Application of long non-coding RNA lncCSRNP3 in EGFR-TKI acquired resistance of non-small cell lung cancer

Abstract

The present invention belongs to the technical field of non-small cell lung cancer treatment, and specifically relates to the application of long non-coding RNA lncCSRNP3 in the EGFR-TKI acquired resistance of non-small cell lung cancer. The purpose of the present invention is to provide a new idea for solving the EGFR-TKI resistance in NSCLC treatment. The technical solution of the present invention is the application of long non-coding RNA lncCSRNP3 in the EGFR-TKI acquired resistance of non-small cell lung cancer, and the nucleotide sequence of the lncCSRNP3 is as shown in SEQ ID No.1. The lncCSRNP3 provided by the present invention is up-regulated in NSCLC cells with EGFR-TKI acquired resistance and in the peripheral blood of patients. Knocking out this molecule in NSCLC cells with EGFR-TKI acquired resistance can significantly improve the sensitivity of the cells to EGFR-TKI. It is an ideal intervention target and has high potential for clinical application development.

Description

Technical Field

[0001] The present invention belongs to the technical field of non-small cell lung cancer treatment, and particularly relates to the application of a long non-coding RNA lncCSRNP3 in the acquired resistance of non-small cell lung cancer to EGFR-TKI. Background Art

[0002] In recent years, epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) such as gefitinib and osimertinib that have been marketed can effectively prolong the overall survival rate and progression-free survival period of patients with non-small cell lung cancer (NSCLC). However, most patients will develop acquired drug resistance after 9-14 months of using this type of drug, and the sensitivity to the drug decreases, resulting in treatment failure and disease progression. However, the molecular mechanism of this phenomenon has not been thoroughly studied at present, and there is a lack of effective biomarkers and intervention means for monitoring and reversing this phenomenon clinically. Therefore, finding drug resistance biomarkers with high sensitivity and specificity, exploring effective intervention targets and drug resistance reversal programs is of great significance for improving the prognosis of NSCLC patients. Summary of the Invention

[0003] The purpose of the present invention is to provide a new idea for solving the EGFR-TKI drug resistance in NSCLC treatment.

[0004] The technical solution of the present invention is the application of a long non-coding RNA lncCSRNP3 in the acquired resistance of non-small cell lung cancer to EGFR-TKI, and the nucleotide sequence of the lncCSRNP3 is as shown in SEQ ID No.13.

[0005] Nucleotide sequence of SEQ ID No.13 lncCSRNP3

[0006] GAGACAGAGTCTTGCTCTGTCACCAGGCTGGAGTGCAATGGTGCAATCTCGGCTCACTGCAACCTCCGCTTCCCGGGTTCAAGTGATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGGACTACAGGCGCACATCACCATGCCCACCTAATTTATGTATTTTTAGTAGAAATGAGGTTTCACCATGTTGGCCAGGCCAGGCACAGTAGCTCATGCCTTTAATCCCAGCACTTTGGGAGGCCAAGGCGGGCAGATCACGAAGTCAAAAAACTTTACCACCTGTACAAAACCCTGTCCAAAGTAAGAAATAATTAAATCTT

[0007] Furthermore, the application is the application of down-regulating the expression of lncCSRNP3 in reducing the resistance of non-small cell lung cancer to EGFR-TKI.

[0008] Specifically, the down-regulation of the expression of lncCSRNP3 is to knockout lncCSRNP3 in target cells.

[0009] The present invention also provides an expression vector, which contains an element for down-regulating the expression of lncCSRNP3, and the nucleotide sequence of lncCSRNP3 is shown as SEQ ID No.13.

[0010] Furthermore, the element is an element for knocking out the expression of lncCSRNP3.

[0011] Specifically, the crispr / cas9 technology is used to knockout lncCSRNP3.

[0012] Preferably, the nucleotide sequence of the shown sgRNA is shown as SEQ ID No.14-17.

[0013] The present invention also provides a host containing the above expression vector.

[0014] The present invention also provides a method for improving the resistance of non-small cell lung cancer cells to EGFR-TKI, which includes the following steps: down-regulating the expression of lncCSRNP3 in cells; the nucleotide sequence of lncCSRNP3 is shown as SEQ ID No.13.

[0015] Specifically, the down-regulation of the expression of lncCSRNP3 is to knockout lncCSRNP3 in target cells.

[0016] Among them, the crispr / cas9 technology is used to knockout lncCSRNP3.

[0017] Preferably, the nucleotide sequence of the shown sgRNA is shown as SEQ ID No.14-17.

[0018] The beneficial effects of the present invention: Through analysis, the present invention finds that the expression of lncCSRNP3 is up-regulated in NSCLC cells with acquired resistance to EGFR-TKI and in the peripheral blood of patients, which has high sensitivity and specificity and is an ideal drug resistance biomarker; at the same time, knocking out this molecule in NSCLC cells with acquired resistance to EGFR-TKI can significantly improve the sensitivity of the cells to EGFR-TKI, which is an ideal intervention target and has high potential for clinical application development. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1, lncCSRNP3 is highly expressed in drug-resistant cell lines. A. Volcano plot of differentially expressed lncRNAs in sensitive and drug-resistant cell lines; B. A total of 9 lncRNAs were significantly differentially expressed in both GR and OR; C. qRT-PCR was used to verify the TOP5 significantly up-regulated lncRNAs (the abscissa represents 5 lncRNAs, and the ordinate represents the relative expression of lncRNA / GAPDH); D. qRT-PCR was used to detect the expression of lnc-CSRNP3 in 50 plasma samples from gefitinib-sensitive and 50 gefitinib-resistant patients (the abscissa represents sensitive and resistant patients, and the ordinate represents the difference in Ct values between lnc-CSRNP3 and GAPDH); E. ROC curve of lncCSRNP3 expression (the abscissa is 1-specificity, and the smaller the value, the greater the specificity; the ordinate represents sensitivity); F. Correlation analysis of lncCSRNP3 expression and gefitinib resistance: Using the median expression of lncCSRNP3 as the cut-off value, patients were divided into the high lncCSRNP3 expression group (High lncCSRNP3 expression) and the low expression group (Low lncCSRNP3 expression), and the proportions of patients with high and low lncCSRNP3 expression in sensitive and drug-resistant patients were calculated; G. qRT-PCR was used to detect the expression of lncCSRNP3 in 15 plasma samples from osimertinib-sensitive and 15 osimertinib-resistant patients; H. ROC curve of lncCSRNP3 expression; I. Correlation analysis of lncCSRNP3 expression and gefitinib resistance.

[0020] Figure 2 , In vitro experiments verified that lncCSRNP3 promoted the generation of EGFR-TKI acquired resistance. A. CCK-8 method was used to verify the effect of lncCSRNP3 on cell IC 50 ; B. The effect of lncCSRNP3 on cell colony formation ability under the treatment of 1 μM gefitinib and 0.5 μM osimertinib; C and D. Flow cytometry was used to verify the effect of lncCSRNP3 on cell cycle distribution under the treatment of 1 μM gefitinib and 0.5 μM osimertinib. Group description: Empty vector is the control empty vector group, OE lncCSRNP3 is the overexpression lncCSRNP3 group, and sg-lncCSRNP3 is the crispr-cas9-mediated sgRNA knockout group.

[0021] Figure 3, in vivo experiments verified that lncCSRNP3 promoted the generation of EGFR-TKI resistance. A. Schematic diagram of tumor size after 30 days of drug administration; B. Line chart of body weight changes during the nude mouse experiment; C. Line chart of tumor volume changes during the experimental period; D. Histogram of tumor weight after 30 days of drug administration; E. Schematic diagram of the H&E staining results of tumors after 30 days of drug administration. Group description: EV is Empty Vector, which is the control empty vector group; OE is Over-expression, which is the over-expression lncCSRNP3 group; NC is negative control, which is the negative control group; sg is sg-lncCSRNP3, which is the crispr-cas9 mediated sgRNA knockout group. Detailed implementation manners

[0022] The present invention will be further described in detail below in conjunction with the accompanying drawings and implementation manners:

[0023] Example 1 LncCSRNP3 is highly expressed in NSCLC with acquired EGFR-TKI resistance

[0024] Human non-small cell lung cancer cell lines PC-9 and HCC827 (purchased from the Cell Bank of the Chinese Academy of Sciences) were cultured in a disposable T25 cell culture flask with RPMI-1640 medium containing 10% FBS and placed in an incubator at 37 °C and 5% CO 2 . PC-9 and HCC827 contain EGFR del19 mutations and are sensitive to EGFR-TKI drugs, that is, PC-9 and HCC827 sensitive strains. In the sensitive strains (PC-9 and HCC827), gradient drug addition method (PMID: 32805491 reference) was used to construct gefitinib- and osimertinib-resistant cell lines PC-9 / GR, HCC827 / GR, PC-9 / OR, and HCC827 / OR (GR means Gefitinib resistance, OR means Osimertinib resistance). They were cultured in conditioned medium containing 1 μM gefitinib or 0.5 μM osimertinib to maintain their drug-resistant phenotypes.

[0025] Total RNA of NSCLC sensitive and drug-resistant strains was extracted by TRIzol, and DNA impurities were degraded using the Turbo DNA-free kit. Subsequently, the Ribo-Zero Gold kit and RNase R were used to purify the RNA. Finally, the Agilent 2100 Bioanalyzer was used to evaluate the RNA integrity. For the qualified RNA, the TruSeq Stranded Total RNA and Ribo-Zero Gold were used to construct the library. Then, these libraries were sequenced on the Illumina sequencing platform, and paired-end reads of 150 bp / 125 bp were generated. All the previous sequencing and subsequent data analysis were carried out in Shanghai OE Biotech Co., Ltd. During data analysis, the fold change (FC) ≥ 2 and P < 0.5 between the drug-resistant group (experimental group) and the sensitive group (control group) were defined as significantly different with statistical significance. The RPM (spliced reads per million, RPM) method was used to calculate the expression level of lncRNAs, and a volcano plot of differentially expressed lncRNAs was drawn, where the red dots represent lncRNAs showing significant differences (FC ≥ 2, P value < 0.05). Figure 1 A). In addition, there were 9 lncRNAs with co-differential changes in EGFR-TKIs sensitive and drug-resistant cell lines, among which 5 genes were co-upregulated (i.e., lnc-ABCA12-8:1, lnc-ABCA12-5:1, lnc-CSRNP3-6:1, lnc-FAM150B-3:1, and lnc-LRRFIP2-2:2), and the analysis results were represented by a Venn diagram Figure 1 B).

[0026] RNA in NSCLC sensitive and drug-resistant cell lines was extracted by TRIzol, and the above 5 significantly upregulated lncRNAs were compared using the fluorescence quantitative polymerase chain reaction (PCR) method. The primers (Table 1) were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0027] Table 1 Primer information

[0028]

[0029] The RNA reverse transcription system is shown in Table 2, and the reaction conditions are as follows: 37 °C for 15 min (reverse transcription reaction), 85 °C for 5 sec (inactivation reaction of reverse transcriptase), 4 °C (directly placed on ice after the reverse transcription ends).

[0030] Table 2 Preparation of the reverse transcription system (performed on ice)

[0031]

[0032]

[0033] The fluorescence quantitative PCR system is shown in Table 3, and the reaction program is as follows: 95°C for 30 s; 95°C for 5 s, 60°C for 30 s, for 40 cycles.

[0034] Table 3 Fluorescence quantitative PCR system

[0035] Reagent Dosage SYBR Premix Ex Taq II (2×) 10 μL PCR Forward Primer (10 μM) 1 μL PCR Reverse Primer (10 μM) 1 μL Template 1 μL <![CDATA[ddH 2 O]]> 7 μL Total volume 20 μL

[0036] The results of qRT-PCR showed that lnc-CSRNP3-6:1 (hereinafter abbreviated as lnc-CSRNP3) was significantly up-regulated in drug-resistant cell lines; compared with sensitive cell lines, the expression level of lncCSRNP3 was increased by 11.8-fold in NSCLC cell lines with acquired resistance to gefitinib and by 8.9-fold in NSCLC cell lines with acquired resistance to osimertinib ( Figure 1 C).

[0037] RNA was extracted from the peripheral blood of NSCLC patients (15 patients each) sensitive and with acquired resistance to EGFR-TKIs (gefitinib, osimertinib) using a plasma RNA extraction kit (QIAGEN miRNeasy Serum / Plasma Kit), and the detailed steps are as follows:

[0038] (1) Prepare 200 μL of plasma sample: The whole blood sample was centrifuged twice within four hours to obtain the plasma sample (centrifuged at 2000 g at 4°C for 10 min, and then centrifuged at 12,000×g at 4°C for 10 min);

[0039] (2) Add 1 mL of QIAzol Lysis Reagent, vortex or invert to mix well, and incubate at room temperature for 5 min;

[0040] (3) Add 200 μL of chloroform, vortex for 15 s, and let stand at room temperature for 2 - 3 min;

[0041] (4) Centrifuge at 12,000×g at 4°C for 15 min. After returning to room temperature, aspirate 600 μL of the supernatant;

[0042] (5) Transfer the supernatant to a new 1.5 mL EP tube, being careful not to aspirate the middle white precipitate, and add 900 μL of absolute ethanol, then invert to mix well;

[0043] (6) Aspirate 700 μL into the RNeasy MinElute spin column collection tube, centrifuge at 8000×g at room temperature for 15 s, and discard the lower layer of liquid;

[0044] (7) Repeat step 6 until all the liquid has been transferred and centrifuged;

[0045] (8) Add 700 μL of Buffer RWT to the column, centrifuge at 8000×g for 15 s at room temperature, and discard the lower liquid.

[0046] (9) Add 500 μL of Buffer RPE to the column, centrifuge at 8000×g for 15 s at room temperature, and discard the lower liquid.

[0047] (10) Add 500 μL of 80% ethanol (prepared with DEPC water), centrifuge at 8000×g for 2 min at room temperature, and discard the lower liquid.

[0048] (11) Place the column into a new 2 mL EP tube, centrifuge at high speed for 5 min to dry the membrane, and discard the lower liquid.

[0049] (12) Place the column in a new 1.5 mL collection tube, add 14 μL of RNase-free water, and centrifuge for 1 min.

[0050] Similarly, the fluorescence quantitative PCR method (the operation and implementation of fluorescence quantitative PCR are the same as before) was used to compare the expression levels of lncCSRNP3 in the peripheral blood of NSCLC patients with EGFR-TKI (gefitinib, osimertinib) sensitivity and acquired resistance. It was found that compared with sensitive patients, the expression level of lncCSRNP3 in the peripheral blood of NSCLC patients with gefitinib-acquired resistance increased by 8.69 times ( Figure 1 D - F). In the peripheral blood of NSCLC patients with osimertinib-acquired resistance, it increased by 10.32 times. The area under the receiver operating characteristic (ROC) curve was 0.9008, and both the sensitivity and specificity were 86.67% ( Figure 1 G - I). The above indicates that the expression of LncCSRNP3 is increased in NSCLC with EGFR-TKI acquired resistance and can be used as a resistance biomarker.

[0051] Example 2 LncCSRNP3 can reduce the sensitivity of non-small cell lung cancer cells to EGFR-TKI

[0052] Using EGFR-TKI-sensitive and acquired-resistant NSCLC cell lines, NSCLC cell lines with overexpression or knockout of lncCSRNP3 were constructed by plasmid transfection and virus infection for subsequent experiments. The detailed steps for establishing the overexpression and knockout models of lnc-CSRNP3 are as follows:

[0053] Overexpression model construction:

[0054] (1) The full-length gene sequence of lnc-CSRNP3 was constructed onto the GV146 plasmid vector (provided by Shanghai Genechem Co., Ltd.), and this step was completed by Shanghai Genechem Co., Ltd.; the nucleotide sequence of lnc-CSRNP3 is shown in SEQ ID No. 13;

[0055] SEQ ID No. 13 The nucleotide sequence of lncCSRNP3

[0056] GAGACAGAGTCTTGCTCTGTCACCAGGCTGGAGTGCAATGGTGCAATCTCGGCTCACTGCAACCTCCGCTTCCCGGGTTCAAGTGATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGGACTACAGGCGCACATCACCATGCCCACCTAATTTATGTATTTTTAGTAGAAATGAGGTTTCACCATGTTGGCCAGGCCAGGCACAGTAGCTCATGCCTTTAATCCCAGCACTTTGGGAGGCCAAGGCGGGCAGATCACGAAGTCAAAAAACTTTACCACCTGTACAAAACCCTGTCCAAAGTAAGAAATAATTAAATCTT

[0057] (2) The steps of plasmid transfection are as follows:

[0058] ① Seeding: Seed the cells in a 6-well plate at a density of about 30%, remove the double antibiotics 24 hours before starting transfection, and use the medium containing only serum; observe the seeding situation under the microscope, and start transfection when the cell confluence reaches 30%-50%;

[0059] ② Before transfection: Prepare Solution A, add siRNA (or plasmid DNA) to a 1.5 mL EP tube containing 200 μL of basal medium, and let it stand at room temperature for 5 min; prepare Solution B, dilute lipo2000, add 2 μL of lipo2000 to a 1.5 mL EP tube containing 200 μL of basal medium, and let it stand at room temperature for 5 min. Note to dilute siRNA (or plasmid DNA) first and then dilute lipo2000. These 5 min are mainly for it to be fully diluted, and the amount of siRNA (or plasmid DNA) / lipo2000 needs to be determined through preliminary experiments;

[0060] (3) Transfection: Mix solution A and solution B, gently mix well (just flick the tube wall with your finger), and let it stand at room temperature for 20 min; Take out the 6-well plate, aspirate the culture medium, wash twice with PBS, add 2 mL of basal medium to each well, evenly drop the AB mixed solution into the well plate, gently shake and mix well, and place it in the cell culture incubator; After 4 - 6 h, replace the basal medium with complete medium; RNA can be extracted after 48 h, and protein can be extracted after 96 h for subsequent analysis.

[0061] (3) Since GV146 has neomycin resistance, select the antibiotic G418 for screening. The surviving monoclonal cells after 10 - 14 days are subjected to proliferation culture, which is the lnc-CSRNP3 stable overexpression cell line.

[0062] CRISPR / Cas9 knockout of lnc-CSRNP3:

[0063] (1) Shanghai GeneChem Co., Ltd. designed and synthesized the sgRNA plasmid and cas9 expression plasmid. sgRNA sequences:

[0064] SEQ ID No.14F1

[0065] GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC

[0066] SEQ ID No.15F2

[0067] GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC

[0068] SEQ ID No.16R1

[0069] GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC

[0070] SEQ ID No.17R2

[0071] GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC.

[0072] (2) Cas9 lentivirus infection:

[0073] ① Calculate the lentivirus volume according to MOI = 50 (Cas9 = 25 μL; sg-lncRNA = 2.5 μL; sg-con = 5 μL);

[0074] ② Treat the cells: Seed PC-9 / GR and HCC827 / GR cells in 12-well plates. Change the medium the next day and perform transfection (25 μL of Cas9 virus per well). The volume of the culture medium is 500 μL. Infect at 37°C for 4 h, and then supplement the culture medium to 1 mL per well after 4 h;

[0075] ③ Antibiotic screening: Infect the cells in the 12-well plates with the virus for 48 h, and then passage the cells to a 10 cm 2 culture dish. Add 5 μg / mL puromycin for screening for 4 - 6 days. Wait until the control group cells (not infected with the virus) are all killed by puromycin, and there is no more cell death in the experimental group (infected with the virus). Then reduce the puromycin concentration to the maintenance concentration (1 μg / mL), and continue to screen and expand the infected cells to construct PC-9 / GR and HCC827 / GR stably expressing Cas9 protein (PC-9 / GR-Cas9 cells, HCC827 / GR-Cas9 cells), and freeze and preserve the cell lines;

[0076] (3) Transfection of sgRNA virus:

[0077] ① Explore the optimal killing concentration of G-418: Seed PC-9 / GR-Cas9 and HCC827 / GR-Cas9 cells in 12-well plates, and set the G-418 concentration gradient: 0, 300, 600, 900, 1200, 150, 1800 μg / mL. Treat for 12 - 14 days to screen out the lowest G-418 concentration that can kill all the cells, which is determined as the screening concentration for subsequent experiments;

[0078] ② Virus transfection: Seed PC-9 / GR-Cas9 and HCC827 / GR-Cas9 cells in 12-well plates. Change the medium the next day. The volume of the culture medium is 500 μL. Perform transfection (sg-lncRNA = 2.5 μL; sg-con = 5 μL), infect at 37°C for 4 h, and then supplement the culture medium to 1 mL per well after 4 h;

[0079] ③ Antibiotic screening: After 48 - 72 h of lentivirus infection (70% - 80% confluence), culture the cells in a medium containing an appropriate concentration of G-418, and change the G-418-containing culture medium every 3 - 4 days until the control group (not infected with the virus) cells are all killed by G-418, and there is no more cell death in the experimental group (infected with the virus). Then reduce the G-418 concentration to the maintenance concentration (1 / 2 - 1 / 4 of the screening concentration), and continue to screen and expand the infected cells. At the same time, collect the cells and extract RNA for qPCR verification of the knockout model.

[0080] Through in vitro drug sensitivity-related experiments (the detailed experimental procedures are as follows), the changes in the sensitivity of EGFR-TKI drugs after overexpression or knockout of lncCSRNP3 were detected;

[0081] CCK-8 assay

[0082] (1) Seeding plates: Take cells in the logarithmic growth phase, prepare a cell suspension, and adjust the cell density to 5×10 3 / well, inoculate into 96-well plates for culture, 200 μL per well;

[0083] (2) Incubate overnight in an incubator at 37 °C and 5% CO 2 After the cells adhere, add gefitinib at different concentration gradients (0, 0.01, 0.1, 1, 10, and 100 μM) and treat for about 48 hours;

[0084] (3) After 48 hours, aspirate the medium containing gefitinib, and add CCK-8 reagent to the 96-well plates at 100 μL / well (90 μL fresh medium + 10 μL CCK-8 reagent), and continue to culture in the incubator for 2 - 4 hours;

[0085] (4) Use a multifunctional microplate reader to detect the absorbance (OD) value at 450 nm and draw the gefitinib drug killing curve (the experiment was independently repeated at least 3 times).

[0086] Cell colony formation assay

[0087] (1) Cell treatment: According to the experimental design, add 1 μM gefitinib or DMSO to treat the cells;

[0088] (2) Preparation of cell suspension: Take cells in the logarithmic growth phase, digest with trypsin, centrifuge, and count;

[0089] (3) Inoculation and plating:

[0090] ① Mixing: After inoculating the cells (density 500 - 1000 cells / well) into 6-well plates, mix them crosswise. If necessary, observe again after placing them in the incubator for 1 h to check if they are uniform;

[0091] ② Observation: Observe under the microscope after inoculation. If 500 cells are plated in a 6-well plate, then 1 - 2 cells can be seen in one field of view under a 100× microscope;

[0092] ③ Culture: For 10 - 14 days;

[0093] (4) Fixation and staining:

[0094] ① Fixation: Wash twice with PBS and fix with methanol for 15 min;

[0095] ②Staining: Stain with crystal violet for 5 min, wash with ddH 2 O, air dry, take pictures and count the number of cell clones;

[0096] (5) Calculate cell survival rate: Colony formation rate = (number of formed colonies / number of seeded cells) × 100%.

[0097] Cell cycle assay

[0098] (1) Seeding plates: Take cells in the logarithmic growth phase, make a cell suspension, adjust the cell density to 1×10 5 / well, inoculate into 6-well plates, 2 mL per well;

[0099] (2) After the cells adhere to the wall, add 1 μM gefitinib or DMSO and treat for 48 h;

[0100] (3) Fixation: After washing the cells with PBS 2 times, digest and centrifuge (800 rpm, 5 min) to collect the cells. First, resuspend the cells with 200 μL PBS and transfer them to a 1.5 mL EP tube. Finally, add 800 μL of pre-cooled absolute ethanol drop by drop, vortex and mix well, and fix for more than 6 h before sending the sample;

[0101] (4) Cell staining: Treat the cells with 100 μL RNase A and stain with 400 μL PI staining solution in the dark at 4°C for 30 min;

[0102] (5) Detect the cell cycle distribution with a CytoFLEX flow cytometer.

[0103] Through the above experiments, the growth, proliferation, colony formation and cycle changes of cells under drug treatment conditions were detected. The results showed that overexpression of lncCSRNP3 in EGFR-TKI-sensitive cells could increase the half-maximal inhibitory concentration (IC 50 ) of gefitinib on cells from 0.0083 μM in the control group to 0.484 μM, and increase the IC 50 of osimertinib from 0.871 μM in the control group to 10.53 μM; while when lncCSRNP3 was knocked out in EGFR-TKI acquired drug-resistant cells, the IC 50 of gefitinib decreased from 2.449 μM in the control group to 1.035 μM, and the IC 50 of osimertinib decreased from 76.820 μM in the control group to 10.440 μM ( Figure 2 A). In addition, lncCSRNP3 could also significantly improve the colony formation ability of NSCLC cells under EGFR-TKI treatment and inhibit its cell cycle arrest phenomenon ( Figure 2 B–D).

[0104] After overexpressing or knocking out lncCSRNP3 in EGFR-TKI-sensitive and acquired drug-resistant NSCLC cell lines respectively, cells were injected subcutaneously into nude mice to establish a subcutaneous tumor-bearing model (in vivo experiment). When the tumor volume reached 100 mm 3 Figure 3 After that, gefitinib or osimertinib was administered by oral gavage for 30 consecutive days (see the detailed operation steps below). The results showed that compared with the control group, overexpression of lncCSRNP3 significantly promoted tumor growth and increased tumor volume, while knocking out lncCSRNP3 had the opposite effect (A-E).

[0105] Detailed operating procedures for in vivo experiments:

[0106] (1) Prepare experimental supplies: sterilized PBS, 0.25% trypsin, cell counting chamber, 75% disinfected alcohol, 1 mL syringe, etc.;

[0107] (2) Prepare experimental mice: 20 female BALB / c-nu nude mice, 4-5 weeks old, weighing 14-16 g;

[0108] (3) Steps:

[0109] ① After digesting and centrifuging the cells with trypsin, count the cells and adjust the cell density to approximately 1×10 5 cells per 100 μL of sterile PBS, place on ice, and inoculate as soon as possible;

[0110] ② After disinfecting the right axilla of the nude mice with 75% disinfected alcohol, use a 1 mL syringe to aspirate 100 μL of cell suspension and inoculate it subcutaneously in the right axilla;

[0111] ③ After successful inoculation, record the tumor formation situation and the body weight of the nude mice every day;

[0112] ④ When the tumor size reaches 100 mm 3

[0113] After that, randomly divide the nude mice into a sensitive strain / DMSO group, a sensitive strain / Gef group, a drug-resistant strain / DMSO group, and a drug-resistant strain / Gef group, and perform DMSO or gefitinib gavage treatment at a dose of 30 mg / kg / day for 30 days; in addition, for the in vivo drug sensitivity experiment of osimertinib, the mice are divided into a sensitive strain / DMSO group, a sensitive strain / Osi group, a drug-resistant strain / DMSO group, and a drug-resistant strain / Osi group, and perform DMSO or osimertinib gavage treatment at a dose of 15 mg / kg / day for 30 days;⑤ After the entire treatment is completed, euthanize the mice with 300 μL / 100 g of 10% chloral hydrate, and surgically dissect the subcutaneous transplanted tumors of the nude mice in the laminar flow hood;

[0114] ⑥Draw the tumor growth curve according to the volume and weight of the dissected tumor mass. Retain a part of the tumor tissue for embedding and subsequent immunohistochemical detection; extract the tumor protein and RNA from the other part for subsequent biological analysis.

Claims

1. Use of long non-coding RNA lncCSRNP3 in the preparation of a product for diagnosing acquired resistance to EGFR-TKI in non-small cell lung cancer, Characterized in that: The nucleotide sequence of the lncCSRNP3 is as shown in SEQ ID N0.13; The EGFR-TKI is osimertinib.

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