Research methods for pemetrexed resistance in non-small cell lung cancer

Abstract

The present invention discloses a method for studying the drug resistance of non-small cell lung cancer to pemetrexed. The method realizes the study of the drug resistance of non-small cell lung cancer to pemetrexed by studying the differentially expressed proteins in the non-small cell lung cancer pemetrexed A549 / PEM drug-resistant cell line. The specific operation steps include: S1: construction and identification of the non-small cell lung cancer pemetrexed A549 / PEM drug-resistant cell line; S2: iTRAQ analysis of the proteome of the A549 / PEM drug-resistant cell line; S3: quantitative analysis of the protein of the A549 / PEM drug-resistant cell line; S4: comparative analysis of the results in step S2 and step S3, and verification of the differential expression of the protein in the A549 / PEM drug-resistant cell line. S5: analysis of the verified differential proteins in step S4, and screening out two differential proteins for drug sensitivity study. In the present invention, the study of differentially expressed proteins provides a basis for the subsequent screening of genes that affect the drug resistance of A549 / PEM cells, and provides a theoretical basis for the clinical treatment of PEM-resistant NSCLC patients.

Description

Technical Field

[0001] The present invention relates to the technical field of non-small cell lung cancer, and in particular to a method for studying the drug resistance of non-small cell lung cancer to pemetrexed. Background Art

[0002] Lung cancer is a common malignant tumor and the leading cause of cancer-related death. Non-small cell lung cancer (NSCLC) accounts for about 85% of all newly diagnosed lung cancers, including adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. NSCLC patients do not show typical clinical symptoms in the early stage, and most patients have developed into advanced lung cancer when diagnosed, which greatly increases the difficulty of clinical treatment for patients. In recent years, with the continuous deepening of research, the early diagnosis and treatment technology of NSCLC has also improved, but the 5-year survival rate of patients is still not optimistic. Cancer recurrence and high metastasis rate are also the main reasons for the failure of NSCLC treatment. Chemotherapy is a common first-line or second-line treatment for NSCLC. Among them, pemetrexed (MTA) drug chemotherapy is a widely used chemotherapy drug in recent years. It mainly destroys the normal metabolic process of intracellular folate dependence and inhibits cell replication, thereby inhibiting tumor growth. Although this treatment method has improved the treatment effect of patients to a certain extent, as the treatment time goes by, more and more patients develop drug resistance to it, resulting in treatment failure. Therefore, understanding the potential drug resistance mechanism of NSCLC and studying new molecular treatment technologies to improve the sensitivity of NSCLC cancer to MTA chemotherapy drugs are the focus of current research. Summary of the invention

[0003] In view of the above problems, the present invention aims to provide a method for studying the resistance of non-small cell lung cancer to pemetrexed by studying the differentially expressed proteins in the pemetrexed A549 / PEM resistant cell line of non-small cell lung cancer.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0005] The method for studying the drug resistance of non-small cell lung cancer to pemetrexed is characterized by: studying the drug resistance of non-small cell lung cancer to pemetrexed by studying the differentially expressed proteins in the pemetrexed A549 / PEM drug-resistant cell line of non-small cell lung cancer.

[0006] Further, the study of differentially expressed proteins in the pemetrexed-resistant A549 / PEM cell line of non-small cell lung cancer specifically includes the following steps:

[0007] S1: Construction and identification of pemetrexed-resistant A549 / PEM cell line for non-small cell lung cancer;

[0008] S2: Analysis of differentially expressed proteins in A549 / PEM resistant cell lines using iTRAQ technology;

[0009] S3: Screening out several proteins of interest from the differentially expressed proteins analyzed in step S2 for PRM verification;

[0010] S4: Analyze the proteins of interest verified in step S3, and further screen out two target differential proteins for drug sensitivity study.

[0011] Furthermore, the specific operations of step S1 include:

[0012] S11. Cell culture: The parent cell line A549 of non-small cell lung cancer was cultured in RPMI-1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin, and incubated in a constant temperature incubator at 37° C. and 5% CO 2 ;

[0013] S12, IC50 value determination of NSCLC parental cell line A549:

[0014] S13: The PEM-resistant NSCLC cell line A549 / PEM was constructed by high-concentration repeated intermittent induction method;

[0015] S14: Determination of drug resistance index of A549 / PEM resistant cell line.

[0016] Furthermore, the specific operations of step S12 include:

[0017] S121: Take A549 cells in logarithmic growth phase with good growth status and add 4×10 3 Inoculate into 96-well plates, set up 3 replicate wells for each group, and culture in a cell culture incubator at 37°C and 5% CO2;

[0018] S122: After the cells adhered to the wall, 1000 ng / ml, 500 ng / ml, 250 ng / ml, 125 ng / ml, 62.5 ng / ml, and 31.25 ng / ml of PEM were added to the NSCLC parental cell line A549, respectively;

[0019] S123: After continuing to culture for 48 hours, discard the culture medium, add 10 μL of freshly prepared toxicity detection solution CCK8 to each well, and incubate the culture plate in an incubator for 1-4 hours;

[0020] S124: Measure the absorbance at a wavelength of 450 nm using an enzyme-labeled instrument and calculate the half-maximal inhibitory concentration (IC50) of the parent strain.

[0021] Furthermore, the specific operations of step S13 include:

[0022] S131: Select pemetrexed with a final concentration of 1 / 3 of the half inhibitory concentration IC50 of the parent strain to impact the A549 parent strain in the logarithmic growth phase;

[0023] S132: After 24 hours of drug action, discard the drug-containing culture medium and replace it with fresh culture medium without drug;

[0024] S133: When the cells grow to 70%-80%, repeat the original dose shock, and repeat this for 8 generations. The cell line that finally survives is the A549 / PEM resistant cell line.

[0025] Furthermore, the specific operations of step S14 include:

[0026] S141: Take the A549 / PEM drug-resistant cell line with good growth status and add 4×10 3 Inoculate into 96-well plates, set up 3 replicate wells for each group, and culture in a cell culture incubator at 37°C and 5% CO2;

[0027] S142: After the cells adhered to the wall, the A549 / PEM resistant cell line was treated with PEM at concentrations of 4000 ng / ml, 2000 ng / ml, 1000 ng / ml, 500 ng / ml, 250 ng / ml, and 125 ng / ml, respectively;

[0028] S143: After continuing to culture for 48 hours, discard the culture medium, add 10 μL of freshly prepared toxicity detection solution CCK8 to each well, and incubate the culture plate in an incubator for 1-4 hours;

[0029] S144: Use an ELISA reader to measure the absorbance at a wavelength of 450 nm and calculate the half-maximal inhibitory concentration IC50 of the A549 / PEM resistant cell line;

[0030] S145: The drug resistance index of A549 / PEM is calculated using the formula RI = half inhibitory concentration IC50 of the A549 / PEM resistant cell line / half inhibitory concentration IC50 of the parental line.

[0031] Furthermore, the specific operations of step S2 include:

[0032] S21: extracting total protein from samples of the parental cell line A549 and the drug-resistant cell line A549 / PEM;

[0033] S22: An equal amount of protein from each sample was digested with trypsin, and the enzymatically digested peptides were desalted;

[0034] S23: Label and group different proteins in samples of parental cell line A549 and A549 / PEM resistant cell line;

[0035] S24: Analysis of proteins in parental cell line A549 and A549 / PEM cell samples by liquid chromatography-mass spectrometry;

[0036] S25: The data obtained by the liquid chromatography-mass spectrometry analysis in step S24 is subjected to a series of optimization processes, and then the data is compared with the database for similarity scoring to perform protein identification.

[0037] Furthermore, the specific operations of step S3 include:

[0038] S31: 16 proteins of interest were screened from the differentially expressed proteins identified in step S2;

[0039] S32: extracting total protein from samples of the parental cell line A549 and the drug-resistant cell line A549 / PEM;

[0040] S33: An equal amount of protein from each sample was digested with trypsin, and the enzymatically digested peptides were desalted;

[0041] S34: HPLC-MS analysis of proteins of interest in parental cell line A549 and A549 / PEM cell samples;

[0042] S35: Quantitative information of the protein of interest is obtained by LC-MS / MS detection;

[0043] S36: Analyze the quantitative information of the protein of interest obtained in step S34.

[0044] Furthermore, the specific operation of extracting total protein from the parental cell line A549 and the A549 / PEM drug-resistant cell line samples includes:

[0045] S231: Add protein lysate to the parental cell line A549 and A549 / PEM resistant cell line samples, mix well, incubate on ice for 5 min, add DTT with a final concentration of 10 mM, and sonicate on ice for 15 min;

[0046] S232: Take the mixture in step S231, centrifuge at 13000g, 4°C for 20 min, and transfer the supernatant into a new centrifuge tube;

[0047] S233: Add 4 volumes of cold acetone to the centrifuge tube and let stand at -20°C overnight;

[0048] S234: Collect protein precipitate by centrifugation, air dry, add 8M urea / 100mM TEAB solution to re-dissolve the protein, add DTT to a final concentration of 10mM, and perform reduction reaction at 56℃ water bath for 30min;

[0049] S235: IAM was added to a final concentration of 55 mM, and the mixture was placed in the dark at room temperature for 30 min for alkylation reaction. The protein concentration was determined by the Bradford method.

[0050] Furthermore, the protein is digested with trypsin, and the specific operation of desalting the enzymatically hydrolyzed peptides includes:

[0051] S236: Take 100 μg of protein and dilute the protein solution 5-fold with 100 mM TEAB;

[0052] S237: Add trypsin at a ratio of 1:50 and enzymatically digest at 37°C overnight;

[0053] S238: The enzymatically hydrolyzed peptides were desalted using a C18 column and the desalted peptides were vacuum freeze-dried.

[0054] The beneficial effects of the present invention are:

[0055] 1. The present invention constructs a non-small cell lung cancer pemetrexed A549 / PEM resistant cell line, and uses iTraq and PRM analysis techniques to analyze differentially expressed proteins in sensitive A549 cells and A549 / PEM resistant cells. These differential proteins may affect the resistance of non-small cell lung cancer to pemetrexed; and screen out proteins of interest related to resistance for PRM proteomics technology for verification. Through the study of differentially expressed proteins, a basis is provided for the subsequent screening of genes that affect the resistance of A549 / PEM cells, and a theoretical basis is provided for the clinical treatment of PEM resistant NSCLC patients; through the study of pemetrexed resistance of non-small cell lung cancer in the present invention, it is expected to explore feasible gene therapy methods for non-small cell lung cancer and promote the development of social science and technology.

[0056] 2. In the present invention, IGF2BP2 and FOLR1 were screened from the differentially expressed proteins in the iTraq and PRM analysis results to carry out relevant functional studies. The results showed that interference with IGF2BP2 and FOLR1 can enhance the sensitivity of A549 / PEM-resistant cells to PEM. Therefore, the present invention is expected to provide reliable biological targets, namely IGF2BP2 and FOLR1, for the clinical treatment of patients with pemetrexed-resistant non-small cell lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 The figure shows the survival of NSCLC parent cell line A549 after adding different concentrations of PEM to A549 in the present invention;

[0058] Figure 2 The survival status of the A549 / PEM drug-resistant cell line after the A549 / PEM drug-resistant cell line was treated with different concentrations of PEM in the present invention;

[0059] Figure 3 The SDS-PAGE electrophoresis diagram of total protein in different A549 / PEM drug-resistant cell line samples measured by Bradford method during iTRAQ analysis of the proteome of A549 / PEM drug-resistant cell line in the present invention;

[0060] Figure 4 The SDS-PAGE electrophoresis diagram of the total protein in different A549 / PEM drug-resistant cell line samples determined by Bradford method in the quantitative analysis of PRM protein in the A549 / PEM drug-resistant cell line of the present invention;

[0061] Figure 5 The PRM spectrum obtained after data collection for each sample using the optimized PRM mass spectrometry acquisition method in the present invention;

[0062] Figure 6 This is a bar graph comparing the difference results between PRM quantification and iTRAQ quantification of the comparison group A549 / PEM resistant cell line and A549 in the present invention;

[0063] Figure 7 The results of qRT-PCR detection of IGF2BP2 mRNA expression in cells transfected with si-NC, si-IGF2BP2#1, si-IGF2BP2#2, and si-IGF2BP2#3, and FOLR1 mRNA expression in cells transfected with si-NC, si-FOLR1#1, si-FOLR1#2, and si-FOLR1#3 in the present invention;

[0064] Figure 8 This is the IC50 value of the cells after adding different concentrations of pemetrexed to the si-NC, si-IGF2BP2 and si-FOLR1 cells in the present invention. DETAILED DESCRIPTION

[0065] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0066] The research method of non-small cell lung cancer resistance to pemetrexed is to study the differentially expressed proteins in non-small cell lung cancer pemetrexed A549 / PEM resistant cell line to achieve the research of non-small cell lung cancer resistance to pemetrexed.

[0067] Specifically, the study of differentially expressed proteins in pemetrexed-resistant A549 / PEM non-small cell lung cancer cell lines specifically includes the following steps:

[0068] S1: Construction and identification of pemetrexed-resistant A549 / PEM cell line for non-small cell lung cancer;

[0069] Specifically, cell culture: the parental cell line A549 of non-small cell lung cancer was cultured in RPMI-1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin, and incubated in a constant temperature incubator at 37° C. and 5% CO 2 ;

[0070] S12, IC50 value determination of NSCLC parental cell line A549:

[0071] A549 cells in logarithmic growth phase with good growth status were selected and 4×10 3 Inoculate into 96-well plates, set up 3 replicate wells for each group, and culture in a cell culture incubator at 37°C and 5% CO2;

[0072] After the cells adhered, 1000ng / ml, 500ng / ml, 250ng / ml, 125ng / ml, 62.5ng / ml, and 31.25ng / ml of PEM were added to the NSCLC parental cell line A549 respectively; after continuing to culture for 48h, the culture medium was discarded, and 10μL of freshly prepared toxicity detection solution CCK8 was added to each well; the culture plate was incubated in an incubator for 1-4 hours; the absorbance at a wavelength of 450nm was measured with an enzyme marker, and the half-maximal inhibitory concentration IC50 of the parental line was calculated.

[0073] After adding different concentrations of PEM to the NSCLC parental cell line A549, the survival of A549 was as follows Figure 1 As shown, according to the attached Figure 1 From the data, it can be concluded that after adding different concentrations of PEM to the NSCLC parental cell line A549, the drug concentration corresponding to the A549 survival rate of 50% is shown in Table 1. It can be seen from Table 1 that the drug concentration corresponding to the A549 survival rate of 50% is 285ng / ml, that is, the half-maximal inhibitory concentration IC50 value of the parental line is 285ng / ml.

[0074] Table 1 IC50 values ​​of parent strains

[0075] log(inhibitor)vs.normalized response-Variable slope Best-fit values LogIC50 2.455 HillSIope -1.327 IC50 285

[0076] S13: The PEM-resistant NSCLC cell line A549 / PEM was constructed by high-concentration repeated intermittent induction method;

[0077] Specifically, a final concentration of pemetrexed of 1 / 3 of the half inhibitory concentration IC50 of the parent strain (1 / 3*285=95ng / ml) was selected to impact the A549 parent strain in the logarithmic growth phase; after 24 hours of drug action, the drug-containing culture medium was discarded and replaced with fresh culture medium without the drug; when the cells grew to 70%-80%, the original dose impact was repeated, and this was repeated for 8 generations, and the cell line that finally survived was the A549 / PEM resistant cell line.

[0078] S14: Determination of A549 / PEM resistance index.

[0079] Specifically, A549 / PEM drug-resistant cell lines with good growth status were selected and 4×10 3 The cells were inoculated into 96-well plates, with 3 replicate wells in each group, and cultured in a cell culture incubator at 37°C and 5% CO2; after the cells adhered to the wall, the A549 / PEM resistant cell line was treated with PEM at concentrations of 4000ng / ml, 2000ng / ml, 1000ng / ml, 500ng / ml, 250ng / ml, and 125ng / ml, respectively; after continuing to culture for 48h, the culture medium was discarded, and 10μL of freshly prepared toxicity detection solution CCK8 was added to each well; the culture plate was incubated in an incubator for 1-4 hours; the absorbance at a wavelength of 450nm was measured with an enzyme marker, and the half-inhibitory concentration IC50 of the A549 / PEM resistant cell line was calculated; the resistance index of A549 / PEM was calculated using the formula RI=half-inhibitory concentration IC50 of the A549 / PEM resistant cell line / half-inhibitory concentration IC50 of the parental strain.

[0080] Specifically, after the A549 / PEM resistant cell line was treated with different concentrations of PEM, the survival of the A549 / PEM resistant cell line was as shown in the attached figure. Figure 2 As shown, from Figure 2 It can be concluded that after the A549 / PEM resistant cell line was treated with different concentrations of PEM, the drug concentration corresponding to the survival rate of the A549 / PEM resistant cell line was 50% is shown in Table 2. From Table 2, it can be seen that the drug concentration corresponding to the survival rate of the A549 / PEM resistant cell line was 50% was 5182 ng / ml, that is, the half inhibition concentration IC50 value of the A549 / PEM resistant cell line was 5182 ng / ml.

[0081] Table 2 IC50 values ​​of A549 / PEM resistant cell lines

[0082]

[0083] Combining Table 1 and Table 2, it can be concluded that the A549 / PEM resistant cell line can only inhibit cell growth by half when the drug concentration is 5182ng / ml, but the A549 cell can inhibit cell growth by half when the drug concentration is 285ng / ml. Comparing the IC50 values ​​of the two cell lines together, it can be said that the A549 / PEM resistant cell line was successfully constructed.

[0084] The resistance index of A549 / PEM resistant cell line was calculated using the formula RI = A549 / PEM resistant cell line half inhibitory concentration IC50 / parental strain half inhibitory concentration IC50 = 5182 / 285 = 18.2.

[0085] Further, step S2: using iTRAQ technology to analyze differentially expressed proteins in A549 / PEM resistant cell lines;

[0086] Specifically, S21: taking the parental cell line A549 and the A549 / PEM drug-resistant cell line successfully constructed in step S1, and extracting total protein from the parental cell line A549 and the A549 / PEM drug-resistant cell line samples;

[0087] Specifically, 2 groups of 6 samples were used, transported with dry ice, and stored in a refrigerator at -80°C. The detailed identification of the samples is shown in Table 3 below.

[0088] Table 3 Sample name correspondence table

[0089]

[0090] Protein lysis buffer (7M Urea / 2M Thiourea / 4% SDS / 40mM Tris-HCl, pH 8.5 / 1mM PMSF / 2mM EDTA) was added to each cell sample, mixed and incubated on ice for 5 min, DTT was added to a final concentration of 10mM, and ice-bath ultrasonication was performed for 15 min;

[0091] Take the above mixture, centrifuge at 13000g, 4℃ for 20min, take the supernatant and transfer it to a new centrifuge tube; add 4 volumes of cold acetone to the centrifuge tube, let it stand at -20℃ overnight; collect the protein precipitate by centrifugation and air dry; add 8M urea / 100mM TEAB solution to re-dissolve the protein, add DTT to a final concentration of 10mM, and perform reduction reaction in a 56℃ water bath for 30min;

[0092] IAM was added to a final concentration of 55 mM and placed in the dark at room temperature for 30 min for alkylation reaction. The protein concentration was determined by Bradford method. The results are shown in Table 4. The protein SDS-PAGE electrophoresis of different samples is shown in the attached figure. Figure 3 shown.

[0093] Table 4 Results of Bradford method for determination of different protein samples

[0094]

[0095] S22: An equal amount of protein from each sample was used for trypsin digestion; 100 μg of protein was used for trypsin digestion, the protein solution was diluted 5 times with 100 mM TEAB, trypsin was added at a mass ratio of 1:50, and the digestion was carried out at 37°C overnight. The peptides after digestion were desalted using a C18 column, and the desalted peptides were freeze-dried in vacuum.

[0096] S23: iTRAQ was used to label and group different proteins; peptides were dissolved in 0.5 M TEAB and labeled according to the instructions of the iTRAQ-8 labeling kit (SCIEX). After labeling, the samples were mixed, and then the mixed peptides were fractionated using the Ultimate 3000 HPLC system (Thermo DINOEX, USA). The chromatographic column used was a Durashell C18 column (5 μm, 4.6×250mm). Peptide separation was achieved by increasing the ACN concentration under alkaline conditions, with a flow rate of 1 ml / min and one tube collected every minute. A total of 42 secondary fractions were collected and combined into 12 components, which were desalted on a Strata-X column and dried in vacuo.

[0097] S24: Analysis of proteins in A549 / PEM cell samples by liquid chromatography-mass spectrometry; mass spectrometry data were collected using a TripleTOF 5600+ liquid chromatography-mass spectrometry system (SCIEX). The peptide samples were dissolved in 2% acetonitrile / 0.1% formic acid and analyzed using a TripleTOF 5600plus mass spectrometer coupled to an Eksigent nanoLC system (SCIEX, USA). The peptide solution was added to a C18 capture column (5 μm, 100 μm×20 mm) and gradient eluted on a C18 analytical column (3 μm, 75 μm×150 mm) with a time gradient of 90 min and a flow rate of 300 nL / min. The two mobile phases were buffer A (2% acetonitrile / 0.1% formic acid / 98% H2O) and buffer B (98% acetonitrile / 0.1% formic acid / 2% H2O). For IDA (Information Dependent Acquisition), the primary mass spectra were scanned with an ion accumulation time of 250 ms, and the secondary mass spectra of 30 precursor ions were collected with an ion accumulation time of 50 ms. MS1 spectra were collected in the range of 350-1500 m / z, and MS2 spectra were collected in the range of 100-1500 m / z; the precursor ion dynamic exclusion time was set to 15 s.

[0098] S25: The data obtained by the liquid chromatography-mass spectrometry analysis in step S24 is subjected to a series of optimization processing, and then the similarity is compared and scored with the database to perform protein identification. The basic process of proteome identification based on mass spectrometry is adopted, that is, the MS / MS mass spectrometry data is subjected to a series of optimization processing and then compared and scored with the database for similarity to perform protein identification. This method has the advantages of high identification accuracy, high throughput, and no need for manual sequence analysis. In the present invention, a search engine supporting ABSciex 5600plus, ProteinpilotTM V4.5, is used. Proteinpilot takes into account all possible modification types during the search, and adds an automatic fault-tolerant matching function. Under the premise of ensuring the credibility of the identification results, it can retrieve more results than similar software. For the identification results of proteinpilot, the present invention has made further filtering. For the identified proteins, the unused score ≥ 1.3 (that is, the confidence level is above 95%), and each protein contains at least one unique peptide segment. The protein that does not meet this condition is not included in this report; for the identified peptides and protein quantification, we filter with conf ≥ 95, that is, the confidence level is above 95% and the credible peptides are considered for protein quantification. The peptides that do not meet this condition are not included in this report.

[0099] Further, step S3: screening several proteins of interest from the differentially expressed proteins analyzed in step S2 for PRM verification;

[0100] Specifically, S31: 16 proteins of interest were screened from the differentially expressed proteins identified in step S2, namely, sp|P07203|GPX1_HUMAN, sp|P08183|MDR1_HUMAN, sp|P08195|4F2_HUMAN, sp|P12236|ADT3_HUMAN, sp|P12956|XRCC6_HUMAN, sp|P15144|AMPN_HUMAN, sp|P15328|FOLR1_HUMAN, sp |P31327|CPSM_HUMAN,sp|P32320|CDD_HUMAN,sp|P46459|NSF_HUMAN,sp|P49585|PCY1A_HUMAN,sp|Q9BRA2|TX D17_HUMAN, sp|Q9NPH2|INO1_HUMAN, sp|Q9NQC3|RTN4_HUMAN, sp|Q9Y3B8|ORN_HUMAN, sp|Q9Y6M1|IF2B2_HUMAN;

[0101] S32: extracting total protein from samples of the parental cell line A549 and the drug-resistant cell line A549 / PEM;

[0102] Two groups of six samples were used, transported with dry ice, and stored in a -80°C refrigerator. The detailed identification of the samples is shown in Table 5 below.

[0103] Table 5 Sample name correspondence table

[0104]

[0105] Take an appropriate amount of each sample for protein extraction; add protein lysis buffer (7M Urea / 2M Thiourea / 4% SDS / 40mM Tris-HCl, pH 8.5 / 1mM PMSF / 2mM EDTA) to the sample, mix well and incubate on ice for 5 minutes, add DTT with a final concentration of 10mM, ultrasonicate on ice for 15 minutes, then centrifuge the above mixture at 13000g, 4℃ for 20 minutes, take the supernatant and transfer it to a new centrifuge tube; add 4 times the volume of cold acetone to the centrifuge tube and let it stand at -20℃ overnight. Collect the protein precipitate by centrifugation and air dry. Add 8M urea / 100mM TEAB (pH 8.0) solution to dissolve the protein again, add DTT to a final concentration of 10mM, and carry out reduction reaction at 56℃ water bath for 30 minutes. Subsequently, add IAM to a final concentration of 55mM and place it at room temperature in the dark for 30 minutes for alkylation reaction. The protein concentration was determined by Bradford method. The results are shown in Table 6. The protein SDS-PAGE electrophoresis diagrams of different samples are shown in the attached figure. Figure 4 shown.

[0106] Table 6 Results of Bradford method for determination of different protein samples

[0107]

[0108] S33: Digest an equal amount of protein from each sample with trypsin and desalt the peptides after digestion; Digest an equal amount of protein from each sample with trypsin. Take 100 μg of protein for trypsin digestion. Dilute the protein solution 5 times with 100 mM TEAB, add trypsin at a mass ratio of 1:50, and digest at 37°C overnight. Desalt the peptides after digestion with a C18 column, and freeze-dry the desalted peptides in a vacuum.

[0109] S34: HPLC-MS analysis of proteins of interest in parental cell line A549 and A549 / PEM cell samples;

[0110] LC-MS / MS detection was used to obtain quantitative information of proteins; the mass spectrometry data was collected using a TripleTOF 5600+ LC-MS system (SCIEX). The peptide sample was dissolved in 2% acetonitrile / 0.1% formic acid, first onto a C18 capture column (5μm, 100μm×20mm), and then gradient eluted from a C18 analytical column (3μm, 75μm×150mm) at a flow rate of 300nL / min. The two mobile phases were aqueous Buffer A (2% acetonitrile / 0.1% formic acid / 98% H2O) and organic Buffer B (98% acetonitrile / 0.1% formic acid / 2% H2O). During IDA (information-dependent acquisition) detection, the primary mass spectra were scanned with an ion accumulation time of 250ms, and the secondary mass spectra of 30 precursor ions were collected with an ion accumulation time of 100ms. MS1 acquisition was performed in the range of 350-1500 m / z, and MS2 acquisition was performed in the range of 100-1500 m / z. The precursor ion dynamic exclusion time was set to 15 s. For PRM (parallel reaction monitoring) data acquisition, the protein peptide list after screening and optimization was added to the inclusion list, and the mass spectrometer selected and fragmented the peptides of different sizes in the list one by one, and collected the secondary ion spectrum.

[0111] Take all the peptide samples obtained after enzymatic hydrolysis, first take a part of each and mix them for mass spectrometry DDA detection, after searching the Proteinpilot database, import them into the Skyline software to establish a spectra library, and screen the peptides of the target protein from them to establish the method. Design a PRM detection method, add the target peptide m / z to the inclusionlist to establish a mass spectrometry acquisition method. Then use the PRM method to collect PRM data for the mixed sample to adjust and optimize the PRM acquisition method to form the final PRM method for sample data acquisition.

[0112] Each sample was dissolved in Loading Buffer (0.1% formic acid / 3% acetonitrile), and the supernatant was taken for spotting after centrifugation. The optimized PRM mass spectrometry acquisition method was used to acquire data for each sample, and 6 PRM spectrum files were obtained, as shown in the attached file. Figure 5 As shown, attached Figure 5 The peak area size (sum of all product ion peak areas) directly represents the content of the peptide in the corresponding sample. By extracting and analyzing these PRM spectrum files, the quantitative information of the protein can be obtained.

[0113] S36: Analyze the quantitative information of the protein of interest obtained in step S34; after obtaining mass spectrometry data by LC-MS / MS, use ProteinPilot software to search the database, and the protein database used is the uniprot-taxonomy_Homo_sapiens database downloaded from the Uniprot website. Use the search engine ProteinpilotTM V4.5 that is compatible with AB Sciex 5600plus. Proteinpilot considers all possible modification types during the search, and adds an automatic fault-tolerant matching function. Under the premise of ensuring the credibility of the identification results, it can retrieve more results than similar software. We further filtered the identification results of proteinpilot. For the identified proteins, we considered unusedscore ≥ 1.3 (i.e., the confidence level was above 95%) and each protein contained at least one unique peptide to be credible proteins. Proteins that did not meet this condition were not included in this report. For the identified peptides and protein quantification, we filtered by conf ≥ 95, i.e., peptides with a confidence level of more than 95% were considered credible for protein quantification. Peptides that did not meet this condition were not included in this report. The specific search parameters are shown in Table 7 below.

[0114] Table 7 Proteinpilot search parameters

[0115]

[0116] Specifically, the specific values ​​of the 16 differentially expressed proteins verified by the iTraq and PRM technologies are shown in Tables 8 and 9 below.

[0117] Table 8 iTraq differential protein expression

[0118]

[0119] Table 9 PRM differential protein expression

[0120]

[0121] The 16 differentially expressed proteins verified by iTraq and PRM were compared. The difference between PRM quantification and iTRAQ quantification of A549 / PEM resistant cell line and A549 is shown in the attached bar graph. Figure 6 As shown in the attached Figure 6 It can be seen that the differential proteins analyzed by PRM and iTraq technology are basically consistent. These differential proteins verified by the two technologies may be involved in the resistance of non-small cell lung cancer to pemetrexed.

[0122] Further, S4: analyzing the proteins of interest verified in step S3, and further screening out two target differential proteins for drug sensitivity study.

[0123] Specifically, the present invention also screened two differentially expressed proteins (IGF2BP2, FOLR1) from the iTraq and PRM analysis results to conduct relevant functional studies and analyze the effects of IGF2BP2 and FOLR1 on the drug sensitivity of A549 / PEM drug-resistant cells. The specific operations include:

[0124] (1) Construction of IGF2BP2 and FOLR1 small interfering RNA (si-IGF2BP2 and FOLR1) and negative control RNA (si-NC);

[0125] Three interference fragments were designed for each gene, namely si-IGF2BP2#1, si-IGF2BP2#2, si-IGF2BP2#3, si-FOLR1#1, si-FOLR1#2, and si-FOLR1#3. The interfering RNA constructs were designed and synthesized by Wuhan Baiqi Biotechnology Co., Ltd.

[0126] (2) A549 / PEM cell transfection;

[0127] a. Collect cells, inoculate the cells into 6-well cell culture plates, and culture them in a cell culture incubator at 37°C. When the cell density is about 70%, use them for cell transfection;

[0128] b. 2 hours before cell transfection, replace the cell culture medium in the 6-well plate with serum-free cell culture medium;

[0129] c. Take 10 μL of Lipofectamine 2000 transfection reagent and 200 μL of serum-free cell culture medium, mix them evenly, react for 5 minutes at room temperature, and prepare Lipofectamine 2000 working solution;

[0130] d. Mix si-NC, si-IGF2BP2#1, si-IGF2BP2#2, si-IGF2BP2#3, si-FOLR1#1, si-FOLR1#2, and si-FOLR1#3 with 200 μL of serum-free cell culture medium, respectively, and react at room temperature for 5 minutes to prepare a plasmid dilution solution;

[0131] e. After the Lipofectamine 2000 working solution and the above-mentioned interfering RNA dilution solution are fully mixed, they are incubated at room temperature for 20 minutes to prepare a liposome-plasmid complex;

[0132] f. Add the liposome-plasmid complex to a 6-well plate and continue culturing for 6 hours. Then, replace the serum-free cell culture medium with serum-containing cell culture medium and continue culturing.

[0133] g. The next day, observe the cell growth status and change the medium after 24 hours of cell culture for subsequent experiments.

[0134] (3) Real-time fluorescence quantitative PCR (qRT-PCR) detection of IGF2BP2 and FOLR1 gene interference efficiency;

[0135] qRT-PCR was used to detect the expression of IGF2BP2 mRNA in cells transfected with si-NC, si-IGF2BP2#1, si-IGF2BP2#2, and si-IGF2BP2#3, and the expression of FOLR1 mRNA in cells transfected with si-NC, si-FOLR1#1, si-FOLR1#2, and si-FOLR1#3. The results are shown in the attached figure. Figure 7 shown.

[0136] From the attached Figure 7 It can be seen that after transfection with si-IGF2BP2#3 and si-FOLR1#3, the expression level of the corresponding genes decreased most significantly, indicating that their interference efficiency is good. In subsequent studies, si-IGF2BP2#3 and si-FOLR1#3 transfected cells were used to carry out sensitivity studies on non-small cell lung cancer.

[0137] (4) CCK-8 was used to measure the A549 / PEMIC50 value of si-IGF2BP2#3 or si-FOLR1#3 transfected cells, and the si-NC transfected group was used as a negative control;

[0138] The cells were seeded into 96-well plates, with 2000 cells in each well, and cultured in an incubator. 250, 500, 1000, 2000, 4000, and 8000 ng / ml of pemetrexed were added for culture, and the IC50 values ​​of each group of cells were determined. The results are shown in the attached figure. Figure 8 shown.

[0139] From the attached Figure 8 It can be seen that compared with the si-NC group, the IC50 values ​​of the si-IGF2BP2 and si-FOLR1 transfection groups were significantly reduced, indicating that IGF2BP2 and FOLR1 interference can enhance the sensitivity of A549 / PEM to pemetrexed and make A549 / PEM cells non-resistant, further confirming that IGF2BP2 and FOLR1 have potential clinical application value.

[0140] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. Use of small interfering RNA that reduces the mRNA level of IGF2BP2 in the preparation of a drug for enhancing the sensitivity of non-small cell lung cancer to pemetrexed.

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