL RT / RI Enzyme Mix were added to 20 μL of DEPC-H2O. The reaction conditions were 42°C for 30 min and 85°C for 5 min. The reverse transcribed cDNA was stored at −20°C.

[0062] 1.2.2 qPCR detection

[0063] (1) qPCR primer sequences:

[0064] Primer pair 1 (SEQ ID No: 1): MZF1 L mRNA common region primer (NM_198055.2, size 339 bp) upstream primer: 5'-GGGGGCATCTTCTCCCCA-3'; downstream primer: 5'-CACCTTGCCACATACATCGC-3'.

[0065] Primer pair 2 (SEQ ID No: 2): MZF1 S mRNA primer (NM_001267033.2, size 116 bp) upstream primer: 5'-CCGTGCTGGACCAGATCTTT-3'; downstream primer: 5'-GGCCCCTGGGGAGAAGA-3'.

[0066] Wherein, the internal reference primer used for normalization is a primer using Beta-Aactin as an internal reference,

[0067] Upstream primer: 5′-TTAGTTGCGTTACACCCTTTC-3′;

[0068] Downstream primer: 5′-ACCTTCACCGTTCCAGTTT-3′.

[0069] (2) Reaction system for qPCR amplification

[0070] As shown in Table 1.

[0071] Table 1 qPCR amplification reaction system

[0072]

[0073] Note: 2×SYBR-Green: Zymo Research, USA, Cat. No. E2004.

[0074] (3) PCR reaction conditions are as follows:

[0075] 50℃ 2min, 95℃ 10min, 95℃ 15s; 60℃ 1min; 40 cycles, collecting fluorescence; 95℃ 15s, 60℃ 1min, 95℃ 15s, 60℃ 15s, to make melting curve. -ΔΔCt The data were analyzed by the method, and the melting curve ensured the specificity of the product.

[0076] 1.2.3 Western blotting analysis

[0077] (1) Sample preparation

[0078] The lung cancer cells to be tested were inoculated into a culture dish with a diameter of 10 cm. When the cells grew to about 60-80%, 3-5 culture dishes were collected from each group.

[0079] (2) Collect and wash samples

[0080] Wash cells 1-2 times with room temperature preheated PBS, then treat with 1% formalin at 37°C for 10 minutes. Wash cells twice with ice-cold PBS, then use a cell scraper to remove cells into 1 ml of ice-cold PBS. Centrifuge at 3,000 rpm for 2 minutes at 4°C, and discard the supernatant. Resuspend the cell pellet in 400 μl of lysis buffer and incubate on ice for 10 minutes.

[0081] (3) Western blotting

[0082] After quantification, the sample was loaded at 30 μg / well. The sample was added to 5× SDS gel loading buffer (Genstar, China, catalog number: E153-05), heated at 100°C for 10 minutes, and the sample was loaded sequentially. SDS-PAGE electrophoresis was performed at 120V. After electrophoresis, the membrane was transferred to a wet electroporator. After transfer, the PVDF membrane (Millipore, USA, catalog number: IPVH00010) was removed. After blocking with 5% skim milk for 1 hour, the primary antibody: MZF1 was incubated. L Antibodies (Wuhan Dian Biotechnology Co., Ltd., antigen immunization: RPFACAECGQSFRQRSNLTQHRRIHTGERPFACAECGKAFRQRPTLTQHLRVHTGEKPFACPECGQRFSQRLKLTRHQRTHTGEKPYHCGECGLGFTQVSRLTEHQRIHTGERPFACPECGQSFRQHANLTQHRRIHTGERPYACPECGKAFRQRPTLTQHLRTHRREKPFACQDCGRRFHQSTKLIQHQRVHSAE), MZF1 S Antibodies (Wuhan Dian Biotechnology Co., Ltd., antigen immunoassay: LGAGWLGAAVAMYVARC) were added overnight at 4°C. Secondary antibodies labeled with anti-rabbit horseradish peroxidase (abcam, UK, cat. no. ab6721) were added and incubated for 1 hour. ECL chemiluminescence kit (ThemoFisher, USA, cat. no. 32132) was added for color development, and images were developed using the Smart Gel Image Analysis System.

[0083] 1.2.4 Multicolor immunohistochemistry analysis

[0084] Multicolor immunohistochemical analysis of tissue arrays was performed using the Opal 7-color fluorescent immunohistochemistry kit (Akoya Biosciences, USA, catalog number: NEL797001KT). 4 μm tissue sections were baked at 70°C for 1 hour. Dewaxed in xylene twice every 20 minutes. Hydrated in gradient ethanol (100%, 95%, 80%) for 5 minutes. The tissue sections were placed in citric acid repair solution, repaired under high temperature and high pressure, cooled to room temperature, and washed three times with 1×PBS for 5 minutes each. Incubated with 3% hydrogen peroxide solution at room temperature for 15 minutes, rinsed with double distilled water, and washed three times with 1×PBS for 5 minutes each. Blocked with 5% skim milk powder for 1 hour, poured off the milk, and washed three times with 1×PBS for 5 minutes each. Incubation primary antibody: CK antibody (Suzhou Baidao Medical Technology Co., Ltd., catalog number: PA125), MZF1 LAntibodies (Wuhan Dian Biotechnology Co., Ltd., antigen immunization: RPFACAECGQSFRQRSNLTQHRRIHTGERPFACAECGKAFRQRPTLTQHLRVHTGEKPFACPECGQRFSQRLKLTRHQRTHTGEKPYHCGECGLGFTQVSRLTEHQRIHTGERPFACPECGQSFRQHANLTQHRRIHTGERPYACPECGKAFRQRPTLTQHLRTHRREKPFACQDCGRRFHQSTKLIQHQRVHSAE), MZF1 S Antibodies (Wuhan Dian Biotechnology Co., Ltd., antigen immunoassay: LGAGWLGAAVAMYVARC), β-actin antibody (Sigma-Aldrich, USA, catalog number: A45551). Incubate at 4°C overnight. The next day, place at room temperature for half an hour and wash three times with 1× PBS, each time for 5 minutes. Add secondary antibody, incubate at room temperature for 30 minutes, and wash three times with 1× PBS, each time for 5 minutes. Multicolor fluorescence images were acquired using the TissueFAXS Spectra S system (tissuegnostic, USA), and protein expression was quantitatively analyzed using an automatic quantitative analysis system (tissuegnostic, USA, software version: StrataQuest7.0.1.165).

[0085] 1.3 Results and Analysis

[0086] First, the expression level of MZF1 in EGFR-TKIs sensitive cell line (HCC827) and resistant cell lines (HCC827 / GR and HCC827 / OR) was detected. The results showed that MZF1 L MZF1 is the predominantly expressed splice variant in drug-resistant cell lines. S The major expressed splice variant ( Figure 1 A).

[0087] Bronchoalveolar lavage fluid samples were collected for MZF1 detection in patients with inflammation (CP, n=10), before treatment (BT, n=22), gefitinib resistance (GR, n=13), and osimertinib resistance (OR, n=12). L and MZF1 S The expression ( Figure 1 BD), the results showed that before treatment, MZF1 L The expression of α-glucose kinase inhibitors was higher than that of gefitinib-resistant and osimertinib-resistant groups (p<0.01, Figure 1 C); MZF1 in the pre-treatment group S The expression of α-glucose was lower in the gefitinib-resistant and osimertinib-resistant groups (p<0.01, Figure 1 C).

[0088] We further investigated the clinical characteristics and MZF1 expression in 47 tumor samples (22 before treatment, 13 gefitinib-resistant, and 12 osimertinib-resistant). L with MZF1 S The expression of Figure 1 D) MZF1 L The expression of MZF1 is related to tumor invasion depth, lymph node metastasis, distant metastasis and clinical stage. S The expression of α-glucose is associated with lymph node metastasis, distant metastasis and clinical stage ( Figure 1 D, see Table 2 for details).

[0089] Table 2 Relationship between MZF1 expression and clinical characteristics

[0090]

[0091]

[0092] To further validate the correlation between the expression of MZF1 splice variants and clinical phenotypes, multiplex immunohistochemistry (mIHC) was performed using antibodies against MZF1 isoforms and cytokeratin (CK) in a cohort of 180 tissue samples, including those with inflammation (CP, n=61), before treatment (BT, n=47), gefitinib-resistant (GR, n=38), and osimertinib-resistant (OR, n=33).

[0093] The experimental results showed that MZF1 was expressed in patients with CP and BT. L Splice variant expression is higher than MZF1 S , MZF1 in patients in GR and OR groups S Splice variant expression is higher than MZF1 L ( Figure 1 E and F). Fluorescence quantitative analysis showed that MZF1 expression in tumor tissues was significantly higher than that in adjacent adjacent tissues. L Reduced expression of MZF1 S Expression increased, and MZF1 in the tumor tissue area of the drug-resistant group L+ MZF1 S- The expression was significantly lower than that in the sensitive group ( Figure 1 G).

[0094] These results indicate that MZF1 splicing variants are differentially expressed in EGFR-TKIs-resistant cell lines and tissues, that is, in the sensitive group, MZF1 L MZF1 is the main splice variant expressed in the drug-resistant group. SThe major expressed splice variants, *p<0.05, **p<0.01.

[0095] Example 2 Detection of the biological function of MZF1 splicing variants in sensitive and resistant lung cancer cells

[0096] The non-small cell lung cancer cells used for the test were HCC827 and PC9; the cells were purchased from the National Biomedical Experimental Cell Resource Bank and cultured and passaged under normal conditions in the applicant's laboratory.

[0097] 2.1 Overexpression of MZF1 in HCC827 and PC9 cells

[0098] Universal empty plasmid, purchased from GeneCopoeia, USA, catalog number: EX-NEG-M02; MZF1 L Plasmid, purchased from GeneCopoeia, catalog number: EX-T3148-M02-5. ; MZF1 S Plasmid, NM_001267033.2, was synthesized by Shanghai GeneChip.

[0099] HCC827 and PC9 cell lines, both sensitive and resistant to non-small cell lung cancer, were stably transfected. Cells were seeded in 6-well cell culture plates. When cells reached 60% confluency, 200 μL of DMEM was added to 20 nM expression plasmid and empty plasmid, respectively. Mix well and incubate at room temperature for 5 minutes. Then, 200 μL of DMEM was added to 6 μL of Lipofectamine 2000, mixed well, and incubated at room temperature for 5 minutes. 200 mL of Lipofectamine 2000 (Thermo Fisher Scientific, USA, Catalog No. 11668019) diluted solution was added to 200 μL of expression plasmid, mixed well, and incubated at room temperature for 20 minutes. 400 μL of the mixture was added to the cell culture plate and cultured at 37°C without serum for 8 hours. The cells were then replaced with 10% FBSDMEM and incubated at 37°C for 24 hours before analysis by RT-qPCR and Western blot.

[0100] 2.2 Cell biology function experiments

[0101] (1) MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) experiment

[0102] Cell lines containing the MZF1 gene from HCC827 and PC9 cells (both in the logarithmic growth phase and overexpressing the sensitive and resistant MZF1 genes, as constructed in step 1) were digested and counted. The cell suspension concentration was adjusted, and 100 μL was added to each well of a 96-well cell culture plate. 2000 cells were plated per well in eight replicates and observed for four consecutive days. 10 μL of MTT (Sigma, USA, Cat. No. M2128) was added to each well daily to a final concentration of 5 μg / mL. After incubation for 4 hours, the medium was discarded. After four days, 150 μL of dimethyl sulfoxide (DMSO, Sigma, USA, Cat. No. D2650) was added to each well. After gently shaking in the dark for 10 minutes, the absorbance of each well was measured at OD 490 nm using an enzyme-linked immunosorbent assay (ELISA) and the data were analyzed.

[0103] (2) Cell clone formation experiment

[0104] Logarithmically growing cells harboring either empty vectors or overexpressing sensitive and resistant HCC827 and PC9 MZF1 genes were digested and counted, seeded in 6-well cell culture plates at 100 cells per well, and the medium was changed every three days. Once cell colonies reached a visible size, the medium was aspirated and the cells were washed twice with 1× PBS. The cells were fixed with 4% paraformaldehyde for 30 minutes and washed twice with 1× PBS. The cells were stained with 0.5% crystal violet for 30 minutes, rinsed, air-dried, photographed, and the number of colonies was counted.

[0105] (3) Migration experiment

[0106] The cell lines containing empty vectors and overexpression of sensitive and resistant HCC827 and PC9 MZF1 genes constructed in step 1 were digested and counted, and added to the upper chamber of 8.0 μm pore size Transwell chamber (Corning, USA, catalog number: CLS3422), 200 μL of serum-free cell suspension was added to each upper chamber, and 2×10 4 cells, add 600 μL complete culture medium to the lower chamber; culture in a 37°C cell culture incubator for 24 hours, carefully wash several times with 1× PBS; fix with 4% paraformaldehyde for 30 minutes, wash three times with 1× PBS; stain with 0.5% crystal violet for 30 minutes, rinse until the rinse solution is colorless, carefully remove the cells in the upper chamber with a cotton swab, and place under a microscope (100×) to count the number of migrated cells.

[0107] 2.3 Statistical analysis

[0108] The experiments were repeated at least three times. The results were analyzed using a two-sided t-test, and the results were expressed as mean ± standard deviation. *p < 0.05 indicates a significant statistical difference, and **p < 0.01 indicates an extremely significant statistical difference.

[0109] 2.4 Experimental Results

[0110] A functional screening model was established to clarify the molecular basis of the role of MZF1 splice variants in acquired resistance to EGFR-TKIs in non-small cell lung cancer. MZF1 was stably overexpressed in EGFR-TKI-sensitive and resistant lung cancer cell lines. L and MZF1 S ( Figure 2 A).

[0111] In HCC827 and PC9 lung cancer cells that exogenously overexpress MZF1 splice variants, the cell biological functions were significantly different ( Figure 2 BD); cells overexpressing MZF1 L After MZF1 overexpression, the proliferation, colony formation and migration were significantly reduced compared with the control group (p < 0.01). S The proliferation, colony formation and migration of tumor cells were significantly increased (p<0.01).

[0112] Example 3 Effects of MZF1 splicing variants on cell function and acquired resistance to EGFR-TKIs

[0113] The non-small cell lung cancer cells used for the test: HCC827 and PC9, were purchased from the National Biomedical Experimental Cell Resource Bank and cultured and passaged under normal conditions in the applicant's laboratory.

[0114] 3.1 MZF1 gene expression in overexpressed HCC827 and PC9 cells

[0115] Universal empty plasmid, purchased from GeneCopoeia, USA, catalog number: EX-NEG-M02; MZF1 L Plasmids were purchased from GeneCopoeia, catalog number: EX-T3148-M02-5; MZF1 S Plasmid, NM_001267033.2, was synthesized by Shanghai GeneChip.

[0116] HCC827 and PC9 cell lines, both sensitive and resistant to non-small cell lung cancer, were stably transfected. Cells were seeded in 6-well cell culture plates. When cells reached 60% confluency, 200 μL of DMEM was added to 20 nM of the expression plasmid and empty plasmid, respectively, mixed, and incubated at room temperature for 5 minutes. Then, 200 μL of DMEM was added to 6 μL of Lipofectamine 2000, mixed, and incubated at room temperature for 5 minutes. 200 mL of Lipofectamine 2000 (Thermo Fisher Scientific, USA, Cat. No. 11668019) diluted solution was added to 200 μL of the expression plasmid, mixed, and incubated at room temperature for 20 minutes. 400 μL of the mixture was added to the cell culture plate and incubated at 37°C serum-free for 8 hours. The cells were then replaced with 10% FBSDMEM and incubated at 37°C for 24 hours. Gefitinib (1 μM) and osimertinib (0.1 μM) were then added and analyzed by RT-qPCR and Western blot.

[0117] 3.2 Cell biology function experiments

[0118] (1) MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) experiment

[0119] Cell lines containing the MZF1 gene in HCC827 and PC9 cells (both in the logarithmic growth phase and in the overexpression phase) were digested and counted. The cell suspension concentration was adjusted, and 100 μL was added to each well of a 96-well cell culture plate. 2000 cells were plated in eight replicates and observed for four consecutive days. Gefitinib (1 μM) / osimertinib (0.1 μM) and 10 μL MTT (Sigma, Cat. No. M2128) were added to each well daily to a final concentration of 5 μg / mL. After incubation for 4 hours, the medium was discarded. After four days, 150 μL of dimethyl sulfoxide (DMSO, Sigma, Cat. No. D2650) was added to each well. After gently shaking in the dark for 10 minutes, the absorbance of each well was measured at 490 nm using an enzyme-linked immunosorbent assay (ELISA) and the data were analyzed.

[0120] (2) Cell clone formation experiment

[0121] Logarithmically growing cells harboring either empty vectors or overexpressing sensitive and resistant HCC827 and PC9 MZF1 genes were digested and counted. Cells were seeded in 6-well cell culture plates at 100 cells per well and treated with gefitinib (1 μM) or osimertinib (0.1 μM). The culture medium was changed every three days. After cell colonies reached a visible size, the culture medium was aspirated and the cells were washed twice with 1× PBS. The cells were fixed with 4% paraformaldehyde for 30 minutes and washed twice with 1× PBS. The cells were stained with 0.5% crystal violet for 30 minutes, rinsed, air-dried, and photographed. The number of colonies was then counted.

[0122] (3) Migration experiment

[0123] The cell lines containing empty vectors and overexpression of sensitive and resistant HCC827 and PC9 MZF1 genes constructed in step 1 were digested and counted, and added to the upper chamber of 8.0 μm pore size Transwell chamber (Corning, USA, catalog number: CLS3422), 200 μL of serum-free cell suspension was added to each upper chamber, and 2×10 4 cells, added 600 μL complete culture medium to the lower chamber; added gefitinib (1 μM) / osimertinib (0.1 μM), cultured in a 37°C cell culture incubator, cultured cells for 24 hours, carefully washed several times with 1× PBS; fixed with 4% paraformaldehyde for 30 minutes, washed three times with 1× PBS; stained with 0.5% crystal violet for 30 minutes, rinsed until the rinse solution was colorless, carefully removed the cells in the upper chamber with a cotton swab, and placed under a microscope (100×) to count the number of migrated cells.

[0124] 3.3 Cell membrane, cytoplasm and nuclear protein extraction experiments

[0125] Wash the cells twice with ice-cold PBS using the Animal Cell Nuclear and Plasma Protein Isolation Kit (Beijing Aoqing Biotechnology Co., Ltd., Cat. No. AQ805-50T) and the Membrane Protein Extraction Kit (Beijing Aoqing Biotechnology Co., Ltd., Cat. No. AQ802-50T). 2 Culture area (about 0.5~1×10 7cells) add 500μL of ice-cold solution A, place on ice and shake slowly for 5 minutes. Pipette the supernatant into a new centrifuge tube. The supernatant should be clear and free of cells and debris. Scrape the cells with a cell scraper, transfer to a centrifuge tube, and centrifuge at 500×g and 4℃ for 10 minutes. Carefully aspirate the supernatant and mix it with the previous step. Extract cell membrane protein: estimate the volume of the precipitate, add 500μL of solution B, pipette to resuspend the precipitate, and shake evenly on ice for 30 minutes. Centrifuge at 5000×g and 4℃ for 10 minutes, save the supernatant, which is the membrane protein. The precipitate is used to extract the cell nucleus. Extract nuclear protein: resuspend the precipitate with 250μL of solution C, and homogenize at medium speed 5 times with a homogenizer. Place on ice and shake evenly for 10 minutes. Centrifuge at 7000×g and 4℃ for 10 minutes, save the supernatant, which is the nuclear protein.

[0126] 3.4 Western blotting

[0127] After quantification, the sample was loaded at 30 μg / well. The sample was added to 5× SDS gel loading buffer (Genstar, China, catalog number: E153-05), heated at 100°C for 10 minutes, and the sample was loaded sequentially. SDS-PAGE electrophoresis was performed at 120V. After electrophoresis, the membrane was transferred to a wet electroporator. After transfer, the PVDF membrane (Millipore, USA, catalog number: IPVH00010) was removed. After blocking with 5% skim milk for 1 hour, the primary antibody: MZF1 was incubated. L Antibodies (Wuhan Dian Biotechnology Co., Ltd., antigen immunization: RPFACAECGQSFRQRSNLTQHRRIHTGERPFACAECGKAFRQRPTLTQHLRVHTGEKPFACPECGQRFSQRLKLTRHQRTHTGEKPYHCGECGLGFTQVSRLTEHQRIHTGERPFACPECGQSFRQHANLTQHRRIHTGERPYACPECGKAFRQRPTLTQHLRTHRREKPFACQDCGRRFHQSTKLIQHQRVHSAE), MZF1 SAntibodies (Wuhan Dian Biotechnology Co., Ltd., antigen immunoassay: LGAGWLGAAVAMYVARC), p-EGFR antibody (abcam, UK, catalog number: ab40815), EGFR antibody (Proteintech, China, catalog number: 66455-1-Ig), and β-actin antibody (Sigma-Aldrich, USA, catalog number: A45551) were used. Incubate overnight at 4°C. Anti-rabbit horseradish peroxidase (abcam, UK, catalog number: ab6721)-conjugated secondary antibody was added and incubated for 1 hour. The ECL chemiluminescence kit (Themo Fisher Scientific, USA, catalog number: 32132) was used for color development, and images were visualized using the Smart Gel Image Analysis System.

[0128] 3.5 Statistical analysis

[0129] The experiments were repeated at least three times. The results were analyzed using a two-sided t-test, and the results were expressed as mean ± standard deviation. *p < 0.05 indicates a significant statistical difference, and **p < 0.01 indicates an extremely significant statistical difference.

[0130] 3.6 Experimental Results

[0131] To investigate the effect of MZF1 splice variants on acquired resistance to EGFR-TKIs, we added gefitinib (1 μM) or osimertinib (0.1 μM) to a cell model overexpressing MZF1 splice variants. MTT and colony formation assays were used to determine whether overexpression of MZF1 splice variants after gefitinib or osimertinib treatment could inhibit the proliferation of resistant cells.

[0132] MTT results showed that compared with the drug-resistant cell lines transfected with empty vector, the overexpression of MZF1 L It can significantly inhibit the ability of drug-resistant cells to promote proliferation (p < 0.01, Figure 3 A) The results of clone formation assay showed that overexpression of MZF1 L It can significantly inhibit the ability of drug-resistant cells to promote clone formation (p < 0.01, Figure 3 B) Transwell assay was used to examine the effect of overexpression of MZF1 splice variants on the invasion and migration of drug-resistant cells after addition of gefitinib or osimertinib. The results showed that compared with the control, overexpression of MZF1 L It can significantly inhibit the ability of drug-resistant cells to promote invasion and migration (p < 0.01, working basis - Figure 3 C).

[0133] GR / OR cells overexpressing MZF1 splice variants were treated with gefitinib / osimertinib and the expression of EGFR-related proteins was detected. The results showed that gefitinib / osimertinib could not reduce the level of p-EGFR in GR / OR cells. However, overexpression of MZF1 L , the expression of p-EGFR was significantly reduced after drug treatment, and overexpression of MZF1 S The opposite results were found (Working basis - Figure 3 D).

[0134] To further investigate the effect of MZF1 splicing variants on EGFR membrane translocation after EGFR-TKIs resistance, the nucleus, cytoplasm, and membrane of drug-resistant cells were isolated. L Overexpression of EGFR increased the expression of mEGFR and decreased the expression of cEGFR and nEGFR ( Figure 3 E). These results indicate that overexpression of MZF1 L The sensitivity of cells to EGFR-TKIs can be increased by regulating the localization of EGFR on the cell membrane.

Claims

1. Use of a reagent for detecting drug-resistant lung cancer in the preparation of a reagent for diagnosing drug-resistant lung cancer patients, wherein the reagent for detecting drug-resistant lung cancer is a reagent for detecting MZF1 L and MZF1 S When MZF1 is detected in lung cancer patients L Low expression, MZF1 s When the expression level is high and MZF1 s Expression level relative to MZF1 L When the expression level of MZF1 is high, the patient can be judged as a drug-resistant patient; L The nucleotide sequence of MZF1 is shown in SEQ ID NO. L The amino acid sequence of MZF1 is shown in SEQ ID NO. 2; s The nucleotide sequence is shown in SEQ ID NO. 3; MZF1 s The amino acid sequence is shown in SEQ ID NO. 4; the drug-resistant lung cancer patients include gefitinib-resistant patients and osimertinib-resistant patients.

Images