Application of KLF13 in preparation of detection reagent for detecting lung adenocarcinoma ferroptosis and cis-platinum sensitivity, detection reagent and kit

Abstract

The invention discloses application of KLF13 in preparation of a detection reagent for detecting lung adenocarcinoma ferroptosis and cis-platinum sensitivity, the detection reagent and a kit. Based on a series of molecular biology experiments, it is proved that high expression of KLF13 promotes lung adenocarcinoma ferroptosis and increases chemotherapy sensitivity. The invention proposes that KLF13 can be used as a biomarker for judging the sensitivity of lung adenocarcinoma to ferroptosis and cis-platinum for the first time; the sensitivity of the lung adenocarcinoma to ferroptosis and cis-platinum is judged by detecting the expression of the KLF13, and the kit has the advantages of simplicity and convenience in operation, high sensitivity and specificity, so that the kit has clinical popularization and application values.

Description

Technical Field

[0001] The present invention relates to molecular diagnostic technology, and in particular to application of KLF13 in detecting ferroptosis and chemotherapy sensitivity of lung adenocarcinoma and a kit. Background Art

[0002] Ferroptosis is a new form of iron-dependent regulated cell death caused by the accumulation of reactive oxygen species (ROS) and lipid peroxidation. Compared with other forms of regulated cell death (RCD), it has characteristic changes in morphology, biochemistry and genetics, including mitochondrial outer membrane rupture, membrane density condensation, mitochondrial cristae reduction or disappearance, and increased intracellular lipid peroxidation, increased ROS, and increased ferrous ions. Although there are currently a variety of treatments for lung cancer, chemotherapy resistance has greatly limited the improvement of prognosis in advanced cases. Recently, increasing evidence has shown that pharmacological approaches using natural compounds to trigger ferroptosis in drug-resistant cancers have great potential in improving the efficacy of chemotherapy. In multiple preclinical studies, inducing ferroptosis by blocking lipid peroxidation to regulate key targets showed strong anti-tumor efficacy against several types of human malignancies. In summary, a deep understanding of the specific regulatory mechanisms of ferroptosis and the development of corresponding new drugs are expected to improve the efficacy of tumor treatment. Therefore, in the clinical treatment of patients with lung adenocarcinoma, accurate detection of ferroptosis and its sensitivity to chemotherapy will play a key guiding role and help improve the treatment effect.

[0003] Lung cancer has become the most common tumor disease, and lung adenocarcinoma is the most common pathological type. Although there are many treatments, chemotherapy resistance has greatly limited the prognosis of many advanced cases. It is particularly important to find new mechanisms and new therapies to improve chemotherapy resistance. Ferroptosis is a form of cell death that depends on iron metabolism disorders and lipid peroxidation. Research on finding new ferroptosis mechanisms and targets to improve lung cancer resistance is currently very promising.

[0004] KLF13 is a member of the Kruppel-like zinc finger transcription factor family and is involved in a variety of key biological processes in tumors. It is reported that KLF13 can significantly promote or inhibit cancer cell proliferation, migration, autophagy, lipid metabolism and other processes. So far, there has been no research report on the relationship between KLF13 and ferroptosis and chemotherapy sensitivity in lung adenocarcinoma. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide the use of KLF13 in the preparation of detection reagents for detecting ferroptosis and cisplatin sensitivity of lung adenocarcinoma, as well as detection reagents and kits.

[0006] Technical solution: On the one hand, the present invention provides the use of KLF13 in the preparation of a reagent for detecting ferroptosis and cisplatin sensitivity in lung adenocarcinoma.

[0007] Furthermore, the lung adenocarcinoma samples are adenocarcinoma cell lines A549 and PC-9.

[0008] Another aspect of the present invention provides a detection reagent for detecting ferroptosis and cisplatin sensitivity of lung adenocarcinoma, wherein the detection reagent is a reagent for detecting the expression level of KLF13 gene in lung adenocarcinoma samples.

[0009] The third aspect of the present invention provides a kit for evaluating the sensitivity of lung adenocarcinoma to ferroptosis and chemotherapy, comprising at least a reagent for specifically detecting the expression level of the KLF13 gene.

[0010] Furthermore, it also includes instructions, which record the evaluation threshold of the KLF13 gene expression level, and the KLF13 gene expression level is calculated by tissue immunohistochemistry scoring.

[0011] Furthermore, the expression of KLF13 in the samples was scored according to the positive ratio of immunohistochemical staining. A positive ratio greater than 40% was considered high expression, and a positive ratio less than 40% was considered low expression.

[0012] Furthermore, the lung adenocarcinoma samples are adenocarcinoma cell lines A549 and PC-9.

[0013] Furthermore, the lung adenocarcinoma sample is a lung adenocarcinoma cell line or a fresh lung adenocarcinoma tissue tumor sample.

[0014] Furthermore, the test sample is a lung adenocarcinoma cell line or a fresh lung adenocarcinoma tissue tumor sample.

[0015] The present invention provides an application of a detection reagent in the preparation of a kit for evaluating the sensitivity of lung adenocarcinoma to ferroptosis inducers and chemotherapy. The detection reagent is a reagent for detecting the expression amount of the KLF13 gene in a lung adenocarcinoma sample. The detection reagent is the only key component of the kit for determining the sensitivity of lung adenocarcinoma to ferroptosis and chemotherapy.

[0016] The research results of the present invention found that KLF13 may promote ferroptosis in lung adenocarcinoma by inhibiting GPX4, thereby enhancing sensitivity to chemotherapeutic drugs. Targeting KLF13 is a promising and innovative treatment method for personalized treatment of lung adenocarcinoma.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention proposes for the first time that KLF13 can be used as a biomarker to determine the sensitivity of lung adenocarcinoma to ferroptosis and chemotherapy; the present invention reveals through bioinformatics analysis that low expression of KLF13 is significantly correlated with ferroptosis and chemotherapeutic properties, and a series of biological experiments confirm the correlation between KLF13 expression and ferroptosis and chemotherapy efficacy, and further proposes to determine the sensitivity of lung adenocarcinoma to ferroptosis and chemotherapy by detecting KLF13 expression, which has simple operation, high sensitivity, specificity, high cost performance and application value, helps to achieve precision medicine for patients with lung adenocarcinoma, and provides a new diagnostic approach for clinically evaluating the sensitivity of patients with lung adenocarcinoma to enhanced ferroptosis and cisplatin treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 KLF13 is involved in ferroptosis in LUAD. AB, E, A549 and PC9 cell lines were treated with different doses of RSL3 (0.5μM, 1μM, and 2μM) and IKE (2μM, 4μM, and 8μM) for 48 h, and the expression levels of KLF13 mRNA and protein were evaluated. CD, F, The expression levels of KLF13 mRNA and protein were evaluated in A549 and PC-9 cell lines treated with 2μM RSL3 or 4μM IKE plus Ferrostatin-1 (ferroptosis inhibitor, 10μM) or Liproxstatin-1 (ferroptosis inhibitor, 20μM) for 48 h;

[0020] Figure 2 KLF13 promotes ferroptosis and improves chemoresistance. A. After A549 and PC-9 cells were treated with different concentrations of RSL3, the relative activities of cells with different KLF13 expressions were determined. B. After A549 and PC-9 cells were treated with different concentrations of IKE, the relative activities of cells with different KLF13 expressions were determined. C. Each group of A549 and PC-9 cells was treated with different doses of CDDP for 24 hours, and then the relative cell viability was measured. D. Each group of A549 and PC-9 cells was treated with different doses of PEM for 24 hours, and then the relative cell viability was measured. EF, each group of A549 and PC-9 cells were treated with DMSO, RSL3 (2 μM) or IKE (4 μM), RSL3 / IKE + Fer-1 (10 μM), RSL3 / IKE + Lip-1 (20 μM), RSL3 / IKE + Z-VAD-FMK (10 μM), and RSL3 / IKE + Necro (1 μM) for 6 h, and then the lipid peroxidation level was determined by C11-BODIPY;

[0021] Figure 3KLF13 promotes ferroptosis and improves chemoresistance in lung adenocarcinoma cells by inhibiting GPX4. A, mRNA expression levels of GPX4 in A549 and PC-9 cells with different KLF13 expression groups. B, Protein expression levels of MAPK8, STAT3, and GPX4 in A549 and PC-9 cells with different KLF13 expression groups. CE, qRT-PCR and western blot confirmed the stable overexpression of GPX4 in KLF13-CTRL and KLF13-OE groups, and the stable knockout of GPX4 in KLF13-CTRL and KLF13-SH1 groups. FG, Dose-toxicity curves show the relative activities of GPX4-transfected control, overexpressed, or knocked-out A549 cells in different KLF13 expression groups after RSL3 or IKE treatment. HI, Dose-toxicity curves show the relative activities of GPX4-transfected control, overexpressed, or knocked-out A549 cells in different KLF13 expression groups after CDDP and PEM treatment. J, C11-BODIPY staining assay showed the relative lipid peroxidation levels in GPX4-transfected control, overexpression or knockout A549 cells in different KLF13 expression groups after 6 h of RSL3 (2 μM) treatment;

[0022] Figure 4 KLF13 promotes ferroptosis and improves chemoresistance in lung adenocarcinoma cells by inhibiting GPX4AC, qRT-PCR and western blot confirmed the stable overexpression of GPX4 in KLF13-CTRL and KLF13-OE groups, and the stable knockout of GPX4 in KLF13-CTRL and KLF13-SH1 groups in PC-9 cells.DE, Dose-toxicity curves show the relative activities of GPX4-transfected control, overexpressed or knocked-out PC-9 cells in different KLF13 expression groups after RSL3 or IKE treatment.FG, Dose-toxicity curves show the relative activities of GPX4-transfected control, overexpressed or knocked-out PC-9 cells in different KLF13 expression groups after CDDP and PEM treatment.HI, C11-BODIPY staining assay shows the relative lipid peroxidation levels of GPX4-transfected control, overexpressed or knocked-out PC-9 cells in different KLF13 expression groups after 6 hours of RSL3 (2 μM) treatment;

[0023] Figure 5KLF13 promotes ferroptosis and chemosensitivity in vivo, is highly expressed in tumor tissues, and is associated with better prognosis. A, Differential expression of KLF13 in LUAD tumor tissues and normal tissues in the GEPIA database. B, Representative images of immunohistochemical staining of 110 LUAD patient samples. C, Kaplan-Meier survival analysis of LUAD patients according to KLF13 expression in the Kaplan-Meier Plotter database. D, Kaplan-Meier survival analysis and log-rank test showed that OS and RFS of LUAD patients with high or low KLF13 expression. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be further described below.

[0025] (I) KLF13 is involved in ferroptosis of lung adenocarcinoma cells

[0026] First, the lung adenocarcinoma cell lines A549 and PC-9 were treated with ferroptosis inducers IKE and RSL3, respectively, and it was found that KLF13 expression was increased ( Figure 1 AB, E). Subsequently, the ferroptosis inhibitors Ferrostatin-1 and Liproxstatin-1 were used for combined treatment and the cells were divided into DMSO group, RSL3 group, RSL3+Ferrostatin-1 group, RSL3+Liproxstatin-1 group, DMSO group, IK group, IKE+Ferrostatin-1 group, IKE+Liproxstatin-1 group, and it was found that KLF13 expression was decreased ( Figure 1 CD, F).

[0027] (II) KLF13 increases ferroptosis and chemotherapy sensitivity in lung adenocarcinoma cells:

[0028] KLF13 overexpression and knockout stable cell lines were constructed, and it was found that high expression of KLF13 could increase the sensitivity of A549 and PC9 to ferroptosis inducers and cisplatin ( Figure 2 AF). These results suggest that KLF13 can promote ferroptosis in lung adenocarcinoma cells and increase chemotherapy sensitivity.

[0029] (III) KLF13 increases ferroptosis and chemotherapy sensitivity of lung adenocarcinoma cells by inhibiting GPX4 expression:

[0030] Using qRT-PCR ( Figure 3 A) and western blot( Figure 3B) After KLF13 overexpression and knockout, we detected the changes in key ferroptosis molecules and found that the expression of ferroptosis core molecule GPX4 was inhibited by KLF13. Subsequently, we restored the expression of GPX4 in the above-mentioned stable cell lines ( Figure 3 CE, Figure 4 AC), found that after GPX4 restoration, the sensitivity of A549 and PC-9 to ferroptosis inducers, cisplatin, and pemetrexed was significantly reduced ( Figure 3 FJ, Figure 4 DI). The above results further indicate that KLF13 promotes ferroptosis of lung adenocarcinoma cells and increases their chemosensitivity by inhibiting the expression of ferroptosis core molecule GPX4.

[0031] (IV) KLF13 is lowly expressed in lung adenocarcinoma tissue and positively correlated with patient prognosis:

[0032] The TCGA database and the lung adenocarcinoma patient cohort of Zhongshan Hospital Affiliated to Fudan University were analyzed. The Zhongshan Hospital cohort detected the expression of KLF13 in the cancer and adjacent tissues of lung adenocarcinoma patients by immunohistochemistry, and divided the expression of KLF13 into high and low expression groups according to the positive threshold of 40%. The results showed that the expression of KLF13 in lung adenocarcinoma tissue was significantly lower than that in adjacent normal lung tissue ( Figure 5 AB). Further prognostic analysis showed that patients with lung adenocarcinoma with low KLF13 expression had a poor prognosis, while patients with high KLF13 expression had a better prognosis ( Figure 5 CD).

[0033] The information of lung adenocarcinoma patients who received postoperative cisplatin chemotherapy in the database of the Department of Thoracic Surgery, Zhongshan Hospital Affiliated to Fudan University was analyzed. Patients with recurrence or metastasis of the primary tumor or death caused by tumor within 1 year were defined as cisplatin-resistant group, while patients with no recurrence or metastasis and death caused by tumor after 5 years of follow-up were defined as cisplatin-sensitive. Frozen fresh lung adenocarcinoma tissue samples were obtained from 26 cisplatin-resistant patients and 27 cisplatin-sensitive patients for immunohistochemistry detection. The specific steps are as follows:

[0034] ① Fresh tissue specimens were further fixed with formalin and embedded in paraffin to make tissue sections, followed by immunohistochemical staining using KLF13 antibody.

[0035] ② Ask two pathologists to score the above staining results and evaluate the proportion of KLF13 positive cells

[0036] ③ The above scoring results were divided into KLF13 high expression group and KLF13 low expression group according to the threshold of 40%. KLF13 in the high expression group was sensitive to cisplatin, while KLF13 in the low expression group was resistant to cisplatin.

[0037] Table 1 Sensitivity and specificity of the scoring model in the collected lung adenocarcinoma patients

[0038] Cisplatin resistance Cisplatin-sensitive sum Predicted to be cisplatin-resistant 19 7 26 Predicted to be cisplatin sensitive 7 20 27 sum 26 27 53

[0039] Results: Sensitivity = 73.1%, specificity = 74.1%;

[0040] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any technician in the relevant technical field, without departing from the scope of the technical solution of the present invention, makes any form of equivalent replacement or modification to the technical solution and technical content disclosed in the present invention, which does not depart from the content of the technical solution of the present invention and still falls within the protection scope of the present invention.

Claims

1. A use of KLF13 in the preparation of a reagent for detecting ferroptosis and cisplatin sensitivity in lung adenocarcinoma.

2. The use according to claim 1, characterized in that The lung adenocarcinoma samples were adenocarcinoma cell lines A549 and PC-9.

3. A detection reagent for detecting ferroptosis and cisplatin sensitivity of lung adenocarcinoma, characterized in that: The detection reagent is a reagent for detecting the expression amount of KLF13 gene in lung adenocarcinoma samples.

4. A kit for evaluating the sensitivity of lung adenocarcinoma to ferroptosis and chemotherapy, characterized in that: The method at least comprises a reagent for specifically detecting the expression level of the KLF13 gene.

5. The kit according to claim 4, characterized in that The method also includes instructions, which record the evaluation threshold of the KLF13 gene expression level, and the KLF13 gene expression level is calculated by tissue immunohistochemistry scoring.

6. The kit according to claim 4, characterized in that The expression of KLF13 in the samples was scored according to the positive ratio of immunohistochemical staining. A positive ratio greater than 40% was considered high expression, and a positive ratio less than 40% was considered low expression.

7. The kit according to claim 4, characterized in that The lung adenocarcinoma samples were adenocarcinoma cell lines A549 and PC-9.

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