CN112175997B

CN112175997B - Application of targeting exosome PKM2 in improvement of cisplatin resistance of non-small cell lung cancer

Info

Publication number: CN112175997B
Application number: CN202010934818.6A
Authority: CN
Inventors: 杨浩; 黄钢; 王栋梁; 解伟; 张坤驰; 孔平; 梁蓓蓓
Original assignee: Shanghai University of Medicine and Health Sciences
Current assignee: Shanghai University of Medicine and Health Sciences
Priority date: 2020-09-08
Filing date: 2020-09-08
Publication date: 2021-06-15
Anticipated expiration: 2040-09-08
Also published as: CN112175997A

Abstract

The invention relates to an application of a targeting exosome PKM2 in improving non-small cell lung cancer cisplatin resistance. The tumor hypoxia area is usually accompanied with the occurrence of drug resistance, and based on that the expression of pyruvate kinase M2 type (PKM2) in cisplatin-resistant non-small cell lung cancer cells is higher than that of sensitive cells, the invention increases the level of active oxygen in the cisplatin-resistant cells and induces apoptosis by interfering with PKM2 gene in the drug-resistant cells, realizes the sensitization effect on the drug-resistant cell treatment, and has important clinical application value.

Description

Application of targeting exosome PKM2 in improvement of cisplatin resistance of non-small cell lung cancer

Technical Field

The invention relates to the technical field of biology, in particular to application of a targeting exosome PKM2 in improvement of cisplatin resistance of non-small cell lung cancer.

Background

Lung cancer is one of the health and life threatening malignancies in humans, of which about 85% of patients are non-small cell lung cancer (NSCLC). Chemotherapy is the primary treatment for lung cancer, and platinum-based chemotherapy has become the standard treatment for advanced NSCLC patients. However, clinical resistance remains a challenge, greatly hindering therapeutic efficacy. Therefore, a more comprehensive understanding of the mechanisms of drug resistance is of great interest for the treatment of NSCLC patients.

Exosomes play an important role in tumorigenesis of lung cancer. Exosomes are late endosomal produced membrane-bound vesicles, ranging in size from 40-160nm, that are released from the cell into the extracellular space by fusion with the plasma membrane. Exosomes comprise different biomolecules, including various types of proteins, DNA and RNA, that affect cellular function by acting as messengers for cellular communication in the microenvironment. It has been reported that exosome delivery of proteins such as EphA2 can enhance gemcitabine resistance in pancreatic cancer. Thus, exosomes may be able to affect cell function by transferring biomolecules directly to the cell or modifying the microenvironment.

Hypoxia is a common phenomenon in most malignancies and is considered to be a complex factor in the tumor microenvironment associated with tumor resistance and tumorigenesis. This phenomenon is due to the lack of oxygen supply to the tumor cells caused by irregular or excessively long distance between tumor vessels. Tumor cells can adapt to this hostile hypoxic environment through a variety of cellular mechanisms, thereby making tumor cells more resistant and viable, which is one of the reasons for their resistance to chemotherapy. One of the important changes is the change in glucose metabolism, the shift of tumor cells from oxidative phosphorylation to glycolysis to meet the energy requirements of tumor cells, and this reprogramming of tumor metabolism, known as the Warburg effect, is considered a hallmark feature of tumors. Pyruvate kinase type M2 (PKM2) is an important regulator of the Warburg effect and has received much attention in recent years because of its role in promoting metabolic reprogramming. PKM2 participates in the Warburg effect by catalyzing the synthesis of pyruvate from phosphoenolpyruvate (PEP) to promote hypoxic glycolysis, which contributes to tumor cell growth, leading to a malignant characterization of lung cancer.

The exosomes secreted by hypoxic tumors play an important role in the interaction between tumor cells and their microenvironment. It has been reported that exosomes from hypoxic tumor cells are rich in immunosuppressive proteins and can promote tumor progression by mediating M2 polarization by mononuclear macrophages. In addition, hypoxia can increase the production of exosomes, thereby promoting cell-to-cell communication in tumors, suggesting that exosomes play an important role in the malignant phenotype of hypoxic tumors, including the development of chemotherapy resistance. Since this exosome and PKM2 have a positive effect on the development of tumor resistance.

Disclosure of Invention

In view of the important role of exosomes and PKM2 in mediating the development of non-small cell lung cancer chemotherapy resistance under hypoxic conditions, the invention provides a method and application for intervening PKM2 or PKM2 in exosomes in improving non-small cell lung cancer cisplatin resistance.

The purpose of the invention can be realized by the following technical scheme:

the invention firstly provides application of a substance targeting an exosome PKM2 in preparing a medicine for improving non-small cell lung cancer cisplatin resistance, wherein the substance targeting an exosome PKM2 is a PKM2 inhibitor or a substance containing an shRNA sequence targeting PKM 2.

Further, an application of an expression vector containing the PKM2 knocked-down shRNA in preparation of a medicine for improving the cisplatin resistance of the non-small cell lung cancer is provided. Agents containing shRNA sequences targeting PKM2 can interfere with the expression of PKM 2.

The substance containing the shRNA sequence targeting the PKM2 can be an expression vector containing the shRNA sequence targeting the PKM2 or a lentivirus containing the shRNA sequence targeting the PKM 2.

Further, the first nucleotide sequence of the shRNA sequence of the target PKM2 is shown as SEQ ID NO. 2 and SEQ ID NO. 3, and the second nucleotide sequence is shown as SEQ ID NO. 4 and SEQ ID NO. 5.

Further, the expression vector containing the shRNA sequence targeting PKM2 is a shuttle plasmid carrying the PKM2 shRNA sequence.

Further, lentiviruses containing shRNA sequences targeting PKM2 are: a lentivirus obtained by packaging a shuttle plasmid into a lentivirus, also known as PKM2 shRNA lentivirus.

Further provides an application of the inhibitor of PKM2 in preparing a medicament for improving the cisplatin resistance of the non-small cell lung cancer. Inhibitors of PKM2 may interfere with the activity of PKM 2.

The invention also provides an application of a substance targeting the exosome PKM2 and cisplatin in preparing a non-small cell lung cancer medicament, wherein the substance targeting the exosome PKM2 can interfere in the expression of PKM2 or interfere in the activity of PKM2, and can enhance the chemotherapy sensitivity of cells to cisplatin under the condition of simultaneously using cisplatin.

The present invention also provides a method of evaluating the effect of the targeting exosome PKM2 on non-small cell lung cancer cisplatin resistance, comprising the steps of:

(1) constructing a eukaryotic expression vector for over-expression of PKM 2;

(2) transfecting the PKM2 overexpression vector obtained in the step (1) into a cisplatin-sensitive non-small cell lung cancer cell and culturing;

(3) collecting a culture solution obtained by culturing the cell line over-expressed by the PKM2 obtained in the step (2), and purifying the exosome by an ultracentrifugation method to obtain the exosome over-expressed by the PKM 2;

(4) constructing two shRNA expression vectors knocked down by PKM2, and packaging the shRNA expression vectors into lentiviruses;

(5) infecting the PKM2 shRNA lentivirus obtained in the step (4) into a cisplatin-resistant non-small cell lung cancer cell and culturing;

(6) collecting a culture solution obtained after the PKM2 shRNA knocked-down drug-resistant cell line is cultured in the step (5), and purifying an exosome by an ultracentrifugation method to obtain a PKM2 knocked-down exosome;

(7) the PKM2 overexpression and knockdown cell line obtained in the step (2) and the step (5) is used for evaluating the influence of PKM2 on the cisplatin resistance of the non-small cell lung cancer;

(8) and (4) carrying out co-culture with normal sensitive cells by using the exosomes with over-expression and knockdown of the PKM2 obtained in the step (3) and the step (6), and applying cisplatin treatment to evaluate the influence of the PKM2 carried by the exosomes on cisplatin resistance of the non-small cell lung cancer.

Further, the PKM2 overexpression eukaryotic expression vector in the step (1), wherein the nucleotide sequence of the PKM2 overexpression is shown as SEQ ID NO: 1.

Preferably, the cisplatin-sensitive non-small cell lung cancer cell of step (2) is an A549 cell line.

Preferably, the cisplatin-resistant non-small cell lung cancer cell in step (5) is an A549/CR cell line induced by A549 at a low concentration of cisplatin for a long time.

Preferably, the A549 cell line is culturedThe culture medium is 90% DMEM basal medium added with 10% fetal calf serum, and the culture conditions are 37 ℃ and 5% CO₂Normoxia (20% O)₂)。

Preferably, the culture solution of the A549/CR cell line is 90% DMEM basal medium plus 10% fetal bovine serum, and the culture conditions of the A549/CR cell line are set to 37 ℃ and 5% CO in view of the correlation between tumor hypoxia and drug resistance₂Hypoxic (1% O)₂)。

The invention also provides a method for improving the cisplatin resistance of the non-small cell lung cancer by targeting the exosome PKM2, which comprises the following steps:

the shRNA of PKM2 or the PKM2 inhibitor is combined with cisplatin to be used for inhibiting the proliferation of a drug-resistant cell line or the growth of subcutaneous tumors of mice.

Further, the shRNA of the PKM2 realizes the inhibition effect on the drug-resistant cells by inhibiting the transcription of mRNA of genes related to carbohydrate metabolism, promoting the apoptosis of the drug-resistant cells and increasing the active oxygen level in the drug-resistant cells.

Preferably, the primer for fluorescent quantitative PCR detection of mRNA of the sugar metabolism-related gene, wherein:

the nucleotide sequence of the upstream primer of PDK1 is shown as SEQ ID NO. 6, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 7;

the nucleotide sequence of the upstream primer of MYC is shown as SEQ ID NO. 8, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 9;

the nucleotide sequence of the upstream primer of GLUT1 is shown as SEQ ID NO. 10, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 11;

the nucleotide sequence of the upstream primer of LDHA is shown as SEQ ID NO. 12, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 13;

the nucleotide sequence of the upstream primer of CCND1 is shown as SEQ ID NO. 14, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 15.

The invention adopts the over-expression vector of PKM2 to transfect the cisplatin-sensitive A549 cell line, and simultaneously uses cisplatin to treat the cell, and analyzes that the over-expression of PKM2 can improve the cisplatin resistance of the A549 cell line. In a cisplatin-resistant A549/CR cell line, the PKM2 shRNA is used for interfering the expression of PKM2, or an inhibitor of PKM2 is used for interfering the activity of PKM2, the chemotherapy sensitivity of cells to cisplatin can be enhanced under the condition of simultaneously using cisplatin, and the cisplatin-resistant A549/CR cell line can be applied to in vitro cell experiments and in vivo mouse tumorigenesis experiments.

According to the invention, the PKM2 expression in a cisplatin-resistant A549/CR cell line is reduced through the PKM2 shRNA, an exosome secreted by the PKM2 expression is extracted, and the drug-resistant cell is treated by using the exosome knocked down by the PKM2, so that the chemotherapy sensitivity of the cell to cisplatin can be enhanced under the condition of simultaneously using the cisplatin. In addition, PKM2 knockdown exosomes have the following uses:

(1) inhibiting the mRNA expression of glucose metabolism related genes GLUT1, MYC, CCND1, PDK1 and LDHA;

(2) inhibiting apoptosis of A549/CR cells induced by cisplatin;

(3) inhibiting the generation of active oxygen in A549/CR cells under the induction of cisplatin.

Based on that the expression of pyruvate kinase M2 type (PKM2) in cisplatin-resistant non-small cell lung cancer cells is higher than that of sensitive cells, the invention increases the active oxygen level in the cisplatin-resistant cells and induces apoptosis by interfering with PKM2 genes in the drug-resistant cells, realizes the sensitization effect on the drug-resistant cell treatment, and has important clinical application value.

Compared with the prior art, the application of the targeting exosome PKM2 in improving the non-small cell lung cancer cisplatin resistance has the following beneficial effects:

(1) the shRNA is used for knocking down the content of PKM2, so that the treatment sensitivity of the cis-platinum to the drug-resistant non-small cell lung cancer cell is enhanced;

(2) the inhibitor is used for reducing the activity of PKM2 and enhancing the treatment sensitivity of cisplatin to the drug-resistant non-small cell lung cancer cell;

(3) the use of PKM 2-knocked-down exosomes enhanced the sensitivity of cisplatin treatment to drug-resistant non-small cell lung cancer cells.

Drawings

FIG. 1A is a graph showing cell growth viability of the cisplatin-sensitive A549 cell line (A549/SEN) and the cisplatin-resistant A549/CR cell line (A549/CR) under treatment with cisplatin at different concentrations;

FIG. 1B is a graph of the reactivity of the A549 cell line sensitive to cisplatin under normoxic and hypoxic conditions to various concentrations of cisplatin;

FIG. 1C is a graph of the reactivity of the cisplatin-resistant A549/CR cell line to different concentrations of cisplatin under normoxic and hypoxic conditions;

FIG. 2A is a graph showing the effect of over-expressing PKM2 on cell proliferation of a549 cell line sensitive to cis-platin;

FIG. 2B shows that two shRNAs knock down PKM2, respectively, and the effect on cell proliferation of a cisplatin-resistant A549/CR cell line is detected;

FIG. 3A is a graph showing the sensitivity of the A549 cell line sensitive to cis-platin overexpressing PKM2 to treatment with different concentrations of cis-platin;

FIG. 3B is a graph showing the effect of two shRNAs on the respective knockdown of the cisplatin sensitivity of PKM2 on a cisplatin-resistant A549/CR cell line;

FIG. 4A is an electron micrograph of exosomes secreted by the cisplatin-sensitive A549 cell line (SENEXo), exosomes secreted by the cisplatin-resistant A549/CR cell line (CRexo), exosomes secreted by the cisplatin-resistant A549/CR cell line in hypoxic culture (hCRNEXo);

FIG. 4B is a graph of particle size analysis of exosomes secreted by the above three cell lines;

FIG. 4C is a marker protein assay for exosomes secreted by the three cell lines described above;

FIG. 4D shows the measurement of the expression level of PKM2 in exosomes secreted by the above three cell lines;

FIG. 5A shows the change in expression of PKM2 in sensitive cells after co-culture with the three exosomes described above;

FIG. 5B is the change in mRNA expression of the metabolism-related gene in sensitive cells after co-culture with the above three exosomes;

FIG. 5C shows that exosomes after shRNA knockdown of PKM2 affect drug resistance in drug-resistant cells;

FIG. 5D shows that exosomes after shRNA knockdown of PKM2 affect the change in mRNA expression of metabolic-related genes;

FIG. 6A shows that exosomes after shRNA knockdown of PKM2 affects drug-resistant apoptosis;

FIG. 6B shows that exosomes after shRNA knockdown of PKM2 affect expression of apoptosis-related proteins in drug-resistant cells;

FIG. 6C shows that exosomes after shRNA knockdown of PKM2 affect the active oxygen content in drug-resistant cells;

FIG. 7A is a graph of the effect of a PKM2 inhibitor in combination with cisplatin on the proliferation of a cisplatin-resistant A549/CR cell line;

FIG. 7B is a graph showing the effect of a PKM2 inhibitor in combination with cisplatin on the inhibition of subcutaneous tumors in mice;

FIG. 7C is a graph of tumor volume measurements taken in mice as described above;

FIG. 7D is a graph of tumor weight measurements in mice as described above;

FIG. 7E is an image of tumor biopsies from the above-mentioned mice.

Detailed Description

The invention is described in detail below with reference to the figures and specific embodiments.

Experimental materials: human NSCLC A549, H1299 and PC9 cell lines were obtained from American Type Culture Collection (ATCC), DMEM basal medium, fetal bovine serum, streptomycin were obtained from Gibco, cisplatin, PKM2 inhibitor were obtained from Selleck, cisplatin-resistant A549/CR cell line was obtained by treating A549 cell line with increasing concentrations of cisplatin for 10 months, CCK8 kit was obtained from BimakeK, plasmid extraction kit was obtained from Qiagen, RNA extraction kit was obtained from Biomiga, reverse transcription reagent and fluorescent quantitative PCR reagent were obtained from Nanjing Nozam Biochemical, apoptosis detection kit was obtained from BD, and active oxygen detection kit was obtained from Nanjing Biochemical research institute.

Example 1 Induction of cisplatin-resistant cells and detection of cisplatin resistance under hypoxic conditions

(1) Non-small cell lung cancer cell A549 is used as a parent cell strain, cisplatin is added to the final concentration of 2ng/ml during each subculture, then the passage and the medicine addition are repeated until the cells are basically adapted and do not die, the concentration of the cisplatin is gradually increased, and the cisplatin-resistant cell strain A549/CR is obtained after 10 months.

(2) The cisplatin half-number inhibition rate (IC50) of sensitive cells A549 and drug-resistant cells A549/CR is detected, the survival rate and the inhibition rate of each cell at various concentrations are firstly obtained by a CCK8 method, then an inhibition dose-response curve with variable slopes is made by Graphpad Prism 7 software, and an IC50 value is calculated, and the result is shown in figure 1A.

(3) The method for detecting the cell survival rate by CCK8 comprises the following steps: the cells were treated with 10⁴The density of individual cells/well was plated in 96-well plates and incubated at 37 ℃ in an incubator while cisplatin was added to the corresponding final concentration for 48 hours. The culture solution was discarded, and CCK8 working solution was added thereto and reacted at 37 ℃ for 1 hour, and then absorbance at 450nm was measured with a microplate reader as cell survival data. The viability of the cells of each group was calculated with reference to the control group and the cells of each group were independently repeated 3 times.

(4) Sensitive cells A549 and drug-resistant cells A549/CR are respectively cultured in the presence of normal oxygen (20% O)₂) And hypoxia (1% O)₂) Cell viability was measured by the CCK8 method with cisplatin added at the corresponding concentration for 48 hours under ambient conditions, and the results are shown in FIG. 1B and FIG. 1C.

Example 2 overexpression of PKM2 in sensitive cells and detection of cell proliferation

(1) The sequence encoding PKM2 gene (SEQ ID NO:1) was cloned into pcDNA4TO-Flag vector, and after confirmation of sequencing, a plasmid overexpressing PKM2 was obtained, and the plasmid was extracted according to the instructions of the manufacturer.

(2) Sensitive cells A549 were planted in 6-well plates at 4X 10⁵One cell per well, transfected with 2 μ g of PKM2 overexpression plasmid or control empty plasmid, respectively. After 24 hours, the cells were digested and pressed to 10⁴The density of individual cells/well was then plated in 96-well plates and cell proliferation was detected by the method of CCK 8.

(3) Cell proliferation is detected by a clone formation method, which comprises the following steps: sensitive cells a549 were seeded at 200 cells/well in 12-well plates and transfected with 1 μ g of either PKM2 overexpression plasmid or control unloaded plasmid, respectively, for 14 days. The culture solution was discarded, 200. mu.l of 4% paraformaldehyde was added to fix the cells, the solution was discarded, the cells were stained with 0.1% crystal violet stain for 30min, and finally unbound crystal violet was washed off with PBS, and the stained cells were photographed and counted, and the results are shown in FIG. 2A.

Example 3 knock-down of PKM2 in drug-resistant cells and detection of cell proliferation

(1) Two target PKM2 shRNA sequences (shRNA sequence #1 is SEQ ID NO:2 and SEQ ID NO:3, shRNA sequence #2 is SEQ ID NO:4 and SEQ ID NO:5) are designed, a DNA fragment is synthesized and inserted into an LV3 shuttle vector, and a shuttle plasmid carrying the PKM2 shRNA sequence is obtained. The shuttle plasmid and packaging plasmids (pGag/Pol, pRev, pVSV-G) were co-transfected into 293T cells, and lentiviruses in the supernatant were collected 72 hours after transfection and the virus titer was measured.

(2) And infecting lentivirus carrying PKM2 shRNA sequence into drug-resistant cells A549/CR, and screening by puromycin to obtain an A549/CR cell line stably knocking down PKM 2.

(3) A549/CR cells and control cells stably knocked-down with PKM2 were plated at 10 per well⁴The CCK8 experiments were performed in 96-well plates, and the colony formation experiments were performed in 12-well plates with 200 cells per well. By the CCK8 method and the clone formation method, compared with the shNC control group, the proliferation condition of the drug-resistant cells A549/CR is detected to be changed after the shRNA knockdown PKM2, and the result is shown in figure 2B.

Example 4 drug sensitivity assay for cisplatin

(1) The control unloaded and PKM2 plasmids were separately overexpressed in A549 cells while treating with 1. mu.g/ml, 2. mu.g/ml, 4. mu.g/ml, 8. mu.g/ml of cisplatin for 48 hours, and cell viability was examined at each concentration by the method of CCK 8. The results are shown in fig. 3A, where overexpression of PKM2 promotes cisplatin resistance in a549 cells.

(2) A549/CR cells and control cells stably knocked-down with PKM2 were plated at 10 per well⁴Each of the cells was inoculated into a 96-well plate and simultaneously treated with 1. mu.g/ml, 5. mu.g/ml, 10. mu.g/ml, 50. mu.g/ml of cisplatin for 48 hours, and the cell viability at each concentration was examined by the method of CCK 8. The results are shown in fig. 3B, and the knockdown of PKM2 restored cisplatin sensitivity in a549/CR cells.

(3) Exosomes secreted from a549/CR cells stably knocked-down with PKM2 were co-cultured with a549 cells in 96-well plates while treated with 1, 2, 4 μ g/ml of cisplatin for 48 hours, and cell viability was examined at each concentration by the method of CCK 8. The results are shown in fig. 5C, and knockdown of PKM2 in exosomes promoted cisplatin sensitivity of a549 cells.

Example 5 extraction and characterization of exosomes of cisplatin-resistant cells

(1) Exosomes were obtained from conditioned media of exosomes of a549, a549/CR and hypoxic cultured a 549/CR. The culture supernatant was filtered through a 0.22 μm filter, centrifuged at 120,000g for 90 minutes, washed with PBS, and centrifuged at 120,000g for another 90 minutes to collect the exosome pellet. The exosome pellet was finally resuspended in PBS and stored in a-80 ℃ freezer.

(2) The exosome weight suspension was dropped on a copper mesh, then fixed with 50 μ l of 1% glutaraldehyde for 5 minutes, 50 μ l of uranyl acetate was dropped on the copper mesh, incubated for 5 minutes, and the excess liquid was absorbed with filter paper and dried. The exosome images were observed by transmission electron microscopy (FEI Tecnai G2 Spirit TEM), and the results are shown in fig. 4A.

(3) Exosomes were injected into a ZetaView PMX instrument to measure the size distribution and concentration of exosomes, the results are shown in figure 4B, and data analysis was performed using ZetaView 8.04.02 software.

(4) The expression of the marker proteins CD63 and TSG101 in exosomes was detected by western blot and the results are shown in fig. 4C.

Example 6western blot detection of expression of PKM2 in cisplatin-resistant exosomes and cells

(1) And (3) carrying out protein gel separation on 10 mu g of exosome protein sample or 10 mu g of cell protein sample co-cultured with exosome by SDS-PAGE electrophoresis, wherein the voltage of the concentrated gel is 80V, and the voltage of the separation gel is 120V.

(2) And transferring the protein gel onto the PVDF membrane after the methanol pretreatment under the constant current of 240V for 90 min.

(3) PVDF membrane in 5% skim milk blocking for 1 hours, then in the corresponding primary antibody 4 degrees C were incubated overnight. PKM2 and β -actin antibodies were purchased from Cell Signaling Technology, and CD63 and TSG101 antibodies were purchased from Proteitech.

(4) After 3 washes with 1 × TBST, incubation with the corresponding secondary antibody for 1 hour was performed and finally visualized by a gel imaging system. The results are shown in fig. 4D and fig. 5A, PKM2 is highly expressed in the drug-resistant extracellular exosomes, and PKM2 expression in sensitive cells is again enhanced after the drug-resistant extracellular exosomes are taken up by the sensitive cells.

Example 7 expression of mRNA of genes involved in glycometabolism by fluorescent quantitative PCR

(1) And (3) extracting RNA of the A549 cells treated by the exosomes (including sensitive cell exosomes, drug-resistant cell exosomes knocked-down PKM2 and corresponding control groups), performing the RNA treatment according to the instruction of a reagent maker, and quantifying the RNA.

(2) After RNA is reversely transcribed into cDNA, the expression of genes related to glycometabolism is subjected to fluorescent quantitative PCR detection, the specific process is carried out according to the instruction of a reagent manufacturer, the primer sequences of the detection gene PDK1 are SEQ ID NO. 6 and SEQ ID NO. 7, the primer sequences of the detection gene MYC are SEQ ID NO. 8 and SEQ ID NO. 9, the primer sequences of the detection gene GLUT1 are SEQ ID NO. 10 and SEQ ID NO. 11, the primer sequences of the detection gene LDHA are SEQ ID NO. 12 and SEQ ID NO. 13, and the primer sequences of the detection gene CCND1 are SEQ ID NO. 14 and SEQ ID NO. 15. As a result, as shown in fig. 5B and fig. 5D, the exosomes secreted by the drug-resistant cells were able to enhance the expression of mRNA of the genes associated with glycometabolism, whereas the exosomes knocked down in PKM2 were able to restore the expression of mRNA.

Example 8 detection of apoptosis following exosome and cisplatin-treated cells

(1) Drug-resistant cell exosomes and control exosomes knocked-down PKM2 were co-cultured with a549/CR, treated with 2 μ g/ml cisplatin, examined for apoptosis before and after treatment, and subjected to data analysis using FlowJo software.

(2) Drug-resistant cell exosomes knocked down by PKM2 significantly promote apoptosis of A549/CR cells induced by cisplatin, and the results are shown in FIGS. 6A and 6B as proved by western blot detection data of apoptosis-related proteins.

Example 9 reactive oxygen species detection following exosome and cisplatin treatment of cells

(1) Drug-resistant cell exosomes and control exosomes with knockdown PKM2 were co-cultured with a549/CR, treated with 2 μ g/ml cisplatin, assayed for levels of active oxygen in cells before and after treatment using DCFH-DA as an active oxygen probe according to the instructions of the reagent manufacturer, and subjected to data analysis using FlowJo software.

(2) Drug-resistant extracellular exosomes knocked-down PKM2 significantly promoted active oxygen production under cisplatin induction, with the results shown in fig. 6C.

Example 10 combination therapy of PKM2 inhibitor and cisplatin in mice with subcutaneous drug resistant tumors

(1) A549/CR resistant cells were plated in 96-well plates and treated with 20. mu.g/ml cisplatin, 2. mu.M PKM2 inhibitor, and combinations thereof, respectively, and CCK8 was detected 48 hours later. The results are shown in figure 7A, where the combined use of cisplatin and PKM2 inhibitors had a more pronounced inhibitory effect on drug-resistant cells than either drug alone.

(2) For in vivo experiments in mice, 5X 10⁶A549/CR drug-resistant cells marked by luciferase are inoculated to nude mice subcutaneously, and 20 mice with approximate tumor size are selected for drug combination treatment about 20 days. The dose used was 4mg/kg of cisplatin and 2mg/kg of PKM2 inhibitor administered 3 times a week via the tail vein.

(3) 20 days after dosing, mice were examined for in vivo imaging and tumor size and weight were recorded. The results are shown in figures 7B, 7C, 7D and 7E, where the combined use of cisplatin and PKM2 inhibitor had a more significant inhibitory effect on mouse subcutaneous resistant tumors than either drug alone.

The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.

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aattcaaaaa aaggcagagg ctgccatcta tctcttgaat agatggcagc ctctgcct 58

<210> 6

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gagagccact atggaacacc a 21

<210> 7

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ggaggtctca acacgaggt 19

<210> 8

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

acacccttct cccttcg 17

<210> 9

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ccgctccaca tacagtcc 18

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ctccggtatc gtcaacacgg 20

<210> 11

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

aagccaaaga tggccacgat 20

<210> 12

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gtcagcatag ctgttccact ta 22

<210> 13

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

agctgatcct ttagagttgc ca 22

<210> 14

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gcgaggaaca gaagtgc 17

<210> 15

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gagttgtcgg tgtagatgc 19

Claims

1. The application of a substance targeting an exosome PKM2 in preparing a medicine for improving the cisplatin resistance of the non-small cell lung cancer is characterized in that the substance targeting an exosome PKM2 is a substance containing an shRNA sequence targeting PKM 2;

the sequence of the shRNA of the target PKM2 is two, the upstream nucleotide sequence of the shRNA of the first target PKM2 is shown as SEQ ID NO. 2, and the downstream nucleotide sequence of the shRNA of the first target PKM2 is shown as SEQ ID NO. 3; the upstream nucleotide sequence of the shRNA sequence of the second targeting PKM2 is shown as SEQ ID NO. 4, and the downstream nucleotide sequence of the shRNA sequence of the second targeting PKM2 is shown as SEQ ID NO. 5.

2. The use of an exosome targeting PKM2 substance as claimed in claim 1 in the preparation of a medicament for improving non-small cell lung cancer cisplatin resistance, wherein the substance containing the shRNA sequence targeting PKM2 is an expression vector containing the shRNA sequence targeting PKM2, or a lentivirus containing the shRNA sequence targeting PKM 2.

3. The application of the exosome PKM 2-targeting substance in preparing a medicine for improving the cisplatin resistance of the non-small cell lung cancer according to claim 2, wherein the expression vector containing the shRNA sequence targeting PKM2 is a shuttle plasmid carrying the PKM2 shRNA sequence.

4. The use of an exosome targeting PKM2 substance as claimed in claim 2 in the manufacture of a medicament for improving non-small cell lung cancer cisplatin resistance, wherein the lentivirus containing shRNA sequence targeting PKM2 is: packaging a shuttle plasmid carrying the PKM2 shRNA sequence into lentivirus obtained from lentivirus.

5. The application of the substance targeting the exosome PKM2 in preparing the medicine for improving the cisplatin resistance of the non-small cell lung cancer according to claim 1, wherein the shRNA of the PKM2 realizes the inhibition effect on the drug-resistant cell by inhibiting the transcription of the mRNA of the gene related to the glycometabolism, promoting the apoptosis of the drug-resistant cell and increasing the active oxygen level in the drug-resistant cell.

6. The use of an exosome targeting substance, PKM2, according to claim 5 in the preparation of a medicament for improving non-small cell lung cancer cisplatin resistance,

the sugar metabolism related genes comprise GLUT1, MYC, CCND1, PDK1 and LDHA,

a primer for fluorescent quantitative PCR detection of mRNA of a gene related to sugar metabolism, wherein:

7. The application of a substance targeting an exosome PKM2 and cisplatin in preparing a non-small cell lung cancer drug, wherein the substance targeting an exosome PKM2 can interfere in the expression of PKM2 or interfere in the activity of PKM2, and can enhance the chemotherapy sensitivity of cells to cisplatin under the condition of simultaneously using cisplatin; the substance targeting the exosome PKM2 is a substance containing an shRNA sequence targeting PKM 2;

8. A method of targeting the effect of exosome PKM2 on cisplatin resistance in non-small cell lung cancer, comprising the steps of:

(1) constructing a eukaryotic expression vector for over-expression of PKM 2;

(8) the exosomes with over-expression and knockdown of the PKM2 obtained in the step (3) and the step (6) are co-cultured with normal sensitive cells, and cisplatin treatment is applied to evaluate the influence of the PKM2 carried by the exosomes on cisplatin resistance of the non-small cell lung cancer;

in the step (4), the upstream nucleotide sequence of the first PKM 2-knocked-down shRNA is shown as SEQ ID NO. 2, the downstream nucleotide sequence of the first PKM 2-knocked-down shRNA is shown as SEQ ID NO. 3, the upstream nucleotide sequence of the second PKM 2-knocked-down shRNA is shown as SEQ ID NO. 4, and the downstream nucleotide sequence of the second PKM 2-knocked-down shRNA is shown as SEQ ID NO. 5.

9. The method for targeting the influence of the exosome PKM2 on the cisplatin resistance of the non-small cell lung cancer according to the claim 8, characterized in that, the PKM2 overexpression eukaryotic expression vector in the step (1), the nucleotide sequence of the overexpression of the PKM2 is shown as SEQ ID NO: 1;

the cisplatin-sensitive non-small cell lung cancer cell in the step (2) is an A549 cell line;

the cisplatin-resistant non-small cell lung cancer cell in the step (5) is an A549/CR cell line obtained by long-term low-concentration cisplatin induction of A549;

the culture solution of the A549 cell line is 90 percentDMEM basal medium added with 10% fetal calf serum under the conditions of 37 ℃ and 5% CO₂、20％O₂；

The culture solution of the A549/CR cell line is 90 percent DMEM basal medium and 10 percent fetal bovine serum, and the culture conditions of the A549/CR cell line are set to 37 ℃ and 5 percent CO in view of the correlation between tumor hypoxia and drug resistance₂、1％O₂。