CN115094134B

CN115094134B - Application of PCSK9 in macrophage M2 type polarization and related diseases thereof

Info

Publication number: CN115094134B
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: 2023-06-30
Anticipated expiration: 2042-04-13
Also published as: CN115094134A

Abstract

The invention provides an application of PCSK9 in macrophage M2 type polarization and related diseases thereof, belonging to the technical fields of biological medicine and molecular biology. The invention discovers that the down regulation of PCSK9 in colon cancer cells or the use of PCSK9 inhibitor can promote M2 to M1 phenotype transition of macrophages co-cultured with the PCSK9 inhibitor and inhibit invasion and metastasis of colon cancer cells, and the mechanism of the down regulation of PCSK9 inhibitor is possibly related to the inhibition of the Warburg effect (glycolysis) of colon cancer cells and the inhibition of lactic acid formation. The research shows that the PCSK9 inhibitor can be used as a macrophage phenotype regulator, and can inhibit the growth of tumors by inducing the phenotype polarization of tumor-related macrophages from M2 to M1, 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 fields of biological medicine and molecular biology, and particularly relates to application of PCSK9 in macrophage M2 type polarization and related diseases thereof.

Background

The information disclosed in the 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 admission or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.

Colorectal cancer (colorectal cancer, CRC) is a common malignant tumor of the digestive system, and the latest cancer burden data worldwide in 2020 shows that the incidence rate and the death rate of colorectal cancer are three first in the world, and the new incidence rate of colorectal cancer in China becomes the second largest cancer next to lung cancer. In addition, patients with metastatic colorectal cancer have poor prognosis, severely threatening the health of humans. The pathogenesis of colorectal cancer is complicated and is not completely clear up to now. With the recent progress in research, new therapeutic regimens continue to emerge, and the overall prognosis and recurrence rate of metastatic CRC patients are still not ideal, although the median survival of colorectal cancer patients is somewhat prolonged. Therefore, it is necessary to search for new potential therapeutic targets by further studying the molecular mechanisms of colorectal cancer occurrence and development.

Currently, in tumor therapy, means for targeting macrophages (TAMs) in the tumor microenvironment are of increasing interest. Tumor cells release signals such as chemokines and cytokines, recruit macrophages and induce the macrophages to differentiate into TAMs, thereby promoting tumor invasion, metastasis and immune escape, and treatments with TAMs as targets are mainly focused on inhibiting TAMs recruitment, inducing TAMs polarization, TAMs elimination and the like. The article published in Cancer cell in 2017, 12, indicates that complete inhibition or elimination of TAMs in tumors causes a massive 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 targeting TAMs phenotypic polarization may be a better choice.

TAMs have been shown to be highly plastic cells and the antitumor effect can be enhanced by modulating the conversion of TAMs from the M2 phenotype to the M1 phenotype. The M1 type TAMs can distinguish tumor cells from normal cells and play an anti-tumor role through an immune recognition function; and the M2 type TAMs are involved in the cancer promotion processes such as tumor growth, invasion and metastasis, immunosuppression, chemotherapy drug resistance and the like. Studies have shown that TAMs in colon cancer patients do not consist of a simple M1 or M2 phenotype, and that the function of TAMs in colon cancer progression depends on tumor stage, TAMs type, and the duty cycle of M1, M2 typing. Therefore, if certain key processes of polarization of TAMs can be reasonably interfered, and the TAMs are regulated to develop to the M1 phenotype polarization in a proper colon cancer development period, the M2 TAMs can be promoted to abandon the cancer promotion property, so that the offset polarization imbalance is turned, and a new thought is provided for treating colon cancer.

Proprotein convertase subtilisin 9 (proprotein convertase subtilisin/kexin type 9, PCSK 9) is a member of the proprotein convertase family, encoded by the PCSK9 gene. This gene was originally discovered to encode neuronal apoptosis-related invertase 1 and is now widely recognized as a key regulator of low density lipoprotein metabolism, involved in blood lipid metabolism and maintenance of cholesterol homeostasis in vivo. It is noted, however, that PCSK9 is not only involved in blood lipid metabolism, but is also involved in a number of other biological processes including cell cycle, inflammation, apoptosis, and viral infection. In recent years, as the relationship between cholesterol metabolism and tumors has been studied intensively, researchers have begun to look at the role PCSK9 plays in the occurrence and progression of cancer. Several studies have shown that PCSK9 may have a regulatory role in a variety of tumors such as hepatocellular carcinoma, lung cancer, breast cancer, prostate cancer, etc., and is associated with risk of developing cancer and prognosis in patients. However, these studies on the relationship of PCSK9 to tumors are controversial, and furthermore, there are few studies on PCSK9 in colorectal cancer, and most of these studies are based on preclinical studies, so that more intensive studies are required to elucidate the role of PCSK9 in colorectal cancer and its molecular mechanism.

Disclosure of Invention

In view of the shortcomings in the prior art, the invention aims to provide the application of PCSK9 in macrophage M2 type polarization and related diseases. The invention discovers that the down regulation of PCSK9 in colon cancer cells or the use of PCSK9 inhibitor can promote M2 to M1 phenotype transition of macrophages co-cultured with the PCSK9 inhibitor and inhibit invasion and metastasis of colon cancer cells, and the mechanism of the down regulation of PCSK9 inhibitor is possibly related to the inhibition of the Warburg effect (glycolysis) of colon cancer cells and the inhibition of lactic acid formation. Studies show that the PCSK9 inhibitor can be used as a macrophage phenotype modulator to inhibit tumor growth by inducing the phenotype polarization of tumor-associated macrophages from M2 to M1. Based on the above results, the present invention has been completed.

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

In a second aspect of the invention there is provided the use of a substance which inhibits PCSK9 gene and its expression products and/or reduced activity in at least one of the following a 1) to a 6):

a1 Inhibiting TAMs M2-like polarization, promoting TAMs M1-like polarization or preparing a product that inhibits TAMs M2-like polarization, promoting TAMs M1-like polarization;

a2 Inhibition of the tumor cell Warburg effect (glycolysis) or the preparation of a product that inhibits the tumor cell Warburg effect (glycolysis);

a3 Inhibiting tumor cell lactic acid formation or preparing a product that inhibits tumor cell lactic acid formation;

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

a5 A product that inhibits tumor growth or inhibits tumor growth;

a6 Tumor treatment or preparation of a product for tumor treatment.

The product may be a drug or an experimental reagent that may be used for basic research. For example, the product can be used for in vitro induced regulation of macrophage polarization, so that an efficient and economical macrophage polarization experimental model is established; also, for example, the product can be used for inhibiting the Warburg effect (glycolysis) of tumor cells in vitro, thereby inhibiting the lactic acid formation of the tumor cells, and further researching the relationship between the lactic acid formation and the related mechanism and tumorigenesis.

Wherein the tumor is a solid tumor, further colorectal cancer. In a specific embodiment of the invention, the tumor cells are human colorectal cancer cells HCT 116 and HT-29; the macrophage is THP-1 source macrophage.

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

The tumor cells are human colorectal cancer cells HCT 116 and HT-29; the macrophage is THP-1 source macrophage.

In a third aspect of the invention, there is provided the use of a substance which inhibits the PCSK9 gene and its expression products and/or reduced 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, promoting TAMs M1-like polarization;

a2 Inhibition of tumor cell Warburg effect (glycolysis);

a3 Inhibiting tumor cell lactate formation;

a4 Inhibiting tumor cell invasion and metastasis;

a5 Inhibiting tumor growth;

a6 Tumor treatment).

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

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

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

the above technical scheme reports for the first time that down-regulating PCSK9 in colon cancer cells or using a PCSK9 inhibitor can promote M2 to M1 phenotype transition of macrophages co-cultured with the PCSK9 inhibitor and inhibit invasion and metastasis of colon cancer cells, and the mechanism of the PCSK9 inhibitor is possibly related to blocking the Warburg effect (glycolysis) of colon cancer cells and inhibiting lactic acid formation. Studies show that the PCSK9 inhibitor can be used as a macrophage phenotype modulator to inhibit tumor growth by inducing the phenotype polarization of tumor-associated macrophages from M2 to M1.

The technical scheme provides a new mechanism research for colorectal cancer occurrence and development and a promising treatment strategy for colorectal cancer patients, so that the method has good potential practical application value.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.

FIG. 1 shows that PMA induces THP-1 cells to differentiate into macrophages in the examples of the present invention.

FIG. 2 is a graph showing the effect of PCSK9 on the modulation of the phenotypic polarization of TAMs in examples of the present invention; RT-PCR to detect the mRNA expression level of the M1 and M2 macrophage marker molecules after the CRC cells and THP-1 source macrophages are co-cultured for 48 h; c, d, detecting protein expression levels of an M1 marker iNOS and an M2 marker CD163 after co-culturing CRC cells and THP-1 source macrophages for 48 hours by using a western blot; e, detecting the expression condition of the M1 macrophage surface differentiation antigen CD86-AF647 after the CRC cells and the THP-1 source macrophages are co-cultured for 48 hours by a flow cytometry method. All experiments were repeated 3 times, the experimental results were represented by mean ± standard error (mean ± SEM), and comparisons between the two groups were performed using a two-tailed t-test, P <0.05 was considered statistically significant; * P <0.05; * P <0.01; * P <0.001.

FIG. 3 shows that PCSK9 expression levels affect the levels of lactate-modified protein in CRC cells in an example of the invention; wherein, after down-regulating the expression of CRC cell PCSK9, HCT 116 and HT-29 lactate modified protein level is obviously reduced; after up-regulating the expression of CRC cell PCSK9, HCT 116 and HT-29 lactate modified protein levels are obviously increased. All experiments were repeated 3 times, the experimental results were represented by mean ± standard error (mean ± SEM), and comparisons between the two groups were performed using a two-tailed t-test, P <0.05 was considered statistically significant; * P <0.05; * P <0.01; * P <0.001.

FIG. 4 is a graph showing glycolysis level detection in an embodiment of the present invention; wherein, lactic acid detection kit analysis finds that knocking down PCSK9 significantly reduces the level of lactic acid product in colon cancer cell culture supernatant, p <0.01; WB detection found that knockdown of PCSK9 inhibited the expression level of the key enzyme of glycolysis TPI1 in colon cancer cells, consistent with proteomic detection results.

FIG. 5 shows the expression level of PCSK9 and the PCSK 9-to-Apc ratio in tumor tissue of a colon cancer patient according to an embodiment of the invention ^Min/+ Regulation and control effect of colon tumor growth of mice; wherein A is the detection of cancer, paracancerous and distal tissues (more than 5cm from cancer tissues) in pathological tissue specimens of 75 colon cancer patients by an Immunohistochemical (IHC) method The result is consistent with the TCGA database search result. PCSK9 is expressed at higher levels in cancer tissues than in paracancerous and distant tissues (p<0.05 No significant difference in expression levels between paracancerous and distant tissues; b, C, D are mice over-expressing PCSK9 and Apc ^Min/+ The mice are hybridized and bred to obtain Apc ^Min/+ PCSK9 (KI) mice, PCSK9 overexpression was found to promote Apc ^Min/+ Growth and malignant transformation of colon tumor of mice; and to Apc ^Min/+ Mice were subcutaneously dosed with the PCSK9 inhibitor Evolocumab and inhibition of PCSK9 was found to reduce Apc ^Min/+ Number, size, and malignancy of colon tumors in mice. E is IHC method detection to find that PCSK9 over-expression increases the level of M2 type TAMs molecular markers CD206 and Arg1 in colon tumor tissue, reduces the level of M1 type molecular markers CD64 and CCR7, and controls Apc ^Min/+ Mice reverse this effect (200×), p using subcutaneous injection of the PCSK9 inhibitor Evolocumab<0.05，**p<0.01。

FIG. 6 shows PCSK9 knockdown in an example of the invention inhibiting invasion and metastasis of colon cancer cells cultured in vitro.

Detailed Description

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the invention. 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 exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.

The invention will now be further illustrated with reference to specific examples, which are given for the purpose of illustration only and are not intended to be limiting in any way. If experimental details are not specified in the examples, it is usually the case that the conditions are conventional or recommended by the reagent company; reagents, consumables, etc. used in the examples described below are commercially available unless otherwise specified.

In one exemplary embodiment of the invention, there is provided the use of a substance for detecting PCSK9 gene and its expression products in the manufacture of a product for diagnosing, detecting, monitoring or predicting the progression of colorectal cancer.

The product is capable of diagnosing, detecting, monitoring or predicting the progression of colorectal cancer by detecting the expression level of the PCSK9 gene and/or PCSK9 gene expression product (e.g., PCSK9 protein). Immunohistochemistry (IHC) method detected PCSK9 expression in cancer, paracancerous and distant tissues (greater than 5cm from cancerous tissue) in pathological tissue specimens of 75 colon cancer patients. PCSK9 was expressed at higher levels in the cancer tissues than in the paracancerous and distal tissues (p < 0.05), with no significant difference in expression levels between the paracancerous and distal tissues.

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

In yet another embodiment of the invention, transcription of PCSK9 in colorectal cancer samples is detected using techniques including, but not limited to, liquid phase hybridization, northern hybridization methods, miRNA expression profiling chips, ribozyme protection analysis techniques, RAKE methods, in situ hybridization; PCSK9 protein in colon cancer samples is detected using a protocol including, but not limited to, immunohistochemistry (IHC), ELISA, colloidal gold test strips, and protein chips.

In yet another embodiment of the present invention, there is provided the use of a substance inhibiting PCSK9 gene and its expression products and/or reduced activity in at least one of the following a 1) to a 6):

a5 A product that inhibits tumor growth or inhibits tumor growth;

a6 Tumor treatment or preparation of a product for tumor treatment.

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

Wherein the substances that inhibit PCSK9 gene and its expression products and/or activity reduction include, but are not limited to, RNA interference molecules or antisense oligonucleotides against PCSK9, small molecule inhibitors, shRNA (small hairpin RNA), small interfering RNA (siRNA), substances that perform lentiviral infection or gene knockout, and specific antibodies against PCSK9 itself or its upstream and downstream molecules, including anti-PCSK 9 antibodies (e.g., ebo You Shan anti-Evolocumab).

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

In yet another embodiment of the invention, there is provided the use of a substance that inhibits PCSK9 gene and its expression products and/or reduced activity in the preparation of a product.

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

a1 Inhibiting TAMs M2-like polarization, promoting TAMs M1-like polarization;

a2 Inhibition of tumor cell Warburg effect (glycolysis);

a3 Inhibiting tumor cell lactate formation;

a4 Inhibiting tumor cell invasion and metastasis;

a5 Inhibiting tumor growth;

a6 Tumor treatment).

The product may be a drug or an experimental reagent that may be used for basic research.

Wherein the substances that inhibit PCSK9 gene and its expression products and/or activity reduction include, but are not limited to, RNA interference molecules or antisense oligonucleotides against PCSK9, small molecule inhibitors, shRNA (small hairpin RNA), small interfering RNA (siRNA), substances that perform lentiviral infection or gene knockout, and specific antibodies against PCSK9 itself or its upstream and downstream molecules, such as anti-PCSK 9 antibodies (e.g., ebo You Shan anti-Evolocumab).

The sequence of the small interfering RNA is shown as 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. Further, the composition can be formulated into various dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, etc., for oral administration, external use, suppositories, and sterile injectable solutions according to a usual method.

The non-pharmaceutically active ingredients, such as carriers, excipients and diluents, which may be included, are well known in the art and can be determined by one of ordinary skill in the art to meet clinical criteria.

In yet another embodiment of the present invention, the carriers, excipients and diluents include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil and the like.

In yet another embodiment of the invention, the medicament of the invention may be administered to the body in a known manner. For example, by intravenous systemic delivery or local injection into the tissue of interest. Alternatively via intravenous, transdermal, intranasal, mucosal or other delivery methods. Such administration may be via single or multiple doses. It will be appreciated 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 yet another embodiment of the present invention, the subject to be administered can be human or non-human mammal, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, gorillas, etc.

In yet another embodiment of the present invention, there is provided a method of tumor treatment, the method comprising: administering to the subject a substance that inhibits the PCSK9 gene and its expression product and/or activity is reduced.

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

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

Examples

Cytology related experiment method

1. Cell transfection

1.1siRNA transient transfection

(1) Dissolving and split charging siRNA: the PCSK9-siRNA and NC-siRNA powder were centrifuged at 2500rpm for 2 minutes to aggregate them at the bottom of the tube, the tube lid was gently opened, and RNase-free H was added ₂ O was dissolved to prepare a 20. Mu.M solution, which was dispensed into RNase-free EP tubes and stored at-20 ℃.

(2) Taking CRC cells in logarithmic growth phase, and adjusting cell concentration to 1x10 with complete culture medium ⁵ cells/ml, 2ml per well into six well plates, at 37℃with 5% CO ₂ Is cultured in an incubator for 24 hours.

(3) The next day, when the cells had grown on the wall to 30-50% confluency, the medium in the wells was aspirated, 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) According to lipo 2000 instruction, 5 μl siRNA is dissolved in 250 μl opti-mem, and the mixture is gently blown and sucked for 3-5 times and mixed uniformly; another RNase-free EP tube was prepared by dissolving 5. Mu.l lipo 2000 in 250. Mu.l opti-mem, gently sucking by air, and mixing the mixture for 3 to 5 times. Standing at room temperature for 5 minutes.

(5) Lipo 2000 and siRNA dilutions were mixed, gently swirled and mixed, and left to stand at room temperature for 20 minutes.

(6) The transfection compound is dripped into a six-hole plate, the six-hole plate is gently shaken and is evenly mixed, and then the mixture is placed into an incubator, and fresh complete culture medium can be replaced after 4 to 6 hours. Cells are collected 24h after transfection for functional experiments, RNA is extracted 24-48h, and protein is extracted 48-72 h.

siRNA sequence:

1.2 transient transfection of plasmids

(1) Dissolution of plasmid: the plasmid DNA and the control plasmid empty vector were centrifuged at 10000rpm for 10-15s and then carefully opened, and a proper amount of ddH was added to each ₂ O is fully vibrated and dissolved to prepare 1 mug/mul solution, and the solution is properly packaged and stored at the temperature of minus 20 ℃.

(2) Taking CRC cells in logarithmic growth phase, and adjusting cell concentration to 2×10 with complete culture medium ⁵ cells/ml, 2ml per well, were seeded into six well plates and cultured in an incubator at 37℃with 5% CO2 for 24 hours.

(3) The next day, when the cell wall-attached growth reached 70-90% confluency, the medium in the wells was aspirated, each well was washed with serum-free McCOY's 5A medium, and then 1.5ml serum-free McCOY's 5A medium was added to each well.

(4) According to lipo 2000 instruction, 4. Mu.g plasmid DNA is dissolved in 250. Mu.l opti-mem, and the mixture is gently blown and sucked for 3 to 5 times and mixed uniformly; another sterile EP tube was taken and 10. Mu.l lipo 2000 was dissolved in 250. Mu.l opti-mem and gently sucked 3-5 times for mixing. Standing at room temperature for 5 minutes.

(5) Lipo 2000 and plasmid DNA dilutions were mixed, gently swirled and mixed, and allowed to stand at room temperature for 20 minutes.

(6) The compound is gently dripped into a six-hole plate, the six-hole plate is gently shaken, and the mixture is placed into an incubator after being evenly mixed, and fresh complete culture medium can be replaced after 4-6 hours. Cells are collected 24h after transfection for functional experiments, RNA is extracted 24-48h, and protein is extracted 48-72 h.

1.3 establishment of stably silenced PCSK9 cell lines

(1) Lentiviruses were purchased from hanheng biotechnology (Shanghai) limited.

(2) CRC cells grown in log phase with good status are adjusted to 3x10 concentration ⁵ cells/ml, 500. Mu.l each well was inoculated in 24 well plates at 37℃in 5% CO ₂ Culturing overnight in an incubator.

(3) The next day, when the cell growth density reaches 30-50%, the virus infection is carried out by adopting a 1/2 small-volume infection method. The virus was removed from the freezer and slowly thawed on ice, the original medium of the cells was aspirated, and 250. Mu.l fresh complete medium (containing 5. Mu.g/ml polybrene) was added. Infection was performed by adding an appropriate volume of virus stock according to moi=10. Viral load per well (μl) =moi x cell number/viral titer (TU/ml) x 1000. The culture medium was replenished to 500. Mu.l 4h after lentiviral infection.

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

(5) After 48-72 hours of infection, fluorescence was observed by a fluorescence microscope to preliminarily determine the infection efficiency.

(6) After the cell state is stable, 2.0 mug/ml of puromycin (HCT 116) or 8.0 mug/ml of puromycin (HT-29) is added, and the stable infected cell strain can be obtained after 3-4 weeks of screening. Subsequent experiments were performed after the amplification culture.

2. Co-culture of cells

2.1 Induced differentiation of THP-1 cells

Collecting THP-1 cell suspension, and adjusting cell concentration to 7×10 ⁵ cells/ml, 2ml per well, were seeded in 6-well plates, stimulated with 100ng/ml PMA for 48h, and allowed to differentiate into macrophages. After washing twice with PBS 2ml of fresh RPMI-1640 complete medium was replaced for the subsequent experiments.

2.2 Co-culture of CRC cells and macrophages

In vitro cell co-culture experiments were performed in a Transwell co-culture chamber (6-well, 0.4um pore size). Gently place the co-culture chamber with sterile forceps into 6-well plates containing THP-1-derived macrophages; after 24h transfection of HCT 116 and HT-29siRNA, cells were collected, resuspended in RPMI-1640 complete medium and adjusted to a cell concentration of 2 x 105cells/ml, 1.8ml of cell suspension per well was added to the upper layer of the chamber, the lid of the 6 well plate was closed, and placed in 37℃C, 5% CO ₂ Is cultured in an incubator of (a). After 48h co-culture, macrophages were collected for correlation detection.

2.3 RT-PCR

2.3.1 extraction of Total RNA

(1) The cell culture plates were removed, the medium discarded, and washed 2 times with pre-chilled PBS.

(2) 1ml of Trizol lysate was added to each well, and the cells were repeatedly lysed by blowing, and allowed to stand at room temperature for 5min.

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

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

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

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

(7) 1ml of 75% ethanol was added to each tube, the tube bottom was flicked off to precipitate, centrifuged at 12000rpm at 4℃for 5min, and this step was repeated 3 times.

(8) Discarding supernatant, retaining precipitate, inverting on absorbent paper, air drying, adding appropriate amount of RNase-free H ₂ O dissolves RNA.

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

2.3.2 reverse transcription reactions

The reverse transcription reaction was performed using Evo M-MLV reverse transcription kit II (AG). All reaction mixtures were formulated on ice.

(1) Removing genomic DNA, preparing a reaction solution according to the following table, and performing a genomic DNA removal reaction.

Reaction conditions: 42 ℃ for 2min

4℃

(2) Reverse transcription: the reaction solution was prepared in accordance with the following table, and the reverse transcription reaction was performed.

Reaction conditions: 37 ℃ for 15min

85℃ 5sec

4℃

The cDNA of the reverse transcription product can be directly used for the subsequent PCR reaction, and can also be preserved at the temperature of minus 20 ℃ for standby.

2.3.3 RT-PCR reaction

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

The operation was performed on ice, taking care of light protection.

(1) The PCR primer is designed and synthesized by Qingdao biological science and technology Co., ltd, and the primer sequence is as follows:

(2) The following reaction system (20 μl) was prepared:

reaction conditions:

(3) Calculating the expression quantity: utilization 2 ^-ΔΔCT The difference in gene expression was calculated by the method.

2.4 Western blot

2.4.1 extraction of cell Total proteins

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

(2) 150 μl RIPA lysate containing protease phosphatase inhibitor cocktail was added to each well and lysed on ice for 15min.

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

(4) The protein supernatant was collected into another new EP tube, and 1/5 volume of 5 Xloading buffer was added to denature the protein in a water bath at 100℃for 10 min.

(5) Preserving at-80 ℃ for standby.

2.4.2 Protein concentration determination by BCA method

(1) Dilution of BSA Standard was diluted to 1mg/ml according to the kit instructions and stored for a long period of time at-20 ℃.

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

(3) Protein concentration determination: a standard curve was prepared by adding 200. Mu.l BCA working solution to each well and adding BSA standard solution as shown in the following Table. Sample wells were filled with 10. Mu.l protein sample per well and 10. Mu.l PBS was added. Incubate at 37℃for 30min and measure absorbance at 562nm with a microplate reader. The protein concentration of the samples was calculated from the standard curve.

2.4.3 SDS-PAGE gel electrophoresis

(1) Washing the glass plate with distilled water, and airing for standby.

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

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

the 5% concentrate was prepared as follows (4 ml for example, unit: ml):

(3) And (3) glue preparation: after the separation gel was mixed, about 7ml of the separation gel was rapidly added to the middle of the clamped glass plate, and 1ml of isopropyl alcohol was added to press the gel. After the separation gel is solidified, the isopropanol is discarded, and the comb is vertically inserted immediately after the concentrated gel is filled up, so that bubbles are avoided.

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

(5) Electrophoresis: the electrophoresis tank is filled with 1x electrophoresis buffer solution and subjected to 80V constant-pressure electrophoresis. After the sample enters the separation gel, the sample is adjusted to 120V, and constant pressure electrophoresis is carried out until the target protein is completely separated.

(6) Transferring: the gel containing the band of interest was cut according to the marker molecular weight. PVDF membrane cut to corresponding size is put into methanol for activation for 1min, and then put into membrane transfer buffer solution for standby. The membrane clips were assembled in the order of negative electrode (black), sponge, 3 layers of filter paper, gel, PVDF membrane, 3 layers of filter paper, sponge, positive electrode (white), taking care that no air bubbles were present between the gel and the membrane. And placing the film transfer clamp into a film transfer groove, and filling film transfer liquid. Constant-current 250mA ice bath film transfer is carried out, and the film transfer time is determined according to the molecular weight of the protein.

(7) Closing: after the film transfer is completed, marking the PVDF film, putting the PVDF film into 5% skimmed milk powder, and sealing for 1.5 hours at room temperature, wherein the sealing process is carried out on a shaking table.

(8) Incubating primary antibodies: after the completion of the blocking, the eluate of 1 XTBE was washed 3 times 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 anti-dilution ratio was determined according to the antibody instructions.

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

(10) Color development: after the antibody incubation was completed, the antibody was washed 3 times with 1 XTBST eluate for 10min each. The ECL chemiluminescent color-developing solution A, B was mixed in equal amounts under light-protected conditions. The PVDF film is placed on an operation table of a gel imager, a proper amount of color development liquid is dripped to start exposure, the recorded result is observed, and the gray value analysis is carried out by using Image J.

2.5 flow cytometry

After 48h of co-culture of THP-1-derived macrophages with CRC cells, different groups of macrophages were collected. Cells were washed twice with PBS and resuspended in 100. Mu.l PBS to make a single cell suspension. According to the antibody instructions, 5. Mu.l of the corresponding antibody labeled with fluorescent substance and isotype control thereof were added to each tube, gently mixed, and incubated at 4℃for 30min in the absence of light. After washing the cells twice with PBS, the cells were resuspended with 300-500. Mu.l PBS. Filtering with 300 mesh nylon net, and loading onto machine to detect cell surface differentiation antigen expression.

2.6 invasion and metastasis experiments

2.6.1 scratch test

(1) Cell concentration was adjusted to 6X 10 by resuspension after cell digestion and centrifugation ⁵ cells/ml, 2ml of cell suspension per well, was seeded in 6-well plates and when cells were full, scored "cross" at the bottom of the plates with a sterile 200ul gun head.

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

(3) The scratch condition is observed under a microscope, and the scratch healing degree is recorded by photographing for 0h and 48h.

2.6.2 Transwell migration experiment

(1) Cell digestion centrifugation with serum-free McCOY' sThe 5A medium resuspended cells and the cell concentration was adjusted to 5 x 10 ⁵ cells/ml (HCT 116) or 7.5 x 10 ⁵ cells/ml(HT-29)。

(2) The Transwell chamber (24 wells, 8 μm pore size) was removed, 600. Mu.l of medium containing 15% FBS was added to the lower chamber, 200. Mu.l of cell suspension was added vertically to the upper chamber, and no air bubbles were noted between the upper and lower chambers. Placing into an incubator for culturing for 48 hours.

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

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

(5) 5 fields were randomly selected under a 200 x microscope to take pictures and counted.

2.6.3 Transwell invasion assay

(1) The matrigel was taken out of the-20 ℃ refrigerator in advance and placed on ice for melting, while pre-cooling the gun head and EP tube. Diluting the melted matrigel with serum-free culture medium according to the ratio of 1:8, sucking 100 μl of diluted matrigel, spreading onto the upper layer of a Transwell cell (24-hole, 8 μm aperture), standing in an incubator for half an hour, and removing gel without sucking, and standing in the cell for use.

(2) Cell digestion centrifugation, resuspension of cells with serum-free McCOY's 5A medium and adjustment of cell concentration to 5.multidot.10 ⁵ cells/ml (HCT 116) or 7.5 x 10 ⁵ cells/ml(HT-29)。

(3) The chamber was removed, 600. Mu.l of medium containing 15% FBS was added to the lower chamber, 200. Mu.l of cell suspension was added vertically to the upper chamber, and no air bubbles were noted between the upper and lower chambers. Placing into an incubator for culturing for 48 hours.

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

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

(7) 5 fields were randomly selected under a 200 x microscope to take pictures and counted.

Animal science related experimental method

1. Experimental animal

1.1 animal hybridization propagation

The sexual maturity 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/+ Male mice were caged and offspring mice were bred by hybridization. Ear tag marking is carried out on newborn mice until the newborn mice reach 4 weeks of age, and Apc required by experiments is screened out through a genotyping method ^Min/+ And Apc ^Min/+ PCSK9 (KI) mice.

1.2 animal identification

1.2.1DNA extraction

2-3mm of the tip of the tail of the mouse is cut and put into a 1.5mL EP tube; to an EP tube containing the tail of the mouse, 500. Mu.L of lysate and 5. Mu.L of proteinase K (10 mg/mL) were added; sealing the EP pipe by using a sealing film (preventing the EP pipe from being heated to open so as to reduce the cracking liquid), placing the EP pipe on an EP pipe frame, and then placing the EP pipe on a constant-temperature water bath (55 ℃) for overnight digestion; taking out the EP pipe the next day, shaking forcefully, and uniformly mixing; adding 500 mu L of a mixed solution of phenol and chloroform (1:1) into each tube, forcibly mixing, and centrifuging at 12000rpm for 15min; about 300. Mu.L of supernatant was aspirated from the centrifuged EP tube into a fresh EP tube using a 1000. Mu.L pipette and the number was clearly indicated; 100% ethanol, 2 times the volume of the supernatant (about 600. Mu.L), was added to the tube, gently mixed (turned upside down several times) until white filamentous DNA was seen, centrifuged at 12000rpm for 10min, and the supernatant was discarded; adding 800 mu L of 70% ethanol into the tube, and fully and uniformly mixing; centrifuging at 12000rpm for 5min, discarding supernatant, standing upside down on filter paper, sucking residual ethanol, and air drying (t >30 min) until no alcoholic smell; 100. Mu.L of TE Buffer was added to the EP tube, and the mixture was shaken at 37℃to dissolve the DNA sufficiently and store it at 4 ℃.

1.2.2 PCR amplification

(1) Apc amplification

Reaction system and program

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(2) PCSK9 amplification

Reaction system and program

1.2.3 gel electrophoresis

(1) And (3) glue preparation:

1% (2%) agarose solution: 1g (2 g) of agarose is weighed and put into a conical flask, 100mL of 1 xTAE electrophoresis buffer solution is added, and then the flask is put into a microwave oven, and the flask is heated with high fire until the solution boils (about 2 min) until the agarose is completely dissolved, thus obtaining the agarose transparent solution with the concentration of 1% (2%).

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

(2) Loading:

and taking 10 mu L of PCR amplified products, blowing and mixing uniformly, and loading the samples, wherein the samples correspond to DNA markers with equal volumes. Apc amplification products were loaded on 2% agarose gel and PCSK9 amplification products were loaded on 1% agarose gel. During loading, the overflow of the sample is avoided, 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, electrophoresis conditions (2% agar) were setSugar gel: the voltage is 90V for 45min;1% agarose gel: voltage 140v,25 min). After electrophoresis is completed, the gel is put into an imaging analysis system for scanning, and photographing and analysis are carried out. By the method, apc is screened out ^Min/+ Mice and Apc ^Min/+ PCSK9 (KI) mice, the latter being a model of PCSK9 over-expressing mice.

2. Animal test

2.1 histopathological examination

(1) Paraffin section was prepared

Fixing: the intestinal tumor tissue blocks of the mice are placed in 10% formalin solution for fixation for 24 hours;

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

and (3) transparency: 1:1 mixed solution of xylene and absolute ethanol (60 min), pure xylene 1 (30 min), pure xylene 2 (30 min);

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

embedding: preheating paraffin wax with the melting point identical to that of the hard paraffin wax 2, pouring the paraffin wax into a wax tank, and putting the tissue block after the paraffin dipping into the wax tank for cooling at room temperature;

slicing: cutting the paraffin-embedded tissue block into sections having a thickness of about 4-5 μm using a microtome;

Spreading: fully spreading the wax sheet in warm water at 45 ℃, taking out the wax sheet from the water by using a glass slide coated with a glycerin film, and drying in a 38 ℃ incubator.

(2) h.E. staining

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

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

dehydration and transparency: 70% ethanol (10 s) →80% ethanol (10 s) →90% ethanol (10 s) →absolute ethanol (10 s) →xylene 1 clear (5 min) →xylene 2 clear (5 min);

sealing piece: naturally airing the slices, dripping neutral gum, and sealing the slices with a cover glass; the sections were observed under a microscope, the nuclei were blue and the cytoplasm was pink or red.

2.2 immunohistochemical experiments

(1) Dewaxing and hydration

The chip is put in a constant temperature box at 60 ℃ for baking for 2 hours; soaking in xylene I for 10min, then soaking in xylene II for 10min, and dewaxing; sequentially soaking in anhydrous ethanol, 95% ethanol, 85% ethanol, 70% ethanol, and distilled water for 5min, and hydrating.

(2) Antigen retrieval and blocking

Heating 0.01M citric acid buffer solution with pH of 6.0 with microwave to boil, placing the chip into the boiled buffer solution, and heating with high fire for 10-15min; cooling to room temperature, washing with distilled water twice, and washing with PBS for 5min; by 3%H ₂ O ₂ The methanol solution is sealed for 10min at room temperature to eliminate the influence of endogenous catalase; washing with PBS for 3 times, each time for 5min, dripping goat serum blocking solution, and blocking at room temperature for 20min.

(3) Immunoreaction and staining

After the sealing is completed, the superfluous liquid is wiped off, the primary antibody is dripped, and the mixture is put into a wet box for 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 time for 5min, dripping secondary antibody, and incubating for 30min at room temperature; washing with PBS for 3 times, each time for 5min, dripping DAB color development liquid, and grasping the dyeing degree under a mirror; washing with distilled water to terminate dyeing; hematoxylin counterstain for 10s and hydrochloric acid ethanol solution differentiate for 5s.

(4) De-water sealing sheet

Sequentially adding into 70% ethanol, 85% ethanol, 95% ethanol and absolute ethanol, and dehydrating; sequentially placing into dimethylbenzene I and dimethylbenzene II, performing transparency; the sheet was sealed with a neutral resin, observed under a mirror, and photographed.

2.3 determination of immunohistochemical outcome

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

3. Grouping scheme for clinical colon cancer patient data

Age: two groups of less than or equal to 55 years old and more than 55 years old; gender: male and female; tumor size: two groups of less than or equal to 4cm and more than 4 cm; pathological grading: class I and class II groups; class III groups; n stage: group N0; groups N1 and N2; TNM staging: TNM1 and TNM2 groups; TNM3 group.

3.1 immunohistochemical assay methods were as before.

3.2 statistical analysis

Experimental data were statistically analyzed with SPSS 19.0 and PCSK9 was tested in chi-square for differential expression analysis in cancerous and paracancerous tissues; correlation between PCSK9 expression levels and patient clinical pathology parameters was analyzed using a chi-square test. P <0.05 is statistically significant.

Experimental results

1. Down-regulating PCSK9 expression in CRC cell can inhibit TAMs M2-like polarization and promote TAMs M1-like polarization

In CRC cells, PCSK9 can regulate the expression level of intracellular lactate modified protein and MIF protein, and lactic acid and MIF are closely related to anti-tumor immunity and participate in regulating the phenotypic polarization of TAMs in tumor microenvironment, so next we studied the effect of PCSK9 on the phenotypic polarization of TAMs.

Tumor-associated macrophages (TAMS) refer to macrophages infiltrated in the tumor microenvironment, primarily from monocytes in the blood that are chemotactic by chemokines in the tumor microenvironment. TAMs can be classified into classical active (M1) and alternative active (M2) types according to the immune response, with M1 exhibiting pro-inflammatory and anti-tumor effects and M2 exhibiting anti-inflammatory and pro-tumor effects. Numerous studies have demonstrated that TAMs have a greater tendency to polarize to an M2-like phenotype, with the ability to promote tumor progression and accelerate tumor malignancy. Currently, targeting TAMs for immunotherapy is becoming a hotspot in tumor therapy research, wherein one of the research ideas is to regulate the functional phenotype of the TAMs, try to reverse the M2-like polarization of the TAMs, and promote the TAMs to M1-like polarization.

In CRC cells, PCSK9 has a regulatory effect on the expression of lactate modified proteins and MIF, and interfering with PCSK9 expression alters the intracellular lactate modified proteins and MIF expression levels, both of which are associated with TAMs phenotype polarization. Therefore, to further verify the regulatory effect of PCSK9 secreted by CRC cell synthesis on TAMs phenotype polarization, PCSK 9-silenced CRC cells HCT 116 and HT-29 were Transwell co-cultured with THP-1 derived macrophages and changes in macrophage M1/M2 markers were detected by RT-PCR, western blot and flow cytometry.

As shown in FIG. 1, THP-1 cells were induced by 100ng/ml PMA for 48 hours, the cells changed from a suspension state to an adherent state, and the cells stopped proliferating, and changed from round cells to a spindle shape or an irregular shape, suggesting that monocytes differentiated into macrophages. RT-PCR analysis of the changes in M1 and M2 marker mRNA expression in macrophages after 48h of co-culture shows that the mRNA expression levels of M1 type marker IL-6, IL-1A, IL-1B, CXCL9, CD64 and TNF-alpha in macrophages of siRNA-PCSK9 group are obviously up-regulated and the mRNA expression levels of M2 type marker IL-10, IL-13, arg-1 and TGF-beta are obviously reduced compared with that of siRNA-NC group (FIG. 2). Protein expression levels of macrophage M1 marker iNOs and M2 marker CD163 after 48h co-culture were detected by Western blot, and the results show 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 with the siRNA-NC group. The positive expression rate of the macrophage M1 type marker CD86 after 48h of co-culture is detected by flow cytometry, and the result shows that compared with the siRNA-NC group, the positive rate of the macrophage M1 type marker CD86 of the siRNA-PCSK9 group is obviously improved (figure 2), which shows that the number of the macrophage M1 type is increased. These experimental results confirm that PCSK9 secreted by CRC cells is involved in the phenotypic polarization process that regulates TAMs, PCSK9 promotes TAMs to M2-type polarization, inhibits TAMs M2-like polarization when expression of PCSK9 in CRC cells is down-regulated, and promotes TAMs M1-like polarization.

2. Regulatory effect of PCSK9 on lactic acid metabolism

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

Since proteomic analysis results suggest that PCSK9 is closely related to intracellular various metabolic processes, and lactate modification as a post-translational modification of protein newly found in recent two years has a non-negligible effect in tumor progression, and in addition, the "aerobic glycolysis phenomenon" in tumor cells can lead to the generation of a large amount of lactic acid to inhibit anti-tumor immunity, we studied whether PCSK9 can have a certain regulatory effect on the intracellular lactic acid level of CRC cells. Western blot results show that after PCSK9 in CRC cells is knocked down, the levels of the lactate modified proteins of HCT116 and HT-29 cells are obviously reduced; in contrast, up-regulation of PCSK9 in CRC cells, the lactate-modified protein levels were significantly elevated (fig. 3). Whereas knockdown of PCSK9 inhibited the level of protein lactogenesis modification in HCT116 cells, reduced lactate production in cell supernatants, and inhibited expression of the key enzyme TPI1 in cells, thus knockdown of PCSK9 reduced the level of glycolysis in colon cancer cells (fig. 4).

3. Expression level of PCSK9 in tumor tissue of colon cancer patient and PCSK 9-Apc ^Min/+ Regulating and controlling effect of colon tumor growth of mice

The immunohistochemical results were observed at random spots under a 40 x and 400 x microscope, respectively, as shown in the figure, PCSK9 was mainly expressed in cytoplasm, and the expression intensity in tissues was cancer tissue > paracancerous tissue > far-end tissue in sequence; and then carrying out chi-square test analysis on the expression difference of PCSK9, and finding that the expression level of PCSK9 in cancer tissues is higher than that of adjacent tissues and far-end tissues beside cancer, wherein the difference has statistical significance, and the result is consistent with the retrieval result of a TCGA database. While PCSK9 was expressed at slightly higher levels in adjacent tissues beside the cancer than in distant tissues, there was no statistical difference (fig. 5).

As previously described, at the in vitro cellular level, the use of siRNA down-regulates PCSK9 levels in colon cancer cells HCT116 and SW620, and it was found that proliferation and clonogenic capacity of colon cancer cells were not significantly altered, but down-regulating PCSK9 in colon cancer cells promoted M2 to M1 phenotype transition in macrophages co-cultured therewith. In addition, with Apc ^Min/+ Apc compared to mice ^Min/+ PCSK9 (KI) mice developed more rapidly in colon tumors, with a change rate of colon adenoma carcinoma increased from 16.7% to 83.3%, PCSK9 overexpression significantly increased the level of the M2-type TAMs molecular marker in tumor tissue, decreased the level of the M1-type TAMs molecular marker, and PCSK9 inhibitor Evolocumab reversed the effect described above (fig. 5). At the same time, PCSK9 knockdown inhibited metastasis of colon cancer cells cultured in vitro (fig. 6).

The above embodiments are provided to illustrate the technical concept and features of the present invention and are intended to enable those skilled in the art to understand the content of the present invention and implement the same, and are not intended to limit the scope of the present invention. All equivalent changes or modifications made in accordance with the spirit of the present invention should be construed to be included in the scope of the present invention.

SEQUENCE LISTING

<110> Jinan City center Hospital

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

<130>

<160> 4

<170> PatentIn version 3.3

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Claims

1. Application of substances for inhibiting PCSK9 gene expression in preparing products for inhibiting TAMs M2-like polarization and promoting TAMs M1-like polarization in colorectal cancer cell-macrophage co-culture system.

2. The use according to claim 1, wherein the product is a pharmaceutical or experimental agent for use in basic research.

3. The use according to claim 1, wherein the colorectal cancer cells are human colorectal cancer cells HCT 116 and HT-29; the macrophage is THP-1 source macrophage.

4. The use according to any one of claims 1 to 3, wherein the substance that inhibits PCSK9 gene expression comprises an antisense oligonucleotide, shRNA, small interfering RNA against PCSK 9;

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