CN105056250B - A kind of applications of microRNA in the medicine for preparing treatment prostate cancer - Google Patents
A kind of applications of microRNA in the medicine for preparing treatment prostate cancer Download PDFInfo
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- CN105056250B CN105056250B CN201510416470.0A CN201510416470A CN105056250B CN 105056250 B CN105056250 B CN 105056250B CN 201510416470 A CN201510416470 A CN 201510416470A CN 105056250 B CN105056250 B CN 105056250B
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Abstract
The present invention relates to applications of the miR 487 in the medicine for preparing treatment prostate cancer, a kind of and pharmaceutical composition with miR 487 for active component, pharmaceutical composition provided by the present invention can suppress the evolution of prostate cancer by miR 487 to the inhibitory action of prostate gland cancer cell transfer ability, suppress efficiency high up to 80%.
Description
Technical field
The present invention relates to biological technical field, specifically, being related to a kind of microRNA is preparing the medicine for the treatment of prostate cancer
Application in thing.
Background technology
Prostate cancer is most common malignant tumour in male genetic, falls ill with the age and increases, is only second to lung cancer, is man
Property is because of the second largest reason of cancer mortality.2009, the U.S. is estimated to be 192280 prostate cancers, and newly to send out patient and 27360 dead
Die patient.Due to China human mortality aging, prostate-cancer incidence increased.Clinically, operation or radio-therapeutic needle to limitation before
Row gland cancer is effective, but be can not be cured for transferrer, is still lacked for metastatic prostate cancer treatment effective
Medicine.MiRNA is the endogenous non-coding microRNA of regulatory gene expression, and gene expression is carried out in post-transcriptional level
Regulation and control, participate in the physiology courses such as cell cycle, apoptosis, development, differentiation and metabolism.MiRNA expresses imbalance meeting in cell
Cause the generation of a variety of diseases including cancer, recent studies have shown that, some miRNA unconventionality expressions in prostate, but which kind of
MiRNA is relevant with generation, the development of prostate cancer not to reach common understanding yet.
It is therefore desirable to find with prostate cancer to occur, develop relevant miRNA, so as to for before clinical treatment metastatic
Row gland cancer provides a kind of effective means.
The content of the invention
It is an object of the invention to provide a kind of pharmaceutical composition containing miRNA for suppressing prostate cancer development, to face
Metastatic prostate cancer offer is treated on bed may.
The present inventor has found miR-487 in forefront during studying the metastasis of prostate cancer
Conspicuousness is expressed in gland cancer epithelial cell line PC-3, DU145 cell to be reduced, while finds that miR-487 can suppress cancer cell increasing
Grow and migrate, promote Apoptosis, miR-487 can suppress the transfer of prostate tumor cells in nude mouse.Prostate cancer is thin
MiR-487 expression quantity height can be as a molecular marked compound of cancer metastasis in born of the same parents.
One aspect of the present invention provides applications of the miR-487 in the medicine for preparing treatment prostate cancer, described
MiR-487 nucleotide sequence is as shown in SEQ ID No.1.
Optionally, the application includes preparing the medicine for suppressing migration of prostate cancer cells, invasion and attack and propagation.
Optionally, the application include after miR-487 is combined with carrier with antineoplastic and/or pharmaceutically acceptable
Pharmaceutical composition is made in auxiliary material compounding.
One aspect of the present invention provides miR-487 and is preparing the high metastatic cell system PC-3 of suppression prostate gland cancer cell
And/or the application in the medicine of DU145 transfer ability.
Present invention also offers a kind of pharmaceutical composition, described pharmaceutical composition using miR-487 as active component, wherein,
The nucleotide sequence of the miR-487 is as shown in SEQ ID No.1.
Optionally, described pharmaceutical composition contains and carrier-bound miR-487 and/or pharmaceutically acceptable auxiliary material.
In pharmaceutical composition provided by the present invention, carrier can be suitable for miRNA in host for commonly used in the art
The kind of carrier expressed in cell, for example, the carrier can be liposome, chitosan or Lentiviral.
In one embodiment of the invention, the expression vector is Lentiviral, it is preferred that the slow disease
Malicious expression vector can be pWPXL, pMD2.G or psPAX2 Lentiviral.
In a preferred embodiment in accordance with this invention, described pharmaceutical composition also optionally comprising it is one or more its
He optionally, contains platinum series antineoplastic medicament, can also wrapped to the effective tumour medicine of prostate cancer in described pharmaceutical composition
Containing other conventional use of tumour medicines in the Combination chemotherapy based on platinum antineoplastic medicine, these other tumour medicines
It is well-known to those skilled in the art.
Optionally, the antineoplastic is cis-platinum or carboplatin.
When miR-487 and platinum series antineoplastic medicament are used in combination, the dosage of platinum series antineoplastic medicament can reduce to
The 20-80% of dosage in normal chemotherapy protocols.
In the present invention, the method for administration of described pharmaceutical composition is intravenous, via arterial infusion or local injection etc., also may be used
To carry out testis or cancer target administration using target administration technology well-known to those skilled in the art.
Pharmaceutical composition provided by the present invention can be made by suppression of the miR-487 to prostate gland cancer cell transfer ability
For suppressing the evolution of prostate cancer, suppress efficiency high up to 80%.See Fig. 5
Brief description of the drawings
Fig. 1 is that miR-487 suppresses cell propagation and clone's balling-up.
Wherein, (A) is that miR-487 transfects PC-3, and Q-PCR detects miR-487 expression quantity；(B) transfected for miR-487
Cell propagation is detected after PC-3；(C) it is clone's balling-up ability detection.
Fig. 2 is that miR-487 can promote apoptosis of prostatic carcinoma cell line result figure.
Wherein, (A) is Annexin V-FITC/PI dyeing detection Apoptosis after miR-487 transfects PC-3；(B) it is to wither
Die cell number quantization figure.
Fig. 3 is that miR-487 suppresses migration of prostate cancer cells and invasion and attack result figure.
Wherein, (A-D) is that miR-487 transfects PC-3 cells, the experiment detection cell migration of cell scratch；(E) it is miR-487
Transfect PC-3 cells, transwell experiment detection cell migrations；(F) after transfecting PC-3 for miR-487, transwell experiment inspections
Survey cell invasion.
Fig. 4 is that Western blot detect cell migration Invasive associated protein molecular change.
Wherein, (A) is that cellular morphology changes after PC-3 transfects miR-487.(B) miR- is detected for western blot
E-cad and N-cad expression change after 487 transfection PC-3.
Fig. 5 be PC-3 cells after converting miR-487 be inoculated into nude mice by subcutaneous into knurl situation
Embodiment
Below will the present invention is described in detail by embodiment.
In the present invention, term " miR-487 " refers to small comprising sequence shown in SEQ ID No.1 or its homologous sequence
RNA.The miR-487 in various sources as is generally known in the art, for example, people, mouse, rabbit etc., these homologous sequences are all contained in the present invention
Term miR-487 in.Also it is substituted, lacks or adds comprising above-mentioned naturally occurring miR-487 sequences in the term of the present invention
Add one or several nucleotides, or still there is the derivative RNA of miR-487 bioactivity after biology chemical modification.This hair
In bright, the artificial synthesized and miR- with miR-487 biological activities that can be obtained by buying commercial goods mode
487 analogies fall within protection scope of the present invention.
In addition, miR-487 of the present invention can also be precursor forms, miR-487 precursors refer to be administered object
Precursor intracellular or that miR-487 can be formed in vivo.The method for obtaining naturally occurring miR-487 precursors is this
Well known to art personnel.
As well known to those skilled in the art, miR-487 initial transcription product forms maturation after a series of processing
miR-487.MiR-487 precursors only just have corresponding biological function after the miR-487 of maturation is processed into.
Composition provided by the present invention can be used for treating prostate cancer.This hair containing effective dose in the composition
Bright miR-487.
In the present invention, the pharmaceutically acceptable auxiliary material includes various excipient, diluent and adjuvant.Auxiliary material itself
It is not necessary active component, and does not have undue toxicity after use.This kind of auxiliary material includes but is not limited to：Physiological saline, delay
Fliud flushing, glucose, water, glycerine, ethanol etc..
In one embodiment of the invention, the form of the composition is suitable for：Direct naked microRNA injections,
Liposome RNA direct injections, breeding unsoundness bacterium carry DNA method or replication defective adenoviral carries target DNA
Method etc..
The miR-487 of present invention effective dose can be entered with the pattern of administration and the order of severity etc. of disease to be treated
The corresponding adjustment of row.The selection of preferable effective dose can be integrated each factor by those of ordinary skill in the art to determine.It is described
Factor includes but is not limited to：MiR-487 pharmacokinetic parameter, the health status of treated patient, body weight, method of administration
Deng.
In the present invention, the method for administration of described pharmaceutical composition is intravenous, via arterial infusion or local injection etc., also may be used
To be administered using medicine-feeding technology well-known to those skilled in the art.
The pharmaceutical composition of the present invention can combine with other treatment means, the treatment for prostate cancer.
Embodiment 1
Cell PC-3 used in example is purchased from ATCC, i.e. American Type Culture Collection, transfection reagent
Lipofectamine 2000 is purchased from invitrogen, and miR-487mimic is purchased from Life Technologies
Corporation (product ID number is MC10648) miR-487 inhibitor are purchased from Life Technologies
Corporation (product ID number is MH10648).
The present embodiment passes through the cell in vitro biological experiment proof miR-487 functions of being shifted to prostate tumor cells
Inhibitory action.
PC-3 cell lines are cultivated, is divided into experimental group and control group and transfects miR-NC (negative control) and miR-487 respectively, are examined
Survey cell propagation, migration and apoptosis change.
(1) miR-487 can suppress cell propagation and clone's balling-up：
MiR-487 analogies miR-487mimics transfections (are used into transfection reagent with lipofectamine 2000
The direct transfections of lipofectamine 2000) prostate cell line PC-3, and its expression quantity such as Figure 1A is detected with Q-PCR, as a result
Show, miR-487 expression quantity conspicuousness rises in cell after miR-487mimics transfections PC-3, it was demonstrated that mimics can be simulated
MiR-487 expression.After miR-487mimics transfects PC-3 cells 24,36,48,72 and 96h, the inspection of external use CCK-8 kits
Survey cell propagation, such as Figure 1B, the results showed that, miR-487 is capable of the propagation of conspicuousness suppression prostate gland cancer cell.Trained with soft agar
Support colony formation detection cell colony dependence and multiplication capacity.Take the logarithm the single-layer culturing cell in growth period, use
0.25% Trypsin Induced simultaneously blows and beats the DMEM/F12 nutrient solutions for into individual cells, cell being suspended in 10% hyclone
In it is standby.The dilution of cell suspension multiple is inoculated in soft agar culture plate, quiescent culture 10-14 weeks, calculates cloning efficiency,
Such as Fig. 1 C, the results showed that miR-487 is capable of conspicuousness suppression PC-3 clone's balling-up ability.
(2) miR-487 can promote apoptosis of prostatic carcinoma cell line.
Annexin V-FITC/PI are dyed after miR-487mimics is transfected into PC-3, Flow cytometry apoptotic cell
Number.
As a result Fig. 2 is seen：MiR-487mimics transfections PC-3 can significantly improve the positive cell numbers of Annexin V, it was demonstrated that
MiR-487 can significantly cause PC-3 Apoptosis,
(3) miR-487 can suppress migration of prostate cancer cells and invasion and attack.
MiR-487mimics transfects PC-3 cells, and cell scratch experiment detects the migration of cell, such as Fig. 3 A-D, used in addition
The migration of transwell Matrigels checking cell and invasive ability, such as Fig. 3 E-F, the results showed that miR-487mimics is transfected
PC-3, cell migration and invasive ability decline.
(4) Western blot detect the related protein molecular change of cell migration invasion and attack
MiR-487mimics transfected PC-3 cells after 24 hours, and cellular morphology changes, and as shown in Figure 4 A, extracted egg
White matter, detection CAP N-caderin (neural cadherins) and E-caderin (E-cadherin) table
Reach, the results showed that, miR-487 can suppress cancer cell surfaces N-caderin, promote E-caderin expression, such as Fig. 4 B institutes
Show, show that miR-487 can suppress the conversion of prostate cancer Epithelial and stromal, suppress cancer cell and shift.
Embodiment 2
The inhibitory action that the present embodiment passes through the experiment in vivo proof miR-487 functions of being shifted to prostate tumor cells.
10 nude mices are randomly divided into two groups, (this is thin by control group tail vein injection expression control miRNA PC-3M-luc
Born of the same parents are to obtain PC-3 cell of the luciferase reporter vector transfection from ATCC).Number of cells is 6 × 106；Experimental group is noted
Firing table reaches miR-487 PC-3M-luc cell lines, and number of cells is 6 × 106；Cell is injected after 5 weeks, every mouse is injected weekly
100 μ l concentration are 30 μ g/ μ l luciferase substrate D-luciferin, then small animal living body imaging luciferase point
Cloth, continuous three weeks statistics cancer metastasis rates.After PC-3 cell line transfections miR-NC and miR-487, examined with transwell methods
The migration and variation of cell is surveyed, is bred with CCK-8 kit detection cells and changed, Apoptosis is detected with Annexin V/PI methods
Change.The organ sites that internal shift experiment is transferred to small animal living body imaging cell, carry out ASSOCIATE STATISTICS.
To taking the tumor section of mouse after 10 weeks, weighed and compared, the PC-3 cells of miR-487 conversions are into knurl size
Significantly lower than control group, it is 80% to suppress efficiency through volume and weighing results statistics.As a result Fig. 5 is seen.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (4)
1.miR-487 the application in the medicine for preparing treatment prostate cancer, the nucleotide sequence such as SEQ ID of the miR-487
Shown in No.1.
2. application according to claim 1, it is characterised in that the application includes preparation suppression prostate gland cancer cell and moved
The medicine for moving, attacking and breeding.
3. application according to claim 1 or 2, it is characterised in that including after miR-487 is combined with carrier with it is antitumor
Pharmaceutical composition is made in medicine and/or pharmaceutically acceptable auxiliary material compounding.
4.miR-487 is preparing the medicine for the transfer ability for suppressing the high metastatic cell system PC-3 and/or DU145 of prostate gland cancer cell
Application in thing, the nucleotide sequence of the miR-487 is as shown in SEQ ID No.1.
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Families Citing this family (5)
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| CN106177995B (en) * | 2016-08-04 | 2020-02-21 | 北京信生元生物医学科技有限公司 | Application of miR-3925 in preparation of medicine for treating pancreatic cancer |
| CN107488733B (en) * | 2017-10-10 | 2019-11-05 | 广州医科大学附属第二医院 | Application of the miR-133b in prostate cancer with osseous metastasis diagnoses, predicts, treats |
| CN107488735B (en) * | 2017-10-10 | 2019-11-05 | 广州医科大学附属第二医院 | MiR-339-5p is inhibiting the application in prostate cancer with osseous metastasis and TGF-β signal path |
| CN107488734B (en) * | 2017-10-10 | 2019-11-05 | 广州医科大学附属第二医院 | MiR-19a-3p is preparing the application in prostate cancer with osseous metastasis diagnostic reagent and therapeutic agent |
| CN108148911B (en) * | 2018-03-02 | 2019-11-05 | 广州医科大学附属第二医院 | Application of the miR-582 in diagnosis, prognosis kit and the drug for preparing prostate cancer with osseous metastasis |
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