CN103083685A - Method for reversing drug resistance of breast cancer by using miR-181a - Google Patents
Method for reversing drug resistance of breast cancer by using miR-181a Download PDFInfo
- Publication number
- CN103083685A CN103083685A CN 201310049732 CN201310049732A CN103083685A CN 103083685 A CN103083685 A CN 103083685A CN 201310049732 CN201310049732 CN 201310049732 CN 201310049732 A CN201310049732 A CN 201310049732A CN 103083685 A CN103083685 A CN 103083685A
- Authority
- CN
- China
- Prior art keywords
- mir
- mcf
- cell
- bcrp
- drug
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention provides the field of medicines for treating cancer, and in particular relates to a method for reversing drug resistance of breast cancer by using miR-181a. The method comprises the following steps of: (1) inhibiting BCRP (Breast Cancer Resistance Protein) expression and drug transport function in drug resisting cells MCF-7/MX of breast cancer by means of a stimulant miR-181amimic of miR-181a, and increasing sensitivity to a BCRP transfer substrate mitoxantrone hydrochloride; (2) performing targeted inhibition on BCRP expression in transplantation tumor of nude mouse with breast cancer induced by MCF-7/MX by means of a stimulant miR-181aagmir lipidosome, and increasing sensitivity to MX by in vivo cancer cells; and (3) up-regulating BCRP expression and drug transport function of drug susceptible cells MCF-7 by means of an inhibitor miR-181ainhibitor of miR-181a, and increasing the drug resistance to MX. The method is used fo reversing drug resistance of breast cancer by using miR-181a.
Description
Technical field:
The invention provides a kind of medicine for treating tumor thing field, the particularly application of a kind of miR-181a in reversing the breast cancer cell drug resistance.
Background technology:
Breast carcinoma is one of modal malignant tumor of women, and the physical and mental health that is having a strong impact on the women is threat to life even.Chemotherapy is the common method of breast cancer treatment, but the drug resistance problem that occurs in chemotherapy process has had a strong impact on clinical efficacy and patient's prognosis.With the closely-related breast drug-resistance protein of breast carcinoma drug-resistant effect (breast cancer resistant protein, BCRP) be that the cross-film of separating with mRNA fingerprint analysis method from the human breast cancer cell strain MCF7/AdrVP of drug resistance is partly transported albumen.Its gene mapping is in human chromosome 4q22, by the ABCG2 gene code, 2.4kb 655 amino acid residues of mRNA translation consist of, contain an ATP in conjunction with territory and a hydrophobicity membrane spaning domain, after multidrug-associated protein (MRP), P glycoprotein (P-gp) and lung drug-resistant protein (LRP), the another focus albumen in the tumor multi-medicine drug-resistant Mechanism Study.BCRP will transport substrate (chemotherapeutics) from contrary Concentraton gradient pump in cell to the extracellular by the hydrolysising ATP energy supply, make mitoxantrone (MX) in breast cancer cell, topotecan isoconcentration descend, thereby cause cell to drug resistance, inducible resistance generation.
The effect of Microrna (microRNA, miRNA) in reversing drug resistance in recent years receives relevant scholar's concern.MiRNA is the approximately non-encoding histone RNA of the endogenous strand of 22nt of length, in the physiological activities such as ontogeny, cell differentiation, propagation, apoptosis and play important regulating and controlling effect in the pathological processes such as tumorigenesis.MiRNA generally by with the expression of 3 ' end non-coding region (3 ' UTR) specific bond regulator gene of target mRNA molecule.Because BCRPmRNA-3 ' UTR length is about 2kb (Genebank:accession no.NM_004827; The UTR data base: UTR database entry UTR:3HSA117529), be longer than the average length 700bp of human mRNA s-3 ' UTR, therefore, the action target spot that prompting BCRPmRNA-3 ' UTR may exist miRNA regulation and control BCRP to express.
Some studies confirm that some miRNAs can regulate by acting on BCRPmRNA-3 ' UTR the expression of BCRP.Wang etc. find that in the research of pancreatic cancer cell miR-520h can lower the protein expression level of BCRP; To KK etc. have confirmed also that in the result of study of colon cancer cell this point: miR-519c has the SI of reduction colon cancer cell endogenous BCRP and transcribes and the protein expression effect; The researchs such as Pan find that the protein expression level of BCRP obviously descends after in the breast cancer cell MCF-7/MX100 of drug resistance transfection miR-328 or miR-519c.As seen miRNA participates in the breast carcinoma drug resistance to Targeted-control BCRP and has played crucial effect.
In the present invention, our Applied Biology information analysis can obtain, the action target spot that BCRPmRNA-3 ' UTR exists miR-181a regulation and control BCRP to express, but whether miR-181a it be not immediately clear the drug resistance that breast cancer cell was regulated and reversed therefrom in the expression of BCRP.therefore, the present invention is in the low expression of breast cancer medicines sensitive cells MCF-7(BCRP) and mdr cell MCF-7/MX(BCRP high expressed) in, difference transfection miR-181a inhibitor and miR-181a mimic, build the reticent breast cancer cell model of expressing and crossing expression of miR-181a, and nude mice is carried out MCF-7/MX cell skin hemostasis, be structured in body transplanted tumor model, after MX processes above-mentioned model, observation of cell and the mice sensitivity to MX, MX changes of contents in the expression of BCRP and cell, inquire into and exsomatize and impact and the mechanism of miRNA-181a on the breast cancer cell drug resistance when the body level.
Summary of the invention:
Technical problem to be solved by this invention is to provide a kind of method that miR-181a of application reverses the breast cancer cell drug resistance.
The present invention is achieved through the following technical solutions:
A kind of method of using miR-181a reverse breast carcinoma drug resistance, described method comprises:
(1) utilize the analogies miR-181a mimic of miR-181a, the BCRP that suppresses in breast carcinoma mdr cell MCF-7/MX expresses and the transport of drug function, increases the sensitivity to BCRP transhipment substrate mitoxantrone hydrochloride MX;
(2) utilize the analogies miR-181a agmir liposome of miR-181a, in the breast cancer xenograft in nude mice that targeting inhibition MCF-7/MX induces, the expression of BCRP, be increased in the body oncocyte to the drug susceptibility of MX;
(3) utilize the BCRP of the inhibitor miR-181a inhibitor rise drug sensitive cell MCF-7 of miR-181a to express and the transport of drug function, increase the drug resistance to MX.
In step (1), miR-181a analogies miR-181a mimic is for comprising the hairpin like fold of miR-181a nucleotide sequence " stem-ring " structure, and main sequence is as follows: 5 '-AACAUUCAACGCUGUCGGUGAGU-3 '.Tumor cell described in step (1) is breast carcinoma mdr cell MCF-7/MX.Breast carcinoma mdr cell MCF-7/MX was expression BCRP breast cancer cell.
The cell that uses in step (3) is breast carcinoma sensitive cells MCF-7; Breast carcinoma sensitive cells MCF-7 is the breast cancer cell that miR-181a crosses expression.
The final concentration scope that in step (1), miR-181a analogies miR-181a mimic is transfected in breast carcinoma mdr cell MCF-7/MX is: 10nM-80nM, the transfection time is 48 hours.
Utilize miR-181a analogies miR-181a agmir liposome in step (2), when acting on the breast cancer xenograft in nude mice that MCF-7/MX induces, the injecting method that adopts is multi-point injection in nude mice tumor week and tumor, the liposome injection volume is 1nmol, frequency injection is jede Woche injection twice, injects altogether five times.
Advantage and effect: in the low expression of breast cancer medicines sensitive cells MCF-7(BCRP) and mdr cell MCF-7/MX(BCRP high expressed) in, detect that miR-181a expresses and protein immunoblot is analyzed BCRP and expressed by real time-RCR, differential expression and the negativity relation of observation miR-181a and BCRP in two cells; By the luciferase reporting analysis, clearly whether there is the action target spot of miR-181a in BCRPmRNA-3 ' UTR district; Further in MCF-7 and MCF-7/MX cell, respectively by transfection miR-181a inhibitor and miR-181a mimic, build the reticent breast cancer cell model of expressing and crossing expression of miR-181a, and nude mice is carried out MCF-7/MX cell skin hemostasis, be structured in body transplanted tumor model, after MX processes above-mentioned model, observation of cell and mice are inquired into and exsomatize and impact and the mechanism of miR-181a on the breast cancer cell drug resistance when the body level MX changes of contents in the expression of the sensitivity of MX, BCRP and cell.
Result confirmation of the present invention, miR-181a suppresses BCRP and expresses by being combined with BCRPmRNA-3 ' UTR targeting.Intervene miR-181a and express and to affect the breast cancer cell drug susceptibility, to targeting BCRP, to affect in cell the MX drug level relevant.Therefore, the present invention can be as the molecular marker of prediction patient with breast cancer to the chemotherapy drug susceptibility degree, for clinical personalized medicine provides foundation, designing and screen for the overriding resistance breast cancer medicines simultaneously provides new target spot, can be developed as again a kind of miRNA small-molecule drug.
Description of drawings:
The expression of miRNAs in two kinds of cells of Fig. 1 miRNAs chip analysis MCF-7 and MCF-7/MX.
Fig. 2 miR-181a targeting suppresses BCRP expresses.The nucleic acid base sequence of A:RNAhybrid theory analysis miR-181a and BCRPmRNA-3 ' UTR, the theory and combining site of predicting both; B: luciferase reporter gene is analyzed the miR-181a targeting in BCRPmRNA-3 ' UTR district, compares with the NC group: * P＜0.05; The expression of miR-181a in MCF-7/MX after C:Real-time PCR detection transfection variable concentrations miR-181a mimic or NC-mimic48h.After D:Real-time PCR detects transfection variable concentrations miR-181a mimic or NC-mimic48h, the expression of the mRNA of BCRP in MCF-7/MX; The expression figure of four kinds of drug-resistant proteins in MCF-7/MX after E:Western Blotting detection transfection miR-181a mimic or NC-mimic48h; F: four kinds of resistance protein levels of statistical analysis.Compare with the NC group:
*P＜0.05
Fig. 3 Western Blotting detects the expression of drug-resistant protein MRP, PGP, LRP and BCRP in MCF-7 and two kinds of cells of MCF-7/MX.A: each organizes representative result figure; B: give variable concentrations MX and process, measure propagation level and the IC50 value of two kinds of cells.
Fig. 4 miR-181a increases the MCF-7/MX cell to MX sensitivity.In A:MCF-7/MX, after transfection miR-181a mimic or NC-mimic48h, MX processed cell 0.5 hour, and Flow Cytometry detects fluorescence intensity in cell; B: cell fluorescence intensity value under statistical analysis different disposal factor, take the NC class value as 1, calculate all the other and process the lower multiple that changes; After transfection miR-181a mimic or NC-mimic48h, after processing cell 48h with variable concentrations MX, detect cell to the variation of drug susceptibility in C:MCF-7/MX; D:(B) in MCF-7/MX after transfection miR-181a mimic or NC-mimic48h, after processing cell 48h with the variable concentrations etoposide, detect cell to the variation of drug susceptibility.Compare with the NC group:
*P＜0.05
In Fig. 5 tumor body, injection miR-181a increases the MX antitumor action.A: different time points is respectively organized tumor growth change in volume curve; B: each organizes tumor tissue weight statistics; C:Real-time PCR detects the expression of respectively organizing miR-181a in tumor
Fig. 6 miR-181a suppresses BCRP expression in breast cancer xenograft in nude mice.A:Real-time RT-PCR detects and respectively organizes MRP, PGP, LRP, four kinds of drug-resistant protein mrna expressions of BCRP in tumor; B:WesternBlot detects and respectively organizes MRP, PGP, LRP, four kinds of resistance proteins of BCRP in tumor; C: statistical software calculates the statistical result of each histone average optical density value.Compare with the NC group:
*P＜0.05
Fig. 7 suppresses the BCRP expression that miR-181a has raised breast cancer medicines sensitive cells MCF-7.The expression of miR-181a in MCF-7 after A:Real-time PCR detection transfection miR-181a inhibitor or NC-inhibitor48h; After B:Real-time PCR detects transfection miR-181a inhibitor or NC-inhibitor48h, the expression of the mRNA of four kinds of drug-resistant protein MRP, PGP, LRP, BCRP in MCF-7; The expression figure of four kinds of drug-resistant proteins in MCF-7 after C:Western Blotting detection transfection miR-181a inhibitor or NC-inhibitor48h; D: four kinds of resistance protein levels of statistical analysis.Compare with the NC group:
*P＜0.05
Fig. 8 suppresses miR-181a increases drug sensitive cell MCF-7 to the drug resistance of MX.In A:MCF-7, after transfection miR-181a inhibitor or NC-inhibitor48h, MX processed cell 0.5 hour, and Flow Cytometry detects fluorescence intensity in cell; B: statistical analysis drug concentration; After transfection miR-181a inhibitor or NC-inhibitor48h, after processing cell 48h with variable concentrations MX, detect cell to the variation of drug susceptibility in C:MCF-7; After transfection miR-181a inhibitor or NC-inhibitor48h, after processing cell 48h with the variable concentrations etoposide, detect cell to the variation of drug susceptibility in D:MCF-7.Compare with the NC group:
*P＜0.05
Embodiment:
A kind of method of using miR-181a reverse breast carcinoma drug resistance, described method comprises:
(1) utilize the analogies miR-181a mimic of miR-181a, the BCRP that suppresses in breast carcinoma mdr cell MCF-7/MX expresses and the transport of drug function, increases the sensitivity to BCRP transhipment substrate mitoxantrone hydrochloride MX;
(2) utilize the analogies miR-181a agmir liposome of miR-181a, in the breast cancer xenograft in nude mice that targeting inhibition MCF-7/MX induces, the expression of BCRP, be increased in the body oncocyte to the drug susceptibility of MX;
(3) utilize the BCRP of the inhibitor miR-181a inhibitor rise drug sensitive cell MCF-7 of miR-181a to express and the transport of drug function, increase the drug resistance to MX.
The present invention is described in further detail below in conjunction with specifically executing example, but be not limitation of the invention:
MiRNAs express spectra difference in embodiment 1:miRNA chip analysis breast carcinoma sensitive cells MCF-7 and mdr cell MCF-7/MX;
Mankind mastopathy cell MCF-7 of the present invention is available from U.S. ATCC cell bank, and is frozen voluntarily by this chamber.MCF-7/MX is by this laboratory-induced.Culture medium is the DMEM culture medium (high sugar) that contains 10% hyclone, wherein adds penicillin 100U/ml and streptomycin 100U/ml, 5%CO
2, cultivate in 37 ℃ of incubators.By miRNAs express spectra difference in miRNA chip analysis breast carcinoma sensitive cells MCF-7 and mdr cell MCF-7/MX, find that there are 2 times of differential expressions (〉 in 23 miRNAs) (seeing Figure 1A and 1B), wherein miR-181a all lowers the most obvious in MCF-7/MX.
Embodiment 2:Real-time PCR detects the differential expression of miR-181a in MCF-7 and MCF-7/MX;
MCF-7 and MCF-7/MX cell culture collecting cell after 24 hours extracts test kit (Bioteke, RP5301) step according to RNA, extracts total RNA, and measures concentration and the quality of RNA with ultraviolet spectrophotometer.Adopt SYBR real-time PCR test kit (Takera, DRR036S), by 10 μ l system (DEPC H
2O0.45 μ l, 5*buffer2 μ l, RRI0.25 μ l, M-MLV0.3 μ l, RT-primmer1 μ l, dNTP1 μ l, RNA5 μ l), reaction condition is: 30 ℃, and 10min; 42 ℃, 1h; 85 ℃, 5min; 5 ℃, 5min; 4 ℃, 2h, reverse transcription gets cDNA.Carry out real-time PCR experiment by 25 μ l systems (deionized water 9 μ l, 2*SYBRgreen12.5 μ l, Rox0.5 μ l, forward primer 0.5 μ l, downstream primer 0.5 μ l, cDNA2 μ l) again, reaction condition is: 95 ℃, and 2min; 95 ℃, 15s; 60 ℃, 30s, 40 circulations; 95 ℃, 1min; 55 ℃, 30s; 95 ℃, 30s.Test minimum triplicate, miR-181a cell inner expression amount is carried out statistical analysis.The expression that real-time PCR testing result of the present invention is presented at miR-181a in the MCF-7/MX cell descends, and wherein the expression of miR-181a descends approximately that 80%(sees Fig. 1 C).
Embodiment 3:Western Blotting detects the differential expression of BCRP in MCF-7 and MCF-7/MX;
MCF-7 and MCF-7/MX cell culture were washed the PBS of cell with pre-cooling one time, according to Membrane protein extraction test kit (Thermo after 24 hours, 89826) operate, add the cell pyrolysis liquid of fresh configuration, scrape cell with cell scraper immediately, the ice bath shaking table shook 30 minutes, fully cell lysis.4 ℃, 13, centrifugal 15 minutes of 000g collects supernatant to new EP pipe, and (protein quantification P0012) is carried out in the green skies with the BCA method.get the equal protein sample, add 5* sample-loading buffer (20% glycerol, 4% sodium lauryl sulphate, 10% beta-mercaptoethanol, 0.05% bromophenol blue, 1.25M Tris – HCl, pH6.8), 95 ℃ of degeneration of metal bath are after 10 minutes, use the 8%SDS-polyacrylamide gel electrophoresis, transfer on pvdf membrane, Tris-HCl buffer salt solution (TBST) with 1% polysorbas20 that contains 5% defatted milk powder sealed two hours, TBST rinses 3 times, each 10 minutes, carry out respectively five hatching egg white mouse source primary antibodie: ABCG2/BCRP(abcam, ab3380), PGP(abcam, ab3366), LRP(Santa, sc23916), MRP (abcam, ab24102), β-actin(SANTA, sc47778), dilute 500 times, incubated at room two hours, 4 ° of C spend the night.TBST rinses three times, each 10 minutes.Add anti-Mus two anti-(1:3000, middle China fir Golden Bridge, ZB2305), incubated at room 1.5 hours, TBST rinses three times, ECL is luminous.Test minimum triplicate, measure gray value, carry out statistical analysis.Use the protein immunoblot analysis and show that four kinds of drug-resistant protein MRP, PGP, LRP, BCRP all increase than expressing in MCF-7/MX in MCF-7, wherein maximum (P＜0.05) (the seeing Fig. 3 A) of BCRP differential expression.It is relevant with the low expression of miR-181a that comprehensive embodiment 2 results suggest of the present invention cause BCRP that the breast cancer cell drug resistance increases to cross expressing.
Embodiment 4: biological information analysis and luciferase reporter gene are analyzed the miR-181a targeting in BCRPmRNA-3 ' UTR district;
There is binding site (Fig. 2 A) in the 3007bp-3029bp that finds miR-181a and BCRPmRNA-3 ' UTR district by RNAhybrid.
In order to verify further whether miR-181a has binding site with BCRPmRNA-3 ' UTR, and the present invention uses the analysis of Luciferase luciferase reporting and detects.PEGFP-BCRP3 ' UTR and pcDNA3.3-miR-181a or pcDNA3.3 empty plasmid (negative control) are pressed lipofectamine
TMAfter 2000 transfection reagent box operating procedure cotransfections enter 293T cell 48h, use the fluorescence detector fluorescence intensity.Test minimum triplicate, the luciferase result is carried out statistical analysis.The present invention as a result finds (seeing 2B): in the 293T cell after cotransfection pcDNA3.3-miR-181a and PGL3-BCRP-3 ' UTR48h, compare enzymatic activity 30% left and right (P＜0.05) that descends with PGL3-BCRP-3 ' UTR with transfection pcDNA3.3 empty plasmid (negative control).There are direct binding site in prompting miR-181a and BCRPmRNA-3 ' UTR district.
The BCRP that embodiment 5:miR-181a suppresses in breast carcinoma mdr cell MCF-7/MX expresses;
Whether the expression of BCRP is had regulating action in order to detect miR-181a, transfection miR-181a mimic in the mdr cell MCF-7/MX of the BCRP high expressed of at first finding in embodiment 2 and embodiment 3 builds miR-181a and crosses the cell model of expression.Its transfection concrete steps are: with breast carcinoma mdr cell MCF-7/MX at six orifice plates (3 * 10
5Individual/hole) or 100mm culture dish (2 * 10
6Individual) the middle cultivation after 24 hours, antibiotic serum-free medium is hungry to be cultivated 1 hour with not containing, and pressed afterwards lipofectamine
TM2000 transfection reagent box operating procedures are transfection miR-181a mimic(Ribobio respectively) and nonspecific negative control plasmid (miR-NC) is (Ribobio), final concentration is respectively 10,20,40,80nM.After 4 hours, replaced former culture medium culturing 48 hours with the fresh DMEM culture medium that contains 10% hyclone, standby.
Press embodiment 2 described by the expression of real-time PCR detection miR-181a in MCF-7/MX, found that 10,20,40,80nM miR-181a mimic transfection is after 48 hours, it is respectively 159,271,335,380 times (seeing Fig. 2 C) in intracellular expression increase, illustrates that miR-181a crosses the success of expression model construction.Further detect the BCRPmRNA expression, find in 20M miR-181amimic transfection after 48 hours, BCRPmRNA express descend respectively 18%, 45%, 30%, 28%(P＜0.05) (seeing Fig. 2 D).As seen, the effect of 20nM miR-181a mimic inhibition BCRPmRNA expression is the most obvious.（P<0.05）。In addition, other three kinds of MRP, PGP, LRP drug-resistant protein mRNA are without significant change.Further detect 20nM miR-181a mimic to the impact of BCRP protein expression in MCF-7/MX by the described Western Blotting that carries out of embodiment 3, the protein expression that found that BCRP 30%(P＜0.05 that descended), and other three kinds of drug-resistant proteins have no significant change (seeing Fig. 2 E and Fig. 2 F).Illustrate that the BCRP that miR-181a energy targeting suppresses in the MCF-7/MX mdr cell expresses.
Embodiment 6:miR-181a can suppress the transport function of BCRP in breast carcinoma mdr cell MCF-7/MX, increases the content of MX in cell;
Press the described transfection method of embodiment 5 MCF-7/MX cell after transient transfection in six orifice plates, blank group is set respectively, negative transfection group, miR-181a mimic group, wherein rear two groups of unitransport substrate mitoxantrone hydrochlorides (MX) that add the BCRP of 3 μ M, washed with cold PBS after 1 hour, add the DMEM culture medium culturing that contains 10% hyclone, 1.5 harvesting after hour with fluorescence intensity in the cells were tested by flow cytometry cell, detects cell to the effect that effluxes of medicine.Test minimum repetition 3 times.Found that and the matched group ratio MX content obviously raise (seeing Fig. 4 A and Fig. 4 B, P＜0.05) in miR-181a transfection group cell.Confirmed that miR-181a can suppress the transport function of BCRP in mdr cell MCF-7/MX, increased the content of MX in cell.
Embodiment 7:miR-181a increases breast carcinoma mdr cell MCF-7/MX to the sensitivity of MX;
Get blank group, negative control group and the miR-181a mimic transfection group cell of MCF-7/MX after transfection 48h, cultivate in 96 orifice plates, every porocyte number is 1 * 10
4giving respectively concentration is 0, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, the MX of 30 μ M and 0, 1, 3, 10, 30, 100, 300, 1000, the etoposide of 3000 μ M was cultivated after 48 hours, add 20 μ l(5mg/ml) MTT, 37 ℃ hatch 4 hours after, the sucking-off supernatant, add 100 μ l/ hole dimethyl sulfoxide (DMSO), shaking table rocks 10min, measure absorbance with microplate reader (Antu) at 570nm, hole (the culture medium that returns to zero is set simultaneously, MTT, dimethyl sulfoxide), control wells (cell, the medicine dissolution medium of same concentrations, culture medium, MTT, dimethyl sulfoxide).Calculate suppression ratio, suppression ratio %=1-(medicine feeding hole-zeroing hole)/(control wells-zeroing hole), use 16.0SPSS computed in software IC50.Test minimum triplicate, experimental result is carried out statistical analysis.The present invention finds: with negative transfection group than (IC50=3.86 ± 0.36 μ M μ M), miR-181a transfection group (IC50=1.57 ± 0.08 μ M) cell obviously increases the sensitivity of MX (sees Fig. 4 C, P＜0.05), and to the sensitivity of etoposide without obviously changing (seeing Fig. 4 D).To sum up result has confirmed that the BCRP that miR-181a suppresses in breast carcinoma mdr cell MCF-7/MX expresses and the transport of drug function, increases the sensitivity to MX.
Embodiment 8:miR-181a has increased the drug susceptibility of breast cancer xenograft in nude mice to MX;
For the impact of further clear and definite miR-181a on the MCF-7/MX drug resistance, carried out nude mice and tested at body.At first carrying out Nude Mouse Model builds: get 4-6 week female athymism BALB/C mice (being purchased from Shanghai Slac Experimental Animal Co., Ltd.), equal aseptic sub-cage rearings under the Experimental Animal Center SPF of Chinese Medical Sciences University level condition, in operating process, water and food abundance give.Adopt cellar culture cell seeding method, the breast carcinoma MCF-7 of the trophophase of taking the logarithm and MCF-7/MX cell make respectively 4 * 10
7The cell suspension of/ml, getting 0.2ml, to be inoculated in every right omoplate of BALB/C Mus subcutaneous, after transplanted tumor grows to 5*5*5mm, to become at random the tumor nude mice to be divided into four organizes greatly: wherein I group (blank group), II group (negative transfection group), III organize (negative transfection+MX processed group) IV group (miR-181a processed group), 6 every group.
Every group of nude mice all adopted multi-point injection processing in tumor week and tumor.I group: normal saline (total amount 200 μ l), II group: contrast liposome 1nmol+ normal saline (total amount 200 μ l), III group: contrast liposome 1nmol+ normal saline (total amount 200 μ l), IV group: miR-181a agmir liposome 1nmol+ normal saline (total amount 200 μ l), jede Woche injection twice is injected five times altogether.Give for the first time intratumor injection after 3 days, according to 1.5mg/kg tail vein injection medicine, I group, II group: normal saline, III group, IV group: mitoxantrone hydrochloride, jede Woche injection twice is injected four times altogether.Before each administration, with vernier caliper measurement tumor maximum gauge and corresponding transverse diameter, according to formula V (mm
3)=π/6 * diameter of tumor (mm) * tumor transverse diameter (mm) * tumor transverse diameter (mm) calculates the gross tumor volume size.Put to death 4 groups of nude mices after fortnight, get tumor.Measure tumor piece quality.Carry RNA and albumen and be used for the related experiment analysis.
The present invention as a result finds: after two weeks, with the matched group ratio, miR-181a group tumor volume and tumor are heavy obviously is suppressed (seeing Fig. 5 A, Fig. 5 B, P＜0.05), shows that miR-181a has increased the drug susceptibility of breast cancer xenograft in nude mice to MX.Further use real-time PCR by the described method of embodiment 2 and detect the expression of respectively organizing miR-181a in tumor, result shows: with the matched group ratio, in miR-181a group tumor, the miR-181a expression obviously raises (P＜0.05), and illustrating can increase MX antitumor action (seeing Fig. 5 C) after in the tumor body, injection miR-181a agmir induces tumor tissue miR-181a high expressed.
Embodiment 9:miR-181a targeting suppresses BCRP in the expression of breast cancer xenograft in nude mice;
The described method of Application Example 2 is carried out real-time PCR and is detected (reverse transcription condition: 95 ℃ of denaturations, 5min; 95 ℃, 30s; 60 ℃, 45s; 72 ℃, 20s, 8 circulations.Real-time PCR condition: 95 ℃, 30s; 56 ℃, 45s; 72 ℃, 20s, 35 circulations; 95 ℃, 1min; 55 ℃, 30s; 95 ℃, 30s.) respectively organize MRP, PGP, LRP, four kinds of drug-resistant protein mrna expressions of BCRP in breast cancer transplantable tumor in embodiment 8.The present invention as a result finds: with the matched group ratio, in miR-181a group tumor, the BCRPmRNA expression obviously reduces (P＜0.05), has concordance (seeing Fig. 6 A) with experiment in vitro result in embodiment 5.The protein immunoblot result shows that also the BCRP protein expression obviously descends (seeing Fig. 6 B and 6C, P＜0.05), and other three kinds of albumen expressions are without obvious change.These inventions have shown under the body environment, and miR-181a still can suppress MCF7/MX cell BCRP to express, and increases the sensitivity to MX.
Embodiment 10: suppress the BCRP expression that miR-181a has raised breast cancer medicines sensitive cells MCF-7;
Whether can raise the BCRP expression and increase breast cancer cell to the drug resistance of MX in order to detect inhibition miR-181a, further hanging down transfection miR-181a inhibitor in the sensitive cells MCF-7 that expresses at BCRP, structure miR-181a hangs down the cell model of expressing.Its transfection concrete steps are: with breast carcinoma sensitive cells MCF-7 at six orifice plates (3 * 10
5Individual/hole) or 100mm culture dish (2 * 10
6Individual) the middle cultivation after 24 hours, antibiotic serum-free medium is hungry to be cultivated 1 hour with not containing, and pressed afterwards lipofectamine
TM2000 transfection reagent box operating procedures are transfection miR-181a inhibitor(Ribobio respectively) and negative control plasmid (Inhibitor-NC), final concentration is 20nmol/l.After 4 hours, replaced former culture medium culturing 48 hours with the fresh DMEM culture medium that contains 10% hyclone, standby.
At first the described Real-time PCR of Application Example 2 detects the expression of miR-181a, and the interior miR-181a of discovery transfection group cell expresses obviously and descends, and expression minimizing approximately 80%(is seen Fig. 7 A, P＜0.05), show that the reticent cell model of miR-181a successfully constructs.Further use Real-time PCR detection and respectively organize MRP, PGP, LRP, four kinds of drug-resistant protein mrna expressions of BCRP in cell, the present invention as a result finds: with the matched group ratio, miR-181a inhibitor transfection group cell BCRPmRNA expresses obviously increases 20%(P＜0.05), other three kinds of albumen mRNA are without significant change (seeing Fig. 7 B).Western Blotting testing result also shows: with the matched group ratio, miR-181a inhibitor transfection group cell BCRP protein expression increases 15%(P＜0.05), other three kinds of albumen expressions are without obviously changing (seeing Fig. 7 C and Fig. 7 D).
Embodiment 11: suppress the transport function that miR-181a has strengthened BCRP in breast cancer medicines sensitive cells MCF-7, reduce the content of MX in cell;
The MCF-7 after transient transfection in six orifice plates respectively organizes cell by the described transfection method of embodiment 10, blank group 1 is set respectively, blank group 2, negative control group, miR-181a inhibitor group, three groups of MX that add 3 μ M wherein, wash with cold PBS after 1 hour, add the DMEM culture medium culturing that contains 10% hyclone, harvesting after 1.5 hours, with fluorescence intensity in the cells were tested by flow cytometry cell, detect cell to the effect that effluxes of medicine.The present invention as a result finds: with the matched group ratio, and MX content obviously descend (seeing Fig. 8 A and Fig. 8 B, P＜0.05) in miR-181a inhibitor transfection group cell.Confirmed to suppress the transport function that miR-181a can strengthen BCRP in breast cancer medicines sensitive cells MCF-7, namely the drug efflux ability increases, and reduces the content of MX in cell.
Embodiment 12: suppressing miR-181a has increased the drug resistance of drug sensitive cell MCF-7 to MX;
Each organizes the MCF-7 cell after transfection 48h, after giving respectively the etoposide cultivation 48h of MX and 0,1,3,10,30,100,300,1000,3000 μ M that concentration is 0,0.01,0.03,0.1,0.3,1,3,10,30 μ M, the variation of observation of cell to drug resistance.The present invention as a result finds: with matched group than (IC50=0.56 ± 0.05 μ M), miR-181a inhibitor transfection group (IC50=2.04 ± 0.08 μ M) cell obviously increases the drug resistance of MX (sees Fig. 8 C, P＜0.05), and to the drug resistance of etoposide without obviously changing (seeing Fig. 8 D).To sum up result confirmed to suppress BCRP that miR-181a raised drug sensitive cell MCF-7 expresses and with the transport of drug function, increase the drug resistance to MX.
Claims (8)
1. use the method that miR-181a reverses the breast carcinoma drug resistance for one kind, described method comprises:
(1) BCRP that utilizes miR-181a analogies miR-181a mimic to suppress in breast carcinoma mdr cell MCF-7/MX expresses and the transport of drug function, increases the sensitivity to BCRP transhipment substrate mitoxantrone hydrochloride (MX);
(2) utilize miR-181a analogies miR-181a agmir liposome targeting to suppress the expression of BCRP in breast cancer xenograft in nude mice that MCF-7/MX induces, be increased in the body oncocyte to the drug susceptibility of MX;
(3) BCRP that utilizes miR-181a inhibitor miR-181a inhibitor to raise drug sensitive cell MCF-7 expresses and the transport of drug function, increases the drug resistance to MX.
2. application miR-181a according to claim 1 reverses the method for breast carcinoma drug resistance, it is characterized in that, in described step (1), miR-181a analogies miR-181a mimic is for comprising the hairpin like fold of miR-181a nucleotide sequence " stem-ring " structure, and main sequence is as follows: 5 '-AACAUUCAACGCUGUCGGUGAGU-3 '.
3. application miR-181a according to claim 1 reverses the method for breast carcinoma drug resistance, it is characterized in that tumor cell described in described step (1) is breast carcinoma mdr cell MCF-7/MX.
4. breast carcinoma mdr cell MCF-7/MX according to claim 3 was expression BCRP breast cancer cell.
5. application miR-181a according to claim 1 reverses the method for breast carcinoma drug resistance, it is characterized in that the cell that uses in described step (3) is breast carcinoma sensitive cells MCF-7.
6. breast carcinoma sensitive cells MCF-7 according to claim 5 is the breast cancer cell that miR-181a crosses expression.
7. application miR-181a according to claim 1 reverses the method for breast carcinoma drug resistance, it is characterized in that the final concentration scope that in described step (1), miR-181a analogies miR-181a mimic is transfected in breast carcinoma mdr cell MCF-7/MX is: 10 nM-80 nM, the transfection time is 48 hours.
8. application miR-181a according to claim 1 reverses the method for breast carcinoma drug resistance, when it is characterized in that utilizing in described step (2) miR-181a analogies miR-181a agmir liposome to act on the breast cancer xenograft in nude mice that MCF-7/MX induces, the injecting method that adopts is multi-point injection in nude mice tumor week and tumor, the liposome injection volume is 1nmol, frequency injection is jede Woche injection twice, injects altogether five times.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201310049732 CN103083685A (en) | 2013-02-07 | 2013-02-07 | Method for reversing drug resistance of breast cancer by using miR-181a |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201310049732 CN103083685A (en) | 2013-02-07 | 2013-02-07 | Method for reversing drug resistance of breast cancer by using miR-181a |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103083685A true CN103083685A (en) | 2013-05-08 |
Family
ID=48197156
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201310049732 Pending CN103083685A (en) | 2013-02-07 | 2013-02-07 | Method for reversing drug resistance of breast cancer by using miR-181a |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103083685A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110172460A (en) * | 2019-04-30 | 2019-08-27 | 厦门大学附属翔安医院 | HTERT-miR-221/222 sponge and its application in the tamoxifen drug resistance for reversing breast cancer |
-
2013
- 2013-02-07 CN CN 201310049732 patent/CN103083685A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110172460A (en) * | 2019-04-30 | 2019-08-27 | 厦门大学附属翔安医院 | HTERT-miR-221/222 sponge and its application in the tamoxifen drug resistance for reversing breast cancer |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Fang et al. | MicroRNA‐29b suppresses tumor angiogenesis, invasion, and metastasis by regulating matrix metalloproteinase 2 expression | |
| Gao et al. | microRNA-126 targeting PIK3R2 promotes rheumatoid arthritis synovial fibro-blasts proliferation and resistance to apoptosis by regulating PI3K/AKT pathway | |
| Mammoto et al. | A mechanosensitive transcriptional mechanism that controls angiogenesis | |
| Lu et al. | Identification and mechanism of regulation of the zebrafish dorsal determinant | |
| Evellin et al. | FOSL1 controls the assembly of endothelial cells into capillary tubes by direct repression of αv and β3 integrin transcription | |
| CN105960459A (en) | Inhibition of NEAT1 for treatment of solid tumors | |
| Ning et al. | Molecular mechanism of microRNA-21 promoting Schwann cell proliferation and axon regeneration during injured nerve repair | |
| Yang et al. | Platelet microparticles-containing miR-4306 inhibits human monocyte-derived macrophages migration through VEGFA/ERK1/2/NF-κB signaling pathways | |
| Liu et al. | MicroRNA-206 regulates cell movements during zebrafish gastrulation by targeting prickle1a and regulating c-Jun N-terminal kinase 2 phosphorylation | |
| Yang et al. | LncRNA RMRP promotes cell proliferation and invasion through miR-613/NFAT5 axis in non-small cell lung cancer | |
| Zhang et al. | Hypoxia preconditioned renal tubular epithelial cell-derived extracellular vesicles alleviate renal ischaemia-reperfusion injury mediated by the HIF-1α/Rab22 pathway and potentially affected by microRNAs | |
| CN103263676A (en) | Method for reversing drug resistance of breast cancer by using miR-487a | |
| CN107988150A (en) | A kind of identification method and system of Myocytes Anoxia-reoxygenation | |
| Zhang et al. | Adipose-derived mesenchymal stem cells ameliorate ulcerative colitis through miR-1236 negatively regulating the expression of retinoid-related orphan receptor gamma | |
| CN104774929A (en) | Applications of miR-455-3p in diagnosis, treatment and prognosis of esophageal squamous cancer | |
| Wang et al. | TEAD1 controls C2C12 cell proliferation and differentiation and regulates three novel target genes | |
| Peng et al. | LncRNA-MALAT1/miRNA-204-5p/Smad4 Axis Regulates Epithelial–Mesenchymal Transition, Proliferation and Migration of Lens Epithelial Cells | |
| CN110251529A (en) | MiR-124-3p and its analog are preparing the application in anti-breast cancer disease medicament | |
| Cappellini et al. | Silencing of Eps8 inhibits in vitro angiogenesis | |
| CN109321656A (en) | Purposes of the protein D EPDC1 as the marker of the negative breast cancer of diagnosis three | |
| CN103826623A (en) | Synthetic lethality and the treatment of cancer | |
| CN103083685A (en) | Method for reversing drug resistance of breast cancer by using miR-181a | |
| CN104056277A (en) | MLH1 gene or application of expression product in colorectal cancer highly expressed by DKK4 | |
| Jaboin et al. | Autophagy in lung cancer | |
| CN100352922C (en) | SiRAN and expression carrier for inhibiting human VEGF gene expression and their pharmaceutical use |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20130508 |