CN107082800B

CN107082800B - Inhibitor of MACC1 gene and application thereof in resisting hepatocellular carcinoma invasion

Info

Publication number: CN107082800B
Application number: CN201710268784.XA
Authority: CN
Inventors: 王莉; 李岩; 孙天娇
Original assignee: Hebei Songnuo Biotechnology Co Ltd
Current assignee: Hebei songnuo Biotechnology Co., Ltd
Priority date: 2017-04-24
Filing date: 2017-04-24
Publication date: 2020-12-11
Anticipated expiration: 2037-04-24
Also published as: CN107082800A

Abstract

The invention discloses an inhibitor of MACC1 gene and application thereof in resisting hepatocellular carcinoma invasion, wherein the inhibitor is a polypeptide which comprises the following amino acid sequence: ETMFWYDAK(Xa) PCFTRVPMA(Xb) GTDEQLPDIWN, respectively; wherein Xa and Xb are selected from M, Y, L, V, W or E. The polypeptide inhibitor provided by the invention can inhibit the expression of MACC1 gene in HepG2 cells from the transcription and translation levels, and is an effective inhibitor of MACC1 gene; meanwhile, the effective inhibitor of the MACC1 gene can obviously inhibit the proliferation and invasion of HepG2 cells, and the effective inhibitor of the MACC1 gene can be used for preparing a medicine for resisting the invasion of hepatocellular carcinoma.

Description

Inhibitor of MACC1 gene and application thereof in resisting hepatocellular carcinoma invasion

Technical Field

The invention belongs to the field of genes, and particularly relates to an inhibitor of MACC1 gene and application thereof in resisting hepatocellular carcinoma invasion.

Background

Primary liver cancer is a malignant tumor with primary focus on liver cells and intrahepatic bile duct epithelial cells, wherein hepatocellular carcinoma (i.e. hepatocellular carcinoma, HCC) is the most predominant pathological type, accounting for over 90% of primary liver cancer. The MACC1 gene is a new gene discovered and named by STEIN et al in 2009. Studies of STEIN and the like find that MACC1 plays an important role in invasion and metastasis of colon cancer as a key regulator of a Hepatocyte Growth Factor (HGF)/C-met signal pathway, and in addition, Liuqingquan and the like find that MACC1 gene possibly plays an important role in invasion and metastasis of hepatocellular carcinoma, and the MACC1 gene possibly becomes one of new targets for liver cancer treatment (Liuqingquan and the like, expression and clinical significance of MACC1 gene in hepatocellular carcinoma, proceedings of Huazhong university of science and technology, 2010, 39: 22-24).

At present, no report of polypeptide inhibitors of MACC1 gene is found.

Disclosure of Invention

The invention aims to provide an inhibitor of MACC1 gene and application thereof in resisting hepatocellular carcinoma invasion.

The technical scheme for realizing the aim of the invention is as follows:

an inhibitor of the MACC1 gene, which inhibitor is a polypeptide comprising an amino acid sequence of formula (i):

ETMFWYDAK(Xa)PCFTRVPMA(Xb)GTDEQLPDIWN； (Ⅰ)

wherein Xa and Xb are selected from M, Y, L, V, W or E.

Preferably, Xa and Xb are selected from M or W.

Preferably, the inhibitor is one of the following polypeptides:

ETMFWYDAKMPCFTRVPMAMGTDEQLPDIWN；

ETMFWYDAKWPCFTRVPMAWGTDEQLPDIWN；

ETMFWYDAKMPCFTRVPMAWGTDEQLPDIWN；

ETMFWYDAKWPCFTRVPMAMGTDEQLPDIWN。

preferably, the polypeptide is chemically modified at the N-and/or C-terminus.

Preferably, the polypeptide is modified by acetylation at the N-terminus and amidation at the C-terminus.

Preferably, the polypeptide is in the form of:

Ac-ETMFWYDAKMPCFTRVPMAMGTDEQLPDIWN-NH₂；

Ac-ETMFWYDAKWPCFTRVPMAWGTDEQLPDIWN-NH₂；

Ac-ETMFWYDAKMPCFTRVPMAWGTDEQLPDIWN-NH₂；

Ac-ETMFWYDAKWPCFTRVPMAMGTDEQLPDIWN-NH₂。

preferably, the polypeptide is in free form or in pharmaceutically acceptable salt form, including hydrochloride, sulfate, phosphate, sulfonate, acetate, citrate, and tartrate.

The inhibitor is applied to the preparation of drugs for resisting proliferation and invasion of hepatocellular carcinoma.

A pharmaceutical composition comprises the above polypeptide inhibitor and pharmaceutically acceptable adjuvants.

The application of the composition in preparing the medicine for resisting proliferation and invasion of hepatocellular carcinoma.

The invention has the outstanding advantages that:

the polypeptide inhibitor provided by the invention can effectively inhibit the expression of MACC1 gene in hepatocellular carcinoma, further effectively inhibit the invasion and metastasis of hepatocellular carcinoma, and can be used for preparing a medicine for resisting the invasion of hepatocellular carcinoma.

Drawings

FIG. 1 is a graph of the effect of polypeptide inhibitors on MACC1mRNA expression;

FIG. 2 is a graph showing the effect of polypeptide inhibitors on MACC1 protein expression.

Detailed Description

The following detailed description of the present invention is provided in connection with the examples, and for reasons of brevity, the description of the experimental procedures is not intended to be exhaustive, and all parts not specifically described in the experiments are routine procedures well known to those skilled in the art.

First, experimental material

The polypeptides involved in the invention are synthesized by standard polypeptide solid phase synthesis technology known in the art, and a fluorenylmethyloxycarbonyl (Fmoc) N-terminal protection strategy is adopted. Connecting corresponding amino acids in sequence according to a resin solid phase synthesis method, removing Fmoc-protecting groups in sequence during the process, cutting peptides to obtain crude products, and separating and purifying the crude products by a C18 column to obtain the polypeptide shown in the formula (I).

HPLC and MS detection confirm the structure of the polypeptide of the invention, and the purity is more than or equal to 98%.

The amino acid sequences of the polypeptides I-1 to I-4 are as follows, the N-and C-termini being chemically unmodified:

ETMFWYDAKMPCFTRVPMAMGTDEQLPDIWN (polypeptide I-1);

ETMFWYDAKWPCFTRVPMAWGTDEQLPDIWN (polypeptide I-2);

ETMFWYDAKMPCFTRVPMAWGTDEQLPDIWN (polypeptide I-3);

ETMFWYDAKWPCFTRVPMAMGTDEQLPDIWN (polypeptide I-4).

The human hepatocellular carcinoma cell line HepG2 was stored in the molecular pharmacology laboratory of this company. Fetal bovine serum was purchased from Hangzhou ilex. Tetramethylazo salts and dimethyl sulfoxide were purchased from Sigma, USA. The rabbit anti-human MACC1 antibody was purchased from Santa Cruz, and the HRP-labeled goat anti-rabbit IgG secondary antibody was purchased from Beijing Zhonghuan gold bridge. Transwell chambers were purchased from Millipore corporation. PCR primers were synthesized in Ruibo, Guangzhou, and RT-PCR kits were purchased from Takara.

Second, Experimental methods

1. Culturing human liver cell cancer cell strain HepG2

The primary liver cancer cell line is cultured in RPMI-1640 culture solution containing 10% fetal calf serum at 37 deg.C and 5% CO₂Culturing, and digesting with 0.25% pancreatin for 2-3d for passage. Cells in logarithmic growth phase were taken for the experiments.

2. MTT method for detecting influence of polypeptide on proliferation capacity of HepG2

HepG2 cells were collected at logarithmic growth phase and made into cell suspension at 5X 10 per well⁴Individual cells were seeded in 96-well plates with 100 μ L cell suspension per well. Divided into a control group, a polypeptide I-1 group, a polypeptide I-2 group, a polypeptide I-3 group and a polypeptide I-4 group. Adding 2, 4, 8, 16 and 32 mu M of polypeptide into the polypeptide group respectively, adding an equal volume of reagent into a control group, culturing for 24 hours, taking out a 96-well culture plate, discarding a culture solution, washing with PBS, adding 100 mu L of RPMI-1640 culture solution containing MTT (with a final mass concentration of 5g/L), continuously culturing cells for 4 hours, discarding the culture solution, adding 100 mu L of dimethyl sulfoxide into each well, and shaking a shaker to dissolve crystals. The OD per well was determined at 490nm with DMSO zeroing. The inhibition rate was calculated according to the following formula, and the IC50 value of each polypeptide was calculated by linear regression. Growth inhibition (%) was (1-dose group OD value/control group OD value) × 100%

3. Cell invasion experiment for detecting influence of polypeptide on invasion capacity of HepG2

Taking 4 ℃ pre-cooled Matrigel and serum-free RPMI-1640 cell culture solution, and mixing the components in a volume ratio of 1: 4, fully and uniformly mixing. To the upper chamber of the Transwell plate, 30. mu.L of the above-mentioned mixture was added per well, covering the entire polycarbonate membrane in the chamber, and after standing in an incubator at 37 ℃ for 3 hours, 100. mu.L of cell suspension (cell density 1X 10) was added per well⁵mL) and 200. mu.L serum-free RPMI-1640 medium; at the same time, 500. mu.L of RPMI-1640 medium containing 30% by volume of fetal bovine serum was added to each well of the Transwell plate. Then, HepG2 cells were treated with 0, 2, 4, 8, 16, 32. mu.M of the polypeptide for 24h, respectively, and then the chamber was taken out, the culture solution was aspirated, fixed with methanol for 30min, stained with crystal violet for 20min, inverted, observed with a Leica DC 300F upright microscope, and photographed. 5 pieces of the upper, lower, left, right and center of the bottom surface of the small chamber are selectedCells were counted in visual field and invasion rate was calculated. The experiment was repeated 3 times with 10 replicates per experimental point.

4. RT-PCR detection of the Effect of Polypeptides on MACC1mRNA expression in HepG2 cells

After digestion and counting of HepG2 cells, the cells were added to a 6-well plate at a cell density of 5X 10⁴and/mL, after the cells are attached, adding 4 and 16 mu M of polypeptide to culture for 24h, collecting the cells, and measuring the expression of MACC1mRNA in HepG2 cells. A control group was also provided, and no polypeptide was added to the control group. Extracting total RNA of cells, carrying out reverse transcription to obtain cDNA, and carrying out PCR by using the cDNA as a template. The primers are as follows:

MACC1 upstream primer: 5'-CCTTCGTGGTAATAATGCTTCC-3'

MACC1 downstream primer: 5'-AGGGCTTCCATTGTATTGAGGT-3'

Beta-actin upstream primer: 5'-ACGCACCCCAACTACAACTC-3'

Beta-actin downstream primer: 5'-TCTCCTTAATGTCACGCACGA-3'

Preparing a 50 mu L reaction system in a sterile enzyme-free PCR tube, wherein the reaction conditions are 94 ℃ for 60s, 60 ℃ for 50s and 72 ℃ for 90s, and after 40 cycles, the extension is carried out for 10min at 72 ℃; stored at 4 ℃ for 20g/L agarose gel electrophoresis. The experiment was repeated 3 times.

5. Western blot detection of influence of polypeptide on expression of MACC1 protein in HepG2 cells

After digestion and counting of HepG2 cells, the cells were added to a 6-well plate at a cell density of 5X 10⁴and/mL, after the cells are attached, adding 4 and 16 mu M of polypeptide to culture for 24h, collecting the cells, and measuring the expression of the MACC1 protein in HepG2 cells. A control group was also provided, and no polypeptide was added to the control group. The total cell protein was quantitatively extracted by BCA method, and a loading buffer was added to the total cell protein sample and sufficiently denatured with boiling water. Separating protein by polyacrylamide gel electrophoresis, performing chemiluminescence after protein membrane transfer, blocking, hybridization of primary antibody (diluted by 1: 5000) and secondary antibody (diluted by 1: 10000). Beta-actin is used as an internal reference. Finally, the strip is subjected to gray scale scanning. Relative expression of MACC1 is MACC1 integrated optical density of the band/beta-actin integrated optical density of the band.

Third, experimental results

1. Effect of Polypeptides on the proliferative Capacity of HepG2

Compared with a control group, the proliferation of HepG2 cells of each polypeptide administration group is obviously inhibited, the proliferation inhibition rate is related to the concentration of the polypeptide, and the higher the concentration is, the stronger the inhibition effect is, and the obvious concentration dependence is realized. The results are shown in Table 1.

TABLE 1 proliferation inhibition (%), of different concentrations of polypeptide on HepG 2%

	2μM	4μM	8μM	16μM	32μM
						Polypeptide group I-1	13.55	24.32	40.69	64.95	78.32
Group I-2 Polypeptides	18.60	28.58	45.46	69.36	84.58
						Group I-3 Polypeptides	10.84	21.72	38.06	59.84	72.44
Polypeptide group I-4	11.56	22.85	39.73	62.08	75.73

The results show that the polypeptide I-1, the polypeptide I-2, the polypeptide I-3 and the polypeptide I-4 have obvious inhibition effect on the proliferation of HepG2 cells, the inhibition effect presents a certain concentration dependence relationship, the IC50 is about 12 mu M, and the drug effect is excellent.

2. Effect of Polypeptides on the invasive Capacity of HepG2

Each polypeptide can obviously inhibit the invasion of HepG2 cells, and the invasion rate of HepG2 cells is reduced along with the increase of the concentration of the polypeptide.

The results are shown in Table 2.

TABLE 2 Effect of different concentrations of polypeptide on the invasion Rate (%) of HepG2

	2μM	4μM	8μM	16μM	32μM
						Polypeptide group I-1	83.48	62.69	44.94	23.84	11.25
Group I-2 Polypeptides	80.16	61.20	43.04	22.28	9.64
						Group I-3 Polypeptides	86.85	65.74	47.35	26.52	13.12
Polypeptide group I-4	85.26	64.92	46.03	25.06	12.03

Transwell invasion experiments prove that the polypeptide I-1, the polypeptide I-2, the polypeptide I-3 and the polypeptide I-4 have obvious inhibition effect on the invasion of HepG2 cells, and the inhibition effect presents a certain concentration dependence relationship.

3. Effect of the Polypeptides on MACC1mRNA expression in HepG2 cells

After 24 hours of administration treatment of the polypeptide I-1, the polypeptide I-2, the polypeptide I-3 and the polypeptide I-4, the MACC1mRNA expression level in HepG2 cells is obviously reduced, and the higher the polypeptide concentration is, the more obvious the MACC1mRNA expression level is reduced.

The relative expression levels of MACC1mRNA in each group are shown in FIG. 1.

The results show that the polypeptide I-1, the polypeptide I-2, the polypeptide I-3 and the polypeptide I-4 have obvious inhibiting effect on the transcription level of MACC1 genes of HepG2 cells.

4. Effect of the Polypeptides on MACC1 protein expression in HepG2 cells

After the polypeptide I-1, the polypeptide I-2, the polypeptide I-3 and the polypeptide I-4 are treated by administration for 24 hours, the MACC1 protein expression level in HepG2 cells is obviously reduced, and the MACC1 protein expression level is obviously reduced when the polypeptide concentration is higher.

The relative expression of MACC1 protein in each group is shown in FIG. 2.

The results show that the polypeptide I-1, the polypeptide I-2, the polypeptide I-3 and the polypeptide I-4 have obvious inhibition effect on the translation level of MACC1 gene of HepG2 cells.

The following conclusions can be drawn by combining the above experiments: the polypeptide I-1, the polypeptide I-2, the polypeptide I-3 and the polypeptide I-4 can inhibit the expression of the MACC1 gene in HepG2 cells from the transcription and translation levels, and are effective inhibitors of the MACC1 gene; meanwhile, the effective inhibitor of the MACC1 gene can obviously inhibit the proliferation and invasion of HepG2 cells, and the effective inhibitor of the MACC1 gene can be used for preparing a medicine for resisting the invasion of hepatocellular carcinoma.

The applicant also tested the pharmacological effects of the above-mentioned polypeptide after terminal modification (N-terminal modified by acetylation and C-terminal modified by amidation), and the results demonstrated that the above-mentioned polypeptide after terminal modification has similar pharmacological effects and potency to those of the unmodified form.

The foregoing embodiments are provided to illustrate the present invention more fully, but those skilled in the art will appreciate that the scope of the present invention should not be limited to the specific embodiments described above.

Claims

1. An inhibitor of the MACC1 gene, which is a polypeptide characterized by the amino acid sequence of one of the following sequences:

sequence 1: ETMFWYDAKMPCFTRVPMAMGTDEQLPDIWN, respectively;

sequence 2: ETMFWYDAKWPCFTRVPMAWGTDEQLPDIWN, respectively;

and (3) sequence: ETMFWYDAKMPCFTRVPMAWGTDEQLPDIWN, respectively;

and (3) sequence 4: ETMFWYDAKWPCFTRVPMAMGTDEQLPDIWN are provided.

2. Use of the inhibitor of claim 1 for the preparation of a medicament for the treatment of proliferation and invasion of hepatocellular carcinoma.