CN107446023A - It is a kind of can antagonism HuR protein rna binding activity polypeptide HIP 13 and its application - Google Patents

Abstract

The invention provides it is a kind of can antagonism HuR protein rna binding activity polypeptide and its application, its amino acid sequence such as SEQ ID NO:Shown in 1；A kind of antineoplastic polypeptide and its application are further related to, the antineoplastic polypeptide includes tumor cytotoxicity domain and wears spanning domain, the amino acid sequence such as SEQ ID NO of tumor cytotoxicity domain:Shown in 1.The spanning domain of wearing of the antineoplastic polypeptide of the present invention does not have cytotoxicity in itself, but after connection tumor cytotoxicity domain, there is the obvious effect for suppressing tumor proliferation, migrating invasion and attack.The antineoplastic polypeptide of the present invention, it can not only be expected to combine other treatment mode to suppress tumour separately as antitumor biotherapeutics.

Description

It is a kind of can antagonism HuR protein rna binding activity polypeptide HIP-13 and its application

Technical field

The present invention relates to neoplasm targeted therapy field, more specifically it relates to which one kind can antagonism HuR protein rna binding activity Polypeptide and its application.

Background technology

Cancer is to cause the underlying cause of death of human death, is a kind of major disease for currently endangering human health.According to state Border Agency for Research on Cancer statistics, the whole world in 2012 shares 14,100,000 new cases and 8,200,000 deaths.Although in the past tens Nian Zhong, because operation, chemicotherapy the methods of progress, oncotherapy achieves significant progress, but these methods are in the same for the treatment of When, often side effect is very big, and most of cancers still have high relapse rate and mortality risk.Therefore, new treatment is sought Measure is very necessary and urgent to improve the survival rate of cancer and cure rate.

The molecular targeted therapy of rising in recent years is that on a molecular scale, for clearly carcinogenic target spot, design is accordingly Medicine, specifically select carcinogenic target spot to play a role, killing tumor cell, without influence normal tissue cell.In recent years Come, novel molecular targeted drug achieves the effect of notable in clinical practice, has pushed the treatment of cancer to a new stage. Targeted polypeptide is presently considered to be more satisfactory tumor cells targeted therapy means.Searching may be with some spies in tumour cell The small peptide that different gene specific combines, the purpose of targeting therapy on tumor can be reached.

Human antigen R (human antigen R, HuR) is embryonic death abnormal vision (embryonic in rna binding protein Lethal abnormalvision, ELAV) protein family member, high expression, such as breast in a variety of mankind tumor tissues and cell Gland cancer, stomach cancer, colorectal cancer, cervical carcinoma, oophoroma and lung cancer are related to tumour progression, transfer and prognosis.HuR can regulate and control growth The mRNA stability of the factor and cell factor, such as p21, c-fos, c-Myc, p53, cyclinA, cyclin E1, COX-2, regulation and control Growth of tumour cell, propagation and apoptosis.HuR activity forms and the grade malignancy of kinds of tumors are into positive correlation.Using HuR as target spot Antineoplastic will provide new thinking for oncotherapy, and new breakthrough is brought for the diagnosis and treatment of tumour.However, do not have still at present There is the relevant report of targeting HuR antineoplastic.

Therefore, it is necessary to build one kind can targets neoplastic cells, and can efficiently enters the novel polypeptide of born of the same parents.

The content of the invention

To solve problem above, inventor is prepared for a kind of biologically active peptide, and the polypeptide is passed through with cell-penetrating peptide Covalent bond connects, and reaches existing targets neoplastic cells, has the effect for efficiently entering born of the same parents again.

Based on the research, the invention provides it is a kind of can antagonism HuR protein rna binding activity polypeptide, its amino acid sequence Row such as SEQ ID NO:Shown in 1.It is demonstrated experimentally that the polypeptide contestable antagonism promotion sensitivity gene HuR and RNA combination, and thin Born of the same parents' level shows obvious tumor inhibition effect.

Present invention also offers it is above-mentioned can antagonism HuR protein rna binding activity polypeptide in antineoplastic is prepared Using.

Present invention also offers a kind of antineoplastic polypeptide, and it includes tumor cytotoxicity domain and wears spanning domain, institute State the amino acid sequence such as SEQ ID NO of tumor cytotoxicity domain:Shown in 1.

Preferably, the amino acid sequence for wearing spanning domain such as SEQ ID NO:Shown in 2.

Preferably, the N-terminal worn spanning domain and be connected to the tumor cytotoxicity domain

Present invention also offers application of the above-mentioned antineoplastic polypeptide in antineoplastic is prepared.

It is an advantage of the current invention that the spanning domain of wearing of the antineoplastic polypeptide of the present invention does not have cytotoxicity in itself, but even After connecing HuR RNA binding activity peptide fragments, obvious tumor inhibitory effect can be observed in cellular level.The present invention's is antitumor Polypeptide, it can not only be expected to combine other treatment mode to suppress tumour separately as antitumor biotherapeutics.

Brief description of the drawings

Fig. 1 is the schematic diagram of HIP-13 competitive antagonism HuR albumen and RNA interaction principles；

Fig. 2 is that control peptide and HIP-13 handle fluorescent microscopy images of the HeLa cells after 48 hours；

Fig. 3 is the MTT colorimetric statistical charts that HeLa is handled with the HIP-13 or control peptide of various concentrations；

Fig. 4 is the MTT colorimetric statistical charts after 20 μm of ol/L HIP-13 or control peptide processing HeLa cell different times；

Fig. 5 is that plate clone forms experiment photo；

Fig. 6 is the statistical chart according to Fig. 5 number of cell clones calculated；

Fig. 7 is Transwell cell invasion experiment photos；

Fig. 8 is the transport number purpose statistical chart according to Fig. 7 tumour cells calculated；

Fig. 9 is RNA co-immunoprecipitation experiments (RIP) photo；

Figure 10 is the PCDHB2mRNA level statistic figures calculated according to Fig. 9；

Figure 11 is the SLC2A4mRNA level statistic figures calculated according to Fig. 9.

Embodiment

The principle and feature of the present invention are described below in conjunction with example, the given examples are served only to explain the present invention, and It is non-to be used to limit the scope of the present invention.

1. the synthesis of antineoplastic polypeptide

A kind of antineoplastic polypeptide is synthesized by solid-phase synthesis, it includes a tumor cytotoxicity domain and one is worn Spanning domain, wherein tumor cytotoxicity domain sequence such as SEQ ID NO:Shown in 1, spanning domain sequence such as SEQ ID are worn NO:Shown in 2, the N-terminal of tumor cytotoxicity domain is connected to, resulting sequence is：Amino acid sequence is YGRKKRRQRRR-VAGHSLGYGFVNK(SEQ ID NO:3), it is named as HIP-13.In order to study conveniently, we are antitumor The C-terminal connection marked by fluorescein isothiocyanate FITC of polypeptide, N-terminal connection biotin labeling Biotin, shone by force biological section by Shanghai Skill Co., Ltd synthesizes, and through high performance liquid chromatography and Mass Spectrometric Identification, purity is more than 95%.Polypeptide is dissolved in sterile PBS bufferings Liquid, concentration 1mmol/L, lucifuge freezes standby in -80 DEG C.

HIP-13 competitive antagonism HuR albumen and the principle that RNA interacts are as shown in Figure 1.

2.HIP-13 cellular localization detection

Pancreatin digestion HeLa cells after, be resuspended cell in complete medium, fully piping and druming, be allowed into single cell suspension, Count.Entered with the cell density kind in 20000/hole in 24 well culture plates.It is first according to the size of creep plate when cell suspension is added dropwise A small amount of culture medium is dripped in the position for preparing to put creep plate in each hole, and then creep plate is placed on drop, compressed, makes creep plate and culture Ware is bonded together by the tension force of culture medium.It is (dense eventually using the targeting peptides and control peptide of FITC marks after cell is affixed on creep plate Spend for 20 μm of ol/L) HeLa cells are incubated, 4% paraformaldehyde (PBS preparations) is added after 48 hours and fixes 20 minutes.Remove poly Formaldehyde, cell is rinsed 3 × 3 minutes using PBS.With DiI (cell membrane red fluorescence probe, green skies company, article No. C1036) with The μ l of final concentration 5 are dyed to cell membrane, and nucleus is dyed with DAPI dyestuffs, and mounting is after under Laser Scanning Confocal Microscope Observe intake and polypeptide positioning of the tumour cell to polypeptide.

Experimental result is as shown in Fig. 2 HIP-13 can be effectively by cellular uptake, and polypeptide accumulates in tumour cell after 48 hours Nucleus, the dyeing of strong karyon is presented.

3.MTT colorimetrically analysings detect inhibitory action of the HIP-13 to growth of tumour cell

The HeLa cells in exponential phase are taken, 96 orifice plates are inoculated in the density of 3000 cells/wells, according to experiment Need with various concentrations polypeptide processing cell, every group of 6 multiple holes；Untreated fish group is arranged to control group.Cultivate as required not The same time.The μ l/ holes of 0.5%MTT 20 are separately added into above-mentioned each group cancer cell, continues culture 4 hours, supernatant discarding, adds The μ l/ holes of DMSO 100, at room temperature vibration determine absorbance (A) value at ELIASA 490nm wavelength immediately to crystallization dissolving.Often Secondary experiment is repeated 3 times.Cancer cell multiplication maximum inhibition (%)=(1- experimental group mean light absorbency A values/control group average absorbance Spend A values) × 100%.

As a result as shown in Figures 3 and 4, compared with control group polypeptide, the vigor of the tumour cell of HIP-13 polypeptides processing is notable Reduce, and the effect is in time and dose dependent, illustrates that the novel polypeptide can effectively reduce the cell viability of tumour cell； When processing cell stage is identical, peptide concentration is that 15-25 μm of ol/L cell viability inhibiting rate is higher；When processing cell concentration When identical, polypeptide processing time is higher for the cell viability inhibiting rate of 24-48 hours

4. plate clone forms inhibitory action of the experiment detection HIP-13 to proliferative activity o f tumor

Take the logarithm the monolayer cultivation HeLa cells in growth period, with 0.25% Trypsin Induced and blow and beat into individual cells, Cell is suspended in standby in nutrient solution.Make the dilution of gradient multiple after cell suspension is counted, with appropriate cell density 300 Individual/hole is inoculated in culture plate () per the visible 5-6 cell in the visual field preferably.After it is thin it is uniformly adherent after, need to handle according to experiment Cell, and untreated control group is set.Put 37 DEG C, 5%CO₂And in the environment of saturated humidity, quiescent culture 2-3 weeks.Work as culture When occurring macroscopic clone in ware, culture is terminated, abandoning supernatant, is carefully embathed with PBS 2 times.Add pure methanol, fix 15 Minute.Fixer is removed, adds appropriate coomassie brilliant blue staining liquid dye 10-30 minutes, slowly washes away dyeing liquor with flowing water, air is done It is dry.Plate is inverted and imaged, clone is with the naked eye directly counted or Counting software calculates clone's number, or counted in microscope (low power lens) Number is more than clone's number of 10 cells.

Experimental result is as it can be seen in figures 5 and 6, compared with control group polypeptide, after experimental group HIP-13 processing, gram of tumour cell Grand Colony forming significantly reduces, and the effect is in dose dependent.

Influences of the 5.Transwell experiments detection HIP-13 to tumor cell migration activity

24 well culture plate lower floors add the μ l of culture medium 500 containing 20% serum, gently put cell, do not produce alveolar cells After counting, fully mix, upper strata adds 10000 cells per hole, adds 5% blood serum medium totally 200 μ l, and multiple holes add per hole Measure equal to ensure uniform pressure, need to handle cell according to experiment, and untreated control group is set.Put 37 DEG C, 5%CO₂And It is incubated overnight in the environment of saturated humidity, cell upper cell is dabbed with swab stick, is added the μ l of methanol 500 and fix 10-15 minutes, PBS, violet staining are more than 15 minutes, PBS, photograph, count each random high-powered fields cell migration number.

As a result as shown in FIG. 7 and 8, compared with control group polypeptide, after experimental group HIP-13 processing, the migration of tumour cell Number is obvious to be reduced.

7.HIP-13 suppresses the mechanism of action of tumour

Suppress the mechanism of action of tumour using RNA co-immunoprecipitation experiments (RIP) research HIP-13.

Collect cell pyrolysis liquid：HeLa cells are cultivated in culture dish, cell length are treated to 70-80% fusions, in two tissue cultures Support and control peptide and HIP-13 are separately added into cell, after cultivating 48 hours, cold PBS culture dish twice, is used after adding cold PBS Cell, which is scraped, scrapes off cell, collects to EP and manages, 1500rpm, and 4 DEG C centrifuge 5 minutes, abandon supernatant, collect cell, with cell etc. Cell is resuspended in the RIP lysates of volume, and piping and druming uniformly stands 5 minutes after on ice, and often pipe dispenses 200 μ l cell pyrolysis liquids, storage It is stored in -80 DEG C.

Magnetic bead is resuspended, sample includes control peptide and HIP-13 treatment group purpose samples (Anti-HuR), negative control (IgG) And positive control (Input).Draw the suspension containing magnetic beads after 50 μ l are resuspended to manage in each EP, often pipe adds 500 μ l RIP Wash Buffer, be vortexed concussion, and EP pipes are placed on magnetic frame, and 15 ° of left-right rotation makes magnetic bead absorption in alignment, removes supernatant, It is repeated once, magnetic bead is resuspended with 100 μ l RIP Wash Buffer, adds about 5 μ g corresponding antibodies in each sample.Room temperature It is incubated 30 minutes, EP pipes is placed on magnetic frame, abandon supernatant, add 500 μ l RIP Wash Buffer, is abandoned after the concussion that is vortexed Clearly, it is repeated once, adds 500 μ l RIP Wash Buffer, is placed on ice after the concussion that is vortexed.

Rna binding protein immunoprecipitation：Often pipe adds 900 μ l RIP immunoprecipitation buffers, rapid defrosting first step system Standby cell pyrolysis liquid, 14000rpm, 4 DEG C centrifuge 10 minutes.Magnetic bead-antibody that 100 μ l supernatants are drawn in previous step is compound In thing so that cumulative volume 1ml, 4 DEG C are incubated 3 hours to overnight, of short duration centrifugation, EP pipes are placed on magnetic frame, abandon supernatant, are added Enter 500 μ l RIP Wash Buffer, EP pipes are placed on magnetic frame after the concussion that is vortexed, abandon supernatant, repeated washing 6 times.

RNA is purified and detection：Above-mentioned magnetic bead-antibody complex is resuspended with 150 μ l proteinase K buffers, 55 DEG C are incubated 30 Minute, after being incubated, EP pipes are placed on magnetic frame, supernatant is sucked in a new EP pipes, are added in every pipe supernatant Enter 250 μ l RIP Wash Buffer, 400 μ l phenol are added in every pipe：Chloroform：Isoamyl alcohol, be vortexed concussion 15 seconds, at room temperature 14000rpm is centrifuged 10 minutes, the careful μ l upper strata aqueous phases of absorption 350, another new EP pipes of suction, 400 μ l chlorine is added in every pipe Imitative, be vortexed concussion 15 seconds, and 14000rpm is centrifuged 10 minutes at room temperature, carefully draws 300 μ l upper strata aqueous phases, is sucked another new EP is managed, and often pipe adds 50 μ l Salt Solution I, 15 μ l Salt Solution II, 5 μ l Precipitate Enhancer, 850 μ l absolute ethyl alcohols (no RNase), in -80 DEG C keep 1h to overnight, 14000rpm, 4 DEG C centrifuge 30 minutes, it is small The heart removes supernatant, and, 14000rpm, 4 DEG C centrifuge 15 minutes, carefully remove supernatant, are dried in air, 10- with 80% alcohol flushing once 20 μ l DEPC water dissolve, -80 DEG C of preservations, or the laggard performing PCR detection detection of reverse transcription.

As a result as shown in figs. 9-11, compared with control group polypeptide, HIP-13 polypeptides can obviously reduce HuR and RNA combination Level, illustrate that the polypeptide of design of the embodiment of the present invention can significantly antagonism HuR combinations RNA ability, and then reduce HuR in the cell With reference to, influence RNA biological activity function, play the function of tumor suppression.

The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all the present invention spirit and Within principle, any modification, equivalent substitution and improvements made etc., it should be included in the scope of the protection.

Sequence table

<110>Wuhan Union Hospital

<120>It is a kind of can antagonism HuR protein rna binding activity polypeptide HIP-13 and its application

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<170> PatentIn version 3.5

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Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Val Ala Gly His Ser

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Claims (6)

1. it is a kind of can antagonism HuR protein rna binding activity polypeptide, it is characterised in that amino acid sequence such as SEQ ID NO:1 institute Show.

2. described in claim 1 can antagonism HuR protein rna binding activity application of the polypeptide in antineoplastic is prepared.

3. a kind of antineoplastic polypeptide, it is characterised in that including tumor cytotoxicity domain and wear spanning domain, the tumour is thin Born of the same parents kill the amino acid sequence such as SEQ ID NO of domain:Shown in 1.

4. antineoplastic polypeptide according to claim 3, it is characterised in that the amino acid sequence for wearing spanning domain is such as SEQ ID NO:Shown in 2.

5. the antineoplastic polypeptide according to claim 3 or 4, it is characterised in that the spanning domain of wearing is connected to described swell The N-terminal of cytotoxic effect domain.

6. application of the antineoplastic polypeptide any one of claim 3-5 in antineoplastic is prepared.