A human lung normal stable cell strain overexpressing MLT1L and a construction method and application thereof

Abstract

This invention discloses a stable human lung cell line overexpressing MLT1L, its construction method, and its applications. This invention belongs to the field of biomedicine, specifically relating to a stable human lung cell line overexpressing MLT1L, its construction method, and its applications. The substance promoting MLT1L expression of this invention can be applied to: (A1) improving the migration ability of normal lung cells; (A2) preparing products for improving the migration ability of normal lung cells; (A3) improving the invasive ability of normal lung cells; (A4) preparing products for improving the invasive ability of normal lung cells; wherein MLT1L is a long non-coding RNA with a nucleotide sequence as shown in SEQ ID No:1. This invention provides a method for constructing a stable human lung cell line overexpressing MLT1L, and overexpression of MLT1L-2034 can significantly increase the viability, proliferation, and invasive ability of Beas-2B cells.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a stable human lung cell line overexpressing MLT1L, its construction method, and its application. Background Technology

[0002] Lung cancer is the leading cause of cancer incidence and mortality worldwide, seriously threatening human health. According to the latest global cancer burden data for 2022, there were 2.5 million new cases of lung cancer globally, accounting for 12.4% of all new cancer cases. During the same period, 1.8 million people died from lung cancer globally, accounting for 18.7% of all cancer deaths. From a pathological and treatment perspective, lung cancer can be divided into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), with NSCLC accounting for approximately 80%-85%, including histological subtypes such as adenocarcinoma, squamous cell carcinoma, and large cell lung cancer. Although significant progress has been made in lung cancer treatment, most patients are diagnosed at an advanced stage, and the 5-year survival rate remains unsatisfactory. Therefore, improving the early diagnosis rate and treatment effectiveness of lung cancer is crucial, and the discovery and clinical application of highly effective lung cancer biomarkers is one of the important means to achieve this. A growing body of research has found abnormal expression of human endogenous retroviruses (HERVs) during the development and progression of lung cancer, suggesting that they may play an important role in the development of lung cancer.

[0003] HERVs originated from exogenous retroviruses (XRVs) that infected the germline throughout evolution. They subsequently integrated into the genome of germ cells, co-evolved with the host, and became fixed in offspring populations, vertically propagating as proviruses through Mendelian inheritance, comprising approximately 8% of the human genome. The HERV genome structure consists of long terminal repeats (LTRs) at both ends and four open reading frames (gag, pro, pol, and env) in the middle. LTRs contain promoters and enhancers essential for transcription, regulating gene expression. HERVs initially integrated into proviral sequences, ranging from complete proviruses to highly fragmented proviral remnants. HERVs can be silenced by destructive mutations, recombination, methylation, histone modifications, and other factors. However, HERVs can be reactivated by various factors, such as infectious agents, exogenous viruses, radiation, aging-related processes, epigenetic drugs, cytokines, or mitogens. Studies have shown that aberrantly expressed HERVs may be involved in tumorigenesis and development. MLT1L is a long non-coding RNA (lncRNA) derived from HERV LTR. Previous studies have shown that interfering with MLT1L can inhibit the proliferation and migration of lung cancer cells. lncRNAs have high tissue specificity, high efficiency, and high stability, and are expected to become potential therapeutic targets and biomarkers for diagnosis and prognosis. Therefore, in-depth exploration of the molecular mechanisms of MLT1L in lung cancer progression and the search for effective potential therapeutic targets are of great significance for the treatment of lung cancer. Summary of the Invention

[0004] The main problem to be solved by this invention is how to obtain a stable human lung cell line that overexpresses MLT1L.

[0005] To address the above problems, the present invention provides the use of substances that promote MLT1L expression in any of the following: (A1) Enhances the migration ability of normal lung cells; (A2) Prepare products to enhance the migration ability of normal lung cells; (A3) Enhances the invasive ability of normal lung cells; (A4) Prepare products for enhancing the invasive ability of normal lung cells; The MLT1L is a long non-coding RNA with a nucleotide sequence as shown in SEQ ID No:1.

[0006] In the above applications, the substance may be any of the following: (C1) is capable of being transcribed into the DNA molecule described in MLT1L; (C2) An expression cassette containing the DNA molecule described in (C1); (C3) A recombinant plasmid containing the DNA molecule described in (C1); (C4) A recombinant lentiviral vector containing the DNA molecule described in (C1).

[0007] In the above applications, the nucleotide sequence of the DNA molecule is shown in SEQ ID No:4.

[0008] The DNA molecules mentioned above also fall within the scope of protection claimed in this invention.

[0009] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cosmids), and viral vectors (such as retroviruses (including lentiviruses), adenoviruses, rabies virus, adeno-associated virus, herpesviruses (such as herpes simplex virus), baculoviruses, or vaccinia virus, etc.). Any plasmid and vector can be used as long as it can stably replicate in the host cell. Those skilled in the art can utilize DNA recombination technology and other techniques to construct expression vectors containing the nucleic acid molecular composition described in this invention, suitable transcription and translation regulatory sequences, promoters, and selective marker genes, among other specific elements. The vector plasmid used in this invention is pcDNA3.1(+).

[0010] In one specific embodiment, the recombinant vector is pcDNA3.1(+)-MLT1L-2034. The structure of the recombinant vector pcDNA3.1(+)-MLT1L-2034 is described as follows: [The text then describes the structure of the vector pcDNA3.1(+) at the 5'-ctagcgtttaaactt...] aagctt -3' and 5'-tgctggatatctgca gaattc A DNA fragment with sequence SEQ ID No:4 was inserted between the -3' fragments, and the recombinant vector was obtained by keeping the other sequences of the pcDNA3.1(+) vector unchanged. The pcDNA3.1(+)-MLT1L-2034 vector can express MLT1L.

[0011] The present invention also provides a biomaterial, which may be any of the following: 1) Recombinant viruses containing the recombinant vector described above; 2) Recombinant microorganisms containing the recombinant vector described above; 3) Recombinant cells containing the recombinant vector described above; 4) Animal cell lines containing the recombinant vectors described above; 5) Animal tissues containing the recombinant vector described above; 6) Animal organs containing the recombinant vectors described above.

[0012] This invention also provides the following method for constructing a normal lung cell model: F1) A method for preparing a lung normal cell model with enhanced migration ability in vitro, comprising the following steps: increasing the expression of MLT1L in recipient lung normal cells to obtain a lung normal cell model with enhanced migration ability; F2) A method for preparing an in vitro lung normal cell model with enhanced invasiveness, comprising the following steps: increasing the expression of MLT1L in recipient lung normal cells to obtain a lung normal cell model with enhanced invasiveness; The MLT1L is a long non-coding RNA with a nucleotide sequence as shown in SEQ ID No:1.

[0013] In the above method, the expression of MLT1L is enhanced by transfecting DNA molecules expressing the MLT1L transcript into normal lung cells of the recipient.

[0014] The normal lung cells mentioned can be human normal lung cells Beas-2B (CL-0496).

[0015] The present invention also provides a lung cell model prepared by the method described above.

[0016] This invention also provides the application of the lung cell model described above in drug screening or in the preparation of products for drug screening; the drug having any of the following functions: (D1) Treatment and / or prevention of lung cancer; (D2) Inhibits the migration of normal lung cells; (D3) Inhibits the invasion of normal lung cells.

[0017] In the above method, the lung cells are lung cells with relatively stronger migration and / or invasion capabilities.

[0018] This invention employs a seamless cloning method when constructing the overexpression vector, and the construction process is not limited by enzyme restriction sites. This invention achieves a high success rate by overexpressing MLT1L in normal lung cell lines, resulting in stable MLT1L-overexpressing lung cell lines that promote cell viability, proliferation, and invasion. The cell lines of this invention stably express MLT1L, overcoming the short duration of exogenous gene overexpression in transient transfection experiments, thus laying the foundation for in-depth research into the molecular mechanisms by which MLT1L plays a role in the development and progression of lung cancer. Attached Figure Description

[0019] Figure 1The images show the RACE PCR amplification of the full-length MLT1L transcript. A is the RACE PCR electrophoresis image, and B is the partial sequencing result of the RACE PCR. Lanes 1 and 3 in A are 2000 DNA markers, lane 2 is the 5' RACE PCR amplification band, and lane 4 is the 3' RACE PCR amplification band. The upper part of B shows the first-generation sequencing result of the 5' RACE PCR, and the lower part shows the first-generation sequencing result of the 3' RACE PCR.

[0020] Figure 2 The images show the PCR electrophoresis and restriction enzyme digestion electrophoresis images of MLT1L-2034. Image A shows the PCR electrophoresis image of MLT1L-2034, where lane 1 contains the 5000 DNA marker, lanes 2-5 contain the MLT1L-2034 transcript, and there are 4 replicates. Image B shows the pcDNA3.1(+) restriction enzyme digestion electrophoresis image, where lane 1 contains the 5000 DNA marker, lanes 2-5 contain the pcDNA3.1(+) linearized vector, and there are 4 replicates.

[0021] Figure 3 Partial sequencing results of pcDNA3.1(+)-MLT1L-2034-OE plasmid Figure 4 To detect the relative expression level of MLT1L-2034 overexpression using RT-qPCR.

[0022] Figure 5 Overexpression of MLT1L-2034 led to an increase in the viability of normal lung cells.

[0023] Figure 6 The expression of MLT1L-2034 leads to an increase in the proliferation capacity of normal lung cells. A shows the staining pattern of stable lung normal cells overexpressing MLT1L-2034; B shows the cell count pattern of the colonies.

[0024] Figure 7 This study demonstrates how overexpression of MLT1L-2034 increases the invasive ability of normal lung cells. Image A shows the staining of stable normal lung cells overexpressing MLT1L-2034 across the Transwell chamber basement membrane; image B shows the cell count of cells crossing the Transwell chamber basement membrane. Detailed Implementation

[0025] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0027] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0028] The human lung normal cells Beas-2B and lung squamous cell carcinoma cells SK-MES-1 in the following examples were purchased from Wuhan Pronosei Life Science Technology Co., Ltd., with product numbers CL-0496 and CL-0213, respectively.

[0029] The pcDNA3.1(+) vector in the following examples is described in: Jin X, Xu XE, Jiang YZ, et al. The endogenous retrovirus-derived long noncoding RNA TROJAN promotes triple-negative breast cancer progression via ZMYND8 degradation. Sci Adv. 2019 Mar6;5(3):eaat9820. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention and may not be used for any other purpose.

[0030] The following examples use GraphPad Prism 8 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. t tests were used, with P < 0.05 (*) indicating a significant difference, P < 0.01 (**) indicating a highly significant difference, P < 0.001 (***) indicating a highly significant difference, and P < 0.001 (****) indicating a highly significant difference.

[0031] Example 1: RACE PCR amplification of MLT1L transcripts 1. Cell Culture Lung squamous cell carcinoma cell line SK-MES-1 (Pronosai, CL-0213) was routinely cultured in DMEM high-glucose medium (Gibco, 11995065) containing 10% inactivated fetal bovine serum (Gibco, 10099-141) and 1% penicillin-streptomycin (Gibco, 15140-122); and then cultured statically in a CO2 incubator (Thermo, 320) at 37°C, 5% CO2, and saturated humidity.

[0032] When the cells reached approximately 80% confluence in the culture flask, they were passaged. The original culture medium was aspirated, and the cells were washed with 1×PBS (Gibco, C10010500CP). An appropriate amount of 0.25% trypsin-EDTA (Gibco, 25200056) was added to digest the cells. The cell status was observed under an inverted microscope (OLYMPUS, CKX53). Once the cells were round, the 0.25% trypsin-EDTA was aspirated, and 10 mL of DMEM high-glucose complete medium was added to terminate the digestion. The cells were repeatedly pipetted to disperse them, and then seeded at a 1:2 ratio into DMEM high-glucose complete medium. The flask was then incubated in a CO2 incubator for further culture.

[0033] 2. Extract total RNA from cells (1) Cell treatment: Remove SK-MES-1 cells from the CO2 incubator, remove the original culture medium, and wash the cells with 1×PBS. Add an appropriate amount of 0.25% trypsin-EDTA to the cell culture flask, digest the cells for 1 min, discard the 0.25% trypsin-EDTA, add 1 mL of complete culture medium, pipette the cells to detach them, transfer the cells to 1.5 mL Eppendorf tubes (Thermo, 3448PK), centrifuge at 1500 rpm for 3 min using a microcentrifuge (Eppendorf, Centrifuge 5425), and wash the cells once with 1×PBS.

[0034] (2) Total RNA was extracted from cells according to the instructions of TaKaRa MiniBEST Universal RNA Extra (TAKARA, 9767).

[0035] (3) Determination of RNA concentration: The concentration of eluted RNA was determined using an ultra-micro UV-Vis spectrophotometer.

[0036] (4) Agarose gel electrophoresis to identify RNA integrity: Prepare 2% agarose gel, set the voltage to 120 V, and electrophoresis for 10 min; if 28S:18S≥2, the total RNA quality is considered to be good.

[0037] 3. Add a polyadenosine tail (poly A tail) to the 3' end of the RNA. E.coli Poly(A) Polymerase (Nanjing Novizan Biotechnology Co., Ltd., DD4111-PC).

[0038] (1) Prepare the reaction system according to Table 1 below: Table 1. Reaction system with polyA tailing

[0039] (2) The reaction time is 37℃ for 30 min.

[0040] 4. RACE PCR amplification Following the instructions of the HiScript-TS 5' / 3' RACE Kit (Vazyme, RA101), we efficiently reversed full-length cDNA using RNA as a template and rapidly amplified its 5' or 3' ends using cDNA as a template.

[0041] 5. Electrophoresis, purification, ligation, transformation, sequencing (1) Electrophoresis: The PCR amplification products were subjected to agarose gel electrophoresis and the PCR products were purified according to the Wizard® SV Gel and PCRClean-Up System (Promega, A9282) instructions.

[0042] (2) Cloning: According to the instructions of the 5 min TA / Blunt-Zero Cloning Kit (Vazyme, C601), prepare the cloning reaction system as shown in Table 2 below: Table 2. Connection Reaction System

[0043] Gently tap the bottom of the centrifuge tube to mix, then centrifuge briefly at low speed to collect all liquid at the bottom of the tube. Incubate at room temperature for 5 minutes. After the reaction is complete, place the centrifuge tube on ice.

[0044] (3) Transformation: Take 50 μL of TransStbl3 competent cells (Beijing TransGen Biotech, CD521-01) thawed on ice, add the ligation product from step 5 (2), gently mix, and place in an ice bath for 30 min. Heat shock in a 42℃ water bath for 60 sec, then quickly transfer the tube to an ice bath for 3 min, without shaking the centrifuge tube during this process. Add 500 μL of sterile SOC medium to each centrifuge tube, mix well, and incubate at 37℃ and 200 rpm for 1 h to allow the bacteria to recover. Take out the sample and centrifuge at 5000 rpm for 2 min. Take 100 μL of culture medium, gently discard the remaining culture medium, resuspend with 100 μL of culture medium, add 100 μL of culture to a plate containing ampicillin antibiotic, spread the culture medium evenly, place the plate at 37℃ until the liquid is absorbed, invert the plate, and incubate overnight at 37℃. Single colonies were picked and inoculated into LB medium containing 100 μg / mL ampicillin. The culture was carried out overnight at 37°C and 180 rpm. Plasmids were extracted according to the instructions of the endotoxin-free plasmid small-scale extraction kit (Tiangen Biotech Co., Ltd., DP118) and sent to Beijing Nuosai Genome Research Center Co., Ltd. for Sanger sequencing.

[0045] The nucleotide sequence of MLT1L is SEQ ID No:1 in the sequence listing. The results are as follows Figure 1 As shown: RACE PCR amplification of the full-length MLT1L transcript, in which... Figure 1 Image A is a RACE PCR electrophoresis image. Figure 1 Figure B shows the partial sequencing results of the RACE PCR. By comparing the sequencing sequences, the 5' RACE sequence length was found to be 1047 bp (nucleotide sequence is SEQ ID No:2 in the sequence listing); the 3' RACE sequence length was found to be 582 bp (nucleotide sequence is SEQ ID No:3 in the sequence listing). By splicing the three sequences in the 5' to 3' direction, MLT1L-2034 was obtained, with the nucleotide sequence being SEQ ID No:4 in the sequence listing.

[0046] Example 2: Construction of a stable human lung cell line overexpressing MLT1L-2034 Cell culture and extraction of total RNA from SK-MES-1 cells were performed in the same manner as in Example 1.

[0047] The human lung normal cell line Beas-2B (Pronosa, CL-0496) was routinely cultured in MEM medium (Gibco, 11095-080) containing 10% inactivated fetal bovine serum (Gibco, 10099-141) and 1% penicillin-streptomycin (Gibco, 15140-122).

[0048] RNA reverse transcription was performed according to the PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa, RR047A) instructions. MLT1L-2034 was obtained by PCR amplification using PrimeSTAR Max DNA Polymerase (TaKaRa, R045A).

[0049] Using cDNA from SK-MES-1 cells as a template, primers were designed and synthesized to amplify the full-length MLT1L-2034 sequence. The primers contain homologous arms, restriction endonuclease Hind III and EcoRI sites, and amplify the full-length sequence. The PCR primer sequences are as follows: Homologous arm+ Hind III restriction site +MLT1L-2034-Forwoard (Primer F for short): 5'-ctagcgtttaaactt aagctt CCTTTTTGTAAAACCATACCAGGC-3'; Homologous arm+ EcoRI restriction site +MLT1L-2034-Reverse (Primer R for short): 5'-tgctggatatctgca gaattc CCTCTAAAGCACTCTGCTTGGTC-3'.

[0050] 1. PCR amplification reaction (1) Prepare the PCR amplification reaction system according to Table 3 below: Table 3. PCR amplification reaction system

[0051] (2) The PCR procedure is shown in Table 4: Table 4. PCR Amplification Procedure

[0052] The PCR amplification products were subjected to agarose gel electrophoresis and the PCR products were recovered using a gel recovery kit to obtain the target gene MLT1L-2034 containing homologous arms, restriction endonuclease Hind III and EcoRI restriction sites.

[0053] 2. pcDNA3.1(+) digestion The pcDNA3.1(+) vector was double-digested with the restriction endonucleases Hind III (NEB, R0101L) and EcoRI (NEB, R0101) at 37°C for 4 h. The digested pcDNA3.1(+) vector formed a linearized vector. The digestion products were subjected to 1% agarose gel electrophoresis, followed by purification using a gel extraction kit. The digested PCR products and the linearized vector were then analyzed.

[0054] (1) The enzyme digestion system is shown in Table 5: Table 5. pcDNA3.1(+) digestion system

[0055] (2) Enzyme digestion conditions: React at 37°C for 4 hours; then cool to 4°C or immediately place on ice to cool.

[0056] The results are as follows Figure 2 As shown, Figure 2 In section A, the MLT1L-2034 transcript was amplified by PCR. Figure 2 B is a linearized plasmid obtained by digesting pcDNA3.1(+) with enzymes.

[0057] 3. Connection and Conversion (1) Seamless DNA cloning According to the instructions for the ClonExpress® II One Step Cloning Kit (Vazyme, C112-01).

[0058] Calculation of the amount of vector fragment used: The molar ratio of vector to inserted fragment is 1:2.

[0059] The recombination reaction system is shown in Table 6 below: Table 6. Recombination Reaction System

[0060] React at 37°C for 30 minutes, then cool to 4°C or immediately place on ice to cool.

[0061] (2) Transformation and sequencing were performed in the same manner as in Example 1, and the results are as follows: Figure 3 As shown: By comparing the sequencing results, MLT1L-2034 was successfully cloned into the pcDNA3.1(+) plasmid, and the correct recombinant vector pcDNA3.1(+)-MLT1L-2034 was obtained.

[0062] The structure of the recombinant vector pcDNA3.1(+)-MLT1L-2034 is described as follows: At the 5'-ctagcgtttaaactt of the starting vector pcDNA3.1(+) aagctt -3' and 5'-tgctggatatctgca gaattc A DNA fragment with sequence SEQ ID No:4 was inserted between the -3' fragments, and the recombinant vector was obtained by keeping the other sequences of the pcDNA3.1(+) vector unchanged. The pcDNA3.1(+)-MLT1L-2034 vector can express MLT1L.

[0063] 4. Transfecting cells 1) One day before transfection, remove Beas-2B cells from the CO2 incubator, aspirate the original culture medium, and wash the cells with 1×PBS. Add 3 mL of 0.25% trypsin-EDTA and digest the cells for 1 min. Discard the 0.25% trypsin-EDTA, add 10 mL of MEM, and pipette to detach the cells. Count the Beas-2B cells and adjust the medium to 2.2×10⁶ cells / mL using MEM high-glucose complete medium containing 10% fetal bovine serum and free of antibiotics. 5 The cells were seeded into 12-well cell culture plates (Thermo, 150628), with 1 mL of culture medium per well. The cell culture plates were shaken to spread the medium evenly across the bottom of the wells. The plates were then placed on a workbench and left to stand for a while. The cells were then observed under an inverted microscope to check the uniformity of the cell distribution. Finally, the plates were placed in a CO2 incubator for cell culture.

[0064] 2) Culture the cells for 24 hours to achieve a confluence of 70% at the time of transfection.

[0065] 3) Preparation of Lipofectamine 3000 dilution: Dilute 3 μL of Lipofectamine 3000 (Thermo Fisher, L3000015) with 50 μL of serum-free Opti-MEM I serum-reduced medium (Gibco, 31985-070) per well, mix gently to obtain Lipofectamine 3000 dilution.

[0066] 4) Experimental group (pcDNA3.1(+)-MLT1L-2034): First, 1 μg / μL pcDNA3.1(+)-MLT1L-2034 was diluted with serum-free Opti-MEM I serum-reduced medium, and then 2 volumes of P3000™ reagent (ThermoFisher, L3000015) was added to dilute the plasmid. The mixture was incubated at room temperature for 5 min, and then Lipofectamine 3000 dilution was added. The mixture was gently mixed and incubated at room temperature for 15 min.

[0067] 5) Control group (pcDNA3.1(+)): 1 μg / μL pcDNA3.1(+) was diluted with serum-free Opti-MEM I serum-reduced medium, and then diluted with 2 times the volume of P3000™ reagent. The mixture was incubated at room temperature for 5 min, and then Lipofectamine 3000 dilution solution was added. The mixture was gently mixed and incubated at room temperature for 15 min.

[0068] 6) Pipette 100 μL of pcDNA3.1(+)-MLT1L-2034 and pcDNA3.1(+) into each well containing cell culture medium. Gently mix by shaking the plate back and forth.

[0069] 7) After culturing cells for 6 hours, remove the medium containing the transfection complex and replace it with MEM high glucose complete medium containing 10% inactivated fetal bovine serum and 1% penicillin antibiotics.

[0070] 8) After incubating the cells at 37°C for 24 h in a CO2 constant temperature incubator, the culture medium was replaced with complete medium (Thermo Fisher, 10131027) containing the selective antibiotic Geneticin (final concentration of 1200 μg / mL) for pressure screening to obtain a stable Beas-2B cell line overexpressing MLT1L-2034, named Beas-2B-pcDNA3.1(+)-MLT1L-2034-OE.

[0071] 5. Expression of MLT1L-2034-OE in normal stable human lung cell lines Two-step real-time quantitative PCR was performed using TB Green® Premix Ex Taq™ (TaKaRa, RR420A) (LightCycler 480 System. Roche). The experimental procedures are described in the kit instructions.

[0072] Two pairs of RT-qPCR primers were designed for SEQ ID No:1 (Table 7), and the amplified fragments were named MLT1L-1 and MLT1L-2.

[0073] Table 7. Primer Information for Real-Time Quantitative PCR

[0074] Each group had 3 replicates, and each group underwent 3 independent tests. β-actin As an internal reference gene, using 2^ -△△CT The formula is used for calculation, where △CT = CT. 实验组 / 对照组 - CT β-actin The standardized data for each group were analyzed using a one-way ANOVA and plotted using GraphPad Prism 8.0. A p-value < 0.05 was considered statistically significant.

[0075] The results are as follows Figure 4 As shown, compared with the empty vector control group (pcDNA3.1(+)), the expression of MLT1L-2034 transcript was significantly increased in the pcDNA3.1(+)-MLT1L-2034-OE overexpression group (P<0.05).

[0076] Example 3: Effects of MLT1L-2034 overexpression on Beas-2B function in normal lung cells 1. Beas-2B cell culture is the same as in Example 1.

[0077] 2. ATPase activity (1) Cell plating Beas-2B-pcDNA3.1(+)-MLT1L-2034-OE cells were digested as described in Example 1. Cells were counted and adjusted to 6.0 × 10⁶ cells / day using MEM complete medium containing 10% fetal bovine serum and free of antibiotics. 4 The cells were seeded into 96-well cell culture plates (Thermo, 150628), with 100 μL of culture medium per well. The cell culture plates were shaken to spread the cells evenly across the bottom of the wells. The plates were then placed on a workbench and left to stand for a while. The cells were then observed under an inverted microscope to check the uniformity of the cell distribution. Finally, the plates were placed in a CO2 incubator for cell culture.

[0078] (2) ATPase activity detection 1) Thaw the frozen luminescence detection reagent and equilibrate to room temperature.

[0079] 2) Remove the cell culture plate and allow it to equilibrate at room temperature for 10 min.

[0080] 3) Prepare the test solution. Add 15 μL of test reagent to each sample: Mix the culture medium and test reagent first.

[0081] 4) Take the cell culture medium from the 96-well plate and add 100 μL of detection reagent.

[0082] 5) Shake at room temperature for 2 min to promote cell lysis.

[0083] 6) Let it sit at room temperature for 10 minutes to allow the luminescence signal to stabilize.

[0084] 7) Use a multi-functional microplate reader for chemiluminescence detection at a wavelength of 450 nm.

[0085] 8) Calculate the relative viability of cells based on chemiluminescence readings.

[0086] 3. Colony Formation Experiment (1) Beas-2B-pcDNA3.1(+)-MLT1L-2034-OE cells were digested according to Example 1.

[0087] (2) Cell plate counting: Inoculate 2 ml of cell suspension into each well of a 6-well plate at a concentration of 2000 cells per well. Gently shake the cell culture plate in a cross direction to disperse the cells evenly.

[0088] (3) Place in a CO2 constant temperature incubator for 1-2 weeks, and replace with fresh culture medium as needed according to the pH change of the culture medium.

[0089] (4) When visible clones appear in the culture dish, stop the culture, discard the culture medium, carefully wash twice with PBS, and air dry. Fix with 4% paraformaldehyde for 25 min, discard the 4% paraformaldehyde, carefully wash twice with PBS, and air dry. Stain with crystal violet for 10 min, slowly wash away the stain with running water, and air dry.

[0090] (5) Take pictures using a microscope.

[0091] 4. Transwell experiment (1) Before conducting the experiment, the pre-packaged Matrigel (Gibco, A1413301) was placed in a 4°C refrigerator overnight from -80°C. The Matrigel melted from a solid state to a liquid state.

[0092] (2) Coating the basement membrane: Matrigel was diluted 1:99 with DMEM / F12 medium (Gibco, 11320-032) and coated on the upper surface of the bottom membrane of the Transwell chamber. The chamber was then placed in a CO2 incubator and incubated at 37°C for 3 h.

[0093] (3) Hydration of the basement membrane: Aspirate the residual liquid in the chamber, add 50 μL of FBS-free and antibiotic-free culture medium to each well, and incubate at 37°C for 30 min.

[0094] (4) Following the steps in Example 1, Beas-2B-pcDNA3.1(+)-MLT1L-2034-OE cells were digested, resuspended in serum-free, antibiotic-free DMEM medium, counted using a cell counting chamber, and diluted to a concentration of 1×10⁻⁶ cells in serum-free, antibiotic-free DMEM medium. 5 / mL, for later use.

[0095] (5) Take 200 μL of diluted cell suspension and add it to the upper chamber of the Transwell chamber. Add 700 μL of medium containing 10% FBS (containing antibiotics) to the lower chamber of the 24-well culture plate.

[0096] (6) The culture plates were placed in a CO2 incubator at 37°C for 24 and 48 h.

[0097] (7) Remove the chamber, rinse twice with PBS, fix in 4% paraformaldehyde for 20 min in a 24-well plate, rinse twice with PBS, stain with crystal violet solution for 15 min, and carefully wipe away the cells in the upper layer of the microporous membrane of the chamber with a cotton swab.

[0098] (8) Take pictures under an inverted microscope, randomly count 10 fields of view for each sample, take the average value, and perform statistical analysis.

[0099] turn out: 1) In the ATPase activity assay, compared with the empty vector control group (pcDNA3.1(+)), the cell viability of the pcDNA3.1(+)-MLT1L-2034-OE overexpression group was increased ( Figure 5 ).

[0100] 2) In the colony formation assay, compared with the empty vector control group (pcDNA3.1(+)), the cell proliferation capacity of the pcDNA3.1(+)-MLT1L-2034-OE overexpression group was increased. Figure 6 (A and B in the middle).

[0101] 3) In the invasion assay, compared with the empty vector control group (pcDNA3.1(+)), the number of cells that crossed the basement membrane of the transwell chamber that had been coated with matrix gel was significantly increased in the pcDNA3.1(+)-MLT1L-2034-OE overexpression group. Figure 7 ).

[0102] In summary, this invention provides a method for constructing a stable human lung cell line overexpressing MLT1L. Overexpression of MLT1L-2034 significantly increases the viability, proliferation, and invasion ability of Beas-2B cells. Targeting the long non-coding RNA MLT1L-2034 can be used to develop novel anti-lung cancer gene drugs and prepare lung cancer detection kits, which has significant implications, broad application prospects, and enormous economic value.

[0103] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Application of substances that promote MLT1L expression in any of the following: (A1) Enhances the migration ability of normal lung cells; (A2) Prepare products to enhance the migration ability of normal lung cells; (A3) Enhances the invasive ability of normal lung cells; (A4) Prepare products for enhancing the invasive ability of normal lung cells; The MLT1L is a long non-coding RNA with a nucleotide sequence as shown in SEQ ID No:

1.

2. The application according to claim 1, characterized in that: The substance is any one of the following: (C1) is capable of being transcribed into the DNA molecule described in MLT1L; (C2) An expression cassette containing the DNA molecule described in (C1); (C3) A recombinant plasmid containing the DNA molecule described in (C1); (C4) A recombinant lentiviral vector containing the DNA molecule described in (C1).

3. The application according to claim 2, characterized in that: The nucleotide sequence of the DNA molecule is shown in SEQ ID No:

4.

4. The DNA molecule as described in claim 3.

5. Methods for constructing normal lung cell models: F1) A method for preparing a lung normal cell model with enhanced migration ability in vitro, comprising the following steps: increasing the expression of MLT1L in recipient lung normal cells to obtain a lung normal cell model with enhanced migration ability; F2) A method for preparing an in vitro lung normal cell model with enhanced invasiveness, comprising the following steps: increasing the expression of MLT1L in recipient lung normal cells to obtain a lung normal cell model with enhanced invasiveness; The MLT1L is a long non-coding RNA with a nucleotide sequence as shown in SEQ ID No:

1.

6. The method according to claim 5, characterized in that: The expression of MLT1L is enhanced by transfecting DNA molecules expressing the MLT1L transcript into normal lung cells of the recipient.

7. A lung cell model prepared by the method of claim 5 or 6.

8. The use of the lung cell model of claim 7 in screening drugs or preparing products for drug screening; wherein the drug has any of the following functions: (D1) Treatment and / or prevention of lung cancer; (D2) Inhibits the migration of normal lung cells; (D3) Inhibits the invasion of normal lung cells.

9. The application according to claim 8, characterized in that: The lung cells are lung cells with relatively stronger migration and / or invasion capabilities.

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