CN102652837A - Medicament for treating colorectal cancer - Google Patents

Abstract

The invention relates to a medicament for treating colorectal cancers, which adopts small interfering RNA capable of inhibiting the expression of Gankyrin genes as an active component, and is prepared into injections or freeze-dried powder. The medicament prepared in the invention can inhibit the proliferation activity of colorectal cancer cells, block the growth cycle of colorectal cancer cells, induce apoptosis of colorectal cancer cells, significantly inhibit p53 protein ubiquitination and Rb protein phosphorylation, and has effective therapeutic effect on colorectal cancer transplantation tumors.

Description

A kind of medicine of treating colorectal cancer

Technical field

The present invention relates to a kind of medicine of treating colorectal cancer, relating in particular to a kind of active component is the medicine of the treatment colorectal cancer of siRNA.

Background technology

Colorectal cancer is the general name of colon cancer and rectal cancer; Be meant the malignant change that big epithelium of intestinal mucosa takes place under multiple carcinogenic factor effects such as environment or heredity; Be one of malignant tumor and modal malignant tumor of digestive tract of big epithelium of intestinal mucosa origin, poor prognosis, mortality rate are high.According to incompletely statistics, 2005, colorectal cancer became the deputy malignant tumor of sickness rate.

About incidence of colorectal age aspect data,, be main with 40 ~ 50 years old according to domestic statistics; But person below 40 years old accounts for about 1/3 of whole cases; And China's colorectal cancer sent out age ratio well abroad ahead of time 10 ~ 15 years old, and person below 30 years old accounts for 11% ~ 13%, especially; Along with going deep into of epidemiological study, the evidence of colorectal cancer and genetic correlation is more and more.Therefore, the treatment of colorectal cancer can not be ignored.

Early stage excision cancerous protuberance is a kind of effective ways of radical cure colorectal cancer, but cancer generally is difficult for early discovery, makes operating effect reduce greatly; The relapse rate of chemotherapy method is high, still has recurrence of 50% ratio and transfer after the treatment, and big to human harm; As the supplementary means of operation and chemotherapy, curative effect can not be satisfactory usually for radiotherapy; Tumor tissues can effectively killed and wounded and destroy to cryotherapy also, but can bring huge misery, needs to take the circumstances into consideration to use.

In order to study the medicine and the method for treatment colorectal cancer, people have paid a large amount of effort, and have obtained some achievements.Wherein, Chinese medicine such as patent CN1806841A disclose a kind of medicine that is used to treat gastric cancer, colorectal cancer and rectal cancer, adopt the dense extracting juice of Chinese crude drug slow fire Ao such as Radix Salviae Miltiorrhizae, the Rhizoma Pinelliae, Endothelium Corneum Gigeriae Galli to prepare; Western medicine such as patent WO2009/089900A1 disclose a kind of imidazole quinoline derivant, can regulate the treatment that local application carries out early stage colon cancer, rectal cancer; The disclosed pharmaceutical composition that is used to treat colorectal cancer of patent CN101292992A, the compositions that adopts 5-nitrogen deoxycytidine and rapamycin is as active component; The combination treatment of the disclosed a kind of colorectal cancer of patent WO2010/ 143986A1 adopts vitamin D-derivatives to carry out dietetic therapy, cytostatics, 5-fluorouracil and/or its precursor as Drug therapy.But conventional medicament is in the application of treatment colorectal cancer, or side effect is big, or weak effect, is difficult to satisfactory.

Along with development of biology; The medicine and the method for new treatment colorectal cancer have caused people's extensive concern; Patent JP2009-278976 (A) discloses a kind of gene and polypeptide that is used to treat colon cancer, mainly is that the gene and the polypeptide of human gene WDRPUH, LRZFPUH, PPILI and APCDD1 coding is used for detecting cytopathy and is used for preparing the medicine of treating colon cancer; Patent US2010/0284918A1 discloses a kind of treatment method for cancer and compositions, adopt to integrate the obtain medical treatment medicine of gastric cancer, colorectal cancer etc. of plain β 4, affinity reagent (like antibody) and carrier or diluent combined; Patent US2010/0310559A1 discloses a kind of recombinant monoclonal antibodies, forms mainly to comprise the CDR (complementary determining region) that contains following aminoacid sequence: SASSSISYMY, DTSKLAS, HQRDSYPWT, SKFGVN, VIWGDGSTSYNSGLIS, CVKPGGDY.

The discovery of RNA interference phenomenon is biological in the last few years most important progress; RNA perturbation technique (RNAi) be with endogenous mRNA coding region homologous small fragment external source double-stranded RNA transfered cell in; Cause the reticent a kind of technology of specific gene; Can be in mammal deactivation or reduce the expression of specific gene, produce the effect that is similar to gene knockout; As the possible treatment means of human diseasess such as the relevant lymphoma of HIV, AMD, the correlational study of RNA perturbation technique has got into I phase and II clinical trial phase, has opened up new approach for the treatment of entity tumor undoubtedly at present.And some reports are also arranged about the research of adopting RNA perturbation technique preparation treatment colorectal cancer medicine aspect; (" China digests 2006 the 3rd phases of journal " mediated rnai is led the reticent colorectal cancer cells that suppresses of transforminggrowthfactor-and grown " like people such as Ran Zhihua; 167 ~ 170 pages) reported double-stranded RNA (siRNA) transfection colorectal cancer HCT116 cell in the literary composition with small fragment TGF β 1, can suppress the expression of tumor cell TGF β 1; People such as Yuan Chaojun have reported in the literary composition that in " the RNA perturbation technique is to the inhibitory action of colorectal cancer cells human telomerase reverse transcriptase gene " (" 2009 the 4th phases of Chinese experimental surgery journal) targeting can reticent colorectal cancer cells hTERT gene of specificity and anticancer growing multiplication in the siRNA of hTERT, thus promotion colorectal cancer cells apoptosis; People such as Sun Haibo disclose pSilencer 3.0 H1 siRNA STAT3 recombiant plasmid in " RNA disturbs the influence of reticent STAT3 gene expression to the behavior of human colorectal cancer cytobiology " (" Chinese gerontology journal 2010 the 30th volume the 17th phase) propagation of colorectal cancer cells have been had the obvious suppression effect in the literary composition; The application of the disclosed a kind of siRNA of patent CN101606948A in preparation treatment colorectal cancer medicine, the siRNA that main employing can suppress mTOR gene expression is as active component.

But the pathogenic factor of colorectal cancer is also indeterminate, and therefore above-mentioned technology can not thoroughly solve the treatment difficult problem of colorectal cancer, and the further research of treating colorectal cancer medicine and method is remained important problem.

Summary of the invention

The invention provides a kind of medicine of treating colorectal cancer; Suppress Gankyrin (Gann ankyrin repeats through the RNA perturbation technique; Carcinous ankyrin repeat; Gene I: 5716) expression of gene, thereby curative effect and all relatively poor technical problem of prognosis of patients when solving the available technology adopting chemotherapy and treating the middle and advanced stage colorectal cancer provide a kind of new selection for the treatment colorectal cancer simultaneously.

The medicine of treatment colorectal cancer according to the invention, active component is one or more any mixture in the double-chain small disturbance RNA that can suppress carcinous ankyrin repeat (Gankyrin gene) and express; Perhaps active component is one or more any mixture in carrier, cell or the host bacterium that contains said siRNA; Or any mixture of said siRNA and said carrier, cell or host bacterium.Wherein, according to the inventionly can suppress Gankyrin gene expression, referring to said siRNA can combine with Gankyrin gene mRNA nucleotide, and cutting, degraded Gankyrin gene, and then suppresses said Gankyrin expression of gene; Said carrier, cell and host bacterium refer to the available carrier in present technique field, cell and host bacterium; Therefore, the implication of " can suppress Gankyrin gene expression " according to the invention and " carrier, cell and host bacterium " is clear and definite to those skilled in the art.

Wherein, in said siRNA, total length double-chain small disturbance RNA content is preferably >=and 97%, i.e. in the preparation process, said siRNA is purified to 97% ~ 100%.

Preferably, said siRNA be can with one or more any mixture in the bonded siRNA of Gankyrin gene mRNA; And further be preferably one or more any mixture since the bonded siRNA of the 313rd, 661 or 1381 site nucleotide; Especially be preferably from Gankyrin gene mRNA the 1381st site nucleotide and begin bonded siRNA.

SiRNA according to the invention is made up of positive-sense strand (Sense Strand) and antisense strand (Antisense Strand); Generally speaking, said positive-sense strand and antisense strand are 21 ~ 23 base length, and the present invention is preferably 21 bases.Wherein, 21 bases of said positive-sense strand are to be held 3 ' by the gene target sequence of 19 bases to add that the outstanding head of two bases obtains, and general siRNA justice catenary suspension head can be TT or UU (T is a thymus pyrimidine, and U is a uracil); 21 bases of said antisense strand and Gankyrin gene target complement sequence.

Preferably, the present invention treats the medicine of colorectal cancer, and active component comprises one or more the mixture in the following siRNA:

The positive-sense strand sequence is that SEQ ID NO:1, antisense strand sequence are the siRNA of SEQ ID NO:2;

The positive-sense strand sequence is that SEQ ID NO:3, antisense strand sequence are the siRNA of SEQ ID NO:4;

The positive-sense strand sequence is that SEQ ID NO:5, antisense strand sequence are the siRNA of SEQ ID NO:6.

Preferably, said siRNA carries out the modification of 2 '-methoxy, to increase siRNA stability.

Wherein, the dosage form of the medicine of above-mentioned treatment colorectal cancer is a freeze dried powder.

Said freeze dried powder medicine is dissolved in the solvent and can uses (as processing injection), and said solvent can be water, D/W, normal saline or DEPC water (being that pyrocarbonic acid diethyl ester is handled the also MiliQ level pure water of autoclave sterilization).Said D/W mass concentration is preferably 5% ~ 10%.

Cancer protein Gankyrin is positioned human chromosome Xq22.3, is made up of 226 aminoacid, forms 7 ankyrin repetitive sequences on the structure, and the encode protein molecule amount is 25 ~ 28KDa.Gankyrin can participate in the incidence and development of tumor through promoting cancer suppressor protein p53 ubiquitinization and Rb phosphorylation.Because antioncogene p53 and Rb bring into play central role in oncobiology, the inactivation of these two paths is essential condition that tumor forms, so Gankyrin can become the target spot of oncotherapy as p53 and the common down regulator of Rb.

The present invention uses the siRNA fragment that can suppress the Gankyrin gene as active component, has prepared a kind of medicine that can treat colorectal cancer.After siRNA is transfected into cell; Promptly start the RNA interference effect stage; SiRNA unwinds into positive-sense strand and antisense strand under the effect of intracellular rna unwindase, continue by antisense siRNA again with body in some enzymes (comprising restriction endonuclease, excision enzyme, unwindase etc.) combine to form the inductive reticent complex of RNA (RISC); RISC carries out specificity with the homology region of the mRNA of expression of exogenous genes and combines, and at the position cutting mRNA that holds 12 bases apart from 3 ' of siRNA.Fracture mRNA after being cut degrades immediately, thereby brings out the degradation reaction of host cell to these mRNA.SiRNA can not only guide RISC cutting homology strand mRNA; And can be used as primer and target RNA knot and be incorporated in the down synthetic more how new double-stranded RNA (dsRNA) of RNA polymerase (RdRP) effect; New synthetic dsRNA produces a large amount of secondary siRNA by the Cobra venom endonuclease in the kytoplasm (Dicer) cutting again; Thereby the effect of RNAi is further amplified, and said target mrna is degraded fully the most at last.

Through above-mentioned mechanism; The medicine of the treatment colorectal cancer of siRNA preparation of the present invention; Can suppress the colorectal cancer cells proliferation activity, retardance colorectal cancer cells growth cycle is induced the colorectal cancer cells apoptosis; Significantly suppress p53 albumen ubiquitinization (ubiquitination) and Rb protein phosphorylation, colorectal cancer transplanted tumor is had the efficacious therapy effect.

The RNA perturbation technique does not find to have toxic and side effects as yet simultaneously, so the drug safety of the present invention's preparation is better.

Description of drawings

Fig. 1 is the siRNA of the present invention Gankyrin protein expression testing result figure that mourns in silence;

Fig. 2 suppresses colorectal cancer cells proliferation activity testing result figure for siRNA1381 transfection of the present invention;

Fig. 3 induces colorectal cancer cells PARP albumen spliced body (89kDa) for siRNA1381 transfection of the present invention and forms testing result figure;

Fig. 4 raises the p53 protein expression and suppresses Rb protein phosphorylation testing result figure for siRNA1381 transfection of the present invention;

Fig. 5 suppresses p53 albumen ubiquitin testing result figure for siRNA1381 transfection of the present invention;

Fig. 6 suppresses nude mice colorectal cancer growth of xenografted testing result figure for siRNA1381 of the present invention;

Fig. 7 suppresses nude mice colorectal cancer transplanted tumor Gankyrin protein expression testing result figure for siRNA1381 transfection of the present invention.

The specific embodiment

The present invention treats the medicine of colorectal cancer, is freeze dried powder; Wherein active component especially begins bonded siRNA from Gankyrin gene mRNA the 313rd, 661,1381 site nucleotide for suppressing the double-chain small disturbance RNA of Gankyrin gene expression.

Said medicine can add available adjuvant, as in injection, adding entry, normal saline, 5% D/W, DEPC water equal solvent.

SiRNA positive-sense strand according to the invention and antisense strand all are preferably 21 bases, and positive-sense strand adds that at 3 ' end 2 UU obtain as outstanding head by the said gene target sequence of 19 bases; 21 bases of antisense strand and Gankyrin gene target complement sequence.

Through three pairs of siRNAs the present invention is described in detail below and describe, so that better understand content of the present invention, but following embodiment does not limit the scope of the invention.

Embodiment 1

From the molecular weight of the Gankyrin gene mRNA bonded siRNA of the 313rd site nucleotide (being designated as siRNA313) is 13320.74 (g/mole); Its positive-sense strand and antisense strand sequence (being designated as siRNA313-S and siRNA313-A respectively) be as follows:

siRNA313-S：GGUUGGUCUCCUCUUCAUATT（SEQ?ID?NO：1）；

siRNA313-A：UAUGAAGAGGAGACCAACCTG（SEQ?ID?NO：2）。

Embodiment 2

From the molecular weight of the Gankyrin gene mRNA bonded siRNA of the 661st site nucleotide (being designated as siRNA661) is 13331.72 (g/mole); Positive-sense strand and antisense strand sequence (being designated as siRNA661-S and siRNA661-A respectively) be as follows:

siRNA661-S：GCCUGGGUUUAAUACUCAATT（SEQ?ID?NO：3）；

siRNA661-A：UUGAGUAUUAAACCCAGGCCA（SEQ?ID?NO：4）。

Embodiment 3

From the molecular weight of the Gankyrin gene mRNA bonded siRNA of the 1381st site nucleotide (being designated as siRNA1381) is 13275.71 (g/mole); Positive-sense strand and antisense strand sequence (being designated as siRNA313-S and siRNA313-A respectively) be as follows:

siRNA1381-S：GAAUGUCAAGCUUGUUAAATT（SEQ?ID?NO：5）；

siRNA1381-A：UUUAACAAGCUUGACAUUCTT（SEQ?ID?NO：6）。

Said three kinds of siRNA are synthetic and production according to the ISO9000 standard, and HPLC is purified to the double-stranded siRNA content of total length 97% ~ 100%; In order to increase stability, above-mentioned three kinds of siRNA can also carry out 2 '-methoxy and modify.Above-mentioned siRNA is processed freeze dried powder ,-20 ℃ of preservations.Processing injection with dissolution with solvents when freeze dried powder uses gets final product.

 

1. experiment in vitro

Experiment material:

Human large intestine cancer cell line HCT116, SW1116, SW480, HT29 (all available from cellular biochemical institute of the Chinese Academy of Sciences); RPMI1640 culture medium, McCOY ' S 5A culture medium (all available from U.S. Sigma company);

Above-mentioned three kinds of siRNA (seeing table 1), and negative control (Shanghai JiMa pharmacy Technology Co., Ltd is synthetic);

DharmaFECT 4 (T-2004-03), DharmaFECT 1 (T-2004-03) siRNA transfection reagent, 5 times of dilution siRNA buffer solution (all available from Dharmacon company); Triton X-100, propidium iodide (Sigma company); RNase A (Roche company); CCK-8 test kit (Japanese colleague's chemistry institute); Immunoprecipitation is with Protein A/G PLUS-Agarose (sc-2003, Santa Cruz Biotechnolog company); The two staining cell apoptosis detection kit (Beckman company) of Annexin V/PI; Test used antibody and see table 2;

Chemiluminescence detection is used test kit: Super Signal West Femto Maximum Sensitivity Substrate (34095); Super Signal West Pico Chemilu-minescent Substrate (34077) (Pierce company);

Cell culture consumptive material (Greiner company); Pvdf membrane Milipore (Bedford company); 8%SDS PAGE, 12%SDS PAGE (PIERCE company); ELIASA Microplate Reader, Model 550 (Bio Rad company); Flow cytometer EPICS-XL (Beckman Coulter company); The Mini Protein of protein electrophoresis system II (BIO-RAD company); Superclean bench YJ-875 (Suzhou cleaning equipment company); CO2 gas incubator SHELLAB MODEL2300 (Sheldon Manufacturing Inc company); Low temperature supercentrifuge Eppendorf Centrifuge 5417R.

The implication of table 2 Chinese and English abbreviation is following:

CST:Cell Signaling Tech company;

WB: Western blotting experiment (Western blot);

IHC: immunohistochemical assay (Immunohistochemistry);

IP: immunoprecipitation experiment (Immunoprecipitation).

External siRNA transient transfection and Gankyrin gene silencing effect measuring

Cell culture:

The culture medium of HCT116, HT29 cell is a McCOY ' S 5A MEDIUM complete culture solution (10% hyclone), and the culture medium of SW1116, SW480 cell is RPMI1640 complete culture solution (10% hyclone), and condition of culture is 95% air, 5%CO ₂, 95% humidity, 37 ℃ of constant temperature.

The siRNA transient transfection:

The blank group is set respectively, and the transfection reagent matched group (Vehicle Control, VC), negative control group (Negative Control, NC), siRNA interference group.

Become single cell suspension with 0.25% trypsin digestion and cell, be inoculated in the Tissue Culture Dish of diameter 6cm, cultivate 20h, make the cell fusion degree reach 50%.Get the siRNA 80 μ l of 20uM, 1 times of siRNA Buffer (buffer solution), 720 μ l, 800 μ l serum-free mediums, totally 1600 μ l, mixing, room temperature was placed 5 minutes, dissolving siRNA.

With the same method dissolving negative control (NC) sequence.

HCT116, HT29 cell are with DharmaFECT 4 transfections, and SW1116, SW480 cell are with DharmaFECT 1 transfection.

Get DharmaFECT 1/ DharmaFECT 4 totally 192 μ l (16 * 12), add 4608 μ l (384 * 8) serum-free mediums, totally 4800 μ l, mixing, room temperature was placed 5 minutes.DharmaFECT 1/ DharmaFECT 4 is mixed with the siRNA equal-volume, and room temperature was placed 20 minutes.Abandon the liquid in the culture dish, add the complete culture solution that 3.2ml contains serum with PBS washing back, the mixture of 800 μ l DharmaFECT 1/4 and siRNA, making the siRNA final concentration is 100nM.The transfection reagent matched group only adds DharmaFECT 1/ DharmaFECT 4, final concentration identical with RNA interference group (4 μ l/ml).Continue to detect Gankyrin albumen behind the cultivation 48h by normal condition.

The extracting of total protein of cell:

Preparation cell lysis buffer solution (RIPA buffer): 150mM NaCl, 1% NP-40,0.5% NaTDC, 50 mM Tris-HCl (pH value 8.0).With preceding adding PMSF, protease inhibitor, phosphorglase inhibitor, with the frozen cell mass of RIPA lysis buffer suspension, 0.1ml/ (1 * 10 ⁶~ 1 * 10 ⁷) cell; Vortex 30s leaves standstill 5min on ice, repeats 3 times; 4 ℃, centrifugal 4 minutes of 14000rpm rotating speed is drawn supernatant, and Coomassie brilliant blue method (Bradford method) protein quantification test kit carries out protein quantification.

Western?blot：

Preparation HEPES electrophoretic buffer: HEPES 23.8g, Tris 12.1g, SDS 1.0g adds deionized water dissolving, is settled to 1000ml.

Preparation Tris-glycine electrotransfer buffer (pH value 8.3): Tris 3.03g, glycine 14.4g, methanol 200ml adds deionized water dissolving, is settled to 1000ml.

The preparation confining liquid: calf serum and TBS solution (deionized water is settled to 1000ml for Tris 12.1g, NaCl 9g, regulates pH to 7.5) are made into the confining liquid that contains 10% calf serum with 1: 9 ratio.

Kind buffer solution (loading buffer) in protein sample after quantitatively and the 5 times of dilutions is mixed; In 100 ℃ of heat denatured 3 ~ 5min, simple centrifugal back order adds in the well, arrives the separation gel bottom with 150V voltage electrophoresis until bromophenol blue; Take out gel; An application of sample amount and electrophoresis situation that is used for coomassie brilliant blue staining with observation sample wherein, another piece is used to change film, balance 10 ~ 15min in glycine electrotransfer buffer.Cut out size and gel phase 6 filter paper and 1 pvdf membrane together.Pvdf membrane soaks with methanol pretreatment 15s, is soaked in Tris-glycine electrotransfer buffer jointly with filter paper.The one side of gel is contacted with filter membrane, be sandwiched between 6 filter paper, neatly be stacked in the electrotransfer device, pvdf membrane is near anode one side.Add Tris-glycine electrotransfer buffer, on ice with 0.65mA/cm ²Current intensity Constant Electric Current transferase 12 ~ 3h, pvdf membrane in confining liquid 4 ℃ spend the night, discard confining liquid, (1ml) vibration was washed film 5 minutes to TTBS for 999mlTBS, Tween-20, and with Gankyrin one anti-incubated at room 2 ~ 3h, TTBS concussion is washed film 3 times, each 15min; With two anti-incubated at room 1 ~ 2h of horseradish peroxidase (HRP) labelling, the TTBS concussion is washed film 3 times, each 15min.Carry out chemiluminescence with the ECL test kit, in the darkroom with A, B luminescent solution equal proportion diluted mixture.Film is with deionized water rinsing a little, and filter paper pastes the angle and blots, and anti-subsides method is overlying on A, the B mixed liquor, places the preservative film internal fixation in film magazine, covers film rapidly, closes the glue box, makes public according to the finding fluorescence intensity.Take out film and immerse 1 ~ 2min in the developer solution immediately fully, be placed on after the clear water rinsing in the fixative solution to the complete photographic fixing of egative film, clear water washes down and dries, and demarcates Marker, analyzes and scans.

Use liposome transfection human large intestine cancer cell line HCT116, HT29, SW1116, SW480; Use Western blot method behind the transfection 48h and detect the Gankyrin protein expression; With internal reference Tubulin protein band density is standard, carries out the sxemiquantitative of Gankyrin protein band density.Among Fig. 1, A is the testing result of human large intestine cancer cell line SW1116, and B is the testing result of human large intestine cancer cell line SW480, and C is the testing result of human large intestine cancer cell line HCT116, and D is the testing result of human large intestine cancer cell line HT29.Can know by Fig. 1, all can significantly suppress the Gankyrin protein expression of four colorectal carcinoma cell lines to the siRNA fragment (siRNA313, siRNA661, siRNA1381) of 3 Gankyrin gene target sites, the most obvious with the effect of siRNA1381.(Mock) compares with the transfection reagent matched group, and siRNA1381 effect group Gankyrin expressing quantity reduces more than 90%.

Experiment detects cell viability and changes

Cell inoculation:

Cell culture is to growing into monolayer, and with 0.125% trypsinization, to contain McCOY ' the S 5A culture medium furnishing single cell suspension of 10% hyclone, concentration is 2 ' 10 ⁴Individual/ml, be inoculated in 96 well culture plates by 100 μ l/ holes.

Transfection:

Inoculate to inhale after 24 hours and remove culture fluid; Add 200 μ l complete culture solutions, SiRNA concentration 100nM wherein, DharmaFECT 1/4 concentration 4 μ l/ml; Solvent control group (Vehicle Control) only adds DharmaFECT 1/4, and the blank group adds complete culture solution.Place old terms to continue to cultivate 24h, 48h, 72h respectively.Establish 5 secondary holes for every group.Every group of experiment triplicate.

Colour developing: every hole adds CCK-8 solution 20 μ l, places 37 ℃ to continue to cultivate 1 ~ 4h.ELIASA is measured the OD value at 450 nm places.The cell viability computing formula is following:

Cell viability (%)=[A (dosing)-A (blank)]/[A (0 dosing)-A (blank)] * 100

Wherein:

A (dosing): absorbance with hole of cell, CCK-8 solution and siRNA solution;

A (blank): have culture medium and CCK-8 solution and do not have the absorbance in the hole of cell;

A (0 dosing): have cell, CCK-8 solution and do not have the absorbance in the hole of siRNA solution.

The test triplicate, data show with

± SD.(NC) compares with negative control group, * P<0.05, * * P<0.01.

Fig. 2 has shown that the siRNA1381 transfection suppresses the testing result figure of colorectal cancer cells proliferation activity.Use liposome transfection siRNA1381 the pure man colorectal carcinoma cell line, 24h, 48h, 72h use CCK-8 kit detection cell vigor after the transfection.Wherein, Fig. 2 A is the testing result of human large intestine cancer cell line SW1116, and Fig. 2 B is the testing result of human large intestine cancer cell line HCT116.Can know by Fig. 2; Compare with the NC group; Transfection siRNA1381 (100nM) back 48h can significantly suppress SW1116 cell and HCT116 cell viability, and (the siRNA1381 group is 64.88 ± 10.7 vs. 93.41 ± 4.914 with the contrast of NC group SW1116 competent cell, and P < 0.01; HCT116 competent cell contrast is 70.5656 ± 8.68 vs. 96.18 ± 3.8, P 0.01), and 72h to the transfection, its effect is more obvious, and (it is 50.775 ± 11.05 vs. 87.91 ± 9.42 that the SW1116 competent cell contrasts, and P < 0.01; The contrast of HCT116 competent cell is 60.737 ± 9.9 vs. 92.797 ± 6.8, P 0.01).

The Flow cytometry cell cycle

The exponential phase cell is cultivated synchronizing them of 24h with serum-free medium.

SiRNA transient transfection, final concentration are 100nM; If negative sequence contrast, blank.

Continue in the complete medium to cultivate 48h, with 0.125% trypsinization collecting cell, the PBS washing after 4 ℃ of dehydrated alcohol are fixing, processes about 10 ⁶The single cell suspension of individual/ml is hatched 10min jointly with the Tris-HCl buffer that contains the 1%RNA enzyme (pH value 7.4).After propidium iodide (PI) dyeing, carry out flow cytometer and detect, according to the cell distribution maps of different relatively dna contents, the percentage rate of each cycle cell of match.3 samples of every group of repetition measurement, the test triplicate.Data show with

± SD.(NC) compares with negative control group, * P < 0.01.

The result shows: 48h behind the transfection siRNA1381; Compare with the NC group; Transfection siRNA1381 group G2/M phase cell quantity significantly increases; Explain that (the siRNA1381 group is 23.667 ± 2.75 vs. 5.965 ± 1.742 with NC group G2/M phase SW1116 cell ratio to siRNA1381 in the G2/M phase with SW1116, HCT116 cell cycle arrest P<0.01; HCT116 cell ratio is 22.375 ± 4.005309 vs. 7.57 ± 1.221, P<0.01).Compare simultaneously with the NC group, 48h behind the transfection siRNA1381, SubG0G1 phase cell quantity significantly increase (SW1116 cell ratio is 10.66 ± 3.782 vs. 1.58 ± 0.358, P<0.01; HCT116 cell ratio is 36.12 ± 5.47 vs. 1.807 ± 0.175, P<0.01), explain that siRNA1381 can bring out apoptosis.

Two dyeing, poly polymerase PARP detect apoptosis

With siRNA1381 (100nM) and negative control sequence (100nM) transfection SW1116, HCT116 cell, after the transfection 48h in 4 ℃ with centrifugal 5 minutes collecting cells of 1300rpm/min; The PBS washed cell of 1ml pre-cooling; Add 200 μ l Binding buffer suspension cells, add 10 μ l FITC-Annexin-V and 5 μ l PI, incubation is 15 ~ 30 minutes under the lucifuge room temperature; Get the cell precipitation smear, coverslip sealing, microscopy immediately.Viable apoptotic cell presents green fluorescence (being shown as white among Fig. 3 A), and the non-viable apoptotic cell film is green fluorescence (being shown as white among Fig. 3 A), examines the fluorescence (the darker part of white area color among Fig. 3 A) that takes on a red color.Detect to find, the experimental group of transfection siRNA1381, but the morphological change of significant apoptosis takes place in inducing cell after 48 hours.

PARP (poly ADP-ribose polymerase) be first certified in apoptosis by the proteolysis substrate of Caspase-3 and the degraded of other cysteine proteinases and foot sign property.The proteolysis of PARP is considered to an apoptotic early molecule sign as the activated molecular events of cysteine proteinase.With the cell harvesting of 48h after the transfection, and the extracting total protein of cell, to use Western blot method and detect PARP albumen spliced body (89kDa), the concrete operations step is said with preamble, and used antibody is as shown in table 2.

Fig. 3 A is the testing result of human large intestine cancer cell line SW1116 transfection negative control sequence; Fig. 3 B is the testing result of human large intestine cancer cell line SW1116 transfection siRNA1381; By finding out among the figure, compare with NC group, cell apoptosis quantity in early stage and late period obviously increases behind the transfection siRNA; The testing result figure that Fig. 3 C induces colorectal cancer cells PARP albumen spliced body (89kDa) to form for the siRNA1381 transfection; 48h behind the transfection siRNA1381; The ratio of PARP albumen spliced body (89kDa) significantly increases; And the PARP of 116kDa is cut into the fragment of 85 ~ 89kDa by caspase-3, is apoptotic key character.

Gene silencing is to the influence of p53 and RB protein content

Use the variation of Western blot method detection Gankyrin gene silencing front and back p53 and RB protein content, the concrete operations step is the same.With siRNA1381 (100nM) and negative control siRNA (100nM) transfection HCT116, SW1116 cell, extracting total protein of cell behind the 48h detects p53, pan-Rb, pho-Rb with Western blot ^Ser780, with GAPDH as internal reference.Testing result is seen Fig. 4, and Fig. 4 A is the testing result of HCT116 cell; Fig. 4 B is the testing result of SW1116 cell.

Can know that by Fig. 4 the siRNA1381 transfection can significantly increase the p53 protein level, reduce Rb protein phosphorylation level (pho-Rb), not change total Rb protein content.Because p53 and Rb are antioncogene; P53 is often by the ubiquitin degraded in the tumor generating process; RB albumen by phosphorylation modification after inactivation, therefore, the reduction of the increase of p53 protein level and Rb protein phosphorylation level can make the cancer suppressing action of p53 and Rb strengthen behind the Gankyrin gene silencing.

Gene silencing is to the influence of p53 ubiquitin degraded

The change of p53 ubiquitin level before and after the Application of I P-Western blot technology for detection Gankyrin gene silencing, Western blot concrete operations step is the same, and the IP operating procedure is following:

With reference to 1.1, RIPA method extracting total protein of cell.Prepare Protein A/G agarose Beads, PBS washes twice, and is diluted to 50% concentration.Add 100 μ l Protein A/G agarose beads (50%) in every 1ml total protein, 4 ℃ of horizontal shaking table 10min to remove non-specific foreign protein, reduce background.

4 ℃, the centrifugal 15min of 14000g transfers to supernatant in the new centrifuge tube, removes Protein A/G agarose Beads; The Bradford method is done the protein standard curve, measures protein concentration.

With PBS dilution total protein to 1 μ g/ μ l.Add the anti-human P 53 of 15 μ l rabbits antibody to 500 μ l total protein, 4 ℃ of slow shaken over night; Add 100 μ l Protein A/G agarose Beads capture antigen antibody complexes, 4 ℃ of slow shaken over night; The centrifugal 5s of 14000rpm collects sepharose 4B-antigen antibody complex, removes supernatant, washs 3 times with the RIPA buffer of pre-cooling; With 2 times of dilutions of 60 μ l sample-loading buffer sepharose 4B-antigen antibody complex is hanged, mixing boils 5min, and is centrifugal, collects supernatant, continues to use Western Blot method and detects ubiquitin (Ubiquitin).

Testing result is seen Fig. 5, and Fig. 5 A is the testing result of HCT116 cell, and Fig. 5 B is the testing result of SW1116 cell.The amount of p53-Ub (Ubiquitin) complex can be significantly lowered in the siRNA1381 transfection, increases the proteic total amount of p53, and the degraded of p53 ubiquitin reduces the increase of p53 protein level behind the prompting Gankyrin gene silencing.Because p53 is an antioncogene, the minimizing of p53 ubiquitinization and the increase of protein content can strengthen its cancer suppressing action.

Research in the body

Experiment material: laboratory animal: the BALB/c nude mice, male, in 4 ages in week, inbred line available from zoopery base, Chinese Academy of Sciences Songjiang (SCXK (Shanghai) 2003-0003), is raised in the IVC of Medical College, Shanghai Communication Univ. Animal Lab. (SYXK (Shanghai) 2003-0007);

SiRNA1381 shown in the siRNA application table 1; Negative control (Negative Control) sequence is carried out methoxy by Shanghai Ji Ma company and is modified, and chemosynthesis;

Transfection reagent: in vivo-jetPEI (201-50G, French Alsaca Polyplus Transfection company); Test used antibody and see table 2; TUNEL method apoptosis test regent box: Roche, 1684809a; 50 μ l microsyringes: the sky, Shanghai is Science and Technology Ltd.; Slide gauge: Shanghai precision instrumentation company limited; Ultrasonic cell disruptor (JY92-II): NingBo XinZhi Biology Science Co., Ltd; Paraffin slicing machine (RM21459), embedding machine (EG1140H), automatic staining machine: Leica company.

Transfection suppresses the growth of human large intestine cancer transplanted tumor in nude mice model

Set up human large intestine cancer transplanted tumor in nude mice model: recovery human large intestine cancer cell strain SW1116, going down to posterity is cultured to the third generation, with 0.25% trypsin digestion and cell, centrifugal collection, with the PBS washed twice, suspending is that single cell suspension, density are 1 * 10 ⁸Individual/ml, it is subcutaneous to be inoculated in nude mice left side armpit with microsyringe, and 100 μ l/ only.1 week of inoculation back forms macroscopic cancerous node, the starting rna i experiment in the 10th day of inoculation back.Laboratory animal is divided 3 groups, and 6 every group, as follows:

Solvent control group (Vehicle control group, BC/Mock): intratumor injection in vivo-jetPEI;

Negative sequence matched group (Negative control group, NC): intratumor injection Negative Control siRNA;

SiRNA1381 interference group (siRNA-Gank group): intratumor injection siRNA1381.

The siRNA transfection: the D/W of 5% mass concentration dissolves in vivo-jetPEI, siRNA, siRNA1381 respectively; In vivo-jetPEI after the dissolving is added siRNA or siRNA1381; The ratio of in vivo-jetPEI and siRNA or siRNA1381 is 1.6 μ l/10 μ g, and cumulative volume is 30 μ l.Room temperature leaves standstill 15min behind the vortex mixing.In the laminar flow superclean bench, divide 3 injections with aseptic microsyringe in tumor by local, 10 μ l/ points.3 groups of nude mice difference intratumor injection in vivo-jetPEI (1.6 μ l/30 μ l/, the BC group), siRNA _NC(10 μ g/30 μ l/, the NC group), siRNA1381 (10 μ g/30 μ l/, the RNAi group).Repeated aforesaid operations once at a distance from two days, cotransfection 6 times, the 2nd day execution laboratory animal after the last transfection.

The gross tumor volume measuring and calculating

Use slide gauge and measure tumor maximum gauge (a) and minimum diameter (b) respectively at the 0th, 4,8,12,16 day of RNAi experiment; Calculate gross tumor volume, data are represented (n=6) with

± SD; The gross tumor volume computing formula is following:

Can know that by Fig. 6 siRNA1381 transfection group growth of tumor speed significantly is lower than transfection reagent matched group (BC) and negative sequence matched group (NC).After the transfection the 16th day, the gross tumor volume of siRNA1381 transfection group (594.3481 ± 87.85729) significantly less than NC matched group (1602.757 ± 227.4018) ( P<0.01) and the BC matched group (1559.264 ± 214.1224, P<0.01), no significant difference between NC and BC matched group.It is thus clear that siRNA1381 can significantly suppress nude mice colorectal cancer growth of xenografted.

Transfection is to the influence of tumor tissues Gankyrin protein expression

Extracting tumor tissues total protein: the observation period finishes the back and adopts the cervical vertebra dislocation method to put to death and the anatomy experiment animal, obtains tumor tissues, removes slough, in liquid nitrogen, it is pulverized into powder.In the RIPA lysis buffer of pre-cooling on ice, every 300mg sample adds the 1ml lysate with powder transfer, and maximum (top) speed homogenate is 30 seconds in the homogenizer.Homogenate is put on ice, shaken 20min on the shaking table, the centrifugal 5min of 150g shifts supernatant to new centrifuge tube.4 ℃ of recentrifuge 30 min of 18000rpm get supernatant and measure protein concentration.

Western blot: the Western blot step of describing according to preamble detects the variation of tumor tissues Gankyrin protein level, with GAPDH as internal reference; Used antibody is as shown in table 2.Testing result figure is as shown in Figure 7.

The SABC method detects tumor tissues Gankyrin protein level: to the laboratory observation terminal point, put to death laboratory animal with the cervical vertebra dislocation method, dissect and peel off tumor, clean with the PBS buffer, remove slough, with the neutral formalin fixed overnight; According to conventional method draw materials, dehydration, FFPE, 4 μ m serial section, 70 ℃ of baking 2h; Paraffin section is with xylene dewaxing 2 times, 10min/ time.

Gradient ethanol aquation (100%, 95%, 75%, 50%), 5min/ time, distilled water rinsing 5min; Contain 0.01M PBS (pH value 7.4) rinsing 2 times of 0.1%Tween-20,5min/ time, drip the hydrogen peroxide methanol solution eliminating endogenous peroxidase activity, incubated at room 20min, the distillation washing, PBS soaks 2 times, 5min/ time; 0.01mol/L citrate buffer solution (pH value is 6.0) microwave is repaired 10min, is cooled to room temperature, distillation washing 2 times, and PBS washes 2 times; 3min/ time, drip sealing with two anti-host's serum, incubated at room 20min, serum deprivation inclines; Do not wash, drip an anti-working solution, 37 ℃ of incubation 1h.PBS flushing 3 times, 5min/ time; It is anti-to drip biotin labeling two, 37 ℃ of incubation 30min; PBS flushing 3 times, 5min/ time; Drip .37 ℃ of incubator 30min of ABC complex; PBS flushing 3 times, 5min/ time; Drip freshly prepared BCIP/NBT liquid lucifuge colour developing 5 ~ 30min, microscopically is observed to occurring positive painted; Distilled water is fully washed and is stopped dyeing, and the nuclear fast red is redyed karyon, and distilled water is fully washed; Gradient ethanol (50%, 75%, 95%, 100%) dehydration, 5min/ time; Transparent 2 times of xylene, 10min/ time; Dry back neutral gum mounting; Application OLYMPUS 1X51 inverted microscope is observed and is taken pictures.The original image amplification: 400 *.

Fig. 7 testing result shows; Compare with matched group; The siRNA1381 transfection can significantly reduce the proteic expression of Gankyrin in the nude mice colorectal cancer transplanted tumor tissue, no significant difference between each matched group, and promptly using siRNA1381 in vivo is the effective means that can suppress the Gankyrin protein expression.The SABC testing result has also confirmed this conclusion simultaneously.

2.3. TUNEL method in situ detection apoptosis

Place color jar, xylene to embathe 2 times paraffin organization section, each 5min, 100% ethanol embathes 2 times, respectively embathes once with 95%, 85%, 70% and 50% ethanol successively, at every turn 3min; PBS embathes 5min, places 4% paraformaldehyde solution or contains fixing 15 min of PBS solution of 10% formalin, and PBS embathes secondary, each 5 min; The microscope slide taking-up is placed horizontal surface, unload the unnecessary liquid on the slide with careful suction of filter paper, the E.C. 3.4.21.64 working solution that drips the new preparation of 100 μ l covers on the sample areas; Room temperature held 10 ~ 30min, PBS embathes 5min then, places 4% paraformaldehyde solution again or contains fixing 5 min of PBS solution of 10% formalin; Embathe secondary with PBS, each 5min unloads the unnecessary liquid on the slide with careful suction of filter paper; The Equilibration Buffer that drips 100 μ l is on sample areas, and equilibrium at room temperature 5 ~ 10min is during the microscope slide balance; Biotin-11-dUTP put on ice thaw, cofabrication TdT enzyme reaction solution is preserved for use on ice.

Remove sample areas unnecessary liquid on every side with careful suction of filter paper, the TdT enzyme reaction solution of Dropwise 50 μ l is evenly distributed to guarantee reactant liquor in the conversion zone covered on sample areas; In 37 ℃, wet environment, react 60 ~ 90min, remove coverslip, adds 2 times and dilute SSC solution 50 μ l cessation reactions; Room temperature is placed 15min; PBS embathes three times, each 5min, the H with 0.3% ₂O ₂Embathe 3 ~ 5min, PBS embathes three times then, each 5min; The Streptavidin-AKP solution that drips 100 μ l dilution is on sample areas, and room temperature reaction 3-5min, PBS embathe three times; Each 5min is in the sample areas dropping BCIP/NBT of microscope slide working solution 100 μ l, up to presenting light blue background; Rinsed with deionized water 2 times is redyed with the nuclear fast red; Rinsed with deionized water 2 ~ 3 times; Successively with 70%, 90%, 100% ethanol embathe, each 3min, 100% ethanol embathes twice, xylene embathes fixing, observes under the resin mounting, optical microscope and takes pictures, blueness is an apoptotic cell.

5 visuals field are got in every section, and Application of I mage Pro Plus 5.0 software calculate positive cell rate.

Detect and find; Compare with matched group; SiRNA1381 transfection group apoptosis rate significantly increases, and explains that the siRNA1381 transfection can induce nude mice colorectal cancer transplanted tumor tissue that significant apoptosis takes place, and siRNA1381 can suppress nude mice colorectal cancer growth of xenografted through cell death inducing.

Can find out that through above-mentioned experiment the freeze dried powder medicine that the present invention can suppress the siRNA preparation of Gankyrin gene expression can effectively suppress the growth of colorectal cancer transplanted tumor.

In the foregoing of the present invention, measurement unit nM refers to 10 ^-9Mol/L; MM refers to 10 ^-6Mol/L; KDa is kilodalton (Dalton).

More than specific embodiment of the present invention is described in detail, but it is just as example, the present invention is not restricted to the specific embodiment of above description.To those skilled in the art, any equivalent modifications that the present invention is carried out with substitute also all among category of the present invention.Therefore, not breaking away from impartial conversion and the modification of being done under the spirit and scope of the present invention, all should contain within the scope of the invention.

SEQ?ID

 

< 110>The three people Hospital, Shanghai Jiaotong University School of Medicine

 

< 120>a kind of medicine of treating colorectal cancer

 

 

<160> 6

 

<170> PatentIn?version?3.3

 

<210> 1

<211> 21

<212> RNA

< 213>artificial sequence

 

<220>

< 223>siRNA positive-sense strand

 

<400> 1

gguuggucuc?cucuucauat?t 21

 

 

<210> 2

<211> 21

<212> RNA

< 213>artificial sequence

 

<220>

< 223>siRNA antisense strand

 

<400> 2

uaugaagagg?agaccaacct?g 21

 

 

<210> 3

<211> 21

<212> RNA

< 213>artificial sequence

 

<220>

< 223>siRNA positive-sense strand

 

<400> 3

gccuggguuu?aauacucaat?t 21

 

 

<210> 4

<211> 21

<212> RNA

< 213>artificial sequence

 

<220>

< 223>siRNA antisense strand

 

<400> 4

uugaguauua?aacccaggcc?a 21

 

 

<210> 5

<211> 21

<212> RNA

< 213>artificial sequence

 

<220>

< 223>siRNA positive-sense strand

 

<400> 5

gaaugucaag?cuuguuaaat?t 21

 

 

<210> 6

<211> 21

<212> RNA

< 213>artificial sequence

 

<220>

< 223>siRNA antisense strand

 

<400> 6

uuuaacaagc?uugacauuct?t 21

Claims (10)

1. a medicine of treating colorectal cancer is characterized in that, said active constituents of medicine is for suppressing one or more any mixture in the double-chain small disturbance RNA of Gankyrin gene expression, the carrier that contains said siRNA, cell or the host bacterium.

2. medicine according to claim 1 is characterized in that, in the said siRNA, and total length double-chain small disturbance RNA content >=97%.

3. medicine according to claim 1 is characterized in that, said siRNA be can with one or more any mixture in the bonded siRNA of Gankyrin gene mRNA.

4. medicine according to claim 3 is characterized in that, said siRNA is for beginning one or more any mixture the bonded siRNA from Gankyrin gene mRNA the 313rd, 661 or 1381 site nucleic acid.

5. medicine according to claim 4 is characterized in that, said siRNA positive-sense strand and antisense strand are 21 bases.

6. medicine according to claim 5 is characterized in that, said siRNA justice catenary suspension head is UU or TT, 21 bases of said antisense strand and Gankyrin gene target complement sequence.

7. medicine according to claim 1 is characterized in that, said active constituents of medicine comprises one or more the mixture in the following siRNA:

The positive-sense strand sequence is that SEQ ID NO:1, antisense strand sequence are the siRNA of SEQ ID NO:2;

The positive-sense strand sequence is that SEQ ID NO:3, antisense strand sequence are the siRNA of SEQ ID NO:4;

The positive-sense strand sequence is that SEQ ID NO:5, antisense strand sequence are the siRNA of SEQ ID NO:6.

8. according to above-mentioned any described medicine of claim, it is characterized in that said siRNA carries out 2 '-methoxy and modifies.

9. medicine according to claim 1 is characterized in that, the dosage form of said medicine is freeze dried powder or injection.

10. medicine according to claim 9 is characterized in that, said injection solvent is one or more the mixture in water, normal saline, 5% D/W, the DEPC water.

Images