Application of LLPH as lung cancer diagnosis marker and treatment target

Abstract

The invention relates to the technical field of biology, and particularly discloses application of LLPH as a lung cancer diagnosis biomarker and a treatment target spot, an LLPH expression inhibitor is an LLPH gene knockout plasmid and a genetic engineering construct containing sgRNA and a carrier, the LLPH expression inhibitor is suitable for CRISPR / Cas system mediated gene editing, the sgRNA sequence is 5 '-CTAAAGCTTACGGAGTAAG-3', the carrier is pX459M, and the enzyme cutting site is BbsI-BbsI; by detecting the relative expression quantity of LLPH in normal lung bronchial epithelial cells and lung cancer cell lines, it is found that LLPH is highly expressed in the lung cancer cell lines, and it is shown that LLPH is closely related to lung cancer development. The invention can provide a new biomarker for diagnosis of lung cancer. Lung cancer A549 and H1299 cells are used as models, and the LLPH gene is knocked out, so that the in-vitro malignant proliferation capacity of the lung cancer cells A549 and H1299 can be remarkably inhibited, and LLPH can be used as a potential treatment target for inhibiting lung cancer proliferation.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to the application of LLPH as a diagnostic biomarker and therapeutic target for lung cancer. Background Art

[0002] According to global cancer statistics for 2022, lung cancer remains the malignant tumor with the highest morbidity and mortality in my country, posing a serious threat to public health. Therefore, improving early screening and accurate diagnosis of lung cancer is key to improving patient survival. Ribosome-associated proteins are a class of proteins that interact with ribosomes but do not directly constitute their core structure. They play a key role in ribosome biogenesis, translation regulation, ribosome stress response, and tumor metabolism. Ribosome-associated proteins typically interact with ribosomal subunits or translation factors to regulate mRNA selective translation, ribosome function, ribosome degradation, and stress adaptation, and are crucial for cellular protein synthesis and growth. In recent years, studies have found that abnormal expression or mutations of ribosome-associated proteins are closely associated with the occurrence and progression of various tumors. Cancer cells promote their own proliferation by enhancing ribosome biogenesis and regulating protein synthesis, and modulate the tumor microenvironment to promote invasion and metastasis, suggesting that ribosome-associated proteins may become new tumor biomarkers.

[0003] LLPH is a ribosome-associated protein that plays a key role in neural development by regulating the translation efficiency of long coding sequence mRNAs. Its functional mechanism involves stable binding to ribosomal subunits and coordinated regulation of multiprotein complexes, providing important clues for understanding translation dynamics and cell fate determination. However, the role of LLPH in lung cancer remains understudied, especially its potential as a diagnostic marker or therapeutic target. Given that LLPH may promote cancer cell proliferation and survival by regulating translational adaptability, in-depth exploration of its functional mechanisms in lung cancer will provide new research directions and potential clinical applications for the precise diagnosis and targeted treatment of lung cancer. Summary of the Invention

[0004] This invention aims to address technical issues in lung cancer diagnosis and targeted therapy, provide LLPH as a biomarker for lung cancer diagnosis and a target for promoting lung cancer growth, and its applications, and reveal the key role of LLPH in lung cancer. Specifically, it includes the following:

[0005] In order to solve the above technical problems, the present invention provides any of the following uses of an LLPH expression inhibitor:

[0006] (1) Application in the preparation of diagnostic markers for lung cancer;

[0007] (2) Use in the preparation of drugs for the prevention, diagnosis and / or treatment of lung cancer.

[0008] Optionally, the LLPH expression inhibitor is an LLPH gene knockout plasmid, a genetically engineered construct comprising an sgRNA and a vector, suitable for CRISPR / Cas system-mediated gene editing, the sgRNA sequence is 5'-CTAAAAGCTTACGGAGTAAG-3', the vector used is pX459M, and the enzyme cutting site is BbsⅠ-BbsⅠ.

[0009] The present invention also provides a composition for preventing and / or treating lung cancer, which comprises: an LLPH expression inhibitor and a pharmaceutically acceptable adjuvant; the adjuvant comprises a carrier, a solvent and / or a buffer.

[0010] Optionally, the LLPH expression inhibitor is a LLPH gene knockout plasmid.

[0011] The purpose of the present invention is to show that LLPH can be used as a diagnostic marker for lung cancer growth as well as a therapeutic target.

[0012] The technical solution adopted by the present invention is as follows: Western blotting was used to detect differential expression of LLPH in lung cancer cell lines. By constructing an LLPH gene knockout vector and establishing A549 LLPHKO and H1299 LLPHKO cell lines, the effect of LLPH deficiency on the malignant proliferation of lung cancer cells was investigated using a soft agar colony formation assay.

[0013] Beneficial effects:

[0014] By detecting the relative expression of LLPH in normal lung bronchial epithelial cells and lung cancer cell lines, the present invention found that LLPH is highly expressed in lung cancer cell lines, indicating that LLPH is closely related to the development of lung cancer. The present invention can provide a new biomarker for the diagnosis of lung cancer.

[0015] The present invention further uses lung cancer A549 and H1299 cells as models, and knocking out the LLPH gene can significantly inhibit the in vitro malignant proliferation ability of lung cancer cells A549 and H1299, indicating that LLPH can be used as a potential therapeutic target for inhibiting lung cancer proliferation. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The specific embodiments of the present invention are further described in detail below with reference to the accompanying drawings.

[0017] Figure 1 LLPH is highly expressed in lung cancer cell lines;

[0018] Figure 2 LLPH deficiency can significantly inhibit the in vitro malignant proliferation ability of lung cancer cells A549 and H1299. DETAILED DESCRIPTION

[0019] The present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto:

[0020] The attached figure below, Figure 1 In Figure A, the relative expression of LLPH in normal lung bronchial epithelial cells and lung cancer cell lines was detected using Western Blotting technology;

[0021] Figure 2 middle,

[0022] B: After knocking out the LLPH gene in A549 and H1299 cells, the gene knockout efficiency was identified by Western blotting technique;

[0023] C and D show the effect of LLPH deficiency on the malignant proliferation ability of lung cancer cells in vitro verified by soft agar experiment.

[0024] Example 1: Detection of LLPH expression in lung cancer cell lines

[0025] Western blotting was used to detect the relative expression of LLPH in normal lung bronchial epithelial cells and lung cancer cell lines. Figure 1 As shown in A, LLPH is highly expressed in lung cancer cell lines. The specific implementation steps are as follows:

[0026] 1) Cell protein extraction

[0027] a. Cells were cultured to approximately 1×10 7 After discarding the culture medium, PBS buffer was added to wash the cell surface, and the PBS was discarded.

[0028] b. Add cell lysis buffer BB and place on ice for 1 minute to allow the cells to fully lyse. Use a clean cell scraper to remove the cells. Use a pipette to transfer the cell lysis buffer to a 1.5 mL centrifuge tube and place on ice for about 5 minutes. The composition of the cell lysis buffer is as follows:

[0029]

[0030] c. Boil the protein lysate in a metal bath at 100°C for 5 minutes and cool on ice.

[0031] d. Ultrasonic cell lysis: Select an appropriate ultrasonic frequency and set the total program time to 2 minutes, 1 second per cycle, with 1 second intervals between cycles. During the sonication process, maintain the protein sample at 4°C and clean the probe after each sample.

[0032] e. Measure the protein concentration and volume of the sample: Use a NanoDrop 2000 spectrophotometer to measure the sample concentration, using ddH2O as a blank control. Measure each sample twice and take the average value. Then use a pipette to measure the sample volume.

[0033] f. Sample volume adjustment: After determining the sample concentration and volume, use the formula to dilute the samples from the same experimental group to the same concentration by adding BB and 6×SB. Vortex the samples evenly and heat them in a metal bath at 100°C for 5 minutes. The resulting protein sample can be used or stored at -20°C. The formula for 6×SB is as follows:

[0034]

[0035] 2) Preparation of SDS-PAGE gel

[0036] a. Prepare a 12% separating gel solution and stir until uniform. Add APS and TEMED immediately before pouring the gel, mix quickly, and pour the mixture onto the gel plate to approximately 2 / 3 of the height of the glass plate. Gently add 1-2 mm of methanol to the gel surface and let it stand for 40 minutes until it completely solidifies. The 12% separating gel formula is as follows:

[0037]

[0038]

[0039] b. After the separation gel has solidified, discard the upper isopropanol / water layer and absorb any remaining liquid with filter paper. Add APS and TEMED to the stacking gel mixture and pour it onto the gel plate until it reaches the top of the glass plate. Quickly insert a comb (to avoid air bubbles) and let it sit for 15-20 minutes until it completely solidifies. The formula for 15% separation gel is as follows:

[0040]

[0041] 3) SDS-PAGE electrophoresis

[0042] a. Prepare 1× Running buffer. Place the gel plate in the electrophoresis tank and pour in 1× Running buffer. Remove the sample comb and pipette through the sample wells to ensure there is no residual gel in the wells. The formula for 1× Running buffer is as follows:

[0043]

[0044] b. Add samples to the sample wells and perform electrophoresis on the stacking gel at 80V and the separating gel at 120V.

[0045] 4) Transfer

[0046] Activate the PVDF membrane in methanol for approximately 15 seconds and then rinse the methanol in the equilibration solution. Remove the gel from the gel sheet and scrape off any excess gel. Place the membrane in a transfer cassette, following the order of sponge, gel, PVDF membrane, and sponge, from positive to negative electrodes. Transfer the membrane to a transfer apparatus. After transfer, remove the PVDF membrane and wash it in TBS buffer for 3-5 minutes.

[0047] 5) Closed

[0048] Add 5% skim milk to the PVDF membrane and block it on a shaker at room temperature for one hour. After blocking, discard the milk and wash the membrane three times with TBS, each for 5 minutes. The formula for 5% skim milk is as follows:

[0049]

[0050] 6) Primary antibody incubation

[0051] Pour LLPH (ABclonal, Catalog No. A15534) and α-Tubulin (Proteintech, Catalog No. 11224-1-AP) primary antibodies at a ratio of 1:1000, enough to cover the surface of the PVDF membrane, and incubate on a shaker at 4°C overnight. After the primary antibody is recovered to the original tube, add TBS and wash three times for 5 minutes each at room temperature on a shaker.

[0052] 7) Secondary antibody incubation

[0053] Discard the TBS and add the secondary antibody of the species corresponding to the primary antibody at a dilution ratio of 1:1000. Incubate on a shaker at 4°C for 2-3 hours. After the secondary antibody incubation is completed, return the secondary antibody to the original tube and wash three times with TBS on a shaker at room temperature for 5 minutes each. Then, wash three times with TBST buffer on a shaker for 15 minutes each, and finally wash with TBS for 10 minutes.

[0054] 8) Development

[0055] First, dilute the ECF developer by adding approximately 7 mL of TBS to 500 μL of ECF developer and mix thoroughly. Immerse the PVDF membrane in the developer. Shake the membrane evenly in the developer for 0.5–1 minute, depending on the protein expression level and detection sensitivity of each sample. Place the membrane on a plastic film. Set the exposure time and area on a Typhoon 7000 (GE) scanner, then place the plastic film holder on the scanner for development.

[0056] Example 2: Investigate the effect of LLPH on the malignant proliferation ability of lung cancer cells.

[0057] 1) Construction of LLPH gene knockout cell line for lung cancer

[0058] A549 cells and H1299 cells were selected for construction, and the pX459M plasmid vector was used to construct the LLPH gene knockout plasmid. The LLPH gene was knocked out in A549 and H1299 cells. Stable transfected cells A549 LLPHKO, H1299 LLPHKO and their controls A549 Vector and H1299 Vector were established. Western blotting experiments were performed to verify the LLPH gene knockout efficiency. The results are shown in Figure 2. Figure 2 As shown in AB, LLPH gene knockout stable cell lines were successfully constructed in A549 and H1299 cells.

[0059] The LLPH gene knockout plasmid was constructed in-house using the pX459M vector with the BbsⅠ-BbsⅠ restriction enzyme sites. The pX459M plasmid is 9211 bp long, and the sgRNA sequence is 5'-CTAAAAGCTTACGGAGTAAG-3'.

[0060] The specific steps to build A549 LLPHKO and H1299 LLPHKO are as follows:

[0061] a. Lipofectamine transfection of A549 and H1299 cells: PolyJet TM A549 and H1299 cells were transfected with lipofectamine using the reagent. Before transfection, cells were grown to 80% density by adherence. Serum-free DMEM medium was replaced 30 minutes before transfection to optimize cell status. The transfection complex was prepared by dissolving 1 μg of LLPH gene knockout plasmid in 50 μL serum-free DMEM and adding 3 μL of PolyJet TM Dissolve the reagent in 50 μL of serum-free DMEM, then quickly mix the two and let them stand for 15 minutes to form the plasmid-liposome complex. Add 100 μL of the transfection mixture dropwise to the cell culture dish, gently shaking to ensure even distribution. After 12 hours, observe the cells under a light microscope and replace the culture medium with 10% FBSDMEM.

[0062] b. Antibiotic selection of A549 LLPHKO and H1299 LLPHKO cell lines: Cells were cultured in DMEM supplemented with 10% FBS for 24 hours, followed by selection with puromycin at a final concentration of 1 μg / mL for 3 days. After selection, 100 surviving cells were counted and evenly plated in 96-well plates to isolate single clones. Selected single clones were expanded and cultured, and Western blotting was performed to verify LLPH gene knockout to ensure efficacy.

[0063] 1) Soft agar colony formation experiment was used to evaluate the effect of LLPH deficiency on the malignant proliferation ability of lung cancer cells.

[0064] The soft agar colony formation assay was used to evaluate the anchorage-independent malignant proliferation ability of tumor cells A549 LLPHKO and H1299 LLPHKO compared with the control A549 Vector and H1299 Vector cells. Figure 2 As shown in CD, LLPH knockout can significantly inhibit the anchorage-independent malignant proliferation of lung cancer cells. The specific steps are as follows:

[0065] a. Prepare 1.25% agarose solution and medium: Autoclave the required 1.25% agarose solution and medium and preheat them in a 45°C water bath. The medium formula is as follows:

[0066]

[0067] b. Prepare the bottom agar: Prepare three replicate wells for each cell type. Add 1.2 mL of 1.25% agarose solution and 1.8 mL of medium to each well in a 15 mL centrifuge tube. Gently pipette to mix thoroughly, then add to the wells of a 6-well plate. Let dry at room temperature for 2 h.

[0068] c. Prepare the top agar: After cell digestion and centrifugation, count the cells. Take the corresponding volume of single-cell suspension and add it to a mixture of 1.25% agarose solution and medium. Gently pipette to mix thoroughly. Quickly add 2 mL of the mixture to the top agar. After standing at room temperature for 2-3 hours, seal the six-well plate with sealing film to maintain moisture and place it in a 37°C, 5% CO2 cell culture incubator for continued incubation. The top agar recipe is as follows:

[0069]

[0070]

[0071] d. Photography and Counting: After approximately 15 days of culture, observe the growth status and take photos under a light microscope. Five photos were taken randomly per well using a 5x magnification microscope. The colony formation rate was calculated and data was analyzed and plotted using GraphPad software. (*) indicates a significant difference compared to the control group (p < 0.05).

[0072] The preferred embodiments of the present invention disclosed above are intended only to help illustrate the present invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the present invention to the specific embodiments described. Obviously, many modifications and variations are possible based on the content of this specification. These embodiments are selected and described in detail in this specification to better explain the principles and practical applications of the present invention, thereby enabling those skilled in the art to better understand and utilize the present invention. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. Any of the following uses of an inhibitor of LLPH expression: (1) Application in the preparation of diagnostic markers for lung cancer; (2) Use in the preparation of drugs for the prevention, diagnosis and / or treatment of lung cancer.

2. The use according to claim 1, characterized in that The LLPH expression inhibitor is a LLPH gene knockout plasmid.

3. A composition for preventing and / or treating lung cancer, characterized in that: The invention comprises the LLPH expression inhibitor according to claim 1 or 2 and a pharmaceutically acceptable adjuvant; the adjuvant comprises a carrier, a solvent and / or a buffer.

4. The composition for preventing and / or treating lung cancer according to claim 3, characterized in that: The LLPH expression inhibitor is a LLPH gene knockout plasmid.

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