Application of MNT in detection of ferroptosis and chemotherapy sensitivity of lung adenocarcinoma

Abstract

The application discloses application of MNT in detection of ferroptosis and chemotherapy sensitivity of lung adenocarcinoma. The application is based on a series of molecular biology experiments, and reveals that high expression of MNT inhibits ferroptosis of lung adenocarcinoma and increases chemotherapy sensitivity. Based on the results of the molecular biology experiments, the application first proposes a new use of MNT in detection of ferroptosis and chemotherapy sensitivity of lung adenocarcinoma; the expression amount of MNT of lung adenocarcinoma is detected to judge the sensitivity of lung adenocarcinoma patients to ferroptosis inducer and chemotherapy treatment, and the method has the advantages of simple operation, high sensitivity, specificity, high cost performance and application value, and provides a new way for clinically evaluating the sensitivity of lung adenocarcinoma patients to ferroptosis and cisplatin treatment.

Description

Technical Field

[0001] This invention relates to the application of MNT in detecting ferroptosis and chemosensitivity in lung adenocarcinoma, and belongs to the field of molecular diagnostic technology. Background Technology

[0002] Ferroprelation is a regulatory cell necrosis induced by iron ions and reactive oxygen species-induced lipid peroxidation, which is significantly different from other forms of regulatory cell necrosis such as apoptosis, necrosis, and autophagy at the morphological, biological, and genetic levels. Studies have confirmed that ferroptosis is involved in various biological processes, such as development, aging, and immunity. Recent research indicates that the loss of ferroptosis can promote tumorigenesis and proliferation. More importantly, ferroptosis has been shown to be associated with chemotherapy resistance. It is well known that lung adenocarcinoma patients are prone to developing drug resistance during chemotherapy, affecting treatment efficacy; inducing ferroptosis in lung cancer can improve chemotherapy sensitivity. Therefore, accurate detection of ferroptosis and chemotherapy sensitivity will provide guidance for the clinical treatment of lung adenocarcinoma patients, thereby improving treatment outcomes.

[0003] MNT, a member of the MXD family, is a relatively little studied transcription factor that primarily functions in transcriptional regulation. It has been reported that MNT is aberrantly expressed in various common tumor tissues and can promote tumor development and progression. To date, there are no reported studies on the relationship between MNT and ferroptosis and chemosensitivity in lung adenocarcinoma. Summary of the Invention

[0004] The purpose of this invention is to provide a new application of MNT in detecting ferroptosis and chemotherapy sensitivity in lung adenocarcinoma. By detecting the expression level of the MNT gene or protein in lung adenocarcinoma, the sensitivity of lung adenocarcinoma patients to ferroptosis inducers and chemotherapy is determined. This invention is simple to operate, highly sensitive, specific, cost-effective, and has application value.

[0005] To achieve the above objectives, the present invention provides the application of a detection reagent in the preparation of a kit for assessing the sensitivity of lung adenocarcinoma to ferroptosis and chemotherapy. The detection reagent is a reagent for detecting the expression level of MNT in lung adenocarcinoma samples. The detection reagent is the only key component of the kit for determining the sensitivity of lung adenocarcinoma to ferroptosis and cisplatin.

[0006] Preferably, the kit also includes an instruction manual, which describes the assessment threshold and assessment criteria for MNT expression level. The MNT expression level is calculated by tissue immunohistochemical scoring. The MNT expression level in the sample is scored according to the positive rate of immunohistochemical staining. When the positive rate is greater than or equal to 40%, it is judged as resistance to ferroptosis and chemotherapy; while when the positive rate is less than 40%, it is judged as sensitivity 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 MNT expression level by immunohistochemistry, specifically including an antibody for immunohistochemistry detection; the detection reagent can also be a reagent used to detect MNT expression level by RT-qPCR, specifically including RNA extraction reagents or kits (e.g., TRIzol reagent), reverse transcription reagents or kits, and qPCR reagents or kits.

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

[0010] Preferably, the reagent for specifically detecting MNT expression can be a reagent used to detect MNT expression using an immunohistochemical detection method, specifically including an antibody for immunohistochemical detection; the detection reagent can also be a reagent used to detect MNT expression using an RT-qPCR method, specifically including RNA extraction reagents or kits (e.g., TRIzol reagent), reverse transcription reagents or kits, and qPCR reagents or kits.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0012] This invention proposes a novel application of MNT in detecting ferroptosis and chemotherapy sensitivity in lung adenocarcinoma. By detecting the expression level of the MNT gene or protein in lung adenocarcinoma, the sensitivity of lung adenocarcinoma patients to ferroptosis inducers and chemotherapy is determined. This method is simple to operate, highly sensitive, specific, cost-effective, and has application value, providing a new approach for clinically assessing the sensitivity of lung adenocarcinoma patients to ferroptosis and cisplatin treatment. Attached Figure Description

[0013] Figure 1 MNT inhibits the sensitivity of lung adenocarcinoma cells to ferroptosis inducers and chemotherapy; MNT 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: MNT expression inhibits the ferroptosis sensitivity of lung adenocarcinoma cells; IJ: MNT inhibits the chemosensitivity of lung adenocarcinoma cells; K: MNT inhibits lipid peroxidation in lung adenocarcinoma cells;

[0014] Figure 2MNT inhibits ferroptosis in lung adenocarcinoma by suppressing SAT1 expression; AB: qPCR showed that MNT inhibits SAT1 expression; CD: Western blot results showed that MNT inhibits SAT1 expression; EH: qPCR results after restoring SAT1 expression; IJ: Western blot results after restoring SAT1 expression; KN: Restoring SAT1 expression increased the ferroptosis sensitivity of lung adenocarcinoma cell lines; OP: Restoring SAT1 expression increased the sensitivity of lung adenocarcinoma to chemotherapeutic drugs; Q: Restoring SAT1 expression increased the lipid peroxidation level in lung adenocarcinoma cells;

[0015] Figure 3 MNT is highly expressed in lung adenocarcinoma tissues and is negatively correlated with the prognosis of lung adenocarcinoma patients; A: A retrospective analysis of tissue samples from 150 lung adenocarcinoma patients at Zhongshan Hospital showed that MNT expression was upregulated in cancerous tissues; B: The prognosis of 150 lung adenocarcinoma patients at Zhongshan Hospital was negatively correlated with MNT expression. Detailed Implementation

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

[0017] Example

[0018] (I) MNT inhibits ferroptosis and chemotherapy sensitivity in lung adenocarcinoma cells:

[0019] First, lung adenocarcinoma cell lines A549 and PC9 were treated with the ferroptosis inducers RSL3 and IKE, respectively, and MNT expression was found to be increased. Figure 1 AD). MNT overexpression and knockdown stable cell lines were constructed, and it was found that MNT knockdown increased the sensitivity of A549 and PC9 cells to ferroptosis inducers and cisplatin. Figure 1 EJ). MNT expression inhibits lipid peroxidation in lung adenocarcinoma cells ( Figure 1 K). This indicates that MNT can inhibit ferroptosis in lung adenocarcinoma cells and reduce chemosensitivity.

[0020] (II) MNT inhibits ferroptosis and chemosensitivity in lung adenocarcinoma cells by suppressing SAT1 expression:

[0021] Using qRT-PCR ( Figure 2 A) and western blot ( Figure 2 B) The changes in key ferroptosis molecules after MNT overexpression and knockdown were examined, and it was found that the expression of the key ferroptosis molecule SAT1 was inhibited by MNT. Figure 2 AD). Subsequently, SAT1 expression was restored in the above stable cell lines (AD). Figure 2EJ) found that after SAT1 recovery, A549 and PC9 showed significantly increased sensitivity to ferroptosis inducers and cisplatin. Figure 2 The above results further demonstrate that MNT inhibits ferroptosis in lung adenocarcinoma cells and increases their chemosensitivity by suppressing the expression of the key ferroptosis molecule SAT1.

[0022] (III) MNT is highly expressed in lung adenocarcinoma tissues and is negatively correlated with patient prognosis:

[0023] An analysis of a cohort of lung adenocarcinoma patients at Zhongshan Hospital affiliated with Fudan University was conducted. Immunohistochemical staining was used to detect the expression of microneedle adenocarcinoma (MNT) in cancerous and adjacent normal lung tissues. MNT expression was divided into high and low expression groups based on a 40% positive threshold. Results showed that MNT expression in lung adenocarcinoma tissues was significantly higher than that in adjacent normal lung tissues. Figure 3 A). Further prognostic analysis showed that patients with lung adenocarcinoma who expressed low levels of MNT had a better prognosis, while those with high levels of MNT had a worse prognosis. Figure 3 B).

[0024] Information on lung adenocarcinoma patients who received postoperative cisplatin-based chemotherapy was analyzed from the Thoracic Surgery Database of Zhongshan Hospital, Fudan University. Patients who experienced primary tumor recurrence or metastasis or tumor-related death within one year were defined as cisplatin-resistant, while those who remained relapse-free or metastatic and did not die from tumor-related death after 5 years or more 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 immunohistochemical analysis. The specific steps are as follows:

[0025] ① After further formalin fixation, fresh tissue specimens were embedded in paraffin, tissue sections were prepared, and then immunohistochemical staining was performed using MNT antibody;

[0026] ② The proportion of MNT positive expression was divided into high MNT expression and low MNT expression groups based on a 40% threshold. The low expression group was sensitive to cisplatin chemotherapy, while the high expression group was resistant to cisplatin chemotherapy. Compared with the actual cisplatin chemotherapy sensitivity results of patients, the sensitivity was calculated as 14 / 20 = 70%, and the specificity as 16 / 20 = 80% (Table 1).

[0027] Table 1 shows the sensitivity and specificity of prediction in patients with lung adenocarcinoma collected from various sources.

[0028] In reality, it was cisplatin-resistant chemotherapy. Actually, it is cisplatin-sensitive to chemotherapy. sum Predicted to be cisplatin resistance 14 4 18 Predicted to be cisplatin sensitive 6 16 22 sum 20 20 30

[0029] The above embodiments are merely 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 those skilled in the art can make several improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. The use of a detection reagent in the preparation of a kit for evaluating the sensitivity of lung adenocarcinoma to ferroptosis inducers and cisplatin treatment, characterized in that, The ferroptosis inducers are RSL3 and IKE; The detection reagent is used to detect the expression level of MNT in lung adenocarcinoma samples. The detection reagent is the only key component of the kit for determining the sensitivity of lung adenocarcinoma to ferroptosis and cisplatin.

2. The application as described in claim 1, characterized in that, The kit also includes instructions, which describe the assessment threshold and criteria for MNT expression. The MNT expression level is calculated by tissue immunohistochemical scoring. The MNT expression level in the sample is scored based on the positive rate of immunohistochemical staining. When the positive rate is greater than or equal to 40%, it is judged as resistance to ferroptosis and cisplatin; while when the positive rate is less than 40%, it is judged as sensitivity to ferroptosis and cisplatin.

3. The application as described in 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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