CN116200340B - Aftetinib-resistant human lung adenocarcinoma cell line PC9-AR and application thereof - Google Patents

Abstract

The invention belongs to the technical field of medical biology, and particularly discloses an African-resistant human lung adenocarcinoma cell line PC9-AR with a preservation number of CCTCC NO: C2022220. The PC9-AR resistant cell strain can stably grow and passaged in a culture system with the action concentration of 1umol/L of the amotinib, the drug Resistance Index (RI) of the amotinib is 1094.2, and the PC9-AR cell strain has 19 exon deletion mutation, KRAS amplification and no C797S mutation; therefore, the anti-ametinib human lung adenocarcinoma cell strain PC9-AR constructed by the invention provides a drug-resistant cell model for researching the morphological and biological characteristics of the ametinib drug-resistant human non-small cell lung cancer cells, the research and analysis of the drug-resistant mechanism of the non-small cell lung cancer to the ametinib, the analysis of the drug-resistant related signal path of the non-small cell lung cancer, the sensitivity analysis of anti-tumor drugs, the preparation of anti-tumor drugs, the screening of tumor drug resistance reversal drugs, the research of more effective tumor treatment methods and the like, and has higher scientific research and production application values.

Description

Amitinib-resistant human lung adenocarcinoma cell line PC9-AR and its application

technical field

The invention belongs to the technical field of biomedicine. It specifically relates to an amitinib-resistant human lung adenocarcinoma cell line PC9-AR and its application.

Background technique

Lung cancer is one of the most common malignancies worldwide and the leading cause of cancer-related death. Lung cancer is divided into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) according to its pathological type. The development of molecular pathological detection technology and the application of targeted therapy have significantly improved the survival period and quality of life of lung cancer patients. Among them, EGFR-TKIs therapy has a significant effect in NSCLC patients with EGFR activating mutations, but the inevitable generation of drug resistance limits its application.

T790M mutation is the main drug resistance mechanism of first- and second-generation EGFR-TKI therapy resistance, and this mutation occurs in about 60% of patients. The 790th threonine residue of the EGFR receptor is located at the entrance of the hydrophobic pocket at the rear of the ATP-binding cleft, which is critical for the specific binding of tyrosine kinase inhibitors (TKIs), and the T790M mutation causes threonine The acid is replaced by methionine, causing steric hindrance to interfere with the binding of TKIs. At the same time, the T790M mutation resulted in an increased affinity of the mutant for ATP. The third-generation EGFR-TKIs are selective and irreversible tyrosine kinase inhibitors developed for EGFR sensitive mutations and T790M mutations. Alimertinib is a new, irreversible, highly selective third-generation EGFR-TKI that can inhibit EGFR sensitive mutations and T790M mutations. At the same time, alectinib has been approved in China for the first-line treatment of adult patients with locally advanced or metastatic NSCLC with EGFR exon 19 deletion or exon 21 L858R mutation. However, the drug resistance mechanism of alectinib used in first-line treatment is not completely clear, and there are no relevant reports on the drug resistance mechanism of alectinib at home and abroad.

Contents of the invention

In view of the problems and deficiencies in the prior art, the object of the present invention is to provide an amitinib-resistant human lung adenocarcinoma cell line PC9-AR and its application.

For realizing the purpose of the invention, the technical scheme adopted in the present invention is as follows:

The first aspect of the present invention provides an amitinib-resistant human lung adenocarcinoma cell line PC9-AR, named as amitinib-resistant human lung adenocarcinoma cell line PC9-AR; the amitinib-resistant human lung adenocarcinoma Cell line PC9-AR, preserved in China Center for Type Culture Collection (CCTCC), the preservation date is September 7, 2022, the preservation number is CCTCC NO:C2022220, and the preservation address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province Wuhan University.

The amotinib-resistant human lung adenocarcinoma cell line PC9-AR adopts the in vitro concentration gradient method, that is, by gradually increasing the concentration of amotinib drug, it continuously acts on the PC9 cell line with EGFR exon 19 deletion to induce the establishment of human lung adenocarcinoma cell line PC9-AR. Adenocarcinoma almotinib-resistant cell lines. Its specific construction method is:

(1) Place the human lung adenocarcinoma cell line PC9 with EGFR exon 19 deletion (stock in our laboratory) in RPMI1640 medium containing 10% fetal bovine serum, at 37°C, 5% CO ₂ , saturation Cultivate in a humid incubator, and use trypsin digestion solution (0.25% Trypsin-EDTA) to digest and pass;

(2) Inoculate PC9 in a 60mm culture dish at a cell concentration of 1×10 ⁶ /mL. After 24 hours of attachment, add complete medium containing 5 nmol/L alimetinib, and place at 37°C, 5% CO ₂ , Cultivate in an incubator with saturated humidity; replace the medium containing alimetinib every 2-3 days (the concentration of alimetinib in the medium remains unchanged), and after the cells tolerate the concentration of alimetinib and grow stably, the medium The concentration of alimertinib in the medium is doubled on this basis, and the above steps are repeated continuously until PC9-AR can still grow stably and be passed down in the medium containing 1umol/L alimertinib, and then the aramectinib-resistant amitinib of the present invention can be obtained. Nigerian lung adenocarcinoma cell line PC9-AR.

The amolitinib-resistant human lung adenocarcinoma cell line PC9-AR constructed by the present invention can grow stably and pass passage in a culture system with an action concentration of 1 umol/L amolitinib, and the resistance index (RI) to amoxitinib is 1094.2, to study the morphology and biological characteristics of amitinib-resistant human non-small cell lung cancer cells, the mechanism of non-small cell lung cancer resistance to amitinib, the research and analysis of non-small cell lung cancer resistance-related signaling pathways, anti-tumor Drug sensitivity analysis, preparation of antitumor drugs, screening of drugs for reversing tumor drug resistance, and research on more effective tumor treatment methods provide drug-resistant cell models, which have high research and production application values.

The second aspect of the present invention provides the application of the amitinib-resistant human lung adenocarcinoma cell line PC9-AR in screening drugs for reversing tumor drug resistance.

The third aspect of the present invention provides the application of the amitinib-resistant human lung adenocarcinoma cell line PC9-AR in the preparation of antitumor drugs.

The fourth aspect of the present invention provides the application of the amitinib-resistant human lung adenocarcinoma cell line PC9-AR in constructing an in vitro tumor drug-resistant cell model.

According to the above application, preferably, the tumor is lung cancer. More preferably, the lung cancer is non-small cell carcinoma. Most preferably, the lung cancer is lung adenocarcinoma.

Compared with the prior art, the beneficial technical effect that the present invention obtains is as follows:

The amolitinib-resistant human lung adenocarcinoma cell line PC9-AR constructed by the present invention can grow stably and pass passage in a culture system with an action concentration of 1 umol/L amolitinib, and the resistance index (RI) to amoxitinib is 1094.2, moreover, the PC9-AR cell line has exon 19 deletion mutation, KRAS amplification, and no C797S mutation; The morphology and biological characteristics of drug-resistant human non-small cell lung cancer cells, the mechanism of non-small cell lung cancer resistance to amitinib, the research and analysis of non-small cell lung cancer resistance-related signaling pathways, the sensitivity analysis of anti-tumor drugs, the preparation of anti-tumor drugs Tumor drugs, screening of tumor drug resistance reversal drugs, and research on more effective tumor treatment methods provide drug-resistant cell models, which have high scientific research and production application value, and are expected to produce good scientific research, economic and social benefits.

Description of drawings

Fig. 1 is the morphological observation result figure of amitinib-resistant cell line PC9-AR and parental cell line PC9 of the present invention;

Fig. 2 is the detection result of the survival rate of the alectinib-resistant cell line PC9-AR of the present invention and the parental cell line PC9 under the action of different concentrations of alectinib;

Fig. 3 is the detection result of the proliferative ability of the amitinib-resistant cell line PC9-AR and the parental cell line PC9 of the present invention; wherein, A represents the difference in the proliferation ability of PC9 and the amitinib-resistant cell line PC9-AR detected by the SRB cell proliferation experiment ; B represents the plate cloning assay to detect the difference in proliferation ability between PC9 and the amoxitinib-resistant cell line PC9-AR;

Fig. 4 shows the detection results of the invasion and migration ability of the alectinib-resistant cell line PC9-AR and the parental cell line PC9 Transwell of the present invention;

Fig. 5 is the western blot experiment result of the expression of EGFR and its downstream proteins in the amitinib-resistant cell line PC9-AR and the parental cell line PC9 of the present invention;

Fig. 6 is the detection result of KRAS gene fluorescent quantitative PCR of amitinib-resistant cell line PC9-AR and parental cell line PC9 of the present invention;

Fig. 7 is a graph showing the detection results of KRAS gene copy numbers in the alimectinib-resistant cell line PC9-AR and the parental cell line PC9 of the present invention.

Detailed ways

The following detailed description is exemplary and intended to provide further explanation of the present invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It should be noted that the terminology used here is only for describing specific embodiments, and is not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly dictates otherwise, the singular is intended to include the plural, and it should also be understood that when the terms "comprising" and/or "comprising" are used in this specification, they mean There are features, steps, operations, components and/or combinations thereof.

The experimental methods that do not indicate specific conditions in the following examples all adopt conventional techniques in this technical field, or according to the conditions suggested by the manufacturer; the reagents or instruments used that do not indicate the manufacturer are all commercially available. product.

In order to enable those skilled in the art to understand the technical solution of the present invention more clearly, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

Example 1: Construction of amitinib-resistant human lung adenocarcinoma cell line PC9-AR

The amotinib-resistant human lung adenocarcinoma cell line PC9-AR adopts the in vitro concentration gradient method, that is, by gradually increasing the concentration of amotinib drug, it continuously acts on the PC9 cell line with EGFR exon 19 deletion to induce the establishment of human lung adenocarcinoma cell line PC9-AR. Adenocarcinoma almotinib-resistant cell lines. Its specific construction method is:

(1) Put human lung adenocarcinoma cell line PC9 with EGFR exon 19 deletion (stock in our laboratory) in RPMI1640 medium containing 10% fetal bovine serum, at 37°C, 5% CO ₂ , cultured in an incubator with saturated humidity, and digested and passaged with trypsin digestion solution (0.25% Trypsin-EDTA);

(2) Get the PC9 cells in the logarithmic growth phase or when the PC9 cell density grows to 50-60%, discard the old medium and add the complete medium containing Alectinib, the concentration of Alectinib in the medium is 10nmol/L, and the cells were cultured in an incubator at 37°C, 5% CO ₂ , and saturated humidity; the medium containing alimetinib was replaced every 2-3 days (the concentration of alimetinib in the medium remained unchanged). When the cell density grows to 80-90%, the cells are digested and subcultured according to the ratio of 1:3, and after the cells adhere to the wall, they are placed in a newly prepared complete medium containing the original concentration of amitinib to continue culturing, 2 -Change the medium every 3 days. Again, when the cell density grows to 80-90%, the cells are digested, subcultured according to the ratio of 1:3, and after the cells adhere to the wall, they are placed in a complete medium with a concentration of alimertinib of 20nmol/L to continue culturing; 2- The medium was changed every 3 days. When the cell density grew to 80-90%, the cells were digested, passaged at a ratio of 1:3, and placed in complete medium with a concentration of alimertinib of 20nmol/L to continue culturing. The concentration of amitinib in the medium is increased in this order: 10nmol/L---20nmol/L---50nmol/L---100nmol/L---200nmol/L---300

nmol/L---400nmol/L---500nmol/L---600nmol/L---800nmol/L---1000nmol/L, for cultivation. Each drug concentration is subcultured at least twice until the cells can still grow stably in the culture medium containing 1000nmol/L amotinib, and then the amotinib-resistant human lung adenocarcinoma cell line PC9- AR. The cell line was deposited in the China Center for Type Culture Collection (CCTCC) on September 7, 2022, with the preservation number CCTCC NO: C2022220, and the preservation address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

Example 2: Cryopreservation and recovery of amitinib-resistant human lung adenocarcinoma cell line PC9-AR

Cryopreservation: Place the drug-resistant cells obtained in Example 1 into the serum-free cryopreservation solution of the cryopreservation tube, and then store the cryopreservation tube in liquid nitrogen. Serum-free freezing solution was purchased from Suzhou Xinsaimei Suzhou Xinsaimei Biotechnology Co., Ltd. After freezing at -80 overnight, put them into liquid nitrogen for storage.

Resuscitation: Take out the cryopreservation tube containing the cells from the liquid nitrogen, immediately place it in a water bath at 37°C and shake it gently, so that the cryopreservation tube can be thawed within 1 minute. Put the thawed cell suspension in a 15ml sterile centrifuge tube, add 5ml PC9 medium, centrifuge at 1000rpm/min for 5 minutes, discard the supernatant, add 8mL complete medium, blow and mix to form a mixed solution, and suck to mix Put the liquid into a 100mm cell culture dish, put it into a carbon dioxide incubator, and cultivate it under the conditions of 37°C, 5% CO ₂ , and saturated humidity. The medium is replaced once every 2-3 days, and the cell density reaches 80-90%. Digested over time and subcultured at 1:3 ratio.

Example 3: Morphological observation of amitinib-resistant human lung adenocarcinoma cell line PC9-AR

1. Experimental cell line materials: the alimectinib-resistant cell line PC9-AR constructed in Example 1 and its parental human lung adenocarcinoma cell line PC9.

2. Experimental method: the amitinib-resistant cell line PC9-AR and its parental human lung adenocarcinoma cell line PC9 were inoculated in 60mm culture dishes, and the cells were grown to the logarithmic growth phase, and the living cells were observed under an inverted phase-contrast microscope form and take pictures.

3. Experimental results:

The experimental results of the morphological observation are shown in Fig. 1 . It can be seen from Figure 1 that, compared with the parental cell PC9, the cells of the amitinib-resistant cell line PC9-AR constructed in the present invention have obvious cell atypia, and the cells are mostly spindle-shaped, multinucleated cells are more common, and vacuoles often appear in the multinucleated cells , possibly due to drug treatment. This shows that the PC9-AR cell line resistant to alectinib of the present invention has changed in morphology.

Example 4: Determination of drug resistance index of amitinib-resistant human lung adenocarcinoma cell line PC9-AR

1. Experimental method:

The alimetinib-resistant human lung adenocarcinoma cell line PC9-AR and the parental cell PC9 in the logarithmic growth phase were seeded in 96-well plates at 3000 cells/100 μL/well, respectively. Add 200 μL PBS solution to each well around the edge of the 96-well plate to reduce medium evaporation during the experiment. After plating, gently tap the edge of the 96-well plate, three times each up, down, left, and right, so that the cells are evenly distributed in the wells, and placed in the cell culture incubator to continue incubation. About 24 hours after the cells were plated, when the cells were completely adhered to the wall, 100 μL of prepared complete medium containing alimetinib in different concentrations was added to each well (the concentration gradient of alimetinib in the complete medium was set as: 0.1 nM, 1 nM, 10 nM , 100nM, 1000nM, 10000nM, etc.), set 3-6 replicate wells for each concentration to reduce errors; set up a blank control group at the same time (the blank control group only adds complete medium, and the medium does not contain drugs). After adding the drug, put it into the cell incubator and continue to incubate for 72h. The cell culture medium was discarded before detection, and 100 μL PBS solution was added to each well to wash twice. Then 100 μL/well of pre-cooled 10% trichloroacetic acid solution (10 g of trichloroacetic acid powder dissolved in 100 mL of ddH ₂ O) was added, and after standing for 5 minutes, it was fixed in a 4-degree refrigerator for 1 h. The trichloroacetic acid solution was discarded, and the 96-well plate was washed four times with running water. When cleaning the orifice plate, the action should be gentle to avoid the water flowing too fast, which will cause the cells fixed on the bottom surface of the orifice plate to fall off. Allow to dry at room temperature after washing. Add 0.4% SRB solution at 100 μL/well (dissolve 0.4 g of SRB powder into 1% acetic acid solution and dilute to 100 mL), and shake for 20 min in the dark. After staining, the plate was washed four times with 1% acetic acid solution (1 mL of 100% acetic acid was added to ddH ₂ O and the volume was adjusted to 100 mL) to completely remove unbound dye and dried at room temperature. Finally, add 10mM Tris-base alkali solution (dissolve 0.1211g Tris powder in ddH ₂ O, and make the volume to 100mL) according to 100μL/well, shake and mix for 5min, so that the protein-bound SRB dye is completely dissolved in Tris- base alkali solution. Or dissolve at room temperature for 30 minutes. Use a microplate reader to detect the absorbance at 540 nm. After correcting the OD value, the cell survival rate of each well was calculated according to the formula, and the drug dose-effect curve was drawn using GraphpadPrism 9.0 software, and the half inhibitory concentration (IC50) of PC9-AR and parental cell NCI-PC9 to amitinib was calculated. and resistance index (RI). Wherein, the calculation formula of cell survival rate is: cell survival rate (%)=experimental group OD value/control group OD value×100%; The calculation formula of drug resistance index (RI) is: drug resistance index (RI)=drug resistance Cell IC50/parental cell IC50.

2. Experimental results:

The results of the detection of the survival rate of the alimertinib-resistant cell line PC9-AR under the action of different concentrations of alectinib are shown in Figure 2 . It can be seen from Figure 2 that under the same concentration of alectinib, the survival rate of PC9-AR cells did not change significantly, while the survival rate of PC9 decreased significantly. According to calculation, the drug resistance index of the alimetinib-resistant human lung adenocarcinoma cell line PC9-AR of the present invention is 1094.2.

Example 5: Detection of proliferation ability of amitinib-resistant human lung adenocarcinoma cell line PC9-AR

1. SRB cell proliferation assay was used to detect the proliferation ability of the amoxitinib-resistant cell line PC9-AR:

(1) Experimental method: The parental cell line PC9 and the drug-resistant cell line PC9-AR in the logarithmic growth phase were respectively inoculated in a 96-well plate at 2000 cells/100 μl/well. After the cells adhered to the wall overnight, the medium was discarded at 0h, 24h, 48h, 72h, and 96h, and the cells were fixed with 10% trichloroacetic acid solution. After the 96-well plate was dried at room temperature, 100 μL of 0.4% (w/v) SRB staining solution (prepared with 1% acetic acid) was added to each well. Wash 4 times to remove unbound dye, and dry at room temperature; dissolve the dye bound to cell protein with 100 μL of unbuffered Tris-base lye (10 mM, pH=10.5), shake for 20 min, and measure the light absorption at 540 nm with a microplate reader value. Graphpad Prism 9.0 software was used to draw growth curves to observe and compare cell proliferation.

(2) Experimental results:

The test results are shown in A in Figure 3. It can be seen from A in Figure 3 that the proliferation ability of the drug-resistant cell line PC9-AR is weaker than that of the parental cell line PC9.

2. Plate cloning assay to detect the proliferation ability of the alimetinib-resistant cell line PC9-AR:

(1) Experimental method:

Select the alimectinib-resistant human lung adenocarcinoma cell line PC9-AR and its parental cell line PC9 in the logarithmic growth phase, inoculate them in a 6-well plate at a density of 500-1000 cells/well, shake gently and mix well before placing Into the cell culture incubator, incubated for 7-14 days. Observe the growth of the cells, and discard the medium to terminate the culture when clones visible to the naked eye appear in the well plate. After rinsing twice with PBS, the cells were fixed with paraformaldehyde for 30 minutes. The fixative was discarded, stained with crystal violet, dried at room temperature, and counted.

(2) Experimental results:

The test results are shown in B in Figure 3. It can be seen from B in Figure 3 that the proliferation ability of the drug-resistant cell line PC9-AR is weaker than that of the parental cell line PC9.

Example 6: Detection of invasion and migration ability of amitinib-resistant human lung adenocarcinoma cell line PC9-AR

1. Experimental method:

(1) Migration experiment:

a. Prepare the Transwell chamber and 24-well plate with a pore size of 8 μm required for the experiment. The drug-resistant cell line PC9-AR and its parental cell line PC9 in the logarithmic growth phase were selected, and were starved for 12-24 hours with serum-free medium before the experiment. After trypsinization, the cells were resuspended and collected by centrifugation at 1000rpm for 5min. Discard the supernatant and resuspend in serum-free 1640 medium.

b. After resuspending and fully mixing the cells, adjust the cell concentration to (1-10)×10 ⁵ cells/mL to obtain a cell suspension. Take 200 μL of the cell suspension and add it to the upper chamber of the Tranwell chamber. And add 800 μL medium containing 20% FBS to the corresponding wells. The medium should cover the lower surface of the Transwell chamber, and there should be no air bubbles between it and the lower surface. Placed in a cell culture incubator for 24 h.

c. Discard the medium in the upper chamber and the medium in each corresponding well, and terminate the culture. Gently wash twice with PBS, and gently remove non-penetrated cells on the upper chamber surface using a moistened cotton swab. After fixed with paraformaldehyde for 30 min, stained with crystal violet for 30 min. Finally placed under an inverted microscope to observe, take pictures, and count.

(2) Invasion experiment:

The prepackaged Matrigel matrigel was melted in a 4-degree refrigerator, and the serum-free 1640 medium was used to dilute the matrigel at a ratio of 1:5. 60 μL/well was evenly spread on the upper chamber of the Transwell chamber, and then placed in a 37-degree cell culture incubator for about 2-3 hours to make the Matrigel gel. The rest of the cell preparation, culture, fixation, and staining steps were the same as the Transwell cell migration assay.

2. Experimental results:

The test results are shown in Figure 4. It can be seen from Figure 4 that the invasion and migration ability of amotinib-resistant lung adenocarcinoma PC9-AR was significantly higher than that of the parental cell PC9.

Example 7: Detection of EGFR and its downstream pathway proteins in amitinib-resistant human lung adenocarcinoma cell line PC9-AR

Extract the protein lysate of the amitinib-resistant human lung adenocarcinoma cell line PC9-AR of the present invention; at the same time, perform EGFR, p-EGFR, Akt, p-Akt, Erk, p-Erk protein immunoblotting experiments on the extracted protein lysate detection.

1. Experimental method:

Prepare cell lysate before extracting protein samples: take the required amount of RIPA lysate, add protease inhibitor mixture and phosphatase inhibitor mixture at a ratio of 100:1 before use, and mix well. The entire protein extraction process was performed on ice.

After the cells were treated according to the experimental requirements, the medium was discarded, the cells were washed twice with PBS, and the cell lysate was directly dropped on the cell surface (150-250 μL per well for a six-well plate) and the lysate was added. Pipet several times with a gun (be careful not to cross-contaminate between different samples), so that the lysate is evenly distributed on the cell surface. Lyse on ice for 10 minutes, then use a spatula to collect protein samples into EP tubes. Centrifuge at 12000g for 5 minutes at 4°C, take the supernatant into a new EP tube, and then add deionized water and 5× protein loading buffer to prepare the protein sample to a uniform concentration after subsequent protein concentration determination. Afterwards, routine electrophoresis, membrane transfer, antibody incubation, and machine exposure were performed according to the western blotting method.

2. Experimental results:

The Western blot results of EGFR and its downstream pathway proteins in the amitinib-resistant human lung adenocarcinoma cell line PC9-AR are shown in Figure 5.

It can be seen from Figure 5 that the phosphorylation of EGFR protein in the parental cell line PC9 was significantly weaker, and 50nmol/L alimertinib could inhibit the expression of p-EGFR, p-Akt and p-Erk proteins; The expression of p-EGFR protein was not detected in the cell line PC9-AR, and 50 to 500 nmol/L alimertinib could not significantly reduce the expression of p-Akt and p-Erk protein, suggesting that there is a bypass in the drug-resistant cell line activation.

Example 8: Detection of relative expression of KRAS in amitinib-resistant human lung adenocarcinoma cell line PC9-AR

1. Experimental method:

(1) RNA extraction:

a. Take PC9 and PC9-AR cells in the logarithmic growth phase, discard the medium and wash twice with PBS. According to 1mL Trizol/10cm2, add Trizol to lyse the cells. Pipette repeatedly at room temperature to completely lyse the cells, and collect the cell lysate into an enzyme-free EP tube.

b. Add chloroform to each sample according to 200μL chloroform/1mL Trizol, invert up and down 15 times or oscillate to mix, and then stand at room temperature for 5 minutes. Centrifuge at 4 degrees, 12000rpm for 15min. After centrifugation, the sample was separated into three layers: the upper aqueous phase (containing RNA and a small amount of DNA), the middle white thin layer (containing protein and DNA), and the lower organic phase (containing chloroform and protein).

c. Transfer the upper aqueous phase to another enzyme-free EP tube, and be careful not to absorb the middle layer. And add isopropanol according to 500μL isopropanol/1mL Trizol, mix thoroughly, and let stand at room temperature for 10min. Centrifuge at 4 degrees, 12000rpm for 10min. After centrifugation, the supernatant was discarded and the RNA was pelleted at the bottom of the tube.

d. Add 75% ethanol according to 1mL 75% ethanol/1mL Trizol, shake gently, and suspend the RNA pellet. And centrifuge at 4 degrees, 7500rpm for 5min. After centrifugation, the supernatant was discarded, dried at room temperature, an appropriate amount of enzyme-free sterile water was added to dissolve the RNA precipitate, and the RNA concentration was determined using Nanodrop 2000.

(2) RNA reverse transcription:

After the RNA extraction was completed, the RNA concentration of each sample was measured, and the RNA concentration was adjusted to 200 ng/μL. a. Removal of genomic DNA. Configure RNA-free ddH ₂ O 12 μL, 4×gDNA wiper Mix 4 μL, and RNA 4 μL according to the following system, and set the PCR instrument at 42°C for 2 minutes.

b. After the reaction in the previous step is completed, directly add 4 μL of 5×HiScript III RT SuperMix to the reaction tube, and set the reaction conditions as 37°C for 15 minutes; 85°C for 5s. The reaction product can be used immediately in qPCR reaction.

(3) Fluorescent quantitative PCR:

This operation was carried out according to Novizym real-time fluorescent quantitative PCR kit ChamQ Universal SYBR qPCR MasterMix. The reverse transcription reaction products of each group of samples were diluted, and after dilution, 2 μL samples were taken from each well as the cDNA template volume, and each sample in each group was set in three replicate wells to reduce errors.

The primers used in this experiment were synthesized by Sangon Bioengineering Co., Ltd. The primer sequences are as follows:

ACTIN Forward Primer: TGGCACCCAGCACAATGAA,

ACTIN Reverse Primer: CTAAGTCATAGTCCGCCTAGAAGCA;

KRAS Forward Primer: ACAGAGAGTGGAGGATGCTTT;

KRAS Reverse Primer: TTTCACACAGCCAGGAGTCTT.

After the reaction, the average Ct value of the three replicate wells of each group of samples was calculated. Taking ACTIN as the internal reference, according to the formula (relative expression of the target gene in each sample=2^-△△Ct, △△Ct=(Ct value of the target gene in the experimental group-Ct value of the internal reference gene)-(Ct value of the target gene in the control group-internal reference Gene Ct value)) Calculate the relative expression of target gene mRNA in each sample.

2. Experimental results:

The results of quantitative PCR detection of KRAS gene in the amitinib-resistant human lung adenocarcinoma cell line PC9-AR are shown in FIG. 6 .

It can be seen from Figure 6 that the relative expression of KRAS gene in the amoxitinib-resistant lung adenocarcinoma cell line PC9-AR was significantly higher than that in the parental cell PC9.

Example 9: Detection of KRAS gene copy number in amitinib-resistant human lung adenocarcinoma cell line PC9-AR

1. Experimental method:

Take the cell pellets of the drug-resistant cell line PC9-AR and its parental cell line PC9 in the logarithmic growth phase, add 200ul PBS, 20ul Proteinase K, 200ul Buffer BCL in sequence, vortex and mix, and bathe in 56°C for 10min; Water and ethanol, vortex and mix; transfer the mixture to the adsorption column, centrifuge at 12000rpm for 1min, and remove impurities such as protein, salt ions, ethanol, etc.; finally add 50ul Elution buffer, let stand at room temperature for 2-5min, and centrifuge at 12000rpm for 1min to elute the genome . And use Nanodrop 2000 to measure the DNA concentration, and then perform fluorescent quantitative PCR to detect the KRAS gene copy number.

2. Experimental results:

The results of KRAS gene copy number detection are shown in Figure 7.

It can be seen from Figure 7 that the copy number of KRAS gene in the alimetinib-resistant cell line PC9-AR was significantly higher than that in the parental cell PC9, suggesting that there was KRAS amplification in PC9-AR.

The purpose of the above-mentioned embodiments is to illustrate the substantive content of the present invention, but not to limit the protection scope of the present invention. Those skilled in the art should understand that the technical solution of the present invention can be modified or equivalently replaced without departing from the essence and protection scope of the technical solution of the present invention.

Claims (4)

1. The anti-ametinib human lung adenocarcinoma cell line PC9-AR is characterized in that the preservation number of the anti-ametinib human lung adenocarcinoma cell line PC9-AR is CCTCC NO: C2022220.

2. Use of an ametinib-resistant cell line according to claim 1 for screening a drug that reverses resistance to lung adenocarcinoma.

3. Use of an ametinib-resistant cell line according to claim 1 for screening and preparing an anti-lung adenocarcinoma drug.

4. Use of an ametinib-resistant cell line according to claim 1 in constructing an extracorporal lung adenocarcinoma drug-resistant cell model.