CN115094134A

CN115094134A - Application of PCSK9 in macrophage M2 type polarization and related diseases thereof

Info

Publication number: CN115094134A
Application number: CN202210385918.7A
Authority: CN
Inventors: 王潞; 丁珊珊; 孟文; 祝捷; 王功富; 苏广珠
Original assignee: JINAN CENTER HOSPITAL
Current assignee: JINAN CENTER HOSPITAL
Priority date: 2022-04-13
Filing date: 2022-04-13
Publication date: 2022-09-23
Anticipated expiration: 2042-04-13
Also published as: CN115094134B

Abstract

The invention provides application of PCSK9 in macrophage M2 type polarization and related diseases, and belongs to the technical field of biological medicine and molecular biology. According to the invention, through research, the mechanism that PCSK9 in colon cancer cells is down-regulated or PCSK9 inhibitors are used for promoting M2 to M1 phenotype transformation of macrophages co-cultured with the PCSK9 cells and inhibiting colon cancer cell invasion and metastasis is probably related to that PCSK9 inhibitors block Warburg effect (glycolysis) of colon cancer cells and inhibit lactic acid formation. Researches show that the PCSK9 inhibitor can be used as a macrophage phenotype regulator, and can inhibit tumor growth by inducing tumor-associated macrophages to polarize from M2 to M1 phenotype, so that the PCSK9 inhibitor has good practical application value.

Description

Application of PCSK9 in macrophage M2 type polarization and related diseases thereof

Technical Field

The invention belongs to the technical field of biomedicine and molecular biology, and particularly relates to application of PCSK9 in macrophage M2 type polarization and related diseases thereof.

Background

The information disclosed in this background of the invention is only for enhancement of understanding of the general background of the invention and is not necessarily to be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

Colorectal cancer (CRC) is a common malignant tumor of the digestive system, and the latest global cancer burden data show that the incidence and mortality of colorectal cancer are three leading in the world in 2020, and the new disease number of colorectal cancer in China is the second largest cancer after lung cancer. In addition, the prognosis of patients with metastatic colorectal cancer is poor, and the health of human beings is seriously threatened. The pathogenesis of colorectal cancer is complicated and not completely clear up to now. With the continuous and intensive research in recent years, new treatment schemes are emerging, and although the median survival of colorectal cancer patients is somewhat prolonged, the overall prognosis and the recurrence rate of metastatic CRC patients are still not ideal. Therefore, it is necessary to search for new potential therapeutic targets by deeply studying the molecular mechanism of colorectal cancer development.

Currently, in tumor therapy, means for targeting macrophages (TAMs) in the tumor microenvironment are gaining increasing attention. Tumor cells release signals such as chemotactic factors and cytokines, macrophages are recruited and induced to be differentiated into the TAMs, so that tumor invasion, metastasis and immune escape are promoted, and treatment taking the TAMs as targets mainly focuses on inhibiting recruitment of the TAMs, inducing polarization of the TAMs, removing the TAMs and the like. A publication published in Cancer cell in 2017, 12 months, states that complete inhibition or elimination of TAMs in tumors causes a large accumulation of immunosuppressive cells at the tumor site, and does not inhibit tumor growth. This suggests that we cannot effectively control tumors by eliminating TAMs, and that targeting TAMs phenotypic polarization may be a better option.

TAMs have been shown to be highly plastic cells and by regulating the transformation of TAMs from the M2 phenotype to the M1 phenotype, anti-tumor effects can be enhanced. M1 type TAMs can distinguish tumor cells from normal cells and play an anti-tumor role through the immune recognition function; while M2-type TAMs are involved in the cancer promotion processes such as tumor growth, invasion and metastasis, immunosuppression, chemotherapy resistance and the like. Studies have shown that TAMs in colon cancer patients do not consist of the pure M1 or M2 phenotype, and that the function of TAMs in colon cancer progression depends on the tumor stage, the type of TAMs, and the proportion of M1, M2 typing. Therefore, if reasonable intervention can be performed on certain key processes of TAMs polarization, the control of TAMs to develop towards M1 phenotype polarization in a proper colon cancer development period can cause M2 type TAMs to abandon the cancer promotion property, so that the deviated polarization imbalance is twisted, and a new idea is provided for treating colon cancer.

Proprotein convertase subtilisin/kexin type 9 (PCSK 9) is a member of the proprotein convertase family, encoded by the PCSK9 gene. The gene was originally found to encode the neuronal apoptosis-related invertase 1, now widely regarded as a key regulator of low density lipoprotein metabolism, and is involved in the metabolism of blood lipids and the maintenance of cholesterol homeostasis in vivo. It is noted that PCSK9 is not only involved in blood lipid metabolism, but also in many other biological processes, including cell cycle, inflammation, apoptosis, and viral infection. In recent years, with the intensive research on the relationship between cholesterol metabolism and tumors, researchers have focused on the role of PCSK9 in the development and progression of cancer. Many studies indicate that PCSK9 may have a certain regulatory effect in various tumors such as hepatocellular carcinoma, lung cancer, breast cancer, and prostate cancer, and is related to the risk of cancer occurrence and the prognosis of patients. However, these studies on the relationship of PCSK9 to tumors are still controversial, and furthermore PCSK9 has been less studied in colorectal cancer, and most of these studies are based on preclinical studies, so that more intensive studies are needed to elucidate the role of PCSK9 in colorectal cancer and its molecular mechanism.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide the application of PCSK9 in macrophage M2 type polarization and related diseases. According to the invention, through research, the mechanism that PCSK9 in colon cancer cells is down-regulated or PCSK9 inhibitors are used for promoting M2 to M1 phenotype transformation of macrophages co-cultured with the PCSK9 cells and inhibiting colon cancer cell invasion and metastasis is probably related to that PCSK9 inhibitors block Warburg effect (glycolysis) of colon cancer cells and inhibit lactic acid formation. Studies have shown that PCSK9 inhibitors can act as modulators of macrophage phenotype, inhibiting tumor growth by inducing tumor-associated macrophages to phenotypically polarize from M2 to M1. Based on the above results, the present invention was completed.

In a first aspect of the invention, there is provided the use of an agent that detects the PCSK9 gene and its expression products in the manufacture of a product for use in diagnosing, detecting, monitoring or prognosticating the progression of colorectal cancer.

In a second aspect of the invention, the application of the substance for inhibiting the reduction of the PCSK9 gene and the expression product and/or activity thereof in at least one of the following a1) -a6) is provided:

a1) inhibiting TAMs M2-like polarization, promoting TAMs M1-like polarization or preparing products inhibiting TAMs M2-like polarization and promoting TAMs M1-like polarization;

a2) inhibiting the Warburg effect (glycolysis) of the tumor cells or preparing products for inhibiting the Warburg effect (glycolysis) of the tumor cells;

a3) inhibiting the formation of tumor cell lactic acid or preparing a product for inhibiting the formation of tumor cell lactic acid;

a4) inhibiting tumor cell invasion and metastasis or preparing a product for inhibiting tumor cell invasion and metastasis;

a5) products that inhibit tumor growth or inhibit tumor growth;

a6) treating tumor or preparing tumor treating product.

The product may be a pharmaceutical or a test agent, which may be used for basic research. For example, the product can be used for inducing and regulating macrophage polarization in vitro, so that a high-efficiency and economical macrophage polarization experimental model is established; for another example, the product is used for inhibiting the Warburg effect (glycolysis) of tumor cells in vitro so as to inhibit the formation of lactic acid of the tumor cells, thereby being used for further researching the relationship between the formation of lactic acid and related mechanisms thereof and tumorigenesis.

Wherein the tumor is a solid tumor, further a colorectal cancer. In a particular embodiment of the invention, the tumor cells are human colorectal cancer cells HCT116 and HT-29; the macrophage is THP-1 derived macrophage.

Thus, the application may be: application of substances for inhibiting PCSK9 gene and expression products and/or activity reduction thereof in preparing products for inhibiting TAMs M2-like polarization and promoting TAMs M1-like polarization in a tumor cell-macrophage co-culture system.

The tumor cells are human colorectal cancer cells HCT116 and HT-29; the macrophage is THP-1 derived macrophage.

In a third aspect of the invention, the invention provides the use of a substance inhibiting the reduction of the PCSK9 gene and its expression product and/or activity in the manufacture of a product.

The function of the product is any one or more of the following:

a1) inhibiting TAMs M2-like polarization and promoting TAMs M1-like polarization;

a2) inhibition of the Warburg effect (glycolysis) of tumor cells;

a3) inhibiting the formation of tumor cell lactic acid;

a4) inhibiting tumor cell invasion and metastasis;

a5) inhibiting tumor growth;

a6) and (3) treating tumors.

In a fourth aspect of the invention, there is provided a method of tumour therapy, the method comprising: administering to the subject an agent that inhibits the reduction of the PCSK9 gene and its expression products and/or activity.

Wherein the tumor is a solid tumor, further a colorectal cancer.

The beneficial technical effects of one or more technical schemes are as follows:

the technical scheme reports that the PCSK9 in the colon cancer cell is down-regulated or the PCSK9 inhibitor is used for promoting the macrophage co-cultured with the colon cancer cell to generate the phenotype conversion from M2 to M1 and inhibiting the invasion and metastasis of the colon cancer cell, and the mechanism of the mechanism is probably related to that the PCSK9 inhibitor blocks the Warburg effect (glycolysis) of the colon cancer cell and inhibits the formation of lactic acid. Studies have shown that PCSK9 inhibitors can act as modulators of macrophage phenotype, inhibiting tumor growth by inducing tumor-associated macrophages to phenotypically polarize from M2 to M1.

The technical scheme provides a new mechanism research for the occurrence and development of the colorectal cancer and a promising treatment strategy for colorectal cancer patients, thereby having good potential practical application value.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are included to explain the illustrative embodiments of the invention and the description of the invention and are not intended to limit the invention unduly.

FIG. 1 shows the differentiation of THP-1 cells into macrophages induced by PMA in the present example.

FIG. 2 is a graphical representation of the modulating effect of PCSK9 on polarization of TAMs phenotype in an embodiment of the present invention; wherein, a, b, RT-PCR is used for detecting the mRNA expression level of M1 and M2 macrophage marking molecules after the CRC cells and the THP-1-derived macrophages are co-cultured for 48 hours; c, d, detecting the protein expression level of an M1 marker iNOS and an M2 marker CD163 after the CRC cells and the THP-1-derived macrophages are co-cultured for 48 hours by using western blot; e, detecting the expression condition of the M1 macrophage surface differentiation antigen CD86-AF647 after the CRC cells and the THP-1-derived macrophages are co-cultured for 48 hours by flow cytometry. All experiments are repeated for 3 times, the experimental results are represented by mean +/-standard error (mean +/-SEM), the two-tailed t test is applied to carry out comparison between two groups, and the P <0.05 is considered to have statistical significance; p < 0.05; p < 0.01; p < 0.001.

FIG. 3 shows that the expression level of PCSK9 affects the level of a modified protein for the intracellular phosphorylation of CRC in an example of the present invention; wherein, a, after the expression of PCSK9 of CRC cells is reduced, the protein levels of HCT116 and HT-29 lactate modification proteins are obviously reduced; b, after the expression of PCSK9 of CRC cells is up-regulated, the HCT116 and HT-29 lactate modification protein level is obviously increased. All experiments are repeated for 3 times, the experimental results are represented by mean +/-standard error (mean +/-SEM), the two-tailed t test is applied to carry out comparison between two groups, and the P <0.05 is considered to have statistical significance; p < 0.05; p < 0.01; p < 0.001.

FIG. 4 is a glycolysis level detection in an embodiment of the invention; wherein, the assay of the lactate assay kit found that knocking-down PCSK9 significantly reduced the level of lactate product in the culture supernatant of colon cancer cells, < 0.01; the WB detection shows that the knocking-down of PCSK9 inhibits the expression level of a glycolysis key enzyme TPI1 in colon cancer cells, and the result is consistent with the proteomic detection result.

FIG. 5 shows PCSK9 expression levels in tumor tissue of colon cancer patients and PCSK9 vs Apc ^Min/+ Regulating and controlling the growth of colon tumor of mouse; wherein, A is the expression of PCSK9 in cancer, paracancerous and distant tissues (more than 5cm from cancer tissues) in pathological tissue specimens of 75 colon cancer patients detected by an Immunohistochemical (IHC) method, and the result is consistent with the result of TCGA database search. Expression level of PCSK9 in cancer tissue is higher than that in paracancer and distal tissue (p)<0.05), there was no significant difference in expression levels between paracancerous and distant tissues; b, C and D represent that PCSK9 overexpression mice and Apc ^Min/+ Hybrid breeding of mice to obtain Apc ^Min/+ PCSK9(KI) mice, PCSK9 overexpression was found to promote Apc ^Min/+ Growth and malignant transformation of colon tumor of mouse; to Apc ^Min/+ Mice were administered with PCSK9 inhibitor evococcus ab by subcutaneous injection and inhibition of PCSK9 was found to reduce Apc ^Min/+ Number, size and malignancy of colon tumors in mice. E is detected by an IHC method, and the over-expression of PCSK9 is found to increase the levels of M2 type TAMs molecular markers CD206 and Arg1 in colon tumor tissues, reduce the levels of M1 type TAMs molecular markers CD64 and CCR7, and control the Apc ^Min/+ Mice reversed this effect (200 ×) using subcutaneous administration of PCSK9 inhibitor evocolumab<0.05，**p<0.01。

FIG. 6 shows that PCSK9 knockdown inhibits colon cancer cell invasion and metastasis in vitro in an example of the present invention.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. 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 invention 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 in accordance with the invention. 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.

The present invention will now be further described with reference to specific examples, which are provided for the purpose of illustration only and are not intended to be limiting. If the experimental conditions not specified in the examples are specified, the conditions are generally as usual or as recommended by the reagents company; reagents, consumables and the like used in the following examples are commercially available unless otherwise specified.

In a typical embodiment of the invention, there is provided the use of an agent that detects the PCSK9 gene and its expression products in the manufacture of a product for use in diagnosing, detecting, monitoring or prognosticating the progression of colorectal cancer.

The product can diagnose, detect, monitor or predict the progression of colorectal cancer by detecting the expression level of the PCSK9 gene and/or the PCSK9 gene expression product (e.g., PCSK9 protein). Immunohistochemistry (IHC) detected PCSK9 expression in pathological tissue specimens of 75 colon cancer patients in cancer, paracancerous and distant tissues (greater than 5cm from the cancerous tissue). The expression level of PCSK9 in cancer tissues is higher than that in paracancer and far-end tissues (p <0.05), and the expression level between the paracancer and the far-end tissues is not significantly different.

Wherein the product comprises a substance for detecting the transcription of PCSK9 in a colorectal cancer sample based on high-throughput sequencing methods and/or based on quantitative PCR methods and/or based on probe hybridization methods; or a substance for detecting the condition of the expression product of PCSK9 in a sample based on an immunodetection method.

In yet another embodiment of the present invention, PCSK9 transcripts in colorectal cancer samples are detected by methods including, but not limited to, liquid phase hybridization, Northern hybridization, miRNA expression profiling, ribozyme protection analysis, RAKE, in situ hybridization; PCSK9 protein status in colon cancer samples was detected using a protocol including, but not limited to, Immunohistochemistry (IHC), ELISA, colloidal gold dipstick, protein chip.

In still another embodiment of the present invention, there is provided a use of a substance inhibiting reduction in the PCSK9 gene and its expression product and/or activity in at least one of the following a1) -a 6):

a5) products that inhibit tumor growth or inhibit tumor growth;

a6) treating tumor or preparing tumor treating product.

The product may be a pharmaceutical or a test agent, which may be used for basic research. For example, the product can be used for inducing and regulating macrophage polarization in vitro, so that a high-efficiency and economical macrophage polarization experimental model is established; for example, the product is used for inhibiting the Warburg effect (glycolysis) of tumor cells in vitro so as to inhibit the formation of lactic acid of the tumor cells, and further research on the relationship between the formation of the lactic acid and related mechanisms thereof and tumorigenesis.

Wherein the tumor is a solid tumor, further colorectal cancer. In a particular embodiment of the invention, the tumor cells are human colorectal cancer cells HCT116 and HT-29; the macrophage is THP-1 derived macrophage.

Therefore, in another embodiment of the present invention, the application is: application of substances for inhibiting PCSK9 gene and expression products and/or activity reduction thereof in preparing products for inhibiting TAMs M2-like polarization and promoting TAMs M1-like polarization in a tumor cell-macrophage co-culture system.

The substance for inhibiting the PCSK9 gene and its expression product and/or activity reduction includes, but is not limited to, RNA interference molecules or antisense oligonucleotides against PCSK9, small molecule inhibitors, shRNA (small hairpin RNA), small interfering RNA (sirna), substances for performing lentiviral infection or gene knock-out, and specific antibodies against PCSK9 itself or molecules upstream and downstream thereof, including anti-PCSK 9 antibodies (e.g., elouzumab, evocumab).

The sequence of the small interfering RNA is shown in SEQ ID NO. 1-2.

In another embodiment of the invention, the use of a substance that inhibits the reduction of the PCSK9 gene and its expression product and/or activity in the manufacture of a product is provided.

The function of the product is any one or more of the following:

a2) inhibition of the Warburg effect (glycolysis) of tumor cells;

a3) inhibiting the formation of tumor cell lactic acid;

a4) inhibiting tumor cell invasion and metastasis;

a5) inhibiting tumor growth;

a6) and (4) treating tumors.

The product may be a pharmaceutical or a test agent, which may be used for basic research.

The substance for inhibiting PCSK9 gene and its expression product and/or activity reduction includes, but is not limited to, RNA interference molecule or antisense oligonucleotide against PCSK9, small molecule inhibitor, shRNA (small hairpin RNA), small interfering RNA (sirna), substance for performing lentiviral infection or gene knockout, and specific antibody against PCSK9 itself or molecules upstream and downstream thereof, such as anti-PCSK 9 antibody (e.g. elouzumab, evocumab).

The sequence of the small interfering RNA is shown in SEQ ID NO. 1-2.

According to the invention, when the product is a medicament, the medicament further comprises at least one pharmaceutically inactive ingredient.

The pharmaceutically inactive ingredients may be carriers, excipients, diluents and the like which are generally used in pharmacy. The composition can be prepared into oral preparations such as powder, granule, tablet, capsule, suspension, emulsion, syrup, and spray, external preparations, suppositories, and sterile injectable solutions according to a conventional method.

Such pharmaceutically inactive ingredients, which may include carriers, excipients and diluents, are well known in the art and can be determined by one of ordinary skill in the art to meet clinical criteria.

In still another embodiment of the present invention, the carrier, excipient and diluent include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.

In still another embodiment of the present invention, the medicament of the present invention may be administered into the body by a known means. For example, by intravenous systemic delivery or local injection into the tissue of interest. Optionally via intravenous, transdermal, intranasal, mucosal or other delivery methods. Such administration may be via a single dose or multiple doses. It will be understood by those skilled in the art that the actual dosage to be administered in the present invention may vary greatly depending on a variety of factors, such as the target cell, the type of organism or tissue thereof, the general condition of the subject to be treated, the route of administration, the mode of administration, and the like.

In still another embodiment of the present invention, the subject to which the medicament is administered may be human and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, chimpanzees, and the like.

In another embodiment of the present invention, there is provided a method of tumor therapy, the method comprising: administering to the subject an agent that inhibits the reduction of the PCSK9 gene and its expression products and/or activity.

In yet another embodiment of the invention, the tumor is a solid tumor, further a colorectal cancer.

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The following examples are test methods in which specific conditions are indicated, and are generally carried out under conventional conditions.

Examples

Cytology related experimental method

1. Cell transfection

1.1 transient transfection of siRNA

(1) Dissolving and subpackaging siRNA: the PCSK9-siRNA and NC-siRNA powders were centrifuged at 2500rpm for 2 minutes to cause aggregation at the bottom of the tube, the cap was gently opened, and RNase-free H was added ₂ Dissolving O to obtain 20 μ M solution, subpackaging in an EP tube without RNase, and storing at-20 deg.C.

(2) Taking CRC cells in logarithmic growth phase, adjusting cell concentration to 1X10 with complete culture medium ⁵ cells/ml, 2ml per well in six well plates, 5% CO at 37 ℃% ₂ The cultivation in the incubator of (1) is carried out for 24 hours.

(3) The next day, when cells grew adherent to 30-50% confluency, the medium in the wells was aspirated away, each well was washed with serum-free McCOY's 5A medium, and then 1.5ml of serum-free McCOY's 5A medium was added to each well.

(4) Dissolving 5 mul of siRNA in 250 mul of opti-mem according to lipo 2000 instructions, and lightly blowing and sucking for 3-5 times to mix uniformly; another RNase-free EP tube was taken, and 5. mu.l of lipo 2000 was dissolved in 250. mu.l of opti-mem, and gently aspirated 3 to 5 times to mix well. The mixture was allowed to stand at room temperature for 5 minutes.

(5) Mix lipo 2000 and siRNA diluent, gently blow and mix well, and stand for 20 minutes at room temperature.

(6) And (3) dripping the transfection compound into a six-hole plate, slightly shaking the six-hole plate, uniformly mixing, placing in an incubator, and replacing a fresh complete culture medium after 4-6 hours. And collecting cells 24h after transfection to perform a functional experiment, extracting RNA 24-48h, and extracting protein 48-72 h.

siRNA sequence:

1.2 transient transfection of plasmids

(1) Plasmid lysis: centrifuging plasmid DNA and control plasmid empty vector at 10000rpm for 10-15s, carefully opening, and adding appropriate amount of ddH ₂ And O, fully shaking and dissolving to prepare a solution with the concentration of 1 mu g/mu l, and storing at the temperature of-20 ℃ after being properly subpackaged.

(2) Taking CRC cells in logarithmic growth phase, adjusting cell concentration to 2x10 with complete culture medium ⁵ cells/ml, 2ml per well were plated into six-well plates and incubated at 37 ℃ in an incubator with 5% CO2 for 24 h.

(3) The next day, when the cells grew adherent to 70-90% confluency, the medium in the wells was aspirated away, each well was washed with serum-free McCOY's 5A medium, and then 1.5ml of serum-free McCOY's 5A medium was added to each well.

(4) Dissolving 4 mu g of plasmid DNA in 250 mu l of opti-mem according to lipo 2000 instructions, and gently sucking for 3-5 times to mix uniformly; another sterile EP tube is taken, 10 mu l of lipo 2000 is dissolved in 250 mu l of opti-mem, and the mixture is gently sucked and blown for 3-5 times and mixed evenly. The mixture was allowed to stand at room temperature for 5 minutes.

(5) Mix lipo 2000 and plasmid DNA diluent, gently blow and mix well, and stand for 20 minutes at room temperature.

(6) And (3) lightly dripping the compound into a six-hole plate, lightly shaking the six-hole plate, uniformly mixing, placing in an incubator, and replacing a fresh complete culture medium after 4-6 hours. And collecting cells 24h after transfection to perform a functional experiment, extracting RNA 24-48h, and extracting protein 48-72 h.

1.3 establishment of stably silenced PCSK9 cell line

(1) Lentiviruses were purchased from hanchang biotechnology (shanghai) ltd.

(2) Well-conditioned log-phase grown CRC cells were adjusted to a concentration of 3 × 10 ⁵ cells/ml, 500. mu.l per well, in 24-well plates, at 37 ℃ with 5% CO ₂ The culture was carried out overnight in an incubator.

(3) The next day, when the cell growth density reached 30-50%, the virus infection was performed by 1/2 small volume infection. The virus was removed from the freezer and placed on ice to thaw slowly, the cell stock medium was aspirated, and 250. mu.l of fresh complete medium (containing 5. mu.g/ml polybrene) was added. According to MOI 10, the infection is performed by adding a suitable volume of virus stock. Add virus amount (μ l) per well-MOI x cell number/virus titer (TU/ml) x 1000. The medium was replenished to 500. mu.l 4h after lentivirus infection.

(4) After 24h of infection, the virus-containing medium was aspirated, replaced with fresh complete medium, and the culture was continued.

(5) And after 48-72h of infection, observing fluorescence by a fluorescence microscope to preliminarily determine the infection efficiency.

(6) After the cell state is stable, 2.0 mu g/ml puromycin (HCT116) or 8.0 mu g/ml puromycin (HT-29) is added, and the cell strain is screened for 3-4 weeks to obtain the stable infected cell strain. Subsequent experiments were performed after the amplification culture.

2. Cell co-culture

2.1 induced differentiation of THP-1 cells

Collecting THP-1 cell suspension, adjusted cell concentration to 7 x10 ⁵ cells/ml, 2ml per well were seeded in 6-well plates and 100ng/ml PMA was added for 48h to differentiate into macrophages. After washing twice with PBS, 2ml of fresh RPMI-1640 complete medium was replaced for subsequent experiments.

2.2 Co-culture of CRC cells with macrophages

In vitro cell co-culture experiments were performed using Transwell co-culture chambers (6 wells, 0.4um pore size). Gently placing the co-culture chamber into a 6-well plate containing THP-1-derived macrophages with sterile forceps; after 24h transfection of HCT116 and HT-29siRNA to human colorectal cancer cells, cells were harvested, resuspended in complete RPMI-1640 medium and adjusted to a cell concentration of 2 × 105cells/ml, 1.8ml cell suspension per well was added to the upper layer of the chamber, the 6-well plate was covered, placed at 37 ℃ with 5% CO ₂ Cultured in an incubator. After 48h of co-culture, macrophages were harvested for relevant detection.

2.3 RT-PCR

2.3.1 extraction of Total RNA

(1) The cell culture plate was removed, the medium was discarded, and washed 2 times with pre-cooled PBS.

(2) Adding 1ml Trizol lysate into each well, repeatedly blowing and cracking the cells, and standing for 5min at room temperature.

(3) The lysate was collected into an RNase free EP tube, added with 1/5 volume of chloroform (1ml Trizol plus 0.2ml chloroform) in suspension volume, shaken vigorously for 15s and allowed to stand at room temperature for 3 min.

(4) Centrifuge at 12000rpm at 4 ℃ for 15 min. After centrifugation the sample was divided into three layers: the lower layer is a red phenol-chloroform layer, the middle layer is a white organic compound, the upper layer is a colorless aqueous phase, and RNA is mainly in the upper aqueous phase.

(5) The upper aqueous phase was transferred to a new rnase-free EP tube, an equal volume of isopropanol was added and shaken upside down for 1 min.

(6) Centrifuge at 12000rpm at 4 ℃ for 15 min. The supernatant was discarded, and the white precipitate was RNA.

(7) Add 1ml 75% ethanol to each tube, flick the bottom of the tube to precipitate, centrifuge at 12000rpm at 4 ℃ for 5min, repeat this step 3 times.

(8) Discarding the supernatant, retaining the precipitate, inverting for absorbing waterAir drying the paper, adding appropriate amount of RNase-free H ₂ O dissolves the RNA.

(9) The RNA concentration was determined and stored at-80 ℃ until use.

2.3.2 reverse transcription

The reverse transcription reaction is completed by an Evo M-MLV reverse transcription kit II (AG). All reaction mixtures were formulated on ice.

(1) Removing genome DNA, preparing reaction solution according to the following table, and performing genome DNA removing reaction.

Reaction conditions are as follows: at 42 ℃ for 2min

4℃

(2) Reverse transcription: the reaction solution was prepared according to the following table, and reverse transcription was performed.

Reaction conditions are as follows: 15min at 37 DEG C

85℃ 5sec

4℃

The reverse transcription product cDNA can be directly used for subsequent PCR reaction, and can also be stored at-20 ℃ for later use.

2.3.3 RT-PCR reaction

RT-PCR reaction was completed by SYBR Green Pro HS premix qPCR kit II (AG).

The operation was performed on ice, taking care to avoid light.

(1) The PCR primers were designed and synthesized by Soukuwa Biotechnology GmbH, and the sequences of the primers were as follows:

(2) the following reaction system (20. mu.l) was prepared:

reaction conditions are as follows:

(3) calculating the expression amount: by use of 2 ^-ΔΔCT The difference in gene expression was calculated.

2.4 Western blot

2.4.1 extraction of Total cellular proteins

(1) The 6-well plate was placed on ice, the medium was discarded, and washed twice with cold PBS.

(2) Mu.l RIPA lysate containing protease phosphatase inhibitor cocktail was added to each well and lysed on ice for 15 min.

(3) The lysate was collected into 1.5ml EP tube, centrifuged at 12000rpm for 15min at 4 ℃.

(4) The protein supernatant was collected into another new EP tube, and 1/5 volumes of 5 Xloading buffer were added and the protein denatured by a 10min 100 ℃ water bath.

(5) Storing at-80 deg.C for use.

2.4.2 BCA assay for protein concentration

(1) And (3) diluting the BSA standard substance, namely diluting the BSA standard substance to 1mg/ml according to the kit instructions, and storing the BSA standard substance at-20 ℃ for a long time.

(2) Preparing a BCA working solution: determining the volume of the required BCA reagent according to the number of samples, and mixing the BCA reagents A and B according to the volume ratio of 50: 1, and fully and uniformly mixing to obtain the BCA working solution.

(3) Protein concentration determination: first, 200. mu.l of BCA working solution was added to each well, and BSA standard solutions were added as shown in the following table to prepare a standard curve. Mu.l protein sample was added to each well and supplemented with 10. mu.l PBS. Incubate at 37 ℃ for 30min, and measure the absorbance at 562nm with a microplate reader. The protein concentration of the sample was calculated from the standard curve.

2.4.3 SDS-PAGE gel electrophoresis

(1) The glass plate was washed with distilled water and dried for use.

(2) Preparation of SDS-PAGE: the concentration of the separation gel is selected according to the molecular weight of the target protein.

Then preparing the separating gel according to the kit instruction, taking 15ml of separating gel as an example, adding the following components (unit: ml) into the separating gel with different concentrations:

the preparation of 5% concentrated gum is as follows (in case of preparation of 4ml, unit: ml):

(3) preparing glue: after the separation gel is mixed evenly, about 7ml of separation gel is rapidly added into the middle of the clamped glass plate, and 1ml of isopropanol is added for pressing gel. After the separation gel is solidified, the isopropanol is discarded, and the comb is vertically inserted immediately after the concentration gel is filled, so that bubbles are prevented from being generated.

(4) Sample adding: and (3) after the gel is solidified, filling 1x of electrophoresis buffer solution in the inner chamber, slowly and vertically pulling out the comb, and adding the same amount of sample to be detected and protein marker according to the detected protein concentration.

(5) Electrophoresis: the electrophoresis tank was filled with 1 Xelectrophoresis buffer and electrophoresed at 80V at constant pressure. After the sample enters the separation gel, the temperature is adjusted to 120V, and electrophoresis is carried out at constant pressure until the target protein is completely separated.

(6) Film transferring: the gel containing the band of interest was cut according to the marker molecular weight. PVDF membranes with corresponding sizes are cut and put into methanol for activation for 1min, and then put into a membrane transfer buffer solution for standby. The turning membrane clips were assembled in the order of negative (black), sponge, 3 layers of filter paper, gel, PVDF membrane, 3 layers of filter paper, sponge, positive (white) electrode, taking care that there were no air bubbles between the glue and the membrane. And (4) putting the film transferring clamp into a film transferring groove, and fully adding the film transferring liquid. And (3) carrying out constant-current 250mA ice bath film transfer, wherein the film transfer time is determined according to the molecular weight of the protein.

(7) And (3) sealing: after the film transfer is finished, marking the PVDF film, putting the PVDF film into 5% skimmed milk powder, sealing for 1.5h at room temperature, and performing the sealing process on a shaking bed.

(8) Incubating the primary antibody: after blocking was complete, wash 3 times with 1 × TBST eluent for 5min each. The PVDF membrane was then placed in an antibody incubation cassette containing the corresponding primary antibody and incubated overnight at 4 ℃. The primary dilution ratio was determined according to the antibody specification.

(9) Incubation of secondary antibody: the next day, primary antibody was recovered. The PVDF membrane was removed and washed 3 times with 1 × TBST eluent for 10min each time. Then put into an antibody incubation box containing corresponding secondary antibody (1: 10000) and incubated for 1-2h at room temperature.

(10) Color development: after the antibody incubation was complete, the cells were washed 3 times with 1 × TBST eluent for 10min each time. ECL chemiluminescence developing solution A, B solution was mixed in equal amount under dark condition. And (3) placing the PVDF film on an operation table of a gel imager, dropwise adding a proper amount of developing solution to start exposure, observing a recorded result, and analyzing a gray value by using Image J.

2.5 flow cytometry

After co-culturing THP-1 derived macrophages with CRC cells for 48h, different groups of macrophages were collected. After washing the cells twice with PBS, the cells were resuspended in 100. mu.l PBS to make a single cell suspension. According to the antibody specification, 5 μ l of the corresponding antibody labeled with the fluorescent substance and isotype control thereof are added into each tube, mixed gently, and incubated at 4 ℃ for 30min in the dark. After washing the cells twice with PBS, the cells were resuspended with 300-. Filtering with 300 mesh nylon net, and detecting the expression of cell surface differentiation antigen.

2.6 invasion transfer experiment

2.6.1 scratch test

(1) Cell digestion, centrifugation, resuspension, cell concentration adjustment to 6X 10 ⁵ cells/ml, 2ml of cell suspension per well were seeded in 6-well plates and, when the cells grew full, "crossed" at the bottom of the plate using a sterile 200ul pipette tip.

(2) The shed cells were gently washed with PBS and 2ml serum-free medium was added to each well.

(3) The scratch condition is observed under a microscope, and the healing degree of the scratch is recorded by taking pictures at 0h and 48 h.

2.6.2 Transwell migration experiment

(1) Cell digestion centrifugation, cell resuspension in serum-free McCOY's 5A medium and cell concentration adjustment at 5 × 10 ⁵ cells/ml (HCT116) or 7.5 x10 ⁵ cells/ml(HT-29)。

(2) The Transwell chamber (24 wells, 8 μm pore size) was removed, 600 μ l of medium containing 15% FBS was added to the lower chamber, and 200 μ l of cell suspension was added vertically to the upper chamber, taking care that no air bubbles were present between the upper and lower chambers. Putting into an incubator to be cultured for 48 h.

(3) The upper chamber medium was discarded, and the cells were gently washed with PBS and fixed with 4% paraformaldehyde at room temperature for 20 minutes.

(4) The chamber was properly air dried, stained with 0.1% crystal violet solution at room temperature for 20 minutes, rinsed with PBS and the upper layer of non-migrated cells was gently wiped off with a cotton swab.

(5) Randomly selected 5 fields under 200 × microscope for photographing and counting.

2.6.3 Transwell invasion test

(1) Taking the matrigel out of a refrigerator at the temperature of-20 ℃ in advance, putting the matrigel on ice for melting, and precooling the gun head and the EP tube at the same time. Diluting the melted matrigel and serum-free medium according to the proportion of 1:8, sucking 100 mul of diluted matrigel, spreading the matrigel on the upper layer (24 holes and 8 mu m aperture) of a Transwell chamber, placing the matrigel in an incubator for half an hour, sucking and removing the matrigel, and keeping the chamber for later use.

(2) Cell digestion centrifugation, resuspension with serum-free McCOY's 5A MediumCells, and adjusting the cell concentration to 5x 10 ⁵ cells/ml (HCT116) or 7.5 x10 ⁵ cells/ml(HT-29)。

(3) The chamber was removed and 600. mu.l of medium containing 15% FBS was added to the lower chamber and 200. mu.l of cell suspension was added vertically to the upper chamber, taking care that there were no air bubbles between the upper and lower chambers. Putting into an incubator to be cultured for 48 h.

(4) The upper chamber medium was discarded, gently rinsed with PBS and fixed with 4% paraformaldehyde at room temperature for 20 minutes.

(5) The chamber was properly air dried, stained with 0.1% crystal violet solution at room temperature for 20 minutes, rinsed with PBS and the upper layer of non-migrated cells was gently wiped off with a cotton swab.

(7) Randomly selected 5 fields under 200 × microscope for photographing and counting.

Zoology related experimental method

1. Laboratory animal

1.1 animal Cross-breeding

The sexual maturation period of the mice is 7-8 weeks, and PCSK9(KI) female mice and Apc are used when the first generation mice are about 8 weeks old ^Min/+ And (5) combining the male mice and hybridizing to breed offspring mice. Marking the newborn mice with ear tags at 4 weeks of age, and screening the Apc required by the experiment by a genotype identification method ^Min/+ And Apc ^Min/+ PCSK9(KI) mice.

1.2 animal identification

1.2.1DNA extraction

Shearing 2-3mm of the tip of the mouse, and putting the tip into a 1.5mL EP tube; to an EP tube containing mouse tail, 500. mu.L of lysate and 5. mu.L of proteinase K (10mg/mL) were added; placing the EP tube seal (preventing cracking liquid from being reduced due to opening of the EP tube by heating) on an EP tube frame by using a sealing film, and then placing the EP tube seal in a constant-temperature water bath (55 ℃) for overnight digestion; the next day, the EP tube is taken out and shaken vigorously to mix the EP tube and the EP tube evenly; adding 500 μ L of phenol/chloroform (1:1) mixed solution into each tube, mixing, and centrifuging at 12000rpm for 15 min; using 1000 uL pipette to suck about 300 uL supernatant from the centrifuged EP tube into a new EP tube and marking the number clearly; adding 100% ethanol 2 times the volume of the supernatant (about 600. mu.L) to the tube, gently mixing (inverting several times) until white filamentous DNA is seen, centrifuging at 12000rpm for 10min, and discarding the supernatant; adding 800 μ L70% ethanol into the tube, and mixing well; centrifuging at 12000rpm for 5min, discarding supernatant, standing the centrifuge tube upside down on filter paper, sucking off residual ethanol, and air drying (t >30min) until no ethanol smell is detected; add 100. mu.L of TE Buffer to the EP tube, shake it up at 37 ℃ to dissolve the DNA sufficiently, and store it at 4 ℃.

1.2.2 PCR amplification

(1) Apc amplification

Reaction system and program

(2) PCSK9 amplification

Reaction system and program

1.2.3 gel electrophoresis

(1) Preparing glue:

1% (2%) agarose solution: weighing 1g (2g) of agarose, placing the agarose into a conical flask, adding 100mL of 1 XTAE electrophoresis buffer solution, placing the agarose into a microwave oven, heating the agarose with high fire until the solution is boiled (about 2min) until the agarose is completely dissolved, and obtaining the agarose transparent solution with the concentration of 1% (2%).

Cooling the solution for 2min at room temperature, adding 7 mu L of nucleic acid dye Gold View II, fully shaking up, pouring into an electrophoresis gel chamber with a comb placed in advance, after the solution naturally solidifies to become white, slightly pulling out the comb along the vertical direction to obtain agarose gel, and adding 1 XTAE electrophoresis buffer solution of the same batch as the gel preparation until the liquid level is about 2mm higher than the gel.

(2) Loading:

and (3) taking 10 mu L of PCR amplification product, blowing, uniformly mixing, and then loading, wherein the loading corresponds to a DNA Marker with the same volume as the loading. Apc amplification products were loaded on 2% agarose gel and PCSK9 amplification products were loaded on 1% agarose gel. When the sample is loaded, the sample is prevented from overflowing, the gun head is prevented from puncturing the hole wall, and the sample number corresponding to each hole is marked clearly.

(3) Electrophoresis:

after the above operation was completed, electrophoresis was started by setting the conditions for electrophoresis (2% agarose gel: voltage 90V, time 45 min; 1% agarose gel: voltage 140V, 25 min). After the electrophoresis is finished, the gel is placed into an imaging analysis system for scanning, and is photographed and analyzed. Screening Apc by the above method ^Min/+ Mouse and Apc ^Min/+ A PCSK9(KI) mouse, namely a PCSK9 overexpression mouse model.

2. Animal testing

2.1 histopathological examination

(1) Making Paraffin sections

Fixing: fixing the intestinal tumor tissue blocks of the mice in 10% formalin for 24 hours;

and (3) dehydrating: 70% ethanol (60min) → 80% ethanol (30min) → 90% ethanol (30min) → 95% ethanol (30min) → absolute ethanol 1(30min) → absolute ethanol 2(30 min);

and (3) transparency: mixed solution of xylene and absolute ethyl alcohol 1:1 (60min) → pure xylene 1(30min) → pure xylene 2(30 min);

wax dipping: soft wax 1(30min) → soft wax 2(30min) → hard wax 1(30-60min) → hard wax 2(60-120 min);

embedding: selecting paraffin with the same melting point as that of the hard wax 2, preheating, pouring the paraffin into a wax groove, putting the tissue block subjected to wax dipping into the wax groove, and cooling at room temperature;

slicing: using a microtome, cutting the paraffin-embedded tissue block into sections approximately 4-5 μm thick;

exhibition of slices: spreading the wax sheet in 45 deg.C warm water, taking out the wax sheet from the water with glass slide coated with glycerol film, and oven drying in 38 deg.C thermostat.

(2) H.E. dyeing

Dewaxing and hydrating: xylene 1(10min) → xylene 2(10min) → absolute ethanol (5min) → 90% ethanol (2min) → 80% ethanol (2min) → 70% ethanol (2min) → triple distilled water (2 min);

dyeing: soaking in hematoxylin for 5-10min, washing with tap water to remove excess dye solution (about 10min), washing with triple distilled water for several seconds, differentiating in 1% hydrochloric acid alcohol differentiation solution for several seconds, washing with tap water for 10min, and soaking with eosin for 2 min;

dehydrating and transparentizing: 70% ethanol (10s) → 80% ethanol (10s) → 90% ethanol (10s) → anhydrous ethanol (10s) → xylene 1 clear (5min) → xylene 2 clear (5 min);

sealing: naturally drying the slices, and sealing the slices by using a cover glass after adding neutral gum dropwise; the sections were observed under a microscope, and the nuclei were blue and the cytoplasm was pink or red.

2.2 immunohistochemical assay

(1) Dewaxing and hydrating

Baking the chip in a constant temperature oven at 60 ℃ for 2 h; soaking in xylene I for 10min, and then soaking in xylene II for 10min for dewaxing; soaking in anhydrous ethanol, 95% ethanol, 85% ethanol, 70% ethanol, and distilled water for 5min, respectively, and hydrating.

(2) Antigen retrieval and blocking

Heating 0.01M citric acid buffer solution pH6.0 with microwave to boiling, placing the chip in the boiling buffer solution, and heating with high fire for 10-15 min; cooling to room temperature, washing with distilled water twice, and washing with PBS for 5 min; with 3% H ₂ O ₂ Blocking the methanol solution at room temperature for 10min to eliminate the effect of endogenous catalase; washing with PBS for 3 times, each for 5min, adding dropwise goat serum blocking solution, and sealing at room temperature for 20 min.

(3) Immunoreaction and staining

After sealing, wiping off excessive liquid, dripping primary antibiotics, and putting into a wet box at 4 ℃ overnight; the next day, taking out the chip from the wet box, and standing at room temperature for 30min for rewarming; washing with PBS for 3 times, each for 5min, adding secondary antibody dropwise, and incubating at room temperature for 30 min; washing with PBS for 3 times, each time for 5min, dripping DAB color developing solution, and mastering the dyeing degree under a mirror; washing with distilled water to stop dyeing; hematoxylin counterstain for 10s, and hydrochloric acid ethanol solution differentiation for 5 s.

(4) Dehydration seal

Sequentially adding 70% ethanol, 85% ethanol, 95% ethanol and anhydrous ethanol, and dehydrating; sequentially putting the mixture into dimethylbenzene I and dimethylbenzene II for transparency; the gel was mounted on a neutral resin, observed under a mirror, and photographed.

2.3 immunohistochemical outcome determination

Scoring according to staining intensity: 0 point (undyed), 1 point (light yellow), 2 points (tan), 3 points (tan); scoring by staining positive rate: 0 point (0-25%), 1 point (26-50%), 2 points (51-75%), 3 points (76-100%); the above two scores are multiplied together to obtain a total score.

3. Grouping scheme for clinical colon cancer patient data

Age: two groups are less than or equal to 55 years old and more than 55 years old; sex: male and female; tumor size: two groups of which are less than or equal to 4cm and more than 4 cm; and (3) pathological grading: class I and class II groups; group III; and N staging: group N0; group N1 and N2; TNM staging: TNM1 and TNM2 groups; group TNM 3.

3.1 immunohistochemical assay methods as before.

3.2 statistical analysis

The experimental data are statistically analyzed by SPSS 19.0, and the expression difference analysis of PCSK9 in cancer tissues and paracarcinoma tissues is tested by chi-square test; the correlation between expression levels of PCSK9 and patient clinical pathology parameters was analyzed using the chi-square test. P <0.05 was statistically significant.

Results of the experiment

1. The down-regulation of PCSK9 expression in CRC cells can inhibit TAMs M2-like polarization and promote TAMs M1-like polarization

In CRC cells, PCSK9 regulates the expression levels of intracellular lactate-modifying proteins and MIF proteins, both of which are closely associated with anti-tumor immunity and involved in the regulation of phenotypic polarization of TAMs in the tumor microenvironment, so we next investigated the effect of PCSK9 on phenotypic polarization of TAMs.

Tumor-associated macrophages (TAMS) refer to macrophages that infiltrate the tumor microenvironment, primarily due to chemotactic of monocytes in the blood by chemokines in the tumor microenvironment. According to the difference of immune response, TAMs can be divided into two types of classical activation type (M1 type) and alternative activation type (M2 type), wherein M1 type plays a role in proinflammatory and antitumor, and M2 type plays a role in anti-inflammatory and tumor promotion. Numerous studies have demonstrated that TAMs tend to be more polarized to the M2-like phenotype, with the ability to promote tumor progression and accelerate tumor malignant progression. At present, immunotherapy targeting TAMs is becoming a hot spot in tumor therapy research, wherein one of the research ideas is to regulate the functional phenotype of TAMs, try to reverse M2-like polarization of TAMs, and promote TAMs to be polarized to M1-like.

In CRC cells, PCSK9 has a regulatory effect on expression of lactate modified protein and MIF, and interference with PCSK9 alters expression levels of intracellular lactate modified protein and MIF, which are both associated with polarization of the TAMs phenotype. Therefore, to further verify the regulation of TAMs phenotypic polarization by PCSK9 secreted by the synthesis of CRC cells, PCSK 9-silenced CRC cells HCT116 and HT-29 were co-cultured with THP-1-derived macrophages in Transwell, and changes in macrophage M1/M2 marker were detected by RT-PCR, western blot and flow cytometry.

As shown in FIG. 1, THP-1 cells were induced with 100ng/ml PMA for 48h, the cells changed from suspension to adhesion, and the cells stopped proliferating and changed from round cells to fusiform or irregular cells, indicating that monocytes differentiated into macrophages. The RT-PCR analysis shows that the mRNA expression level of M1 and M2 marker molecules in macrophages after 48h of co-culture is obviously up-regulated, and the mRNA expression level of M2 marker molecules IL-10, IL-13, Arg-1 and TGF-beta is obviously reduced compared with the siRNA-NC group, wherein the mRNA expression level of the M1 marker molecules IL-6, IL-1A, IL-1B, CXCL9, CD64 and TNF-alpha of the macrophages in the siRNA-PCSK9 group is obviously up-regulated, and the mRNA expression level of the M2 marker molecules IL-10, IL-13, Arg-1 and TGF-beta is obviously reduced (figure 2). Protein expression levels of macrophage M1 marker iNOs and M2 marker CD163 after 48h of co-culture were measured by Western blot, and the results showed that the expression of M1 marker iNOs was up-regulated and the expression of M2 marker CD163 was down-regulated in the siRNA-PCSK9 group compared to the siRNA-NC group. The flow cytometry detects the positive expression rate of macrophage M1 type marker CD86 after 48h of co-culture, and the result shows that the positive rate of the siRNA-PCSK9 group CD86 is obviously improved compared with the siRNA-NC group (figure 2), which indicates that the number of M1 type macrophages is increased. These experimental results confirm that PCSK9 secreted by the synthesis of CRC cells is involved in the regulation of the phenotypic polarization process of TAMs, PCSK9 promotes the polarization of TAMs to M2 type, and the expression of PCSK9 in CRC cells is down-regulated to inhibit the M2-like polarization of TAMs and promote the M1-like polarization of TAMs.

2. Regulation of lactate metabolism by PCSK9

Metabolic reprogramming is one of the important markers for cancer cells, which increase glucose uptake and produce more lactate even under aerobic conditions. Cancer cells export lactate to prevent intracellular acidification, not only increasing lactate levels but also creating an acidic PH in the extracellular environment. Lactic acid in a tumor microenvironment is used as a signal molecule, has special effects of promoting tumor proliferation and growth, and has important effects on tumor metastasis, angiogenesis, immunologic escape, chemotherapy drug resistance and the like. Lactic acid accumulation in solid tumors is a key and early event in the development of malignancies.

As the results of proteomics analysis indicate that PCSK9 is closely related to various intracellular metabolic processes, and the lactic acid modification as a protein posttranslational modification mode newly discovered in recent two years has a non-negligible effect in tumor progression, and in addition, the aerobic glycolysis phenomenon in tumor cells can cause the generation of a large amount of lactic acid to inhibit anti-tumor immunity, we discuss whether PCSK9 has a certain regulation effect on the lactic acid level in CRC cells. Western blot results show that after PCSK9 in CRC cells is knocked down, the levels of lactic acid modified proteins of HCT116 and HT-29 cells are obviously reduced; in contrast, PCSK9 was upregulated in CRC cells and lactate modified protein levels were significantly elevated (fig. 3). While the knock-down of PCSK9 inhibited the level of modification of protein lactate in HCT116 cells, reduced lactate production in the cell supernatant, and inhibited the expression of the glycolytic key enzyme TPI1 in the cells, therefore, the knock-down of PCSK9 reduced the glycolytic level of colon cancer cells (fig. 4).

3. PCSK9 expression levels in tumor tissues of colon cancer patients and PCSK9 vs Apc ^Min/+ Regulation of colon tumor growth in mice

Randomly spotting and observing immunohistochemical results under a 40 × microscope and a 400 × microscope respectively, as shown in the figure, PCSK9 is mainly expressed in cytoplasm, and the expression intensity in the tissues is cancer tissue > cancer adjacent tissue > distal tissue in turn; and further performing chi-square test analysis on the expression difference of the PCSK9, and finding that the expression level of the PCSK9 in the cancer tissue is higher than that of adjacent tissues and remote tissues beside the cancer, the difference has statistical significance, and the result is consistent with the result of TCGA database search. While PCSK9 was expressed in the near tissue at the precancerous site at slightly higher levels than in the distant tissue, there was no statistical difference (fig. 5).

As described above, at the cellular level in vitro, PCSK9 levels in HCT116 and SW620 of colon cancer cells were downregulated using siRNA, and it was found that there was no significant change in the proliferation and clonogenic capacity of colon cancer cells, but down-regulation of PCSK9 in colon cancer cells promoted a phenotypic shift of M2 to M1 in macrophages co-cultured therewith. In addition, with Apc ^Min/+ Mouse comparison, Apc ^Min/+ PCSK9(KI) mice progressed faster with colonic tumors increasing from 16.7% to 83.3% and PCSK9 overexpression significantly increased the levels of M2-type TAMs molecular markers in tumor tissues, decreased the levels of M1-type TAMs molecular markers, whereas PCSK9 inhibitor evocumcolumn reversed the above effect (figure 5). At the same time, PCSK9 knockdown inhibited colon cancer cell invasion metastasis in vitro cultures (fig. 6).

The above embodiments are merely illustrative of the technical concepts and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

SEQUENCE LISTING

<110> Jinan City center Hospital

<120> application of PCSK9 in macrophage M2 type polarization and related diseases thereof

<130>

<160> 4

<170> PatentIn version 3.3

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Claims

1. Use of a substance that detects the PCSK9 gene and its expression products in the manufacture of a product for diagnosing, detecting, monitoring or prognosticating the progression of colorectal cancer.

2. The application of a substance for inhibiting the reduction of the PCSK9 gene and the expression product and/or activity thereof in at least one of the following a1) -a 6):

a2) inhibiting the tumor cell Warburg effect or preparing a product for inhibiting the tumor cell Warburg effect;

a5) products that inhibit tumor growth or inhibit tumor growth;

a6) treating tumor or preparing tumor treating product.

3. The use of claim 2, wherein the product is a medicament or a test agent for use in basic research.

4. Use according to claim 2, wherein the tumour is a solid tumour, preferably colorectal cancer;

preferably, the tumor cells are human colorectal cancer cells HCT116 and HT-29; the macrophage is THP-1 derived macrophage.

5. The application of claim 2, wherein the application is: the application of the substance for inhibiting the PCSK9 gene and the expression product and/or activity reduction thereof in preparing products for inhibiting the TAMs M2-like polarization and promoting the TAMs M1-like polarization in a tumor cell-macrophage co-culture system;

6. The use according to any one of claims 2 to 5 wherein the agent which inhibits the PCSK9 gene and expression products and/or reduction in activity thereof comprises an RNA interfering molecule or antisense oligonucleotide directed to PCSK9, a small molecule inhibitor, shRNA, small interfering RNA, an agent which effects lentiviral infection or gene knock-out, and an antibody specific for PCSK9 itself or a molecule upstream or downstream thereof, including anti-PCSK 9 antibody;

wherein the sequence of the small interfering RNA is shown as SEQ ID NO. 1-2;

the anti-PCSK 9 antibody comprises eloitumumab.

7. The application of a substance for inhibiting the reduction of the PCSK9 gene and the expression product and/or activity thereof in preparing products;

the function of the product is any one or more of the following:

a2) inhibiting the Warburg effect of tumor cells;

a3) inhibiting the formation of tumor cell lactic acid;

a4) inhibiting tumor cell invasion and metastasis;

a5) inhibiting tumor growth;

a6) and (3) treating tumors.

8. The use of claim 7, wherein the product is a medicament or a test agent for use in basic research.

9. The use according to claim 7, wherein the tumour is a solid tumour, further a colorectal cancer;

the substances for inhibiting the PCSK9 gene and the expression products and/or activity reduction thereof comprise RNA interference molecules or antisense oligonucleotides against PCSK9, small molecule inhibitors, shRNA, small interfering RNA, substances for performing lentiviral infection or gene knockout, and specific antibodies against PCSK9 itself or molecules downstream thereof;

wherein the sequence of the small interfering RNA is shown as SEQ ID NO. 1-2;

the anti-PCSK 9 antibody comprises eloitumumab.

10. The use according to claim 7, wherein when the product is a medicament, the medicament further comprises at least one pharmaceutically inactive ingredient.