Application of CX43 as detection target and treatment target in lung adenocarcinoma

Abstract

The invention discloses application of CX43 as a detection target and a treatment target in lung adenocarcinoma, and proves that the CX43 can be used as a multifunctional biomarker and a treatment target in lung adenocarcinoma for the first time: the expression level of the CX43 is positively correlated with clinical staging of tumors, so that staging diagnosis can be assisted; the function of the CX43 gene is necessary for promoting tumor growth, and the CX43 gene knockout or drug inhibition can significantly inhibit the growth of tumors in vivo; meanwhile, high expression of CX43 is remarkably related to poor prognosis of non-small cell lung cancer patients. Therefore, CX43 provides a new target and strategy for diagnosis, prognosis evaluation and treatment of lung adenocarcinoma.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of CX43 as a detection and therapeutic target in lung adenocarcinoma. Background Technology

[0002] Lung cancer is a leading cause of cancer-related deaths worldwide, and its incidence continues to rise, making it a major public health issue. Non-small cell lung cancer (NSCLC) is the most prevalent pathological type of lung cancer, with lung adenocarcinoma being the most common subtype. Because lung cancer often develops insidiously, early diagnosis presents significant challenges, and many patients are diagnosed at an advanced stage, losing the opportunity for radical surgical resection and resulting in a poor overall prognosis.

[0003] Accurate staging and effective treatment are crucial for improving the prognosis of patients with lung adenocarcinoma. Currently, there is a lack of highly sensitive and specific molecular biomarkers for accurate staging and prognostic assessment of lung adenocarcinoma, especially early-stage lung adenocarcinoma. There is also an urgent need to develop new therapeutic targets. Identifying biomarkers that can assist in staging, predict prognosis, and serve as potential therapeutic intervention targets is of great significance for improving the clinical management and survival rate of lung adenocarcinoma patients. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of the prior art by providing the application of CX43 as a detection and therapeutic target in lung adenocarcinoma.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect is the application of reagents for detecting CX43 expression levels in the preparation of products used to assist in the staging of lung adenocarcinoma.

[0006] Furthermore, the expression level of CX43 in the product was detected by immunohistochemistry or real-time quantitative PCR.

[0007] The second aspect is to provide the application of CX43 inhibitors in the preparation of drugs for the treatment of lung adenocarcinoma.

[0008] Furthermore, the CX43 inhibitor is a nucleic acid molecule that can reduce CX43 gene expression, or a peptide inhibitor that can inhibit CX43 channel function.

[0009] Furthermore, the nucleic acid molecule is an sgRNA targeting the CX43 gene, and its nucleotide sequence is shown in SEQ ID No. 1 or / and SEQ ID No. 2; the peptide inhibitor is GAP26.

[0010] The third aspect is to provide a pharmaceutical composition for treating lung adenocarcinoma, comprising the aforementioned sgRNA or GAP26, and a pharmaceutically acceptable carrier.

[0011] The fourth aspect is the application of reagents for detecting CX43 expression levels in the preparation of products for assessing the prognosis of patients with non-small cell lung cancer.

[0012] Furthermore, the product is achieved by detecting the expression level of CX43, where high expression of CX43 is associated with poor patient prognosis.

[0013] Furthermore, the expression level of CX43 in the product was detected by immunohistochemistry or real-time quantitative PCR.

[0014] The present invention adopts the above technical solution and has the following technical effects compared with the prior art: This invention demonstrates for the first time that the cell gap junction channel protein CX43 can serve as a multifunctional biomarker and therapeutic target in lung adenocarcinoma: its expression level is positively correlated with the clinical stage of the tumor, aiding in staging diagnosis; its function is essential for promoting tumor growth, and gene knockout or drug inhibition of CX43 can significantly inhibit tumor growth in vivo; simultaneously, high expression of CX43 is significantly associated with poor prognosis in patients with non-small cell lung cancer. Therefore, CX43 provides a new target and strategy for the diagnosis, prognostic assessment, and treatment of lung adenocarcinoma. Attached Figure Description

[0015] Figure 1 This is an image of CX43 immunohistochemical staining in Example 1.

[0016] Figure 2 The relative expression levels of CX43 mRNA in lung adenocarcinoma tissues at different stages are shown in Example 2.

[0017] Figure 3 Images of tumor tissue and tumor volume curves from Example 3 are shown.

[0018] Figure 4 Images of tumor tissue and tumor volume curves from Example 4.

[0019] Figure 5 Images of tumor tissue and tumor volume curves from Example 5.

[0020] Figure 6 The Kaplan-Meier survival curve is shown in Example 6. Detailed Implementation

[0021] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the invention. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of the present invention can be combined with each other.

[0022] Experimental methods in the following examples, unless otherwise specified, were performed under standard conditions or as recommended by the manufacturer. Unless otherwise stated, all reagents and materials used in the following examples were commercially available.

[0023] Example 1: CX43 is highly expressed in high-stage lung adenocarcinoma tumor tissues. Immunohistochemical analysis was performed on lung adenocarcinoma tumor tissues from high-stage (111B) and low-stage (1A) patients (10 cases in each group). All tissue samples were obtained from patients with postoperative pathological diagnosis at Shanghai Pulmonary Hospital, and the staging was determined according to the 9th edition of the IASLC (International Association for the Study of Lung Cancer) staging criteria. The immunohistochemical methods are as follows: 1) Preparation of paraffin sections: Select lung or tumor tissue samples and fix them with 4% formaldehyde solution. After fixation, wash the samples three times with phosphate-buffered saline (PBS), 5 minutes each time. Treat the samples sequentially with 70%, 80%, and 90% ethanol solutions for 30 minutes each, then treat them twice with 95% and 100% ethanol solutions for 20 minutes each time to achieve tissue dehydration. Subsequently, treat the samples with a 1:1 mixture of 100% ethanol and xylene for 15 minutes, and then treat them with pure xylene until the tissue is completely transparent.

[0024] After the tissue was fully immersed in pre-melted paraffin wax, it was quickly transferred to a mold and cooled to obtain a paraffin-embedded block. The paraffin block was sectioned using a microtome, with the section thickness set to approximately 3 μm. The resulting sections were flattened in warm water, picked up with a glass slide, and dried in a 45°C slide dryer for later use.

[0025] 2) Immunohistochemical staining: The dried sections were placed in a 60°C oven for 2 hours. After dewaxing with xylene solutions, the ethanol concentration was gradually reduced to 70%, and finally washed with PBS. The sections were treated with 3% hydrogen peroxide solution for 20 minutes to block endogenous peroxidase activity, followed by washing with PBS. The sections were then placed in boiling 1×EDTA buffer for 20 minutes for retrieval, allowed to cool naturally to room temperature, and then washed with PBS.

[0026] Blocking buffer containing 5% bovine serum albumin (BSA) and 0.5% Triton X-100 was incubated at room temperature for 1 hour. Primary antibody solution was then added, and the slides were incubated overnight at 4°C in a humidified chamber. The next day, after returning to room temperature, the slides were washed with PBS, and the corresponding secondary antibody solution was added. After incubation at room temperature for 1 hour, the slides were washed again with PBS. A DAB chromogenic reaction was performed, and the reaction was terminated with double-distilled water when a brownish-yellow positive signal appeared. Sections were counterstained with hematoxylin for approximately 1 minute, rinsed with tap water, treated with differentiation solution for approximately 30 seconds, and then rinsed. Subsequently, the slides were dehydrated with ethanol solutions of different concentrations, and finally cleared with xylene. After drying, the slides were mounted with neutral resin to complete the immunohistochemical staining preparation.

[0027] Figure 1 The images show immunohistochemical staining of CX43. The results indicate that CX43 is significantly more expressed in high-stage lung adenocarcinoma tumor tissues compared to lower-stage tumor tissues (deeper brown staining in immunohistochemistry represents higher expression).

[0028] Example 2: CX43 is highly expressed in high-stage lung adenocarcinoma tumor tissues. The real-time quantitative PCR method is as follows: 1) Total RNA extraction from samples: Total RNA was extracted from frozen lung adenocarcinoma tissue samples using Trizol reagent. The procedure was performed according to the instructions from Acrel Biotech, ensuring RNA purity (260 / 280 ratio between 1.8 and 2.0). 2) Using the extracted total RNA as a template, reverse transcription was performed using the Aikerui Biotechnology Reverse Transcription Kit to synthesize complementary DNA (cDNA). 800 ng of total RNA and 5* reverse transcription solution were mixed together with RNase-free water and incubated at 37°C for 15 min, followed by heating at 85°C for 5 s to inactivate enzyme activity, thus obtaining the cDNA product.

[0029] 3) Real-time quantitative PCR (qPCR) reaction: qPCR was performed using Aikerui SYBR Green Mix. The qPCR primer sequences are as follows: CX43 upstream primer: 5′-GGTGACTGGAGCGCCTTAG-3′ (SEQ ID No. 3); CX43 downstream primer: 5′-GCGCACATGAGAGATTGGGA-3′ (SEQ ID No. 4); The primer sequences for the internal reference gene GAPDH are as follows: Upstream primer: 5′-CATGAGAAGTATGACAACAGCCT-3′ (SEQ ID No. 5); Downstream primer: 5′-AGTCCTTCCACGATACCAAAGT-3′ (SEQ ID No. 6).

[0030] The relative expression level of the target gene CX43 was calculated using the 2^-ΔΔ Ct method and normalized using the internal reference gene GAPDH.

[0031] Results analysis: The relative expression level of CX43 mRNA in high-stage lung adenocarcinoma tissue group was statistically compared with that in low-stage lung adenocarcinoma tissue group.

[0032] Figure 2 The results showed that the expression level of CX43 mRNA in high-stage lung adenocarcinoma tumor tissues was significantly higher than that in low-stage tumor tissues (P<0.05), suggesting that high expression of CX43 may be related to the malignancy of lung adenocarcinoma.

[0033] Example 3: Knockout of CX43 expression inhibits tumor growth 1. Construction of LLC cell line with CX43 knockout: LLC cell line with CX43 gene knockout (CX43-KO) and non-specific control cell line (WT) were constructed using the CRISPR / Cas9 system.

[0034] The nucleotide sequence of the sgRNA targeting the Cx43 gene is as follows: 5′-GCAGACCGACGGGGTCAACG-3′ (SEQ ID No. 1); 5′-GCGCTGATCCACGATAGCTAA-3′ (SEQ ID No. 2).

[0035] The following steps were taken to construct Cx43 gene knockout cell lines and control cell lines: 1) Construction of transfected cell lines: In LLC cells (mouse lung adenocarcinoma cell line, purchased from the Chinese Academy of Sciences Cell Bank), cas9 plasmids carrying the CX43sgRNA gene were transfected (control transfection with empty cas9 vector).

[0036] 2) 72 hours after transfection, use puromycin for selection. After the GFP fluorescence intensity under the microscope reaches more than 80%, plant single colonies to allow them to grow.

[0037] 3) The knockout effect was assessed two weeks later. The assessment criterion was that CX43 was not expressed by Western blotting, indicating successful knockout. Cells that were successfully knocked out and in good condition were selected for subsequent experiments.

[0038] 2. Subcutaneous tumor formation experiment in immunocompetent mice.

[0039] 1) Experimental Animals: Ten healthy male C57BL / 6J mice (5-6 weeks old) were selected. The animals were obtained from the Shanghai Laboratory Animal Center. All nude mice were housed in a specific pathogen-free (SPF) laboratory animal facility provided by Shanghai Pulmonary Hospital, with the ambient temperature maintained between 22℃ and 26℃, and the relative humidity maintained between 50% and 60%, under a 12-hour day-night alternating light and shadow regime. Before the experiment, the mice were randomly divided into two groups of 5 mice per cage: an experimental group (CX43 KO) and a control group (WT).

[0040] 2) Cell preparation: On the day of tumor cell inoculation, the cells are digested and collected, and a cell suspension is prepared with a concentration of 1×10⁻⁶. 7 Cells / μL.

[0041] 3) Subcutaneous inoculation: Take 100μL of the prepared cell suspension with a 1ml sterile syringe, lift the mouse skin with the syringe and inject. After injection, slowly withdraw the needle and press the injection site for a few seconds to avoid leakage of liquid. 4) Observe tumor formation: Starting from the first week after inoculation, observe tumor growth and measure tumor size every other day.

[0042] Tumor formation was detected on day 8. The long and short diameters of the tumor were measured every 2 days to calculate the volume and growth rate. Mice were sacrificed and the tumors were removed on day 20. Figure 3 The images of tumor tissue and the tumor volume curve are shown. The results show that the tumor growth rate of the control group (WT) cells is significantly stronger than that of the CX43 KO knockout group (experimental group, CX43 KO).

[0043] Example 4: Knockout of CX43 inhibits tumor growth in immunodeficient mice 1. Transfecting cell lines: The method for constructing the knockout cell line is the same as in Example 3, and will not be repeated here.

[0044] 2. Subcutaneous tumor formation experiment in nude mice.

[0045] 1) Laboratory Animals: Ten healthy male Balb / c nude mice (5-6 weeks old) were selected. The animals were obtained from the Shanghai Laboratory Animal Center. All mice were housed in a specific pathogen-free (SPF) laboratory animal facility provided by Shanghai Pulmonary Hospital, with the ambient temperature maintained between 22℃ and 26℃, and the relative humidity maintained between 50% and 60%, under a 12-hour day-night alternating light and shadow regime. Before the experiment, the mice were randomly divided into two groups of five per cage: an experimental group (CX43 KO) and a control group (WT).

[0046] 2) Cell preparation is the same as in Example 3.

[0047] 3) Subcutaneous inoculation, same as in Example 3; 4) Observe tumor formation: Starting from the first week after inoculation, observe tumor growth and measure tumor size every other day.

[0048] Tumor formation was detected on day 8. The long and short diameters of the tumor were measured every 2 days to calculate the volume and growth rate. Mice were sacrificed and the tumors were removed on day 20. Figure 4 The images of tumor tissue and tumor volume curves are shown. The results show that the tumor growth rate of the control group (WT) cells in immunodeficient mice was significantly stronger than that of the CX43 KO knockout group (experimental group, CX43 KO).

[0049] Example 5: CX43 inhibitors inhibit tumor growth 1. Cell line: LLC cells 2. Subcutaneous tumor formation and drug intervention experiments in mice: 1) The experimental animals are the same as in Example 3; 2) Cell preparation and subcutaneous inoculation are the same as in Example 3; 3) CX43 inhibitor intervention: Drug intervention began when the subcutaneous tumor became visible to the naked eye (usually around 7 days after inoculation). Inhibitor source: The CX43 inhibitor GAP26 used in the experiment was purchased from MCE. Administration was via intratumoral injection; the experimental group received an injection of GAP26 inhibitor (200 μg / 80 μL), while the control group received an equal volume of PBS. During injection, the drug was uniformly injected into the tumor at three points. Injections were performed every other day.

[0050] 4) Observe tumor formation: After the start of drug administration, the long and short diameters of the tumor were measured every other day to calculate the volume and growth rate.

[0051] Figure 5 The results show that the tumor growth rate of the control group (PBS-treated) cells in immunodeficient mice was significantly stronger than that of the experimental group treated with the CX43 inhibitor GAP26, suggesting that the CX43 inhibitor GAP26 can effectively inhibit tumor growth in vivo.

[0052] Example 6: Analysis of CX43 expression and survival prognosis in NSCLC patients Data on NSCLC patients were retrieved using the Kaplan-Meier Plotter online database. Based on the optimal cutoff value for CX43 expression in NSCLC patients, all patients were divided into a high-expression group (CX43-High, expression level above the cutoff threshold) and a low-expression group (CX43-Low, expression level below or equal to the cutoff threshold), comprising 369 and 135 samples respectively. Kaplan-Meier survival curves were then used to compare the overall survival (OS) of these two groups, and the hazard ratio (HR) and P-value of the Logrank test for each gene were calculated to assess the impact of their expression levels on survival prognosis.

[0053] Figure 6 The Kaplan-Meier survival curves showed that the hazard ratio (HR) for the CX43 high expression group was 1.34 (95% CI: 0.98–1.83), and the logrank test p-value was 0.07, indicating a difference in survival rates between the CX43 high expression group (CX43-High, n=369) and the low expression group (CX43-Low, n=135), suggesting a significant negative correlation between CX43 expression and NSCLC patient prognosis. Furthermore, the survival curve for the CX43 high expression group (CX43-High) was below that of the low expression group (CX43-Low), indicating that the survival rate of patients with high CX43 expression was significantly lower than that of patients with low expression.

[0054] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the content and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of reagents for detecting CX43 expression levels in the preparation of products for assisting in the staging of lung adenocarcinoma.

2. The application as described in claim 1, characterized in that, The expression level of CX43 in the product was detected by immunohistochemistry or real-time quantitative PCR.

3. Application of CX43 inhibitors in the preparation of drugs for the treatment of lung adenocarcinoma.

4. The application as described in claim 3, characterized in that, The CX43 inhibitor is a nucleic acid molecule that can reduce CX43 gene expression, or a peptide inhibitor that can inhibit CX43 channel function.

5. The application as described in claim 4, characterized in that, The nucleic acid molecule is an sgRNA targeting the CX43 gene, and its nucleotide sequence is shown in SEQ ID No. 1 or / and SEQ ID No. 2; the peptide inhibitor is GAP26.

6. A pharmaceutical composition for treating lung adenocarcinoma, characterized in that, It comprises the sgRNA or GAP26 as described in claim 5, and a pharmaceutically acceptable vector.

7. Application of reagents for detecting CX43 expression levels in the preparation of products for assessing the prognosis of patients with non-small cell lung cancer.

8. The application as described in claim 7, characterized in that, The product is achieved by detecting the expression level of CX43, where high expression of CX43 is associated with poor patient prognosis.

9. The application as described in claim 7, characterized in that, The expression level of CX43 in the product was detected by immunohistochemistry or real-time quantitative PCR.

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