Lung cancer cell strain with stable and low expression of Integrin alpha6 and construction method of lung cancer cell strain

Abstract

The invention relates to the field of biomedical research, and provides a lung cancer cell strain (H1299) capable of stably and low-expressing Integrin alpha6 protein and a construction method of the lung cancer cell strain (H1299). The preparation method comprises the following steps: designing and synthesizing an shRNA (short hairpin Ribonucleic Acid) sequence for specifically knocking down gene expression aiming at a human Integrin alpha6 gene, forming a double-chain fragment through annealing, and inserting the shRNA sequence into a pLKO.1-puro lentiviral vector by adopting a connection independent cloning (LIC) method, so as to construct a pLKO.1-ITG alpha6-shRNA recombinant plasmid. Furthermore, the recombinant plasmid and lentivirus packaging plasmids psPAX2 and pVSVG are co-transfected to an HEK293T cell by utilizing a lentivirus packaging system, and the lentivirus is produced by packaging. And infecting a target lung cancer cell strain H1299 by using the obtained lentivirus, and then carrying out puromycin resistance screening and continuous subculture to finally obtain the stable and continuous low-expression Integrin alpha6 protein H1299 cell strain. A fluorescence microscope, a real-time fluorescent quantitative PCR (Polymerase Chain Reaction) and an immunoblotting technology are used for verifying that the gene silencing efficiency of the Integrin alpha6 in the cell strain can reach more than 80%. The stable cell strain constructed by the invention provides a reliable and efficient tool cell model for researching the molecular mechanism of the Integrin alpha6 in the processes of occurrence and development, invasion and metastasis, energy metabolism, immune escape and the like of lung cancer, and also provides a new experimental material for screening antitumor drugs targeting the Integrin alpha6.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically to a stable lung cancer cell line with low expression of Integrin α6 protein and a method for constructing the same. Background Technology

[0002] Lung cancer is the most common and deadliest malignant tumor, and its increasing incidence and mortality rates make it a serious public health problem. However, the complexity of its pathogenesis has limited the development of effective clinical methods. Therefore, exploring the molecular mechanisms of lung cancer development and progression is of significant practical importance for early diagnosis and the identification of therapeutic targets.

[0003] Integrins are transmembrane glycoprotein receptors widely distributed on the cell surface, mediating bidirectional signal transduction between cells and the extracellular matrix (ECM) and between adjacent cells. Integrins consist of α and β subunits linked non-covalently to form heterodimers. Currently, 18 α subunits and 8 β subunits have been identified, which can combine to form at least 24 functional dimers with different ligand specificities. Recent studies have shown that integrins not only participate in cell adhesion, migration, proliferation, and survival, but also play a crucial regulatory role in signal transduction, tissue development, immune responses, and tumorigenesis and development. In various malignant tumors, such as lung cancer, breast cancer, colorectal cancer, and liver cancer, the expression of specific integrin isoforms is often abnormally upregulated or functionally activated, regulating tumor invasion, metastasis, angiogenesis, and treatment resistance. Integrin α6 (CD49f) primarily pairs with the β1 or β4 subunits to form α6β1 or α6β4 dimers, and is highly expressed in various epithelial tumors (such as lung cancer). As a high-affinity receptor for laminin, it is closely related to tumor stem cell characteristics, epithelial-mesenchymal transition (EMT), and poor prognosis. Therefore, Integrin α6 holds promise as a novel biomarker for lung cancer, providing new strategies for early detection, monitoring, diagnosis, treatment, and prognosis of lung cancer, and thus becoming a target for clinical translational research on lung cancer biomarkers.

[0004] This study focuses on the function of Integrin α6 in tumors, especially lung cancer. By constructing different lung cancer cell models with stable low expression of Integrin α6 through a lentiviral transfection system, this study provides a useful tool for studying the molecular mechanisms of Integrin α6 in lung cancer development, energy metabolism homeostasis, and biological functions. It also provides experimental evidence and cellular resources for targeted therapy strategies against Integrin α6 in clinical practice. Summary of the Invention

[0005] The purpose of this invention is to provide a lung cancer cell line with stable low expression of integrin α6 (ITGα6) protein and its lentiviral transfection construction method. This cell line can be used as an in vitro cell model to study the regulatory mechanism of ITGα6 in the development and progression of lung cancer and its impact on biological functions such as proliferation, migration, energy metabolism reprogramming, and immune escape. It also provides a stable and reliable tool cell for screening anti-tumor drugs targeting Integrin α6.

[0006] This invention provides a stable lung cancer cell line with low expression of ITGα6 protein. Using a human lung cancer cell line (H1299) as the host, a plasmid was constructed using a ligation-independent cloning (LIC) method. The expression of the ITGα6 gene was specifically knocked down using lentivirus-mediated shRNA interference technology. The expression vector and viral packaging plasmid were packaged into lentivirus using HEK293T cells to obtain the desired viral solution. After infecting the target cell line, the cell line underwent drug screening for the corresponding antibiotic (puromycin) and amplification culture to ultimately obtain a lung cancer cell line that stably and continuously expresses low levels of ITGα6. The ITGα6-shRNA sequence targets a conserved region of ITGα6 mRNA, efficiently and continuously reducing ITGα6 transcription and protein expression. The transfection efficiency of the recombinant plasmid can be detected by real-time quantitative PCR and Western blotting.

[0007] This invention provides a method for constructing a stable lung cancer cell line with low expression of ITGα6 protein. Specifically, the shRNA plasmid is constructed using the pLKO.1 vector (puromycin resistant). An shRNA interference sequence corresponding to the target gene is inserted after the U6 promoter. Age I and EcoRI double restriction sites are selected. The expression vector, viral packaging plasmid psPAX2, pVSVG, and transfection reagent PEI form a complex and are co-transfected into HEK293T cells for lentiviral packaging. The resulting viral solution infects the target cell line, followed by drug screening for corresponding resistance. A stable cell line is obtained in approximately 7 weeks. The specific steps are as follows:

[0008] 1. Preparation of lentiviruses

[0009] (1) Primer annealing: Two specific shRNA sequences were designed based on the coding sequence of the human ITGα6 gene. Two complementary, phosphorylated single-stranded oligonucleotides (Oligos) were synthesized for the selected effective shRNA sequences. The two Oligo sequences were pre-added with single-stranded overhang sequences that were completely complementary to the ends of the specifically treated LIC-compatible vector and did not contain restriction enzyme sites. The two complementary shRNA Oligos were mixed with T4 polynucleotide kinase (T4 PNK) and denatured in boiling water, then naturally cooled to room temperature to anneal the two primers to form phosphorylated double-stranded fragments with sticky ends, which were directly used for ligation with the linearized vector.

[0010] (2) Vector digestion: A LIC-compatible lentiviral vector (pLKO.1-puro-LIC) suitable for shRNA expression was selected, whose multiple cloning site includes Age I and EcoRI restriction sites. The vector was double-digested using high-fidelity restriction endonucleases Age I and EcoRI. The reaction system was incubated at 37°C for 1-2 hours to completely linearize the circular plasmid, producing linear DNA molecules with sticky ends. The digestion products were verified by agarose gel electrophoresis and purified by gel extraction.

[0011] (3) Directional ligation: The double-stranded shRNA insert prepared in step (1) is mixed with the linearized vector in step (2), T4 DNA Ligase and T4 Buffer and incubated at room temperature for 30 minutes to 1 hour.

[0012] (4) Bacterial transformation: Add competent cells (DH-5α) to the DNA ligation reaction fragment in step (3), mix thoroughly, incubate on ice for 30 minutes, heat shock at 42°C in a constant temperature water bath for 45 seconds, continue in an ice bath for 5 minutes, and then use for subsequent plating.

[0013] (5) LB agar plate coating: In a microbial clean bench, the above ligation products are evenly coated onto the corresponding resistance LB agar plates using coating beads. The plates are then inverted and incubated at 37°C for 16 hours until single colonies are formed.

[0014] (6) Sequencing verification: In a microbial clean bench, use a sterile pipette tip to pick up 3 or more single-clone colonies and quickly inoculate them into TB medium. Place the medium in a 37°C constant temperature shaker. After the bacterial solution becomes turbid, take 200 μl of the bacterial solution and send it to Shanghai Sangon Biotech for sequencing. If the sequencing results are as expected, the next step of plasmid extraction can be carried out.

[0015] (7) Plasmid extraction: Plasmids were extracted using a commercial plasmid extraction kit, and the plasmid concentration and purity were determined using a multifunctional microplate.

[0016] (8) Lentiviral preparation: HEK293T cells in good growth condition were pre-coated in culture dishes. Transfection with plasmids was only performed when the cells were in good growth condition and reached a density of 70-80%. The recombinant target plasmid ITGα6 shRNA obtained in step (7) was mixed with the lentiviral packaging plasmids psPAX2 and pVSVG in Opti-MEM medium at a mass ratio of 10:9:1. Simultaneously, linear polyethyleneimine (PEI, 1 mg / mL) was diluted in another tube of Opti-MEM medium. A mixed suspension was prepared according to the ratio of plasmid (μg): transfection reagent: serum-free DMEM medium = 1:4:100. After mixing the two solutions, incubation at room temperature for 15 minutes to form a DNA-PEI complex. The complex was then added to the prepared HEK293T cells.

[0017] (9) Collecting virus solution: Collect the virus-containing supernatant 48 hours after transfection. Filter the virus solution through a 0.22μm disposable filter into a 15ml sterile centrifuge tube for the infection of the target cell line (the infection aid polybrene can be selected according to the characteristics of the cell line, with a final concentration of 5μg / ml).

[0018] 2. Lentiviral infection of lung cancer cells and screening of stable cell lines

[0019] (1) Selection of stable lung cancer cell lines: The expression level of ITGα6 protein in normal lung epithelial cell line HBE and nine human lung cancer cell lines A549, H1299, H460, H2030, H23, H1944, H1437, H292 and H1703 was detected by immunoblotting. The lung cancer cell line with the highest expression level was selected to construct a stable lung cancer cell line with low expression of ITGα6.

[0020] (2) Lung cancer cell seeding: H1299 cells were seeded in 6-well plates one day before the virus transfection experiment, with 2 × 10⁶ cells per well. 5 Each cell ensures a cell density of 30-40% the next day.

[0021] (3) Virus infection of target cells: Take out the lung cancer cells inoculated in step (1) from the cell culture incubator, remove the original cell culture medium, add the collected virus solution to the above lung cancer cell culture dish, add the virus solution: complete culture medium = 1:10, place it in a 37℃, 5% CO2 incubator, and continue to culture for 24 hours.

[0022] (4) Change the culture medium: After 24 hours, remove the culture medium containing the virus and replace it with complete culture medium (RPMI-1640+10% FBS+1×P / S), place it in a 37℃, 5% CO2 incubator, and continue to incubate for 48-72 hours.

[0023] (5) Screening of stable cell lines: Puromycin, an antibiotic corresponding to the backbone resistance, was selected for positive cell screening. The final concentration of Puromycin was 2.0 g / ml. Puromycin was changed every three days according to the cell status. The presence of green fluorescent cells was observed under a fluorescence microscope to preliminarily assess the transfection efficiency.

[0024] (6) Identification of positive cell lines: A portion of the stable selected cell lines were taken out, and total RNA and total protein were extracted using Trizol RNA extraction reagent and protein lysis buffer, respectively. The ITGα6 knockdown efficiency was detected by real-time quantitative PCR and Western blotting. If the ITGα6 knockdown efficiency reached more than 80% and the cells continued to stably and persistently express ITGα6 after passage, it meant that the lung cancer cell line with stable low expression of ITGα6 was successfully constructed. Beneficial effects

[0025] Compared with existing technologies, this invention has the following advantages: First, compared with existing transient knockdown methods such as RNA interference or chemical transfection, this patent uses lentivirus-mediated shRNA interference technology to stably embed the interference sequence targeting ITGα6 into the host genome, ensuring long-term stable inhibition of the target gene and avoiding the short-lived gene silencing effect, rebound expression, and batch-to-batch silencing differences. Second, this patent uses a ligation-independent cloning (LIC) method to construct recombinant vectors, which can improve the efficiency and specificity of gene knockdown. Simultaneously, the introduction of GFP and Puromycin resistance selection markers into the vector enables real-time monitoring of transfection efficiency and rapid enrichment of stable low-expression positive cell lines. Finally, this method for constructing stable low-expression lung cancer cell lines is applicable to the construction of stable cell lines in various cancer types and other disease models, providing experimental tools for studying tumor stem cell characteristics, malignant biological behavior, energy metabolism reprogramming, and immune escape, and providing favorable support for drug screening and clinical targeted therapy targeting TGα6. Attached Figure Description

[0026] Figure 1 Enzyme digestion diagram of the pLKO.1-ITGα6-shRNA recombinant plasmid.

[0027] Figure 2 Fluorescence identification of H1299 stable cell line with low expression of human ITGα6 gene under an inverted fluorescence microscope.

[0028] Figure 3 : Expression levels of ITGα6 protein in normal lung epithelial cells (HBE) and nine lung cancer cell lines (A549, H1299, H460, H2030, H23, H1944, H1437, H292, H1703) using Western blotting. ACTIN is an internal control.

[0029] Figure 4 Figure: Identification of ITGα6 mRNA expression level in the H1299 stable cell line with low expression of human ITGα6 gene by real-time quantitative PCR. GAPDH was used as an internal control.

[0030] Figure 5 Image showing the expression level of ITGα6 protein in the H1299 stable cell line with low expression of human ITGα6 gene, as determined by Western blotting. ACTIN was used as an internal control. Detailed Implementation

[0031] The present invention will now be described in further detail with reference to the accompanying drawings. Example 1: Construction of ITGα6 recombinant expression vector

[0032] (1) shRNA sequence design and synthesis: Based on the coding sequence of the human ITGα6 gene, two specific shRNA sequences (ITGα6-sh1 and ITGα6-sh2) were designed. Phosphorylated complementary single-stranded oligonucleotides (Oligos) containing AgeⅠ / EcoRI sticky ends were synthesized by Shanghai Sangon Biotech Co., Ltd. The sequences are as follows:

[0033] ITGα6-sh1:

[0034] Forward Oligo:

[0035] 5'-CCGGCGAGAAGGAAATCAAGACAAACTCGAGTTTGTCTTGATTTCCTTCTCGTTTTTG-3';

[0036] Reverse Oligo:

[0037] 5'-AATTCAAAAACGAGAAGGAAATCAAGACAAACTCGAGTTTGTCTTGATTTCCTTCTCG-3';

[0038] ITGα6-sh2:

[0039] Forward Oligo:

[0040] 5'-CCGGTTCTTTAACTGCCGTAATTTACTCGAGTAAATTACGGCAGTTAAAGAATTTTTG-3';

[0041] Reverse Oligo:

[0042] 5'-AATTCAAAAATTCTTTAACTGCCGTAATTTACTCGAGTAAATTACGGCAGTTAAAGAA-3';

[0043] (2) Oligonucleotide annealing: Dissolve each Oligo in 1×TE to 10 μM, take 1 μl of each and mix with 1 μl of T4 polynucleotide kinase (T4 PNK), add ddH2O to 10 μl. Denature in boiling water bath and then cool naturally to room temperature to form a double-stranded fragment with phosphorylated sticky ends (ds-shRNA).

[0044] (3) Vector linearization by restriction enzyme digestion: The lentiviral shRNA expression backbone pLKO.1-puro was selected and double-digested with high-fidelity restriction enzymes Age I / EcoRI. The reaction system (50 μl) consisted of: 2 μg of vector plasmid, 5 μl of 10×FastDigest Buffer, 20 U each of Age I / EcoRI, and ddH2O to a final volume of 50 μl. The mixture was incubated at 37 °C for 1-2 hours. The linearized bands were verified by 1% agarose gel electrophoresis. Based on the size of the indicator band on the DNA Marker (TAKARA DL5000), the target band was quickly excised under UV light, dissolved in 500 μl of Buffer MB, recovered, and its concentration was determined by NanoDrop.

[0045] (4) Directional ligation: Mix the ds-shRNA obtained in step (2) with the linearized pLKO.1 in step (3) at a molar ratio of 3:1, add 1 μl of T4 DNA ligase (ThermoFisher) and 2 μl of 10×T4 ligation buffer, add ddH2O to 20 μl, and ligate at room temperature for 1 hour. Example 2: Bacterial Transformation, Plasmid Extraction, and Virus Preparation

[0046] (1) Bacterial transformation: Take 5 μl of the ligation product from step (4) and add it to 50 μl of DH5α competent cells. Incubate on ice for 30 minutes, heat shock at 42°C for 45 seconds, and then incubate on ice for 5 minutes.

[0047] (2) Plate coating: The ligation product was evenly coated onto LB plates containing 100 μg / ml ampicillin using coating beads. The plates were incubated upside down in a 37°C incubator for 16 hours to observe the formation of single colonies.

[0048] (3) Pick single colonies and verify: Use a sterile pipette tip to pick 3 single colonies and place them in 5 mL of TB medium. Place them in a 37°C constant temperature shaker and culture. After the bacterial solution becomes turbid, take 200 μl of the bacterial solution and send it to Shanghai Sangon Biotech Co., Ltd. for sequencing to confirm that the inserted sequence is correct.

[0049] (4) Plasmid extraction: Collect bacterial culture, centrifuge, invert to drain excess supernatant, use plasmid extraction kit (TaKaRa MiniBEST Plasmid Purification Kit) to extract endotoxin-free plasmids, and NanoDrop to determine OD (260 / 280) value and plasmid concentration.

[0050] (5) Lentiviral solution preparation: One day before the experiment, well-grown HEK293T cells were seeded into 10cm culture dishes, with 1×10 cells per dish. 6 For each HEK293T cell, add 10 mL of DMEM medium containing 10% fetal bovine serum and 1×P / S, and incubate at 37°C in a 5% CO2 incubator. The cell density will reach 70-80% the next day. Mix the recombinant target plasmid pLKO.1-ITGα6-shRNA with the packaging plasmids psPAX2 and pVSVG at a mass ratio of 10:9:1 (total plasmid 6 μg), and add Opti-MEM medium to a final volume of 600 μl. In another tube, add 24 μL of PEI transfection reagent (1 mg / mL) to 576 μL of Opti-MEM. Mix the two tubes and incubate at room temperature for 15 minutes to form a DNA-PEI complex. Add the complex to the prepared HEK293T cells, gently shake the culture dish in a figure-eight motion to mix, and incubate at 37°C in a 5% CO2 incubator for 48 hours. Collect the supernatant, filter it through a 0.22 μm filter membrane, and transfer the supernatant to a 15 mL sterile centrifuge tube to obtain the lentivirus stock solution. The virus solution can be used directly or aliquoted and stored at -80℃ for long-term use.

[0051] Example 3: Construction of H1299 lung cancer cell line with stable low expression of ITGα6

[0052] (1) Selection of stable lung cancer cell lines: Nine human lung cancer cell lines, A549, H1299, H460, H2030, H23, H1944, H1437, H292, and H1703, and the normal human lung epithelial cell line HBE were seeded in 10cm culture dishes, with 1×10⁻⁶ cells per dish. 6 After the cells were stably grown, total cell protein was extracted, and protein quantification was performed using the BCA method. The expression of ITGα6 in each cell line was detected by Western blotting. The cell line with the highest ITGα6 expression level was selected to construct a stable lung cancer cell line with low ITGα6 expression.

[0053] (1) H1299 lung cancer cell line seeding: The day before the experiment, well-grown H1299 cells were seeded into 6-well plates at a density of 2 × 10⁶ cells per well. 5 Add 3 mL of 1640 medium containing 10% fetal bovine serum and 1×P / S to each well of cells and incubate at 37°C with 5% CO2. The cell density will reach 30-40% the next day.

[0054] (2) Infection of H1299 lung cancer cell line: The next day, remove the original cell culture medium, wash twice with 2 mL PBS per well, add 300 μl of virus stock solution and Polybrene (final concentration 5 μg / ml) to 3 mL of complete culture medium per well, and incubate at 37℃ in a 5% CO2 incubator. After 24 hours, remove the virus-containing culture medium and replace it with complete culture medium (RPMI-1640 + 10% FBS + 1×P / S), and continue culturing for 48-72 hours. During this period, monitor the cells for green fluorescence in real time under a fluorescence microscope.

[0055] (3) Screening of stable low-expression ITGα6 cell lines: More than 60% of cells were found to express GFP under a fluorescence microscope. Puromycin (final concentration 2.0 μg / ml) was then added to screen positive cells. The medium was changed every 3 days and the screening was continued for more than 7 weeks until all untransfected control group cells died and the experimental group formed a stable cell line.

[0056] (4) Identification of ITGα6 stable low-expression positive cell lines: ① mRNA level verification: A portion of cells were taken, total RNA was extracted with Trizol, and cDNA was synthesized by reverse transcription and then detected by real-time quantitative PCR. Nucleic acid extraction reagents, reverse transcription and qPCR detection reagents were obtained from Takara Bio's RNAiso Plus, iScience's All-in-one FirstStrand Synthesis Mastermix reverse transcription kit and One Step SYBR PrimeScrip RT-PCR Kit, respectively. The operation steps were in accordance with the product instructions. (Primer sequences are as follows:)

[0057] ITGα6-F 5′-AGAGAATTCGGATCCACCATGGCCGCCGCCGGGCAGCTG-3′,

[0058] ITGα6-R 5′-CTTCCATGGCTCGAGTGCATCAGAAGTAAGCCTCTCTTTA-3′;

[0059] (2) Protein level verification: Total cell protein was extracted, protein quantification was performed using the BCA method, and the expression of ITGα6 in the H1299 cell line was detected by Western blotting. ITGα6 antibody from Proteintech (rabbit anti-ITGα6, 1:1000 dilution) was used as the internal control, and β-Actin antibody (mouse anti-β-Actin, 1:5000 dilution) was used for incubation, followed by ECL imaging. The silencing efficiency was over 80%, indicating that the H1299 lung cancer cell line with low ITGα6 expression was successfully constructed. This cell line can be expanded and cultured, and then stored in liquid nitrogen for a long time. It can be frozen in liquid nitrogen to establish a cell bank for subsequent functional studies.

Claims

1. A method for constructing a lung cancer cell line with stable low expression of Integrin α6, characterized in that, Includes the following sequential steps: (1) Based on the coding sequence of the human Integrin α6 gene, two shRNA sequences that specifically knock down the expression of the Integrin α6 gene were designed and synthesized, and phosphorylated double-stranded fragments with sticky ends were formed by annealing. (2) The lentiviral expression vector pLKO.1-puro was selected and linearized by double digestion with Age I and EcoR I. The double-stranded shRNA fragment obtained in step (1) was directionally ligated with the linearized vector using T4 DNA ligase. After bacterial transformation, single-clone colony picking, verification and plasmid extraction, the pLKO.1-ITGα6-shRNA recombinant plasmid was finally obtained. (3) The recombinant plasmid obtained in step (2) was mixed with lentiviral packaging plasmids psPAX2 and pVSVG at a mass ratio of 10:9:1 and transfected into HEK293T cells. After culturing for 48 hours, the supernatant containing the virus was collected and filtered through a 0.22 μm filter membrane to obtain Integrin α6-shRNA lentiviral solution. (4) Infect the pre-inoculated H1299 lung cancer cells with the virus solution obtained in step (3), add Puromycin at a final concentration of 2.0 μg / ml for positive cell screening, change the fresh culture medium containing Puromycin every three days, and continue screening for more than 7 weeks until a stable low expression of Integrin α6 H1299 cell line is obtained. (5) The expression of green fluorescence in cells was monitored in real time by fluorescence microscopy, and the mRNA and protein expression levels of Integrin α6 were detected by real-time quantitative PCR and immunoblotting. The silencing efficiency of Integrin α6 was confirmed to be above 80%, and H1299 lung cancer cell line with stable low expression of Integrin α6 was obtained.

2. The method according to claim 1, characterized in that: The design and synthesis of the two shRNA sequences in step (1) are as follows: (1) Based on the NCBI reference sequence, two independent shRNA sequences targeting the conserved region of human Integrin α6 mRNA were designed; CCGG was added to the 5′ end of the sense strand and AATTC was added to the 5′ end of the antisense strand to form 4-nt sticky ends that are completely complementary to pLKO.1-puro after Age I / EcoRI digestion; the sequences are as follows: ITGα6-sh1: Forward Oligo: '-CCGGCGAGAAGGAAATCAAGACAAACTCGAGTTTGTCTTGATTTCCTTCTCGTTTTTG-3'; Reverse Oligo: '-AATTCAAAAACGAGAAGGAAATCAAGACAAACTCGAGTTTGTCTTGATTTCCTTCTCG-3'; ITGα6-sh2: Forward Oligo: '-CCGGTTCTTTAACTGCCGTAATTTACTCGAGTAAATTACGGCAGTTAAAGAATTTTTG-3'; Reverse Oligo: '-AATTCAAAAATTCTTTAACTGCCGTAATTTACTCGAGTAAATTACGGCAGTTAAAGAA-3'; (2) 10 μl of reaction solution contains 1 μl (10 μM) each of positive and antisense strands and 1 μl of T4 PNK, with the remainder being ddH2O. After denaturation in boiling water bath, the solution is naturally cooled to room temperature to form a double-stranded fragment (ds-shRNA) with phosphorylated sticky ends.

3. The method according to claim 1, characterized in that: Step (2) of vector linearization, ligation, transformation, and plasmid extraction is performed as follows: (1) Double enzyme digestion system: 50 μl system contains 2 μg pLKO.1-puro, 5 μl 10×FastDigest buffer, 20 U Age I, 20 U EcoR I, and the remainder is made up with ddH2O. Incubate at 37 °C for 1-2 hours to completely linearize the circular plasmid and produce linear DNA molecules with sticky ends. (2) Gel recovery: The linearized vector was separated by 1% agarose gel electrophoresis and purified using a gel recovery kit, with the A260 / 280 ratio controlled at 1.8–2.0; (3) Ligation system: 20 μl system contains ds-shRNA and linear vector (molar ratio 3:1), 1 μl T4 DNA ligase, 2 μl 10×T4 ligation buffer, ligation at room temperature for 1 hour; (4) Thermal conversion: Add 5 μl of ligation product to 50 μl of DH5α competent cells, incubate on ice for 30 minutes, heat shock at 42 °C for 45 seconds, and incubate on ice for 5 minutes; (5) Plate preparation, single colony picking and verification: Plate the colonies on LB plates containing 100 μg / ml ampicillin, incubate upside down in a 37 °C incubator for 16 hours, pick ≥3 single colonies, verify the positive bands by colony PCR, and send them for sequencing to confirm that the inserted sequence is correct and there are no mutations.

4. The method according to claim 1, characterized in that: The lentivirus packaging and concentration in step (3) shall be performed as follows: (1) Cell preparation: Seed 1×10 cells in a 10 cm dish 6 HEK293T cells were cultured in DMEM + 10% fetal bovine serum + 1×P / S medium at 37 °C with 5% CO2 until 70–80% confluence. (2) Plasmid ratio: target plasmid pLKO.1-ITGα6-shRNA: packaging plasmid psPAX2: packaging plasmid pVSVG = 10:9:1, total DNA 6μg; (3) PEI preparation: 24 μl of PEI transfection reagent (1 mg / ml) was dissolved in 576 μl of Opti-MEM and allowed to stand at room temperature for 5 minutes; plasmid DNA was dissolved in 600 μl of Opti-MEM, and the two were mixed in equal volumes and allowed to stand at room temperature for 15 minutes to form a DNA-PEI complex; (4) Transfection: Add 1.2 ml of this DNA-PEI complex to the prepared HEK293T cells and incubate at 37°C and 5% CO2 for 48 hours; (5) Virus collection and concentration: After sterilization by 0.22μm filter membrane, the supernatant of the virus solution is collected to obtain a clear virus stock solution; the endotoxin content is <0.1 EU / μg to ensure the efficiency of virus packaging; dispense 100μl / tube, store at -80 °C in the dark, and repeat freeze-thaw cycles ≤2 times; If required for the experiment, the virus solution can be added to an ultracentrifuge tube, centrifuged at 20,000g at 4 °C for 2 hours, the supernatant discarded, and the virus resuspended in PBS to 1 / 100 of the original volume to obtain concentrated virus with a titer ≥1×10^7 TU / ml.

5. The method according to claim 1, characterized in that: The H1299 cell line infection and screening in step (4) shall be performed as follows: (1) Cell seeding: 2 × 10^ 2 cells per well in a 6-well plate 5 H1299 cells, RPMI-1640 + 10% fetal bovine serum + 1×P / S, cultured at 37 °C and 5% CO2 until 30–40% confluence; (2) Virus infection of H1299 cell line: The virus solution was mixed with the complete culture medium at a ratio of 1:10, with a total volume of 3 mL. Polybrene (final concentration 5 μg / mL) was added to prepare the virus infection solution, which was then added to H1299 cells and incubated at 37°C in a 5% CO2 incubator. The next day, the old culture medium was removed and replaced with normal complete culture medium. The cells were incubated for another 48-72 hours, and the green fluorescence of the cells was observed under a fluorescence microscope. (3) Screening stable low-expression cell lines: Add Puromycin (final concentration 2.0 μg / ml) to screen positive cells. Change the medium every 72 hours with fresh medium containing the same concentration of Puromycin. Continue screening for more than 7 weeks to obtain stable low-expression cell lines.

6. The method according to claim 1, characterized in that: The verification of the stable low-expression H1299 cell line in step (5) shall be performed as follows: (1) mRNA level verification: Total RNA was extracted from cells, and the silencing efficiency of ITGα6 transcription was detected by real-time quantitative PCR (primer sequences are as follows: ITGα6-F 5′-AGAGAATTCGGATCCACCATGGCCGCCGCCGGGCAGCTG-3′, ITGα6-R 5′-CTTCCATGGCTCGAGTGCATCAGAAGTAAGCCTCTCTTTA-3′) (2) Protein level verification: Total cell protein was extracted and the ITGα6 translation silencing efficiency was detected by immunoblotting. The silencing efficiency was over 80%, indicating that the H1299 lung cancer cell line with low ITGα6 expression was successfully constructed.

7. A lung cancer cell line with stable low expression of Integrin α6 obtained by the construction method according to any one of claims 1 to 6, characterized in that: This cell line, after continuous passage in vitro, maintained Integrin α6 protein expression at ≤20% of the control level and was free of mycoplasma contamination. It is suitable for studies on Integrin α6 loss of function, tumor energy metabolism, EMT, and targeted drug screening.

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