CN110042101B - siRNA for reducing ROGDI gene expression, recombinant vector and application thereof - Google Patents
siRNA for reducing ROGDI gene expression, recombinant vector and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular biology and genetic engineering, and relates to siRNA for reducing ROGDI gene expression, a recombinant vector and application thereof. In particular, it relates to a siRNA that specifically reduces ROGDI gene expression. The siRNA provided by the invention can specifically reduce ROGDI gene expression, so that the siRNA can be applied to preparation of medicaments for treating and/or preventing breast cancer.
Description
Technical Field
The invention belongs to the technical field of molecular biology and genetic engineering, and particularly relates to siRNA for reducing ROGDI gene expression, shRNA corresponding to the siRNA, DNA for encoding the shRNA, a recombinant vector comprising the DNA, a recombinant lentivirus comprising the recombinant vector, a host cell comprising the siRNA, the shRNA, the DNA for encoding the shRNA, the recombinant vector and the recombinant lentivirus, and applications of the siRNA, the shRNA, the DNA for encoding the shRNA, the recombinant vector and the recombinant lentivirus.
Background
Breast cancer is one of common malignant tumors of women worldwide, and the new incidence and mortality of breast cancer are the first of all types of cancer. Although the incidence of breast cancer is relatively low in China, the population is large, the total incidence is large, the incidence rate tends to rise year by year, and the incidence age tends to be younger. Breast cancer presents great complexity and heterogeneity in cell origin, histological morphology, disease grading, clinical presentation, treatment response, metastatic potential and other aspects, and the effectiveness and the universality of the existing breast cancer treatment method are limited. There is still a lack of clinical targets for breast cancer therapy.
ROGDI (Rogdi), also known as KTZS. The gene coded by the protein is positioned at 16p13.3, has 11 exons and is usually expressed in a large amount in tissues and organs such as human brain, bone marrow, heart and the like. Through further studies, the protein was found to localize in the nucleus, cytoplasm and mitochondria. However, to date, no biological function of ROGDI has been reported.
Disclosure of Invention
The invention aims to provide a siRNA for reducing ROGDI gene expression, a recombinant vector and application thereof, so as to specifically reduce ROGDI gene expression, and thus, breast cancer can be treated and/or prevented.
In order to achieve the above object, a first aspect of the present invention provides an siRNA that specifically reduces the expression of the ROGDI gene.
The present invention focuses on providing a novel breast cancer therapeutic target, and is not limited to a specific siRNA sequence, and siRNA directed to the target can be designed according to various methods conventional in the art. The siRNA generally has a nucleotide sequence of 19-27 bp.
Specifically, the nucleotide sequence of the siRNA at least comprises the following group of nucleotide sequences:
(1) a first set of nucleotide sequences
The first group of nucleotide sequences are shown as SEQ ID NO 1 and SEQ ID NO 2, the SEQ ID NO 1 is 5'-GCUGGUCAACGUCUACAUCAA-3', and the SEQ ID NO 2 is 5'-UUGAUGUAGACGUUGACCAGC-3';
(2) second set of nucleotide sequences
The second group of nucleotide sequences are shown as SEQ ID NO 3 and SEQ ID NO 4, the SEQ ID NO 3 is 5'-CCAUGUGAGCCAAGCCAUUUA-3', and the SEQ ID NO 4 is 5'-UAAAUGGCUUGGCUCACAUGG-3';
(3) a third group of nucleotide sequences
The third group of nucleotide sequences are shown as SEQ ID NO 5 and SEQ ID NO 6, wherein the SEQ ID NO 5 is 5'-CCCACCUAACACAUUUGCACU-3', and the SEQ ID NO 6 is 5'-AGUGCAAAUGUGUUAGGUGGG-3'.
The second aspect of the present invention provides a shRNA which is a single-stranded RNA having a stem-loop structure, wherein the nucleotide sequence of the shRNA at least comprises the following group of nucleotide sequences:
(1) nucleotide sequence of the fifth group
The fifth group of nucleotide sequences are shown as SEQ ID NO 9 and SEQ ID NO 10, wherein the SEQ ID NO 9 is 5'-CCGGGCUGGUCAACGUCUACAUCAACUCGAGUUGAUGUAGACGUUGACCAGCUUUUUG-3', and the SEQ ID NO 10 is 5'-AAUUCAAAAAGCUGGUCAACGUCUACAUCAACUCGAGUUGAUGUAGACGUUGACCAGC-3';
(2) nucleotide sequence of group VI
The sixth group of nucleotide sequences are shown as SEQ ID NO. 11 and SEQ ID NO. 12, wherein the SEQ ID NO. 11 is 5'-CCGGCCAUGUGAGCCAAGCCAUUUACUCGAGUAAAUGGCUUGGCUCACAUGGUUUUUG-3', and the SEQ ID NO. 12 is 5'-AAUUCAAAAACCAUGUGAGCCAAGCCAUUUACUCGAGUAAAUGGCUUGGCUCACAUGG-3';
(3) nucleotide sequence of group seven
The seventh group of nucleotide sequences are shown as SEQ ID NO. 13 and SEQ ID NO. 14, wherein the SEQ ID NO. 13 is 5'-CCGGCCCACCUAACACAUUUGCACUCUCGAGAGUGCAAAUGUGUUAGGUGGGUUUUUG-3', and the SEQ ID NO. 14 is 5'-AAUUCAAAAACCCACCUAACACAUUUGCACUCUCGAGAGUGCAAAUGUGUUAGGUGGG-3'.
In a third aspect of the present invention, there is provided a DNA encoding the shRNA provided in the second aspect of the present invention, wherein the nucleotide sequence of the DNA comprises at least one of the following nucleotide sequences:
(1) nucleotide sequence of the ninth group
The ninth group of nucleotide sequences are shown as SEQ ID NO 17 and SEQ ID NO 18, wherein the SEQ ID NO 17 is 5'-CCGGGCTGGTCAACGTCTACATCAACTCGAGTTGATGTAGACGTTGACCAGCTTTTTG-3', and the SEQ ID NO 18 is 5'-AATTCAAAAAGCTGGTCAACGTCTACATCAACTCGAGTTGATGTAGACGTTGACCAGC-3';
(2) tenth group of nucleotide sequences
The tenth group of nucleotide sequences are shown as SEQ ID NO. 19 and SEQ ID NO. 20, wherein the SEQ ID NO. 19 is 5'-CCGGCCATGTGAGCCAAGCCATTTACTCGAGTAAATGGCTTGGCTCACATGGTTTTTG-3', and the SEQ ID NO. 20 is 5'-AATTCAAAAACCATGTGAGCCAAGCCATTTACTCGAGTAAATGGCTTGGCTCACATGG-3';
(3) nucleotide sequence of the eleventh group
The eleventh group of nucleotide sequences are shown as SEQ ID NO 21 and SEQ ID NO 22, wherein the SEQ ID NO 21 is 5'-CCGGCCCACCTAACACATTTGCACTCTCGAGAGTGCAAATGTGTTAGGTGGGTTTTTG-3', and the SEQ ID NO 22 is 5'-AATTCAAAAACCCACCTAACACATTTGCACTCTCGAGAGTGCAAATGTGTTAGGTGGG-3'.
The fourth aspect of the present invention provides a recombinant vector obtained by inserting, for example, a DNA encoding shRNA provided in the third aspect of the present invention into the multiple cloning sites AgeI and EcoRI of the GV493 plasmid (available from Kjekay Gene science and technology, Inc., of Shanghai).
In a fifth aspect, the invention provides a recombinant lentivirus obtainable by co-transfecting a mammalian cell with the recombinant vector provided in the fourth aspect of the invention and the viral packaging helper plasmid phelprer 1.0 vector and the viral packaging helper plasmid phelprer 2.0 vector.
The sixth aspect of the present invention provides a host cell comprising at least one of the siRNA provided by the first aspect of the present invention, the shRNA provided by the second aspect of the present invention, the DNA encoding the shRNA provided by the third aspect of the present invention, the recombinant vector provided by the fourth aspect of the present invention, and the recombinant lentivirus provided by the fifth aspect of the present invention. The present invention is not particularly limited with respect to the specific type of the host cell, and examples thereof include 293T cells.
The seventh aspect of the invention provides a use of at least one of the siRNA provided by the first aspect of the invention, the shRNA provided by the second aspect of the invention, the DNA encoding the shRNA provided by the third aspect of the invention, the recombinant vector provided by the fourth aspect of the invention, and the recombinant lentivirus provided by the fifth aspect of the invention in the preparation of an agent for inhibiting the expression of the ROGDI gene.
An eighth aspect of the present invention provides a use of at least one of the siRNA provided by the first aspect of the present invention, the shRNA provided by the second aspect of the present invention, the DNA encoding the shRNA provided by the third aspect of the present invention, the recombinant vector provided by the fourth aspect of the present invention, and the recombinant lentivirus provided by the fifth aspect of the present invention in the preparation of a medicament for inhibiting growth and/or proliferation of a breast cancer cell.
The ninth aspect of the present invention provides an application of at least one of the siRNA provided by the first aspect of the present invention, the shRNA provided by the second aspect of the present invention, the DNA encoding the shRNA provided by the third aspect of the present invention, the recombinant vector provided by the fourth aspect of the present invention, and the recombinant lentivirus provided by the fifth aspect of the present invention in the preparation of a medicament for treating and/or preventing breast cancer.
The siRNA provided by the invention can specifically reduce ROGDI gene expression, so that the siRNA can be applied to preparation of medicaments for treating and/or preventing breast cancer.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Drawings
The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings.
Fig. 1 shows the difference in expression of ROGDI in normal and breast cancer cells. Wherein MCF-10A (abbreviated as 10A) is normal cell, MCF7, MDA-MB-231 (abbreviated as 231), BT-474 and SK-BR-3 are breast cancer cells.
Figure 2 shows the effect of knockdown of ROGDI on breast cancer cell proliferation. Wherein shCtrl is a control group, shROGDI is an experimental group, the upper curve is a control group, and the lower curve is an experimental group.
Figure 3 shows the effect of knockdown of ROGDI on breast cancer growth in tumor-bearing mice. Wherein NC is a control group, and shrogDI is an experimental group.
Detailed Description
Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the present invention, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein. The examples, in which the specific conditions are not specified, were conducted under the conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The human ROGDI referred to in the examples below is numbered Gene ID 79641.
In the examples below, statistical analysis was performed using GraphPad Prism 6.0 software. All in vitro experiments were repeated three more times. Data are expressed as mean ± Standard Deviation (SD). P <0.05 was considered statistically significant.
Example 1
This example serves to illustrate the high expression of ROGDI in breast cancer cell lines.
RNA extraction and reverse transcription quantitative PCR (RT-qPCR).
1. Total RNA extraction: this example was carried out at low temperature. Culturing cells in 6-well plate, removing culture medium, rinsing with PBS for 3 times, adding 1000 μ l Trizol per well, and shaking for 10 min; collecting into 1.5ml centrifuge tube, adding 200 μ l chloroform into each tube, mixing vigorously for 30sec, standing for 15min, and centrifuging at 4 deg.C 12000rpm for 15 min; gently sucking 400 μ l of the supernatant liquid into another new centrifuge tube, adding isopropanol with the same volume, gently inverting and mixing, and centrifuging at 12000rpm at 4 ℃ for 10 min; discarding the supernatant, adding 1ml of 75% alcohol to wash the precipitate, and centrifuging at 4 ℃ and 12000rpm for 10 min; the supernatant was discarded as much as possible, air-dried at room temperature for 10min, 10. mu.l of RNase-free water was added to each tube, dissolved, and quantified with a spectrophotometer.
2. Reverse transcription: each 25. mu.l reverse transcription system contained 100pmol random primer, 2. mu.g total RNA, 1. mu.l M-MLV reverse transcriptase, 0.625. mu.l RNase inhibitor, 1.25. mu.l dNTPs (10mM), 5. mu.l 5 XM-MLV buffer, and the remainder ddH2The volume of O is filled to 25 μ l. The reaction conditions are as follows: 1h at 37 ℃ and 5min at 95 ℃.
3. Quantitative PCR: each 20. mu.l reaction system contained 2 XPCR Mix (ABI) 10. mu.l, upstream and downstream primers 0.4. mu.l each, cDNA 1. mu.l, ddH2O8.2. mu.l. The reaction conditions are as follows: 94 ℃ for 2min, 94 ℃ for 15s, 60 ℃ for 40s, 40 cycles. The sequences of the primers used in the experiments are shown in Table 1.
TABLE 1 fluorescent quantitative RT-PCR primer sequences
Fig. 1 shows the difference in expression of ROGDI in normal and breast cancer cells. Wherein MCF-10A (abbreviated as 10A) is normal cell, MCF7, MDA-MB-231 (abbreviated as 231), BT-474 and SK-BR-3 are breast cancer cells. As shown in fig. 1, the RT-qPCR results show: the expression of ROGDI gene is obviously increased in MCF7, MDA-MB-231, BT-474 and SK-BR-3 breast cancer cells compared with that of MCF-10A cells of a control group (P is less than 0.01, P is less than 0.05 and P is less than 0.05).
Example 2
This example demonstrates that knocking down ROGDI gene expression can inhibit breast cancer proliferation and/or growth.
First, preparation of RNAi lentivirus clone
1. Target design
Aiming at the ROGDI gene sequence, 3 RNAi target sequences, nucleotide sequences of 3 siRNAs of the ROGDI gene and nucleotide sequences of Negative Control (NC) are designed according to the RNAi sequence design principle. The names corresponding to the 3 kinds of siRNA and the negative control are ROGDI-si-1a, ROGDI-si-1b, ROGDI-si-2a, ROGDI-si-2b, ROGDI-si-3a, ROGDI-si-3b, NC-si-a and NC-si-b respectively, which are sequences designed for the negative control group. See table 2 for details.
TABLE 23 nucleotide sequences of RNA interference targets (siRNA) and negative controls
Table 3 shows the nucleotide sequences of 3 kinds of shRNAs used in the examples, wherein the shRNAs used in the examples are the nucleotide sequences of ROGDI-sh-1a, ROGDI-sh-1b, ROGDI-sh-2a, ROGDI-sh-2b, ROGDI-sh-3a and ROGDI-sh-3b, and NC-sh-a and NC-sh-b are the nucleotide sequences of a control group. See table 3 for details.
TABLE 33 nucleotide sequences of shRNA and negative controls
The nucleotide sequences of the DNAs used to encode the 3 shRNAs are shown in Table 4. The DNAs encoding shRNAs used in the examples were the nucleotide sequences of ROGDI-d-1a, ROGDI-d-1b, ROGDI-d-2a, ROGDI-d-2b, ROGDI-d-3a, ROGDI-d-3b, and NC-d-a and NC-d-b were the nucleotide sequences of the control group. See table 4 for details.
DNA and nucleotide sequence of negative control of the 43 shRNAs in Table
2. Vector cleavage
A50. mu.l digestion system was prepared according to Table 5. Sequentially adding various reagents according to the sequence of a list, lightly blowing and uniformly mixing by using a pipette, carrying out instantaneous centrifugation, and reacting for 3 hours at 37 ℃. And (4) carrying out agarose gel electrophoresis on the vector enzyme digestion product, and recovering a target band.
TABLE 5 vector cleavage System
3. DNA annealing of shRNA to form double-stranded DNA
And (3) dissolving the DNA dry powder of the synthesized paired shRNA in an annealing buffer solution, carrying out water bath at 90 ℃ for 15min, and naturally cooling to room temperature.
4. Carrier attachment
The double-cut linearized vector and the annealed double-stranded DNA were ligated by means of T4 DNA ligase (T4 DNA ligase) for 1-3h at 16 ℃.
TABLE 6 Carrier attachment System
| Reagent | Volume (μ l) |
| Linearized vector (100 ng/. mu.l) | 1 |
| Double-stranded DNA (100 ng/. mu.l) | 1 |
| 10 |
2 |
| T4 DNA ligase | 1 |
| Double distilled water (ddH)2O) | Make up to 20 |
5. Transformation of
Adding 10 μ L of the ligation reaction product into 100 μ L of competent cells, flicking the tube wall, mixing, and standing on ice for 30 min; heat shock is carried out for 90s at 42 ℃, and incubation is carried out for 2min in ice bath; adding 500 μ L LB culture medium, and shake culturing at 37 deg.C for 1 h; taking a proper amount of bacterial liquid, uniformly coating the bacterial liquid on a flat plate containing corresponding antibiotics, and carrying out inverted culture in a constant-temperature incubator for 12-16 h.
6. Sequencing identification
And (3) inoculating the identified positive clone transformant into a proper amount of LB liquid culture medium containing corresponding antibiotics, culturing for 12-16h at 37 ℃, and taking a proper amount of bacterial liquid for sequencing and identifying.
7. Plasmid transfection and lentivirus harvesting
Viral packaging involves a total of three plasmids: the vector comprises a tool vector plasmid GV493 vector (purchased from Shanghai Jikai gene science and technology Co., Ltd.), a virus packaging Helper plasmid Helper 1.0 vector (purchased from Shanghai Jikai gene science and technology Co., Ltd.) and a virus packaging Helper 2.0 vector (purchased from Shanghai Jikai gene science and technology Co., Ltd.). 293T cells were co-transfected with the three plasmids described above.
24h before transfection, 293T cells in the logarithmic growth phase were trypsinized and cell density was adjusted to about 5X 10 in medium containing 10% serum615ml, reseeding in 10cm cell culture dish, 37 deg.C, 5% CO2Culturing in an incubator; the cell can be used for transfection after 24 hours when the cell density reaches 70-80%; replacing the medium with a serum-free medium 2h before transfection; adding DNA solutions (GV493 plasmid 20 μ g, pHelper 1.0 vector plasmid 15 μ g, pHelper 2.0 vector plasmid 10 μ g) into a sterilized centrifuge tube, mixing with corresponding volume of Gecky transfection reagent, adjusting total volume to 1ml, and incubating at room temperature for 15 min; the mixed solution is slowly dripped into 293T cell culture solution, mixed evenly and treated at 37 ℃ with 5% CO2Culturing in a cell culture box; after 6h incubation the medium containing the transfection mixture was discarded, washed once with 10ml PBS, gently shaken to wash the remaining transfection mixture and discarded.
Slowly adding 10% serum-containing cell culture medium 20ml, and heating at 37 deg.C with 5% CO2Culturing in the incubator for 48-72 h.
8. Lentivirus concentration and purification and quality control
Collecting 293T cell supernatant 48h after transfection (which can be counted as 0h after transfection) according to cell states; centrifuging at 4000g for 10min at 4 deg.C to remove cell debris; filtering the supernatant with a 0.45 μm filter in a 40ml ultracentrifuge tube; respectively balancing samples, putting the ultracentrifuge tubes with virus supernatant into a Beckman ultracentrifuge one by one, centrifuging for 2h at 4 ℃ and 25000 rpm; after the centrifugation is finished, removing the supernatant, removing the liquid remained on the tube wall as much as possible, adding a virus preservation solution, and lightly and repeatedly blowing and resuspending; after full dissolution, centrifuging at high speed 10000rpm for 5min, taking the supernatant and subpackaging according to the requirement.
The main points of the quality control of lentiviruses comprise physical state detection, sterility detection and virus titer detection.
Second, Lentiviral transfection
In order to ensure the gene interference efficiency, this example designs 3 RNA interference targets (siRNA) for the ROGDI gene, and mixes 3 plasmids carrying different targets in equal proportion for lentiviral packaging, thereby ensuring the knockdown efficiency of the target gene after the virus infects cells.
After 12-16 hours of cell subculture into 6-well plates: mixing 0.15ml of virus solution with fresh cell culture solution uniformly, and adding 0.5ml of fresh culture solution and 0.65 μ l of polybrene (final concentration 4ng/ml) into the virus supernatant with the proportion of 0.15 ml; the premixed virus infection liquid is added into a target cell culture dish, and the cell density is not more than 50%. After overnight culture, the culture medium was replaced with fresh medium.
Cells in the logarithmic growth phase were taken 3 days after infection and subjected to cell proliferation assay.
Third, cell proliferation experiment
Pancreatin digestion is carried out on the control group and the cells in the logarithmic growth phase with the ROGDI expression being knocked down to prepare cell suspension; the cell suspension (cell number about 3000) was inoculated into a 96-well plate, and the cell number was counted on days 1, 2, 3, 4, and 5, respectively, to draw a growth curve.
Figure 2 shows the effect of knockdown of ROGDI on breast cancer cell proliferation. Wherein shCtrl is a control group, shROGDI is an experimental group, the upper curve is a control group, and the lower curve is an experimental group. As shown in fig. 2, the results of cell proliferation experiments show that the knocking down of the expression of the ROGDI gene significantly inhibits the proliferation of the breast cancer cell MCF7 both on day 4 and day 5 (P <0.001 ).
Tumor bearing experiment of nude mouse breast cancer
The control group and the MCF7 breast cancer cell line with the ROGDI knocked down are respectively prepared into cell suspensions, and the nude mice are planted subcutaneously. Each group of 6 mice was inoculated with 100. mu.l of 5X 106And (4) cells. After 1.5 months, tumor size and volume were measured and statistically analyzed.
Figure 3 shows the effect of knockdown of ROGDI on breast cancer growth in tumor-bearing mice. Wherein NC is a control group, and shrogDI is an experimental group. As shown in fig. 3, the tumor-bearing experimental results of mice showed that the tumor volume of the breast cancer in the ROGDI-low expression group is significantly lower than that in the control group (P < 0.01). Therefore, the reduction of the expression of the ROGDI gene can obviously inhibit the growth of breast cancer cells.
Having described embodiments of the present invention, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.
Sequence listing
<110> Jiangsu medical profession college
<120> siRNA for reducing ROGDI gene expression, recombinant vector and application thereof
<141> 2019-03-18
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<210> 21
<211> 58
<212> DNA
<213> Artificial Sequence
<400> 21
ccggcccacc taacacattt gcactctcga gagtgcaaat gtgttaggtg ggtttttg 58
<210> 22
<211> 58
<212> DNA
<213> Artificial Sequence
<400> 22
aattcaaaaa cccacctaac acatttgcac tctcgagagt gcaaatgtgt taggtggg 58
<210> 23
<211> 57
<212> DNA
<213> Artificial Sequence
<400> 23
ccggttctcc gaacgtgtca cgtttcaaga gaacgtgaca cgttcggaga atttttg 57
<210> 24
<211> 57
<212> DNA
<213> Artificial Sequence
<400> 24
aattcaaaaa ttctccgaac gtgtcacgtt ctcttgaaac gtgacacgtt cggagaa 57
<210> 25
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 25
tgtggcacag accaggtgaa 20
<210> 26
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 26
aggtaaatgg cttggctcac a 21
<210> 27
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 27
ggcacccagc acaatgaaga 20
<210> 28
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 28
actcctgctt gctgatccac 20
Claims (3)
1. Use of an siRNA for the manufacture of a medicament for inhibiting the growth and/or proliferation of breast cancer cells MCF7, wherein the siRNA specifically reduces ROGDI gene expression, and the siRNA is a combination consisting of the following nucleotide sequences:
(1) a first set of nucleotide sequences
The first group of nucleotide sequences are shown as SEQ ID NO 1 and SEQ ID NO 2, the SEQ ID NO 1 is 5'-GCUGGUCAACGUCUACAUCAA-3', and the SEQ ID NO 2 is 5'-UUGAUGUAGACGUUGACCAGC-3';
(2) second set of nucleotide sequences
The second group of nucleotide sequences are shown as SEQ ID NO 3 and SEQ ID NO 4, the SEQ ID NO 3 is 5'-CCAUGUGAGCCAAGCCAUUUA-3', and the SEQ ID NO 4 is 5'-UAAAUGGCUUGGCUCACAUGG-3';
(3) a third group of nucleotide sequences
The third group of nucleotide sequences are shown as SEQ ID NO 5 and SEQ ID NO 6, wherein the SEQ ID NO 5 is 5'-CCCACCUAACACAUUUGCACU-3', and the SEQ ID NO 6 is 5'-AGUGCAAAUGUGUUAGGUGGG-3'.
2. The application of shRNA in preparing a medicament for inhibiting growth and/or proliferation of breast cancer cells MCF7 is characterized in that the shRNA is a combination consisting of the following nucleotide sequences:
(1) nucleotide sequence of the fifth group
The fifth group of nucleotide sequences are shown as SEQ ID NO 9 and SEQ ID NO 10, wherein the SEQ ID NO 9 is 5'-CCGGGCUGGUCAACGUCUACAUCAACUCGAGUUGAUGUAGACGUUGACCAGCUUUUUG-3', and the SEQ ID NO 10 is 5'-AAUUCAAAAAGCUGGUCAACGUCUACAUCAACUCGAGUUGAUGUAGACGUUGACCAGC-3';
(2) nucleotide sequence of group VI
The sixth group of nucleotide sequences are shown as SEQ ID NO. 11 and SEQ ID NO. 12, wherein the SEQ ID NO. 11 is 5'-CCGGCCAUGUGAGCCAAGCCAUUUACUCGAGUAAAUGGCUUGGCUCACAUGGUUUUUG-3', and the SEQ ID NO. 12 is 5'-AAUUCAAAAACCAUGUGAGCCAAGCCAUUUACUCGAGUAAAUGGCUUGGCUCACAUGG-3';
(3) nucleotide sequence of group seven
The seventh group of nucleotide sequences are shown as SEQ ID NO. 13 and SEQ ID NO. 14, wherein the SEQ ID NO. 13 is 5'-CCGGCCCACCUAACACAUUUGCACUCUCGAGAGUGCAAAUGUGUUAGGUGGGUUUUUG-3', and the SEQ ID NO. 14 is 5'-AAUUCAAAAACCCACCUAACACAUUUGCACUCUCGAGAGUGCAAAUGUGUUAGGUGGG-3'.
3. Use of a DNA encoding an shRNA according to claim 2 in the manufacture of a medicament for inhibiting the growth and/or proliferation of breast cancer cells MCF7, wherein the DNA is a combination consisting of the nucleotide sequences:
(1) nucleotide sequence of the ninth group
The ninth group of nucleotide sequences are shown as SEQ ID NO 17 and SEQ ID NO 18, wherein the SEQ ID NO 17 is 5'-CCGGGCTGGTCAACGTCTACATCAACTCGAGTTGATGTAGACGTTGACCAGCTTTTTG-3', and the SEQ ID NO 18 is 5'-AATTCAAAAAGCTGGTCAACGTCTACATCAACTCGAGTTGATGTAGACGTTGACCAGC-3';
(2) tenth group of nucleotide sequences
The tenth group of nucleotide sequences are shown as SEQ ID NO. 19 and SEQ ID NO. 20, wherein the SEQ ID NO. 19 is 5'-CCGGCCATGTGAGCCAAGCCATTTACTCGAGTAAATGGCTTGGCTCACATGGTTTTTG-3', and the SEQ ID NO. 20 is 5'-AATTCAAAAACCATGTGAGCCAAGCCATTTACTCGAGTAAATGGCTTGGCTCACATGG-3';
(3) nucleotide sequence of the eleventh group
The eleventh group of nucleotide sequences are shown as SEQ ID NO 21 and SEQ ID NO 22, wherein the SEQ ID NO 21 is 5'-CCGGCCCACCTAACACATTTGCACTCTCGAGAGTGCAAATGTGTTAGGTGGGTTTTTG-3', and the SEQ ID NO 22 is 5'-AATTCAAAAACCCACCTAACACATTTGCACTCTCGAGAGTGCAAATGTGTTAGGTGGG-3'.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101433724A (en) * | 2007-11-16 | 2009-05-20 | 天津医科大学附属肿瘤医院 | Use of Akt2-siRNA fusion expression vector in preparing medicament for inhibiting metastasis of human breast cancer cell |
| CN108949827A (en) * | 2018-07-20 | 2018-12-07 | 深圳市南山区疾病预防控制中心 | The special shRNA Lentiviral for inhibiting lung cancer PPARd gene expression and its construction method and application |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101433724A (en) * | 2007-11-16 | 2009-05-20 | 天津医科大学附属肿瘤医院 | Use of Akt2-siRNA fusion expression vector in preparing medicament for inhibiting metastasis of human breast cancer cell |
| CN108949827A (en) * | 2018-07-20 | 2018-12-07 | 深圳市南山区疾病预防控制中心 | The special shRNA Lentiviral for inhibiting lung cancer PPARd gene expression and its construction method and application |
Non-Patent Citations (1)
| Title |
|---|
| Downregulation of a novel human gene,ROGDI,increases radiosensitivity in cervical cancer cells;Yi-Fan Chen等;《Cancer Biology and Therapy》;20161231;第17卷(第10期);摘要，第1076页右栏第1段 * |
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