CN104667299A - Application of double-stranded RNA and anti-tumor composition related to double-stranded RNA in preparation of tumor cell inhibitor - Google Patents
Application of double-stranded RNA and anti-tumor composition related to double-stranded RNA in preparation of tumor cell inhibitor Download PDFInfo
- Publication number
- CN104667299A CN104667299A CN201510091139.6A CN201510091139A CN104667299A CN 104667299 A CN104667299 A CN 104667299A CN 201510091139 A CN201510091139 A CN 201510091139A CN 104667299 A CN104667299 A CN 104667299A
- Authority
- CN
- China
- Prior art keywords
- stranded rna
- double
- tumor
- anticc
- ome
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Abstract
The invention discloses an application of double-stranded RNA and an anti-tumor composition related to double-stranded RNA in preparation of a tumor cell inhibitor. Active components of the anti-tumor composition consist of A and chemotherapy medicines, wherein A is double-stranded RNA, or a modifier of double-stranded RNA, or a biomaterial related to double-stranded RNA; the name of double-stranded RNA is AntiCC, the sequence of one chain of double-stranded RNA is SEQ ID No. 1 in a sequence table, the sequence of the other chain is SEQ ID No. 2 in the sequence table, and SEQ ID No. 1 and SEQ ID No. 2 are reversely complementary; and the modifier of double-stranded RNA is obtained by modifying 2'-OH of ribose of double-stranded RNA. Proven by experiments, by adopting the anti-tumor composition and AntiCC disclosed by the invention, the tumor cell proliferation can be inhibited, and/or the tumor cell apoptosis can be promoted.
Description
Technical field
The present invention relates to biomedical sector double center chain RNA and relevant anti-tumor compositions is preparing the application in inhibiting tumour cells agent.
Background technology
Small nucleic acid (miRNA) is a kind of non-coding tiny RNA comprising 21 ~ 25 nucleotide that is endogenous, wide expression in vivo, is one of material causing RNA interference phenomenon immediate cause.MiRNA is generated after Dicer enzyme is processed by the single stranded RNA precursor of about 70 ~ 90 base sizes with hairpin structure, and high conservative in evolution, carries out post-transcriptional control to gene expression.Each miRNA can have multiple regulation and control (target) gene, and several miRNA also can regulate same gene, and by inference, miRNA regulates the gene of one of trichotomy.The physiology that miRNA conditioner body weight is wanted and pathological process, at cell differentiation, play a great role in biological development and disease development process, and have very strong tissue specificity, caused the concern of increasing research worker.
Cervical cancer (Cervical Cancer) is a kind of malignant tumor of serious threat women's health.The data display of World Health Organization (WHO), the mortality rate of cervical cancer is only second to breast carcinoma in female tumor kind, accounts for second.About there are 28.8 ten thousand cases death in the whole world, 510,000 new cases every year, and wherein have the case of 80% in developing country, the annual new cases of China is about 13.5 ten thousand people, and death about has 3 ~ 50,000 people.
Summary of the invention
Technical problem to be solved by this invention is how inhibition tumor cell, and described inhibition tumor cell can be inhibition tumor cell propagation and/or promotes apoptosis of tumor cells.
For solving the problems of the technologies described above, the present invention provide firstly anti-tumor compositions.
Anti-tumor compositions provided by the present invention, its active component is made up of A and chemotherapeutics, and described A is double-stranded RNA or described double-stranded RNA trim or the biomaterial relevant to described double-stranded RNA.
In above-mentioned anti-tumor compositions, described double-stranded RNA, its name is called AntiCC, the chain of described AntiCC hold the nucleotides sequence to 3 ' end to be classified as SEQ ID No.1 sequence table from 5 ', another chain of described AntiCC hold the nucleotides sequence to 5 ' end to be classified as SEQ ID No.2 sequence table from 3 '.
Wherein, SEQ ID No.1 is made up of 20 ribonucleotides, SEQ ID No.2 is made up of 20 ribonucleotides, the 1-20 position ribonucleotide of SEQ ID No.1 and the 1-20 position ribonucleotide reverse complemental of SEQ ID No.2, by the double-stranded RNA called after AntiCC that the two is formed.
In above-mentioned anti-tumor compositions, described double-stranded RNA trim specifically can be modifies to described AntiCC the double-stranded RNA trim obtained, and its name is called AntiCC mimic.In described double-stranded RNA trim, various method of modifying all can be selected, and comprises the combination etc. of one or more be selected from ribose modification, base modification and phosphate backbones modification.As can to as described in AntiCC carry out following A 1) A2) or A3) modification obtain as described in double-stranded RNA trim:
A1) 2 '-OH of the ribose of described AntiCC is modified;
A2) phosphodiester bond of the connection nucleotide of described AntiCC is modified;
A3) 5 ' end of the ribose of described AntiCC is connected to the modification of cholesterol.
In above-mentioned anti-tumor compositions, 2 '-OH of the described ribose to described AntiCC is modified to and described 2 '-OH methoxyl group or fluorine is replaced or carry out deoxidation modification to described 2 '-OH; The phosphodiester bond of the described connection nucleotide to described AntiCC is modified to and is replaced by the oxygen sulfur of described phosphodiester bond.
In one embodiment of the invention, described double-stranded RNA trim is that 2 '-OH of all cytidylic acids (C) and all adenylic aciies (A) in chain shown in the SEQ ID No.2 by described AntiCC is all substituted by methoxyl group, shown in SEQ ID No.2, other nucleotide of chain is not modified, and chain does not carry out modifying the trim obtained shown in SEQ ID No.1, its name is called 2 '-OMe-AntiCC.
In above-mentioned anti-tumor compositions, the biomaterial that described and described double-stranded RNA is relevant is following B1)-B12) in any one:
B1) to encode the DNA molecular of described AntiCC;
B2) containing B1) expression cassette of described DNA molecular;
B3) containing B1) recombinant vector of described DNA molecular;
B4) containing B2) recombinant vector of described expression cassette;
B5) containing B1) recombinant microorganism of described DNA molecular;
B6) containing B2) recombinant microorganism of described expression cassette;
B7) containing B3) recombinant microorganism of described recombinant vector;
B8) containing B4) recombinant microorganism of described recombinant vector;
B9) containing B1) the transgenetic animal cell system of described DNA molecular;
B10) containing B2) the transgenetic animal cell system of described expression cassette;
B11) containing B3) the transgenetic animal cell system of described recombinant vector;
B12) containing B4) the transgenetic animal cell system of described recombinant vector.
In above-mentioned anti-tumor compositions, the expression cassette (AntiCC expression cassette) of the DNA molecular containing coding AntiCC B2) refers to the DNA that can express AntiCC in host cell, this DNA not only can comprise the promoter starting AntiCC and transcribe, and also can comprise the terminator stopping AntiCC and transcribe.Further, described expression cassette also can comprise enhancer sequence.
Available existing expression vector establishment contains the recombinant vector of described AntiCC expression cassette.
In above-mentioned anti-tumor compositions, described carrier can be plasmid, glutinous grain, phage or viral vector.
In above-mentioned anti-tumor compositions, B5)-B8) described in microorganism can be yeast, antibacterial, algae or fungus.
In above-mentioned anti-tumor compositions, B9)-B12) described in transgenetic animal cell system do not comprise propagating materials.
In above-mentioned anti-tumor compositions, described chemotherapeutics can be camptothecine.
The structural formula of described camptothecine is such as formula 1:
In above-mentioned anti-tumor compositions, the mol ratio of described A and described chemotherapeutics can be 1 × 10
-4: 4 × 10
4.
For solving the problems of the technologies described above, present invention also offers the application of following M or N:
M, described anti-tumor compositions are preparing the application in inhibiting tumour cells agent or tumor inhibitor;
N, described A are preparing the application in inhibiting tumour cells agent or tumor inhibitor.
In above-mentioned application, described inhibiting tumour cells agent can be the product (as medicine) of inhibition tumor cell propagation and/or promotes that the product (as medicine or vaccine) of apoptosis of tumor cells, described tumor inhibitor can be the product (as medicine or vaccine) of Tumor suppression generation and/or the product (as medicine or vaccine) of Tumor suppression growth.
For solving the problems of the technologies described above, present invention also offers the product (as medicine or vaccine) treating and/or preventing tumor.
The product (as medicine or vaccine) treating and/or preventing tumor provided by the present invention, its active component is described anti-tumor compositions or described AntiCC or described AntiCC mimic or described biomaterial.
Above-mentionedly treat and/or prevent in the product (as medicine or vaccine) of tumor, described medicine can also comprise other pharmaceutically acceptable carrier." pharmaceutically acceptable carrier " used herein should be compatible with the double stranded rna molecule in medicine of the present invention.Described " pharmaceutically acceptable carrier " refers to transfection reagent in body, as polymine (PEI), and jetPEI (L-PEI), liposome, transferrins, folic acid, nano-emulsion, nanoparticle etc.Other examples that can be used as some materials of pharmaceutically acceptable carrier or its component are freeze drying protectant saccharides, as lactose, dextrose plus saccharose; Starch, as corn starch and potato starch; Tragakanta powder; Fructus Hordei Germinatus; Gelatin; Talcum; Kollag, as stearic acid and magnesium stearate; Calcium sulfate; Vegetable oil, as Oleum Arachidis hypogaeae semen, Oleum Gossypii semen, Oleum sesami, olive oil, Semen Maydis oil and cupu oil; Polyhydric alcohol, as glycerol, mannitol; Alginic acid; Emulsifying agent, as Tween; Phospholipid, as lecithin, soybean phospholipid, PHOSPHATIDYL ETHANOLAMINE, phosphatidyl glycerol, phosphatidylinositols, Phosphatidylserine, stearmide; Cholesterol; Macromolecular polymer, as polymine, chitosan, hyaluronic acid; Wetting agent, as sodium lauryl sulfate; Coloring agent; Flavoring agent; Tablet agent, stabilizing agent; Antioxidant; Antiseptic; Apirogen water; Isotonic saline solution; With phosphate buffer etc.; Normal saline, glycerol and phosphate buffered saline (PBS).
For solving the problems of the technologies described above, present invention also offers described AntiCC or described AntiCC mimic or described biomaterial.
In the present invention, described tumor can be cervical cancer, intestinal cancer, nasopharyngeal carcinoma, hepatocarcinoma, gastric cancer, breast carcinoma, pulmonary carcinoma, nonsmall-cell lung cancer or carcinoma of endometrium.
Experiment proves, anti-tumor compositions of the present invention can improve the apoptosis ratio of human cervical carcinoma cell: through the early apoptosis ratio of the human cervical carcinoma cell of 2 '-OMe-AntiCC-CPT process, late apoptic ratio and apoptotic cell ratio are respectively 4.07 times of the human cervical carcinoma cell through 2 '-OMe-AntiCC-DMSO process, 1.05 times and 2.51 times, be respectively 1.71 times of the human cervical carcinoma cell processed through water-CPT, 1.21 times and 1.57 times, be respectively 1.54 times of the human cervical carcinoma cell through 2 '-OMe-NC-CPT process, 2.18 times and 1.64 times, be respectively 7.90 times of the human cervical carcinoma cell through 2 '-OMe-NC-DMSO process, 1.33 times and 3.83 times, be respectively 11.61 times of the human cervical carcinoma cell processed through water-DMSO, 3.04 times and 7.21 times, be respectively 10.57 times of untreated human cervical carcinoma cell, 2.38 times and 6.07 times.
Experiment proves, AntiCC of the present invention can improve the apoptosis ratio of human cervical carcinoma cell: be respectively 1.71 times, 1.21 times and 1.57 times of the human cervical carcinoma cell processed through water-CPT through the early apoptosis ratio of human cervical carcinoma cell of 2 '-OMe-AntiCC-CPT process, late apoptic ratio and apoptotic cell ratio, be respectively 1.54 times, 2.18 times and 1.64 times of the human cervical carcinoma cell through 2 '-OMe-NC-CPT process, be respectively 10.57 times of untreated human cervical carcinoma cell, 2.38 times and 6.07 times; 1.94 times, 1.27 times and 1.53 times of the human cervical carcinoma cell through 2 '-OMe-NC-DMSO process are respectively through the early apoptosis ratio of human cervical carcinoma cell of 2 '-OMe-AntiCC-DMSO process, late apoptic ratio and apoptotic cell ratio, be respectively 2.85 times, 2.90 times and 2.87 times of the human cervical carcinoma cell processed through water-DMSO, be respectively 2.60 times of untreated human cervical carcinoma cell, 2.27 times and 2.42 times.
Experiment proves, the propagation of AntiCC of the present invention to human cervical carcinoma cell is inhibited: the absorbance of 3 days 2 '-OMe-AntiCC cell liquid to be measured is respectively 0.42 times of the absorbance of 0.53 times, the 3 days liquid to be measured of cell in contrast of the absorbance of 3 days 2 '-OMe-NC cell liquid to be measured.
Experiment proves, can utilize anti-tumor compositions of the present invention and AntiCC inhibition tumor cell propagation and/or promote apoptosis of tumor cells.
Accompanying drawing explanation
Fig. 1 be through 2 '-OMe-AntiCC process human cervical carcinoma cell to be measured to the sensitivity Detection result of chemotherapeutics.Wherein, AntiCC+CPT represents the human cervical carcinoma cell through 2 '-OMe-AntiCC-CPT process, AntiCC represents the human cervical carcinoma cell through 2 '-OMe-AntiCC-DMSO process, CPT represents the human cervical carcinoma cell processed through water-CPT, NC+CPT represents the human cervical carcinoma cell through 2 '-OMe-NC-CPT process, NC represents the human cervical carcinoma cell through 2 '-OMe-NC-DMSO process, and DMSO represents the human cervical carcinoma cell processed through water-DMSO, and Normal represents untreated human cervical carcinoma cell.
Fig. 2 is through the human cervical carcinoma cell of 2 '-OMe-AntiCC process and the photo of after third time transfection the 48th hour of human cervical carcinoma cell under inverted phase contrast microscope through 2 '-OMe-NC process.
Fig. 3 is the result after the human cervical carcinoma cell through 2 '-OMe-AntiCC process and the human cervical carcinoma cell dyeing in after third time transfection the 48th hour through 2 '-OMe-NC process.
Fig. 4 measures 2 '-OMe-AntiCC cell liquid to be measured, 2 '-OMe-NC cell liquid to be measured and the absorbance of the liquid to be measured of cell in contrast under 450nm wavelength respectively.Wherein, BL represents cell liquid to be measured in contrast, and NC represents 2 '-OMe-NC cell liquid to be measured, and AntiCC represents 2 '-OMe-AntiCC cell liquid to be measured.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the present invention is further described in detail, the embodiment provided only in order to illustrate the present invention, instead of in order to limit the scope of the invention.Experimental technique in following embodiment, if no special instructions, is conventional method.Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Human cervical carcinoma cell (Hela cell) in following embodiment is ATCC product, and article No. is
cCL-2
tM, the public can obtain from Shenzhen Graduate School of Tsinghua University, and this biomaterial related experiment of the present invention of only attaching most importance to again is used, not can be used as other purposes and uses.
The preparation of embodiment 1, anti-tumor compositions
The chain-ordering of AntiCC is another chain-ordering of SEQ ID No.1, AntiCC in sequence table is SEQ ID No.2 in sequence table.Wherein, SEQ ID No.1 is made up of 20 ribonucleotides, and SEQ ID No.2 is made up of 20 ribonucleotides, the 1-20 position ribonucleotide of SEQ ID No.1 and the 1-20 position ribonucleotide reverse complemental of SEQ ID No.2, the two can form double-stranded RNA, i.e. AntiCC.The present invention selects random ds RNA as negative control, the name of this random ds RNA is called NC, a chain-ordering of NC is SEQ ID No.3 in sequence table, another chain-ordering of NC is SEQ ID No.4 in sequence table, in double-stranded RNA shown in SEQ ID No.3 and SEQ ID No.4, t represents deoxythymidine acid, and a, g, c and u all represent ribonucleotide.2 '-OH of all cytidylic acids (C) and all adenylic aciies (A) in chain shown in the SEQ ID No.2 of AntiCC is all substituted by methoxyl group, shown in SEQ ID No.2, other nucleotide of chain is not modified, and chain shown in SEQ ID No.1 does not carry out the trim called after 2 '-OMe-AntiCC modifying the AntiCC obtained.2 '-OH of all cytidylic acids (C) and all adenylic aciies (A) in chain shown in the SEQ ID No.4 of NC is all substituted by methoxyl group and chain shown in SEQ ID No.3 does not carry out modifying the trim called after 2 '-OMe-NC of the NC obtained.
Anti-tumor compositions of the present invention is made up of 2 '-OMe-AntiCC and chemotherapeutics camptothecine, and the structural formula of camptothecine is such as formula 1:
2 '-OMe-AntiCC and 2 '-OMe-NC entrusts lucky agate (GenePharma) Pharmaceutical Technology Inc. in Shanghai synthesis (Wincott F, DiRenzo A, Shaffer C, GrimmS, Tracz D, Workman C, Sweedler D, Gonzalez C, Scaringe S and Usman N.Synthesis, deprotection, analysis and purification of RNA and ribozymes.Nucleic Acids Res.1995,23:2677-84).
The anti-tumor compositions of embodiment 2, embodiment 1 is on the impact of human cervical carcinoma cell apoptosis
In triplicate, the step at every turn repeating to test is as follows in experiment:
1,2 '-OMe-AntiCC powder dissolution embodiment 1 obtained, in the sterilized water without RNA enzyme, makes its final concentration be 20pmol/L, obtains 2 '-OMe-AntiCC solution.
2, (in 2 '-OMe-AntiCC solution of this 5 μ L step 1, the content of 2 '-OMe-AntiCC is 1 × 10 to get 2 '-OMe-AntiCC solution of 5 μ L steps 1 respectively
-4the lipofectamine 2000 (Invitrogen) of pmol) and 5 μ L, it is diluted in respectively in the serum-free medium (Opti-MEM) of 245 μ L, obtains 2 '-OMe-AntiCC serum-free medium and lipofectamine 2000 serum-free medium respectively; Lipofectamine 2000 serum-free medium is at room temperature hatched 5 minutes, then 2 '-OMe-AntiCC serum-free medium is mixed with lipofectamine 2000 serum-free medium, obtain 2 '-OMe-AntiCC-liposome complex culture fluid 500 μ L, and this 2 '-OMe-AntiCC-liposome complex culture fluid is left standstill 20 minutes in room temperature.
3, cultivate human cervical carcinoma cell with the DMEM culture fluid (GIBCO product) containing 10% hyclone, collect the human cervical carcinoma cell culture fluid being in exponential phase; Be in cell concentration in the human cervical carcinoma cell culture fluid of exponential phase with the above-mentioned DMEM culture fluid adjustment containing 10% hyclone, obtain dilution human cervical carcinoma cell culture fluid, in dilution human cervical carcinoma cell culture fluid, the concentration of cell is 30000/mL; This dilution human cervical carcinoma cell culture fluid of 100 μ L is added respectively in 6 60mm culture dishs, in every ware, cell content is 3000, and with the above-mentioned DMEM culture fluid containing 10% hyclone, the volume of culture fluid in every ware is mended to 2mL, obtain the culture dish containing cervical cancer cell liquid; Culture dish containing human cervical carcinoma cell liquid is placed in containing 5%CO
2the incubator of 37 DEG C in cultivate 12 hours, make cell attachment, obtain the culture dish after cell attachment.
4,2 '-OMe-AntiCC-liposome complex culture fluid after leaving standstill respectively to the room temperature adding 500 μ L above-mentioned steps 2 in the culture dish after each above-mentioned cell attachment of step 3 and 1500 μ L serum-free DMEM culture fluid carry out transfection, obtain the 2 '-OMe-AntiCC cell culture fluid of 4mL; 2 '-OMe-AntiCC the cell culture fluid of this 4mL is placed in containing 5%CO
2the incubator of 37 DEG C in cultivate 6 hours, culture medium is changed to 10%FBS-DMEM culture medium, obtains the 2 '-OMe-AntiCC cell culture fluid of 2mL, the 2 '-OMe-AntiCC cell culture fluid of this 2mL is continued under normal operation cultivate.In incubation after 48 hours as stated above transfection once, cotransfection 3 times, obtains the system of 2 '-OMe-AntiCC, three transfections of 4mL.The system of 2 '-OMe-AntiCC, three transfections of this 4mL is placed in containing 5%CO
2the incubator of 37 DEG C in cultivate after 36 hours, chemotherapeutics camptothecine (CPT) is added in the system of 2 '-OMe-AntiCC, three transfections of this 4mL, obtain the system of 2 '-OMe-AntiCC, three transfections containing camptothecine of 4mL, the concentration of camptothecine in the system of 2 '-OMe-AntiCC, three transfections containing camptothecine of this 4mL is 10 μMs, and (i.e. the content of the camptothecine in the system of 2 '-OMe-AntiCC, three transfections containing camptothecine of this 4mL is 4 × 10
4pmol), the system of 2 '-OMe-AntiCC, three transfections containing camptothecine of this 4mL is placed in containing 5%CO
2the incubator of 37 DEG C in cultivate 20h, obtain through 2 '-OMe-AntiCC-CPT process human cervical carcinoma cell.
According to the method for above-mentioned steps 1-4, CPT is replaced with DMSO, the concentration of volume percent of DMSO in the system of 2 '-OMe-AntiCC, three transfections containing DMSO is 0.2%, and all the other steps are constant, obtains the human cervical carcinoma cell through 2 '-OMe-AntiCC-DMSO process.
According to the method for above-mentioned steps 1-4,2 '-OMe-AntiCC is replaced with 2 '-OMe-NC, all the other steps are constant, obtain the human cervical carcinoma cell through 2 '-OMe-NC-CPT process.
According to the method for above-mentioned steps 1-4,2 '-OMe-AntiCC is replaced with 2 '-OMe-NC, CPT is replaced with DMSO, the concentration of volume percent of DMSO in the system of 2 '-OMe-AntiCC, three transfections containing DMSO is 0.2%, all the other steps are constant, obtain the human cervical carcinoma cell through 2 '-OMe-NC-DMSO process.
According to the method for above-mentioned steps 2-4,2 '-OMe-AntiCC solution of step 1 is replaced with the sterilized water without RNA enzyme, and all the other steps are constant, obtain the human cervical carcinoma cell processed through water-CPT.
According to the method for above-mentioned steps 2-4,2 '-OMe-AntiCC solution of step 1 is replaced with the sterilized water without RNA enzyme, CPT is replaced with DMSO, the concentration of volume percent of DMSO in system is 0.2%, all the other steps are constant, obtain the human cervical carcinoma cell processed through water-DMSO.
According to the method for above-mentioned steps 2-4,2 '-OMe-AntiCC solution of step 1 is replaced with the sterilized water without RNA enzyme, the step adding CPT deleted, all the other steps are constant, obtain untreated human cervical carcinoma cell.
With FITC Annexin V Apoptosis Detection Kit I (BD, Cat No.556547) and according to the operating procedure of description respectively to the above-mentioned human cervical carcinoma cell through 2 '-OMe-AntiCC-CPT process, through the human cervical carcinoma cell of 2 '-OMe-NC-CPT process, through the human cervical carcinoma cell of 2 '-OMe-AntiCC-DMSO process, through the human cervical carcinoma cell of 2 '-OMe-NC-DMSO process, through the human cervical carcinoma cell that water-CPT processes, the human cervical carcinoma cell processed through water-DMSO and undressed human cervical carcinoma cell dye, the viable apoptotic cell of the human cervical carcinoma cell of above-mentioned different disposal and the ratio of non-viable apoptotic cell is detected respectively with flow cytometer.Repeat the result once repeating to test in testing for three times as shown in Figure 1, repeat the meansigma methods of result of testing for three times and standard deviation as shown in table 1.
The apoptosis situation of the human cervical carcinoma cell of table 1, different disposal
Result shows, through the early apoptosis ratio of the human cervical carcinoma cell of 2 '-OMe-AntiCC-CPT process, late apoptic ratio and apoptotic cell ratio are respectively 4.07 times of the human cervical carcinoma cell through 2 '-OMe-AntiCC-DMSO process, 1.05 times and 2.51 times, be respectively 1.71 times of the human cervical carcinoma cell processed through water-CPT, 1.21 times and 1.57 times, be respectively 1.54 times of the human cervical carcinoma cell through 2 '-OMe-NC-CPT process, 2.18 times and 1.64 times, be respectively 7.90 times of the human cervical carcinoma cell through 2 '-OMe-NC-DMSO process, 1.33 times and 3.83 times, be respectively 11.61 times of the human cervical carcinoma cell processed through water-DMSO, 3.04 times and 7.21 times, be respectively 10.57 times of untreated human cervical carcinoma cell, 2.38 times and 6.07 times.Show, the anti-tumor compositions of embodiment 1 can improve the apoptosis ratio of human cervical carcinoma cell.
1.71 times, 1.21 times and 1.57 times of the human cervical carcinoma cell processed through water-CPT are respectively through the early apoptosis ratio of human cervical carcinoma cell of 2 '-OMe-AntiCC-CPT process, late apoptic ratio and apoptotic cell ratio, be respectively 1.54 times, 2.18 times and 1.64 times of the human cervical carcinoma cell through 2 '-OMe-NC-CPT process, be respectively 10.57 times of untreated human cervical carcinoma cell, 2.38 times and 6.07 times; 1.94 times, 1.27 times and 1.53 times of the human cervical carcinoma cell through 2 '-OMe-NC-DMSO process are respectively through the early apoptosis ratio of human cervical carcinoma cell of 2 '-OMe-AntiCC-DMSO process, late apoptic ratio and apoptotic cell ratio, be respectively 2.85 times, 2.90 times and 2.87 times of the human cervical carcinoma cell processed through water-DMSO, be respectively 2.60 times of untreated human cervical carcinoma cell, 2.27 times and 2.42 times.Show, 2 '-OMe-AntiCC of embodiment 1 can improve the apoptosis ratio of human cervical carcinoma cell.
The propagation of 2 '-OMe-AntiCC inhibition tumor cell of embodiment 3, embodiment 1
One, 2 '-OMe-AntiCC suppresses human cervical carcinoma cell experiment 1
In triplicate, the concrete steps at every turn repeating to test are as follows in experiment:
2 '-OMe-AntiCC powder dissolution embodiment 1 obtained, in the sterilized water without RNA enzyme, makes its final concentration be 20pmol/L, obtains 2 '-OMe-AntiCC solution.Get the lipofectamine 2000 (Invitrogen) of this 2 '-OMe-AntiCC solution of 5 μ L and 5 μ L respectively, it is diluted in respectively in the serum-free medium (Opti-MEM) of 245 μ L, obtains 2 '-OMe-AntiCC serum-free medium and lipofectamine2000 serum-free medium respectively; Lipofectamine 2000 serum-free medium is at room temperature hatched 5 minutes, then 2 '-OMe-AntiCC serum-free medium is mixed with lipofectamine 2000 serum-free medium, obtain 2 '-OMe-AntiCC-liposome complex culture fluid 500 μ L, and this 2 '-OMe-AntiCC-liposome complex culture fluid is left standstill 20 minutes in room temperature.
Cultivate human cervical carcinoma cell with the DMEM culture fluid (GIBCO product) containing 10% hyclone, collect the human cervical carcinoma cell culture fluid being in exponential phase; Be in cell concentration in the human cervical carcinoma cell culture fluid of exponential phase with the above-mentioned DMEM culture fluid adjustment containing 10% hyclone, obtain dilution human cervical carcinoma cell culture fluid, in dilution human cervical carcinoma cell culture fluid, the concentration of cell is 30000/mL; This dilution human cervical carcinoma cell culture fluid of 100 μ L is added respectively in 6 60mm culture dishs, in every ware, cell content is 3000, and with the above-mentioned DMEM culture fluid containing 10% hyclone, the volume of culture fluid in every ware is mended to 2mL, obtain the culture dish containing cervical cancer cell liquid; Culture dish containing human cervical carcinoma cell liquid is placed in containing 5%CO
2the incubator of 37 DEG C in cultivate 12 hours, make cell attachment, obtain the culture dish after cell attachment.
Respectively to add in the culture dish after each above-mentioned cell attachment the above-mentioned room temperature of 500 μ L leave standstill after 2 '-OMe-AntiCC-liposome complex culture fluid and 1500 μ L serum-free DMEM culture fluid carry out transfection, obtain 2 '-OMe-AntiCC cell culture fluid; Then 2 '-OMe-AntiCC cell culture fluid is placed in containing 5%CO
2the incubator of 37 DEG C in cultivate after 6 hours, be changed to 10%FBS-DMEM culture medium, continue under normal condition to cultivate.In incubation after 48 hours as stated above transfection once, cotransfection 3 times, obtain through 2 '-OMe-AntiCC process human cervical carcinoma cell.
According to the method described above, 2 '-OMe-AntiCC is replaced with 2 '-OMe-NC, all the other steps are constant, obtain the human cervical carcinoma cell through 2 '-OMe-NC process.
Above-mentioned through 2 '-OMe-AntiCC process human cervical carcinoma cell and through 2 '-OMe-NC process human cervical carcinoma cell third time transfection after the 48th hour, take pictures respectively under inverted phase contrast microscope, result is as shown in Figure 2.Take pictures after terminating and fix with cold methanol respectively by the human cervical carcinoma cell processed through 2 '-OMe-AntiCC with through the human cervical carcinoma cell of 2 '-OMe-NC process, and use violet staining respectively, take pictures, result as shown in Figure 3.Above-mentioned through 2 '-OMe-AntiCC process human cervical carcinoma cell and through 2 '-OMe-NC process human cervical carcinoma cell third time transfection after the 48th hour, 2 '-OMe-AntiCC inhibits human cervical carcinoma cell to breed.Show that the propagation of 2 '-OMe-AntiCC to human cervical carcinoma cell is inhibited,
Two, 2 '-OMe-AntiCC suppresses human cervical carcinoma cell experiment 2
In triplicate, the concrete steps at every turn repeating to test are as follows in experiment:
Get 2 '-OMe-AntiCC solution and the 0.2 μ L lipofectamine 2000 (Invitrogen) of 0.2 μ L embodiment 2 respectively, it is diluted in respectively in the serum-free medium (Opti-MEM) of 10 μ L, obtains 2 '-OMe-AntiCC serum-free medium and lipofectamine 2000 serum-free medium respectively; Lipofectamine 2000 serum-free medium is at room temperature hatched 5 minutes, then 2 '-OMe-AntiCC serum-free medium is mixed with lipofectamine 2000 serum-free medium, obtain 2 '-OMe-AntiCC-liposome complex culture fluid, and this 2 '-OMe-AntiCC mimic-liposome complex culture fluid is left standstill 20 minutes in room temperature.
In the 96 every holes of well culture plate, add the dilution human cervical carcinoma cell culture fluid of 100 μ L embodiments 2, make cell content in every hole be 1000, obtain 96 orifice plates containing human cervical carcinoma cell liquid; 96 orifice plates this being contained human cervical carcinoma cell liquid are placed in containing 5%CO
2the incubator of 37 DEG C in cultivate 12 hours, make cell attachment, obtain 96 orifice plates after cell attachment.
Fluid medium in every hole of 96 orifice plates after sucking-off cell attachment, each hole of 96 orifice plates of cell is posted with the PBS washing of preheating at 37 DEG C, after abandoning PBS, fresh 2.5%FBS-DMEM culture medium is added in every hole, 2 '-OMe-AntiCC-liposome complex culture fluid after the room temperature adding 20 μ L the present embodiment afterwards in every hole leaves standstill and 60 μ L serum-free DMEM culture fluid carry out transfection, obtain 2 '-OMe-AntiCC cell culture fluid, 5 repeating holes are set altogether.Then 2 '-OMe-AntiCC cell culture fluid is placed in containing 5%CO
2the incubator of 37 DEG C in cultivate after 6 hours, the culture fluid in every hole is changed to 2.5%FBS-DMEM culture medium, every hole 100 μ L, obtains the human cervical carcinoma cell through 2 '-OMe-AntiCC process; Human cervical carcinoma cell through 2 '-OMe-AntiCC process is continued to cultivate under normal operation, in 24 hours (1 days) of continuing to cultivate, then 96 orifice plates is placed in containing 5%CO
2the incubator of 37 DEG C in cultivate 2 hours, obtain 1 day 2 '-OMe-AntiCC cell liquid to be measured; 1 day 2 '-OMe-AntiCC cell liquid to be measured is continued cultivation 12 hours (2 days), then 96 orifice plates is placed in containing 5%CO
2the incubator of 37 DEG C in cultivate 2 hours, obtain 2 days 2 '-OMe-AntiCC cell liquid to be measured; 2 days 2 '-OMe-AntiCC cell liquid to be measured is continued cultivation 12 hours (2 days), then 96 orifice plates is placed in containing 5%CO
2the incubator of 37 DEG C in cultivate 2 hours, obtain 3 days 2 '-OMe-AntiCC cell liquid to be measured.
According to the method described above, 2 '-OMe-AntiCC solution of embodiment 2 is replaced with 2 '-OMe-NC of embodiment 2, all the other steps are constant, obtain 1 day 2 '-OMe-NC cell liquid to be measured, 2 days 2 '-OMe-NC cell liquid to be measured and 3 days 2 '-OMe-NC cell liquid to be measured.
According to the method described above, 2 '-OMe-AntiCC solution of embodiment 2 is replaced with the sterilized water without RNA enzyme, all the other steps are constant, obtain 1 day liquid to be measured of cell in contrast (i.e. Blank contrast), 2 days liquid to be measured of cell in contrast (i.e. Blank contrast) and the 3 days liquid to be measured of cell in contrast (i.e. Blank contrast).
Under 450nm wavelength, the absorbance (OD value, Fig. 4 and table 2) of 1 day 2 '-OMe-AntiCC cell liquid to be measured, 2 days 2 '-OMe-AntiCC cell liquid to be measured, 3 days 2 '-OMe-AntiCC cell liquid to be measured, 1 day 2 '-OMe-NC cell liquid to be measured, 2 days 2 '-OMe-NC cell liquid to be measured, 3 days 2 '-OMe-NC cell liquid to be measured, 1 day liquid to be measured of cell in contrast, 2 days liquid to be measured of cell in contrast and the 3 days liquid to be measured of cell is in contrast measured respectively by microplate reader.The absorbance of 3 days 2 '-OMe-AntiCC cell liquid to be measured is respectively 0.42 times of the absorbance of 0.53 times, the 3 days liquid to be measured of cell in contrast of the absorbance of 3 days 2 '-OMe-NC cell liquid to be measured.Result shows that the propagation of 2 '-OMe-AntiCC to human cervical carcinoma cell is inhibited.
Absorbance under the 450nm wavelength of table 2, different cell liquid to be measured
| Cell liquid to be measured | Absorbance (OD value) |
| 1 day 2 '-OMe-AntiCC cell liquid to be measured | 0.67±0.01 |
| 2 days 2 '-OMe-AntiCC cell liquid to be measured | 0.59±0.03 |
| 3 days 2 '-OMe-AntiCC cell liquid to be measured | 1.57±0.10 |
| 1 day 2 '-OMe-NC cell liquid to be measured | 0.84±0.05 |
| 2 days 2 '-OMe-NC cell liquid to be measured | 0.87±0.03 |
| 3 days 2 '-OMe-NC cell liquid to be measured | 2.94±0.11 |
| The 1 day liquid to be measured of cell in contrast | 0.84±0.04 |
| The 2 days liquid to be measured of cell in contrast | 1.07±0.05 |
| The 3 days liquid to be measured of cell in contrast | 3.70±0.09 |
Claims (10)
1. anti-tumor compositions, its active component is made up of A and chemotherapeutics, and described A is double-stranded RNA or described double-stranded RNA trim or the biomaterial relevant to described double-stranded RNA;
A chain-ordering of described double-stranded RNA is SEQ ID No.1 in sequence table, and another chain-ordering of described double-stranded RNA is SEQ ID No.2 in sequence table;
Described double-stranded RNA trim is through following A 1) or A2) or A3) modify the double-stranded RNA trim that obtains:
A1) 2 '-OH of the ribose of described double-stranded RNA is modified;
A2) phosphodiester bond of the connection nucleotide of described double-stranded RNA is modified;
A3) 5 ' end of the ribose of described double-stranded RNA is connected to the modification of cholesterol;
The biomaterial that described and described double-stranded RNA is relevant is following B1)-B12) in any one:
B1) to encode the DNA molecular of described double-stranded RNA;
B2) containing B1) expression cassette of described DNA molecular;
B3) containing B1) recombinant vector of described DNA molecular;
B4) containing B2) recombinant vector of described expression cassette;
B5) containing B1) recombinant microorganism of described DNA molecular;
B6) containing B2) recombinant microorganism of described expression cassette;
B7) containing B3) recombinant microorganism of described recombinant vector;
B8) containing B4) recombinant microorganism of described recombinant vector;
B9) containing B1) the transgenetic animal cell system of described DNA molecular;
B10) containing B2) the transgenetic animal cell system of described expression cassette;
B11) containing B3) the transgenetic animal cell system of described recombinant vector;
B12) containing B4) the transgenetic animal cell system of described recombinant vector.
2. anti-tumor compositions according to claim 1, is characterized in that: described chemotherapeutics is camptothecine.
3. anti-tumor compositions described in claim 1 or 2 is preparing the application in inhibiting tumour cells agent or tumor inhibitor.
4. A described in claim 1 is preparing the application in inhibiting tumour cells agent or tumor inhibitor.
5. the application according to claim 3 or 4, is characterized in that: described inhibiting tumour cells agent is the product of inhibition tumor cell propagation and/or the product of promotion apoptosis of tumor cells; Described tumor inhibitor is the inhibitor of Tumor suppression generation and/or the inhibitor of Tumor suppression growth.
6. treat and/or prevent the product of tumor, its active component is anti-tumor compositions described in claim 1 or 2.
7. treat and/or prevent the product of tumor, its active component is A described in claim 1.
8. product described in anti-tumor compositions or the arbitrary described application of claim 3-5 or claim 6 or 7 according to claim 1 or 2, is characterized in that: described tumor can be cervical cancer, intestinal cancer, nasopharyngeal carcinoma, hepatocarcinoma, gastric cancer, breast carcinoma, pulmonary carcinoma, nonsmall-cell lung cancer or carcinoma of endometrium.
9. double-stranded RNA described in claim 1 or described double-stranded RNA trim.
10. biomaterial relevant to described double-stranded RNA described in claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510091139.6A CN104667299A (en) | 2015-02-28 | 2015-02-28 | Application of double-stranded RNA and anti-tumor composition related to double-stranded RNA in preparation of tumor cell inhibitor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510091139.6A CN104667299A (en) | 2015-02-28 | 2015-02-28 | Application of double-stranded RNA and anti-tumor composition related to double-stranded RNA in preparation of tumor cell inhibitor |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104667299A true CN104667299A (en) | 2015-06-03 |
Family
ID=53303230
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510091139.6A Pending CN104667299A (en) | 2015-02-28 | 2015-02-28 | Application of double-stranded RNA and anti-tumor composition related to double-stranded RNA in preparation of tumor cell inhibitor |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104667299A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106668861A (en) * | 2016-10-28 | 2017-05-17 | 清华大学深圳研究生院 | Method for enhancing anti-cervical cancer activity of glutaminase enzyme inhibitor by taking TIGA1 (Transcript Induced by Growth Arrest 1) as target spot |
| CN110358765A (en) * | 2018-04-09 | 2019-10-22 | 湖南师范大学 | Inhibit siRNA and its application of people TNFAIP1 gene expression |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008036776A2 (en) * | 2006-09-19 | 2008-03-27 | Asuragen, Inc. | Mir-15, mir-26, mir -31,mir -145, mir-147, mir-188, mir-215, mir-216 mir-331, mmu-mir-292-3p regulated genes and pathways as targets for therapeutic intervention |
| CN104147616A (en) * | 2014-08-06 | 2014-11-19 | 清华大学深圳研究生院 | Application of dsRNA or modification thereof in preparation of tumor inhibitor |
-
2015
- 2015-02-28 CN CN201510091139.6A patent/CN104667299A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008036776A2 (en) * | 2006-09-19 | 2008-03-27 | Asuragen, Inc. | Mir-15, mir-26, mir -31,mir -145, mir-147, mir-188, mir-215, mir-216 mir-331, mmu-mir-292-3p regulated genes and pathways as targets for therapeutic intervention |
| CN104147616A (en) * | 2014-08-06 | 2014-11-19 | 清华大学深圳研究生院 | Application of dsRNA or modification thereof in preparation of tumor inhibitor |
Non-Patent Citations (2)
| Title |
|---|
| 叶振南 等: "微小RNA与肿瘤信号转导通路", 《国际肿瘤学杂志》 * |
| 孙青 等: "化学修饰小干扰RNA研究进展", 《河南外科学杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106668861A (en) * | 2016-10-28 | 2017-05-17 | 清华大学深圳研究生院 | Method for enhancing anti-cervical cancer activity of glutaminase enzyme inhibitor by taking TIGA1 (Transcript Induced by Growth Arrest 1) as target spot |
| CN106668861B (en) * | 2016-10-28 | 2019-07-09 | 清华大学深圳研究生院 | It is a kind of that TIGA1 is enhanced into the active method of glutamine enzyme inhibitor anti-cervical cancer as target spot |
| CN110358765A (en) * | 2018-04-09 | 2019-10-22 | 湖南师范大学 | Inhibit siRNA and its application of people TNFAIP1 gene expression |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2018214137B2 (en) | MiRNA and its diagnostic therapeutic uses in diseases or conditions associated with melanoma, or in diseases or conditions associated with activated BRAF pathway | |
| US10201556B2 (en) | Combination for use in treating diseases or conditions associated with melanoma, or treating diseases or conditions associated with activated B-raf pathway | |
| CN107073294A (en) | Use the method for targeting TYR or MMP1 exonuclease treatment aging and skin disorder | |
| JP2014503553A (en) | MiRNA for treating diseases and conditions associated with neovascularization | |
| JP2021513508A (en) | Anti-cancer microRNA and its lipid preparation | |
| CN107636003A (en) | Inhibitor of miR 92 and application thereof | |
| CN104667299A (en) | Application of double-stranded RNA and anti-tumor composition related to double-stranded RNA in preparation of tumor cell inhibitor | |
| CN104147616B (en) | The application of double-stranded RNA or its trim in tumor inhibitor is prepared | |
| CN102125696A (en) | Oligomeric ribonucleic acid composite for inhibiting tumor growth and angiogenesis and application thereof | |
| CN109745335A (en) | MiR-218 is preparing the application in mammary cancer chemotherapy drug sensitizer | |
| CN102657878B (en) | Application of small activating RNA (Ribonucleic Acid) of INTS6 (Homo Sapiens Integrator Complex Subunit 6) gene to preparation of prostate cancer fighting medicament | |
| CN102140471B (en) | Oligo-nucleic acid for suppressing tumor growth and application thereof | |
| CN102643810B (en) | The antisense oligonucleotide of people miR-299-5p and application thereof | |
| CN102643806B (en) | The antisense oligonucleotide of people miR-1913 and application thereof | |
| CN102643807B (en) | Antisense oligodeoxyncleotide of human miR-484 and application thereof | |
| RU2551238C2 (en) | Method of inducing apoptosis of malignant tumour cells of colorectal cancer and means for its realisation | |
| CN102643813B (en) | The antisense oligonucleotide of people miR-504 and application thereof | |
| CN102643809B (en) | The antisense oligonucleotide of people miR-1274b and application thereof | |
| CN102140465B (en) | Human miR-1249 antisense nucleic acid and application thereof | |
| CN102140467B (en) | Human miR-365 antisense nucleic acid and application thereof | |
| CN103937800A (en) | Targeted-polygene siRNA (small interfering ribonucleic acid) molecule and application thereof in inhibiting tumors | |
| Berg | Transient silencing of GLUT1, LDHA, and MCT4 affects glucose transport, glycolysis, and chemosensitivity in human pancreatic cancer cells | |
| CN105779452A (en) | Oligonucleotide capable of inhibiting tumor growth and application of oligonucleotide | |
| CN102643812B (en) | The antisense oligonucleotide of people miR-1227 and application thereof | |
| CN102643808B (en) | The antisense oligonucleotide of people miR-1539 and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150603 |