Application of long non-coding RNA lnc070974 in preparing products for treating or diagnosing lung cancer

Abstract

The present invention discloses the application of long non-coding RNA lnc070974 in the preparation of products for treating or diagnosing lung cancer, relates to the field of biomedicine, and specifically relates to the application of a reagent for inhibiting the expression of long non-coding RNA lnc070974 in the preparation of a drug for treating lung cancer, and the application of a reagent for detecting the expression level of lnc070974 in the preparation of products for diagnosing lung cancer. The present invention has found that lnc070974 can promote the growth and metastasis of lung cancer. After the lung cancer cell line is subcutaneously injected into nude mice to form tumors, the siRNA of lnc070974 is injected intratumorally, and the size and weight of the tumor are detected. It is found that the size and weight of the tumor are reduced, indicating that inhibiting lnc070974 can alleviate and treat lung cancer. In addition, lnc070974 can also be used as a marker for the diagnosis of lung cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to application of long-chain non-coding RNA lnc070974 in preparing products for treating or diagnosing lung cancer. Background Art

[0002] The RNA interference (RNAi) phenomenon was first discovered in Caenorhabditis elegans. It is an anti-double-stranded RNA reaction induced by double-stranded RNA (dsRNA), which leads to sequence-specific gene silencing. It was later discovered that this mechanism exists in many eukaryotic organisms. The discovery of the RNAi mechanism provides a new means for studying gene function in basic medical research, allowing the study of gene function to be carried out on a large scale. In terms of clinical medical treatment, the specificity and high efficiency of RNAi make it a promising treatment method. At present, this method has been tried in the treatment of infectious diseases, tumors, and genetic diseases through vectors such as plasmids, adenoviruses, retroviruses, and lentiviruses.

[0003] Lung cancer is a malignant tumor originating from the bronchial mucosa or glands of the lungs. Its morbidity and mortality rates are increasing rapidly, and it is one of the most threatening malignant tumors to people's health and life. Non-invasive diagnosis and treatment of lung cancer have not yet been solved. Therefore, the development of detection and treatment drugs for lung cancer is of great practical significance. Summary of the invention

[0004] The purpose of the present invention is to provide an application of long non-coding RNA lnc070974 in the preparation of a product for treating or diagnosing lung cancer, so as to solve the problems existing in the above-mentioned prior art. The present invention has found that inhibiting lnc070974 can alleviate and treat lung cancer, and detecting the expression level of lnc070974 can realize the diagnosis of lung cancer.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides use of a reagent for inhibiting the expression of long non-coding RNA lnc070974 in preparing a drug for treating lung cancer.

[0007] Furthermore, the reagent is si-lncRNA070974-1, the sense strand is shown in SEQ ID NO.3, and the antisense strand is shown in SEQ ID NO.4.

[0008] Furthermore, the reagent is si-lncRNA070974-2, the sense strand is shown in SEQ ID NO.5, and the antisense strand is shown in SEQ ID NO.6.

[0009] Furthermore, the reagent is the shRNA shown in SEQ ID NO.15.

[0010] Furthermore, the lung cancer is non-small cell lung cancer.

[0011] The present invention also provides a drug for treating lung cancer, comprising si-lncRNA070974-1, si-lncRNA070974-2 or shRNA; the sense chain of the si-lncRNA070974-1 is shown as SEQ ID NO.3, and the antisense chain is shown as SEQ ID NO.4; the sense chain of the si-lncRNA070974-2 is shown as SEQ ID NO.5, and the antisense chain is shown as SEQ ID NO.6; the sequence of the shRNA is shown as SEQ ID NO.15.

[0012] The present invention also provides the use of a reagent for detecting the expression level of lnc070974 in the preparation of a product for diagnosing lung cancer.

[0013] Furthermore, the reagent for detecting the expression level of lnc070974 includes a primer pair as shown in SEQ ID NO.11-12.

[0014] The present invention discloses the following technical effects:

[0015] The present invention explores the role of long noncoding RNA lnc070974 in lung cancer, and identifies that lnc070974 binds to Y-Box Binding Protein 1 (YBX1) through RNA pull-down experiments, mass spectrometry analysis and RNA binding protein immunoprecipitation (RIP) technology. Through CCK-8, plate cloning and other technologies, it is found that knocking down lnc070974 and YBX1 can inhibit the growth of lung cancer cells.

[0016] The present invention first constructs a lung cancer cell line with stable knockdown of lnc070974 by lentiviral transfection technology; then injects the cell line into nude mice subcutaneously, and measures and weighs the size and weight of the subcutaneous tumors of nude mice; in addition, the lung cancer cells with knockdown of lnc070974 and the original lung cancer cells are respectively injected into nude mice through the tail vein to observe the changes of the lung tumors of nude mice; through techniques such as mouse in vivo imaging, it is found that lnc070974 can promote the growth and metastasis of lung cancer. After the lung cancer cell line is subcutaneously injected into nude mice to form tumors, the siRNA of lnc070974 is injected intratumorally, and the size and weight of the tumor are detected, and it is found that the size and weight of the tumor are reduced, indicating that inhibiting lnc070974 can alleviate and treat lung cancer.

[0017] The present invention uses qPCR to detect the level of lnc070974 in lung cancer tissue or adjacent tissue; at the same time, blood is taken from normal people and lung cancer patients, exosomes in the serum are separated, and the level of lnc070974 in the exosomes is detected by qPCR. It is found that the level of lnc070974 in cancer tissue is high, and the level of lnc070974 in the serum exosomes of lung cancer patients is relatively high. Therefore, lnc070974 can be used as a marker for the diagnosis of lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 The results of treating A549 and HCC827 cells with si-NC, si-lnc070974-1 and si-lnc070974-2, respectively, where A is the CCK8 result of A549 cells; B is the CCK8 result of HCC827 cells; C is the two groups of cell plate clones; D is the statistical result of the number of cell clones in the two groups;

[0020] Figure 2 The results of si-NC and si-YBX1 treatment of A549 and HCC827 cells, respectively, where A is RNA pu-down electrophoresis; B: RIP qPCR product electrophoresis; C: RIP qPCR results; D: A549 cell CCK8 results; E: HCC827 cell CCK8 results; F: two groups of cell plate clones; G: two groups of cell clone number statistics;

[0021] Figure 3 The experimental results of lnc070974 shRNA used for in vivo treatment of animals, where A is the measurement result of tumor size; B is the tumor growth curve; C is the tumor weighing result; D is the in vivo imaging of mice; E is the observation result of changes in lung metastatic nodules;

[0022] Figure 4 is the relative expression level of lnc070974 gene in cancer and adjacent tissues of 3 lung cancer patients, with actin gene as control gene;

[0023] Figure 5 These are the experimental results of using lnc070974 siRNA for in vivo treatment of animals, where A is the measurement result of tumor size; B is the tumor growth curve; and C is the tumor weighing result. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] Example 1

[0030] 1. RNA interference experiment

[0031] Add 100 μL of Opti-MEM medium to the 12-well cell plate, add 15 μL of each of the four siRNAs shown in Table 1 (concentration of 20 μM, synthesized by Jima Biotechnology), and then add 6 μL of RNAiMAX reagent (purchased from ThermoFisher) was mixed well and added to the culture dish and allowed to stand for 15 min. 60,000 A549 and HCC827 cells were plated respectively and DMEM culture medium (purchased from Gebico) was added to 1 mL. After mixing well, the culture dish was placed in a 37°C 5% CO 2Incubator for 24 hours.

[0032] Table 1 Interference chain

[0033]

[0034] RNA was extracted and then the SYBR Premix Ex TaqTM II kit was used. The system was added according to the instructions and the following procedure was performed: 95°C for 3 min, (95°C for 5 s, 60°C for 30 s, 72°C for 40 s, 40 cycles), 72°C for 5 min, 95°C for 15 s, 60°C for 1 min, 95°C for 15 s. The relative expression level of the gene after the reaction was calculated according to 2 -△△Ct (△Ct value = Ct target - Ct internal reference, △△Ct value = △Ct experimental group - △Ct control group) was calculated to obtain the relative expression level of the gene. The actin gene was used as the control gene. The interference efficiency of si-lncRNA070974-1 and si-lnc070974-2 was 65% and 80% respectively, and the interference efficiency of si-YBX1 was 90%. These three interference chains were used for subsequent experiments. The primer sequences used in the detection process are shown in Table 2.

[0035] Table 2 Primer sequences

[0036]

[0037]

[0038] 2. Construction of A549 cell line with stable knockdown of lnc070974

[0039] 150,000 A549 cells were plated per well in a 6-well plate and divided into two groups (sh-NC group and sh070974 group). 4 μL of Plent-CMV-luciferase-P2A-puro (nonsense sequence, provided by Shandong Weizhen Biotechnology Co., Ltd.) was added to the sh-NC group, and 4 μL of human lncRNA070974 gene shRNA (SEQ ID NO.15: CCUUCCUGGUUGGCUGUUUTT) was added to the sh070974 group (2×10 5 IU, constructed by Shandong Weizhen Biotechnology Co., Ltd.), the cell medium was changed after 24 hours, and 0.8μg / mL puromycin was added for selection after 48 hours. The selection was continued for 14 days, and the luciferin substrate was added to detect the fluorescence signal. The interference efficiency of the sh070974 group was 60% higher than that of the sh-NC group. The A549 stable transfected cell line with luciferase was successfully constructed and used for subsequent experiments.

[0040] 3. CCK8 detection of cell proliferation

[0041] (1) After 48 h of treatment of A549 and HCC827 cells with si-NC, si-lnc070974-1, si-lnc070974-2, and si-YBX1, the cell density was approximately 70%-80%;

[0042] (2) The cells were trypsinized and 2000 cells were taken and added to a 96-well plate and allowed to adhere to the plate;

[0043] (3) Discard the old culture medium, add 100 μL of 10% CCK-8 reagent to each well, and place the cells back in the incubator for 2 h;

[0044] (4) Use an ELISA reader to measure the absorbance at 450 nm at 0 h, 24 h, 48 h, 72 h, and 96 h to reflect the number of living cells;

[0045] The results showed that the growth of cells in the si-lnc070974-1, si-lnc070974-2 and si-YBX1 groups was inhibited at 24 h of culture, and the inhibitory effect became more obvious as time went on. Figure 1 A and B, Figure 2 D and E).

[0046] 4. Plate cloning experiment

[0047] (1) After 48 h of treatment of A549 and HCC827 cells with si-NC, si-lnc070974-1, si-lnc070974-2, and si-YBX1, the cell density was approximately 70-80%;

[0048] (2) The cells were digested with trypsin and 2000 cells were taken and added to a 6-well plate at 37°C and 5% CO. 2 After 7 days of culture in the incubator, fresh normal culture medium (10% FBS complete culture medium) was replaced and cultured for another 7 days until the clones grew to be visible to the naked eye;

[0049] (3) Wash twice with PBS, add 4% paraformaldehyde for fixation at room temperature for 30 min, discard the fixative, and then stain with 500 μL of crystal violet for 30 min;

[0050] (4) Discard the staining solution, wash away the unbound staining solution with double distilled water, and dry at room temperature;

[0051] (5) Take photos under a microscope and count the number of single clones;

[0052] The results showed that the number of monoclonal clones in the si-lnc070974-1, si-lnc070974-2 and si-BX1 groups was significantly reduced, indicating that reducing lncRNA070974 and YBX1 can significantly inhibit cell growth ( Figure 1 C and D, Figure 2 F and G).

[0053] 5. Animal Experiments

[0054] 5.1 Subcutaneous tumor formation in nude mice

[0055] (1) BALB / C-nu nude mice were purchased at 3 weeks old and were kept in an SPF barrier system with a temperature of 25°C, humidity of 40%-60%, 12h alternating light and dark during the day and night, and noise <60 dB. The cages, bedding, and water dispenser feed used were all sterilized. The animal experiments involved in this project were approved by the Animal Experiment Ethics Committee of Wenzhou Medical University. After one week of feeding, the relevant experiments were carried out.

[0056] (2) Cell preparation: A549-Luc (luciferase expressing A549) cells were used to construct a stable lnc070974 knockdown group (sh070974) and a control group (sh-NC), identified, and expanded in culture dishes (for cell construction methods, see "2. Construction of A549 cell line with stable knockdown of lnc070974").

[0057] (3) Preparation of cell suspension: Take cells in the logarithmic growth phase to obtain cell suspension and count them. Prepare a concentration of 1×10 8 / mL cell suspension and place on ice to slow down cell metabolism.

[0058] (4) Inoculation: Nude mice were randomly divided into two groups. The difference between the two groups should not be too large. The injection site was disinfected with alcohol cotton and 100 μL was slowly injected subcutaneously into the mice using an insulin needle.

[0059] (5) Observe the tumor growth every day and measure the long diameter a (mm) and short diameter b (mm) of the tumor with a vernier caliper every 3 days. The formula V = 1 / 2 × ab 2 Tumor volume was calculated.

[0060] (6) 47 days after cell injection, the nude mice were killed and the tumors were removed. It was observed that the tumor size of the sh070974 group was significantly larger than that of the sh-NC group ( Figure 3 A).

[0061] (7) Drawing of tumor growth curve: Calculate the mean tumor volume ± standard deviation ( Figure 3 B).

[0062] (8) Changes in tumor weight: The statistical results showed that the weight of the tumor in the sh070974 group decreased by 78% compared with the sh-NC group (normal group). Figure 3 C).

[0063] 5.2 Tumor metastasis in nude mice

[0064] (1) BALB / C-nu nude mice, 3 weeks old, were purchased and raised according to the breeding method in 5.1. After one week of breeding, the following related experiments were carried out.

[0065] (2) Cell preparation: A549-Luc (luciferase expressing A549) cells were constructed to stably knock down lnc070974 (sh070974) and control group (sh-NC) cells for expansion culture (for cell construction methods, see "2. Construction of A549 cell line with stable knockdown of lnc070974").

[0066] (3) Preparation of cell suspension: Take cells in the logarithmic growth phase to obtain cell suspension and count them. Prepare a concentration of 1×10 7 / mL cell suspension and place on ice to slow down cell metabolism.

[0067] (4) Inoculation: Nude mice were randomly divided into two groups. The difference between the two groups should not be too large. The tails of the mice were disinfected with alcohol cotton. 200 μL of the solution was taken with an insulin needle and injected into the mice along the tail vein. The status of the mice was observed and recorded.

[0068] (5) Animal in vivo imaging: Protected from light, Luciferin was diluted with DPBS (15 mg / mL), and Luciferin was injected intraperitoneally according to the weight of the mouse. The injection dose was 10 μL / mouse weight (g). After 3 minutes, the mice were anesthetized and imaged using IVIS LμMinaIII animal imaging to observe the growth of tumor cells in the mice. The results showed that the fluorescence intensity in the sh070974 group was lower than that in the sh-NC group ( Figure 3 D).

[0069] (6) On day 45, the mice were euthanized and the lung tissues were removed. The lung tissues were fixed in Bouin's fixative for 24 h, washed twice with PBS, and the changes in lung metastatic nodules were observed. The results showed that the number and size of lung metastatic nodules in the sh070974 group were significantly less than those in the sh-NC group ( Figure 3 E).

[0070] 6. Magnetic Bead Analysis of RNA-Protein Pull-down Experiment

[0071] Experimental procedures refer to Thermo Scientific TM Kit (No:20164), the specific steps are as follows:

[0072] 6.1 Protein preparation:

[0073] (1) Prepare 10-12 10 cm cell culture dishes of HCC827 cells, and lyse the cells on ice for 15 min with 200 μL / dish of IP lysis buffer (1% PMSF);

[0074] (2) Centrifuge at 14000g for 15 min at 4°C, collect the supernatant into a 10 KDa protein ultrafiltration tube, and centrifuge at 4000g for ≥40 min at 4°C;

[0075] (3) When the protein liquid in the ultrafiltration tube is ≤500 μL, stop centrifugation and collect the protein in the concentrator tube and store it at -30°C.

[0076] 6.2 Magnetic Bead Pretreatment

[0077] (1) Take out the magnetic beads from the 4°C refrigerator and vortex at low speed to mix;

[0078] (2) 50 μL of magnetic bead suspension was added to a new 1.5 mL EP tube without enzyme.

[0079] (3) Place the EP tube on the magnetic rack (the magnetic beads will be adsorbed to one side) and remove the waste liquid;

[0080] (4) Add 100 μL of 0.1 M NaOH and 50 mM NaCl mixture to wash the beads twice;

[0081] (5) Wash the magnetic beads once with 100 μL 100 mM NaCl and keep them for subsequent use.

[0082] 6.3 Binding of magnetic beads to labeled RNA (25-100 pmol RNA corresponds to 20-50 μL magnetic beads)

[0083] (1) Take 50 μL of pre-treated magnetic beads, place them on a magnetic rack, and remove the supernatant;

[0084] (2) Add an equal volume (50 μL) of 20 mM Tris (pH 7.5), pipette and mix, and repeat once;

[0085] (3) Place the EP tube on a magnetic rack, allow the magnetic beads to adsorb once, and remove the supernatant;

[0086] (4) Add an equal volume (50 μL) of 1× RNA Capture buffer and resuspend by pipetting;

[0087] (5) Add about 20 μL of biotinylated RNA and gently pipette to resuspend. DEPC water can be added as a blank control.

[0088] (6) After mixing, incubate at room temperature for 15 to 30 minutes, stirring every 5 minutes (you can also rotate and incubate for 15 to 30 minutes).

[0089] 6.4 Biotin-labeled RNA and protein reaction and elution of bound protein

[0090] (1) Place the EP tube from the previous step on a magnetic rack and remove the supernatant;

[0091] (2) Add an equal volume (50 μL) of 20 mM Tris (pH = 7.5) to wash, resuspend by pipetting, place on a magnetic rack, remove the supernatant, and repeat once;

[0092] (3) Dilute 10× protein-RNA binding buffer to 1×, e.g., add 10 μL 10× protein-RNA binding buffer to 90 μL ddHO 2 O in;

[0093] (4) Add 100 μL 1× protein-RNA binding buffer to the magnetic beads and mix well;

[0094] (5) Prepare the reaction solution (Master Mix to RNA-protein binding buffer) as shown in Table 3:

[0095] Table 3

[0096]

[0097] (6) Place the magnetic beads on a magnetic rack and remove the supernatant;

[0098] (7) Add 100 μL Master Mix to RNA-protein binding buffer and mix well;

[0099] (8) Place in a four-dimensional rotator and incubate at 4°C for 60 min;

[0100] (9) Place the EP tube on a magnetic stand and transfer the supernatant to a new 1.5 mL EP tube for subsequent analysis (recorded as F1);

[0101] (10) Add 100 μL of 1× Washing buffer and wash the magnetic beads with a gun for about 30 times;

[0102] (11) Repeat steps (9) and (10) once (if necessary, transfer the eluate to a 1.5 mL EP tube for subsequent analysis);

[0103] (12) Place on a magnetic stand and transfer the supernatant to a 1.5 mL EP tube for subsequent analysis (referred to as F2);

[0104] (13) Add 50 μL of elution buffer to the magnetic beads, vortex to mix, and incubate at 37°C for 15-30 min, stirring every 5 min.

[0105] (14) The magnetic beads were placed on a magnetic stand, the beads were adsorbed to one side, and the supernatant was transferred to a 1.5 mL EP tube (labeled E) for subsequent analysis;

[0106] (15) Measure the protein concentration of F1, F2, and E, add 1 / 4 volume of 5× loading buffer, and boil at 100°C for 5 min;

[0107] (16) Store at -30°C until use for silver staining or Coomassie brilliant blue staining.

[0108] Note: The protein concentration is very low and is detected using the Qubit protein kit from Thermo Fisher Scientific. For specific steps, see the reagent manual (Invitrogen, NO: 2057447).

[0109] 6.5 Silver staining

[0110] Reference Thermo Scientific TM Kit (No: 24612), the specific steps are as follows:

[0111] (1) Prepare SDS-PAGE electrophoresis gel (separation gel concentration is 12%) in advance and add approximately 40 μL of sample to the loading well;

[0112] (2) Electrophoresis: 70 V for 40 min, 110 V for 70 min;

[0113] (3) Cut off the concentrated gel part and wash the separation gel with ddH 2 After gently rinsing with O, transfer to 30% ethanol: 50mL 10% acetic acid solution for blocking and fixation for 15min×2 times, or directly block overnight;

[0114] (4) Wash the separation gel in 10% ethanol (about 100 mL) by shaking for 5 min × 2 times;

[0115] (5) Discard 10% ethanol and add ddH 2 O (about 100 mL) ultrapure water, shake and wash for 5 min × 2 times;

[0116] (6) Pre-prepare the Sensitizer Working Solution: add 100 μL of Sensitizer to 50 mL of ultrapure water;

[0117] (7) Transfer the separation gel to the Sensitizer Working Solution prepared in step (6), shake and incubate for 1 min, and wash the separation gel with ultrapure water for 1 min × 2 times;

[0118] (8) Pre-prepare Stain Working Solution: add 1 mL Enhancer to 50 mL Stain;

[0119] (9) Transfer the separation gel to the Stain Working Solution prepared in step (8) and incubate for 30 min;

[0120] (10) Prepare the Developer Working Solution in advance: add 1 mL of Enhancer to 50 mL of Developer;

[0121] (11) Shake and wash the separation gel with ultrapure water for 20 seconds × 2 times;

[0122] (12) Transfer the separation gel to the Developer Working Solution prepared in step (10) and incubate with shaking until bands appear, generally 2-3 min;

[0123] (13) After the bands appeared, transfer them to 5% glacial acetic acid solution to terminate the color development. After a short wash, replace the 5% glacial acetic acid solution and incubate with shaking for 10 min to further terminate the color development reaction. Perform gel imaging (see Figure 2 A).

[0124] 6.6 Gel excision and protein digestion

[0125] (1) Use a sterile blade to cut the Coomassie Brilliant Blue-stained target protein and place it on a clean EP glove. Cut the protein band into small pieces of about 1.5 cubic millimeters and transfer them to a 1.5 mL EP tube;

[0126] (2) Wash with 500 μL double distilled water for 10 min × 2 times;

[0127] (3) Staining and decolorization: 50 μL 150 mM NH 4 HCO 3 / acetonitrile (CH 3 CN) (1:1), ultrasonic decolorization for 5 min or at 37 °C for 20 min, discard the liquid; if it is a silver-stained gel spot, it can be left undecolored or 15 mM acetonitrile potassium ferrate (K 3 Fe(CN) 6 ) / 50mM sodium thiosulfate (Na 2S 2 O 3 ) Shake gently until it turns light yellow and transparent, then wash repeatedly with water until it becomes colorless;

[0128] (4) Repeat step (3) until the blue color fades;

[0129] (5) Add 50 μL CH 3 CN dehydrates until the particles turn white;

[0130] (6) Add 20 μL 10 mM DTT (25 mM NH 4 HCO 3 ), 56°C water bath for 60 min;

[0131] (7) Cool to room temperature, aspirate dry, and quickly add 20 μL 50 mM iodoacetamide (IAA) (25 mA NH 4 HCO 3 ), placed in a dark room for 45 min;

[0132] (8) Use the following solutions for ultrasonic or suspension cleaning: 25 mM NH 4 HCO 3 (2 times), 25 mM NH 4 HCO 3 +50% CH 3 CN(2 times), CH 3 CN (1 time), 10 min each time, CH 3 CN dehydrates until the particles turn white;

[0133] (9) 0.1 μg / μL enzyme solution was diluted with 25 mM NH 4 HCO 3 Dilute 10-20 times, add 2-3 μL to each tube, centrifuge slightly to allow the enzyme solution to fully contact the colloid particles, place in a 4°C refrigerator for 30 minutes, wait for the enzyme solution to be completely absorbed by the colloid particles, add 25 mM NH 4 HCO 3 , to a total volume of 10-15 μL, incubate in a 37°C water bath overnight;

[0134] (10) Add 0.1% TFA or 50% CH 3 CN + 0.1% trifluoroacetic acid (TFA) was used to terminate the reaction, and the mixture was shaken and evenly mixed. The enzymatic hydrolysate was collected by centrifugation. The enzymatic hydrolysate can be stored at -20°C or directly used for target application.

[0135] Note: Each time the solution is added, the solution must be vortexed to ensure that the particles are fully immersed in the solution. The precipitate must be sucked away before proceeding to the next step.

[0136] 6.7 Protein Identification by Mass Spectrometry

[0137] (1) Prepare the following solutions: 0.1% trifluoroacetic acid (0.1% TFA), 30% acetonitrile (30% ACN), TA30 (3 mL 0.1% trifluoroacetic acid and 7 mL 30% acetonitrile), and use TA30 to prepare a saturated HCCA (α-cyano-4-hydroxycinnamic acid) solution (add HCCA to 500 μL TA30 solution until yellow granules appear);

[0138] (2) Take out the enzymatic hydrolysate at -20°C and thaw it on ice. Pipette 1 μL of the enzymatic hydrolysate or peptide standard and 1 μL of TA30 to prepare a saturated HCCA solution into a 200 μL clean EP tube;

[0139] (3) After mixing by pipetting, pipette 1 μL of the sample into the Ground Steel target plate;

[0140] (4) Allow to dry at room temperature. You can use an ear cleaning bulb to blow and dry.

[0141] (5) Correctly load the Ground Steel target plate into the Bruker Autoflex Speed ​​TOF / TOF mass spectrometer, run the instrument, and identify proteins bound to lnc070974.

[0142] 6.8 RNA binding protein immunoprecipitation (RIP)

[0143] The steps refer to EMD Millipore Corp., (NO:289118), the specific process is as follows:

[0144] 6.8.1 Collection and lysis of cells

[0145] (1) After A549 and HCC827 cells grow to more than 80% in a 10 cm cell culture dish (about 6 dishes of cells are needed in total), discard the culture medium and wash twice with 10 mL PBS;

[0146] (2) Discard PBS, add 6 mL of new PBS, scrape the cells with a sterile cell scraper, and pipette them into a suspension;

[0147] (3) Centrifuge at 2000 rpm and 4°C for 5 min, discard the supernatant, and lyse the cells with 150 μL complete RIP Lysis buffer. The formula of this lysate is as shown in Table 4:

[0148] Table 4

[0149]

[0150] (4) Resuspend the cells, place on ice for 5 min, and store in 200 μL aliquots at -80°C.

[0151] 6.8.2 Immunoprecipitation (IP) and RNA precipitation

[0152] (1) Take out 4°C RIP Wash buffer and mix thoroughly by pipetting;

[0153] (2) Place the EP tube on a magnetic rack, remove the supernatant, and repeat step (1);

[0154] (3) Discard the supernatant, add 100 μL Wash buffer and 5 μg of the corresponding antibody and mix well, including anti-YBX1 in the experimental group and IgG (rabbit source) in the negative control group;

[0155] (4) Place in a four-dimensional rotator and incubate at room temperature for 30 min;

[0156] (5) Split off, place on a magnetic stand, remove the supernatant, and add 500 μL RIP Wash buffer to wash twice;

[0157] (6) Discard the supernatant, add 500 μL RIP Wash buffer, mix well, and place on ice for later use;

[0158] (7) Prepare IP buffer, the components are as shown in Table 5:

[0159] Table 5

[0160]

[0161] (8) Take out the RIP Lysis buffer lysate stored at -80°C, thaw rapidly, and centrifuge at 14000 rpm at 4°C for 10 min;

[0162] (9) Place the EP tube on a magnetic rack, discard the supernatant, add 900 μL IP buffer and 100 μL cell lysis supernatant, and vortex to mix;

[0163] (10) Place on a four-dimensional rotator and incubate at 4°C with vortexing overnight;

[0164] (11) Take 10 μL of cell lysis supernatant, record it as Input, and store it at -80°C for later use;

[0165] (12) Take the EP tube back from 4°C, place it on a magnetic rack, and discard the supernatant;

[0166] (13) Add 500 μL RIP Wash buffer and wash 6 times;

[0167] (14) Place on a magnetic stand, discard the supernatant, add 500 μL RIP Wash buffer, vortex to mix, and place on ice;

[0168] (15) Prepare proteinase K enzymatic buffer, the components of which are as shown in Table 6:

[0169] Table 6

[0170]

[0171] (16) Place on a magnetic stand, discard the supernatant, add 150 μL of proteinase K enzymatic buffer, and mix by pipetting;

[0172] (17) Take out the Input stored at -80°C, add 117 μL Wash buffer, 15 μL 10% SDS, and 18 μL Proteinase K, and mix by pipetting;

[0173] (18) Incubate at 55°C with shaking (250 rpm) for 30 min and vortex every 5 min;

[0174] (19) Split off the tube, place on a magnetic stand, and aspirate the supernatant into a new EP tube;

[0175] (20) Add 250 μL RIP Wash buffer and mix well.

[0176] (21) Add 400 μL of phenol:chloroform:isoamyl alcohol (125:24:1) and mix, then vortex for 15 sec;

[0177] (22) Centrifuge at 14000 rpm and 4°C for 10 min, and transfer 400 μL of the upper aqueous phase to a new EP tube;

[0178] (23) Add 450 μL of chloroform, vortex for 15 s, and centrifuge at 14,000 rpm at 4 °C for 10 min;

[0179] (24) 350 μL of the upper aqueous phase was transferred to a new EP tube, and 50 μL of solution I, 15 μL of solution II, 5 μL of Enhancer, and 850 μL of anhydrous ethanol were added and mixed. The mixture was precipitated at −80 °C overnight.

[0180] 6.8.3 RNA extraction and qPCR

[0181] (1) Take out the RNA precipitated overnight from the -80°C refrigerator and place it on ice until it thaws;

[0182] (2) Centrifuge at 14000 rpm at 4°C for 30 min and carefully discard the supernatant;

[0183] (3) Add 80% ethanol (prepared with DEPC water) to wash the precipitate, centrifuge at 14000 rpm at 4°C for 15 min, carefully discard the supernatant, and dry the precipitate at 42°C for about 3 min;

[0184] (4) Add 10 μL DEPC water to dissolve RNA;

[0185] (5) The reverse transcription and qPCR steps refer to the Nanjing Novozyme kit (NO: R223-01) as follows:

[0186] Reverse transcription

[0187] ①Removal of genomic DNA

[0188] Table 7

[0189]

[0190] As shown in Table 7, the PCR reaction system was prepared, and after gently blowing and mixing with a pipette, the EP tube was placed in the PCR instrument and the program was set to run at 42°C for 2 min.

[0191] ②Configure the reverse transcription reaction system

[0192] Table 8

[0193]

[0194] As shown in Table 8, prepare the reverse transcription reaction system, use a pipette to gently blow and mix the mixture, then briefly centrifuge the EP tube at 2000 rpm for 10 seconds, and place it in a PCR instrument for the next step.

[0195] ③Reverse transcription reaction (reaction procedure see Table 9)

[0196] Table 9

[0197]

[0198] After the PCR process was completed, the cDNA samples were stored in a -20°C refrigerator.

[0199] Real-time quantitative PCR (qRT-PCR)

[0200] Refer to Nanjing Novozyme kit (NO: Q311-02) and the steps are as follows:

[0201] ④Configure the reaction system (see Table 10)

[0202] Table 10

[0203]

[0204] Mark the eight tube strips and add 20 μL of the above mixed solution to each well in order. Put the eight tube strips into the centrifuge and balance them, then centrifuge briefly to collect the reaction solution.

[0205] ⑤PCR reaction (operated on CFX96 Real-Time PCR System), the reaction procedure is shown in Table 11.

[0206] Table 11

[0207]

[0208] The relative expression level of the gene after the reaction was calculated according to 2 -△△Ct (△Ct value = Ct target - Ct internal reference, △△Ct value = △Ct experimental group - △Ct control group) was calculated to obtain the relative expression level of the gene, and Actin gene was used as the control gene to detect the enrichment of YBX1 protein on lnc070974 ( Figure 2 C).

[0209] 6.8.4 Agarose electrophoresis of qPCR products

[0210] (1) Prepare 1% agarose gel according to Table 12:

[0211] Table 12

[0212]

[0213] Pour into a conical flask and heat in a microwave oven to boil. After the outside of the conical flask cools to 60°C, add 1.75μL of GoldView I nucleic acid dye. After fully mixing, pour the liquid into a clean gel tank and insert a comb. Leave at room temperature for 30 minutes until the gel is completely solidified.

[0214] (2) Loading, remove the comb, and soak the agarose gel in 1×TAE buffer. Take 1 μL of 6×Loading buffer and mix with 5 μL of qPCR product by pipetting and add to the gel wells.

[0215] (3) Electrophoresis: 110 V, 25 min.

[0216] (4) Exposure Figure 2 B).

[0217] 7. Lung cancer clinical sample testing

[0218] Detection of cancer and adjacent tissues of lung cancer patients: Cancer and adjacent tissues of 3 lung cancer patients, sera of 8 lung cancer patients, and 4 normal sera were obtained from the Second Affiliated Hospital of Wenzhou Medical University.

[0219] (1) Extraction of tissue RNA and detection of lnc070974: RNA was extracted using the Invitrogen TRIzol method (purchased from ThermoFisher) according to the instructions of the kit; 1000 ng of total RNA was used and the PrimeScript TM Reverse transcription was performed using the RT reagent Kit with gDNA Eraser (refer to the instructions of Nanjing Novozymes kit (NO: R223-01)).

[0220] (2) SYBR Premix Ex TaqTM II kit was used to perform qPCR to analyze the relative expression level of the lnc070974 gene. The actin gene was used as the control gene. The results showed that the expression levels of lnc070974 in cancer tissues of the three patients were 6.59, 5.42, and 4.85 times that of the adjacent tissues. Figure 4 ).

[0221] 8. Isolation of serum exosomes and detection of lnc070974

[0222] (1) Pour the serum sample into a 38.5 mL ultracentrifuge tube, weigh it and balance it strictly, then place it in an ultracentrifuge and centrifuge it at 4°C and 100,000 g for 2 h.

[0223] (2) Obtain exosome precipitate for RNA extraction and detection. Please refer to "7. Lung cancer clinical sample detection" for the steps.

[0224] Analysis showed that lnc070974 expression could be detected in the serum exosomes of lung cancer patients, but not in the serum exosomes of normal subjects (see Table 13).

[0225] Table 13 Content of lnc070974 in exosomes of healthy people and lung cancer patients

[0226]

[0227] Note: >37.00 means it exceeds the detection limit of qPCR

[0228] 9. Noncoding RNA-lnc070974 for in vivo treatment of animals

[0229] (1) For the feeding of nude mice, preparation of cells, and inoculation, refer to the “5. Animal Experimentation” section;

[0230] (2) The status of the mice was observed daily. On the 30th day of subcutaneous tumor formation, six nude mice with uniform tumor size were randomly divided into two groups: si-NC and si-lnc070974, with three mice in each group. The two groups were injected intratumorally with 100 μL of si-NC and si-lncRNA070974-2 (Table 1) (in vivo siRNA, 5 nmol), respectively. Thereafter, the injection was given intratumorally once every three days.

[0231] (3) Tumor size was measured with a vernier caliper every 3 days. After 10 intratumoral injections, the animals were euthanized and the tumors were removed. The tumors in the si-lnc070974 group were smaller than those in the si-NC group. Figure 5 A).

[0232] (4) Drawing of tumor growth curve: Calculate the mean tumor volume ± standard deviation ( Figure 5 B).

[0233] (5) Tumor weight was measured. The weight of the tumor injected with si-lnc070974 group decreased by 34% compared with the si-NC group. Figure 5 C).

[0234] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of an agent for inhibiting the expression of long non-coding RNA lnc070974 in the preparation of a drug for treating lung cancer; It is characterized in that The lung cancer is non-small cell lung cancer; The reagent is si-lncRNA070974-1, si-lncRNA070974-2 or shRNA as shown in SEQ ID NO.15; The sense strand of the si-lncRNA070974-1 is shown in SEQ ID NO.3, and the antisense strand is shown in SEQ ID NO.4; The positive strand of the si-lncRNA070974-2 is shown in SEQ ID NO.5, and the antisense strand is shown in SEQ ID NO.

6.

2. Application of reagents for detecting the expression level of lnc070974 in the preparation of products for diagnosing lung cancer, It is characterized in that The reagent for detecting the expression level of lnc070974 includes a primer pair as shown in SEQ ID NO.11-12.

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