CN116656822A - Application of long-chain non-coding RNA lncCSRNP3 in EGFR-TKI acquired drug resistance of non-small cell lung cancer - Google Patents

Abstract

The invention belongs to the technical field of non-small cell lung cancer treatment, and particularly relates to application of long-chain non-coding RNA lncCSRNP3 in EGFR-TKI acquired drug resistance of non-small cell lung cancer. The invention aims to provide a new idea for solving EGFR-TKI drug resistance in NSCLC treatment. The technical scheme of the invention is the application of long-chain non-coding RNA lncCSRNP3 in the EGFR-TKI acquired drug resistance of non-small cell lung cancer, and the nucleotide sequence of the lncCSRNP3 is shown as SEQ ID No. 1. The lncCSRNP3 provided by the invention is up-regulated in the NSCLC cell with EGFR-TKI acquired drug resistance and peripheral blood of a patient, and the molecules are knocked out from the NSCLC cell with EGFR-TKI acquired drug resistance, so that the sensitivity of the cell to EGFR-TKI can be obviously improved, and the lncCSRNP3 is an ideal intervention target point and has higher clinical application development potential.

Description

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

Technical Field

The invention belongs to the technical field of non-small cell lung cancer treatment, and particularly relates to application of long-chain non-coding RNA lncCSRNP3 in EGFR-TKI acquired drug resistance of non-small cell lung cancer.

Background

In recent years, the epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI) such as gefitinib, ornitinib and the like on the market can effectively prolong the overall survival rate and progression-free survival time of patients with non-small cell lung cancer (NSCLC), but most patients can have acquired drug resistance phenomenon after 9-14 months of application of the drugs, and the sensitivity to the drugs is reduced, so that treatment failure and disease progression are caused. However, the molecular mechanisms underlying this phenomenon are not yet thoroughly studied, and there is a clinically significant lack of effective biomarkers and interventions to monitor and reverse this phenomenon. Therefore, searching for drug-resistant biomarkers with high sensitivity and specificity, exploring effective intervention targets and drug-resistant reversion schemes, and having important significance for improving prognosis of NSCLC patients.

Disclosure of Invention

The invention aims to provide a new idea for solving EGFR-TKI drug resistance in NSCLC treatment.

The technical scheme of the invention is the application of long-chain non-coding RNA lncCSRNP3 in the EGFR-TKI acquired drug resistance of non-small cell lung cancer, and the nucleotide sequence of the lncCSRNP3 is shown as SEQ ID No. 13.

Nucleotide sequence of SEQ ID No.13lncCSRNP3

GAGACAGAGTCTTGCTCTGTCACCAGGCTGGAGTGCAATGGTGCAATCTCGGCTCACTGCAACCTCCGCTTCCCGGGTTCAAGTGATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGGACTACAGGCGCACATCACCATGCCCACCTAATTTATGTATTTTTAGTAGAAATGAGGTTTCACCATGTTGGCCAGGCCAGGCACAGTAGCTCATGCCTTTAATCCCAGCACTTTGGGAGGCCAAGGCGGGCAGATCACGAAGTCAAAAAACTTTACCACCTGTACAAAACCCTGTCCAAAGTAAGAAATAATTAAATCTT

Further, the application is the application of down-regulating the expression of the lncCSRNP3 in reducing the EGFR-TKI resistance of the non-small cell lung cancer.

In particular, the down-regulating expression of lncCSRNP3 is knocking out lncCSRNP3 in the target cell.

The invention also provides an expression vector, which comprises an element for down-regulating the expression of the lncCSRNP3, wherein the nucleotide sequence of the lncCSRNP3 is shown as SEQ ID No. 13.

Further, the element is an element that knocks out lncCSRNP3 expression.

Specifically, the crispr/cas9 technique was used to knock out lncCSRNP3.

Preferably, the nucleotide sequence of the sgRNA is shown in SEQ ID Nos. 14 to 17.

The invention also provides a host comprising the expression vector.

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

In particular, the down-regulating expression of lncCSRNP3 is knocking out lncCSRNP3 in the target cell.

Wherein, the crispr/cas9 technology is adopted to knock out the lncCSRNP3.

Preferably, the nucleotide sequence of the sgRNA is shown in SEQ ID Nos. 14 to 17.

The invention has the beneficial effects that: according to the invention, the expression of lncCSRNP3 in NSCLC cells with EGFR-TKI acquired drug resistance and peripheral blood of a patient is up-regulated through analysis, and the EGFR-TKI acquired drug resistance biological marker has high sensitivity and specificity and is an ideal drug resistance biological marker; meanwhile, the molecule is knocked out from NSCLC cells with EGFR-TKI acquired drug resistance, so that the sensitivity of the cells to EGFR-TKI can be obviously improved, and the molecule is an ideal intervention target point and has higher clinical application development potential.

Drawings

FIG. 1 shows that lncCSRNP3 is highly expressed in drug-resistant cell lines. A. Volcanic map of lncRNA differentially expressed in sensitive and drug-resistant cell lines; total 9 lncRNA with significant differential expression in gr and OR; the qRT-PCR method verifies that TOP5 is significantly up-regulated lncRNA (the abscissa represents 5 lncRNAs and the ordinate represents the relative expression amount of lncRNA/GAPDH); qRT-PCR (quantitative reverse transcription-polymerase chain reaction) detecting the expression level of lnc-CSRNP3 in plasma samples of 50 gefitinib sensitive patients and 50 gefitinib resistant patients (the abscissa represents sensitive and resistant patients, and the ordinate represents the difference between the lnc-CSRNP3 and GAPDHCt values); ROC curve expressed by lncc csrnp3 (abscissa is 1-specificity, smaller value is greater specificity; ordinate represents sensitivity); correlation analysis of lncCSRNP3expression with gefitinib resistance: dividing patients into a lncCSRNP3 High expression group (High lncCSRNP3 expression) and a Low expression group (Low lncCSRNP3 expression) by taking the median of lncCSRNP3expression as a cut off value, and calculating the occupation ratio of the lncCSRNP3 High expression and Low expression patients in sensitive and drug resistant patients; detecting the expression quantity of lncCSRNP3 in 15 octenib sensitive and 15 octenib drug resistant patient plasma samples by using a qRT-PCR; ROC curve expressed by h.lncsrnp 3; correlation analysis of lncCSRNP3expression with gefitinib resistance.

FIG. 2, in vitro experiments, demonstrate that lncCSRNP3 promotes EGFR-TKI acquired resistance. CCK-8 method for verifying lncCSRNP3 pair cell IC ₅₀ Is a function of (1); B. effect of lncCSRNP3 on cell clonality in 1 μM gefitinib and 0.5 μM octenib treatment; c and d flow-through validation of the effect of lncsrnp 3 on cell cycle distribution in 1 μm gefitinib and 0.5 μm octtinib treatments. Grouping description: empty vector is control Empty, OElncCSRNP 3 is over-expressed lncCSRNP3, sg-lncCSRNP3 is crispr-cas 9-mediated sgRNA knockout.

FIG. 3, in vivo experiments, demonstrate that lncCSRNP3 promotes EGFR-TKI resistance. A. Schematic of cell neoplasia size 30 days after administration; B. a weight change line graph of nude mice in the experimental period; C. cell tumorigenic volume change line graph during experimental period; D. cell neoplasia weight bar graph 30 days after dosing; E. tumor H & E staining results are shown schematically 30 days after dosing. Grouping description: EV, empty Vector, is a control Empty group; OE, over-expression, is Over-expressed in the lncCSRNP3 group; NC, negative control, is a negative control group; sg, sg-lncCSRNP3, is the crispr-cas9 mediated sgRNA knockout group.

Detailed Description

The invention is described in further detail below with reference to the attached drawings and embodiments:

EXAMPLE 1 increased expression of LncCSRNP3 in EGFR-TKI acquired resistant NSCLC

Human non-small cell lung cancer cell lines PC-9, HCC827 (purchased from cell bank of China academy of sciences) were cultured in disposable T25 cell culture flasks with 10% FBS in RPMI-1640 medium and placed at 37℃in 5% CO ₂ Is included in the incubator. PC-9 and HCC827 containing EGFR ^del19 Mutations, sensitive to EGFR-TKI drugs, i.e., PC-9, HCC827 sensitive strains. Construction of drug resistant cell lines PC of gefitinib and oxatinib in sensitive strains (PC-9, HCC 827) by gradient drug addition method (PMID: 32805491 reference)-9/GR, HCC827/GR, PC-9/OR, HCC827/OR (GR, gefitinib resistance, OR, osimertinib resistance) were cultured in conditioned medium containing 1. Mu.M gefitinib OR 0.5. Mu.M octenib to maintain its drug resistant phenotype.

NSCLC sensitive and resistant strain total RNA is extracted by TRIzol, and DNA impurities are degraded by using Turbo DNA-free kit. The RNA was then purified using the Ribo-Zero Gold kit and RNase R. Finally, RNA integrity was assessed using an Agilent 2100 bioanalyzer. Qualified RNA was evaluated and libraries were constructed using TruSeq total RNA and Ribo-Zero Gold. These libraries were then sequenced on an Illumina sequencing platform and paired-end reads of 150bp/125bp were generated. All prior sequencing and subsequent data analysis were performed in Shanghai European company. In data analysis, the change times (FC) of a drug resistant group (experimental group) and a sensitive group (control group) are more than or equal to 2, and P is less than 0.5, which is defined as a significant difference with statistical significance. The amount of lncRNAs expressed was calculated using RPM (spliced reads per million, RPM) and volcanic plots of differentially expressed lncRNAs were plotted, with red dots representing lncRNAs exhibiting significant differences (FC. Gtoreq.2, P < 0.05) (FIG. 1A). In addition, 9 lncRNAs with a total of 5 up-regulated genes (i.e., lnc-ABCA12-8:1, lnc-ABCA12-5:1, lnc-CSRNP3-6:1, lnc-FAM150B-3:1, and lnc-LRRFIP 2-2:2) were differentially altered in EGFR-TKIs-sensitive and drug-resistant cell lines, and the analysis results were represented by Wen plots (FIG. 1B).

RNA from NSCLC sensitive and resistant cell lines was extracted by TRIzol and the 5 significantly up-regulated lncRNA was compared using the fluorescent quantitative Polymerase Chain Reaction (PCR) method and primers (Table 1) were synthesized by Shanghai.

TABLE 1 primer information

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

TABLE 2 preparation of reverse transcription System (performed on ice)

The fluorescent quantitative PCR system is shown in Table 3, the reaction procedure is as follows: 95 ℃ for 30s;95℃for 5s,60℃for 30s,40cycles.

TABLE 3 fluorescent quantitative PCR System

Reagent(s)	Usage amount
		SYBR Premix Ex Taq II(2×)	10μL
PCR Forward Primer(10μM)	1μL
		PCR Reverse Primer(10μM)	1μL
Template	1μL
		ddH 2 O	7μL
Total volume of	20μL

qRT-PCR results show that lnc-CSRNP3-6:1 (hereinafter abbreviated as lnc-CSRNP 3) is significantly up-regulated in drug-resistant cell lines; compared to the sensitive strain, the expression level of lncCSRNP3 was increased 11.8-fold in gefitinib-acquired resistant NSCLC cell lines and 8.9-fold in octenib-acquired resistant NSCLC cell lines (FIG. 1C).

The RNA in peripheral blood of EGFR-TKI (gefitinib, orientinib) sensitive and acquired resistant NSCLC patients (15 patients each) was extracted by a Plasma RNA extraction Kit (QIAGEN miRNeasy Serum/Plasma Kit) as follows:

(1) 200 μl plasma samples were prepared: the whole blood sample is centrifuged twice within four hours to obtain a plasma sample (2000 g for 10min at 4 ℃ and 12,000Xg for 10min at 4 ℃);

(2) Adding 1mL QIAzol Lysis Reagent, vortex shaking or reversing, mixing, and incubating at room temperature for 5min;

(3) Adding 200 mu L of chloroform, vortex oscillating for 15s, and standing at room temperature for 2-3 min;

(4) Centrifuging at 12,000Xg and 4 ℃ for 15min, and sucking 600 mu L of supernatant after the temperature is recovered to room temperature;

(5) Transferring the supernatant to a new 1.5mL EP tube, adding 900 mu L of absolute ethyl alcohol without sucking the supernatant to the middle white precipitate, and mixing the mixture upside down;

(6) Sucking 700 mu L to RNeasy MinElute spin column collecting tube, centrifuging at 8000 Xg for 15s at room temperature, and discarding the lower liquid;

(7) Repeating the step 6 until all the liquid is transferred and centrifuged;

(8) Adding 700 μL Buffer RWT into column, centrifuging at 8000 Xg for 15s at room temperature, and discarding the lower layer liquid;

(9) Adding 500 μl Buffer RPE into column, centrifuging at 8000×g at room temperature for 15s, and discarding the lower layer liquid;

(10) Adding 80% ethanol (prepared with DEPC water) 500 μl, centrifuging at 8000×g at room temperature for 2min, and discarding the lower liquid;

(11) Placing column into a new 2mL EP tube, centrifuging at high speed for 5min to dry the membrane, and discarding the lower layer liquid;

(12) Column was placed in a fresh 1.5mL collection tube, and 14. Mu.L RNase-free water was added and centrifuged for 1min.

Also comparing the expression level of lncCSRNP3 in peripheral blood of EGFR-TKI (gefitinib, ornitinib) sensitive and acquired resistant NSCLC patients using the fluorescent quantitative PCR method (operation of fluorescent quantitative PCR and prior to implementation), the expression level of lncCSRNP3 in peripheral blood of gefitinib acquired resistant NSCLC patients was found to be 8.69-fold higher compared to sensitive patients (FIGS. 1D-F). Is increased by 10.32 times in peripheral blood of NSCLC patients with acquired resistance to the Ornitinib. The area under the subject's working characteristics (ROC) curve was 0.9008, with sensitivity and specificity of 86.67% (fig. 1G-I). As described above, lncCSRNP3 has increased expression in EGFR-TKI-acquired drug resistant NSCLC and can be used as a drug resistance biomarker.

Example 2LncCSRNP3 reduces sensitivity of non-small cell lung cancer cells to EGFR-TKI

An NSCLC cell strain with lncCSRNP3 over-expression or knockout is constructed by using EGFR-TKI sensitivity and acquired drug resistance through transfection plasmid and virus infection and is used for subsequent experiments. The detailed steps of the overexpression and knock-out model establishment of the lnc-CSRNP3 are as follows:

and (3) building an overexpression model:

(1) The full-length gene sequence of lnc-CSRNP3 was constructed on a GV146 plasmid vector (supplied by Shanghai Ji Kai Gene science and technology Co., ltd.) and this step was completed by Shanghai Ji Kai; the nucleotide sequence of the lnc-CSRNP3 is shown as SEQ ID No. 13;

nucleotide sequence of SEQ ID No.13lncCSRNP3

GAGACAGAGTCTTGCTCTGTCACCAGGCTGGAGTGCAATGGTGCAATCTCGGCTCACTGCAACCTCCGCTTCCCGGGTTCAAGTGATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGGACTACAGGCGCACATCACCATGCCCACCTAATTTATGTATTTTTAGTAGAAATGAGGTTTCACCATGTTGGCCAGGCCAGGCACAGTAGCTCATGCCTTTAATCCCAGCACTTTGGGAGGCCAAGGCGGGCAGATCACGAAGTCAAAAAACTTTACCACCTGTACAAAACCCTGTCCAAAGTAAGAAATAATTAAATCTT

(2) The plasmid transfection procedure was as follows:

(1) and (3) paving: the cells were seeded in 6-well plates at a density of about 30% and the diabodies were removed 24 hours before starting transfection using serum-only medium; observing the condition of the seed plates by using a microscope, and starting transfection when the cell confluency reaches 30% -50%;

(2) before transfection: preparing solution A, adding siRNA (or plasmid DNA) into a 1.5mL EP tube containing 200 mu L of basal medium, and standing at room temperature for 5min; solution B was prepared, lipo2000 was diluted, 2. Mu.L lipo2000 was added to a 1.5mL EP tube containing 200. Mu.L basal medium, and left to stand at room temperature for 5min. Note that the siRNA (or plasmid DNA) is diluted first and lipo2000 is diluted again, and this 5min is mainly to allow it to be diluted sufficiently, and the amount of siRNA (or plasmid DNA)/lipo 2000 needs to be determined by a preliminary experiment;

(3) transfection: mixing the solution A and the solution B, gently mixing (the finger flicks the tube wall), and standing at room temperature for 20min; taking out a 6-hole plate, sucking out the culture medium, washing with PBS for 2 times, adding 2mL of basic culture medium into each hole, uniformly dripping AB mixed solution into the hole plate, uniformly mixing by shaking, and putting into a cell culture box; after 4-6 hours, the basic culture medium is changed into a complete culture medium; RNA can be extracted after 48 hours, and protein can be extracted after 96 hours for subsequent analysis.

(3) Since GV146 is neomycin resistant, antibiotic G418 was selected for screening. The surviving monoclonal after 10-14 days is subjected to proliferation culture to obtain the lnc-CSRNP3 stable over-expression cell strain.

Crispr/cas9 knock-out lnc-CSRNP3:

(1) Shanghai Ji Kai company designed and synthesized sgRNA plasmid and cas9 expression plasmid. sgRNA sequence:

SEQ ID No.14F1

GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC

SEQ ID No.15F2

GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC

SEQ ID No.16R1

GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC

SEQ ID No.17R2

GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC。

(2) Cas9 lentiviral infection:

(1) lentiviral volumes were calculated as moi=50 (Cas 9=25 μl; sg-lncrna=2.5 μl; sg-con=5 μl);

(2) treating cells: PC-9/GR and HCC827/GR cells were plated in 12-well plates, the next day with a plating, and transfected (25. Mu.L Cas9 virus per well). The volume of the culture solution is 500 mu L, the culture solution is supplemented to 1 mL/hole after 4 hours of infection at 37 ℃;

(3) antibiotic screening: cells in 12-well plates were infected with virus for 48h and the cells were passaged to 10cm ² Screening for 4-6 days by adding 5 mug/mL puromycin into a culture dish, sterilizing cells of a control group (uninfected virus) by puromycin, reducing the puromycin concentration to a maintenance concentration (1 mug/mL) after no cell death of an experimental group (infected virus), continuing screening and amplifying the infected cells, thereby constructing PC-9/GR and HCC827/GR (PC-9/GR-Cas 9 cells and HCC827/GR-Cas9 cells) for stably expressing Cas9 protein, freezing and preserving seeds;

(3) Transfection of sgRNA virus:

(1) optimum killing concentration of G-418 was found: PC-9/GR-Cas9 and HCC827/GR-Cas9 cell seed 12-well plates, G-418 concentration gradients were set: 0. 300, 600, 900, 1200, 150, 1800 mug/mL, for 12-14 days, screening out the lowest G-418 concentration capable of killing the cells completely, and determining the concentration as the screening concentration in the later experiment;

(2) virus transfection: plating 12-well plates on PC-9/GR-Cas9 and HCC827/GR-Cas9 cells, changing the liquid the next day, and the volume of the culture solution is 500 mu L, transfecting (sg-lncRNA=2.5 mu L; sg-con=5 mu L), infecting for 4h at 37 ℃, and supplementing the culture solution to 1 mL/well after 4 h;

(3) antibiotic screening: after 48-72h (70% -80% confluence) of lentivirus infection, cells are cultured in a culture medium containing G-418 with proper concentration, the culture medium containing G-418 is replaced every 3-4 days until the cells of a control group (uninfected virus) are killed by G-418, and after no cells of an experimental group (infected virus) die, the concentration of G-418 is reduced to a maintenance concentration (1/2-1/4 of the screening concentration), the infected cells are continuously screened and amplified, the cells are collected, and RNA is extracted for qPCR verification of a knockout model.

Detecting the change condition of EGFR-TKI drug sensitivity after over-expression or knocking out lncCSRNP3 through an in-vitro drug sensitivity related experiment (detailed experimental steps are as follows);

CCK-8 experiment

(1) Seed plate: taking cells in logarithmic growth phase, preparing cell suspension, and adjusting cell density to 5×10 ³ Well, inoculating into 96-well plate for culture, 200 μl each;

(2) At 37 ℃,5% CO ₂ Incubator culture overnight, after cell attachment, add gefitinib (0, 0.01, 0.1, 1, 10 and 100 μm) at different concentration gradients for about 48 hours;

(3) After 48 hours, the medium containing gefitinib was removed, and the CCK-8 reagent was added to a 96-well plate at 100. Mu.L/well (90. Mu.L fresh medium+10. Mu.L CCK-8 reagent) and incubated in an incubator for 2-4 hours;

(4) The absorbance (OD) value at 450nm was detected using a multifunctional microplate reader, and gefitinib drug killing curves were plotted (experiments were repeated independently at least 3 times).

Cell clone formation assay

(1) Cell treatment: cells were treated with 1 μm gefitinib or DMSO according to the experimental design;

(2) Preparation of cell suspension: taking cells in logarithmic growth phase, digesting and centrifuging by using pancreatin, and counting;

(3) Inoculation and plating:

(1) mixing evenly: the cells are uniformly mixed in a cross way after being inoculated into a 6-hole plate (the density is 500-1000/hole), and if necessary, the cells can be observed again after being placed in an incubator for 1 h;

(2) and (3) observation: after inoculation, observing under a microscope, if a 6-well plate is paved with 500 cells, 1-2 cells can be seen from one view of 100 x of the microscope;

(3) culturing: 10-14 days;

(4) Fixing and dyeing:

(1) fixing: PBS was washed 2 times and methanol was fixed for 15min;

(2) dyeing: crystal violet staining for 5min, ddH ₂ O cleaning and air dryingPhotographing and counting the number of cell clones;

(5) Cell viability was calculated: clone formation rate= (number of formed clones/number of inoculated cells) ×100%.

Cell cycle experiments

(1) Seed plate: taking cells in logarithmic growth phase, preparing cell suspension, and adjusting cell density to 1×10 ⁵ Well, inoculated in 6-well plate, 2mL of each well;

(2) After the cells are attached, adding 1 mu M gefitinib or DMSO for treatment for 48 hours;

(3) Fixing: after washing the cells 2 times by adding PBS, digesting and centrifuging (800 rpm,5 min) to collect the cells, firstly re-suspending the cells by 200 mu L of PBS and transferring the cells to a 1.5mL EP tube, finally dropwise adding 800 mu L of precooled absolute ethyl alcohol, and fixing the cells for more than 6 hours after vortex mixing uniformly for sample feeding;

(4) Cell staining: cells were treated with 100. Mu.L RNase A and stained with 400. Mu.L PI stain for 30min at 4℃in the dark;

(5) Cell cycle distribution was measured using a CytoFLEX flow cytometer.

The growth and proliferation, clone formation and cycle change of cells under the drug treatment conditions were examined by the above experiments. As a result, it was found that overexpression of lncCSRNP3 in EGFR-TKI sensitive cells resulted in half-Inhibitory Concentrations (IC) of gefitinib on cells ₅₀ ) Increasing from 0.0083. Mu.M to 0.484. Mu.M in the control group resulted in an IC of Ornitinib ₅₀ Increase from 0.871 μm to 10.53 μm in the control group; whereas gefitinib IC was obtained after knockout of lncCSRNP3 in EGFR-TKI-acquired drug resistant cells ₅₀ IC of octenib reduced from 2.449 μm to 1.035 μm in control group ₅₀ From 76.820 μm in the control group to 10.440 μm (fig. 2A). In addition, lncCSRNP3 significantly improved the clonogenic capacity and inhibited cycle arrest in NSCLC cells treated with EGFR-TKI (FIGS. 2B-D).

After respectively overexpressing or knocking out lncCSRNP3 in NSCLC cell lines of EGFR-TKI sensitivity and acquired drug resistance, injecting the cells into nude mice to construct a nude mice subcutaneous tumor model (in vivo experiment) until the tumor volume reaches 100mm ³ Administration of gefitinib or octreotide by oral gavage is started thereafterNile, 30 consecutive days (see below for detailed procedure). As a result, it was found that the overexpression of lncCSRNP3 significantly promoted tumor growth, increased tumor volume, and the effect of lncCSRNP3 knockout was reversed compared to the control group (FIGS. 3A to E).

Detailed operation steps of in vivo experiment:

(1) Preparing an experimental article: sterilized PBS, 0.25% pancreatin, cell counting plate, 75% sterilized alcohol, 1mL syringe, etc.;

(2) Mice for experiments were prepared: 20 female BALB/c-nu nude mice, 4-5 weeks old, and weight 14-16 g;

(3) The steps are as follows:

(1) after digestion and centrifugation of the cells with pancreatin, the cells were counted and the cell density was adjusted to approximately 1X 10 cells per 100. Mu.L of sterile PBS ⁵ Individual cells, placed on ice, inoculated as soon as possible;

(2) after disinfecting the right armpit of a nude mouse with 75% disinfecting alcohol, sucking 100 mu L of cell suspension by a 1mL syringe, and inoculating the cell suspension under the skin of the right armpit;

(3) recording the tumor formation condition, the weight of the nude mice and the like every day after successful inoculation;

(4) when the tumor size reaches 100mm ³ Then, the nude mice are randomly grouped into sensitive strains/DMSO groups, sensitive strains/Gef groups, drug-resistant strains/DMSO groups and drug-resistant strains/Gef groups, and DMSO or gefitinib is subjected to gastric lavage treatment for 30 days according to the dosage of 30 mg/kg/day; in addition, in vivo drug sensitivity experiments with octenib mice were divided into sensitive strain/DMSO group, sensitive strain/Osi group, drug resistant strain/DMSO group and drug resistant strain/Osi group, and DMSO or octenib gastric lavage treatment was performed at a dose of 15mg/kg/day for 30 days;

(5) after the whole treatment is finished, the mice are sacrificed by 300 mu L/100g of 10% chloral hydrate, and the subcutaneous transplantation tumor of the nude mice is subjected to operation stripping in an ultra clean bench;

(6) and drawing a tumor growth curve according to the volume weight of the peeled tumor body and the like. A part of tumor body tissue is reserved for embedding and the subsequent immunohistochemical detection is carried out; the other fraction extracts tumor protein and RNA for subsequent biological analysis.

Claims (10)

1. The application of long-chain non-coding RNA lncCSRNP3 in EGFR-TKI acquired resistance of non-small cell lung cancer is characterized in that: the nucleotide sequence of the lncCSRNP3 is shown as SEQ ID No. 1.

2. The use according to claim 1, characterized in that: the application is the application of down-regulating the expression of lncCSRNP3 in reducing the EGFR-TKI resistance of non-small cell lung cancer.

3. The use according to claim 2, characterized in that: the down-regulating expression of lncCSRNP3 is knocking out lncCSRNP3 in the target cell.

4. An expression vector, characterized in that: comprising an element that down-regulates expression of lncCSRNP3, wherein the nucleotide sequence of lncCSRNP3 is shown in SEQ ID No. 1.

5. The expression vector of claim 4, wherein: the element is an element for knocking out the expression of lncCSRNP3.

6. The expression vector of claim 5, wherein: the nucleotide sequence of the sgRNA is shown as SEQ ID No. 14-17.

7. A method for improving the resistance of non-small cell lung cancer cells EGFR-TKI, characterized by: the method comprises the following steps: down-regulating expression of lncCSRNP3 in the cell; the nucleotide sequence of the lncCSRNP3 is shown as SEQ ID No. 13.

8. The method according to claim 7, wherein: the down-regulating expression of lncCSRNP3 is knocking out lncCSRNP3 in the target cell.

9. The method according to claim 8, wherein: the inccsrnp 3 was knocked out using the crispr/cas9 technique.

10. The method according to claim 9, wherein: the nucleotide sequence of the sgRNA is shown as SEQ ID No. 14-17.