CN111793138B

CN111793138B - Fusion peptide targeting breast cancer and application thereof

Info

Publication number: CN111793138B
Application number: CN202010598626.2A
Authority: CN
Inventors: 张虎; 刘雪; 王雨婷; 陈昱锦; 时瑞; 李保路
Original assignee: Jiangsu Vocational College of Medicine
Current assignee: Jiangsu Vocational College of Medicine
Priority date: 2020-06-28
Filing date: 2020-06-28
Publication date: 2021-08-17
Anticipated expiration: 2040-06-28
Also published as: CN111793138A

Abstract

The invention belongs to the field of biomedicine, and relates to a fusion peptide targeting breast cancer and application thereof. The fusion peptide comprises a membrane-penetrating peptide at the N end and a leader peptide at the C section, wherein the amino acid sequence of the leader peptide is shown as SEQ ID NO: 1 is shown. According to the biological center rule, the invention researches a transcription factor PDEF nuclear localization signal key sequence, synthesizes a leader peptide for inhibiting PDEF nuclear transport in a targeted mode according to the design, further prepares a fusion peptide, and experiments prove that the fusion peptide can obviously inhibit the growth of breast cancer cells.

Description

Fusion peptide targeting breast cancer and application thereof

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a fusion peptide targeting breast cancer and application thereof.

Background

At present, new cases of cancer increase year by year worldwide, and Breast Cancer (BC) is still the tumor with the highest morbidity and mortality in women, and the incidence of the cancer is in a trend of younger age, also in china. The breast cancer has high heterogeneity, and can be divided into three subtypes, namely basal-like subtype, luminal subtype and HER2+ subtype, which respectively account for 10-15%, 60-70% and 25-30% according to gene expression profiles. The basal-like breast cancer has high malignancy degree, and no ideal target medicine exists in clinic; the ratio of the luminal breast cancer is about 60-70%, and the Estrogen Receptor (ER) is positive and has better prognosis, and endocrine treatment can be adopted clinically; the HER2+ type breast cancer accounts for about 25-30%, the prognosis is poor, and an anti-HER 2 targeted drug is usually adopted for treatment clinically. However, both luminal and HER2+ breast cancers develop resistance during clinical treatment. Therefore, the existing treatment means cannot meet the requirement of clinical individualized treatment of breast cancer, and the development of novel treatment targets and medicaments is imperative.

Disclosure of Invention

According to the invention, through the integrated analysis of TCGA data, CCLE data, GTEx data and tumor cell growth dependent gene data, the fact that the Prostate-Derived Ets Factor (PDEF) is specifically expressed in breast cancer and Prostate cancer and specifically promotes the growth of luminal and HER2+ type breast cancer cells with high PDEF expression is found, and the PDEF is a potential new target for targeted therapy of breast cancer.

The invention provides a fusion peptide for targeting breast cancer, which comprises a membrane-penetrating peptide at the N end and a leader peptide at the C section, wherein the amino acid sequence of the leader peptide is shown as SEQ ID NO: 1 is shown.

RLWGIRKNRPAMNYDKLSRSIRQYYKKGIIRKPD(SEQ ID NO：1)。

The cell-penetrating peptide in the present invention may be various cell-penetrating peptides conventional in the art, including but not limited to: TAT, Pennetrat, Polyargines, DPV₁₀₄₇MPG, Pep-1, pVEC, ARF (1-22), BPrPr (1-28), MAP, Transportan, p28, VT5, Bac 7(Bac 1-24), C105Y, PFVYLI or Pep-7.

The fusion peptides of the present invention can be synthesized by methods conventional in the art, and can also be obtained commercially.

The second aspect of the invention provides the application of the leader peptide in preparing a breast cancer cell growth inhibitor.

Specifically, the breast cancer cells are MDA-MB-453 cells and/or SK-BR-3 cells.

The invention researches a transcription factor PDEF nuclear localization signal key sequence according to a biological center rule, synthesizes a leader peptide for inhibiting PDEF nuclear transport in a targeted mode according to the design, further prepares a fusion peptide, and experiments prove that the fusion peptide can obviously inhibit the growth of breast cancer cells, such as MDA-MB-453 cells and SK-BR-3 cells.

Additional features and advantages of the invention will be set forth in the detailed description which follows.

Drawings

The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings.

Figure 1 shows the effect of PDEF on cancer cell growth. A: knocking out PDEF to inhibit the growth of PDEF high-expression breast cancer cells in a targeted manner; b: knocking down PDEF targets and inhibits the growth of PDEF high-expression breast cancer cells.

FIG. 2 shows the effect of the PDEF nuclear localization signal NLS on its nuclear import. A: the online tool predicts and displays that the amino acid sequence of the PDEF nuclear localization signal is as follows: RLWGIRKNRPAMNYDKLSRSIRQYYKKGIIRKPD, respectively; b: cell immunofluorescence experiments prove that nuclear localization signal NLS deletion inhibits PDEF nuclear transport.

Figure 3 shows the H15998 unloaded plasmid map.

Fig. 4A and 4B show the inhibitory effect of the fusion peptide of the present invention on the growth of breast cancer cells, respectively. FIG. 4A: the fusion peptide acts on the third day and the fourth day, so that the growth of breast cancer cell MDA-MB-453 is obviously inhibited (P is less than 0.05, and P is less than 0.05); FIG. 4B: the fusion peptide acts on the third day and the fourth day, and the growth of breast cancer cells SK-BR-3 is obviously inhibited (P is less than 0.05, and P is less than 0.05).

Detailed Description

Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the present invention, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein.

Example 1

This example serves to illustrate the intervention of PDEF specifically to inhibit the growth of PDEF-highly expressed breast cancer cells.

Cancer cell growth dependent gene analysis

1. Analysis of whole genome knockout library screening data:

the user logs in a Depmap portal (https:// deppmap. org/portal /) portal, inputs the gene name "PDEF", automatically recognizes the gene name, selects "PDEF", selects the data set "CRISPR (Avana) Public 20Q 2" on the left side, and downloads the result, as shown in FIG. 1A.

2. Analysis of whole genome knockdown library screening data:

logging in a Depmap portal (https:// deppmap. org/portal /) portal, inputting a gene name "PDEF", automatically identifying, selecting "PDEF", selecting a data set "Combined RNAi (Broad, Novartis, Marcotte)" on the left side, and downloading the result, as shown in FIG. 1B.

Each circle in fig. 1 represents a breast cancer cell line, and the size of the circle indicates high and low PDEF expression; color indicates abrupt change; a cell growth dependent parameter CERES or DEMETER2 value < 0 indicates that PDEF promotes cell growth and >0 indicates that PDEF inhibits cell growth. Red line at-1 is the threshold, left side of red line indicates PDEF significantly affects cell growth, right side indicates PDEF does not significantly affect cell growth. As can be seen from fig. 1, the knocking-out of PDEF targets the inhibition of the growth of PDEF-high-expression breast cancer cells, and the knocking-down of PDEF targets the inhibition of the growth of PDEF-high-expression breast cancer cells.

Example 2

This example serves to demonstrate that the PDEF nuclear localization signal peptide affects the nuclear transport of PDEF.

Firstly, predicting a PDEF nuclear localization signal multi-sequence:

logging in an NCBI Protein database (https:// www.ncbi.nlm.nih.gov/Protein), inputting 'PDEF', selecting 'Ets transfer factor PDEF [ Homo sapiens ]', and downloading a P DEF Protein amino acid sequence; logging in a nuclear localization signal prediction tool NLS _ Mapper (http:// NLS-m ap. iab. keio. ac. jp/cgi-bin/NLS _ Mapper _ form. cgi); inputting a PDEF amino acid sequence, and clicking a 'Predict NLS' download result, as shown in the A diagram of FIG. 2; the online tool predicts that the P DEF nuclear localization signal amino acid sequence is: RLWGIRKNRPAMNYDKLSRSIRQYYKK GIIRKPD are provided.

Cloning construction

1. Obtaining an insert:

the full-length PDEF CCDS region and the PDEF nuclear localization signal deletion DNA sequence are synthesized by the company of Biotechnology (Shanghai);

2. and (3) carrying out enzyme digestion on the vector:

a50. mu.l digestion system was prepared according to Table 1. Sequentially adding various reagents according to the sequence of a list, lightly blowing and uniformly mixing by using a pipette, carrying out instantaneous centrifugation, and reacting for 3 hours at 37 ℃. And (4) carrying out agarose gel electrophoresis on the vector enzyme digestion product, and recovering a target band. The H15998 plasmid was purchased from Heyu Biotechnology (Shanghai) Ltd and the map of the unloaded plasmid is shown in FIG. 3.

TABLE 1 vector cleavage System

3. Recombinant connection of the vector:

by passing

The plus One step PCR Cloning Kit (from Novoprotein) was used to directionally clone PDEF DNA fragment into the vector by homologous recombination.

A20. mu.l reaction system was prepared according to Table 2 and reacted at 50 ℃ for 10 min.

TABLE 2 Carrier attachment System

4. Transformation of

Adding 10 μ L of the ligation reaction product into 100 μ L of competent cells, flicking the tube wall, mixing, and standing on ice for 30 min; heat shock is carried out for 90s at 42 ℃, and incubation is carried out for 2min in ice bath; adding 500 μ L LB culture medium, and shake culturing at 37 deg.C for 1 h; taking a proper amount of bacterial liquid, uniformly coating the bacterial liquid on a flat plate containing corresponding antibiotics, and carrying out inverted culture in a constant-temperature incubator for 12-16 h.

5. Sequencing identification

And (3) inoculating the identified positive clone transformant into a proper amount of LB liquid culture medium containing corresponding antibiotics, culturing for 12-16h at 37 ℃, and taking a proper amount of bacterial liquid for sequencing and identifying.

Thirdly, detecting the influence of the nuclear localization signal on the nuclear transport of PDEF through a cell immunofluorescence experiment:

1. cell transfection:

after the MDA-MB-453 cells in the 12-well plate were confluent to 60%, 1.6. mu.l Lipo8000^TM(purchased from Biyuntian Bio Inc.) and 800ng of plasmid were added to 50. mu.l of serum-free medium, mixed, and allowed to stand at room temperature for 5min, followed by additionInto 24-well plates, fresh medium was replaced after 8 h.

2. Staining and Observation of Hoechst

Removing the culture medium from the cells cultured in the 12-well plate, adding 500. mu.l of Hoechst 33258 (purchased from Biyuntian corporation), and covering the cells sufficiently; continuously culturing the cells for 30 min; discarding the staining solution, washing 3 times with PBS; anti-fluorescence quenching liquid seal (available from Biyuntian corporation); the EGFP-PDEF fusion protein nuclear localization was observed by confocal laser microscopy and the photographs were recorded, as shown in panel B of FIG. 2. Cell immunofluorescence experiments prove that nuclear localization signal NLS deletion inhibits PDEF nuclear transport. The unloaded and NLS-deleted PDEF group was predominantly cytoplasmic, and the full-length PDEF group showed that PDEF was predominantly nuclear.

Example 3

This example is used to demonstrate the effect of the fusion peptides of the invention on inhibiting the growth of breast cancer cells.

Synthesis of fusion peptide

The sequences of fusion peptide TAT-AP34 and control polypeptide TAT-Ctrol34 are shown in Table 3 and are assigned to Kinsley Biotech.

TABLE 3

Second, cell proliferation assay

Resuspension of 7.0X 10 cells using 200. mu.l of Opti-MEM⁵Respectively adding 10mg of TAT-AP34 or TAT-Ctrol34 into MDA-MB-453-GFP and SK-BR-3-GFP cells of breast cancer, and incubating for 1h at 37 ℃; after the culture medium is resuspended, 3000 cells/well are inoculated in a 96-well plate, the plate is photographed at regular time every day for 4 days, the fluorescence intensity is calculated, a growth curve is drawn, and statistical analysis is carried out on SPSS 16.0.

FIGS. 4A-4B show the inhibitory effect of the fusion peptide TAT-AP34 on breast cancer cell growth. TAT-AP34 significantly inhibited the growth of breast cancer cell MDA-MB-453 (P < 0.05, FIG. 4A) and the growth of breast cancer cell SK-BR-3 (P < 0.05, FIG. 4B) on the third and fourth days of action.

Having described embodiments of the present invention, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.

Sequence listing

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<120> fusion peptide targeting breast cancer and application thereof

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Claims

1. A fusion peptide, wherein the amino acid sequence of the fusion peptide is as set forth in SEQ ID NO: 2, respectively.

2. Use of the fusion peptide of claim 1 for the preparation of a breast cancer cell growth inhibitor.

3. Use according to claim 2, wherein the breast cancer cells are MDA-MB-453 cells and/or SK-BR-3 cells.