CN105566460B - The polypeptide analog and corresponding code cDNA of inhibition tumor cell proliferation and application - Google Patents

Abstract

The invention discloses a kind of polypeptide analog for inhibiting tumor cell proliferation and corresponding code cDNA and applications.The present invention is based on the establishments that nucleolin Nucleostemin (NS) in tumour and cell cycle protein dependent kinase inhibit albumen (CKI) member p27kip1 (abbreviation p27) interaction domain, have constructed polypeptide analog NS using gene engineering method^118‑288aa.Polypeptide analog of the present invention can bind directly p27, block the interaction of NS albumen and p27, and NS albumen is inhibited to degrade p27, to play the function of inhibiting the hyper-proliferative of tumour cell.Theoretical foundation can be provided for seeking for cancer target, the clinical treatment for being applied to tumour has highly important development prospect.

Description

Polypeptide analog for inhibiting tumor cell proliferation and corresponding coding cDNA and application thereof

technical field

The present invention relates to polypeptide analogs, in particular to a polypeptide analog for inhibiting tumor cell proliferation, the corresponding coding cDNA and its application.

Background technique

NS protein is also called guanine nucleotide binding protein-like 3 (guanine nucleotide binding protein-like3, GNL3), which plays an important role in the process of embryogenesis, tissue regeneration and tumorigenesis. These functions of NS protein and its promotion The functions of cell cycle progression are inextricably linked. The expression of NS protein is closely related to the degree of malignancy of tumors. Interfering with the expression of NS protein can cause a general decrease in the proliferation ability of these tumors. Therefore, NS protein has become one of the important targets in the development of anti-tumor drugs.

In recent years, studies have shown that NS protein can inhibit the function of tumor suppressor genes and promote the development of tumors by interacting with tumor suppressor genes. For example, NS protein interacts with P53 to down-regulate the expression of P53, which can promote the excessive proliferation of cells by inhibiting the transcription of P21 gene, and can also inhibit apoptosis and induce tumors by affecting the function of Bcl-2 protein family.

p27 is an important cell cycle G1 cyclin inhibitor, which can bind to CDK4-Cyclin D complex, inhibit the latter's activity and cell cycle G1-S transition, thereby inhibiting the proliferation of tumor cells. The data published in this patent shows that in tumors, NS protein can interact with p27 to promote the degradation of p27 in tumor cells. Lead to excessive proliferation and development of tumor cells. Therefore, blocking the interaction between NS protein and tumor suppressor gene p27 has become a new strategy for us to find anti-tumor drugs.

Contents of the invention

The object of the present invention is to provide a polypeptide analog and its application for inhibiting tumor cell proliferation, which can at least solve one of the above problems.

To achieve the above object, according to one aspect of the present invention, a polypeptide analogue that inhibits tumor cell proliferation is provided, the amino acid sequence of which is shown in SEQ NO.1 in the sequence listing:. The polypeptide analogs that inhibit tumor cell proliferation are used to interfere with the interaction between NS protein and p27.

Correspondingly, the present invention also provides the application of the above-mentioned polypeptide analogs for inhibiting tumor cell proliferation in the preparation of anti-tumor drugs.

Correspondingly, the present invention also provides the coding cDNA sequence of the above-mentioned polypeptide analogue for inhibiting tumor cell proliferation, the sequence of which is shown in SEQ NO.1 of the Sequence Listing.

Correspondingly, the present invention also provides the application of the coding cDNA sequence of the above-mentioned polypeptide analogue for inhibiting tumor cell proliferation in the preparation of antitumor drugs.

The beneficial effects of the present invention are as follows: the present invention is based on the establishment of the interaction domain between the nucleolar protein Nucleostein (NS) and the cyclin-dependent kinase inhibitor (CKI) member p27kip1 (p27 for short) in tumors, and uses genetic engineering methods to construct Peptide analog NS ^118-288aa . The polypeptide analogue of the present invention can block the interaction between NS protein and p27, and inhibit the degradation of p27 by NS protein, thereby exerting the function of inhibiting the excessive proliferation of tumor cells. It can provide a theoretical basis for the exploration of tumor therapeutic targets, and has very important development prospects for clinical treatment of tumors.

Description of drawings

Figure 1 and Figure 1A are the results of the immunoprecipitation complex detection experiment of p27 and NS protein immunoprecipitation, and Figure 1B is the co-localization of NS protein and p27 in liver cancer cells analyzed by immunofluorescence;

Figure 2 and the upper part of Figure 2 are different truncated fragments of the NS protein, and the lower part of Figure 2 is the detection chart of the immunoprecipitation results;

Fig. 3, the map of recombinant eukaryotic expression vector in step S102;

Fig. 4, the result diagram of the protein expression experiment of the polypeptide in the HepG2 cell line in step S102 proved by immunoblotting;

Fig. 5, Western blotting in step S103 shows that in the HepG2 cell line, the polypeptide analog blocks the interaction experiment results of NS and p27;

Fig. 6, Western blotting in step S104 shows that the peptide analogs in the HepG2 cell line inhibit the degradation of p27 by NS;

Fig. 7 , the diagram of the experimental results of the blockade of cell cycle progression by polypeptide analogues in the HEP3B cell line measured by flow cytometry in step S201;

Fig. 8 is a diagram showing the results of experiments on the inhibition of tumor cell proliferation by polypeptide analogues in cell lines in transfected and untransfected cells in step S202.

Fig. 9 is a graph showing the results of tumor formation experiments in vivo in HepG2 cells that were not transfected and stably transfected with Flag-NS ^118-288aa expression plasmids in step S203.

Detailed ways

The present invention will be described in further detail below in conjunction with the accompanying drawings.

The invention provides a polypeptide analogue for inhibiting tumor cell proliferation, the amino acid sequence of which is shown in SEQ NO.1 in the sequence table. The polypeptide analogs that inhibit tumor cell proliferation are used to interfere with the interaction between NS protein and p27. The above polypeptide analogs for inhibiting tumor cell proliferation can be applied to the preparation of antitumor drugs.

Correspondingly, the present invention also provides the coding cDNA sequence of the above-mentioned polypeptide analogue for inhibiting tumor cell proliferation, the sequence of which is shown in SEQ NO.2 of the Sequence Listing. The present invention also provides the application of the coding cDNA sequence of the polypeptide analogue for inhibiting tumor cell proliferation in the preparation of antitumor drugs.

1. The following is the verification of the interaction between NS protein and p27.

(1) There is a direct interaction and intracellular colocalization between p27 and NS protein in HCC cells.

Liver cancer tissue was lysed with IP lysate (50mM Tris-HCl pH8.0, 150mM NaCl, 1% NP-40, 5mM EDTA, 20mM MgCl2, 1×Roche phosphatase&Protease inhibitor cocktail), and p27 and NS protein were respectively used Immunoprecipitation, detecting NS and p27 in the immunoprecipitation complex. As shown in Figure 1A, the results showed that the two interacted in liver cancer tissues.

The co-localization of NS protein and p27 in HCC cells was analyzed by immunofluorescence. In the Huh7 and Hep3B liver cancer cell lines, the NS-Flag vector was transfected with Lipofectamine 2000, and the co-localization of NS protein and p27 in the liver cancer cells was analyzed by immunofluorescence 36 hours after transfection. The results showed that, as shown in Figure 1B, there was obvious co-localization of NS protein and p27 in liver cancer cells, and both had obvious nucleolus distribution.

(2) NS protein interacts with p27 through its 118-228 fragment.

Different truncated fragments of the NS protein were constructed separately, as shown in the upper part of Figure 2, the NS protein was divided into three truncated fragments of 1-123aa, 118-228aa and 289-550aa according to its domain. All three truncated fragments were constructed into pCMV-N-Flag vector, and the constructed NS protein truncated vector and p27-myc full-length vector were co-transfected into 293T cells. After 36 hours of transfection, the cells were collected for immunoprecipitation experiments, and Flag antibody was used for immunoprecipitation to analyze which fragment of NS could interact with p27. The results of immunoprecipitation showed that, as shown in the lower part of Figure 2, there was a direct interaction between the NS ^118-228aa truncated fragment and p27.

2. The following content is the recombination steps of the eukaryotic expression vector of the polypeptide analogue that inhibits tumor cell proliferation of the present invention.

S101. Cloning vector construction of polypeptide analogs.

The human Huh7 liver cancer cell line was cultured in vitro with DMEM medium supplemented with 10% fetal calf serum. When the cells were in the exponential growth phase, 1ml of Trizol lysate was added to extract the total RNA in Huh7 cells. The total RNA was reverse-transcribed into the human liver cancer cell cDNA library, and the corresponding cDNA sequence was successfully amplified by PCR technology. The amplified fragment was ligated with a plasmid vector, and the ligated product was transformed into competent Escherichia coli, the cloned plasmid was selected on the Amp+ agar plate, and the recombinant plasmid was extracted by alkaline lysis method, and identified by ECOR1 enzyme digestion.

S102. Construction of polypeptide analogue eukaryotic expression vector and its expression detection.

After double digestion of the cloning plasmid of S101 and plasmid pCMV-N-Flag, use the recovery kit to obtain the target fragment and connect it with the vector. The map of the recombinant eukaryotic expression vector Flag-NS ^118-288aa obtained through transformation, extraction and other steps is shown in Fig. 3 . Recombinants were identified by enzyme digestion, and further confirmed by sequencing, the sequencing results. The correctly linked polypeptide eukaryotic expression vector was transfected into tumor cell HepG2 cell line, and samples were collected 48 hours later. As shown in Figure 4, a blank control group was set up, and the results of immunoblotting proved the expression of this polypeptide.

Determination of S103, polypeptide analogs blocking the interaction of NS and p27.

The constructed Flag-NS ^118-288aa was transfected into the tumor cell line HepG2. After 36-48 hours of transfection, the cells were collected and IP lysate (50mM Tris-HCl pH 8.0, 150mM NaCl, 1% NP-40, 5mM EDTA, 20mM MgCl2, 1×Roche phosphatase&Protease inhibitor cocktail) to lyse the cells at 13000g, and centrifuge at 4°C for 15min. Transfer the supernatant to a new tube, keep a part of the sample as Input, add 4 μg of the control Flag antibody and incubate overnight, then add 30 μl Protein A/G beads for immunoprecipitation. After washing 4 times with IP lysate, add 25 μl of loading buffer and boil for 5 min. As shown in Figure 5, a blank control group was set up, and Western blotting confirmed that the interaction between NS protein and p27 was weakened.

S104. Determination that polypeptide analogs inhibit the degradation of p27 by NS.

The constructed Flag-NS ^118-288aa was transfected into tumor cell HepG2 cell line, and the cells were collected 36-48 hours after transfection. As shown in Figure 6, a blank control group was set up, and the results of immunoblotting showed that this polypeptide inhibited the degradation of p27 by NS.

3. The following content is the detection of the anti-tumor function of the polypeptide analogue for inhibiting tumor cell proliferation of the present invention.

S201. Flow cytometric detection of polypeptide analogs blocking cell cycle progression.

Transfect the constructed Flag-NS ^118-288aa into the tumor cell HepG2 cell line, digest with trypsin after 48-72 hours to collect the transfected and non-transfected cells, wash twice with PBS, discard the supernatant, add In 1ml of 70% pre-cooled ethanol, pipette evenly, and fix at 4°C for more than 24 hours. Wash with PBS to remove ethanol, centrifuge at 1000rpm for 5min, wash twice, resuspend the cells in 0.5ml PBS, add PI and RNaseA to a final concentration of 50μg/ml, incubate at 37°C for 30min, measure the cell cycle by flow cytometry to reflect the inhibition of the polypeptide function of proliferation. PI: propidium iodide, made up to 1 mg/ml in PBS, stored at 4°C. RNaseA: 10mg/ml. As shown in FIG. 7 , when a blank control group was set up, the cell cycle progression of the transfected HepG2 cells was obviously blocked.

S202. A monoclonal colony formation assay is used to detect the activity of the polypeptide analog expression plasmid in inhibiting cell proliferation.

Inoculate the transfected and untransfected cells at a density of 300 cells per well into a six-well plate containing 2 mL of 37°C pre-warmed culture solution, and rotate gently to make the cells evenly dispersed. Place them in a cell culture incubator at 37°C, 5% CO2 and saturated humidity for 2 to 3 weeks. Observe frequently, and terminate the culture when colonies visible to the naked eye appear in the culture wells. Discard the supernatant and carefully soak twice with PBS. Add 5 mL of 4% paraformaldehyde to fix the cells for 15 minutes. Then remove the fixative, add an appropriate amount of GIMSA and apply the staining solution to dye for 10-30 minutes, then slowly wash off the staining solution with running water, and air dry. Invert the six-well plate and superimpose a transparent film with a grid, directly count the colonies with the naked eye, and finally calculate the colony formation rate. Colony formation rate=(number of clones/number of seeded cells)×100%. The results are shown in Figure 8, the cell proliferation rate of the transfected cells was significantly slowed down.

S203. In vivo nude mouse tumorigenesis assay analysis Transfection of polypeptide analog expressing plasmid inhibits HCC cell tumorigenesis in vivo.

HepG2 liver cancer cells were transfected with empty plasmid and Flag-NS ^118-288aa expression plasmid, and 48 hours after transfection, the transfected cells were screened with 500 μg/ml G418 (Geneticin). Incubate in a cell culture incubator at 37°C, 5% CO ₂ and saturated humidity, replace the medium every 2 days, and keep the G418 concentration of 500 μg/ml for continuous selection. After the screening, the resistant stable cell lines were collected for expansion. About 2×10 ⁷ cells were collected and subcutaneously injected into the axilla of nude mice. Each nude mouse was injected with 1×10 ⁶ tumor cells in the left and right axilla, and 5 nude mice were injected in the control group and the Flag-NS ^118-288aa expression group. After the injection, the tumor growth was observed every 5 days, and after a total of 40 days, the mice were sacrificed. The tumors in the control group and the Flag-NS ^118-288aa expression group were removed, tumor images were taken, and the length, width and height of the tumor were measured with a vernier caliper, according to the formula:

Tumor volume = tumor length x tumor width x tumor height/2

Calculate the volume of the tumor. Statistical analysis was made to see if there was any statistical difference in the growth of the tumors in the control group and the Flag-NS ^118-288aa expression group. As shown in FIG. 9 , the tumor volume formed in the HepG2 cells transfected with the Flag-NS ^118-288aa expression plasmid was significantly smaller than that of the untransfected HepG2 cells.

What have been described above are only some embodiments of the present invention. For those skilled in the art, without departing from the inventive concept of the present invention, several modifications and improvements can be made, and these all belong to the protection scope of the present invention.

Claims (2)

1. inhibiting the polypeptide analog of tumor cell proliferation, which is characterized in that its amino acid sequence such as sequence table SEQ NO.1 institute Show：PPKGACKVPA QESQDGSGSR PAAPLIGAPANSEDTHLVDPK TDPSDSQTGL AEQCAGIRKR PATDDSSTQN KRANRTEENVSDGSPNAGSV EQTPKKPGLR RRQT。

2. the polypeptide analog described in claim 1 for inhibiting tumor cell proliferation is in the application for preparing anti-tumor drug.