CN105331616B - Tetracycline-induced artificial MicroRNA element - Google Patents
Tetracycline-induced artificial MicroRNA element Download PDFInfo
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- CN105331616B CN105331616B CN201510690643.8A CN201510690643A CN105331616B CN 105331616 B CN105331616 B CN 105331616B CN 201510690643 A CN201510690643 A CN 201510690643A CN 105331616 B CN105331616 B CN 105331616B
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Abstract
The invention discloses a tetracycline-induced artificial MicroRNA element, the nucleotide sequence of which is shown as SEQ NO 1 or SEQ NO 2. The artificial microRNA element can be used for targeting genes of different bladder cancer cell lines, such as a beta-catenin gene, and in the case, the sequence is shown as SEQ NO:1 is shown in the specification; or HIF-1 alpha gene, wherein the sequence is as shown in SEQ NO:2, respectively. The artificial element can effectively inhibit the expression of a target gene in a bladder cancer cell line, can effectively inhibit the proliferation of the bladder cancer cell, promotes the apoptosis of the bladder cancer cell and inhibits the migration of the bladder cancer cell, thereby providing possibility for treating the bladder cancer.
Description
Technical Field
The present application relates to the field of bladder cancer detection and treatment.
Background
RNA interference (RNAi) is known as one of the ten scientific discoveries in the 20 th century, and is a degradation process of homologous mRNA induced by micro RNA (microrna), which can inhibit gene expression. microRNA-based RNA interference therapy may be a safer and more effective tumor therapy. However, the use of natural microRNA is prone to disadvantages such as limited target gene, easy degradation in vivo, short duration of drug action, and easy initiation of interferon response.
The artificial microRNA takes a natural microRNA precursor short hairpin structure as a basic skeleton, replaces a functional sequence of the natural microRNA with an antisense sequence of a target gene which is artificially designed, and obtains the artificial microRNA which has the same structure with the natural microRNA but is completely unrelated to the target sequence of the natural microRNA. The artificial microRNA is generally generated by transcription and processing of an expressive vector, and the generation mechanism and the action path of the artificial microRNA are the same as those of natural microRNA. Compared with natural microRNA, the artificial microRNA has the advantages of low expression cost, high interference efficiency, long duration, low toxicity and the like. The tetracycline switch system gene expression system is a newly developed gene expression system with high efficiency, no toxicity and strict switch function, and has been widely applied in many aspects, especially in gene expression regulation and gene therapy.
With the rapid development of the new science of synthetic biology and the continuous maturation of the engineering principle and technology of synthetic biology, the development of efficient, quantitative and controllable RNA interference tumor treatment becomes possible.
Disclosure of Invention
The invention provides a novel artificial MicroRNA element.
The invention provides a tetracycline-induced artificial MicroRNA element, the nucleotide sequence of which is shown as SEQ NO 1 or SEQ NO 2.
The artificial microRNA element can be used for targeting genes of different bladder cancer cell lines, such as a beta-catenin gene, and in the case, the sequence is shown as SEQ NO:1 is shown in the specification; or HIF-1 alpha gene, wherein the sequence is as shown in SEQ NO:2, respectively.
The application of the element in preparing a reagent for inhibiting the expression of the beta-catenin gene in a bladder cancer cell line is disclosed, and the nucleotide sequence is shown as SEQ NO: 1.
The application of the element in preparing a reagent for inhibiting HIF-1 alpha gene expression in a bladder cancer cell line, wherein the nucleotide sequence is shown as SEQ NO. 2.
The target gene is highly expressed in the bladder cancer cell line, and the expression of the target gene can be effectively inhibited through the artificial element.
The use of one of said elements in the manufacture of a medicament for inhibiting the migration of bladder cancer cells.
The bladder cancer cells were T24 and 5637.
The application of the element in preparing a medicament for promoting the apoptosis of bladder cancer cells.
A non-therapeutic method of promoting apoptosis in a bladder cancer cell, said bladder cancer cell transfected with said element.
The use of one of said elements for the preparation of a medicament for the treatment of bladder cancer.
The use of said element for regulating the expression of a targeted gene in bladder cancer cells.
The target genes are HIF-1 alpha gene and beta-catenin gene.
The invention has the beneficial effects that: the artificial element can effectively inhibit the expression of a target gene in a bladder cancer cell line, can effectively inhibit the proliferation of the bladder cancer cell, promotes the apoptosis of the bladder cancer cell and inhibits the migration of the bladder cancer cell, thereby providing possibility for treating the bladder cancer.
Drawings
FIG. 1 is a schematic diagram showing the construction of a tetracycline-inducible artificial MicroRNA element according to this embodiment;
FIG. 2 is a graph showing the results of quantitative and controllable inhibition of the expression of the related oncogenes by the tetracycline-inducible artificial MicroRNA element of the present embodiment, wherein in section A, B, C, D, the five square bars from left to right represent 0.0. mu.g/ml Dox, 0.1. mu.g/ml Dox, 0.5. mu.g/ml Dox, 2. mu.g/ml Dox, and 10. mu.g/ml Dox, respectively;
FIG. 3 is a diagram showing the results of the tetracycline-inducible artificial MicroRNA element of the present embodiment capable of effectively inhibiting the expression of the target gene downstream of its target gene, wherein in section A, B, the four square columns from left to right represent NC (Dox-), NC (Dox +), β -catenin (Dox-), β -catenin (Dox +); C. in section D, the four square columns from left to right represent NC (Dox-), NC (Dox +), HIF-1 α (Dox-), HIF-1 α (Dox +);
FIG. 4 is a graph showing the results of the tetracycline-inducible artificial MicroRNA element of this embodiment effectively inhibiting bladder cancer cell proliferation;
FIG. 5 is a graph showing the results of the tetracycline-inducible artificial MicroRNA element of the present embodiment effectively inducing apoptosis of bladder cancer cells, in section E, F, the four square bars from left to right represent NC (Dox-), NC (Dox +), β -catenin (Dox-) (or HIF-1 α (Dox-)), β -catenin (Dox +) (or HIF-1 α (Dox +));
FIG. 6 is a graph showing the results of inhibition of migration and motility of bladder cancer cells by the tetracycline-inducible artificial MicroRNA element of the present embodiment, wherein the four squares at the bottom left side from left to right represent NC (Dox-), NC (Dox +), β -catenin (Dox-), β -catenin (Dox +); the four square columns at the bottom right side from left to right represent NC (Dox-), NC (Dox +), HIF-1 alpha (Dox-), HIF-1 alpha (Dox +), respectively.
Detailed Description
Techniques and methods
1. Cell culture
Bladder cancer cell lines T24 and 5637 were purchased from American Type Culture Collection (ATCC, Manassas, USA), and cell Culture broth was formulated from 90% DMEM (Invitrogen, CA), 10% fetal bovine serum (Invitrogen), 1% -2% glutamine, and 0.5% -1% diabody.
2. Construction of tetracycline-induced artificial MicroRNA element
The natural MicroRNA-30 is used as a basic framework, and the functional sequence of mature MicroRNA is replaced by the interference sequence of the selected target gene, so that the artificial MicroRNA of the specific target gene is obtained. Meanwhile, in order to enable the transcribed artificial MicroRNA to be processed and modified more accurately and effectively, ribozyme sequences named RGR are added at two ends of the designed artificial MicroRNA. The transcribed ribozyme RGR fragment can be modified by self-cleavage, so as to help the intermediate target fragment form an effective spatial structure. And then, introducing the constructed artificial MicroRNA into tetracycline-inducible plasmids to obtain a synthetic element capable of generating the tetracycline-inducible artificial MicroRNA.
3. Cell transfection
Day before transfection, 4-5X 104Inoculating the cells on 24-well plate, adding 0.5ml culture medium, placing at 37 deg.C and 5% CO2In the incubator. After 24 hours of growth, the cell confluence reached 70%, 1ug of recombinant plasmid was added to 50ul of serum-free medium and mixed gently. 1ul Lipofectamin2000(Invitrogen, CA) reagent was added to 50ul of serum-free medium, mixed gently, and left at room temperature for 5 min. The diluted plasmid and Lipo2000 were gently mixed wellAnd left at room temperature for 20 minutes to allow the formation of plasmid/lipofectamin complex. The plasmid/lipofectamin mixture was added to 24-well plates and the 24-well plates were gently shaken. Placing the culture medium into an incubator, replacing the culture medium after 4-6 hours, collecting cells after 48 hours, and preparing for the next experiment.
4. Total RNA extraction
Total RNA was extracted using Trizol reagent from Invitrogen, USA. The quality of the extracted total RNA was checked using an ultraviolet spectrophotometer and 2% agarose gel. After quantification, the mixture is stored at-80 ℃ for later use.
5. Real-time quantitative PCR
The real-time quantitative PCR detection selects U6(snRNA U6) as the endogenous control gene. Real-time reverse transcription synthesis of cDNA and quantitative PCR validation were performed using the All-in-one miRNA qRT-PCR Detection Kit (GeneCopoea Inc, MD, USA). First, miRNA reverse transcription is carried out to synthesize cDNA. The reverse transcription Reaction system was total RNA 1. mu.l, 2.5U/. mu.l Poly A Polymerase 1. mu.l, RTase Mix 1. mu.l, 5 × Reaction buffer 5. mu.l, dd H2O (RNase-/DNase free) 17. mu.l. The cells were centrifuged instantaneously, incubated at 37 ℃ for 60min and then enzyme-inactivated at 85 ℃ for 5 min. The real-time quantitative PCR total reaction system was 20. mu.l, including 10. mu.l of 2xAll-in-one (TM) qPCR Mix, 2. mu.l of Universal adapter PCR Primer (2. mu.M), 2. mu.l of All-in-one (TM) qPCR Primer (2. mu.M), 2. mu.l of First-Strand cDNA (diluted in 1:5),50X ROX Reference Dye 0.4. mu.l and 3.6. mu.l of double distilled water. The qPCR reaction and analysis were performed using ABI PRISM 7000 Fluorescent Quantitative PCR System (Applied Biosystems, CA, USA). The PCR reactions were all 3 replicates. The PCR reaction parameters are as follows, (1) pre-denaturation at 95 ℃ for 15 min; (2)40 cycles, each cycle comprising 95 ℃ x15s, 55 ℃ x20s, 70 ℃ x30 s. All-in-OneTMThe miRNA qPCR Primer has the following cargo number: has-miR-210, HmirQP 0317; snRNAU6, HmiRQP 9001. Taking the median of 3 repeats to calculate the relative miRNA expression (delta Ct ═ Ct)medianmiRNA﹣CtmediansnRNAU6). For fold expression 2-ΔΔCtThe method is carried out.
6. Cell proliferation assay
Cell proliferation was detected using the Cell Counting Kit (CCK-8), 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfophenyl) -2H-tetrazole monosodium salt. A single cell suspension was prepared from a culture medium containing 10% fetal calf serum, and the cells were seeded in 96-well plates in a volume of 200. mu.l per well at 5000 cells per well. Corresponding wells were taken for detection at 24, 48 and 72h post-transfection, respectively. Each sample was provided with 3 multiple wells. 10. mu.l of CCK-8 solution (5mg/ml) was added to each well. Incubate for 1h, terminate the culture. The 450nm wavelength was selected, the absorbance of each well was measured using a microplate reader (Bio-Rad, USA), the results were recorded, and the cell growth curve was plotted with time as abscissa and absorbance as ordinate.
Using 5-ethyl-2' -deoxyuridine (Edu), a thymidine analog is capable of penetrating replicating DNA molecules during cell proliferation in place of thymine (T) by being based on the reaction of EdU with
The specific reaction of the fluorescent dye rapidly detects cell proliferation. Single cell suspension was prepared from 10% fetal calf serum in culture medium at 2X 10 per well4The individual cells were seeded in 12-well plates in 1ml volumes per well. Each sample was provided with 3 multiple wells. At 48h after transfection, the corresponding wells were taken for detection. Add 500. mu.L of 50. mu.M EdU medium to each well and incubate for 2 hours, and aspirate the culture supernatant from the wells. Then adding 500 mu L of cell fixing solution (namely PBS containing 4% paraformaldehyde) into each hole, incubating for 30 minutes at room temperature, and removing the fixing solution; next 500. mu.L of 1 Xs were added per well
Incubating the dyeing reaction liquid for 30 minutes in a dark place at room temperature by using a decoloring shaker, and then discarding the dyeing reaction liquid; finally, 500. mu.L of 1X Hoechst33342 reaction solution is added into each hole, and after incubation for 30 minutes in a dark place at room temperature and a decoloration shaking table, the dyeing reaction solution is discarded. Fluorescence photographs were taken with a fluorescence microscope (olympus, japan).
7. Apoptosis detection
Apoptosis was detected using the Hoechst Staining Kit (Hoechst33342) Staining. Single cell suspension was prepared from 10% fetal calf serum in culture medium at 2X 10 per well4The individual cells were seeded in 12-well plates in 1ml volumes per well. Each sample was provided with 3 multiple wells. At 48h after transfection, the corresponding wells were taken for detection. The culture was terminated and the culture medium was discarded. Then theAdding 500 μ L of cell fixing solution (PBS containing 4% paraformaldehyde) into each well, incubating at room temperature for 30min, and discarding the fixing solution; next, 500. mu.L of 1X Hoechst33342 reaction solution was added to each well, and after incubating for 30 minutes in a dark place at room temperature in a decolorized shaker, the staining reaction solution was discarded. Cells were washed 3 times with PBS and fluorescence photographs were taken with a fluorescence microscope (olympus, japan).
Apoptosis was detected using an enzyme-linked immunosorbent assay (ELISA). Single cell suspension was prepared from 10% fetal calf serum in culture medium at 2X 10 per well4The individual cells were seeded in 12-well plates in 1ml volumes per well. Each sample was provided with 3 multiple wells. At 48h after transfection, the corresponding wells were taken for detection. The culture was terminated and the culture medium was discarded. Then adding 500 μ L pancreatin per well to digest cells thoroughly, adding culture solution to stop digestion, collecting cells and 1.5ml EP tube, placing in a refrigerator at-80 deg.C for 30min, placing in a water bath kettle at 37 deg.C for 30min, and repeatedly freezing and thawing for 3 times. The cell fluid was placed in an ELISA chamber, incubated at 37 ℃ for 30min and discarded. 90ul of the reaction solution was added, incubated at 37 ℃ for 30min, and 60ul of stop solution was added. The 450nm wavelength was selected, and the absorbance of each well was measured using a microplate reader (Bio-Rad, USA) and the results were recorded.
8. Cell migration assay
The cell migration motility was examined by cell scratch test. Single cell suspension was prepared from 10% fetal calf serum in culture medium at 5X 10/well5The individual cells were seeded in 6-well plates, 2ml per well volume. Each sample was provided with 3 multiple wells. At 48h after transfection, the corresponding wells were taken for detection. Firstly, a marker pen is used at the back of the 6-hole plate, and then the 6-hole plate is touched by a ruler, so that transverse lines are uniformly drawn and cross through the holes approximately every 0.5-1 cm. Each hole passes through at least 5 lines. About 5X10 was added to the wells5After the cells are full, the tip is used for comparing with the ruler, and the scratches are made on the transverse line at the back as much as possible. Cells were washed 3 times with PBS, the scraped cells were removed, and serum-free medium was added. Put in 37 ℃ 5% CO2And (5) an incubator for culture. Samples were taken at 0, 24 hours and photographed.
The cell migration motility was examined by cell scratch test. Single cell suspension was prepared from 10% fetal calf serum in culture medium at 5X 10/well5Seeding of individual cells to 6Well plates, 2ml per well volume. Each sample was provided with 3 multiple wells. At 48h after transfection, the corresponding wells were taken for detection. After digestion with pancreatin, each experiment and control group was taken 5X103And adding the cells into a transwell chamber, and discarding the culture solution after 20h to detect the migration condition of the cells. The gel was stained with crystal violet for 30min, washed with PBS 3 times, freed of excess dye, air dried and photographed.
9. Statistical analysis
Data analysis was performed using SPSS17.0 statistical software, with data for normal distributions represented by x + -s. The comparison between the two groups is carried out,
independent sample t-test was used. The test level, P <0.05, indicates that the difference is statistically significant.
Results of the experiment
1. Construction of tetracycline-induced artificial MicroRNA element
Aiming at different target genes, firstly designing antisense sequences of RNAi target sequences; the natural MicroRNA-30 is used as a basic framework, and the functional sequence of mature MicroRNA is replaced by the interference sequence of the selected target gene, so that the artificial MicroRNA of the specific target gene is obtained. Meanwhile, in order to enable the transcribed artificial MicroRNA to be processed and modified more accurately and effectively, ribozyme sequences named RGR are added at two ends of the designed artificial MicroRNA. The transcribed ribozyme RGR fragment can be modified by self-cleavage, so as to help the intermediate target fragment form an effective spatial structure. And then, introducing the constructed artificial MicroRNA into tetracycline-inducible plasmids to obtain a synthetic element capable of generating the tetracycline-inducible artificial MicroRNA. Xho1 and Not1 cleavage sites were added at both ends of the knock-down element, and subcloned into the ORF framework of the PEV-Lv208 vector (as shown in FIG. 1). The relevant sequences are shown in Table 1.
TABLE 1 Tetracycline-inducible artificial MicroRNA element and cDNA sequence listing of negative control thereof
In table 1, the bold font is the ribozyme sequence, the underlined portion is the miRNA backbone, and the italic portion is the targeting sequence.
2. The tetracycline-induced artificial MicroRNA element can effectively reduce the expression level of corresponding targeted genes in bladder cancer cell lines
Respectively transfecting the constructed tetracycline-inducible artificial MicroRNA element expression vector and a negative control vector thereof into bladder cancer cell lines T24 and 5637, respectively carrying out Dox treatment with different doses, and detecting the expression quantity of corresponding targeted genes after 48 hours: comparing the treatment results of different doses in different experimental groups and control groups, the expression of the corresponding target gene in the cells after the treatment of different doses of Dox in the experimental groups is inhibited to different degrees (as shown in figure 2), which indicates that the element can quantitatively and effectively inhibit the expression of the corresponding target gene in a controllable manner. Meanwhile, compared with the control group, the expression of the downstream gene of the target gene of the experimental group after Dox treatment is also obviously inhibited, and the fact that the element can effectively inhibit the expression of the target gene is shown in figure 3.
4. The tetracycline-induced artificial MicroRNA element can effectively inhibit the proliferation of bladder cancer cell lines
And (3) transfecting the constructed tetracycline-inducible artificial MicroRNA element expression vector and a negative control vector thereof into bladder cancer cell lines T24 and 5637 respectively, and detecting the proliferation activity of the cells after 24, 48 and 72 hours: compared with a negative control group, the cell proliferation of the tetracycline-induced artificial MicroRNA element transfection group is obviously inhibited (as shown in FIG. 4), which indicates that the element can effectively inhibit the growth of cancer cells by inhibiting the expression of related oncogenes.
5. The tetracycline-induced artificial MicroRNA element can effectively promote the apoptosis of bladder cancer cells
To further confirm that the inhibition of the growth of the bladder cells by the tetracycline-inducible artificial microRNA element is realized by promoting the apoptosis of the cells, the constructed tetracycline-inducible artificial microRNA element expression vector and a negative control vector thereof are respectively transfected into bladder cancer cell lines T24 and 5637, and the apoptosis rate of the cells is detected after 48 hours: compared with a negative control group, the apoptosis rate of the tetracycline-induced artificial MicroRNA element transfection group is obviously increased (as shown in FIG. 5), and the fact that the element can effectively induce the apoptosis of cancer cells by inhibiting the expression of related oncogenes is shown.
6. The tetracycline-induced artificial MicroRNA element can effectively inhibit the migration of bladder cancer cells
Respectively transfecting the constructed tetracycline-inducible artificial MicroRNA element expression vector and a negative control vector thereof into bladder cancer cell lines T24 and 5637, and detecting the migration distance of the cells after 24 hours: compared with a negative control group, the cell migration process of the tetracycline-induced artificial MicroRNA element transfection group is obviously inhibited (as shown in FIG. 6), which indicates that the element can effectively inhibit the migration movement capability of cancer cells by inhibiting the expression of related oncogenes.
The foregoing is a more detailed description of the present invention that is presented in conjunction with specific embodiments, and the practice of the invention is not to be considered limited to those descriptions. It will be apparent to those skilled in the art that a number of simple derivations or substitutions can be made without departing from the inventive concept.
Claims (7)
1. A tetracycline-inducible artificial MicroRNA element is characterized in that the nucleotide sequence of the element is shown as SEQ NO 1 or SEQ NO 2.
2. An application of a tetracycline-induced artificial MicroRNA element in preparing a reagent for inhibiting the expression of a beta-catenin gene in a bladder cancer cell line, wherein the nucleotide sequence of the element is shown as SEQ NO: 1.
3. An application of a tetracycline-induced artificial MicroRNA element in preparing a reagent for inhibiting HIF-1 alpha gene expression in a bladder cancer cell line, wherein the nucleotide sequence of the element is shown as SEQ NO. 2.
4. Use of an element according to claim 1 for the preparation of a medicament for inhibiting migration of bladder cancer cells.
5. The use of claim 4, wherein the bladder cancer cell is T24 and 5637.
6. Use of an element according to claim 1 in the manufacture of a medicament for promoting apoptosis in bladder cancer cells.
7. A method of promoting apoptosis in bladder cancer cells for non-therapeutic purposes, wherein the bladder cancer cells are transfected with the element of claim 1.
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| Lentiviral miR30-based RNA interference against heparanase suppresses melanoma metastasis with lower liver and lung toxicity;Xiao-yan Liu等;《Int J Biol Sci》;20130612;第9卷(第6期);第564-577页 * |
| Synthetic artificial microRNAs targeting UCA1-MALAT1 or c-Myc inhibit malignant phenotypes of bladder cancer cells T24 and 5637;Xing Fu等;《Mol Biosyst》;20150531;第11卷(第5期);第1285-1289页 * |
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