CN114381426A

CN114381426A - Natural killer cell and application

Info

Publication number: CN114381426A
Application number: CN202111532248.9A
Authority: CN
Inventors: 不公告发明人
Original assignee: Ningxia Houze Biomedical Technology Co ltd
Current assignee: Ningxia Houze Biomedical Technology Co ltd
Priority date: 2021-12-14
Filing date: 2021-12-14
Publication date: 2022-04-22

Abstract

The invention discloses a natural killer cell and application thereof, wherein the low-expression lncRNA SNHG1 in the natural killer cell. The invention discovers that the lncRNA SNHG1 gene low expression has an enhancement effect on killing power of natural killer cells, and the lncRNA SNHG1 low expression natural killer cells have a prospect of developing a cell-forming immunotherapy medicament.

Description

Natural killer cell and application

Technical Field

The invention belongs to the field of cell therapy, and particularly relates to a natural killer cell and application thereof.

Background

Biological treatment is a new mode for treating tumors after operations, radiotherapy and chemotherapy. At present, biotherapy includes tumor nonspecific small molecule compound immunotherapy, adoptive immunotherapy, tumor vaccine therapy, tumor monoclonal antibody immunotherapy, molecular targeted therapy, gene therapy, and the like. Wherein, the cellular immunotherapy refers to the tumor treatment by utilizing immune cells according to the immunological principle. This therapy is increasingly being valued. The cellular immunotherapy is mainly divided into two types according to action mechanisms, one type is that immune cells with anti-tumor activity are infused back to a patient and have in vitro activation and amplification to a certain number, and the anti-tumor effect is exerted in vivo, and the cellular immunotherapy belongs to passive cellular immunotherapy; another group is the return of cells, such as dendritic cells, to a patient that can elicit a specific anti-tumor immune response in the patient in vivo, and belongs to active cellular immunotherapy.

NK cells are one of the most important immune cells in passive cellular immunotherapy. NK cells are an important component of the innate immune system, are the first line of defense of the body against invading pathogens and tumor cells, and have a number of advantages. For example, the target cells killed by the NK cells do not need to be sensitized in advance, are not limited by Major Histocompatibility Complex (MHC), can directly and rapidly kill diseased cells, and perform immune clearance and immune monitoring functions.

The in vitro activation, expansion and killing activity improvement of NK cells is an important research field of NK cells for immunotherapy.

miR-718is an important gene, and researches show that the miR-718 plays multiple roles in the body. miR-718 regulates and controls PI3K/Akt signals by directly down-regulating phosphatase and tensin homolog (PTEN), so that phosphorylation of Akt is promoted, and generation of proinflammatory cytokines is reduced; macrophage tolerance to LPS can also be modulated (miR-718 stress secretion production through targeting phospholipid and tensin homolog. journal of Biological chemistry volume 292, Issue 14.2017). miR-718is also involved in the regulation of a variety of tumor cells (MiR-718media and its Expression interaction between genomic DNA SEMA3B-AS1 and PTEN to regulation the Expression of heterologous cells. physiological genomic genes Volume 51, Issue 10.2019; miR-718is included in the genomic of plasmid and plasmid Expression 214, Issue 11.2018; High miR-718 Expression vectors and molecular vector host (PTN) Expression and Expression of extracellular Expression of cancer cell and molecular Expression of cancer cell 2. Expression and Expression of cancer cell.

LncRNA SNHG1 is another gene that has been studied more extensively, involving immune, parkinson, tumor, and cardiovascular fields. Libaohua discovery that LncRNA SNHG1 can regulate memory CD8⁺T cell differentiation (research on the function and mechanism of long-chain non-coding RNA Snhg1 in memory CD8+ T cell differentiation, university of military and military medical science of people liberation military in China, doctor's academic paper). It has been found that LncRNA SNHG1 plays an important role in the development of Parkinson's disease (Long Noncoding RNA SNHG1 knock down animals Apoptosis, Oxidative Stress and Inflammation in Models of Parkinson's disease)Disease by Inhibiting the miR-125b-5p/MAPK1 Axis. LncRNA SNHG1 is involved in the development of various tumors (LncRNA SNHG1 in fuels cells promotion, migration, invasion, and apoptosis of non-small cells in cancer cells via the miR-361-3p/FRAT1 axis. Thorac cancer volume 11, Issue 2.2020; SNHG1 proteins monoclonal progress of diabetes by Targeting miR-140-5p and Regulating PI3K/AKT pathway. cancer Management and research volume 12,2020.). It has also been found that SNHG1 overexpression is a novel mechanism for protecting the function of HUVECs, and this mechanism may delay the progression of atherosclerosis (LncRNA SNHG1 regulation of vascular endothelial cell proliferation and angiogenesis via miR-196a. journal of Molecular HistologyVolume 51, Issue 2.2020.).

The reports of the miR-718 and LncRNA SNHG1 related to the activity of NK cells are less.

Disclosure of Invention

The invention aims to provide NK cells with high proliferation activity and/or high lethality and application thereof.

The above purpose of the invention is realized by the following technical scheme:

the technical scheme A is as follows:

an NK cell with high proliferation activity and high lethality, wherein miR-718is highly expressed in the NK cell.

The application of improving the expression level of miR-718 in improving the proliferation activity and lethality of NK cells.

The use of the above NK cells for cellular immunotherapy.

The technical scheme B is as follows:

a natural killer cell that underexpresses lncRNA SNHG 1.

The natural killer cells are applied to cellular immunotherapy.

Use of an LncRNA SNHG1 inhibitor for increasing natural killer cell lethality.

Has the advantages that:

the invention researches the enhancing effect of miR-718 gene high expression or lncRNA SNHG1 gene low expression on NK cell lethality from three different levels: on a molecular level, the expression levels of Perforin and Granzyme B in the NK cells of the miR-718 mix group are obviously higher than those of the Control-A group and the miR-NC group, and the expression levels of Perforin and Granzyme B in the NK cells of the si-SNHG1 group are obviously higher than those of the Control-B group and the si-NC group; on a cell level, the killing rate of the NK cells of the miR-718mimics group to the tumor cells is obviously higher than that of the Control-A group and the miR-NC group, and the killing rate of the NK cells of the si-SNHG1 group to the tumor cells is obviously higher than that of the Control-B group and the si-NC group; on the animal level, the tumors of the miR-718mimics group are obviously smaller than those of the Control-A group and the miR-NC group, and the tumors of the si-SNHG1 group are obviously smaller than those of the Control-B group and the si-NC group. The invention also discovers that the high expression of the miR-718 gene is beneficial to improving the proliferation activity of the NK cells.

Drawings

FIG. 1 shows the results of agarose gel electrophoresis; wherein, A shows that the expression level of miR-718 in miR-718mimics cells is obviously higher than that of Control-A cells and miR-NC cells, which indicates that the construction of miR-718 high-expression NK cells is successful; b shows that the expression level of lncRNA SNHG1 in the si-SNHG1 group cells is obviously lower than that of the Control-B group and the si-NC group, which indicates that the lncRNA SNHG1 low-expression NK cells are successfully constructed.

FIG. 2 shows the Western blotting result; wherein, A shows that the expression levels of Perforin and Granzyme B in NK cells of miR-718 mix group are obviously higher than those of Control-A group and miR-NC group, and B shows that the expression levels of Perforin and Granzyme B in NK cells of si-SNHG1 group are obviously higher than those of Control-B group and si-NC group.

FIG. 3 shows the killing rate of NK cells on tumor cells in each group (A) and the comparison of detached tumors (B).

Detailed Description

The following examples are given to illustrate the essence of the present invention, but not to limit the scope of the present invention.

1. Material

The human lymphocyte separation solution was purchased from Beijing Solaibao Tech Co. NK cell culture medium was purchased from CellGro, Germany. Recombinant human interleukin 2 was purchased from yaji biotechnology limited, shanghai. FITC-labeled CD3 mAb and APC-labeled CD56 mAb were purchased from Shanghai Komin Biotech, Inc. miR-718mimics, miR-NC, si-SNHG1 and si-NC were purchased from the Gima gene. Lipofectamine 2000, Trizol reagent was purchased from Invitrogen, USA. The RT-PCR kit is purchased from Beijing Solaibao science and technology Limited, and the primers are designed by the Jima gene. HepG2 cells, A549 cells were purchased from ATCC, and Balb/c nude mice were purchased from Kyowa Kavens laboratory animals Co., Ltd.

2. Method of producing a composite material

2.1NK cell culture

Collecting peripheral blood of healthy volunteers, diluting with 4 deg.C precooled PBS buffer solution in equal volume, slowly adding upper layer of lymphocyte separation solution, centrifuging at 650g and 4 deg.C for 20min, collecting white cell layer, separating to obtain mononuclear cells, washing with normal saline, and adjusting initial density to 2.0 × 10 with NK cell culture medium⁶/L。

Adding IL-2500U/mL on day 0, standing at 37 deg.C and 5% CO₂Culturing in incubator, adding 500U/mLIL-2 NK cell culture medium every 3 days, and adjusting cell density to 2.0 × 10⁶/mL。

Taking cells of 0d and cultured to 10d, detecting CD3 with FITC-CD3 and APC-CD56 monoclonal antibody according to conventional flow operation protocol^-CD56⁺Cell ratio. NK cells cultured to 10d were taken for subsequent experiments.

2.2NK cell transfection

2.2.1 construction of miR-718 high expression NK cell

Taking NK cells, adjusting cell density to 2.0 × 10⁶/mL, 5% CO at 37 ℃ in NK cell Medium₂Culturing in a constant temperature incubator. After 24h, the treatment is carried out according to groups. The Control group (Control-A) was not transfected, and the miR-718mimics group and the miR-NC group were transfected into NK cells using Lipofectamine 2000 transfection reagents according to the instructions, respectively. After 24h of transfection, the medium was removed by centrifugation and washed 3 times with 4 ℃ pre-chilled PBS.

2.2.2 construction of LncRNA SNHG1 Low expressing NK cells

Taking NK cells, adjusting cell density to 2.0 × 10⁶/mL, 5% CO at 37 ℃ in NK cell Medium₂Culturing in a constant temperature incubator. After 24h, according to the groupAnd (6) processing. The Control group (Control-B) was not transfected, and the si-SNHG1 group and the si-NC group were transfected with SNHG1 siRNA and si-NC, respectively, into NK cells using Lipofectamine 2000 transfection reagent according to the instructions. After 24h of transfection, the medium was removed by centrifugation and washed 3 times with 4 ℃ pre-chilled PBS.

2.3 verification of transfection Effect

2.3.1 RT-PCR validation of miR-718 high expression NK cells

Taking Control-A, miR-718 mix and miR-NC group cells, culturing with NK cell culture medium at 37 deg.C and 5% CO₂And (3) continuously culturing for 48h in the constant-temperature incubator, extracting total RNA in the cells by using a Trizol method, carrying out reverse transcription on the RNA to obtain cDNA, and configuring a corresponding system according to the PCR kit to carry out PCR amplification. The primers are shown in Table 1, and GAPDH is the internal control. The PCR amplification product is subjected to 1.2% agarose gel electrophoresis, staining by a dye solution and gel imaging analysis.

2.3.2 RT-PCR validation of LncRNA SNHG1 Low expressing NK cells

Taking Control-B, si-SNHG1 and si-NC group cells, culturing with NK cell culture medium at 37 deg.C and 5% CO₂And (3) continuously culturing for 48h in the constant-temperature incubator, extracting total RNA in the cells by using a Trizol method, carrying out reverse transcription on the RNA to obtain cDNA, and configuring a corresponding system according to the PCR kit to carry out PCR amplification. The primers are shown in Table 1, and GAPDH is the internal control. The PCR amplification product is subjected to 1.2% agarose gel electrophoresis, staining by a dye solution and gel imaging analysis.

TABLE 1RT-PCR primers

2.4 cell proliferation Activity assay

Taking Control-A, miR-718 mix, miR-NC group cells and Control-B, si-SNHG1, si-NC group cells, and culturing with NK cell culture medium at 37 deg.C and 5% CO₂Continuously culturing in constant temperature incubator for 48 hr, digesting to obtain the product with concentration of 2.5 × 10⁴The cell suspension was inoculated in a 96-well plate at 200. mu.L/well with 5% CO at 37 ℃%₂Culturing in a constant temperature incubator. After culturing in a 96-well plate for 48 hours, 20. mu.L of MTT solution with the concentration of 5mg/mL is added to each well, the mixture is incubated for 4 hours, and the supernatant is discardedThen, 200. mu.L of DMSO was added to each well, the crystals were sufficiently dissolved by shaking, the blank was set to zero, and OD value at 490nm wavelength was measured in each well of the transfection group and the control group, and the cell proliferation rate of the transfection group was calculated as follows based on the cell proliferation rate of the control group as 100%. 5 replicates per concentration. Wherein, the Control group of the miR-718mimics and miR-NC transfection group is calculated by Control-A, and the Control group of the si-SNHG1 and si-NC transfection group is calculated by Control-B. Cell proliferation rate (%) ═ OD transfection group/OD control group × 100%.

2.5 cell lethality assay

(1) At the molecular level

Taking Control-A, miR-718 mix, miR-NC group cells and Control-B, si-SNHG1, si-NC group cells, and culturing with NK cell culture medium at 37 deg.C and 5% CO₂Continuously culturing for 48h in a constant-temperature incubator, cracking RIPA lysate, measuring the protein concentration by using a BCA method, taking equal amount of total protein for each group, carrying out SDS-PAGE electrophoresis, transferring to a PVDF membrane, sealing by using 5% skimmed milk powder, adding Perforin, Granzyme B and beta-actin primary antibody, incubating at 4 ℃ overnight, washing the membrane by using TBS-T washing liquid, adding secondary antibody, incubating for 1h at room temperature, carrying out color development and exposure by using an ECL method, and taking a picture by using a chemiluminescence imaging system.

(2) Cellular level

Taking Control-A, miR-718 mix, miR-NC group cells and Control-B, si-SNHG1, si-NC group cells, and culturing with NK cell culture medium at 37 deg.C and 5% CO₂Continuously culturing for 48h in a constant-temperature incubator to serve as effector cells; taking HepG2 cells in logarithmic growth phase as target cells, digesting, washing, and preparing into 2 × 10 by DMEM medium containing 10% fetal calf serum⁶/mL、2×10⁵Each 100. mu.L of the cell suspension was inoculated into a 96-well plate at an effective target ratio of 10:1, and a target cell group and an effector cell group were separately prepared. Each group is provided with 3 more holes at 37 ℃ and 5% CO₂Culturing in a cell culture box under the condition; after 24h of culture, 20 mu L of CCK-8 reagent is added into each well, the culture is continued for 4h, the absorbance OD value of each well under the wavelength of 450nm is measured by a microplate reader, and the killing rate is calculated according to the following formula.

The killing rate was [1- (experimental OD value-effector OD value)/target OD value ] × l 00%.

(3) Animal level

Taking 4-week-old male Balb/c nude mice, adaptively feeding for 1 week, and then feeding according to the proportion of 2 × 10⁶100 mu L/100 log phase lung cancer A549 cells are implanted into nude mice by axillary subcutaneous injection until tumor body grows to 100mm³The two groups were divided into 6 groups (Control-A, miR-718mimics, miR-NC group, and Control-B, si-SNHG1, si-NC group) at random, each group containing 3 drugs, and each group was injected into tail vein containing 1 × 10⁷Saline per ml nk cells, 1 time daily, was injected continuously for 5 days, and after 9 days of the last injection, the neck was removed and killed, and the intact tumor was dissected and photographed.

2.6 data processing

Data were processed using SPSS17.0 statistical software and experimental results are presented as mean ± SD. Group comparisons using the t-test, p <0.05 indicated significant differences.

3. Results

3.1NK cell culture

0d、10d CD3^-CD56⁺The proportion of the phenotype NK cells is (3.18 +/-0.97)%, and (43.40 +/-3.72)%, and the proportion of the NK cells is obviously improved, which indicates that the NK cells are successfully cultured.

3.2NK cell transfection

The results of agarose gel electrophoresis are shown in FIG. 1. A in figure 1 shows that the expression level of miR-718 in miR-718mimics group cells is remarkably higher than that of Control-A group cells and miR-NC group cells, and the construction success of miR-718 high-expression NK cells is demonstrated. B in FIG. 1 shows that the expression level of lncRNA SNHG1 in si-SNHG1 group cells is significantly lower than that of Control-B group and si-NC group, which indicates that lncRNA SNHG1 low-expression NK cells are successfully constructed.

3.3 cell proliferative Activity

The OD values and cell proliferation rates of the respective groups are shown in Table 2. The proliferation activity of the NK cells of the miR-718mimics group is obviously higher than that of the Control-A group and the miR-NC group, and the proliferation activity of the NK cells of the si-SNHG1 group is not obviously different from that of the NK cells of the Control-B group and the si-NC group. The result shows that the high expression of the miR-718 gene is beneficial to improving the proliferation activity of the NK cell, and the low expression of the lncRNA SNHG1 gene has no obvious influence on the proliferation activity of the NK cell.

TABLE 2 OD values and cell proliferation rates of the respective groups

Group of	OD value	Proliferation Rate (%)	Group of	OD value	Proliferation Rate (%)
						Control-A	0.382±0.014	100.0	Control-B	0.389±0.015	100.0
miR-718mimics	0.606±0.017	158.6	si-SNHG1	0.394±0.011	101.3
						miR-NC	0.378±0.012	99.0	si-NC	0.397±0.014	102.1

3.4 cell killing power

(1) At the molecular level

Western blotting results are shown in A and B in FIG. 2. The expression levels of Perforin and Granzyme B in the NK cells of the miR-718mimics group are obviously higher than those of the Control-A group and the miR-NC group, and the expression levels of Perforin and Granzyme B in the NK cells of the si-SNHG1 group are obviously higher than those of the Control-B group and the si-NC group. Perforin and Granzyme B are two key proteins with a killing effect on NK cells, and the fact that the high expression of miR-718 gene or the low expression of lncRNA SNHG1 gene is beneficial to improving the killing power of the NK cells can be seen from a molecular level.

(2) Cellular level

The killing rate of each group of NK cells against tumor cells is shown in table 3 and a in fig. 3. Obviously, from the cellular level, the high expression of the miR-718 gene or the low expression of the lncRNA SNHG1 gene is beneficial to improving the lethality of NK cells.

TABLE 3 killing rate of tumor cells by NK cells of each group

Group of	Killing rate (%)	Group of	Killing rate (%)
				Control-A	35.2±3.9	Control-B	33.6±3.5
miR-718mimics	57.4±4.5	si-SNHG1	65.9±4.8
				miR-NC	34.8±4.1	si-NC	34.5±3.3

(3) Animal level

The comparison of the peeled tumors is shown in B in FIG. 3, the tumors of the miR-718mimics group are obviously smaller than those of the Control-A group and the miR-NC group, and the tumors of the si-SNHG1 group are obviously smaller than those of the Control-B group and the si-NC group, which indicates that the high expression of the miR-718 gene or the low expression of the lncRNA SNHG1 gene is beneficial to improving the killing power of NK cells according to the level of an animal.

The above-described embodiments are intended to be illustrative of the nature of the invention, but those skilled in the art will recognize that the scope of the invention is not limited to the specific embodiments.

Claims

1. A natural killer cell, comprising: the lncRNA SNHG1 is low expressed in the natural killer cells.

2. Use of the natural killer cell of claim 1 in cellular immunotherapy.

Use of an LncRNA SNHG1 inhibitor for increasing natural killer cell lethality.