阅读说明：本技术 一种治疗结直肠癌的药物 (Medicine for treating colorectal cancer ) 是由 方媛 邓海军 李永盛 沈智勇 刘玥琛 张展侨 李振康 于 2021-05-25 设计创作，主要内容包括：本发明提供了一种治疗结直肠癌的药物,包括siRNA,所述siRNA的正义链如SEQ ID NO:1或SEQ ID NO:2所示。本发明通过基础实验在细胞水平证实干扰巨噬细胞的MS4A4A表达的siRNA可抑制巨噬细胞向M2型极化。通过体内动物实验发现,干扰MS4A4A表达的siRNA可通过抑制巨噬细胞M2型极化继而促进肿瘤浸润CD8+T细胞的活化,进而发挥抗肿瘤作用。(The invention provides a medicine for treating colorectal cancer, which comprises siRNA, wherein a sense strand of the siRNA is shown as SEQ ID NO. 1 or SEQ ID NO. 2. The invention proves that siRNA interfering MS4A4A expression of macrophages can inhibit the macrophage from polarizing to M2 type at a cellular level through basic experiments. In vivo animal experiments show that siRNA interfering with expression of MS4A4A can inhibit macrophage M2 type polarization, so that activation of tumor infiltrating CD8+ T cells is promoted, and an anti-tumor effect is further exerted.)

1. The drug for treating colorectal cancer is characterized by comprising siRNA, wherein the sense strand of the siRNA is shown as SEQ ID NO. 1 or SEQ ID NO. 2.

Use of the MS4A4A gene as a target in screening or preparing a medicament for treating colorectal cancer.

3. Use of an agent that inhibits or blocks expression of the MS4A4A gene in the manufacture of a medicament for the treatment of colorectal cancer.

4. The use of claim 3, wherein said agent comprises siRNA.

5. The use of claim 4, wherein the sense strand of the siRNA is as shown in SEQ ID NO 1 or SEQ ID NO 2.

Technical Field

The invention relates to a medicine for treating colorectal cancer.

Background

Colorectal cancer (CRC) is one of the clinically common malignancies, with global morbidity at the 3 rd and mortality at the 2 nd position of malignancy. In China, the morbidity and mortality of colorectal cancer tend to rise year by year and to be younger, and the life health of people in China is seriously threatened.

MS4A4A belongs to a member of the MS4A protein family, is specifically expressed in Tumor Associated Macrophages (TAMs), is associated with Dectin-1 receptor function in macrophage lipid rafts, and is required for complete activation of the Syk-dependent signaling pathway. MS4A4A is expressed both in the plasma membrane and in cells of macrophages, and its expression regulates the secretion of certain soluble proteins by macrophages. Recent studies have shown that MS4A4A is likely to be a novel marker of M2-type macrophages and is associated with poor prognosis in gastric and renal cancers. However, in colorectal cancer, the function of MS4A4A in TAM, the molecular mechanisms involved in regulating the immune microenvironment, and the relationship to tumorigenesis and progression remain unknown.

The siRNA (in vivo siRNA) for animals is an siRNA compound which adopts special chemical modification and can be directly injected into animals, and has the advantages of high efficiency, specificity, stability and low toxicity. At present, hundreds of RNAi drugs enter clinical research worldwide, and the first siRNA drug is approved by the U.S. FDA to be listed in 2018. In animal experiments, in vivo siRNA administration methods can be divided into systemic administration (such as intravenous administration, intraperitoneal administration and the like) and local administration (such as subcutaneous injection, intravitreal administration, intrathecal administration and the like). Generally, systemic administration is performed by injecting 5-20 nmol/20g of body weight each time, local administration is performed by injecting 1-5 nmol/20g of body weight each time, administration frequency is 2-3 times a week, and an administration scheme can be flexibly adjusted according to experimental research purposes. A number of in vivo siRNA related research results have been published. Due to the lack of related research, no experimental research results of animal siRNA aiming at MS4A4A gene are reported at present.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings of colorectal cancer treatment technology, provides a medicine for treating colorectal cancer, and provides application of an MS4A4A gene as a target.

In order to realize the purpose, the technical scheme is as follows: a medicine for treating colorectal cancer comprises siRNA, wherein a sense strand of the siRNA is shown as SEQ ID NO. 1 or SEQ ID NO. 2. .

The invention provides application of MS4A4A gene as a target in screening or preparing medicaments for treating colorectal cancer.

The invention provides application of an agent for inhibiting or blocking expression of an MS4A4A gene in preparation of a medicine for treating colorectal cancer.

Preferably, the agent comprises siRNA.

Preferably, the sense strand of the siRNA is shown as SEQ ID NO. 1 or SEQ ID NO. 2.

Has the advantages that:

the invention proves that siRNA interfering MS4A4A expression of macrophages can inhibit the macrophage from polarizing to M2 type at a cellular level through basic experiments. In vivo animal experiments show that siRNA interfering with expression of MS4A4A can inhibit macrophage M2 type polarization, promote activation of tumor-infiltrating CD8+ T cells and further play an anti-tumor role.

Drawings

FIG. 1 shows that the expression of interfering MS4A4A in example 1 of the present invention can inhibit the polarization of macrophages to M2 type, wherein FIG. 1A shows the result of qPCR detection on the expression of interfering macrophages MS4A4A, FIG. 1B shows the result of western blot detection on the expression of interfering macrophages MS4A4A, and FIG. 1C shows the result of the change in the expression level of M2 type macrophage markers.

FIG. 2 is a picture of the effect of interfering with MS4A4A expression on significantly inhibiting the growth of intestinal cancer cells in mice in example 2 of the present invention.

FIG. 3 is a photograph showing the inhibition of tumor growth caused by the reversible MS4A4A siRNA of CD8+ T cells in mice cleared in example 3 of the present invention.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples. In vivo siRNA (siMS4A4A) used in animal experiments in this study was purchased from Ribo Biotech Co., Ltd, product number: siBDMV002, Guangzhou.

Example 1

MS4A4A promoted macrophage M2 polarization. The experimental procedure and results are as follows (results are shown in FIG. 1):

mouse Bone marrow-derived macrophages (Bone marrow derived macrophages, BMDM) were induced into M2-type macrophages using IL4(20ng/ml), BMDM was transfected with MS4A4A siRNA (siRNA1 and siRNA2) and control siRNA (sinc), and results of interference with MS4A4A expression detected by qPCR and western blot are shown in FIG. 1A and FIG. 1B, respectively. mRNA and protein level expression of MS4A4A was significantly inhibited compared to the siNC group. The sense strand of siRNA directed against the MS4A4A gene was as follows:

siRNA1：5’-CCUUGGGAUUGUGCAGAUUTT-3’；

siRNA2：5’-GCAGCCUAGGCUUGAACAUTT-3’。

qPCR experiments were used to detect changes in expression of MS4A4A, M2-type macrophage marker expression levels that interfere with macrophages. As shown in fig. 1C, mRNA expression of M2-type macrophage markers Fizz1, Arg1, and Tgfb1 was significantly inhibited after transfection with MS4A4A siRNA compared to siNC group.

Example 2

Inhibition of MS4A4A expression significantly inhibited tumor cell growth in mice, with the following experimental methods and results (results are shown in fig. 2):

FIGS. 2A-B: a C57BL/6 mouse subcutaneous tumor model is constructed by using a murine colon cancer cell MC 38. The cancer inhibition effect of the intratumoral injection of MS4A4A siRNA is detected, as shown in FIG. 2A-B, and the experimental results of FIG. 2A-B show that 2 MS4A4A siRNAs in vivo have significant inhibition effect on the growth of colon cancer cells.

FIGS. 2C-D: the subcutaneous tumor tissue of the mouse is detected by flow, and the result shows that: compared with the control group, the proportion of CD8+ T cells (LAG3+ CD8+ T cells and TIM3+ CD8+ T cells) exhausted in tumors was significantly reduced, while the proportion of CD11b + F4/80+ macrophages was not significantly different between the two groups.

FIGS. 2E-F: the subcutaneous tumor tissue of the mouse is detected by flow, and the result shows that: the proportion of IFN- γ + CD8+ T cells infiltrating within the tumor and Ki67+ CD8+ T cells was significantly increased compared to the control group.

FIGS. 2G-H: the subcutaneous tumor tissue of the mouse is detected by flow, and the result shows that: the proportion of CD206+ F4/80+ macrophages infiltrated within the tumor was significantly reduced compared to the control group.

Example 3

The tumor growth inhibition due to the reversible interference of MS4A4A expression by clearing CD8+ T cells in mice (results are shown in fig. 3):

FIGS. 3A-B: a C57BL/6 mouse subcutaneous tumor model is constructed by using a murine colon cancer cell MC 38. Mice were treated in groups, experimental mice were intraperitoneally injected with CD8 neutralizing antibody and intratumorally injected with MS4A4A siRNA 2. The control group of mice is injected with IgG antibody in the abdominal cavity and MS4A4A siRNA2 in the tumor, whether the anti-tumor effect of MS4A4A siRNA depends on CD8+ T cells is detected, and the result shows that: in vivo depletion of CD8+ T cells attenuated the anti-tumor effect of MS4A4A siRNA.

FIGS. 3C-D: the flow detection mouse subcutaneous tumor tissue is used for verifying the clearing efficiency of CD8+ T cells in mice of experimental groups, and the result shows that: the proportion of CD8+ T cells within the CD8 neutralizing antibody treated group was significantly reduced compared to the IgG group.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.