CN106822873B - Application of mature form IL-37 in preparing medicine for inhibiting tumor metastasis - Google Patents

Abstract

The invention discloses application of mature IL-37 in preparing Rac1 activity inhibitor and in preparing medicine for inhibiting tumor metastasis. The invention discovers for the first time: the mature form of IL-37 can inhibit the migration of tumor cells by directly inhibiting the activity of the cytoskeletal regulatory protein Rac 1. The invention is proved by experiments that: the mature IL-37 inhibits the cell migration by directly binding Rac1 and further inhibiting the activity and the phosphorylation of downstream effector molecule PAK, and proves that the IL-37 is a novel endogenous Rac1 inhibiting molecule and has the potential of being used for preparing Rac1 activity inhibitors and/or preparing drugs for inhibiting tumor metastasis.

Description

Application of mature form IL-37 in preparing medicine for inhibiting tumor metastasis

Technical Field

The invention relates to the technical field of biomedicine, in particular to application of a mature form IL-37 in preparing a medicament for inhibiting tumor metastasis.

Background

IL-37 is a new anti-inflammatory cytokine of the IL-1 family, and human IL-37 forms 5 different subtypes after alternative splicing: IL-37a-IL-37 e. Since IL-37b contains 5 of the 6 exons, it encodes the largest spliceosome and is widely expressed in multiple organs and tissues in humans, including kidney, brain, heart, testis, bone marrow and peripheral blood mononuclear cells. Therefore, IL-37b is currently the most studied IL-37 subtype.

Current studies indicate that the primary role of IL-37 is to suppress excessive inflammatory responses through a negative feedback regulatory mechanism. Thus, it is involved in a variety of inflammatory diseases, such as: inflammatory skin diseases, inflammatory liver diseases, inflammatory intestinal diseases, autoimmune diseases, metabolic diseases, and the like. However, there have been few studies on IL-37 in tumor progression, particularly in inhibiting tumor metastasis.

In addition, IL-37 is a rare cytokine that can exert biological effects both intracellularly and extracellularly. Previous studies have shown that: IL-37 exerts an anti-inflammatory effect through two pathways, one being the IL-18Ra-IL-1R8(SIGIRR) receptor-mediated extracellular pathway: extracellular IL-37 binds IL-18Ra and utilizes IL-1R8 to activate multiple anti-inflammatory signaling pathways within the cell; the other is the intracellular pathway: intracellular IL-37 binds phosphorylated-Smad3 and promotes nuclear translocation of p-Smad3 which in turn inhibits the production of a variety of inflammatory cytokines. However, it is not clear whether IL-37 can also target other protein molecules within the cell.

Rac1 is a member of the Rho gtpase family and is also an important regulator of the cytoskeleton, and is involved in a variety of cellular processes including cytoskeletal remodeling and cell motility by activating target proteins PAK downstream of the signaling pathway. In addition, it has been shown that Rac1 signals may also promote tumorigenesis. Both overactivation and overexpression of Rac1 can cause invasive growth and other malignant behavior in a variety of tumors. Therefore, specific inhibitors of Rac1 would be promising drugs for tumor therapy.

However, the relation between IL-37 and Rac1 has not been reported in the literature so far; in particular, the study of the relationship between the mature form of IL-37 and Rac1 and tumor metastasis remains a technical gap.

Disclosure of Invention

In view of the above-mentioned deficiencies of the prior art, the present invention aims to study the relationship between IL-37 and the activity of the cytoskeletal regulatory protein Rac1, and further to discover the role of IL-37 in tumor migration and metastasis.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention proves the effect of the tumor cell in cell migration by over-expressing or interfering IL-37 in the tumor cell, and the result shows that the over-expressing IL-37 can obviously inhibit the migration capability of the tumor cell and the silencing expression of IL-37 can obviously improve the migration capability of the tumor cell. The invention also utilizes GST-pull down experiment to find that the silent expression of IL-37 enhances the GTPase activity of Rac1 in tumor cells, and the over-expression of IL-37 obviously inhibits the GTPase activity of Rac1 in tumor cells. The results show that IL-37 inhibits the activation of Rac1 and a downstream effector molecule PAK, the invention also verifies whether the influence of IL-37 on the migration capacity of tumor cells depends on a Rac1 pathway, and a Transwell experiment proves that the change of the migration capacity of the cells caused by IL-37 can be reversed by using a Rac1 specific inhibitor NSC23766 or silencing the expression of Rac 1. The above results indicate that the cytokine IL-37 exerts the effect of inhibiting tumor cell migration through the Rac1 signaling pathway

To further elucidate the exact relationship between IL-37 and Rac1, the present invention first observed the localization of both in tumor cells by immunofluorescence staining. We found that they had significant co-localization, especially at the edges of the cell membrane and cell processes. The invention also detects the combination of the two by co-IP experiment in tumor cells, and the result shows that IL-37 and Rac1 can combine with each other. The present invention also tested whether the binding between IL-37 and Rac1 was direct. Purified recombinant mature human IL-37 protein (amino acid sequence: 46-218) and GST-Rac1 recombinant protein were mixed extracellularly, and it was found that these two proteins could be directly bound to each other by GST-pull down experiment. These results demonstrate that the mature form of IL-37 inhibits its activity by binding directly to Rac 1.

In summary of the above findings, in a first aspect of the invention there is provided the use of mature form IL-37 in the preparation of an inhibitor of Rac1 activity.

The activity of Rac1 can be inhibited by over-expressing IL-37 or adding exogenous recombinant protein, therefore, the invention further provides the application of the promoter of IL-37 generation and/or action in preparing Rac1 activity inhibitor.

In the application, the nucleotide sequence of the IL-37 is shown as SEQ ID NO. 1. The method comprises the following specific steps:

vhtsp kvknlnpkkfsihdqdhkvl vldsgnliav pdknyirpei ffalasslssasaekgspil lgvskgefclycdkdkgqsh pslqlkkekl mklaaqkesa rrpfifyraq vgswnmlesaahpgwfictscncnepvgvt dkfenrkhie fsfqpvckae ms psevsd。(SEQ ID NO.1)

in a second aspect of the invention, there is provided the use of IL-37 in the manufacture of a medicament for inhibiting tumor metastasis.

The over-expression of IL-37 or the use of IL-37 recombinant protein or the enhancement of IL-37 function can inhibit the migration of tumor cells, therefore, the invention further provides the application of the promoter of IL-37 generation and/or function in the preparation of drugs for inhibiting tumor metastasis.

The promoter for the production and/or action of the IL-37 is selected from LPS, IL-1 α -18, TNF α -gamma or TGF β and the like, and the expression of the IL-37 can be obviously up-regulated after being stimulated by the promoter.

In the application, the nucleotide sequence of the IL-37 is shown as SEQ ID NO. 1.

In the application, the tumors such as liver cancer and lung cancer are all tumors.

The invention has the beneficial effects that:

the invention discovers that the cytokine IL-37 can inhibit the migration of tumor cells. In the aspect of action mechanism, the invention firstly discovers that the cytokine IL-37 can inhibit the migration of tumor cells by directly binding with a cytoskeleton regulatory protein Rac1 so as to inhibit the activity of the cytokine and the phosphorylation of a downstream effector molecule PAK. The invention further verifies that the cytokine IL-37 depends on a Rac1 signal channel to exert an inhibiting effect on the migration of tumor cells by using a specific inhibitor NSC23766 of Rac1 or silencing expression of Rac 1. The results prove that the cytokine IL-37 is a novel Rac1 inhibiting molecule and has the potential of being used for preparing Rac1 activity inhibitors and/or preparing medicaments for inhibiting tumor metastasis.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application.

FIG. 1: intracellular IL-37 negatively regulates the migratory capacity of tumor cells. In the figure: A. b represents transwell experiment to detect the influence of over-expressing IL-37 on the migration capacity of BEL-7402 and A549 cells, and Mock in A represents an empty vector which can be used as a control; C. d represents the transwell experiment to examine the effect of interfering IL-37 on the migratory capacity of BEL-7402 and HepG2 cells, and the results are expressed as the mean ± SD of three independent experiments,. P < 0.01; p < 0.001.

FIG. 2: intracellular IL-37 inhibits the activation of Rac 1. In the figure, A represents BEL-7402 and HepG2 cell transfection control siRNA and IL-37siRNA, B represents A549 cell infection control lentivirus LV-NC and experimental group lentivirus LV-IL-37B, cell lysate is used for PAK-GST Pull-down experiment, and western blot detects activated Rac1 and total Rac1 in lysate Pull down; c, D represents western blot detection of expression of phospho (p) -PAK and total PAK; experiments with similar results were repeated at least 3 times.

FIG. 3: the cytokine IL-37 inhibits the migration of tumor cells in a Rac 1-dependent manner. A and B respectively show that BEL-7402 and HepG2 cells are pretreated by NSC23766 or interfere with the expression of IL-37, and transwell assay detects the change of the migration capability of the cells. C and D represent that migration experiments were carried out after co-transfection of sictrl or siIL-37(shNC or shIL-37) and sictrl or siRac1 with BEL-7402 and HepG2, respectively. Data are shown as representative of 3 independent experiments. P < 0.05; p < 0.01; p < 0.001.

FIG. 4: cytokine IL-37 binds directly to Rac1 and inhibits its activation. In the figure, A shows that IL-37 and Rac1 are co-localized in cell membranes and leading edge, HepG2 and BEL-7402 are fixed, then the appointed protein is stained by specific antibodies and analyzed by a confocal microscope photograph, and DAPI stained nuclei are shown as blue; b shows that THP-1 is differentiated by PMA, stimulated by LPS and PDGF for 15min, stained by specific antibody of the specified protein and observed under a confocal microscope, and DAPI stained nuclei are blue; c represents that IL-37 and Rac1 form a complex, a coprecipitation experiment is carried out on total BEL-7402 cell lysate by using an anti-Rac 1 mouse monoclonal antibody and a control mouse IgG antibody, and the expressions of IL-37 and Rac1 in a coprecipitation and cell lysate are detected by using western blot; d represents that THP-1 is treated like B, then lysed cells are subjected to co-immunoprecipitation by using a Rac1 mouse monoclonal antibody and an isotype control IgG antibody, precipitates and cell lysates are subjected to western blot, the expression of IL-37 and Rac1 is detected, and the experiment with similar results is repeated for at least 3 times; e shows GST-tagged Rac1 (0.6. mu.g) bound beads were incubated with rhIL-37 (0.6. mu.g), and samples eluted from the washed beads were immunoblotted with GST and IL-37 specific antibodies.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

As described in the background, the relationship between the cytokine IL-37 and Rac1, especially the relationship between the cytokine IL-37 and Rac1 and tumor migration has not been reported in the literature. Therefore, the present application has conducted intensive studies on this, specifically as follows:

1. effect of the cytokine IL-37 on tumor cell migration

The present invention demonstrates its role in cell migration by overexpressing or interfering with IL-37 in tumor cells. Using transwell experiments, we found that over-expression of IL-37 significantly inhibited the migratory capacity of tumor cells such as BEL-7402 and A549 cells (FIGS. 1A and B). Consistent with this result, migration of tumor cells such as BEL-7402 and HepG2 cells was significantly enhanced after interference of IL-37 with siRNA or shRNA (FIGS. 1C and D).

2. Effect of the cytokine IL-37 on Rac1 Activity

The invention further studies the action mechanism of IL-37 inhibiting the migration of tumor cells, and Rac1 is used as a regulatory protein of cytoskeleton, and can regulate various cell functions, especially cell movement. Therefore, we guess whether IL-37 could inhibit tumor cell migration by affecting the Rac1 signaling pathway. We take the following experiments to demonstrate our guess. First, using GST-pull down experiments, we found that silencing expression of IL-37 enhanced the GTPase activity of Rac1 in tumor cells such as BEL-7402 and HepG2 cells, while over-expression of IL-37 reduced the GTPase activity of Rac1 in tumor cells such as A549 cells (FIG. 2A, B).

Since numerous studies demonstrated that PAK is the major effector molecule downstream of Rac1, we further examined the effect of IL-37 on PAK activation. As shown in FIG. 2C, D, interference with IL-37 significantly enhanced PAK phosphorylation, while overexpression of IL-37 significantly attenuated p-PAK expression. These results indicate that IL-37 inhibits the activation of Rac1 and its downstream effector molecule, PAK.

Study of the action relationship between IL-37 and Rac1

To further confirm whether IL-37 inhibits tumor cell migration through the Rac1 signaling pathway, we manipulated the activity of Rac1 with sirnas specific for Rac1 inhibitors NSC23766 and Rac 1. The results show that 50. mu.M NSC23766 in tumor cells such as BEL-7402 and HepG2 cells reversed the change in cell migration ability due to interference with IL-37 (FIG. 3A, B). Consistent with this result, silencing expression of Rac1 with siRNA almost completely abolished the inhibitory effect of IL-37 on tumor cell migration (fig. 3C, D). These results demonstrate that cytokine IL-37 inhibits tumor cell migration by inhibiting the Rac1 pathway.

To elucidate the exact relationship between IL-37 and Rac1, the present inventors first observed the localization of both in BEL-7402 and HepG2 cells by immunofluorescence staining. We found that they had significant co-localization, especially at the edges of the cell membrane and cell processes (fig. 4A). To demonstrate whether this phenomenon is general, the present invention repeated the same experiment in the monocytic cell line THP-1 of acute leukemia origin, with similar results (fig. 4B).

Next, to confirm whether endogenous IL-37 binds to endogenous Rac 1. The BEL-7402 cell lysate is used for immunoprecipitating Rac1 binding protein by using an anti-Rac 1 monoclonal antibody. When blotted with anti-IL-37 antibody, an IL-37 band could be detected in the pellet (FIG. 4C), indicating that IL-37 and Rac1 proteins are structurally associated in tumor cells. In addition, the same results were obtained in THP-1 cells (FIG. 4D).

In addition, we also tested whether the binding between IL-37 and Rac1 was direct. Purified recombinant mature human IL-37 protein (amino acid sequence: 46-218) and GST-Rac1 were mixed extracellularly and binding of both was detected using co-IP and GST-pull down experiments. We found that these two proteins bind directly to each other (fig. 4E). These results demonstrate that: the mature form of IL-37 inhibits its activity by binding directly to Rac 1.

In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.

The test materials referred to in the examples are as follows:

1. cell lines

(1) Human hepatoma cell lines: HepG2 and BEL-7402 and the human monocyte THP-1 cell line were cultured in 1640 medium (Gibco, Invitrogen, Clasbad, CA, USA) containing 10% heat-inactivated Fetal Bovine Serum (FBS) (Gibco).

(2) Lung cancer cell line a 549: cultured in Ham's F12K medium containing 2mM L-glutamine, 1.5g/L sodium bicarbonate, 10% FBS (Gibco).

The above liver cancer treating medicineThe cell line and the lung cancer cell line both contain 5% CO₂And culturing in a 37 ℃ humid incubator.

2. Plasmids, shRNA, siRNA, lentiviruses and transfections

The N-terminal Myc-tagged pCMV vector, the C-terminal 3 Flag-tagged pcDNA3.1hisC plasmid and the N-terminal Myc-tagged pRK5 plasmid are conventional plasmids in the prior art and can be obtained by commercial purchase.

IL-37shRNA, target sequence CCTCGCTAATTTGAACTAATT, negative control sequence GTTCTCCGAACGTGTCACGT.

The plasmid vector for shRNA was hGPU 6/GFP/Neo.

IL-37 specific siRNA (sense 5'-GGGUGACAGAUAAAUUUGATT-3'; 5'-UCAAAUUUAUCUGUCACCCTT-3', GenePharma).

Rac1(sense 5 '-GCAAACAGUGUGUUCUUDTdT-3' and 5 '-UAAGAACACACUCUGUUUGCTdT-3', GenePharma). Negative control GV358 lentiviral vector (Genechem, Co, Ltd, Shanghai, China).

Full-length human IL-37 and Rac1 were obtained from cDNA clones by PCR and cloned into N-terminal Myc-tagged pCMV vector or C-terminal 3 Flag-tagged pcDNA3.1hisC plasmid and N-terminal Myc-tagged pRK5 plasmid. All IL-37 and Rac1 mutants were obtained from IL-37 and Rac1 plasmids using the KOD-Plus-Mutagenesis kit (Toyobo).

Tumor cells were transfected with plasmid or s iRNA using Lipofectamine2000(Invitrogen, Carlsbad, Calif., USA) according to the instructions. For lentiviral infection, A549 cells were plated in 24-well plates at 12h prior to transfection, 1.5 x 10 x 4 cells per well. The infection process was performed according to the lentivirus infection manual (MOI ═ 20, polybrene (5 μ g/ml). 12h after transfection, the lentivirus-containing medium was replaced with fresh F12K medium containing 10% FBS serum, and at 72h after transfection, the cell infection efficiency was observed under a fluorescent microscope (Olymp us) and expression was identified with western blot.

Other test materials not described in detail are conventional in the art and are commercially available.

Example 1:

western blot experiment

Cells were lysed with RIPA cell lysate plus a mixture of protease inhibitors (Sigma, USA). After high speed centrifugation at 4 ℃ the supernatant was obtained and the protein concentration was measured with BCA protein assay kit (Thermo Scientific) according to the instructions. The same amount of protein was loaded on 10% SDS-PAGE and then transferred to PVDF membrane (Millipore, Billerica, MA, USA). After blocking for 2-3h with 5% BSA, the membrane was incubated overnight at 4 ℃ in diluted primary antibody and then incubated for 1h at room temperature in HRP-conjugated secondary antibody (1: 2000; Jackson Immuno Research, West Grove, Pa.). After washing, eECLWestern Blot Kit (CWBIO). The color developing solution detects the protein band and is observed by an ECL gel imaging system (Millipore).

Example 2: transwell experiment

Cell migration ability was measured by transwells chamber with 8 μm pore size (Costar, Acton, USA). cells were collected (5 × 10)⁴-1×10⁵) Then resuspended in serum-free medium before seeding into the transwell upper chamber. RPMI1640 medium containing 10% fetal bovine serum was added to the lower chamber. In the presence of 5% CO₂After incubation for 10-15h in a 37 ℃ humidified incubator, the cells in the upper chamber were wiped off with a cotton swab, fixed to migrate to the lower chamber with methanol for 10min, stained with crystal violet for 20min at room temperature and counted under an optical microscope.

Example 3: PBD pull down experiment

PBD pull down experiments the expression of GTP-Rac1 was tested according to the instructions. The cells were lysed with lysis buffer and 20. mu.g of p21-activated kinase (PAK) -GST protein beads (Cytoskeleton) were added to the same amount of cell lysis protein and incubated for 1h on a 4 ℃ rotating disk. The GTP-Rac1 was detected by western blotting. The amount of GTP-Rac1 in each individual sample was normalized to the total amount of Rac1 in the cell lysate.

Example 4: GST-pull down experiment

RhIL-37 (0.6. mu.g) and GST-Rac1 (0.6. mu.g) proteins were mixed in 400. mu.l of IP buffer and incubated at 4 ℃ for 3-4h on a rotating disk, followed by addition of 20. mu.l of GST binding Glutathione-agarose (Santa Cruz) and incubation at 4 ℃ overnight on the rotating disk. Samples were eluted from the washed beads and analyzed by immunoblotting with GST and IL-37 specific antibodies.

Example 5: immunofluorescence staining localization experiment

Cells on the slide were fixed with 4% paraformaldehyde for 15min, and then cell membranes were permeabilized with 0.1% Triton X-100. Blocking with 2% BSA for 30min at room temperature to block non-specific binding, followed by incubation with PBS diluted primary antibody in a refrigerator at 4 ℃ overnight: anti-IL-37 antibody (1:400), anti-Rac 1 antibody (1: 200). The group without the primary antibody was the negative control group. After washing with PBS, FITC-conjugated coat-anti-rabbitIgG and TRITC-conjugated coat-anti-mouse IgG (Abcam, Cambridge, UK) diluted with fluorescent secondary antibody were incubated for 1h at 37 ℃ in the absence of light. Actin filaments were stained with TRITC-conjugated phallloidin (Sigma-Aldrich, Louis, USA) for 1 h. Nuclei were stained with 4', 6-diaminodino-2-phenylindole (DAPI) (P36941, life technologies corporation, USA) for 5 min. And (5) washing the PBS and then sealing the anti-fluorescence quenching sealing liquid. Confocal microscopy (Carl Zeiss, LSM780, Oberkochen, Germany) and photographs were taken.

Example 6: co-immunoprecipitation (co-IP) experiments

After high speed centrifugation at 4 ℃ the supernatant was taken and added with 1. mu.l of anti-Rac 1 mouse monoclonal antibody or IgG isotype control antibody, shaken on a 4 ℃ rotating disk for 1h, followed by addition of 40. mu.l of resuspended Protein A/G Plus-agarose (Santa Cruz) and incubated overnight on a 4 ℃ rotating disk in some experiments, the purchased recombinant Protein was used in co-IP experiments, rhIL-37 (0.6. mu.g) (R & D sys) and Rac1 (0.6. mu.g) proteins were mixed in 400. mu. lIP buffer, incubated at 4 ℃ for 3-4h, then 1 or IgG isotype control antibody was added, after incubation at 4 ℃ for 1h, 40. mu.l of resuspended Protein A/G Plus-agarose 4 ℃ was added and the beads were centrifuged (1,000G,5min,4 ℃ and then incubated with 5. mu.l of buffer solution at 25. mu.m.n, and the expression was detected by addition of the supernatant with 5. mu.l of buffer solution of 5. mu.m, 5. mu.n.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

SEQUENCE LISTING

<110> Shandong university

Application of mature form IL-37 in preparation of drugs for inhibiting tumor metastasis

<130>2017

<160>1

<170>PatentIn version 3.5

<210>1

<211>173

<212>PRT

<213> mature form of IL-37

<400>1

Val His Thr Ser Pro Lys Val Lys Asn Leu Asn Pro Lys Lys Phe Ser

1 5 10 15

Ile His Asp Gln Asp His Lys Val Leu Val Leu Asp Ser Gly Asn Leu

20 25 30

Ile Ala Val Pro Asp Lys Asn Tyr Ile Arg Pro Glu Ile Phe Phe Ala

35 40 45

Leu Ala Ser Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly Ser Pro Ile

50 55 60

Leu Leu Gly Val Ser Lys Gly Glu Phe Cys Leu Tyr Cys Asp Lys Asp

65 70 75 80

Lys Gly Gln Ser His Pro Ser Leu Gln Leu Lys Lys Glu Lys Leu Met

85 90 95

Lys Leu Ala Ala Gln Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe Tyr

100 105 110

Arg Ala Gln Val Gly Ser Trp Asn Met Leu Glu Ser Ala Ala His Pro

115 120 125

Gly Trp Phe Ile Cys Thr Ser Cys Asn Cys Asn Glu Pro Val Gly Val

130 135 140

Thr Asp Lys Phe Glu Asn Arg Lys His Ile Glu Phe Ser Phe Gln Pro

145 150 155 160

Val Cys Lys Ala Glu Met Ser Pro Ser Glu Val Ser Asp

165 170

Claims (1)

1. The application of IL-37 in the preparation of an inhibitor of Rac1 activity is characterized in that the amino acid sequence of the IL-37 is shown as SEQ ID NO. 1.

Images