Application of A2M in reducing drug resistance and malignancy of paclitaxel-resistant lung cancer cells

Abstract

The present invention discloses the application of A2M in reducing the drug resistance and malignancy of paclitaxel-resistant lung cancer cells. Experiments have shown that A2M repair inhibits the proliferation and invasion of paclitaxel-resistant lung cancer cells, reactivates the sensitivity of paclitaxel-resistant lung cancer cells to paclitaxel, and can also reduce the malignant characteristics of paclitaxel-resistant lung cancer cells. A2M is a new paclitaxel resistance-related gene, and its reduced expression is closely related to the acquisition of paclitaxel resistance in lung cancer patients. The gradual loss of A2M contributes to the development of paclitaxel resistance in lung cancer, and restoring the expression of A2M in paclitaxel-resistant cells can restore their sensitivity to paclitaxel. Therefore, A2M deficiency can be used as a predictive indicator and therapeutic target for paclitaxel resistance. The present invention has important application value.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to the application of A2M in reducing the drug resistance and malignancy of paclitaxel-resistant lung cancer cells. Background Art

[0002] Lung cancer (LC) is one of the common malignant tumors. Although surgery is the best treatment option, chemotherapy is the main treatment for patients with advanced LC. Among the current chemotherapy drugs, paclitaxel is often used as the first-line treatment for lung cancer and other cancers. It fights against a variety of advanced malignancies by affecting cell mitosis and inhibiting cell growth. However, the emergence of acquired drug resistance causes patients to progress or relapse in a short period of time, limiting the effectiveness of its clinical application. Therefore, exploring the potential mechanism of paclitaxel resistance is of great significance to reversing paclitaxel resistance and improving patient prognosis.

[0003] Several mechanisms of paclitaxel resistance have been reported. For example, P-glycoprotein (P-gp), also known as multidrug resistance protein 1 (MDR1), encoded by the gene ABCB1, is a classic mechanism mediating paclitaxel resistance. Overexpression of P-gp leads to increased paclitaxel efflux, thereby reducing the ability of paclitaxel to kill tumor cells. β-tubulin mutations reduce the affinity of paclitaxel for tubulin, leading to paclitaxel resistance. Upregulation of anti-apoptotic Bcl-2 family members (such as Bcl-1 and Bcl-XL) or downregulation of pro-apoptotic Bax in the apoptotic pathway are also associated with paclitaxel resistance. Despite this, the mechanism of paclitaxel resistance in lung cancer remains unclear.

[0004] The A2M gene encodes alpha-2-macroglobulin (A2M protein). The A2M protein is a large tetrameric secreted protein (Mr 725000) in mammalian blood, composed of identical subunits. The A2M protein was originally identified as a broadly specific plasma protease inhibitor and cytokine transporter that regulates the distribution and activity of most proteases and growth factors, hormones, and cytokines. Recently, A2M has been considered a tumor suppressor because it can inactivate a series of proteases such as plasmin, urokinase plasminogen activator, and metalloproteinases that are associated with tumor cell invasion. Summary of the invention

[0005] The purpose of the present invention is to improve the sensitivity of paclitaxel-resistant cells to paclitaxel.

[0006] The present invention firstly protects the use of A2M mRNA / protein or a substance that increases the expression of A2M mRNA / protein in the preparation of a product; the function of the product may be at least one of A1), A2) and A3):

[0007] A1) Reduce paclitaxel resistance;

[0008] A2) Increased sensitivity to paclitaxel;

[0009] A3) Reduce the malignancy of paclitaxel-resistant cancer cells.

[0010] The present invention also protects the use of A2M mRNA / protein or a substance that increases the expression of A2M mRNA / protein, which may be at least one of A1), A2) and A3):

[0011] A1) Reducing paclitaxel resistance;

[0012] A2) Increased sensitivity to paclitaxel;

[0013] A3) Reduce the malignancy of paclitaxel-resistant cancer cells;

[0014] The application is for non-disease diagnosis and treatment.

[0015] In any of the above applications, reducing the drug resistance of paclitaxel may be reducing the drug resistance of paclitaxel-resistant cells.

[0016] In any of the above applications, the improving the sensitivity to paclitaxel may be improving the sensitivity of paclitaxel-resistant cells to paclitaxel.

[0017] In any of the above applications, the reduction of the malignancy of paclitaxel-resistant cells can be manifested as inhibiting the proliferation ability of paclitaxel-resistant cancer cells.

[0018] In any of the above applications, the reduction of malignancy of paclitaxel-resistant cells can be manifested as reducing the clonal growth of paclitaxel-resistant cancer cells.

[0019] In any of the above applications, the reduction of malignancy of paclitaxel-resistant cells can be manifested as inhibiting the migration ability and / or invasion ability of paclitaxel-resistant cancer cells.

[0020] In any of the above applications, the cells or cancer cells may be lung cancer cells.

[0021] The present invention also protects the use of A2M mRNA / protein as a biomarker, which can be B1) or B2):

[0022] B1) preparing a product for predicting paclitaxel resistance;

[0023] B2) prediction of paclitaxel resistance;

[0024] B3) preparing products for lung cancer screening;

[0025] B4) Lung cancer screening;

[0026] The application is for non-disease diagnosis and treatment.

[0027] In the above application, the judgment principle for predicting paclitaxel resistance can be: if the A2M mRNA / protein expression level decreases, the paclitaxel resistance increases; if the A2M mRNA / protein expression level increases, the paclitaxel resistance decreases.

[0028] In the above application, the judgment principle of lung cancer screening can be: if the A2M mRNA / protein expression level of the subject is lower than that of healthy people, then the subject has or is suspected of having lung cancer.

[0029] The inventors of the present invention established multiple lung cancer cell lines with different degrees of acquired paclitaxel resistance, and then performed transcriptome RNA sequencing to screen genes that were significantly changed in resistant cells. The results showed that in addition to the gradual upregulation of the known paclitaxel resistance-inducing genes ABCB1, TMEM243 and ID1, the A2M gene was also gradually downregulated with the increase of paclitaxel resistance, that is, the expression of A2M was negatively correlated with paclitaxel resistance. Further experiments proved that A2M repair inhibited the proliferation and invasion of paclitaxel-resistant lung cancer cells; A2M repair significantly downregulated the expression of paclitaxel resistance mediators ABCB1, TMEM243 and ID1, and reactivated the sensitivity of paclitaxel-resistant lung cancer cells to paclitaxel; A2M repair can also reduce the malignant characteristics of paclitaxel-resistant lung cancer cells, highlighting the significance of A2M in paclitaxel resistance prediction and therapeutic intervention. In conclusion, A2M is a new paclitaxel resistance-related gene, and its reduced expression is closely related to the acquisition of paclitaxel resistance in lung cancer patients. The gradual loss of A2M contributes to the development of paclitaxel resistance in lung cancer, and restoring the expression of A2M in paclitaxel-resistant cells can restore their sensitivity to paclitaxel. Therefore, A2M deficiency can be used as a predictive indicator and therapeutic target for paclitaxel resistance. The present invention has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 IC50 and resistance index (RI) of paclitaxel-resistant cell lines were determined for CCK8.

[0031] Figure 2 The cell lines were resistant to paclitaxel and showed low proliferation and high invasiveness.

[0032] Figure 3Transcriptome RNA sequencing identified A2M downregulation as a marker for paclitaxel-resistant lung cancer cells. A, Transcriptome RNA sequencing data showed upregulation of paclitaxel resistance-inducing factors ABCB1, TMEM243, and ID1 in paclitaxel-resistant cell lines. B, RT-qPCR detection of relative expression of ABCB1, TMEM243, and ID1 in paclitaxel-resistant cell lines. C, RNA-seq and RT-qPCR analysis showed downregulation of A2M in paclitaxel-resistant cell lines. D, RT-qPCR detection of relative expression of A2M in HCC827 / TAX and A549 / TAX cells. E, Oncomine analysis of A2M in docetaxel-sensitive Shimokunis cell line samples.

[0033] Figure 4 A2M downregulation is associated with lung cancer progression. A is TCGA analysis, B is CPTAC analysis, C is TCGA analysis showing that A2M mRNA levels in LUAD samples are negatively correlated with cancer stage and lymph node metastasis, and D is CPTAC analysis showing downregulation of A2M protein in LUAD samples with higher cancer grade and stage.

[0034] Figure 5 A2M repair helps reduce the malignancy of paclitaxel-resistant cell lines. A is RT-qPCR to detect the expression of A2M in H446-HR-A2M and H446-HR-EV; B is RTCA analysis to evaluate the effect of A2M repair on H446-HR ​​proliferation; C is a clone formation test to determine the effect of A2M repair on H446-HR ​​clone generation and growth; D is a Transwell assay to evaluate the effect of A2M repair on H446-HR ​​migration and invasion, and E is RT-qPCR to detect the expression of EMT markers in H446-HR-A2M and H446-HR-EV.

[0035] Figure 6 To restore A2M, ABCB1, TMEM243 and ID1 were downregulated and paclitaxel-resistant cells were re-sensitized to paclitaxel. A is CCK8 detection of cell viability of H446-HR ​​after A2M recovery in the presence of different concentrations of paclitaxel; B is RT-qPCR detection of the expression levels of ABCB1, TMEM243 and ID1 in H446-HR-A2M and H446-HR-EV. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0037] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0038] In the following examples, * means p<0.05; ** means p<0.01; *** means p<0.001, and **** means p<0.0001.

[0039] Example

[0040] 1. Experimental Materials and Methods

[0041] 1. Cell lines and cell culture

[0042] Lung cancer cell line NCI-H446 (hereafter referred to as NCI-H446), lung cancer cell line A549 (hereafter referred to as A549), and lung cancer cell line HCC827 (hereafter referred to as HCC827) were all obtained from the Chinese Academy of Medical Sciences (Beijing, China).

[0043] NCI-H446 and HCC827 were cultured in RPMI-1640 medium (Gibco, USA) containing 10% (v / v) FBS (Gibco, USA) and 1% penicillin-streptomycin. A549 was cultured in DMEM medium (Gibco, USA) containing 10% (v / v) FBS and 1% penicillin-streptomycin. All cells were cultured in a humidified incubator at 37°C and 5% CO2.

[0044] 2. Establishment of paclitaxel-resistant cell line

[0045] The paclitaxel-resistant cell line was constructed by gradually increasing the concentration of paclitaxel in the culture medium. The details are as follows:

[0046] (1) The parental NCI-H446 sensitive cells (abbreviated as NCI-H446-S or H446-S) were treated with paclitaxel (treatment concentration of 0.02 μg / ml) for about two weeks until no obvious cell death occurred; then the paclitaxel treatment was continued and the treatment concentration of paclitaxel was gradually increased from 0.02 μg / ml to 0.20 μg / ml (the concentrations of paclitaxel were 0.02 μg / ml, 0.04 μg / ml, 0.06 μg / ml, 0.08 μg / ml, 0.10 μg / ml, 0.12 μg / ml, 0.14 μg / ml, respectively). / ml, 0.16μg / ml, 0.18μg / ml, 0.20μg / ml), and the resistant cells were stably cultured for at least two weeks at each concentration; finally, the low-resistance paclitaxel-resistant cell line NCI-H446-LR (abbreviated as NCI-H446-LR or H446-LR) that stably survived in the culture medium containing 0.02μg / ml paclitaxel and the high-resistance paclitaxel-resistant cell line NCI-H446-HR ​​(abbreviated as NCI-H446-HR ​​or H446-HR) that stably survived in the culture medium containing 0.2μg / ml paclitaxel were obtained.

[0047] The parental NCI-H446 sensitive cells are the lung cancer cell line NCI-H446.

[0048] (2) According to the method of step (1), the parental NCI-H446 sensitive cells were replaced with the parental A549 sensitive cells, and the other steps remained unchanged, and finally a paclitaxel-resistant cell line A549 / TAX (abbreviated as A549 / TAX) with low resistance that stably survived in a culture medium containing 0.02 μg / ml paclitaxel was obtained.

[0049] The parental A549 sensitive cells are lung cancer cell line A549.

[0050] (3) According to the method of step (1), the parental NCI-H446 sensitive cells were replaced with the parental HCC827 sensitive cells, and the other steps remained unchanged, and finally a paclitaxel-resistant cell line HCC827 / TAX (abbreviated as HCC827 / TAX) with low resistance that stably survived in a culture medium containing 0.02 μg / ml paclitaxel was obtained.

[0051] The parental HCC827 sensitive cells are lung cancer cell line HCC827.

[0052] 3. Transcriptome RNA sequencing

[0053] Total cellular RNA of NCI-H446-S, NCI-H446-LR, and NCI-H446-HR ​​was obtained, followed by RNAseq by Main Health Technology (Beijing) Co., Ltd. RNA sequence data were uniquely mapped to the reference genome using HISAT 2.0.5. Expression values ​​at the gene level were assigned by HTSeq 0.6.0. Differentially expressed genes were determined by DEGseq 1.18.0 and DESeq1.16.0 based on p < 0.05. mRNA expression was evaluated using fragments per kilobase of transcript per million mapped reads (FPKM).

[0054] 4. Cell Viability Assay

[0055] The cell counting kit (CCK8, Yeasen, China) was used to detect the half inhibitory concentration (IC) of cells (NCI-H446-S, NCI-H446-LR and NCI-H446-HR). 50 The specific steps are as follows:

[0056] (1) 1×10 4 Cells were seeded into 96-well plates and cultured for 48 hours in medium exposed to different concentrations of paclitaxel. NCI-H446-S was exposed to medium containing 0, 0.001, 0.002, 0.004, 0.006, 0.008, 0.012, 0.016 and 0.024 μg / ml paclitaxel, NCI-H446-LR was exposed to medium containing 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.12, 0.16 and 0.24 μg / ml paclitaxel, and NCI-H496-HR was exposed to medium containing 0, 0.2, 0.3, 0.4, 0.6, 0.8, 1.2, 1.6 and 2.4 μg / ml paclitaxel.

[0057] (2) After completing step (1), add culture medium containing 10% CCK8 to each well and incubate at 37°C for 1 hour. Then measure the optical density (OD) at 450 nm using a spectrophotometer. sample , O.D. blank Represents the optical density of the cell-free well at 450 nm, OD control The optical density at 450 nm of the wells with a paclitaxel concentration of 0 μg / ml.

[0058] (3) Calculate the cell survival rate. Survival rate (%) = (OD sample -OD blank ) / (OD control -OD blank )×100%.

[0059] (4) Calculate the half inhibitory concentration (IC) of cells using GraphPad Prism 6 50 ; The cell resistance index (RI) was further calculated.

[0060] Resistance index (RI) = IC of paclitaxel-resistant cells 50 IC of lung cancer cell line NCI-H446 50

[0061] 5. Obtaining A2M Stable Overexpression Cell Line

[0062] The A2M-expressing lentivirus pLV[Exp]-Puro CMV>hA2M (referred to as the A2M-expressing lentivirus) is a product of Cyagen (Suzhou, China), with the catalog number VB180420-1094ysy

[0063] (https: / / en.vectorbuilder.com / vector / VB180420-1094ysy.html).

[0064] The control lentivirus was a product of Cyagen (Suzhou, China), with the catalog number VB160420-1011mqh (https: / / www.vectorbuilder.com / us / en / vector / VB160420-1011mqh.html).

[0065] NCI-H446-HR ​​was seeded into 6-well plates and infected with a lentivirus expressing A2M in the presence of 2 μg / ml polybrene (Gibco, USA) for 48 hours. The cells were then selected with 2 μg / ml puromycin (Gibco, USA) for 1-2 weeks to obtain an A2M stable overexpression cell line (abbreviated as H446-HR-A2M).

[0066] According to the above steps, the lentivirus expressing A2M was replaced with the control lentivirus, and the other steps remained unchanged to obtain a control cell line (abbreviated as H446-HR-EV).

[0067] 6. RNA Extraction and Quantitative RT-PCR

[0068] (1) Total RNA of cells was extracted using TRIzol reagent (Invitrogen, USA), and then 1 μg of total RNA of cells was reverse transcribed using a reverse transcription kit (Yeasen, China) to obtain the first-strand cDNA of cells.

[0069] (2) Real-time quantitative PCR was performed on a 7500 sequence detection system (Applied Biosystems) using the first-strand cDNA of the cells as a template using SYBR Green Premix (Yeasen, China). -ΔΔCT The relative expression level of the target gene in cDNA was detected by the method (with human GAPDH gene as the internal reference gene).

[0070] The primers for detecting the target gene and human GAPDH gene are shown in Table 1.

[0071] Table 1

[0072]

[0073] 7. RTCA and clone formation assay

[0074] Real-time cell analysis system (RTCA) (Agilent, USA) and clonogenic assay were used to evaluate cell proliferation ability.

[0075] (1) RTCA assay

[0076] Cells were seeded at a density of 5000 cells per well in 96-well microplates (E-plates; Roche Diagnostics, Basel, Switzerland), and data were recorded at 15-min intervals for 140 h.

[0077] (2) Clone formation assay

[0078] The cells were seeded into 6-well plates at a density of 500 cells per well. After culturing for 2 weeks, the cell clones were fixed with 4% paraformaldehyde for 15 minutes and then stained with 1% crystal violet for 15 minutes. Finally, the cell clones were photographed and counted using ImageJ software.

[0079] 8. Cell migration and cell invasion

[0080] The migration and invasion abilities of cells were detected by Transwell analysis.

[0081] (1) Cell migration

[0082] (1-1) 5×10 4 The cells were suspended in 100 μl RPMI-1640 medium and then inoculated into the upper chamber of the transwell filter (8 μm / pore), and 600 μl RPMI-1640 medium containing 10% (v / v) FBS was added to the lower chamber and incubated for 24 hours.

[0083] (1-2) After completing step (1-1), the cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature, stained with 1% crystal violet for 15 minutes, and then photographed.

[0084] (2) Cell invasion

[0085] (2-1) Take a transwell and pre-coat the upper chamber with matrigel (Corning, USA) and solidify at 37°C for 2 hours.

[0086] (2-2) After completing step (2-1), 5×10 4 The cells were suspended in 100 μl RPMI-1640 medium and then inoculated into the upper chamber of the transwell filter (8 μm / pore), and 600 μl RPMI-1640 medium containing 10% (v / v) FBS was added to the lower chamber and incubated for 24 hours.

[0087] (2-3) After completing step (2-2), the cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature, stained with 1% crystal violet for 15 minutes, and then photographed.

[0088] 9. Statistical analysis

[0089] GraphPadPrism 6.0 (GraphPad Software) was used for data analysis. Independent sample T test and analysis of variance were used for intra-group and multi-group comparisons, respectively. Quantitative data are expressed as mean ± standard deviation. p < 0.05 was considered statistically significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0090] 2. Experimental Results

[0091] 1. Construction of paclitaxel-resistant cell line

[0092] Paclitaxel-resistant lung cancer cell line NCI-H446 was induced by gradually increasing the concentration of paclitaxel in the culture medium in vitro, and finally the paclitaxel-resistant cell line NCI-H446-LR (referred to as NCI-H446-LR) with low paclitaxel resistance that stably grows in the culture medium containing 0.02μg / ml paclitaxel and the paclitaxel-resistant cell line NCI-H446-HR ​​(referred to as NCI-H446-HR) with high paclitaxel resistance that stably grows in the culture medium containing 0.2μg / ml paclitaxel were obtained. The IC values ​​of NCI-H446-S, NCI-H446-LR and NCI-H446-HR ​​were determined by using Cell Counting Kit-8. 50 .

[0093] Test results see Figure 1The results showed that the IC of NCI-H446-S 50 was 0.008 μg / ml, while the IC 50 They were 0.06μg / ml and 0.83μg / ml respectively. Compared with NCI-H446-S, the resistance index (RI) of NCI-H446-LR was 7.55, and the resistance coefficient of NCI-H446-HR ​​was 103.25.

[0094] 2. Low proliferation and high invasiveness of paclitaxel-resistant cell lines

[0095] The proliferation and motility abilities of NCI-H446-S and NCI-H446-HR ​​were compared to characterize the paclitaxel-resistant cell lines.

[0096] The results of the clone formation assay are shown in Figure 2 Middle A. The results showed that the proliferation capacity of NCI-H446-HR ​​was impaired during the development of paclitaxel resistance, and the cell clones formed by NCI-H446-HR ​​were significantly smaller, that is, the paclitaxel-resistant cell line proliferated more slowly.

[0097] Transwell analysis results are shown in Figure 2 Middle B. The results showed that NCI-H446-HR ​​had enhanced migration and invasion abilities compared with NCI-H446-S.

[0098] RT-qPCR was used to detect the levels of EMT markers. Figure 2 Middle C. The results showed that the mesenchymal markers Snail, ZEB-1, and N-cad were upregulated in NCI-H446-HR ​​compared with NCI-H446-S, accompanied by the downregulation of the epithelial marker ZO1.

[0099] 3. Transcriptome RNA sequencing confirmed that A2M downregulation is a marker of paclitaxel resistance in lung cancer cells

[0100] (1) Transcriptome RNA sequencing analysis was performed using H446-S, H446-LR, and H446-HR ​​to screen genes associated with paclitaxel resistance in cell lines. Three replicates were performed for each cell.

[0101] RNA-seq test results are shown in Figure 3 A and Table 2. The results showed that the known paclitaxel resistance-inducing factors ABCB1, TMEM243, and ID1 were gradually upregulated in H446-LR and H446-HR ​​cells compared with H446-S.

[0102] Table 2

[0103]

[0104] Further RT-qPCR was used to detect the relative expression of ABCB1, TMEM243 and ID1 genes, indicating the reliability and sensitivity of RNA sequence analysis. Figure 3 Middle B.

[0105] (2) In addition to the above three up-regulated genes (i.e., ABCB1, TMEM243, and ID1), the inventors of the present invention also found that the expression of the A2M gene was negatively correlated with the drug resistance of lung cancer cells to paclitaxel in the sequencing results, which was further confirmed by the RT-qPCR test results ( Figure 3 Middle C).

[0106] (3) In order to further verify the negative correlation between A2M and paclitaxel resistance, the inventors of the present invention analyzed the relative expression levels of the A2M gene in A549 / TAX and HCC827 / TAX by RT-qPCR.

[0107] Test results see Figure 3 Middle D. The results showed that compared with the lung cancer cell line A549, the relative expression of A2M gene in A549 / TAX was significantly decreased; compared with the lung cancer cell line HCC827, the relative expression of A2M gene in HCC827 / TAX was significantly decreased.

[0108] (4) In addition, Oncomine (www.Oncomine.org) analysis of Shimokuni cell line transcriptome data grouped by docetaxel sensitivity also showed a negative correlation between A2M and docetaxel resistance ( Figure 3 Middle E).

[0109] The above results indicate that A2M is a marker for paclitaxel resistance in lung cancer cells.

[0110] 4. A2M downregulation is associated with lung cancer progression

[0111] The inventors of the present invention also evaluated the expression of A2M in lung cancer by analyzing the transcriptome data of lung cancer samples from the TCGA database and the proteome data from the CPTCA database using the UALCAN tool.

[0112] TCGA analysis see Figure 4 Figure A, A2M mRNA expression was significantly downregulated in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). CPTCA analysis was performed in Figure 4Figure B, A2M protein was significantly reduced in LUAD compared with normal samples. Further studies showed that in TCGA-LUAD samples, A2M mRNA expression was negatively correlated with advanced cancer stage and lymph node metastasis, while in CPTAC-LUAD samples, A2M protein levels were negatively correlated with higher cancer grade and stage ( Figure 4 C and D). All these suggest that A2M may be an inhibitory protein in lung cancer.

[0113] 5. Restoration of A2M reduces the malignancy of paclitaxel-resistant cell lines

[0114] (1) To determine the role of A2M in paclitaxel-resistant cell lines, the inventors of the present invention restored the expression of A2M in NCI-H446-HR ​​using a lentiviral infection method to obtain H446-HR-A2M and H446-HR-EV (as a control); and confirmed the restoration of A2M by RT-qPCR (see Figure 5 (A).

[0115] (2) RTCA analysis Figure 5 Middle B. The results showed that the proliferation ability of NCI-H446-HR ​​was significantly inhibited compared with NCI-H446-S, while the proliferation potential of H446-HR-A2M was further reduced.

[0116] (3) Colony formation assay Figure 5 Middle C. The results show that the clonogenic growth of H446-HR-A2M was significantly reduced compared with H446-HR-EV. A2M repair significantly reduced the clonogenic growth of H446-HR.

[0117] (4) Transwell analysis Figure 5 Middle D. The results showed that the migration and invasion abilities of H446-HR-A2M were significantly decreased compared with those of H446-HR-EV, that is, the restoration of A2M expression significantly inhibited the migration and invasion abilities of H446-HR.

[0118] (5) RT-qPCR detection of EMT marker levels. Figure 5 Middle E. The results showed that the expression of mesenchymal markers Snail, ZEB-1, and N-cad was significantly reduced, and the expression of epithelial marker E-cad was significantly increased in H446-HR-A2M compared with H446-HR-EV. Restoration of A2M promoted the inactivation of the EMT program in H446-HR.

[0119] The above results indicate that A2M restoration helps to reduce the malignancy of paclitaxel-resistant cell lines.

[0120] 6. Restoration of A2M downregulates ABCB1, TMEM243, and ID1 and resensitizes paclitaxel-resistant cells to paclitaxel

[0121] The inventors of the present invention explored whether A2M repair can help reverse paclitaxel resistance. The cell activity of H446-HR-A2M and H446-HR-EV was detected using culture medium containing different concentrations of paclitaxel. CCK8 analysis showed that ( Figure 6 Middle A), A2M restoration significantly reduced the survival ability of H446-HR ​​cells during paclitaxel treatment: under 0.05μg / ml paclitaxel treatment, 95.43% of H446-HR-EV cells survived, while only 66.44% of H446-HR-A2M cells survived (p<0.001); under 0.5μg / ml paclitaxel treatment, 81.08% of H446-HR-EV cells survived, while the survival rate of H446-HR-A2M cells was only 53.66% (p<0.001); under 1μg / ml paclitaxel treatment, 50.72% of H446-HR-EV cells survived, while the survival rate of H446-HR-A2M cells was 38.53% (p<0.001). It can be seen that restoring A2M can re-sensitize paclitaxel-resistant cells to paclitaxel.

[0122] The inventors of the present invention also studied the effect of A2M restoration on known paclitaxel resistance inducers, including ABCB1, TMEM243, and ID1, which are gradually upregulated during the development of paclitaxel resistance in NCI-H446 cells. Notably, A2M restoration in H446-HR ​​significantly reduced the expression of ABCB1, TMEM243, and ID1 ( Figure 6 Middle B), indicating that A2M may improve sensitivity to paclitaxel by inhibiting the expression of ABCB1, TMEM243 and ID1.

[0123] It will be apparent to those skilled in the art that the present invention may be implemented in a wide range of equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the disclosed scope of this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Use of A2M mRNA / protein or a substance that increases the expression of A2M mRNA / protein in the preparation of a product; the function of the product is at least one of A1), A2) and A3): A1) Reduce the drug resistance of paclitaxel-resistant cells; A2) Increase the sensitivity of paclitaxel-resistant cells to paclitaxel; A3) Reduce the malignancy of paclitaxel-resistant cancer cells; The cell or cancer cell is a lung cancer cell; The substance that increases the expression of A2M mRNA / protein is the lentivirus pLV[Exp]-Puro CMV>hA2M that expresses A2M.

2. The use according to claim 1, characterized in that: The reducing of the malignant manifestation of paclitaxel-resistant cancer cells is to inhibit the proliferation ability of paclitaxel-resistant cells.

3. The use according to claim 1, characterized in that: The method of reducing the malignant manifestation of paclitaxel-resistant cancer cells is to reduce the clonal growth of paclitaxel-resistant cells.

4. The use according to claim 1, characterized in that: The reducing the malignant manifestation of paclitaxel-resistant cancer cells is to inhibit the migration ability and / or invasion ability of paclitaxel-resistant cells.

5. Application of A2M mRNA / protein as a biomarker in the preparation of a product for predicting paclitaxel resistance in lung cancer cells.

6. The use according to claim 5, characterized in that: The judgment principle for predicting paclitaxel resistance is: if the A2M mRNA / protein expression level decreases, the paclitaxel resistance of lung cancer cells increases; if the A2M mRNA / protein expression level increases, the paclitaxel resistance of lung cancer cells decreases.

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