Kit for predicting lung adenocarcinoma intracellular pH value and cisplatin treatment sensitivity, use method and application

Abstract

The invention discloses a kit for predicting the intracellular pH value of lung adenocarcinoma and the sensitivity of cisplatin treatment as well as a use method and application of the kit. According to the kit disclosed by the invention, the pH value in tumor cells of a patient is judged by detecting the expression quantity of ZNF266 in tumor tissues of the patient with lung adenocarcinoma, and the treatment effect of cis-platinum is predicted; the method is used for predicting the prognosis of a patient before treatment and evaluating the curative effect of a treatment scheme and the prognosis of the patient during and after treatment, so that precise medical treatment is realized. According to the invention, the method for predicting the pH value in the tumor cells of the patient and the cis-platinum treatment sensitivity by detecting the ZNF266 expression is proposed for the first time, the treatment scheme effect can be effectively evaluated, the individualized treatment of the lung adenocarcinoma patient is guided, the clinical benefit is improved, and meanwhile, unnecessary medical resource waste is avoided.

Description

Technical Field

[0001] The present invention relates to biomedicine technology, and in particular to a kit for predicting pH value in lung adenocarcinoma cells and cisplatin treatment sensitivity. Background Art

[0002] Lung adenocarcinoma is the most common and most lethal tumor, with both morbidity and mortality ranking among the highest among all tumors. Due to the Warburg effect, tumor cells often exhibit a pH distribution with alkaline intracellular and acidic extracellular pH, which makes tumor cells more prone to proliferation, migration, invasion, and drug resistance, ultimately leading to clinical progression of cancer and poor prognosis for patients. Cisplatin is a first-line drug for the treatment of lung cancer, but different patients have different sensitivities to cisplatin, and drug resistance often occurs during treatment. Not all patients with lung adenocarcinoma can benefit from this treatment. Therefore, evaluating the pH value of tumor cells in patients with lung adenocarcinoma and predicting the sensitivity of cancer lesions to cisplatin treatment are extremely important for predicting treatment efficacy, evaluating patient prognosis, and reducing the burden on society. Summary of the Invention

[0003] Purpose of the invention: In order to solve the technical problem of how to accurately judge the pH value and cisplatin sensitivity of lung adenocarcinoma cells before treatment of lung adenocarcinoma, the purpose of the present invention is to provide a kit for predicting the pH value and cisplatin sensitivity of lung adenocarcinoma cells.

[0004] Technical solution: The kit for predicting the pH value and cisplatin treatment sensitivity in lung adenocarcinoma cells includes a reagent for detecting the expression level of the ZNF266 gene. The reagent determines the pH level and cisplatin treatment sensitivity of the patient's lung adenocarcinoma cells by detecting the level of ZNF266 gene expression.

[0005] Preferably, the reagent is used to detect the expression level of ZNF266 mRNA in tumor tissue of a patient.

[0006] Preferably, the reagent obtains qPCR.Ct.ZNF266 and qPCR.Ct.ACTB values by detecting the mRNA expression levels of ZNF266 and ACTB in the patient's tumor tissue, respectively; the difference is calculated by the formula qPCR.Ct=qPCR.Ct.ZNF266-qPCR.Ct.ACTB; when the difference is less than or equal to 12.0, it indicates that the pH value in the patient's tumor tissue is less than 7.0, the intracellular environment is acidic, and it is sensitive to cisplatin treatment.

[0007] Preferably, the patient comprises a lung adenocarcinoma patient.

[0008] Preferably, the qPCR primer sequence of the internal reference ACTB is:

[0009] Forward primer: 5′-TGACGTGGACATCCGCAAAG-3′ (SEQ ID NO: 3);

[0010] Reverse primer: 5'-CTGGAAGGTGGACAGCGAGG-3' (SEQ ID NO: 4).

[0011] Preferably, the reagent for detecting the expression level of the ZNF266 gene is a qPCR reagent.

[0012] Preferably, the qPCR amplification primer sequence of ZNF266 in the ZNF266 detection system is:

[0013] Forward primer: 5′-CAGTCTGATCTCTTGGCTGGAACA-3′ (SEQ ID NO: 1);

[0014] Reverse primer: 5'-CCTCCGTTGTGGCTTCCTATCATT-3' (SEQ ID NO: 2).

[0015] Preferably, the kit further comprises an mRNA extraction and separation system for extracting and isolating mRNA from tumor tissues of patients with lung adenocarcinoma.

[0016] Preferably, the kit further comprises an mRNA reverse transcription system for reverse transcribing mRNA into cDNA.

[0017] Another aspect of the present invention provides a method for predicting the pH value and cisplatin treatment sensitivity of lung adenocarcinoma cells, which determines the pH value and cisplatin treatment sensitivity of the patient's lung adenocarcinoma cells by detecting the expression level of the ZNF266 gene in the patient's tumor tissue.

[0018] Preferably, the detecting the expression level of the ZNF266 gene in the patient's tumor tissue is detecting the expression level of ZNF266 mRNA in the patient's tumor tissue.

[0019] Preferably, the detection of the expression level of the ZNF266 gene in the patient's tumor tissue is performed by qPCR.

[0020] Preferably, the prediction of the intracellular pH value and prognosis of a patient's lung adenocarcinoma cells is performed by detecting the mRNA expression levels of ZNF266 and ACTB in the patient's tumor tissue to obtain qPCR.Ct.ZNF266 and qPCR.Ct.ACTB values, respectively; calculating the difference using the formula qPCR.Ct = qPCR.Ct.ZNF266 - qPCR.Ct.ACTB; when the difference is less than or equal to 12.0, it indicates that the intracellular pH value of the patient's tumor tissue is less than or equal to 7.0, the intracellular environment is acidic, and the patient is sensitive to cisplatin treatment. Preferably, the patient includes a lung adenocarcinoma patient.

[0021] The third aspect of the present invention provides the use of the kit in predicting the pH value in lung adenocarcinoma cells and the sensitivity of cisplatin treatment.

[0022] The technical solution adopted by the present invention is to provide a kit for predicting the pH value and cisplatin treatment sensitivity in lung adenocarcinoma cells, including a reagent for detecting the expression level of the ZNF266 gene, which determines the pH value and cisplatin treatment sensitivity in lung adenocarcinoma cells by detecting the high or low expression level of the ZNF266 gene; the reagent is used to detect the expression level of ZNF266 mRNA in the patient's tumor tissue; the reagent is a qPCR reagent; the reagent is used to detect the mRNA expression levels of ZNF266 and ACTB in the patient's tumor tissue to obtain qPCR.Ct.ZNF266 and qPCR.Ct.ACTB values respectively; the difference is calculated by the formula qPCR.Ct=qPCR.Ct.ZNF266-qPCR.Ct.ACTB; when the difference is less than or equal to 12.0, it indicates that the pH value in the patient's tumor tissue is less than or equal to 7.0, the intracellular environment is acidic, and the patient is sensitive to cisplatin treatment; the patient includes a lung adenocarcinoma patient.

[0023] The present invention provides a method for predicting the pH value and cisplatin treatment sensitivity in lung adenocarcinoma cells. The method comprises detecting the expression level of the ZNF266 gene in the patient's tumor tissue to determine the pH value and cisplatin treatment sensitivity in the lung adenocarcinoma cells. The method comprises detecting the expression level of the ZNF266 gene in the patient's tumor tissue by detecting the expression level of ZNF266 mRNA in the patient's tumor tissue. The method comprises detecting the expression level of the ZNF266 gene in the patient's tumor tissue by a qPCR method. The method comprises detecting the mRNA expression levels of ZNF266 and ACTB in the patient's tumor tissue to obtain qPCR.Ct.ZNF266 and qPCR.Ct.ACTB values, respectively. The difference is calculated by the formula qPCR.Ct=qPCR.Ct.ZNF266-qPCR.Ct.ACTB. When the difference is less than or equal to 12.0, it indicates that the pH value in the patient's tumor tissue is less than 7.0, the intracellular environment is acidic, and the patient is sensitive to cisplatin treatment. The patient includes a lung adenocarcinoma patient.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention provides a kit for predicting the pH value and cisplatin sensitivity in lung adenocarcinoma cells. By detecting the expression of ZNF266 in lung adenocarcinoma patient tumor tissue, the pH value in the patient's tumor cells can be determined, and the efficacy of cisplatin treatment can be predicted. This kit is used to predict patient prognosis before treatment and to evaluate the efficacy of treatment regimens and patient prognosis during and after treatment, thereby achieving precision medicine. This invention proposes, for the first time, predicting the pH value and cisplatin sensitivity in patient tumor cells by detecting ZNF266 expression. This method can effectively assess the effectiveness of treatment regimens, guide individualized treatment for lung adenocarcinoma patients, improve clinical benefits, and avoid unnecessary waste of medical resources. The present invention is simple to operate, cost-effective, highly accurate, and has good sensitivity and specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Figure 2 shows the intracellular pH level after overexpression of ZNF266 in A549 and PC-9 cells;

[0027] Figure 2 This is a graph showing the cell survival rate after A549 cells overexpressed ZNF266 and were treated with cisplatin;

[0028] Figure 3 This is a graph showing the cell survival rate after overexpression of ZNF266 in PC-9 cells and treatment with cisplatin;

[0029] Figure 4 Figure 3 is a graph showing the survival rate of lung adenocarcinoma organoids after overexpression of ZNF266 and cisplatin treatment. DETAILED DESCRIPTION

[0030] 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.

[0031] 1. The kit for predicting the pH value and cisplatin treatment sensitivity of lung adenocarcinoma cells of this embodiment includes: a ZNF266 detection system for detecting the expression level of ZNF266 mRNA in the tumor tissue of lung adenocarcinoma patients; a vector recording the mathematical model for determining the high / low demarcation standard of ZNF266 expression. When the expression level of ZNF266 mRNA in the patient's lung adenocarcinoma tissue is higher than the standard, it indicates that the pH value in the patient's cancer cells is lower than 7.0, and the intracellular environment is acidic, indicating that the patient is suitable for cisplatin combination therapy, that is, the patient is sensitive to cisplatin treatment; conversely, when the expression level of ZNF266 mRNA in the tumor tissue of the patient's lung adenocarcinoma is lower than the standard, it indicates that the pH value in the patient's cancer cells is higher than 7.0, and the intracellular environment is alkaline, indicating that the patient is not suitable for cisplatin combination therapy, that is, the patient is resistant to cisplatin treatment. The nucleotide sequence number of ZNF266 is: NM 001271314.2.

[0032] Specifically, the mathematical model is as follows: the expression level of ZNF266 mRNA in tumor tissues of patients with lung adenocarcinoma is expressed as qPCR.Ct = qPCR.Ct.ZNF266 - qPCR.Ct.ACTB, that is, the difference in Ct values of ZNF266 extracted from tumor tissues of patients with lung adenocarcinoma by quantitative PCR, with ACTB as the internal reference gene; the standard is as follows: qPCR.Ct takes 12.0 as the critical value; if ≤12.0, ZNF266 is considered to be highly expressed, indicating that the intracellular pH value of the patient's cancer lesion is less than or equal to 7.0, the intracellular environment is acidic, indicating that the patient is sensitive to cisplatin treatment, is unlikely to develop drug resistance, and is suitable for cisplatin combination therapy; if <12.0, ZNF266 is considered to be underexpressed, indicating that the intracellular pH value of the patient's cancer lesion is greater than 7.0, the intracellular environment is alkaline, indicating that the patient is insensitive to cisplatin treatment, is likely to develop drug resistance, and is not suitable for cisplatin combination therapy.

[0033] The internal reference can be ACTB or other internal reference genes such as GAPDH, 18sRNA, etc., with ACTB being more preferred. When the internal reference gene is replaced with another internal reference gene, the qPCR Ct cutoff value needs to be appropriately changed.

[0034] The carrier may be a paper instruction manual; for example, an instruction manual with an APP download window, such as a QR code, or a computer program storage device or system such as a USB flash drive, a network disk, etc.

[0035] In a preferred embodiment, the above-mentioned kit further comprises an mRNA extraction and separation system for extracting and isolating mRNA from tumor tissue of patients with lung adenocarcinoma. For example, the above-mentioned mRNA extraction and separation system can be TRNzol Universal Total RNA Extraction Reagent (DP424) of Tiangen Biochemical Technology (Beijing) Co., Ltd. At the same time, the above-mentioned kit further comprises an mRNA reverse transcription system for reverse transcribing mRNA into cDNA. For example, the above-mentioned mRNA reverse transcription system can be TRNzol Universal Total RNA Extraction Reagent (DP424) of Yisheng Biotechnology (Shanghai) Co., Ltd. II 1st Strand cDNA Synthesis SuperMix for qPCR (gDNAdigester plus) reagent. At the same time, the above kit also contains qPCR reagents for qPCR experiments to detect ZNF266. For example, the above qPCR reagents can be qPCRSYBR Green Master Mix (Low Rox Plus).

[0036] The qPCR amplification primer sequences for ZNF266 in the above ZNF266 detection system are:

[0037] Forward primer: 5′-CAGTCTGATCTCTTGGCTGGAACA-3′;

[0038] Reverse primer: 5′-CCTCCGTTGTGGCTTCCTATCATT-3′.

[0039] The qPCR primer sequence of ACTB used as the internal reference is:

[0040] Forward primer: 5′-TGACGTGGACATCCGCAAAG-3′;

[0041] Reverse primer: 5′-CTGGAAGGTGGACAGCGAGG-3′.

[0042] The kit of the present invention can detect the expression level of ZNF266 in patient samples, predict the pH value and cisplatin treatment sensitivity in lung adenocarcinoma cells, and determine whether the patient is suitable for a cisplatin combination treatment regimen to avoid delaying the patient's treatment and achieve the goal of precision medicine.

[0043] As an inhibitory transcription factor, ZNF266 plays an important role in the development and progression of lung adenocarcinoma. ZNF266 can inhibit the expression of carbonic anhydrase IX (CA9) in lung adenocarcinoma, thereby inhibiting and reversing the alkaline intracellular environment created by malignant tumors through the Warburg effect. Therefore, in lung adenocarcinoma cells with high ZNF266 expression, the intracellular environment is acidic, and the cell proliferation, migration, invasion, and drug resistance are significantly reduced, resulting in a significant increase in the cell sensitivity to chemotherapy drugs such as cisplatin. By detecting ZNF266 expression in tumor tissues of patients with lung adenocarcinoma, the intracellular pH value of the patient's cancer lesions and the sensitivity to cisplatin treatment can be effectively predicted. This can be effectively applied to the development of personalized chemotherapy regimens for patients with lung adenocarcinoma, achieving precision medicine for patients.

[0044] The mRNA extraction and separation system used in the embodiment is TRNzolUniversal total RNA extraction reagent (DP424) of Tiangen Biochemical Technology (Beijing) Co., Ltd., and the mRNA reverse transcription system can be the TRNzolUniversal total RNA extraction reagent (DP424) of Yisheng Biotechnology (Shanghai) Co., Ltd. II 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digesterplus) reagent. qPCR reagents can be obtained from Yisheng Biotechnology (Shanghai) Co., Ltd. qPCR SYBRGreen Master Mix (Low Rox Plus) was performed according to the kit instructions. Primers were synthesized and provided by Sangon Biotech (Shanghai) Co., Ltd.

[0045] Human lung adenocarcinoma cell lines A549 and PC-9 were provided by Zhongshan Hospital Affiliated to Fudan University. The cells were cultured in DMEM (high glucose) medium (Jiangsu KeyGen Biotechnology Co., Ltd.) containing 10% FBS and 1% double-antibody in a 37°C, 5% CO2 incubator (Thermo Fisher Scientific, USA).

[0046] 2. Changes in intracellular pH after overexpression of ZNF266 in lung adenocarcinoma cells:

[0047] After ZNF266 was overexpressed in A549 and PC-9 lung adenocarcinoma cells using lentivirus, the intracellular pH value was detected using a BCECF-AM probe. Figure 1 The study found that the intracellular pH of A549 and PC-9 cells overexpressing ZNF266 decreased significantly compared to controls. This suggests that increased ZNF266 expression reverses the pH in lung adenocarcinoma cells to an acidic state.

[0048] 3. Changes in sensitivity to cisplatin treatment in lung adenocarcinoma cells and organoids after overexpression of ZNF266:

[0049] After overexpressing ZNF266 in A549 and PC-9 lung adenocarcinoma cells and lung adenocarcinoma organoids using lentivirus, the ZNF266-overexpressing cells and organoids were treated with cisplatin. After 48 hours of treatment, the viability of the experimental subjects was measured using Alamar Blue and an organoid viability assay kit. The experimental results are shown in Figure 2. Figure 2-4 The study found that A549 and PC-9 cells overexpressing ZNF266, as well as lung adenocarcinoma organoids, showed significantly increased sensitivity to cisplatin compared to controls. This suggests that increased ZNF266 expression increases the sensitivity of lung adenocarcinoma cells and organoids to cisplatin treatment.

[0050] 4. ZNF266 expression in lung adenocarcinoma tissues and cisplatin treatment efficacy

[0051] Tumor tissues were further collected from 50 patients with stage IIIA lung adenocarcinoma who received cisplatin-based chemotherapy after surgery. Of these, 25 patients survived for more than five years after cisplatin-based therapy, and 25 patients survived for less than three years after cisplatin-based therapy (all samples were obtained from the Department of Thoracic Surgery, Zhongshan Hospital, Fudan University). The assay's effectiveness was initially assessed by measuring ZNF266 mRNA expression levels in the tumor samples and comparing them with the patients' clinical treatment outcomes.

[0052] Quantitative PCR results showed that 19 of the 25 patients who survived longer than 5 years had a qPCR Ct < 12.0, indicating high ZNF266 expression. However, only 4 of the 25 patients who survived less than 3 years had a qPCR Ct ≤ 12.0. Therefore, the sensitivity of this kit was 76% and the specificity was 84%.

[0053] 5. The use process of the present invention:

[0054] ZNF266 expression levels in lung adenocarcinoma tissues were measured using quantitative PCR and compared with the internal reference gene ACTB. The difference between the Ct value of ZNF266 for quantitative PCR and the internal reference Ct value was calculated. The cutoff value for the qPCR Ct was 12.0. If the qPCR Ct value was ≤12.0, ZNF266 was highly expressed, indicating that the intracellular pH of the patient's cancer cells was ≤7.0, the intracellular environment was acidic, and the patient was sensitive to cisplatin treatment, not resistant, and suitable for cisplatin combination therapy. If the qPCR Ct value was >12.0, ZNF266 was low, indicating that the patient was insensitive to cisplatin treatment and resistant, and not suitable for cisplatin combination therapy.

[0055] like Figure 1 Figure 2: Overexpression of ZNF266 in A549 and PC-9 cells revealed a significant decrease in intracellular pH to below 7.0 as detected by BCECF-AM probe. NC represents control cells, and OE represents cells overexpressing ZNF266.

[0056] like Figure 2 Figure 2: Overexpression of ZNF266 in A549 cells reduces cell survival after cisplatin treatment, indicating that overexpression of ZNF266 can enhance the sensitivity of these cells to cisplatin. NC represents control cells, OE represents cells overexpressing ZNF266, and Fold Change represents the fold change.

[0057] like Figure 3 Figure 2: Overexpression of ZNF266 in PC-9 cells reduces cell survival after cisplatin treatment, indicating that overexpression of ZNF266 can enhance the sensitivity of these cells to cisplatin. NC represents control cells, OE represents cells overexpressing ZNF266, and Fold Change represents the fold change.

[0058] like Figure 4 Figure 2: Overexpression of ZNF266 in lung adenocarcinoma organoids reduced organoid survival after cisplatin treatment, indicating that high ZNF266 expression can promote the sensitivity of lung adenocarcinoma organoids to cisplatin treatment. NC represents control lung adenocarcinoma organoids, and OE represents lung adenocarcinoma organoids overexpressing ZNF266.

[0059] The above description is merely a preferred embodiment of the present invention and does not limit the present invention in any way. Any person skilled in the art who, without departing from the scope of the present invention, makes any equivalent substitution, modification, or other changes to the technical solution and technical content disclosed in the present invention shall be deemed to be within the scope of the present invention and still fall within the scope of protection of the present invention.

Claims

1. A kit for predicting pH value and cisplatin sensitivity in lung adenocarcinoma cells, characterized in that: The invention comprises a reagent for detecting the expression level of ZNF266 gene, and the reagent determines the pH level and cisplatin treatment sensitivity of the patient's lung adenocarcinoma cells by detecting the expression level of ZNF266 gene.

2. The kit for predicting pH value and cisplatin treatment sensitivity in lung adenocarcinoma cells according to claim 1, characterized in that: The reagent is used to detect the expression level of ZNF266 mRNA in patient tumor tissue.

3. The kit for predicting pH value and cisplatin treatment sensitivity in lung adenocarcinoma cells according to claim 2, characterized in that: The reagent detects the mRNA expression levels of ZNF266 and ACTB as an internal reference in the patient's tumor tissue to obtain qPCR.Ct.ZNF266 and qPCR.Ct.ACTB values, respectively; the difference is calculated using the formula qPCR.Ct=qPCR.Ct.ZNF266-qPCR.Ct.ACTB. When the difference is less than or equal to 12.0, it indicates that the pH value in the patient's tumor tissue is less than 7.0, the intracellular environment is acidic, and the patient is sensitive to cisplatin treatment.

4. The kit for predicting pH value and cisplatin treatment sensitivity in lung adenocarcinoma cells according to claim 3, characterized in that: The qPCR primer sequences for the internal reference ACTB are: Forward primer: 5′-TGACGTGGACATCCGCAAAG-3′ (SEQ ID NO: 3); Reverse primer: 5'-CTGGAAGGTGGACAGCGAGG-3' (SEQ ID NO: 4).

5. The kit for predicting pH value and cisplatin treatment sensitivity in lung adenocarcinoma cells according to claim 1, characterized in that: The reagent for detecting the expression level of the ZNF266 gene is a qPCR reagent.

6. The kit for predicting pH value and cisplatin treatment sensitivity in lung adenocarcinoma cells according to claim 1, characterized in that: The qPCR amplification primer sequences for ZNF266 in the ZNF266 detection system are: Forward primer: 5′-CAGTCTGATCTCTTGGCTGGAACA-3′ (SEQ ID NO: 1); Reverse primer: 5'-CCTCCGTTGTGGCTTCCTATCATT-3' (SEQ ID NO: 2).

7. The kit for predicting pH value and cisplatin treatment sensitivity in lung adenocarcinoma cells according to claim 1, characterized in that: The kit also includes an mRNA extraction and separation system for extracting and separating mRNA from tumor tissues of patients with lung adenocarcinoma.

8. The kit for predicting pH value and cisplatin treatment sensitivity in lung adenocarcinoma cells according to claim 1, characterized in that: The kit also includes an mRNA reverse transcription system for reverse transcribing mRNA into cDNA.

9. A method for predicting pH value and cisplatin sensitivity in lung adenocarcinoma cells, characterized in that: The kit according to any one of claims 1 to 8 is used to detect the expression level of the ZNF266 gene in the patient's tumor tissue to determine the pH value and cisplatin treatment sensitivity of the patient's lung adenocarcinoma cells.

10. Use of the kit according to any one of claims 1 to 8 in predicting the pH value in lung adenocarcinoma cells and their sensitivity to cisplatin treatment.

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