CN103705924A - Combined application of miR-1255a and tumor treating medicine - Google Patents
Combined application of miR-1255a and tumor treating medicine Download PDFInfo
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- CN103705924A CN103705924A CN201410003495.3A CN201410003495A CN103705924A CN 103705924 A CN103705924 A CN 103705924A CN 201410003495 A CN201410003495 A CN 201410003495A CN 103705924 A CN103705924 A CN 103705924A
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Abstract
The invention relates to the field of biological medicines, in particular relates to a small RNA (Ribose Nucleic Acid) molecule and a medical application thereof, provides combined application of miR-1255a and a tumor treating medicine, and particularly provides a medicine composition capable of influencing a tumor treating effect. The medicine composition comprises 1) a miR-1255a agonist of an effective dose and 2) the tumor treating medicine.
Description
Technical field
The present invention relates to biomedicine field.More specifically, the present invention relates to small RNA molecular and medical usage thereof.
Background technology
The unconventionality expression of microRNA can cause the drug resistance of tumor cell.For example, the discovery patient with breast cancers such as Climent are because the disappearance of miR-125b on 11q chromosome causes the drug resistance of anthracyclines chemotherapeutic to increase, and patient with breast cancer also produces drug resistance to chemotherapeutic such as paclitaxel, 5-fluorouracil, topotecan simultaneously.Existing Patent Application Publication method based on microRNA diagnosis, prognosis and treatment (US2006/029889, the Crouse etc. for breast carcinoma; US2010/0297627, Watson et al.).In patient with breast cancer's test-and-treat, still there are the following problems: chemotherapeutical anti-tumor medicine exists Drug resistance, and cost is high, expensive; MicroRNA testing cost is high.
By metabonomic technology, can obtain a large amount of metabolite qualitative, quantitative results, through screening, be expected to find special, sensitive, to medical diagnosis on disease or treat relevant biomarker, also can be new drug development drug target be provided.For example, Beckonert etc. (Beckonert et al., 2003) utilize 1H-NMR metabonomic technology to analyze breast tumor sample (49) and normal galactophore tissue's sample (39), and find feature difference metabolite.Found that; in tissue, the concentration of lecithin, uridine 5'-diphosphate hexose, phosphoethanolamine increases along with the rising of tumor grade; and the taurine of ultrahigh concentration in malignant tumor sample, can be detected, result show these several lipid metabolism things and high malignancy breast cancer tumour closely related.Yet, up to now, do not relate to the application that utilizes metabonomic technology to find the microRNA of variant variation.
At micromolecule miR-145 in the treatment of the inflammation metabolic disease such as diabetes, through experiment, confirm, in high sugar, high fat situation, the inflammatory disorders that this microRNA participates in the metabolic diseases such as diabetic angiopathy develops, can be used for the inflammation metabolic disease medicines such as preparation treatment inflammation medicine, especially preparation treatment diabetes and preparation and detect diabetes preparation.MiR-145 can become diabetic and detect one of index, and as the treatment target spot of the diseases associated with inflammation such as diabetictrunk angiopathy.Still there is following problem in this kind of scheme: the miR-145 detection method of use, as miRNA chip, cost is too high.
Therefore, in this area, need to solve the detection microRNA relevant to drug susceptibility and divide the period of the day from 11 p.m. to 1 a.m, length consuming time, the problem that cost is high; And need to be provided for the new therapeutant of Breast Cancer Patients Treated, thereby avoid the drug resistance of chemotherapeutics.
Summary of the invention
Inventor is based on mtt assay and metabonomic technology, screened efficiently the microRNA relevant to drug susceptibility, find the microRNA molecule miR-1255a relevant with the drug susceptibility of tumor cell, and therefore prepared a kind of Pharmaceutical composition that affects drug susceptibility.
The inventor finds that microRNA molecule miR-1255a is relevant with the drug susceptibility of tumor cell, and based on metabonomic technology, after breast cancer cell Drug therapy, identifies the perturbation action of this microRNA molecule to cellular metabolism level.
Based on above-mentioned discovery, on the one hand, the invention provides the therapeutic use of microRNA molecule miR-1255a.Described therapeutic use is the concentration that miR-1255a can reduce anti-tumor medicine, thereby saves the cost of anti-tumor medicine and alleviate patient with breast cancer's misery.In a preferred embodiment, described anti-tumor medicine is paclitaxel.
In another aspect, the present invention also provides a kind of Pharmaceutical composition that affects oncotherapy effect that has, for saving the cost of chemotherapeutics and alleviating patient with breast cancer's misery.In this application, to affect oncotherapy effect be inhibition tumor cell drug susceptibility and affect tumor cell metaboilic level.Be that miR-1255a agonist has cooperative effect when using with one of anti-tumor medicine.Described pharmaceutical composition comprise effective dose 1) miR-1255a agonist and 2) anti-tumor medicine.In a preferred embodiment, pharmaceutical composition of the present invention also comprises pharmaceutically acceptable carrier.In a preferred embodiment, described anti-tumor medicine is paclitaxel.
Accompanying drawing explanation
Fig. 1 illustrates the impact of miR-1255a on MCF7 cell taxol drug sensitivity.
Fig. 2 illustrates nuclear magnetic spectrogram and analyzes the impact of pharmaceutical composition on the whole metaboilic level of cell.
Fig. 3 illustrates PCA shot chart and analyzes the impact of pharmaceutical composition on cellular metabolism level.
Fig. 4 illustrates PCA load diagram and analyzes the impact of pharmaceutical composition on cellular metabolism level.
The specific embodiment
The invention provides and a kind ofly there is inhibition tumor cell drug susceptibility and affect the pharmaceutical composition of tumor cell metaboilic level, it comprise effective dose 1) miR-1255a agonist and 2) anti-tumor medicine.In a preferred embodiment, pharmaceutical composition of the present invention also comprises 3) pharmaceutically acceptable carrier.In a preferred embodiment, described anti-tumor medicine is paclitaxel.
In this application, miR-1255a agonist is directly by the mature sequence of miR-1255a (SEQ ID NO:1; The precursor sequence of miR-1255a is SEQ ID NO:2) through special chemical modification (as the cholesterol of the thio-modification of the modification that methylates on full chain, 5 ' end and 3 ' end and 3 ' end is modified), to synthesize, it is by simulating the expression that is used for regulating target gene of endogenous miR-1255a.
In this application, effective dose refers to the amount that is enough to produce bioactive pharmaceutical composition, selected dosage level depends on many factors, comprise pharmaceutical composition activity, route of administration, with other drug or the combination of Therapeutic Method, the patient's that treats the order of severity etc.Preferably, most suitable dosage is evaluated by those skilled in the art.
In this application, pharmaceutical composition of the present invention is prepared under aseptic and apyrogenic condition.The preparation method of described pharmaceutical composition is within those skilled in the art's limit of power, can be with reference to Remington ' s Pharmaceutical Science, the 17th edition, MackPublishing Company, Easton, in Pa. (1985), its disclosure is described.
In this application, suitable pharmaceutically acceptable carrier comprises water, aqueous buffer solution, normal saline, saline of 0.4% etc.Pharmaceutical composition of the present invention comprises conventional drug excipient and/or additive.Suitable drug excipient comprises stabilizing agent, antioxidant, osmotic pressure regulator, buffer agent and pH adjusting agent.Suitable additive comprises adding of for example physiology biocompatibility buffer agent (Tromethamine hydrochloride), chelating agen (DTPA or DTPA-bisamide) or calcium chelate (DTPA calcium, CaNaDTPA-bisamide), calcium or sodium salt (calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate).Pharmaceutical composition of the present invention can liquid form packing or lyophilizing.
Solid composite medicament of the present invention, the mannitol of pharmaceutically acceptable non-toxic carrier such as the pharmaceutical grade of available routine, lactose, starch, cellulose, glucose, sucrose, magnesium carbonate etc.
A lot of for the method for nucleic acid transfered cell is had in this field, it includes but not limited to the conversion of liposome RNA method, electroporation, calcium phosphate mediation, conversion, virus and the non-virus carrier etc. of DEAE-glucosan mediation.
Except as otherwise noted, according to the description of manufacturer, use the commercially available reagent of mentioning in embodiment.Unless otherwise defined, the implication that all technology used herein are understood with one of those skilled in the art conventionally with scientific terminology is identical.Below described exemplary method and material, yet also can be used for implementing or test the present invention with those methods similar or of equal value described herein and material.Described material, method and embodiment are only exemplary, are not intended to scope to limit.
Embodiment
1) Growth of Cells is measured
By the good MCF7 cell of growth conditions with 8 * 10
3the density of individual/mL is inoculated in 96 orifice plates, is placed in 37 ℃, 5%CO
2incubator in incubated overnight, the miRNA transfection description providing with reference to Rui Bo bio tech ltd, Guangzhou proceeds to MCF7 cell by miR-1255a agonist, after transfection 24 hours, paclitaxel (T7402, Sigma-Aldrich) drug treating is 24 hours, then carry out 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl bromination tetrazole blue (being called for short MTT) detects.In brief, in every hole, add the 0.5mg/mL MTT(Sigma of 10 μ L), in 37 ℃ of incubators, continue to hatch 4 hours, finally add 150 μ L DMSO(Amerso), vibrate 10 minutes, by microplate reader, measure the OD value under 570nm wavelength.
2) miR-1255a agonist transfection
MiR-1255a agonist is directly the mature sequence of miR-1255a (SEQ ID NO:1) to be synthesized through special chemical modification (cholesterol of the modification that methylates on full chain, the thio-modification of 5 ' and 3 ' end and 3 ' end is modified), and it is by the expression that is used for regulating target gene of simulation endogenous miR-1255a.MCF7 cell is plated in 96 orifice plates to incubated overnight.When cell density reaches 40-50%, by 49.75 μ L serum-free medium (Opti-MEM) dilution 0.25 μ L20 μ mol/L miR-1255a agonist (miR40005906, Rui Bo bio tech ltd, Guangzhou) storage liquid.Mix gently, make its local concentration reach 100nM, every hole adds the above-mentioned mixed liquor of 50 μ L.After transfection 4-6 hour, the DMEM culture medium that every hole adds 50 μ L to contain 10% hyclone again.
3) 1H-NMR metabolism group experiment
A) cell is processed and grouping
The good MCF7 cell of growth conditions is inoculated in 6 orifice plates, incubated overnight, when cell density is 60-70%, cell is handled as follows:
The 1st group: miR-1255a agonist negative control transfectional cell 24h, transfection method is the same.The taxol drug that adds again 1mg/mL is processed cell 24h;
The 2nd group: the taxol drug of 1mg/mL is processed cell 24h;
The 3rd group: miR-1255a agonist transfectional cell 24h, transfection method is the same.The taxol drug that adds again 1mg/mL is processed cell 24h;
The 4th group: miR-1255a agonist transfectional cell 24h, transfection method is the same;
The 5th group: miR-1255a agonist negative control transfectional cell 24h, transfection method is the same;
The 6th group: blank group.
B) sample pretreatment
Culture medium in 6 orifice plates is taken out, 1000rpm, 4 ℃ of centrifugal 5min, sucking-off 1mL supernatant, to centrifuge tube, and is positioned on ice.Add 1mL acetonitrile to centrifuge tube, mix, 25000rpm, 4 ℃, centrifugal 20min.Draw supernatant, supernatant is placed in to Nitrogen evaporator, blow away organic solvent acetonitrile, be put in-80 ℃ of refrigerators and preserve and spend the night.With freeze dryer, sample is frozen into powdered again.
C) sample treatment
In the sample of lyophilized powder, add 600 μ L D
2o, makes its dissolving.The centrifugal 8min of 14000rpm, gets supernatant 550 μ L, adds and is added with 50 μ L TSP(2,2,3,3-trimethyl silyl propanoic acid) heavy aqueous solution (D
2o, 1mg/mL) 5mm nmr tube in stand-by.
For each sample, adopt respectively the analytical technology of PRESAT(nuclear magnetic spectrogram) pulse train (Presaturation presaturation pulse train) collecting sample data.Spectrum width is 9000Hz, sampling number 32k, and sampling time 4s, accumulative frequency 64 times, relaxation delay is 2s, adopts therebetween low powder pulsed water peak to be carried out to presaturation.After free induction decay (free induction decay, FID) signal data zero filling, add the line broadening factor of 0.5Hz, after Fourier transform, obtain 1H-NMR spectrogram.By TSP calibration, be δ 0.
4) 1H-NMR date processing and analysis
The 1H-NMR spectrogram obtaining through Fourier transformation is after phase place adjustment and baseline correction, spectrum to scope in δ 0.4-9.4 is carried out subsection integral by every section of 0.01ppm, the spectrum of excluded ranges between δ 4.7-5.1, integration is normalized by the total mark intensity of every spectrum, and after processing, gained integration data is saved to Excel.
By the Excel integration data of preservation be input to SIMCA-P+ software (v10.04, Umetrics,
sweden), in, carry out pattern recognition process.After Pareto scale for data acquisition (Pareto scaling) pretreatment, carry out again PCA(principal component analysis) analyze.PCA is a kind of non-supervisory mode identification method, is mainly used in the separation trend observed between group and experiment whether have abnormity point.Result represents with the form of shot chart (scores plot) and load diagram (loadings plot), shot chart represents the distribution trend of data point, between the gathering explanation sample of point, (observational variable) has high similarity, and point discrete illustrates and between observational variable, have obvious difference.Load diagram represents to cause the variable of differences between samples, variable from the space length of initial point more away from, illustrate that the difference that this variable causes sample is larger.And load diagram is carried out to T-test check, and find difference metabolite, result represents with Heatmap diagram form.
After medicine composite for curing MCF7 cell prepared by the present invention, obviously affect the growth of cell.As shown in Figure 1, wherein ago-microRNA is miR-1255a agonist to experimental result; NC is the negative control (miR04201 of miR-1255a agonist, Rui Bo bio tech ltd, Guangzhou), this negative control is a kind of general negative control, through bioinformatic analysis, show that its sequence and the gene order of people, mice, rat have minimum homology (having at least 5 bases not mate), and confirm its feasibility through great many of experiments, can be used for the negative control of the miRNA experiment of people, mice, rat.
Pharmaceutical composition prepared by the present invention affects the metaboilic level of MCF7 cell, experimental result as in Figure 2-4, from nuclear magnetic spectrogram visible (Fig. 2), chemical displacement value peak height in δ 0.8-1.4 section, δ 1.8-2.6 section, δ 3-4 section is all variant, show to compare with negative transfection dosing group, metabolite changes to some extent in miR-1255a transfection dosing group.PCA shot chart result (Fig. 3) illustrates that metabolite has notable difference in two groups, enough by two groups of sample area separately.Load diagram (Fig. 4) also show chemical shift δ 2.73,2.74,1.32,1.33,1.34,1.92 etc. locate from initial point space length away from, the compound of pointing out this place's chemical shift representative may be the metabolite that causes group difference.And in PCA load diagram, by t-test, check (setting P<0.01, FDR<0.01).
Claims (10)
1. there is a Pharmaceutical composition that affects oncotherapy effect, comprise effective dose 1) miR-1255a agonist and 2) anti-tumor medicine.
2. the Pharmaceutical composition of claim 1, described anti-tumor medicine is paclitaxel.
3. claim 1 or 2 Pharmaceutical composition, also comprise 3) pharmaceutically acceptable carrier.
4. claim 1 or 2 Pharmaceutical composition, also comprise drug excipient and/or additive.
5. claim 1 or 2 Pharmaceutical composition, the sequence of described miR-1255a is SEQ ID NO.1.
6. claim 1 or 2 Pharmaceutical composition, described miR-1255a agonist is that the modification that methylates, the thio-modification of 5 ' and 3 ' end and the cholesterol of 3 ' end on full chain modified.
7.miR-1255a agonist is for the preparation of the purposes that reduces the concentration medicine of anti-tumor medicine.
8. the purposes of claim 7, described anti-tumor medicine is paclitaxel.
9. claim 7 or 8 purposes, the sequence of described miR-1255a is SEQ ID NO.1.
10. claim 7 or 8 purposes, described miR-1255a agonist is that the modification that methylates, the thio-modification of 5 ' and 3 ' end and the cholesterol of 3 ' end on full chain modified.
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| CN107884431A (en) * | 2017-10-25 | 2018-04-06 | 长春中医药大学 | It is based on1H NMR metabolism group differentiates that fresh pilose antler fries fine and soft method with heat |
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| CN107884431A (en) * | 2017-10-25 | 2018-04-06 | 长春中医药大学 | It is based on1H NMR metabolism group differentiates that fresh pilose antler fries fine and soft method with heat |
| CN107884431B (en) * | 2017-10-25 | 2019-10-25 | 长春中医药大学 | It is based on1The metabolism group of H-NMR identifies fresh pilose antler and heat fries fine and soft method |
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Application publication date: 20140409 |