Application of KLF11 in detecting ferroptosis and chemotherapy sensitivity in lung adenocarcinoma

Abstract

The present invention discloses the application of KLF11 in detecting ferroptosis and chemotherapy sensitivity in lung adenocarcinoma. Based on a series of molecular biology experiments, the present invention reveals that high expression of KLF11 promotes ferroptosis in lung adenocarcinoma and increases chemotherapy sensitivity. Based on the results of molecular biology experiments, the present invention proposes for the first time a new use of KLF11 in detecting ferroptosis and chemotherapy sensitivity in lung adenocarcinoma; by detecting the expression level of KLF11 in lung adenocarcinoma, it is used to judge the sensitivity of lung adenocarcinoma patients to ferroptosis inducers and chemotherapy treatment. It has simple operation, high sensitivity, specificity, high cost performance and application value, and provides a new way to clinically evaluate the sensitivity of lung adenocarcinoma patients to ferroptosis and cisplatin treatment.

Description

Technical Field

[0001] The present invention relates to application of KLF11 in detecting ferroptosis and chemotherapy sensitivity of lung adenocarcinoma, and belongs to the technical field of molecular diagnosis. Background Art

[0002] Ferroptosis is a type of regulated cell necrosis that relies on iron ions and reactive oxygen species to induce lipid peroxidation. It is significantly different from other forms of regulated cell necrosis, such as apoptosis, necrosis, and autophagy, at the morphological, biological, and genetic levels. Studies have confirmed that ferroptosis is associated with various biological processes, such as development, aging, and immunity. Recent studies have shown that the loss of ferroptosis can promote tumorigenesis and promote tumor proliferation. More importantly, ferroptosis has been shown to be associated with chemotherapy resistance. It is well known that patients with lung adenocarcinoma are prone to drug resistance during chemotherapy, which affects the treatment effect. Inducing ferroptosis in lung cancer can increase the sensitivity of chemotherapy. Therefore, accurate detection of ferroptosis and chemotherapy sensitivity will provide guidance for the treatment of clinical lung adenocarcinoma patients, thereby improving the treatment effect.

[0003] KLF11, a member of the Kruppel-like zinc finger transcription factor family, is a relatively poorly studied transcription factor that primarily functions as a transcriptional regulator. KLF11 has been reported to be aberrantly expressed in a variety of common tumor tissues and to promote tumor proliferation and metastasis. To date, no studies have reported on the relationship between KLF11 and ferroptosis and chemotherapy sensitivity in lung adenocarcinoma. Summary of the Invention

[0004] The purpose of the present invention is to provide a new use of KLF11 in detecting ferroptosis and chemotherapy sensitivity in lung adenocarcinoma. By detecting the expression level of KLF11 gene or protein in lung adenocarcinoma, the sensitivity of lung adenocarcinoma patients to ferroptosis inducers and chemotherapy treatment can be judged. The invention has simple operation, high sensitivity, specificity, high cost performance and application value.

[0005] In order to achieve the above objectives, the present invention provides a detection reagent for use in preparing a kit for evaluating the sensitivity of lung adenocarcinoma to ferroptosis and chemotherapy. The detection reagent is a reagent for detecting the expression level of KLF11 in lung adenocarcinoma samples. The detection reagent serves as the only key component of the kit for determining the sensitivity of lung adenocarcinoma to ferroptosis and cisplatin.

[0006] Preferably, the kit also includes instructions, which record the evaluation threshold and evaluation criteria of KLF11 expression. The KLF11 expression is calculated by tissue immunohistochemistry scoring, and the KLF11 expression in the sample is scored according to the positive ratio of immunohistochemical staining. When the positive ratio is greater than 40%, it is judged to be sensitive to ferroptosis and chemotherapy; and when the positive ratio is less than 40%, it is judged to be resistant to ferroptosis and chemotherapy.

[0007] Preferably, the lung adenocarcinoma sample is a lung adenocarcinoma cell line or a fresh lung adenocarcinoma tissue tumor sample.

[0008] Preferably, the detection reagent can be a reagent used to detect the expression level of KLF11 using an immunohistochemical detection method, specifically, including an antibody for immunohistochemical detection; the detection reagent can also be a reagent used to detect the expression level of KLF11 using an RT-qPCR method, specifically, including an RNA extraction reagent or kit (for example, TRIzol reagent), a reverse transcription reagent or kit, and a qPCR reagent or kit used to extract RNA.

[0009] The present invention also provides a kit for detecting the sensitivity of lung adenocarcinoma to ferroptosis and chemotherapy, which at least comprises a reagent for specifically detecting the expression level of KLF11.

[0010] Preferably, the reagent for specifically detecting the expression level of KLF11 can be a reagent used for detecting the expression level of KLF11 by immunohistochemistry, specifically, including an antibody for immunohistochemistry detection; the detection reagent can also be a reagent used for detecting the expression level of KLF11 by RT-qPCR, specifically, including an RNA extraction reagent or kit (for example, TRIzol reagent), a reverse transcription reagent or kit, and a qPCR reagent or kit used for extracting RNA.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] The present invention proposes for the first time a new use of KLF11 in detecting ferroptosis and chemotherapy sensitivity in lung adenocarcinoma; by detecting the expression level of KLF11 gene or protein in lung adenocarcinoma, it is used to judge the sensitivity of lung adenocarcinoma patients to ferroptosis inducers and chemotherapy treatment. It has simple operation, high sensitivity, specificity, high cost performance and application value, and provides a new way to clinically evaluate the sensitivity of lung adenocarcinoma patients to enhanced ferroptosis and cisplatin treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 :KLF11 promotes the sensitivity of non-adenocarcinoma cells to ferroptosis inducers and chemotherapy; KLF11 expression is upregulated after treatment of lung adenocarcinoma cell lines (A549, PC9) with ferroptosis inducers (RSL3, IKE): AB: qPCR results, CD: western blot results; EH: KLF11 expression increases the ferroptosis sensitivity of lung adenocarcinoma cells; IJ: KLF11 increases the chemotherapy sensitivity of lung adenocarcinoma cells; KL: KLF11 promotes lipid peroxidation in lung adenocarcinoma cells;

[0014] Figure 2: KLF11 inhibits GPX4 expression and promotes ferroptosis in lung adenocarcinoma; AB: qPCR results show that KLF11 inhibits GPX4 expression; CD: Western blot results show that KLF11 inhibits GPX4 expression; EH: qPCR results after restoration of GPX4 expression; IJ: Western blot results after restoration of GPX4 expression; KN: After restoration of GPX4 expression, lung adenocarcinoma cell lines develop ferroptosis resistance; OP: After restoration of GPX4 expression, lung adenocarcinoma develops resistance to chemotherapy; QR: After restoration of GPX4 expression, the level of lipid peroxidation in lung adenocarcinoma cells increases;

[0015] Figure 3 : KLF11 is lowly expressed in lung adenocarcinoma tissues and positively correlated with the prognosis of lung adenocarcinoma patients; A: KLF11 expression is downregulated in tumor tissues of lung adenocarcinoma patients in the TCGA database; B: A review of 150 lung adenocarcinoma patient tissue samples from Zhongshan Hospital showed that KLF11 expression was downregulated in cancer tissues; C: KLF11 expression was positively correlated with patient prognosis in the TCGA database; D: The prognosis of 150 lung adenocarcinoma patients in Zhongshan Hospital was positively correlated with KLF11 expression. DETAILED DESCRIPTION

[0016] To make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0017] Example

[0018] (I) KLF11 increases ferroptosis and chemotherapy sensitivity in lung adenocarcinoma cells:

[0019] First, we treated lung adenocarcinoma cell lines A549 and PC9 with ferroptosis inducers and found that KLF11 expression was increased ( Figure 1 AD). KLF11 overexpression and knockout stable cell lines were constructed, and it was found that high expression of KLF11 could increase the sensitivity of A549 and PC9 to ferroptosis inducers and cisplatin ( Figure 1 EL). These results indicate that KLF11 can promote ferroptosis in lung adenocarcinoma cells and increase their chemotherapy sensitivity.

[0020] (II) KLF11 increases ferroptosis and chemotherapy sensitivity in lung adenocarcinoma cells by inhibiting GPX4 expression:

[0021] Using qRT-PCR ( Figure 2 A) and western blot( Figure 2 B) After KLF11 overexpression and knockout, the changes in key ferroptosis molecules were detected. It was found that the expression of the core ferroptosis molecule GPX4 was inhibited by KLF11 ( Figure 2 AD). Subsequently, the expression of GPX4 was restored in the above-mentioned stable cell lines ( Figure 2EJ) found that after GPX4 restoration, the sensitivity of A549 and PC9 to ferroptosis inducers and cisplatin was significantly reduced ( Figure 2 These results further indicate that KLF11 promotes ferroptosis in lung adenocarcinoma cells and increases their chemosensitivity by inhibiting the expression of the core ferroptosis molecule GPX4.

[0022] (III) KLF11 is lowly expressed in lung adenocarcinoma tissue and positively correlated with patient prognosis:

[0023] The TCGA database and two lung adenocarcinoma patient cohorts from Zhongshan Hospital affiliated to Fudan University were analyzed. The Zhongshan Hospital cohort was tested for KLF11 expression in lung adenocarcinoma and adjacent tissues by immunohistochemistry. The KLF11 expression was divided into high and low expression groups according to a 40% positive threshold. The results showed that both cohorts showed that KLF11 expression in lung adenocarcinoma tissue was significantly lower than that in adjacent normal lung tissue ( Figure 3 AB). Further prognostic analysis showed that patients with lung adenocarcinoma who had low KLF11 expression had a poor prognosis, while those with high KLF11 expression had a better prognosis ( Figure 3 CD).

[0024] The database of the Department of Thoracic Surgery at Zhongshan Hospital, Fudan University, was analyzed for patients with lung adenocarcinoma who received postoperative cisplatin chemotherapy. Patients who experienced primary tumor recurrence or metastasis within 1 year, or who died from the disease, were defined as cisplatin-resistant, while patients who had no recurrence or metastasis or death from the disease after 5 years of follow-up were defined as cisplatin-sensitive. Frozen fresh lung adenocarcinoma tissue samples were obtained from 20 cisplatin-resistant patients and 20 cisplatin-sensitive patients for immunohistochemistry. The specific steps were as follows:

[0025] ① Fresh tissue specimens were further fixed with formalin and embedded in paraffin, and tissue sections were prepared. Immunohistochemical staining was then performed using KLF11 antibodies.

[0026] ② Using a threshold of 40% for KLF11 expression, patients were divided into high-KLF11 and low-KLF11 expression groups. The high-KLF11 expression group was sensitive to cisplatin chemotherapy, while the low-KLF11 expression group was resistant to cisplatin. Compared with the cisplatin sensitivity results in actual patients, the calculated sensitivity was 14 / 20 = 70%, and the specificity was 16 / 20 = 80% (Table 1).

[0027] Table 1 Prediction sensitivity and specificity in patients with lung adenocarcinoma

[0028] Cisplatin chemotherapy resistance Actually sensitive to chemotherapy cisplatin sum Predicted to be cisplatin-resistant 14 4 18 Predicted to be cisplatin-sensitive 6 16 22 sum 20 20 30

[0029] The above embodiments are only preferred embodiments of the present invention and are not intended to limit the present invention in any form or substance. It should be noted that ordinary technicians in this technical field can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. Use of a detection reagent in the preparation of a kit for evaluating the sensitivity of lung adenocarcinoma to ferroptosis and chemotherapy, characterized in that: The detection reagent is a reagent for detecting the expression level of KLF11 in lung adenocarcinoma samples. The detection reagent is the only key component of a kit for determining the sensitivity of lung adenocarcinoma to ferroptosis and cisplatin.

2. The use according to claim 1, characterized in that The kit also includes instructions, which record the evaluation threshold and evaluation criteria of KLF11 expression. The KLF11 expression is calculated by tissue immunohistochemistry scoring, and the KLF11 expression in the sample is scored according to the positive ratio of immunohistochemical staining. When the positive ratio is greater than 40%, it is judged to be sensitive to ferroptosis and chemotherapy; and when the positive ratio is less than 40%, it is judged to be resistant to ferroptosis and chemotherapy.

3. The use according to claim 2, characterized in that The lung adenocarcinoma sample is a lung adenocarcinoma cell line or a fresh lung adenocarcinoma tissue tumor sample.

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