Overexpressed YTHDC2 macrophage and pharmaceutical application thereof

Abstract

The invention discloses an overexpression YTHDC2 macrophage and a pharmaceutical application of the overexpression YTHDC2 macrophage. The overexpressed YTHDC2 macrophage disclosed by the invention is obtained by modifying and reprogramming a YTHDC2 gene. The medicine application comprises an application of the overexpressed YTHDC2 macrophage in preparation of a medicine for killing breast cancer cells; the invention relates to application of overexpressed YTHDC2 macrophages in preparation of breast cancer cell targeting drugs. The invention relates to application of overexpressed YTHDC2 macrophages as a drug carrier for targeting breast cancer cells. Compared with the prior art, the expression quantity of the YTHDC2 gene of the overexpressed YTHDC2 macrophage is 5 times higher than that of the common macrophage, and the overexpressed YTHDC2 macrophage shows an M1 type anti-tumor phenotype. The obvious effects of targeting and killing breast cancer cells are realized.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceuticals, and in particular to a YTHDC2-overexpressing macrophage and a pharmaceutical use thereof. Background Art

[0002] Macrophages (TAMs) are an important component of the tumor microenvironment, playing a role in angiogenesis, extracellular matrix remodeling, tumor cell proliferation and immunosuppression, as well as resistance to chemotherapy. When properly activated, macrophages can mediate phagocytosis of tumor cells and cytotoxic tumor cell killing, and involve effective bidirectional interactions with components of the innate and adaptive immune systems. An increasing number of studies have shown that the tumor microenvironment reprograms the gene expression of TAMs to reshape and maintain immunosuppressive functions. TAMs typically exhibit a range of activation states. Typically, they deviate from the "classically" activated anti-tumor phenotype (called M1) and tend to the "alternative" activated tumor phenotype (M2). However, like macrophages in many other tissues, TAMs show significant functional plasticity and often express markers of two activation states.

[0003] As an mRNA modification, N6-methyladenosine (m6A) has been increasingly shown to be closely related to the occurrence and development of tumors. RNA methylation is an important regulatory factor in the occurrence and development of tumors, and its expression level often directly determines the pathological process of tumors. In different types of tumors, RNA methylation modification status can be both a carcinogenic factor and a tumor suppressor factor. There are still many unknown gaps in the role of m6A-modified macrophages in various specific tumors. Summary of the invention

[0004] The present invention provides an m6A-modified reprogrammed YTHDC2-overexpressing macrophage, and the preparation method thereof is as follows:

[0005] According to the CDS sequence of the YTHDC2 gene in NCBI, a viral packaging plasmid was used to transfect THP.1 cells; 1×105THP.1 was seeded in each well of a 6-well plate, 3μL of virus solution and 40μL of HiTransGA infection enhancement solution were added to each well, the cell solution was centrifuged for 1 hour, and the cells were placed in an incubator for further incubation; 24 hours after infection, the cell solution was centrifuged for 2 minutes to collect the cells, and they were cultured with complete culture medium for 48 hours, and 2μg / mL of puromycin was added to screen the surviving cells to obtain the target cells.

[0006] To obtain the best transfection effect, the virus used in the present invention is LV-YTHDC2 (63966-1) with a concentration of 2E+9TU / ml.

[0007] The YTHDC2 gene expression level of the reprogrammed cells obtained by this method is 5 times higher than that of ordinary macrophages, showing an M1 anti-tumor phenotype.

[0008] Experiments have shown that the YTHDC2-overexpressing macrophages of the present invention have obvious targeting and killing effects on breast cancer cells. Therefore, the present invention further discloses several pharmaceutical uses of the YTHDC2-overexpressing macrophages:

[0009] 1. Use of macrophages overexpressing YTHDC2 in the preparation of drugs that kill breast cancer cells.

[0010] 2. Use of YTHDC2-overexpressing macrophages in the preparation of breast cancer cell-targeted drugs.

[0011] 3. Use of YTHDC2-overexpressing macrophages as drug carriers targeting breast cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is the plasmid map for overexpressing YTHDC2;

[0013] Figure 2 Flow cytometry was used to detect the fluorescence expression in transfected cells;

[0014] Figure 3 is qPCR to test the overexpression of YTHDC2 gene in THP.1;

[0015] Figure 4 Flow cytometry was used to analyze the effect of overexpression of YTHDC2 on the polarization direction of THP.1 cells;

[0016] Figure 5 This is the flow cytometry gate diagram of THP.1 cell polarization;

[0017] Figure 6 This is a fluorescent image of macrophages after co-culture with tumor cells;

[0018] Figure 7 This is a diagram showing the flow cytometry analysis of YTHDC2-THP.1 and NC THP.1 phagocytosis of tumor cells after co-culture of macrophages with tumor cells. DETAILED DESCRIPTION

[0019] The present invention will be further described below in conjunction with specific embodiments.

[0020] 1. Construction of YTHDC2-overexpressing macrophages and expression level analysis

[0021] 1) Construction of macrophages overexpressing YTHDC2

[0022] According to the CDS sequence of the YTHDC2 gene in NCBI (as shown in sequence number (ID): 1), the virus provided by GeneCare (virus type LV-YTHDC2 (63966-1), concentration: 2E+9TU / ml) was used to package the plasmid (plasmid map see Figure 1 ) to transfect THP.1 cells. 1×105THP.1 were seeded in each well of a 6-well plate. After adding 3μL of virus solution and 40μL of HiTransGA infection enhancement solution to each well, the cells were centrifuged at 200g for 1 hour using a flat-angle centrifuge and placed in an incubator for continued incubation. 24 hours after infection, the cells were collected by centrifugation at 200g for 2 minutes. The supernatant was removed and replaced with complete culture medium for further culture. After 48 hours, the transfection efficiency was observed using a fluorescence microscope. 2μg / mL puromycin was added for screening, and surviving cells were continued to be cultured. The successfully transfected cell line is THP.1 cells overexpressing YTHDC2, maintained in RPMI-1640 medium with 10% FBS.

[0023] The control plasmid without the target gene was transfected in the same manner as above to construct THP.1 cells of the negative control group.

[0024] The above cells were incubated at 37°C in a humidified atmosphere containing 5% CO2.

[0025] 2) Detection of plasmid fluorescence expression in cells

[0026] After culturing YTHDC2-THP.1 infected with lentivirus, some cells were taken out, centrifuged to remove the supernatant, and washed with PBA to prepare a single cell suspension. The GFP positive rate was detected by flow cytometry. The results are as follows: Figure 2 As shown, macrophages successfully transfected with the plasmid expressed strong GFP.

[0027] 3) qPCR detection of YTHDC2 gene expression in cells

[0028] Extraction of cellular RNA

[0029] (1) Pour off the culture medium and wash twice with 1× PBS; digest the cells with trypsin, collect the cells by centrifugation at 10,000 rpm / 4°C for 5 min, remove the supernatant, and store the cell pellet in a -80°C refrigerator for later use.

[0030] (2) Add 1 mL of RNAiso Plus to each tube of cells (approximately 10 million cells);

[0031] (3) Resuspend the cells with a pipette and then vortex vigorously until there is no obvious precipitation in the lysate;

[0032] (4) Leave at room temperature for 5 min;

[0033] (5) Add 200 μL of chloroform (1 / 5 of the volume of RNAiso Plus) to the homogenate lysate obtained in the above step, cover the centrifuge tube tightly, and Vortex mix until the solution is emulsified and becomes milky white;

[0034] (6) Leave at room temperature for 5 minutes;

[0035] (7) Centrifuge at 12000 rpm and 4°C for 15 min. (Carefully remove the centrifuge tube from the centrifuge. The homogenate is now divided into three layers: a colorless supernatant (containing RNA), a white protein layer in the middle (containing most of the DNA), and a colored lower organic phase (the bright red organic layer contains proteins, polysaccharides, fatty acids, cell debris, and a small amount of DNA).

[0036] (8) Aspirate the supernatant (500 μL) and transfer it to another new centrifuge tube (do not aspirate the white middle layer to avoid contamination);

[0037] (9) Add 1 ml of isopropanol to the supernatant (the same volume as that of RNAiso Plus added in step 2), mix thoroughly by inverting the tube, and let stand at room temperature for 10 min.

[0038] (10) Centrifuge at 12000g, 4°C for 10 min. A white RNA precipitate can be observed at the bottom of the test tube.

[0039] (11) Carefully discard the supernatant and do not touch the precipitate;

[0040] (12) Add 75% ethanol (1 mL) equal to the amount of RNAiso Plus, gently invert the tube to wash the wall, centrifuge at 7500 g at 4°C for 5 min, and carefully discard the supernatant;

[0041] (13) Repeat step (8) 2 more times;

[0042] (14) After washing, remove the supernatant, centrifuge briefly, and then carefully aspirate with a micropipette and air dry in a clean bench;

[0043] (15) Open the centrifuge tube cap and dry the precipitate on a clean bench for about 10 min;

[0044] (16) After the precipitate is dried, add 20 μ L of DEPC water-dissolved precipitate;

[0045] (17) Take 2 μ L RNA solution was dissolved in 18 μ Add 1 L of TE Buffer, pipette evenly, and measure the absorbance of RNA.

[0046] Reverse Transcript Experiment

[0047] (1) After the extracted total RNA sample is quantified by NanoDrop to determine the concentration and RNA purity, the components in Table 1 are added to a 200 μL PCR tube on ice and mixed (10 μL reaction system):

[0048] Table 1

[0049] Reagents Addition volume (μL) Final concentration 5XRTMaster Mix 2 1X RNA 100ng (up to 1μg) DEPC water Total volume 10μL

[0050] (2) After sufficient mixing, immediately place in a PCR instrument for reverse transcription reaction.

[0051] Set temperature and duration: 37℃...15min; 85℃...5sec; 4℃...∞ after reverse transcription

[0052] The cDNA can be quantified again by spectrophotometer (NanoDrop) to determine the concentration and purity of the cDNA.

[0053] Quantitative Real-time PCR (qPCR) experiment

[0054] (1) Prepare the sample according to Table 2:

[0055] Table 2

[0056]

[0057] A. Primer mixture: Mix PCR forward primer and PCR reverse primer in equal volumes. B. Mix 2x SYBR GreenMaster + primer mixture + ddH2O, and finally add cDNA;

[0058] (2) After adding the sample, place the tubes in an eight-tube centrifuge and centrifuge them, then cover the tubes;

[0059] (3) The procedure is as shown in Table 3:

[0060] Table 3

[0061]

[0062] The results showed that compared with the control group, the gene expression level of macrophages transfected with YTHDC2 overexpressing plasmid was 5 times higher (results as shown in Figure 3 ), thus obtaining macrophages overexpressing YTHDC2.

[0063] 2. Detection of macrophage polarization tendency after reprogramming

[0064] THP.1, NC-THP.1 and overexpressed YTHDC2-THP.1 cells were seeded into 6-well plates (1×106 / well) and treated with 100 ng / ml of 2-acetoxy-1-methoxypropane (PMA) for 48 hours to produce M0 macrophages. The cells were collected into EP tubes using a cell scraper, washed with PBA, and after adding the required fluorescent dye-coupled antibody combination (anti-tumor marker flow dyes including BV785-Cd11b, APC-CD80 and PE-Cy7-CD206), the THP.1 cells to be tested were incubated on ice in the dark for 30 minutes, the antibodies were washed off, and resuspended in 300 μL PBA. Thereafter, flow cytometry analysis was performed on each group of M0 THP.1 cells. The data were analyzed using FlowJo software. The results are shown in Figure 4 and Figure 5 It can be seen that macrophages overexpressing YTHDC2 are more inclined to M1 phase polarization than the control group, and M1 phase macrophages have the ability to inhibit tumor growth.

[0065] 3. Overexpression of YTHDC2 promotes the enhancement of macrophage phagocytosis of tumor cells

[0066] (I) In vitro co-culture experiment of YTHDC2 and breast cancer cell line organoids

[0067] 1×104 breast cancer cells were added to a 96-well low-adhesion U-shaped cell culture plate for 3D suspension culture for 72 hours to allow more cells to aggregate into spheroids. On the third day, M0-stage NC-THP.1 and overexpressed YTHDC2-THP.1 were added to the culture plate at a ratio of 1:2 between tumor cells and macrophages. After 3 days of co-culture, white light and fluorescence images were captured using an inverted fluorescence microscope. The fluorescence images showed that compared with NC-THP.1, YTHDC2-THP.1 was more abundant in the tumor spheroids. Figure 6 This indicates that the reprogrammed macrophages can better infiltrate the internal structure of the tumor spheroids and have better phagocytic and killing effects.

[0068] (II) In vitro killing experiment of macrophages co-cultured with breast cancer cell line organoids

[0069] After microscopic observation, carefully aspirate the organoids and macrophages and digest them with trypsin digestion solution at 37°C for 3 minutes. During this period, use a pipette to continuously blow the organoids to digest them into single cell suspensions as much as possible. Terminate the culture with complete medium, wash with PBA, centrifuge at 2000rpm for 5 minutes, and add CD11b antibody. Place the mixture on ice and incubate in the dark for 30 minutes. Then wash again with PBA to remove the antibody. Add 200μL PBS and detect fluorescence by flow cytometry. Use FlowJo software to analyze the test results. The results are as follows Figure 7 As shown, the number of tumor cells in the YTHDC2 overexpressing macrophage group was reduced more, suggesting that overexpression of the YTHDC2 gene increased the ability of macrophages to kill tumor cells.

Claims

1. A macrophage overexpressing YTHDC2, characterized in that: The cells were prepared as follows: According to the CDS sequence of the YTHDC2 gene in NCBI, a viral packaging plasmid was used to transfect THP.1 cells; 1×105THP.1 was seeded in each well of a 6-well plate, 3μL of virus solution and 40μL of HiTransGA infection enhancement solution were added to each well, the cell solution was centrifuged for 1 hour, and the cells were placed in an incubator for further incubation; 24 hours after infection, the cell solution was centrifuged for 2 minutes to collect the cells, and they were cultured with complete culture medium for 48 hours, and 2μg / mL of puromycin was added to screen the surviving cells to obtain the target cells.

2. The YTHDC2-overexpressing macrophage according to claim 1, characterized in that The virus used in the preparation method of the cells is LV-YTHDC2 (63966-1) with a concentration of 2E+9TU / ml.

3. Use of the YTHDC2-overexpressing macrophages according to claim 1 in preparing a drug for killing breast cancer cells.

4. The use according to claim 2, characterized in that The use includes the use of YTHDC2-overexpressing macrophages in the preparation of breast cancer cell targeting drugs.

5. The use according to claim 3, characterized in that The use includes the use of the YTHDC2-overexpressing macrophages as drug carriers targeting breast cancer cells.

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