CN109096373B

CN109096373B - Synthetic oligopeptides and uses thereof

Info

Publication number: CN109096373B
Application number: CN201811128109.8A
Authority: CN
Inventors: 郑嘉荣
Original assignee: Jiangsu Hengrui Biomedical Technology Co ltd
Current assignee: Jiangsu Hengrui Biomedical Technology Co ltd
Priority date: 2018-09-27
Filing date: 2018-09-27
Publication date: 2022-06-21
Anticipated expiration: 2038-09-27
Also published as: CN109096373A

Abstract

The invention discloses a synthetic oligopeptide and application thereof. The lung cancer is one of the malignant tumors with the highest global morbidity and mortality, the morbidity of the lung cancer is on the rising and younger trend in China in recent years, and the occurrence, development and metastasis of the lung cancer relate to extremely complex polygene regulation abnormal processes. Therefore, the research on the etiology and pathogenesis of the lung cancer has important clinical significance. Invasion and metastasis are the major causes of patient death due to malignancy. The oligopeptide provided by the invention can inhibit proliferation, migration and invasion of lung cancer cells by inhibiting NGAL gene expression, and can be prepared into a medicament for preventing and treating lung cancer metastasis and treating lung cancer.

Description

Synthetic oligopeptides and uses thereof

Technical Field

The invention belongs to the field of chemistry, and relates to synthetic oligopeptides and application thereof.

Background

The lung cancer is one of the malignant tumors with the highest global morbidity and mortality, the morbidity of the lung cancer is on the rising and younger trend in China in recent years, and the occurrence, development and metastasis of the lung cancer relate to extremely complex polygene regulation abnormal processes. Therefore, the research on the etiology and pathogenesis of the lung cancer has important clinical significance.

Invasion and metastasis are the major causes of patient death due to malignancy.

Disclosure of Invention

The present invention aims to provide synthetic oligopeptides and uses thereof.

The technical scheme of the invention is as follows:

an oligopeptide, characterized in that: the Sequence is shown in Sequence No. 2.

Use of the above-mentioned oligopeptide as an inhibitor of NGAL gene expression.

The oligopeptide is used for preparing the medicine for preventing and treating lung cancer metastasis.

The oligopeptide is used for preparing the medicine for treating the lung cancer.

Has the beneficial effects that:

the lung cancer is one of the malignant tumors with the highest global morbidity and mortality, the morbidity of the lung cancer is on the rising and younger trend in China in recent years, and the occurrence, development and metastasis of the lung cancer relate to extremely complex polygene regulation abnormal processes. Therefore, the research on the etiology and pathogenesis of the lung cancer has important clinical significance. Invasion and metastasis are the major causes of patient death due to malignancy. The oligopeptide provided by the invention can inhibit proliferation, migration and invasion of lung cancer cells by inhibiting NGAL gene expression, and can be prepared into a medicament for preventing and treating lung cancer metastasis and treating lung cancer.

Drawings

FIG. 1 shows the effect of oligopeptides 1-5 on NGAL gene expression in lung cancer A549 cells;

FIG. 2 shows the effect of oligopeptides 1-5 on lung cancer A549 cell proliferation;

FIG. 3 shows the effect of oligopeptides 1-5 on the migration ability of lung cancer A549 cells;

FIG. 4 shows the effect of oligopeptides 1-5 on the invasive potential of lung cancer A549 cells.

Detailed Description

The following detailed description of the present invention is provided in connection with the accompanying drawings and examples, but not intended to limit the scope of the invention.

First, experimental material

Oligopeptide 1-5 is synthesized by entrusting an outsourcing mechanism, the purity is not lower than 95%, and the sequence is as follows:

oligopeptide 1: RCRVFYKPWVRHQMRGRYN (Sequence No. 1);

oligopeptide 2: HPWYRKWNYRVRWRGWFMR (Sequence No. 2);

oligopeptide 3: QDRKWSYKSSYFRKRGRSRT (Sequence No. 3);

oligopeptide 4: PHPEGEFRTKMEYRWEYRVR (Sequence No. 4);

oligopeptide 5: PHPEDEFATKFEYRWGYRVR (Sequence No. 5).

Fetal bovine serum and DMEM high-sugar medium are purchased from Gbico company; TRIzol reagent was purchased from Invitrogen; the reverse transcription kit and the quantitative PCR kit are purchased from TAKARA company; the Tanswell invasion cell was purchased from millipore corporation.

RT-PCR primers for NGAL and GAPDH were synthesized by the outsourcing agency.

Second, Experimental methods

1. Cell culture

Lung cancer A549 cell is cultured in DMEM medium containing 10% fetal calf serum at 37 deg.C and 5% CO₂Culturing under the condition.

2. Grouping and administration of drugs

Taking lung cancer A549 cells in logarithmic growth phase, and randomly and group-wise administering:

control group: culturing with DMEM medium containing 10% fetal calf serum;

oligopeptide 1 group: adding 5 mu M oligopeptide 1 on the basis of a control group for culture;

oligopeptide 2 group: adding 5 mu M oligopeptide 2 on the basis of a control group for culture;

oligopeptide 3 group: adding 5 mu M oligopeptide 3 on the basis of a control group for culture;

oligopeptide 4 group: adding 5 mu M oligopeptide 4 on the basis of a control group for culture;

oligopeptide 5 group: 5 mu M oligopeptide 5 was added to the control group for culture.

3. Effect of oligopeptide on NGAL gene expression in lung cancer A549 cells

Culturing lung cancer A549 cell in DMEM medium containing 10% fetal calf serum, digesting and counting cells in logarithmic growth phase, and 2 × 10⁵And (3) paving the cells/hole on a 24-hole plate, after the cells adhere to the wall, grouping according to the grouping method, replacing the cells with corresponding culture media for culture, arranging 3 parallel holes in each group, continuing to culture for 48 hours, removing supernatant, collecting the cells, extracting total RNA of the cells according to the specification of a TRIzol kit, respectively measuring the RNA concentration by using an ultraviolet spectrophotometer, and judging the purity according to the ratio of absorbance OD260 to OD 280. Respectively taking 5 mu g of total RNA of the cells, carrying out reverse transcription to form cDNA according to the specification of a reverse transcription kit, and using the cDNA as a template. The reaction conditions are as follows: preheating at 94 ℃ for 3min, then carrying out amplification at 94 ℃ for 30s, 52 ℃ for 30s and 72 ℃ for 50s for 29 cycles, and carrying out extension at 72 ℃ for 10 min. The PCR product was subjected to 1% agarose gel electrophoresis, and the grayscale value of each electrophoresis band was analyzed by Image J Image analysis software, and the relative expression level of the target gene mRNA was expressed as the ratio of the grayscale value of the target band to the grayscale value of the reference band within GAPDH. The forward primer of NGAL was 5'-GAAGACAAAGACCCGCAAAAG-3', and its reverse primer sequence was 5'-CTGGCAACCTGGAACAAAAG-3'. The sequence of the upstream primer of the internal control GAPDH is 5'-ACCACAGTCCATGCCATCAC-3', and the sequence of the downstream primer is 5'-TCCACCACCCTGTGCTGTA-3'.

4. MTT method for detecting proliferation inhibition effect of oligopeptide on lung cancer A549 cells

Culturing lung cancer A549 cells in DMEM medium containing 10% fetal bovine serumIn the middle, the cells in logarithmic growth phase are digested and counted, 4 × 10⁴And (3) paving the cells/hole on a 96-well plate, after the cells adhere to the wall, grouping according to the grouping method, replacing the cells with corresponding culture media for culture, arranging 6 parallel holes in each group, continuing to culture for 48 hours, removing supernatant, adding MTT (methyl thiazolyl tetrazolium) dissolved in a serum-free culture medium into each hole, incubating for 4 hours, and measuring the absorbance value at 570nm of an enzyme labeling instrument after dissolving the cells in dimethyl sulfoxide.

5. Cell migration ability assay

Culturing lung cancer A549 cells in DMEM medium containing 10% fetal calf serum, digesting cells in logarithmic growth phase, centrifuging, discarding supernatant, suspending cells by DMEM medium containing 10% fetal calf serum in volume fraction to obtain single cell suspension, and performing grouping and administration at 37 deg.C and 5% CO in volume fraction₂Culturing in a saturated humidity incubator for 48 h.

After 48h of culture, the cells were collected by digestion and made into cell suspension in serum-free DMEM medium at 2.5X 10⁴The cells were seeded at a density of one well per well in a 6-well plate, and when the cells were attached to the wall in a monolayer and were close to 100% confluency, the cells were scraped in the 6-well plate using a 10. mu.l sterile pipette tip, washed to suspend the exfoliated cells, and then placed in a serum-free medium at 37 ℃ in 5% CO₂In the incubator, pictures are taken under an inverted optical microscope at 0h and 48h after scratching respectively, the healing rate of the scratching is calculated, and the experiment is repeated for 3 times. The scratch healing rate (scratch distance after 0h scratch distance-48 h)/0 h scratch distance × 100%.

6. Cell invasion capacity assay

After 48h of culture, the cells were collected by digestion and cell suspension was prepared in serum-free DMEM medium at 2.5X 10⁵The density of the pores is spread in the upper chamber of the Transwell cell. Then, DMEM medium containing 20% by volume fetal bovine serum was added to the Transwell cell lower chamber. After 24h incubation, the non-invasive cells were removed from the upper chamber of the Transwell chamber with a cotton swab, the Transwell chamber was taken, washed 3 times with PBS, fixed in methanol for 30min, stained with 0.1% crystal violet for 30min, and counted by observation on an inverted optical microscope. Randomly select 5 field counts.

7. Statistical analysis

The experiment was repeated 3 times using SPSS 19.0 statistical software, data were expressed as mean. + -. standard deviation, and the sample mean comparisons were performed using the t test between groups. P <0.05 is significant.

Third, experimental results

1. Effect of oligopeptide 1-5 on NGAL gene expression in lung cancer A549 cells

The results are shown in table 1 and fig. 1, wherein oligopeptides 1-4 can significantly inhibit the expression level of NGAL gene in lung cancer A549 cells, and oligopeptide 5 has no obvious inhibition effect on the expression of NGAL gene in lung cancer A549 cells.

TABLE 1 Effect of oligopeptides 1-5 on NGAL Gene expression in Lung cancer A549 cells

Neutrophil gelatinase-associated lipocalin (NGAL) is a member of the lipocalin family. The research finds that the NGAL is highly expressed in lung cancer tissues and is possibly a potential target point for treating the lung cancer (reference document: immunohistochemical research, experiment and inspection medicine of NGAL expression in squamous cell lung cancer and adenocarcinoma, 2014 01).

2. Effect of oligopeptide 1-5 on lung cancer A549 cell proliferation

The results are shown in table 2 and fig. 2, oligopeptides 1-4 can obviously inhibit the proliferation of lung cancer A549 cells, and oligopeptide 5 has no obvious inhibition effect on the proliferation of lung cancer A549 cells.

TABLE 2 Effect of oligopeptides 1-5 on Lung cancer A549 cell proliferation

3. Influence of oligopeptide 1-5 on lung cancer A549 cell migration capacity

The results are shown in table 3 and fig. 3, wherein oligopeptides 1-4 can significantly inhibit the migration ability of lung cancer A549 cells, and oligopeptide 5 has no obvious inhibition effect on the migration ability of lung cancer A549 cells.

TABLE 3 Effect of oligopeptides 1-5 on migration ability of Lung cancer A549 cells

4. Effect of oligopeptide 1-5 on lung cancer A549 cell invasion capacity

The results are shown in table 4 and fig. 4, wherein oligopeptides 1-4 can significantly inhibit the invasive ability of lung cancer A549 cells, and oligopeptide 5 has no significant inhibition effect on the invasive ability of lung cancer A549 cells.

TABLE 4 Effect of oligopeptides 1-5 on the invasive potential of Lung cancer A549 cells

The above-described embodiments are intended to be illustrative of the nature of the invention, but those skilled in the art will recognize that the scope of the invention is not limited to the specific embodiments.

Sequence listing

<110> Huaian Anlai Biotechnology Ltd

<120> synthetic oligopeptides and uses thereof

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 19

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Arg Cys Arg Val Phe Tyr Lys Pro Trp Val Arg His Gln Met Arg Gly

1 5 10 15

Arg Tyr Asn

<210> 2

<211> 19

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

His Pro Trp Tyr Arg Lys Trp Asn Tyr Arg Val Arg Trp Arg Gly Trp

1 5 10 15

Phe Met Arg

<210> 3

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Gln Asp Arg Lys Trp Ser Tyr Lys Ser Ser Tyr Phe Arg Lys Arg Gly

1 5 10 15

Arg Ser Arg Thr

20

<210> 4

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Pro His Pro Glu Gly Glu Phe Arg Thr Lys Met Glu Tyr Arg Trp Glu

1 5 10 15

Tyr Arg Val Arg

20

<210> 5

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Pro His Pro Glu Asp Glu Phe Ala Thr Lys Phe Glu Tyr Arg Trp Gly

1 5 10 15

Tyr Arg Val Arg

20

Claims

1. An oligopeptide, characterized in that: the Sequence is shown as Sequence number 2.

2. Use of the oligopeptide of claim 1 for the preparation of a medicament for the treatment of lung cancer.