CN114558138B

CN114558138B - Application of substance for knocking down BACE2 gene expression in resisting tumor and BACE2 inhibitor

Info

Publication number: CN114558138B
Application number: CN202110277211.XA
Authority: CN
Inventors: 刘洁; 阮班来; 陈紫寅; 丁千山
Original assignee: Huachenyang Shenzhen Technology Co ltd
Current assignee: Huachenyang Shenzhen Technology Co ltd
Priority date: 2021-03-15
Filing date: 2021-03-15
Publication date: 2024-01-12
Anticipated expiration: 2041-03-15
Also published as: CN114558138A

Abstract

The invention provides application of a substance for knocking down BACE2 gene expression in resisting tumors and a BACE2 inhibitor, wherein the tumors are colon tumors. The invention obviously reduces the expression of BACE2 gene by selecting specific siRNA, and effectively inhibits the migration, proliferation and invasion capacity of tumor cells. Shows that the proliferation of colon tumor can be effectively inhibited by knocking down BACE2 gene expression, and a new idea is provided for colon tumor treatment.

Description

Application of substance for knocking down BACE2 gene expression in resisting tumor and BACE2 inhibitor

Technical Field

The invention relates to the field of biological medicine, in particular to application of a substance for knocking down BACE2 gene expression in resisting tumors and a BACE2 inhibitor.

Background

Colon tumors are common malignant tumors in the gastrointestinal tract, with incidence inferior to gastric and esophageal cancers, the most common part of colorectal cancer. Colorectal cancer is not uncontrollable, and in fact colorectal cancer is the most self-screening condition, and if early detection is performed, the most curable condition is also found. Treatment of colon tumors generally requires surgical removal of the tumor, along with removal of a range of adjacent bowel and surrounding lymph nodes. However, the operation treatment has the defects of weak pertinence and low cure rate. Since the occurrence of tumors is closely related to abnormal gene expression in cells, the treatment of tumors by regulating intracellular gene expression has become a hot spot of current research in recent years with the development of scientific technology.

Scientific researches find that the BACE2 gene is closely related to the apoptosis, proliferation and migration capability and invasion capability of colon cancer cells. Thus, reducing or inhibiting the expression of the BACE2 gene is one of the effective ways to treat tumors.

Disclosure of Invention

In order to solve the technical problems, a first aspect of the present invention provides an application of a substance for knocking down BACE2 gene expression in anti-tumor, wherein the tumor is colon tumor.

As a preferred embodiment, the substance that knocks down the expression of the BACE2 gene is selected from the group consisting of siRNA, short hairpin RNA or antisense nucleotide that specifically interferes with the expression of the BACE2 gene.

As a preferred embodiment, the substance that knocks down the expression of the BACE2 gene is an siRNA that specifically interferes with the expression of the BACE2 gene.

As a preferred embodiment, the substance for knocking down the expression of the BACE2 gene is selected from the following siRNA molecule sequences: SEQ ID No. 1,SEQ ID No:2,SEQ ID No:3.

The sequence of the siRNA is as follows:

SEQ ID No:1(5'to 3')：CCGCCAACUUCUUGGCCAUTT

AUGGCCAAGAAGUUGGCGGTT

SEQ ID No:2(5'to 3')：GGGAUUAAAUGGAAUGGAATT

UUCCAUUCCAUUUAAUCCCTT

SEQ ID No:3(5'to 3')：GGUGGAAUUGAACCAAGUUTT

AACUUGGUUCAAUUCCACCTT

as a preferred embodiment, the siRNA molecule is carried on a pharmaceutically acceptable transfection vector.

In a second aspect of the invention there is provided a BACE2 inhibitor, the BACE2 inhibitor comprising a substance that knocks down the expression of the BACE2 gene.

The sequence of the siRNA is as follows:

SEQ ID No:1(5'to 3')：CCGCCAACUUCUUGGCCAUTT

AUGGCCAAGAAGUUGGCGGTT

SEQ ID No:2(5'to 3')：GGGAUUAAAUGGAAUGGAATT

UUCCAUUCCAUUUAAUCCCTT

SEQ ID No:3(5'to 3')：GGUGGAAUUGAACCAAGUUTT

AACUUGGUUCAAUUCCACCTT

The beneficial effects are that:

1) The creative discovery of the invention can inhibit the proliferation of colon tumor by knocking down BACE2 gene expression, thereby providing a new idea for colon tumor treatment. The BACE2 gene can be used as a target point of a gene for preventing or treating colon tumor cells, and can be used for screening agents for preventing or treating colon tumors.

2) The invention discovers that the knock-down BACE2 gene can inhibit the proliferation of tumor cells, takes the BACE2 gene as a target, designs a substance with high-efficiency knock-down BACE2 gene expression and/or function in an RNA interference mode by selecting a proper target gene sequence, prepares an inhibitor for effectively inhibiting the proliferation of colon tumor cells, and provides a new technical scheme for the prevention and treatment of colon tumor cells.

3) The specific BACE2 gene expression knocking-down substance and BACE2 inhibitor provided by the invention can achieve good treatment effect on colon tumor, and have good application prospect.

Description of the drawings:

FIG. 1 shows the WB assay of HT29 cell line of example 1, wherein the normal, NC control, siRNA-325, siRNA-672, siRNA-858 groups are shown in the order from left to right;

FIG. 2 is a graph showing the results of the test for the ability of HT29 cell line CCK8 to detect cell proliferation of example 2;

FIG. 3 is a graph showing the results of the scratch test for detecting cell migration function of HT29 cell line of example 3;

FIG. 4 is a graph showing the results of a Transwell test for detecting cell invasion function of HT29 cell line of example 4, wherein the normal group, NC control group, siRNA-325, siRNA-672 and siRNA-858 are sequentially shown from left to right.

FIG. 5 shows the WB detection of the SW620 cell line of example 1, wherein the normal group, NC control group and siRNA-858 are sequentially arranged from left to right;

FIG. 6 is a graph showing the results of the test for the ability of the SW620 cell line CCK8 of example 2 to detect proliferation of cells;

FIG. 7 is a graph showing the results of the scratch test for SW620 cell line in example 3;

FIG. 8 is a graph showing the results of a Transwell test for the detection of cell invasion by the SW620 cell line of example 4, in which the normal group, NC control group and siRNA-858 were sequentially arranged from left to right.

Detailed Description

As a preferred embodiment, the colon tumor comprises one or more of colon cancer cell line SW620, colon cancer cell line HT 29.

The colon tumor is a common malignant tumor in the gastrointestinal tract, and the incidence rate is inferior to that of stomach and esophagus cancers, and is the most common part of colorectal cancer. The reason for tumor generation is closely related to the abnormal expression of intracellular protein amino acid, and the research discovers that BACE2 is highly expressed in colon tumor, so that cells become more malignant and aggressive, and the research also provides a basis for treating the colon tumor by knocking down BACE2 gene expression by taking BACE2 as a target.

The sequence of the siRNA is as follows:

SEQ ID No:1(5'to 3')：CCGCCAACUUCUUGGCCAUTT

AUGGCCAAGAAGUUGGCGGTT

SEQ ID No:2(5'to 3')：GGGAUUAAAUGGAAUGGAATT

UUCCAUUCCAUUUAAUCCCTT

SEQ ID No:3(5'to 3')：GGUGGAAUUGAACCAAGUUTT

AACUUGGUUCAAUUCCACCTT

the substance that knocks down the expression of the BACE2 gene can prevent translation by interfering with post-transcriptional degradation of mRNA of a particular gene expressing a nucleotide sequence complementary to that of the gene. And can be introduced into cells through different transfection techniques, has specific knockdown effect on specific genes, and is one of important tools for inhibiting gene expression.

As a preferred technical solution, the transfection vector is Lip6000 transfection reagent.

The transfection carrier can carry siRNA into cells, and the transfection carrier is used for helping the siRNA to enter the cells, so that the operation is simple, the transfection is efficient, and no toxic or side effect is caused to target cells.

The sequence of the siRNA is as follows:

SEQ ID No:1(5'to 3')：CCGCCAACUUCUUGGCCAUTT

AUGGCCAAGAAGUUGGCGGTT

SEQ ID No:2(5'to 3')：GGGAUUAAAUGGAAUGGAATT

UUCCAUUCCAUUUAAUCCCTT

SEQ ID No:3(5'to 3')：GGUGGAAUUGAACCAAGUUTT

AACUUGGUUCAAUUCCACCTT

The present invention will be specifically described below by way of examples. It is noted herein that the following examples are given solely for the purpose of further illustration and are not to be construed as limitations on the scope of the invention, as will be apparent to those skilled in the art in light of the foregoing disclosure.

In addition, the raw materials used are commercially available unless otherwise indicated.

Examples

The main reagent comprises: DMEM high sugar medium (sigma), serum FBS (ORION), PBS solution (prios), lip6000 (bi cloud) pancreatin (SparkJade), hematoxylin (marhan co), CCK8 kit (subishi bio-tech).

The preparation method of the siRNA solution comprises the following steps: centrifuge tubes containing siRNA followed by slow opening of the tube lid, 62.50ul of DEPC water was added to each tube (0.5OD260/tube) to prepare a 20uM solution.

The sequence of the siRNA is as follows:

SEQ ID No:1(5'to 3'):CCGCCAACUUCUUGGCCAUTT

AUGGCCAAGAAGUUGGCGGTT

SEQ ID No:2(5'to 3'):GGGAUUAAAUGGAAUGGAATT

UUCCAUUCCAUUUAAUCCCTT

SEQ ID No:3(5'to 3'):GGUGGAAUUGAACCAAGUUTT

AACUUGGUUCAAUUCCACCTT

the siRNA used in this patent delegates Ji Ma gene synthesis, which can be ordered by the gene synthesis company as required by the person skilled in the art.

In the following examples and performance tests, SEQ ID No. 1 was designated siRNA-325, SEQ ID No. 2 was designated siRNA-672, and SEQ ID No. 3 was designated siRNA-858.

The colon cancer cell lines selected are HT29 and SW620 cells respectively for testing.

Example 1 WB detection of related protein expression

1. Colon cancer cell lines HT29 and SW620 are selected and cultured respectively, cell suspensions of the colon cancer cell lines HT29 and SW620 are inoculated into six-hole plates respectively, and the number of cells in each hole is about 3 multiplied by 10 ⁵ When the cells enter the logarithmic growth phase, a transfection solution is prepared according to Table 1, and after the cells are transfected for 8 hours, the normal culture medium is replaced and the culture is continued for 48 hours.

2. Six-hole plate is placed on ice, protein samples are extracted and separated, and the expression level of BACE2, bcl-2 and GAPDH proteins in cells is detected by taking GAPDH as an internal reference.

TABLE 1

Group of	DMEM medium	Lip6000	si-RNA solutions
				Normal group	2ml	-	-
NC control group	2ml	10μl	8μl
				siRNA-325	2ml	10μl	8μl(SEQ ID No:1)
siRNA-672	2ml	10μl	8μl(SEQ ID No:2)
				siRNA-858	2ml	10μl	8μl(SEQ ID No:3)

The performance tests show that the SEQ ID No. 1, the SEQ ID No. 2 and the SEQ ID No. 3 knock down BACE2 genes in colon cancer cells, and obviously inhibit the expression condition of HT29 cell proteins; the BACE2 gene in the 3 knockdown colon cancer cells of SEQ ID No. significantly inhibits the protein expression condition of SW620 cells, and even the expression of the colon cancer cells knocked down by siRNA is far lower than that of Bcl-2 genes, so that the low BACE2 gene has an important effect on cancer cell apoptosis by siRNA.

Example 2CCK8 detection of cell proliferation Capacity

1. 100ul of cell suspensions of colon cancer cell lines HT29 and SW620 (the cell suspension being a mixture of serum medium and colon cancer cells) were added to 96 well plates, 2000 cells per well, respectively. The plates were set at 37℃in an incubator with 5% CO ² Is pre-incubated for 24 hours.

2. The transfection solution was prepared as in Table 2, and the cells after preculture were transfected for 6 hours, and after transfection, the cells were replaced with a serum-containing medium and cultured for 24 hours.

TABLE 2

Group of	DMEM medium	Lip6000	si-RNA solutions
				Normal group	0.2ml	-	-
NC control group	0.2ml	1μl	0.8μl
				siRNA-325	0.2ml	1μl	0.8μl(SEQ ID No:1)
siRNA-672	0.2ml	1μl	0.8μl(SEQ ID No:2)
				siRNA-858	0.2ml	1μl	0.8μl(SEQ ID No:3)

3. 10ul of CCK8 solution was added to each well and the plates were placed in an incubator for 2h incubation. The absorbance at 450nm was measured with a microplate reader and further data analysis was performed.

The performance test shows that the proliferation capacity of the normal group and the negative control group of the HT29 cells which are not knocked down is more than 50%; the proliferation capacity of HT29 cells which knock down BACE2 genes in colon cancer cells through SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3 is less than 50%; the proliferation capacity of the SW620 cells of the normal group and the negative control group of the non-knocked down SW620 cells is far greater than that of the SW620 cells of the BACE2 genes in the SEQ ID No. 3 knockdown colon cancer cells, so that the proliferation of the colon cancer can be obviously inhibited by the siRNA pair-knocking down the BACE2 genes.

Example 3 scratch assay to detect cell migration function

1. The marker pen was used to draw lines uniformly across the six-well plate back, approximately every 0.5-1cm, with a straight ruler, across the vias, with each via passing through at least 5 lines.

2. Will be about 3 x 10 respectively ⁵ The individual colon cancer cell lines HT29 and SW620 cells were equally distributed into each of the six well plates. The plates were set at 37℃in an incubator with 5% CO ² Is pre-incubated for 24 hours.

3. Cells after preculture were transfected with the transfection solution (same as in example 1) and after 6 hours of transfection, the cells were replaced with serum-containing medium and cultured for a further 24 hours.

4. After cells are grown, the gun head is compared with the ruler, and is vertical to the transverse line behind the ruler as much as possible, so that the gun head is vertical and does not need to be inclined.

5. The cells were washed 3 times with PBS, the scraped cells were removed, and serum-free DMEM medium was added. Placing at 37deg.C, 5% CO ² Culturing in an incubator. Samples were taken at 0, 12, 24 hours and photographed.

The performance tests show that the SEQ ID No. 1, the SEQ ID No. 2 and the SEQ ID No. 3 knock down BACE2 genes in colon cancer cells, and obviously inhibit the migration capacity of HT29 cells; the BACE2 gene in the 3 knockdown colon cancer cells of SEQ ID No. significantly inhibits the migration capacity of SW620 cells, so that the migration of colon cancer can be significantly inhibited by knocking down the BACE2 gene through siRNA.

Example 4 Transwell detection of cell invasion function

1. Colon cancer cell lines HT29 and SW620 are selected for culture respectively, when cells enter a logarithmic growth period, the cells are transfected by a transfection solution (the same as in example 1) in a six-well plate, after 24 hours of transfection, the cells are digested, the centrifuged cells are obtained for re-suspension, the cells are re-suspended by 1ml of a serum-containing culture medium, cell counting is carried out, and the cell density of the cell suspension is calculated.

2. Opening a Transwell experimental well plate, separating the cell from the outer chamber (placing the cell in a blank hole of a partition wall) by using sterile forceps, adding cell suspension into the cell, and making each hole 2×10 ⁴ Cells were inoculated, 600ul of medium containing 25% serum was added to the outer chamber, the chamber was replaced with the outer chamber to which medium had been added, the lid was closed, and the cells were cultured in a cell incubator for 48 hours.

3. Cell fixation and staining

1) Removing the culture solution, and washing with PBS for 2 times; 500ul of formaldehyde with a mass concentration of 4% is added into the blank hole, the mixture is placed into a small chamber for fixation for 20 minutes, washed 2 times with PBS, and the mixture is inverted on filter paper to remove formaldehyde.

2) A hematoxylin solution with the mass concentration of 0.1% is added into a blank hole, the blank hole is placed into a small chamber for dyeing for 20 minutes, and the blank hole is washed with PBS for 2 times. The cells in the chamber were rubbed with a cotton swab, observed under a microscope at 200X field of view, and photographed.

The performance test shows that the invasion capacity of the normal group and the negative control group of the HT29 cells which are not knocked down is far greater than that of HT29 cells which knock down BACE2 genes in colon cancer cells by SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3; the proliferation capacity of the SW620 cells which are not knocked down in the normal group and the negative control group is far greater than the invasion capacity of the SW620 cells of the BACE2 gene in the colon cancer cells knocked down by SEQ ID No. 3, so that the invasion of the colon cancer can be obviously inhibited by knocking down the BACE2 gene by siRNA.

Results of Performance test

Scientific researches show that the high expression of BACE2 genes in colon tumors can be found by comparing the performance test conditions of normal groups, negative control groups, siRNA-325, siRNA-672 and siRNA-858 through the above examples, and the invention can obviously reduce the expression of BACE2 genes by selecting the siRNA for specifically knocking down the expression of BACE2 genes, thereby effectively inhibiting the migration, proliferation and invasion capacity of tumor cells. Shows that the proliferation of colon tumor can be effectively inhibited by knocking down BACE2 gene expression, and a new idea is provided for colon tumor treatment.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the invention in any way, and any person skilled in the art may make modifications or alterations to the disclosed technical content to equivalent embodiments without departing from the technical content of the present invention, and any simple modification, equivalent changes and alterations to the above embodiments according to the technical substance of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A BACE2 inhibitor, wherein the BACE2 inhibitor comprises a substance that knocks down expression of a BACE2 gene; the substances for knocking down the BACE2 gene expression are siRNA which specifically interfere with the BACE2 gene expression; the substances for knocking down the BACE2 gene expression are selected from the following siRNA molecule sequences: 1,SEQ ID No:2,SEQ ID No:3.

2. The BACE2 inhibitor of claim 1, wherein the siRNA molecule is supported on a pharmaceutically acceptable transfection vector.