CN102321585B - Application of miRNA-106a and miRNA-106a inhibitor in preparing glioblastoma stem cell invasion regulator - Google Patents
Application of miRNA-106a and miRNA-106a inhibitor in preparing glioblastoma stem cell invasion regulator Download PDFInfo
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Abstract
The present invention relates to the field of medicine, specifically to a regulator for regulation of invasion behaviors of glioblastoma (GBM) stem cells. The technical scheme of the present invention is that: an application of the miRNA-106a in preparing a GBM stem cell invasion accelerating agent is provided; an application of a miRNA-106a inhibitor in preparing a GBM stem cell invasion inhibitor is provided; a expression level of the miRNA-106a in glioma stem cells (GSCs) is significantly higher than the expression level of the miRNA-106a in glioma cells (GCs), both the mRNA level and the protein level of TIMP-2 in the GSCs is significantly lower than the mRNA level and the protein level of the TIMP-2 in the GCs; the miRNA-106a inhibitor can significantly inhibit the invasiveness of the GSCs; after the expression of the miRNA-106a is inhibited, the mRNA level and the protein level of the TIMP-2 are significantly increased; luciferase reporter gene experiment results show that: miRNA-106a can negatively regulate the expression of the TIMP-2, and can directly regulate the expression of the TIMP-2 through a combination of the miRNA-106a and 3'UTR of the TIMP-2.
Description
Technical field
The present invention relates to medical field, particularly the adjusting control agent of glioma stem cell invasion and attack behavior regulation and control.
Background technology
(glioblastoma GBM) is the modal primary malignancy cerebral tumor of cns to glioblastoma.The growth of invasion by tumor cells property is that the important biomolecule of glioblastoma is learned characteristic, and this invasion and attack characteristic makes operation be difficult to thorough tumor resection tissue, and the chemotherapy and radiation weak effect, and the postoperative recurrence rate is high, and prognosis is not good.The invasive growth of glioblastoma it be unclear that with the treatment resistance mechanisms.
A large amount of research recently shows that (cancer stem cells's tumor stem cell CSCs) plays a significant role in tumour takes place and develops.Discover exist in the glioblastoma glioma stem cell (glioma stem cells, GSCs), the ability that these GSCs have self ability, multidirectional differentiation potential and form tumour.Find that simultaneously GSCs has high invasiveness, yet concrete molecular mechanism is not clear.
MicroRNA (miRNA) is the non-coding strand microRNA that one type of length of finding recently is about 21～23 Nucleotide, in growth and disease, has participated in physiological and pathologic processes such as propagation, migration, differentiation and apoptosis through regulating target gene expression.More and more evidences shows that dissimilar miRNA has participated in the generation and the transfer of modulate tumor.In addition, different in the at present existing express spectra of discovering miRNA among the CSCs and the normal stem cell, and significant in diagnosis, treatment and the prognosis of tumour.The expression of miRNA possibly participated in the regulation and control of glioblastoma invasion and attack, but and also further further investigation of the relation between the GSCs biological behaviour.
Summary of the invention
One of the object of the invention is to provide a kind of promotor of glioma stem cell invasion and attack, and this promotor provides valid approach for preparation glioma stem cell invasive model.
The application of miRNA-106a in preparation glioma stem cell invasion and attack promotor.
Further, the application of miRNA-106a in preparation TIMP-2 suppressor factor.
Further, the application of miRNA-106a in preparation MMP2 promotor.
Further, the application of miRNA-106a in preparation MMP9 promotor.
Two of the object of the invention is to provide a kind of suppressor factor of glioma stem cell invasion and attack, and this suppressor factor provides new approaches for the treatment glioma.
For realizing such scheme, technical scheme of the present invention is:
The application of the suppressor factor of miRNA-106a in preparation glioma stem cell invasion and attack suppressor factor.
Further, said miRNA-106a is the suppressor factor of TIMP-2.
Further, said miRNA-106a is a MMP2 promotor.
Further, said miRNA-106a is a MMP9 promotor.
Further, the suppressor factor of said miRNA-106a is Anti-miR miRNA-106a inhibitors.
Technique scheme obtains through following method: at first separated people's glioma cell line U87 and former generation glioma sample source CD133
+Glioma cell (glioma cells, GCs), and be tested and appraised confirmed people's glioma cell line U87 and former generation glioma sample source CD133
+Glioma cell has the GSCs characteristic; Then, according to miRNA chip results (miRCURY LNA
TMMicroRNA array (v.10.0), Denmark Exiqon Life Sciences company) found two functional miRNA, one of them is miRNA-106a, has detected miRNA-106a and the expression of TIMP-2 path in GSCs and GCs; At last, through research miRNA-106a to the direct regulation and control effect of TIMP-2 and prove that this is the important mechanisms that miRNA-106a promotes the GSCs invasion and attack.
Beneficial effect of the present invention is: separate the CD133 that obtains
+Cell has the biological characteristics of self, multidirectional differentiation potential, formation tumour etc.; Proof GSC has high aggressive than glioma cell (GC); The expression of miRNA-106a in GSC is significantly higher than GC (P＜0.05), and no matter the mRNA level still is the expression of protein level in GSC to TIMP-2 significantly is lower than GC (P＜0.05); The suppressor factor of miRNA-106a can obviously suppress the invasive ability (P＜0.05) of GSC; After suppressing the expression of miRNA-106a; The expression of TIMP-2mRNA level and protein level significantly raise (P＜0.05); Luciferase reporter gene experiment confirm miRNA-106a not only can negative regulation TIMP-2 expression, and be the expression of the mode direct regulation and control TIMP-2 that combines through 3 ' UTR with TIMP-2.So GSCs has high invasion and attack characteristic, find that the miRNA-106a high expression level has promoted the aggressive of GSCs, its mechanism is expressed through downward modulation TIMP-2, rise MMP2 and MMP9 at least and is realized.
Description of drawings
Fig. 1 behave glioma cell line U87 with former generation glioma sample source the streaming cell node that separates of GSCs really scheme.
Fig. 2 is for being CD133
-Cell was cultivated 36 hours in the tumor stem cell substratum, CD133
+Cell was cultivated 7 days in the tumor stem cell substratum; Observation by light microscope, * 200.
Fig. 3 is CD133
+The expression immunofluorescence dyeing of CD133 and nestin in the cell, laser confocal microscope is observed, DAPI labeled cell nuclear, scale=50 μ m.
Fig. 4 is CD133
+Plastidogenetic cell ball induces differentiation after 7 days, is divided into the cell of expressing GFAP, β-tubulin III and MBP.Immunofluorescence dyeing, laser confocal microscope is observed, DAPI labeled cell nuclear, scale=50 μ m.
Fig. 5 is CD133
+Cell and CD133
-Cell becomes the photo of knurl size in the nude mouse body.
Fig. 6 is CD133
+Cell and CD133
-Cell becomes the quantitative analysis results of knurl size, * P＜0.05, * * P＜0.01 in the nude mouse body.
Fig. 7 is Transwell invasion and attack experimental cell number figure, observation by light microscope, * 200.
Fig. 8 is Transwell invasion and attack experimental cell number quantitative analysis results, * P＜0.05.
Fig. 9 is quantitative PCR detection miRNA-20a and the differential expression of miR-106a in GSCs and GCs, * * P＜0.01, * * * P＜0.001.
Figure 10 be miRNA-20a and miR-106a respectively to the influence of GSCs invasive ability, Transwell invasion and attack experimental cell number figure, observation by light microscope, * 200.
Figure 11 is Transwell invasion and attack experimental cell number quantitative analysis results, * P＜0.05,, wherein miRNA-20aI represents the miRNA-20a suppressor factor, and miRNA-106aI represents the miRNA-106 suppressor factor.
Figure 12 is TIMP-2, MMP2 and the differential expression of MMP9mRNA level in GSCs and GCs, * P＜0.05, * * P＜0.01, * * * P＜0.001.
Figure 13 is TIMP-2, MMP2 and the differential expression of MMP9 protein level in GSCs and GCs.
Figure 14 is that the expression of TIMP-2 behind difference transfection miRNA-20a and the miR-106a suppressor factor, MMP2 and MMP9mRNA level changes * P＜0.05, * * P＜0.01.
Figure 15 is that the expression of TIMP-2 behind difference transfection miRNA-20a and the miR-106a suppressor factor, MMP2 and MMP9 protein level changes.
Figure 16 detects miRNA-20a and miR-106a respectively to direct regulation and control effect * P＜0.05 of TIMP-2 for luciferase reporter gene experiment.
Figure 17 is the sectional drawing of the binding site of the miRNA-20a of information biology prediction and miR-106a and TIMP-2.
U87 is specially glioma cell line U87 in the accompanying drawing, and Case1 specifically refers to former generation glioma cell Case1, and Case2 specifically refers to former generation glioma cell Case2; MiR is writing a Chinese character in simplified form of miRNA.
Embodiment
1 material
The cultivation of former generation people glioma cell
Fresh samples of human glioma is taken from southwestern hospital and big level ground hospital (Chongqing City Third Military Medical University) Neurological Surgery, successively gets 2 examples altogether, is glioblastoma multiforme (WHO IV level).In former generation,, the samples of human glioma piece cleaned 3 times with 0.01M PBS, carefully removed surface fiber and necrotic tissue; Tissue block is immersed in a small amount of DMEM substratum (U.S. Gibco company), and with eye scissors with tumor tissue's repeated shear to 1mm
3Fragment; Add epoxy glue protoenzyme (U.S. Gibco company) digestion 20～30 minutes; Blow and beat with pipettor while digesting, obtain single cell suspension, place DMEM substratum (containing volume(tric)fraction is 10% foetal calf serum) to cultivate; Change liquid after 24 hours and remove red corpuscle, treat that cell grows at 80%～90% o'clock and is used for sorting CD133
+Cell.
The cultivation of glioma cell line
People's glioma cell line U87 is from American Type Culture Collecti (ATCC), and preserving number is HTB-14
TMPeople's glioma cell line U87 uses and to contain the DMEM substratum that volume(tric)fraction is 10% foetal calf serum (U.S. Gibco company), and putting 37 ℃, volume(tric)fraction is 5%CO
2Cultivate in the incubator (relative humidity is 95%).It is 2～3 days that cell changes the liquid time, and the generation time is 3～5 days, and using massfraction before going down to posterity is that 0.25% trypsinase+massfraction is 0.02%EDTA (U.S. Sigma company) mixture slaking liquid digestion 2～4 minutes.
2CD133
+The separation of cell and cultivation
Former generation glioma cell and glioma cell line U87 grow at 80%～90% o'clock, PBS cleans 3 times; Add trypsinase+EDTA Digestive system and digested 5 minutes, piping and druming makes cell detachment gently, becomes single cell suspension; The collecting cell suspension, the centrifugal 5min of 1000rpm; Supernatant discarded, PBS cleans 2 times, the centrifugal 5min of 1000rpm; Using streaming Buffer is 2 * 10 with the cell concn dilution
7/ ml; Per 100 μ l cell suspensions add 10 μ l CD133/2-PE antibody (German Miltenyi company), and control group only adds 10 μ l streaming Buffer; 4 ℃ of lucifuges are hatched 30min, and every 10min piping and druming mixing is once avoided cell precipitation; With the above-mentioned cell suspensions of respectively organizing of 10 times of volume PBS dilution, wash 2 times after, the centrifugal 5min of 1000rpm; Using streaming Buffer re-suspended cell to make concentration is 2 * 10
7/ ml, upflowing cell sorter (U.S. Becton and Dickinson company) detects and sorting; After the sorting; With containing B27 (1 *, U.S. Gibco company), bFGF (20ng/ml, U.S. Upstate company) and EGF (20ng/ml; U.S. Sigma company) cultivate the resuspended back of DMEM/F12 (U.S. Gibco company) stem cell perfect medium, changes liquid and all take half amount to change the liquid mode with going down to posterity.
As shown in Figure 1, before Flow Cytometry detects sorting, CD133
+The ratio that cell accounts for glioma cell line U87 and former generation glioma cell Case1, Case2 is respectively 0.5%, 2.4% and 1.5%.Return after the sorting and survey CD133
+Cell proportion is up to more than 95%, CD133
-CD133's is expressed in below 1% in the cell.
3 immunofluorescence dyeings
Flow Cytometry is detected sorting resulting 5 * 10
4Individual CD133
-Cell and CD133
+Cell inoculation is in DMEM/F12 stem cell perfect medium.Immunofluorescence dyeing detects the CD133 that cultivates
+The expression of stem cell labeling thing CD133 and nestin in the cell.What fix respectively organizes the cell sheet with 0.01M PBS aquation, 5min * 3 time; Drip lowlenthal serum (company of China fir Golden Bridge in Beijing) 37 ℃, sealing 30min inhales and removes unnecessary serum, does not wash; Each group drips mouse anti human CD133 monoclonal antibody (1: 100, German Miltenyi company), mouse anti human nestin monoclonal antibody (1: 100, U.S. Chemicon company) respectively, and 4 ℃ are spent the night; 0.01M PBS washing 5min * 3 time; Each group drips the anti-mouse IgG of FITC labelled goat (1: 100, U.S. Molecular Probes company) respectively, 37 ℃ hatch 30min after, inhale and remove unnecessary two anti-liquid, do not wash; Drip 20 μ l DAPI (U.S. Sigma company) incubated at room 10min; 0.01M PBS washing 5min * 3 time; Drip a little mountant, left-hand thread notes not producing bubble on slide glass, and laser confocal microscope (German Leica company) is observed down, and immunofluorescence dyeing shows, the CD133 that sub-elects
+Cell wide expression NSC affinity tag CD133 and nestin are as shown in Figure 3.Immunofluorescence dyeing, laser confocal microscope is observed, DAPI labeled cell nuclear, scale=50 μ m.
4CD133
+The cell balling-up is cultivated and differentiation
Sorting is obtained 5 * 10
4Individual CD133
-Cell and CD133
+Cell inoculation is cultivated in DMEM/F12 stem cell perfect medium, observes CD133
-Cell and CD133
+Cell forms the ability of cell ball.With CD133
+Plastidogenetic cell ball is inoculated in the DMEM substratum (containing massfraction is 10% foetal calf serum) and induces differentiation, detects the expression of GFAP, β-tubulin III and MBP in the noble cells after 7 days with the immunofluorescence dyeing method.What fix respectively organizes the cell sheet with 0.01M PBS aquation, 5min * 3 time; Drip 37 ℃ of lowlenthal serums, sealing 30min inhales and removes unnecessary serum, does not wash; Each group dripped the anti-people GFAP of rabbit polyclonal antibody respectively (1: 100; U.S. Dako company), the anti-people MBP of rabbit polyclonal antibody is (1: 100; U.S. Chemicon company) and mouse anti human β-tubulinIII monoclonal antibody (1: 100, U.S. Chemicon company), 4 ℃ are spent the night; 0.01M PBS washing 5min * 3 time; Each group dripped the anti-mouse IgG of FITC labelled goat respectively (1: 100; U.S. Molecular Probes company), the anti-rabbit igg of FITC labelled goat (1: 100, U.S. Molecular Probes company), the anti-mouse IgG of Cy3 labelled goat (1: 500, U.S. Molecular Probes company), the anti-rabbit igg of Cy3 labelled goat are (1: 500; U.S. Molecular Probes company); 37 ℃ hatch 30min after, inhale and to remove unnecessary two anti-liquid, do not wash; Drip 20 μ l DAPI incubated at room 10min; 0.01M PBS washing 5min * 3 time; Drip a little mountant, left-hand thread notes not producing bubble on slide glass, and ordinary optical microscope and laser confocal microscope are observed down.
Have shown in Figure 2, under opticmicroscope observation: CD133
+Cell began to form cell ball, 70%～80%CD133 after 7～10 days in 3～4 days
+Cell forms the cell ball of clone's property growth, their rounded or ovals, and similar NSC ball, clear border is suspension growth, and refractivity is good, and is consistent with the prior art report.Under same culture conditions, most of CD133
-Cell is adherent in about 6 hours, the cell of visible differentiation about 24 hours, and cell is fusiformis, and is similar with the parental cell under normal condition is cultivated, and cultivates after 3～4 days, and cell begins dead gradually.
As shown in Figure 4, observation by light microscope is with CD133
+The cell ball is inoculated in the DMEM substratum (containing volume(tric)fraction is 10% foetal calf serum), and the beginning in about about 6 hours of cell ball is adherent, and cell ball outside cell breaks up at first about 16 hours, and the cell of differentiation is the fusiformis growth; Cultivate 3 days left and right sides cell balls and diminish, inboard cell begins differentiation; Cultivate after 7 days, most of cell breaks up in the cell ball.Immunofluorescence dyeing shows that these cells can be expressed astroglia cell mark GFAP, and oligodendrocyte mark MBP and neurone mark β-tubulinIII are as shown in Figure 4.
The foundation of embodiment 2 transplanted tumor animal models
1 laboratory animal source
The female nude mice in ages in animal for research 4～6 week, body weight 15～20g is provided by Third Military Medical University's Experimental Animal Center, and standard conditions are raised.
2 experiments are divided into groups
Experiment is divided into 3 groups, and 5 every group, first group is U87-CD133
-Cell and U87-CD133
+Groups of cells; Second group is former generation Case1-CD133
-Cell and former generation Case1-CD133
+Groups of cells; The 3rd group is: former generation Case2-CD133W cell and former generation Case2-CD133
+Groups of cells.
3 subcutaneous transplantation knurl models
Subcutaneous transplantation knurl model is used to analyze GSCs and becomes the knurl ability.5 * 10
4Individual CD133
+Cell (derive from U87 clone and former generation glioma cell) and CD133
-Cell (derive from U87 clone and former generation glioma cell) is suspended among the 100 μ l PBS, is injected in respectively under the left and right inguinal region of 4～6 week nude mices in age.After the inoculation, animal is all raised in SPF level environment, observes the transplanted tumor size weekly, observes for 6 weeks altogether.
By Fig. 5 and shown in Figure 6, become in the body knurl experiment to show, no matter be the U87 cell or former generation glioma sample source CD133
+Cell becomes the knurl ability to be significantly higher than CD133
-Cell, CD133
-Cell becomes knurl hardly.
The external invasion and attack of embodiment 3GSCs and GCs
The difference of research GSCs and GCs invasive ability, the invasive ability of we have adopted the external invasion and attack experimental observation of Transwell GSCs and GCs.It is 2 groups that the external invasion and attack experiment of Transwell is divided into, and is respectively GSCs group and GCs group, and the concrete operations step is: cell is discarded substratum, PBS washed cell 3 times; Attached cell is centrifugal with trypsinase+EDTA Digestive system digestion back, and after suspension cell was directly centrifugal, resuspended with substratum, the adjustment cell concn was 5 * 10
5/ ml; Take transwell cell (U.S. company BD) packing apart, press from both sides out the transwell cell with aseptic nipper and put into 24 orifice plates; Draw 10 μ l Matrigel glue (U.S. company BD) mixed solutions and spread glue on the transwell film, note the shop notes evenly, be swift in motion and gentleness, Matrigel glue can solidify rapidly in 37 ℃; The cell of completing glue is put into 37 ℃ of incubator 30min together with 24 orifice plates; The chamber adds the complete stem cell media of 500 μ l under cell, does not have bubble to produce between chamber substratum and cell under noting; The above-mentioned cell suspension of respectively organizing of indoor adding 100 μ l on cell is put into 37 ℃ of incubators and is hatched respectively; Took out U87 cell and stem cell in 24 hours, took out former generation glioma cell and stem cell in 48 hours, with last chamber in the cell and the substratum sucking-off of following chamber, PBS washs 3 times; Add chamber 500 μ l, 4% Paraformaldehyde 96, fixedly 20min down; PBS washing 3 times, each 5min; The chamber adds Viola crystallina (the emerging bio tech ltd of Shanghai section) 500 μ l under the cell, dyeing 20min; After dyeing finishes, clean termination dyeing with tap water, cell can not be acutely shaken in attention, in order to avoid cell detachment on the film cleans 4 times; Examine under a microscope and calculate the cell number that passes cell.
Show that like Fig. 7,8 GSCs that derives from U87 clone passes about 360 of the cell number average out to of cell, and corresponding GCs passes the cell number of cell and on average is merely about 20; The GSCs that derives from former generation glioma cell Case 1 passes about 250 of the cell number average out to of cell, and corresponding GCs passes the cell number of cell and on average is merely about 21; The GSCs that derives from former generation glioma cell Case 2 passes about 309 of the cell number average out to of cell, and corresponding GCs passes the cell number of cell and on average is merely about 52.This shows that the quantity of GSCs invasion and attack is significantly more than GCs (P＜0.05).
The differential expression of embodiment 4miRNA-106a in GSCs and GCs
The 1microRNA chip
Adopt Trizol reagent (American I nvitrogen company) to press its process specifications from CD133
-Cell and CD133
+Extract total RNA in the cell.Adopt ELIASA to measure RNA concentration.Total RNA that will extract then is sent to the Haikang and becomes biotechnology ltd, carries out microRNA chip detection (miRCURY LNA
TMMicroRNA array (v.10.0), Denmark Exiqon Life Sciences company).
Adopt miRNA high-throughput chip technology to people's glioma cell line U87 and former generation glioma sample source GSCs and GCs detect the probe of 638 ripe miRNA altogether.62 miRNA expression are arranged among the GSCs in U87 clone source and the GCs, and there were significant differences, and wherein 59 are significantly raised, 3 significantly downward modulations; Former generation glioma sample source GSCs and GCs in have 95 miRNA to express that there were significant differences, wherein 75 are significantly raised, and significantly reduce for 20.Therefrom filter out and in U87 clone and former generation sample, express totally 15 of the miRNA that all there were significant differences (fold >=1.5), be the miRNA of up-regulated in GSCs, the miRNA of downward modulation does not have common factor (table 1).
MiRNA-20a sequence (SEQ ID NO:11): 5 '-uaaagugcuuauagugcagguag-3 '
MiRNA-106a sequence (SEQ ID NO:12): 5 '-aaaagugcuuacagugcagguag-3 '
Express the miRNAs that there were significant differences among table 1GSCs and the GCs
In order to verify the miRNA chip results, we with quantitative PCR detection the differential expression of miRNA-106a in GSCs and GCs.
2qRT-PCR
QRT-PCR detects the expression of miRNA
Adopt Trizol reagent to press its process specifications from CD133
-Cell and CD133
+Extract total RNA in the cell.Adopt ELIASA to measure RNA concentration.Adopt the microRNA Reverse Transcription Kit of U.S. Ambion company
to carry out reverse transcription by its process specifications.Adopt Japanese Takara company
Premix Ex Taq
TMII Kit carries out the qPCR reaction by its process specifications.
According to document (1.Morrison CJ .Cellular activation of MMP-2 (gelatinase A) by MT2-MMP occurs via a TIMP-2-independent pathway.J Biol Chem 2001; 2.Lockwood CJ; Matrix metalloproteinase 9 (MMP9) expression in preeclamptic decidua and MMP9 induction by tumor necrosis factor alpha and interleukin 1beta in human first trimester decidual cells.Biol Reprod 2008.) the qRT-PCR primer sequence of design TIMP-2, MMP2 and MMP9, GAPDH is as the internal reference thing.Synthetic by the handsome Bioisystech Co., Ltd in Shanghai.Primer sequence is following:
Adopt Trizol reagent to press its process specifications from CD133
-Cell and CD133
+Extract total RNA in the cell.Adopt ELIASA to measure RNA concentration.Adopt the Japanese Takara PrimeScript of company
TMRT reagent Kit carries out reverse transcription by its process specifications.Adopt Japanese Takara company
Premix Ex Taq
TMII Kit carries out the qPCR reaction by its process specifications.
Show that like Fig. 9 miRNA-20a has raised about 9 times than GCs in the GSCs in U87 clone source, and two former generation glioma sample source GSCs in raised respectively about 7 times and 15 times; MiR-106a has raised in the GSCs in U87 clone source about 2 times than GCs, and two former generation glioma sample source GSCs in raised respectively about 4 times and 35 times.This shows that miRNA-20a and the miR-106a expression in GSCs is significantly raised than GCs, consistent with above-mentioned microRNA chip results.Shown in figure 12, to compare with GCs, the expression of TIMP-2mRNA in the GSCs in U87 clone and two glioma sample of former generation source all significantly descended about 50%; The expression of MMP2mRNA in GSCs then obviously raised, and is respectively 1.3 times, 3 times and 1.75 times of GCs; The expression of MMP9mRNA in GSCs also obviously raised, and is respectively 2.75 times, 11.5 times and 1.3 times of GCs.
Embodiment 5miRNA-106a is to the regulation and control of GSCs invasion and attack
1miRNA-106a is to the external invasion and attack of GSCs
The concrete operations step of external invasion and attack sees " the external invasion and attack of GSCs and GCs " part for details.
In order to study the influence of miRNA-106a to the GSCs invasion and attack; In GSCs respectively transfection the suppressor factor of miRNA-20a (Anti-miR miRNA-20a inhibitors; Article No. AM10057, U.S. Ambion company) and suppressor factor (Anti-miR miRNA-106a inhibitors, the article No. AM12567 of miR-106a; U.S. Ambion company) after, the variation of GSCs invasive ability of having adopted the external invasion and attack experimental observation of Transwell.
Like Figure 10, shown in 11; In transfection miR inhibitor negative control (article No. AM17010; U.S. Ambion company) control group, the GSCs that derives from U87 clone, former generation glioma cell Case 1 and former generation glioma cell Case 2 pass about 350,245 and 300 of the cell number average out to of cell; Transfection after the miRNA-20a suppressor factor, the cell number that the GSCs that derives from U87 clone, former generation glioma cell Case 1 and former generation glioma cell Case 2 passes cell on average is merely about 101,64 and 81; Transfection after the miR-106a suppressor factor, the cell number that the GSCs that derives from U87 clone, former generation glioma cell Case 1 and former generation glioma cell Case 2 passes cell on average is merely about 99,70 and 90.This shows, behind transfection miRNA-20a and the miR-106a suppressor factor, the quantity of GSCs invasion and attack significantly descend (P＜0.05).
2Western?blot
Whether variant for the expression of studying miRNA-20a and the miR-106a expression in GSCs and GCs there were significant differences its potential target gene TIMP-2 path of back, the expression of TIMP-2, MMP2 and MMP9mRNA level and protein level that adopted quantitative PCR and Western blot technology for detection respectively.Adopt the Mammalian ProteinExtraction Reagent of U.S. Thermo company
to extract total protein of cell by its process specifications.Adopt the BCA ProteinAssay Kit of U.S. Pierce company
to measure total protein concentration, on ELIASA, measure proteic typical curve and concentration by its process specifications.The cellular proteins that extracts is carried out SDS-polyacrylamide gel (SDS-PAGE) electrophoresis; Adopt half-dried electric transferring system (U.S. Bio-Rad company) electricity to change NC film (U.S. Amersham Biosciences company); Use contains the TBST solution room temperature sealing 4 hours that massfraction is 5% skim-milk; With TIMP-2 (1: 200; U.S. Chemicon company), MMP2 is (1: 1500; U.S. Abcam company) and MMP9 (1: 200, U.S. Santa Cruz company) antibody after containing the TBST solution dilution proportion as required that massfraction is 5% skim-milk, hatch with film, 4 ℃ are spent the night; Behind TBST solution washing 3 times, with two anti-be that the TBST solution of 5% skim-milk is after 1: 1500 the dilution proportion according to volume ratio and film is hatched jog on shaking table, room temperature 2 hours with containing massfraction; Behind TBST solution washing 3 times, adopt the U.S. SuperSignal West Pico Chemiluminescent Substrae of Pierce company test kit, operation is recorded as picture with gel imaging appearance (U.S. Bio-Rad company) automatic exposure to specifications.The mRNA level of TIMP-2, MMP2 and MMP9 is consistent among TIMP-2, MMP2 and MMP9 protein level expression and the embodiment 4 in GSCs and GCs, has significant difference at GSCs and GCs, sees Figure 13 for details.
The expression of 3miRNA-20/106a regulation and control TIMP-2 path
In order to study whether miRNA-20a and miR-106a regulate and control TIMP-2 in GSCs expression; Suppressor factor Anti-miR miRNA-20a inhibitors (article No. AM10057 with miRNA-20a and miR-106a; U.S. Ambion company) and Anti-miR miRNA-106a inhibitors (article No. AM12567; U.S. Ambion company) transfection is advanced among the GSCs respectively, adopt quantitative PCR with Western blot technology for detection the expression variation of TIMP-2, MMP2 and MMP9mRNA level and protein level.
The design of TIMP-23 ' UTR binding fragment is with synthetic
According to information biology prediction (Figure 17), the sequence and the mutant nucleotide sequence of design TIMP-23 ' UTR and miRNA-20a and miR-106a binding fragment.By precious biotechnology (Dalian) ltd synthetic (pMD-T Simple-miR-20/106-TIMP2-WT and pMD-T Simple-miR-20/106-TIMP2-MUT).
Fragment sequence is following:
miR-20/106-TIMP2-WT(SEQ?ID?NO：9)：
ggactagttaacatttactcctgtttctgctgattgtttttttaatgttttggtttgtttttgacatcagctgtaatcattcctgtgctgtgttttttattacccttggtaggtattagacttgcacttttttaaaaaaaggtttctgcatcgtggaagcatttgacccagagtggaacgcgtggcctatgcaggtggattccttcaggtctttcctttggttctttgaagcttgg
miR-20/106-TIMP2-MUT(SEQ?ID?NO：10)：
ggactagttaacatttactcctgtttctgctgattgtttttttaatgttttggtttgtttttgacatcagctgtaatcattcctgtgctgtgttttttattacccttggtaggtattagacttgcagatttttaaaaaaaggtttctgcatcgtggaagcatttgacccagagtggaacgcgtggcctatgcaggtggattccttcaggtctttcctttggttctttgaagcttgg
Shown in figure 14; Transfection behind the miRNA-20a suppressor factor; The expression level of TIMP-2mRNA in the GSCs in U87 clone and two glioma sample of former generation source miR inhibitor negative control (the article No. AM17010 that has been transfection respectively; About 1.5 times, 2.05 times and 1.95 times of control group U.S. Ambion company); The MMP2mRNA expression level has then descended about 50%, 48% and 30% respectively than control group, the MMP9mRNA expression level has also descended about 74%, 77% and 38% respectively than control group; Transfection behind the miR-106a suppressor factor; The expression level of TIMP-2mRNA has been transfection respectively miR inhibitor negative control (article No. AM17010; About 1.2 times, 1.65 times and 1.35 times of control group U.S. Ambion company); The MMP2mRNA expression level has then descended about 52%, 48% and 28% respectively than control group, the MMP9mRNA expression level has also descended about 87%, 82% and 25% respectively than control group.And in transfection behind miRNA-20a suppressor factor and the miR-106a suppressor factor; The expression and the mRNA level of TIMP-2, MMP2 and MMP9 protein level are consistent; With transfection the control group of miR inhibitor negative control (article No. AM17010, U.S. Ambion company) compare significant difference (Figure 15) all arranged.
The Mechanism Study of 4miR-106a direct regulation and control TIMP-2
In order to study the whether expression of direct regulation and control TIMP-2 of miR-106a, we have made up the luciferase reporter gene carrier, adopt the luciferase reporter gene experiment to detect.
With Hind III and Spe I (Japanese TaKaRa company) double digestion pMIR-Reporter, pMD-TSimple-miR-20/106-TIMP2-WT and pMD-T Simple-miR-20/106-TIMP2-MUT plasmid; Agarose gel electrophoresis adopts glue to reclaim test kit (U.S. OMEGA company) glue and reclaims the big fragment of pMIR-Reporter plasmid, miR-20/106-TIMP2-WT and miR-20/106-TIMP2-MUT fragment; The big fragment of pMIR-Reporter plasmid that reclaims and miR-20/106-TIMP2-WT, miR-20/106-TIMP2-MUT fragment are connected with T4DNA ligase (Japanese TaKaRa company), and 4 ℃ are spent the night; To connect plasmid and transform DH5 α competent cell, it is dull and stereotyped that the shop contains penbritin LB, 37 ℃ of overnight cultures; After picking pMIR-miR-20/106-TIMP2-WT and 37 ℃ of incubated overnight of pMIR-miR-20/106-TIMP2-MUT mono-clonal bacterial strain, adopt plasmid a small amount of extraction agent box (U.S. OMEGA company) to carry out plasmid extraction; PMIR-miR-20/106-TIMP2-WT and pMIR-miR-20/106-TIMP2-MUT plasmid that extracting goes out are identified with Hind III and Spe I double digestion again.
Plasmid transfection and luciferase reporter gene experiment
Respectively with miRNA-20a suppressor factor and analogue body (miR-20a mimics; Article No. PM10057; U.S. Ambion company), miR-106a suppressor factor and analogue body (miR-106a mimics; Article No. PM12567, U.S. Ambion company) and pMIR-miR-20/106-TIMP2-WT, pMIR-miR-20/106-TIMP2-MUT and pRL-TK plasmid advance CD133 with Lipofectamine2000 (American I nvitrogen company) transfection reagent transfection
+Behind the cell 48h; Adopt the U.S. Dual-Luciferase Reporter Assay System of Promega company test kit to carry out the luciferase reporter gene experiment by its process specifications; Calculate the ratio of sea pansy fluorescence/Lampyridea fluorescence, analyze miRNA-20a and miR-106a regulating and controlling effect TIMP-2.
Shown in figure 16, the report carrier uciferase activity that contains wild-type TIMP-23 ' UTR has significantly reduced about 50% and 40% (P＜0.05) respectively than the uciferase activity of control vector and mutant TIMP-23 ' UTR report carrier.Transfection behind the suppressor factor of miRNA-20a and miR-106a; The report carrier uciferase activity that contains wild-type TIMP-23 ' UTR has significantly increased by about 50% and 33% (P＜0.05); Behind the analogue body of transfection miRNA-20a and miR-106a, the report carrier uciferase activity has then significantly reduced about 35% and 16% (P＜0.05).And the suppressor factor of miRNA-20a and miR-106a and analogue body are to the uciferase activity that contains mutant TIMP-23 ' UTR report carrier do not make significant difference (P＞0.05).
In sum: the discovery of the GSCs that exists in the glioblastoma helps us deeply to be familiar with the generation and the development process of this tumour, particularly the mechanism of GBM invasion and attack.Present GSCs can adopt stem cell surface affinity tag CD133 fluidic cell sorting technology and these two kinds of methods of the experiment of cell balling-up under special stem cell cultivation conditions from glioma cell, to separate and obtain.This group cell can be identified from aspects such as stem cell self ability, multidirectional differentiation potential and tumour formation abilities through testing in external and the body.GSCs has been found in the glioma vasculogenesis and has played an important role with producing in the conventional treatment opposing.In addition, GSCs also has very high invasive ability, possibly be the root of glioma recurrence.
Though the high invasion and attack of GBM cell characteristic possibly cause the failure of present treatment, it is still not fully aware of whether GSCs has participated in the invasion and attack of glioma.Therefore, the present invention separates with the fluidic cell sorting technology GSCs from people's glioma cell line U87 and former generation people samples of human glioma, under the stem cell cultivation conditions of serum-free, cultivate, and detected their invasive ability.The result confirms that it is CD133 that the invasive ability of GSCs is significantly higher than non-GSCs
-Cell, GSCs are the major reasons that invasion and attack take place glioma.
MiRNA is through (3 ' UTR) interact causes the synthetic of said target mrna degraded or arrestin matter, in the regulation and control of tumour cell oncogene and cancer suppressor gene, plays a significant role with target gene mRNA 3 ' non-translational region.The expression level of miRNA in various tumor tissues is obviously unusual; This abnormal expression and tumour cell comprise that the aspects such as propagation, apoptosis, invasion and attack, transfer and vasculogenesis of glioma cell are closely related, even also relevant with the pernicious biological characteristics of GSCs.Yet the effect of miRNA in the GSCs invasion and attack do not illustrated clear yet.
The expression of ripe miR-20/106a that the present invention has adopted miRNA chip and qRT-PCR technology for detection; Find miR-20a and miR-106a (abbreviation miR-20/106a) in GSC than the remarkable high expression level of glioma cell; The result has confirmed for the first time that miR-20a and miR-106a express in GSC increase, and prompting miR-20a and miR-106a possibly bring into play the effect of oncogene in GSC.
MiR-20a and miR-106a have the target gene of many predictions in different cells, why select this path of TIMP-2, MMP2 and MMP9 to be because they have vital role in the invasion and attack of tumour.Big quantity research is illustrated in the tumor invasion transfer, and most crucial steps is that tumour cell is broken through ECM and basement membrane.The ECM degraded of MMPs mediation is in keying action, and it is the major reason that invasion and attack take place tumour cell that MMPs destroys the basilar membrane integrity.The degradation process of ECM mainly relies on MMP2 and the MMP9 that the crucial moity IV Collagen Type VI of basilar membrane is had Degradation.MMP2 and MMP9 belong to the gelatin enzyme in the MMPs family.TIMP-2 is the natural inhibition of MMPs; TIMP-2 combines and suppresses the activity of MMPs with 1: 1 the covalency form and the MMPs of state of activation; Through suppressing the degraded of MMPs, thereby keep the integrity of basilar membrane, be considered to the supressor that one type of tumor invasion shifts ECM.But TIMP-2 comprises that at tumour cell the expression in the glioma cell is lower.
MiRNA can suppress target gene expression through translation of arrestin matter or degraded said target mrna.Therefore, the present invention adopts qRT-PCR and western blot technology to detect the expression whether miR-20a and miR-106a regulate and control TIMP-2.Can find out among the embodiment: after miR-20a and miR-106a were suppressed, TIMP-2 was that mRNA level or protein level all significantly increase, and the mRNA level of the MMP2 in its downstream and MMP9 and protein level all significantly reduce.Therefore, the result shows the expression that miR-20a and miR-106a can negativity regulation and control TIMP-2.Then, through research miR-20a and miR-106a whether can direct regulation and control TIMP-2 expression, adopt luciferase reporter gene to test and detect.Embodiment shows, after miR-20a and miR-106a were suppressed, the report carrier uciferase activity that contains wild-type TIMP-23 ' UTR significantly raise than control vector, and the report carrier of mutant TIMP-23 ' UTR is not then had influence.Therefore, the result has proved the expression that miR-20a and miR-106a can directly reduce TIMP-2 through the mode that combines with TIMP-23 ' UTR for the first time.
It is relevant with the invasive ability of miR-106a and GSCs that the present invention has observed miR-20a, finds that the downward modulation of miR-20a and miR-106a can suppress the invasive ability of GSCs significantly.Therefore, thus we have confirmed that for the first time miR-20/106a can promote the invasive ability of glioma through the expression of TIMP-2 among the direct downward modulation GSCs.
In sum, miRNA-106a possesses the ability of the application in preparation glioma stem cell invasion and attack promotor, and the suppressor factor of miRNA-106a possesses the ability of the application in preparation glioma stem cell invasion and attack suppressor factor.
Explanation is at last; Above embodiment is only unrestricted in order to technical scheme of the present invention to be described; Although through invention has been described with reference to the preferred embodiments of the present invention; But those of ordinary skill in the art should be appreciated that and can make various changes to it in form with on the details, and the spirit and scope of the present invention that do not depart from appended claims and limited.
Claims (9)
1.miRNA-106a the purposes in preparation glioma stem cell invasion and attack promotor.
2. purposes according to claim 1 is characterized in that: the purposes of miRNA-106a in preparation TIMP-2 suppressor factor.
3. purposes according to claim 1 is characterized in that: the purposes of miRNA-106a in preparation MMP2 promotor.
4. purposes according to claim 1 is characterized in that: the purposes of miRNA-106a in preparation MMP9 promotor.
5.miRNA-106a the purposes of suppressor factor in preparation glioma stem cell invasion and attack suppressor factor.
6. purposes according to claim 5 is characterized in that: said miRNA-106a is the suppressor factor of TIMP-2.
7. purposes according to claim 5 is characterized in that: said miRNA-106a is a MMP2 promotor.
8. purposes according to claim 5 is characterized in that: said miRNA-106a is a MMP9 promotor.
9. purposes according to claim 5 is characterized in that, the suppressor factor of said miRNA-106a is Anti-miRmiRNA-106a inhibitors.
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