CN107354159B - Application of siRNA of long-chain non-coding RNA SMAD5-AS1 in ovarian cancer treatment - Google Patents
Application of siRNA of long-chain non-coding RNA SMAD5-AS1 in ovarian cancer treatment Download PDFInfo
- Publication number
- CN107354159B CN107354159B CN201710657458.8A CN201710657458A CN107354159B CN 107354159 B CN107354159 B CN 107354159B CN 201710657458 A CN201710657458 A CN 201710657458A CN 107354159 B CN107354159 B CN 107354159B
- Authority
- CN
- China
- Prior art keywords
- ovarian cancer
- smad5
- sirna
- seq
- lncrna
- 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.)
- Expired - Fee Related
Links
- 206010033128 Ovarian cancer Diseases 0.000 title claims abstract description 67
- 206010061535 Ovarian neoplasm Diseases 0.000 title claims abstract description 63
- 101000689342 Homo sapiens SMAD5 antisense gene protein 1 Proteins 0.000 title claims abstract description 27
- 102100024488 SMAD5 antisense gene protein 1 Human genes 0.000 title claims abstract description 27
- 108020004459 Small interfering RNA Proteins 0.000 title claims abstract description 22
- 238000011282 treatment Methods 0.000 title claims abstract description 13
- 108091027963 non-coding RNA Proteins 0.000 title abstract description 5
- 102000042567 non-coding RNA Human genes 0.000 title abstract description 5
- 239000003814 drug Substances 0.000 claims abstract description 8
- 238000013508 migration Methods 0.000 claims abstract description 8
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 7
- 230000005012 migration Effects 0.000 claims abstract description 7
- 230000009545 invasion Effects 0.000 claims abstract description 5
- 230000035755 proliferation Effects 0.000 claims abstract description 5
- 108091046869 Telomeric non-coding RNA Proteins 0.000 claims description 46
- 230000004048 modification Effects 0.000 claims description 8
- 238000012986 modification Methods 0.000 claims description 8
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 6
- 150000007523 nucleic acids Chemical group 0.000 claims description 6
- 239000000825 pharmaceutical preparation Substances 0.000 claims description 5
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 claims description 2
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 claims description 2
- 241000700605 Viruses Species 0.000 claims description 2
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 claims description 2
- 235000012000 cholesterol Nutrition 0.000 claims description 2
- 229940079593 drug Drugs 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 239000002502 liposome Substances 0.000 claims description 2
- 239000002105 nanoparticle Substances 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 claims description 2
- 239000008194 pharmaceutical composition Substances 0.000 claims 3
- 238000009472 formulation Methods 0.000 claims 1
- 239000007972 injectable composition Substances 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 239000000203 mixture Substances 0.000 claims 1
- 238000011321 prophylaxis Methods 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 6
- 238000011161 development Methods 0.000 abstract description 3
- 230000005764 inhibitory process Effects 0.000 abstract description 3
- 230000002194 synthesizing effect Effects 0.000 abstract 1
- 230000008685 targeting Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 28
- 206010028980 Neoplasm Diseases 0.000 description 23
- 201000011510 cancer Diseases 0.000 description 16
- 210000001519 tissue Anatomy 0.000 description 16
- 238000002474 experimental method Methods 0.000 description 12
- 108090000623 proteins and genes Proteins 0.000 description 12
- 230000001105 regulatory effect Effects 0.000 description 7
- 108020005198 Long Noncoding RNA Proteins 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 238000001890 transfection Methods 0.000 description 6
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 238000003753 real-time PCR Methods 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 4
- 208000007571 Ovarian Epithelial Carcinoma Diseases 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 3
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 3
- 230000033228 biological regulation Effects 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 230000004663 cell proliferation Effects 0.000 description 3
- 230000034994 death Effects 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 230000036210 malignancy Effects 0.000 description 3
- 239000003607 modifier Substances 0.000 description 3
- 238000003068 pathway analysis Methods 0.000 description 3
- 230000019491 signal transduction Effects 0.000 description 3
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 3
- CCEKAJIANROZEO-UHFFFAOYSA-N sulfluramid Chemical group CCNS(=O)(=O)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)F CCEKAJIANROZEO-UHFFFAOYSA-N 0.000 description 3
- 230000003827 upregulation Effects 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 2
- 210000002469 basement membrane Anatomy 0.000 description 2
- 238000003766 bioinformatics method Methods 0.000 description 2
- 230000012292 cell migration Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000007405 data analysis Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 108010082117 matrigel Proteins 0.000 description 2
- 210000004379 membrane Anatomy 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000002547 new drug Substances 0.000 description 2
- 230000002611 ovarian Effects 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 230000001124 posttranscriptional effect Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000006748 scratching Methods 0.000 description 2
- 230000002393 scratching effect Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 239000012124 Opti-MEM Substances 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 101150094827 SMAD5-AS1 gene Proteins 0.000 description 1
- 108700025695 Suppressor Genes Proteins 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 210000001766 X chromosome Anatomy 0.000 description 1
- 230000001594 aberrant effect Effects 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000003491 array Methods 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 208000015606 cardiovascular system disease Diseases 0.000 description 1
- 230000004709 cell invasion Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 230000001973 epigenetic effect Effects 0.000 description 1
- 230000006718 epigenetic regulation Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000002657 hormone replacement therapy Methods 0.000 description 1
- 235000003642 hunger Nutrition 0.000 description 1
- 230000000887 hydrating effect Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 238000010232 migration assay Methods 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 230000037443 ovarian carcinogenesis Effects 0.000 description 1
- 231100001249 ovarian carcinogenesis Toxicity 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 230000000379 polymerizing effect Effects 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 210000004994 reproductive system Anatomy 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000011255 standard chemotherapy Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
- C12N2310/141—MicroRNAs, miRNAs
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Oncology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses application of siRNA of long-chain non-coding RNA (lncRNA) SMAD5-AS1 in ovarian cancer treatment. According to the invention, by designing and synthesizing a siRNA sequence for specifically targeting and inhibiting SMAD5-AS1 and transfecting the siRNA sequence into an ovarian cancer cell strain, the inhibition of SMAD5-AS1 expression is proved to be capable of obviously inhibiting the proliferation, migration and invasion capacity of ovarian cancer cells. The invention provides a new method and a new medicine development direction for treating ovarian cancer.
Description
Technical Field
The invention relates to application of siRNA of LncRNA SMAD5-AS1 in ovarian cancer treatment, belonging to the technical field of biological engineering.
Background
Ovarian malignancies are the leading cause of death in patients with gynecological reproductive system malignancies and also the fifth cause of death in women (CA Cancer J Clin.2017; 67(1): 7-30). Recent data from the U.S. research show that 225500 new cases of ovarian cancer diagnosis are added every year worldwide, and the fatality rate is as high as 62%. Despite the decline in ovarian cancer incidence over the past few decades due to changes in hormone replacement therapy, there has been no significant progress in the research of diagnosis and treatment of ovarian cancer. Epithelial Ovarian Cancer (EOC), commonly known as ovarian cancer, accounts for approximately 90% of ovarian malignancies, is difficult to diagnose early in the united states because of its unobvious early symptoms and lack of effective early screening, and 2016, national institute for cancer research indicates that cases above 2/3 in the united states have progressed to late when diagnosed with ovarian cancer, with often poor prognosis for late stage ovarian cancer, with a 5-year survival rate of 92.1% for stage I ovarian cancer and only 25% for stage III IV (Eur J cancer.2015), and thus ovarian cancer is often referred to by the name "silent killer". At present, the main treatment mode of ovarian cancer is tumor cell debulking operation combined with standard chemotherapy scheme based on platinum, the operation can achieve the maximum tumor debulking, but some metastatic foci cannot be thoroughly solved through the operation. Although the early effective rate of chemotherapy is high, most ovarian cancer patients have chemotherapy drug resistance, which is also the reason that the recurrence rate and the death rate of ovarian cancer are high, and the 5-year survival rate is only about 30%. Therefore, the search for a sensitive early diagnosis index of ovarian cancer and a more effective treatment mode is particularly important for saving the life of ovarian cancer patients.
In recent years, epigenetic studies have greatly expanded our understanding of the disease, where cancer occurs as a result of deletion or abnormal expression of proto-and suppressor genes, where epigenetic regulation plays an irreplaceable role (Nat Rev cancer. 2016; 16(11): 694-707). There is increasing evidence that a key factor in tumor induction is aberrant expression of multiple genes and the intergenic regulatory network. Long non-coding RNAs (lncRNAs) are non-coding RNAs with transcripts larger than 200bp and can exert their biological functions in various ways (Nat Rev Genet.2016; 17(10): 601: 614). A great deal of research finds that lncRNAs are involved in a plurality of important regulation processes such as post-transcriptional activation, X chromosome silencing, post-transcriptional interference and the like, the regulation effects are widely concerned in recent years, 4% -9% of transcripts in mammalian genome sequences are lncRNA, the proportion of protein coding RNA only accounts for 1%, but the functions of most of lncRNA are not disclosed (Nat Rev Genet.2016; 17(1): 47-62).
More importantly, most lncRNAs are tissue specific compared to mRNA encoding the protein, and only about 1% of lncRNAs are expressed in every tissue throughout the body. Recent research results show that the expression or function abnormality of lncRNAs is closely related to the occurrence of human diseases, including serious diseases such as tumor, cardiovascular system diseases, and sexual neurodegenerative diseases, which seriously endanger human health (Nat Rev Genet.2016; 17(10): 601-. To investigate the potential role of lncRNAs in ovarian carcinogenesis, we first examined lncRNAs expression profiles in cancer tissues of epithelial ovarian cancer patients (table 1) using gene chip technology (Arraystar Human LncRNA microarray V3.0). And selecting lncRNAs with the increase or decrease of more than 2 times as a differential expression gene. Thus, we screened 1945 lncRNAs that were significantly elevated and 1582 different lncRNAs that were significantly reduced in ovarian cancer patients, as shown by the hierarchical cluster map (fig. 1) and volcano map (fig. 2), while we found that these aberrantly expressed lncRNAs were associated with multiple tumor signaling pathways (fig. 3). Then, we verified a group of lncRNAs that were significantly up-regulated in the chip results using quantitative PCR, including FOXN3-AS1, CATIP-AS2, RP11-124N14.3, SMAD5-AS1 (FIG. 4). Notably, the results of 6.7-fold upregulation of SMAD5-AS1 in cancer tissues and 6.0-fold upregulation in serum of patients with ovarian cancer indicate that SMAD5-AS1 plays a potentially important role in ovarian cancer, while it is unknown whether SMAD5-AS1 plays a role in disease, and whether inhibition of expression of SMAD5-AS1 gene has a therapeutic effect on disease has not been studied. Based on the current situation of the existing gene therapy technology, the application synthesizes two siRNAs of LncRNA SMAD5-AS1, and the effect of SMAD5-AS1siRNA in inhibiting proliferation, migration and invasion of ovarian cancer cells is verified for the first time in the experimental process, so that a new drug target point is provided for the treatment of ovarian cancer, and a new idea is provided for the clinical diagnosis and treatment of ovarian cancer.
Disclosure of Invention
The invention aims to provide a novel method and a novel medicament for preventing and treating ovarian cancer.
In order to achieve the purpose, the invention adopts the following technical means:
the invention utilizes gene chip technology to detect the expression spectrum of lncRNAs in the cancer tissues of ovarian cancer patients. And (3) performing data analysis by using a bioinformatics analysis method, and selecting lncRNAs which are increased or decreased by more than 2 times as differential expression genes. Thus, 1945 lncRNAs that were significantly elevated and 1582 different lncRNAs that were significantly reduced in ovarian cancer patients were screened, and the results of pathway analysis associated with the different lncRNAs revealed that these abnormally expressed lncRNAs might be involved in regulating multiple ovarian cancer-associated signaling pathways. The invention applies real-time fluorescent quantitative PCR experiments to detect the expression levels of some lncRNAs which are obviously up-regulated in the blood serum of cancer tissues, tissues beside cancer, healthy patients and ovarian cancer patients of ovarian cancer patients, and the result shows that the lncRNA SMAD5-AS1 is obviously increased by 6.7 times in the cancer tissues of the ovarian cancer patients and 6 times in the blood serum of the ovarian cancer patients. Then, the invention respectively detects the influence of SMAD5-AS1siRNA-1 (shown in SEQ ID NO. 2) and SMAD5-AS1siRNA-2 (shown in SEQ ID NO. 3) on cell proliferation, migration and invasion capacity through a CCK8 experiment, a scarification experiment and a Transwell experiment, and the result shows that the siRNA-1 and the siRNA-2 can effectively inhibit the expression of SMAD5-AS1 in SKOV3 cells and have the effect of obviously inhibiting the development of ovarian cancer cells.
Therefore, on the basis of the research, the invention provides the application of the siRNA of LncRNA SMAD5-AS1 in the preparation of the medicine for preventing and treating ovarian cancer, and the sequence of the SMAD5-AS1 is shown AS SEQ ID NO. 1.
Preferably, the siRNA of LncRNA SMAD5-AS1 can inhibit the proliferation, migration and invasion of ovarian cancer cells to treat ovarian cancer.
Wherein, preferably, the sequence of the siRNA of LncRNA SMAD5-AS is shown AS SEQ ID NO.2 or SEQ ID NO. 3.
Furthermore, the invention also provides a pharmaceutical preparation for treating ovarian cancer, which comprises effective dose of siRNA of LncRNA SMAD5-AS or a nucleic acid sequence modifier thereof and a pharmaceutically acceptable carrier, wherein the sequence of the siRNA of LncRNA SMAD5-AS is shown AS SEQ ID NO.2 or SEQ ID NO. 3.
Wherein, the preferred nucleic acid sequence modifier is a nucleic acid sequence modifier obtained by performing one or a combination of several modifications of ribose modification, base modification and phosphate backbone modification of any nucleotide on the basis of the sequence shown in SEQ ID NO.2 or SEQ ID NO. 3; the carrier is virus, nano-particles, cholesterol or liposome.
Wherein, preferably, the pharmaceutical preparation is an injection preparation and an oral preparation.
Furthermore, the invention also provides the application of the pharmaceutical preparation in preparing the medicines for preventing and treating ovarian cancer.
The invention provides a new drug target and a new treatment mode for treating ovarian cancer.
Drawings
FIG. 1 is a hierarchical cluster chart of the data on the chip showing the expression of lncRNAs in cancer and paracancerous tissues of patients with ovarian cancer;
FIG. 2 is a volcano chart showing the difference in expression of lncRNAs between cancer tissues and paracarcinoma tissues of ovarian cancer patients;
wherein the vertical gray lines indicate a 2-fold increase or more (positive x-axis) and a 50% decrease or more (negative x-axis), respectively, and the horizontal gray lines indicate a statistical difference in p-value of 0.05. The black dots on the right represent genes whose expression is significantly upregulated; the left gray point represents a gene whose expression is significantly down-regulated;
FIG. 3 is a pathway analysis relating to differential lncRNAs;
fig. 4 shows the expression levels of lncRNAs significantly up-regulated in the data of the real-time fluorescence quantitative PCR detection chip in tissues and serum of ovarian cancer patients (n ═ 8); p <0.01, p <0.001, indicating that the difference is statistically significant;
fig. 5 shows that CCK8 experiment detects cell proliferation and viability (n-3) of each group; p <0.001vs SiRNA-NC, indicating that the difference is statistically significant;
fig. 6 shows that the cell scratch test detects the migration ability of each group of cells (n is 3); p <0.01vs SiRNA-NC, indicating that the difference is statistically significant;
FIG. 7 shows the ability of each group of cells to migrate and invade by Transwell assay (n is 3); p <0.001vs SiRNA-NC, indicating that the difference is statistically significant.
Detailed Description
The invention is further illustrated and verified by the following examples, all of which are intended to be illustrative only and not limiting to the scope of the invention. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.
Example 1 demonstration of upregulation of LncRNA SMAD5-AS1 expression in ovarian cancer
We examined the expression profiles of lncRNAs in cancer tissues of ovarian cancer patients (Table 1) using GeneChip Exon 1.0 ST Arrays from Affymetrix, Inc. The lncRNA chip comprises transcripts annotated as lncRNA from an authorized power database such as RNAdb, Refseq, NCBI Gene, UCSC Gene and the like, and the data are comprehensive and reliable. A bioinformatics analysis method is used for data analysis, and lncRNAs which are increased or decreased by more than 2 times are selected as differential expression genes. Thus, we screened 1945 lncRNAs that were significantly elevated and 1582 different lncRNAs that were significantly reduced in ovarian cancer patients, with the results shown in the hierarchical cluster map (fig. 1) and volcano map (fig. 2). We also performed pathway analysis related to differential lncRNAs. The results revealed that these abnormally expressed lncRNAs might be involved in the regulation of multiple ovarian cancer-associated signaling pathways (fig. 3).
TABLE 1 chip detection of basic information of ovarian cancer patients
Next, we performed real-time fluorescence quantitative PCR experiments to detect expression levels of some lncRNAs that were significantly up-regulated in the sera of ovarian cancer patients, cancer tissues near the cancer tissues, healthy patients, and ovarian cancer patients, and the results showed that lncRNA SMAD5-AS1 were significantly increased 6.7 times in the cancer tissues of ovarian cancer patients and 6 times in the sera of ovarian cancer patients (fig. 4).
Example 2 demonstration that LncRNA SMAD5-AS1siRNA-1, siRNA-2 inhibit ovarian cancer cell progression
We cultured human epithelial ovarian cancer cell line SKOV3 for experiment, and the conventional culture was performed using RPMI 1640 medium containing 10% FBS at 37 deg.C and 5% CO2Culturing in a saturated humidity incubator. The grouping is as follows: SMAD5-AS1siRNA-1 (shown in SEQ ID NO. 2) transfection group, SMAD5-AS1siRNA-2 (shown in SEQ ID NO. 3) transfection group and siRNA-NC (shown in SEQ ID NO. 4) transfection group. Real-time fluorescent quantitative PCR detection of expression of SMAD5-AS1 in each group of cellsAnd (4) horizontal.
The cell transfection method comprises the following steps: the cells adherent to 60-80% were replaced with serum-free medium, X-tremagene siRNA transfection reagent and siRNAs were diluted with Opti-MEM, mixed and incubated at room temperature for 15-20 minutes, transfection complex was added to the cell culture supernatant to give a final concentration of 100nM, complete medium was replaced 6 hours after transfection, and the experiment was continued for 24-48 h.
The CCK8 experiment detects cell proliferation and activity: groups of cells in logarithmic growth phase were seeded in 96-well cell culture plates (5X 10 per ml)4Cells), 100. mu.l per well. After 24h of culture, adding 10 mul of CCK solution into each hole, continuously incubating for 4h after shaking and mixing uniformly, and measuring the absorbance at 450nm by using an enzyme-linked immunosorbent assay (ELISA) instrument after shaking and mixing uniformly.
Scratch test for cell migration ability: inoculating cells into a six-hole plate, scratching (0.5mm) by using a sterilized 10ul gun head when the cells reach 95% confluence, and immediately replacing a serum-free culture medium for culturing for 48 h; the degree of wound healing was observed by photographing under a phase contrast microscope at 0h and 48h after scratching, respectively. The scratch distance is 0h to 48 h.
The Transwell experiment detects the migration invasion capacity of cells: coating basement membrane, polymerizing Matrigel into gel, hydrating basement membrane. Starving the serum, adding 100 μ l of cell suspension into a Transwell chamber, staining with 0.1% crystal violet for 20min, and observing cell count; the cells were pretreated with 30. mu.g of Matrigel gel, 1X 10 of each group on top of the cell680ul of cells, adding cell culture solution to the lower part of the small chamber for culture; the upper chamber was removed, cells on the lower chamber membrane were fixed with 4% paraformaldehyde, stained with trypan blue, and the number of cells passing through the filtration membrane was counted under a light microscope.
The results show that siRNA-1 and siRNA-2 can effectively inhibit the expression of SMAD5-AS1 in SKOV3 cells, the inhibition efficiency reaches over 75 percent, compared with NC group, the transfected siRNA-1 and siRNA-2 respectively reduce the activity of SKOV3 cells to 62 percent and 58 percent, which indicates that two siRNAs can inhibit the proliferation of ovarian cancer cells by inhibiting the expression of SMAD5-AS1 (figure 5); cell scratch experiment results show that the migration capacity of SKOV3 cells is remarkably reduced after siRNA-1 and siRNA-248 h are transfected (FIG. 6); results of Transwell experiments show that transfection of siRNA-1 and siRNA-2 can significantly inhibit the invasive ability of SKOV3 (FIG. 7). The experimental results show that siRNA-1 and siRNA-2 can efficiently inhibit the expression of SMAD5-AS1 in SKOV3 cells, have the effect of remarkably inhibiting the development of ovarian cancer cells, and show that the two siRNAs can be used AS a new direction for developing ovarian cancer medicaments.
Sequence listing
<110> Harbin university of medicine
<120> application of siRNA of long-chain non-coding RNA SMAD5-AS1 in ovarian cancer treatment
<130> KLPI170522
<160> 4
<170> PatentIn 3.5
<210> 1
<211> 1827
<212> DNA
<213> SMAD5-AS1 sequence
<400> 1
tgatctgaaa ggaatggaag cacaaaatga tgaataaggt atttttaaca aagatacatg 60
ggtaaattaa cagcagtaat gtaaaaaaga ctgagggagc aacaatgtgg aaagggaagg 120
aaaggaagct gtataggaac aatcacacaa ataatcatca aaattaaact ttataaatct 180
aaacatagta tctacaacct ttatgaaggt taagttttgt taaattgtct aagttttgca 240
tttttacaat tttcactgta gagaaaaaaa tattaatagt taccaaagga ataaaagact 300
aaaataattt gttatacatc tgcaaatagc tccttaaatt tggaacacta atttcataat 360
gatatttgga tccataaatg tgaaactgtc acactgaatg taggtactgt tttccccaca 420
atggatacta ctgcattggc atctagcaga aattgtaagc tatttggaat gttactaaac 480
ctactgggac taaggttttc aatcttttgt ctttcaaatt aaaattaaaa actaattcac 540
tctgggcaat tcaggtactc taaggaaatt caagtactct agttttaaat acgaattttc 600
aaaaacgcaa cccaagtacc ctttcctctc aacttctgtg aggccaagcc acgacctcca 660
cctattcaat ctttgctgct agataacaac ccttttggta gtgctaggca acaggcatga 720
tgtgacaaac ctagacatca aaacttttcc acagaaagat aaggtaaaca agatatactt 780
agcgccacac ccatttaaat gcacaagcaa ccaaaattgc tccctccccc cgcaacaccc 840
cccccccccc cccaaagctc cagctggtca ggaaacattg tctttacaat caagataaac 900
atctggctca gggtgttcag ccattcctca cctacaggct tgcctaagcc ccattctcca 960
atgccatcac caccagaacc ggaacattct gttgggaagc cggcaaacgt ccaaataccc 1020
caagtatcct cgcccgagtt ctgcaaccaa aaatctgttt tggctactga gcatgctcag 1080
acttaagacg ggcgcgccac atgggagttt tcaaagctta ggctccaaac tccacgtagt 1140
aacagcagct gaaaggagga gaggggaaaa ggggagggga agagactgga atattaattt 1200
gagccctgat attgaagtat aggacccggc cagcggctga atatggtgaa ggcaaaaaga 1260
cgctgctttg gcattctctt tgcagcacag agaagtttac atgaagtaga agaaatcctt 1320
gcagtggtga aaactgacat ggcaagtggc agaaaccagg tctaactacc agctgattgc 1380
agtagccggt cacaactgcc cagagtgtaa ttcccaccga taagcgcgga tccttatctt 1440
tgaaacacaa agcgacggcg gcacggattc atcaagccat tggacagctt cgcagtgcag 1500
cagcccaggc tgcccggccg agcaccgcag tcctatcaag gtttgctgta gctcagaagg 1560
ctctgcctac atgactactg cataccctac tgggagaatt ggaagaatgt gatgatggta 1620
ggagtcaccc agcatagaag catgagtgaa tgacatccca gagatctgca aaggatcctg 1680
gtagccactt caaagacttt ttctcccttc ctcagacaat catggcacat ggacatcaga 1740
ggaagaaatg gcgaagtttg tatgactcct ccagcagagt ccttggaaac aataaagctt 1800
cctttccaaa aaaaaaaaaa aaaaaaa 1827
<210> 2
<211> 23
<212> DNA
<213> siRNA-1
<400> 2
ccgataagcg cggatcctta tct 23
<210> 3
<211> 23
<212> DNA
<213> siRNA-2
<400> 3
tggcagaaac caggtctaac tac 23
<210> 4
<211> 21
<212> DNA
<213> siRNA-NC
<400> 4
uucuccgaac gugucacgut t 21
Claims (7)
- The application of the siRNA of LncRNA SMAD5-AS1 in the preparation of drugs for preventing and treating ovarian cancer is characterized in that the sequence of the SMAD5-AS1 is shown AS SEQ ID NO. 1.
- 2. The use of claim 1, wherein the siRNA of LncRNA SMAD5-AS1 achieves the treatment of ovarian cancer by inhibiting proliferation, migration and invasion of ovarian cancer cells.
- 3. The use of claim 1, wherein the siRNA of LncRNA SMAD5-AS has the sequence AS shown in SEQ ID NO.2 or SEQ ID NO. 3.
- 4. A pharmaceutical preparation for treating ovarian cancer is characterized by comprising effective dose of siRNA of LncRNA SMAD5-AS or a nucleic acid sequence modification thereof and a pharmaceutically acceptable carrier, wherein the sequence of the siRNA of LncRNA SMAD5-AS is shown AS SEQ ID NO.2 or SEQ ID NO. 3.
- 5. The pharmaceutical preparation according to claim 4, wherein the modified nucleic acid sequence is a modified nucleic acid sequence obtained by modifying one or more of ribose modification, base modification and phosphate backbone modification of any nucleotide based on the sequence shown in SEQ ID NO.2 or SEQ ID NO. 3; the carrier is virus, nano-particles, cholesterol or liposome.
- 6. The pharmaceutical formulation of claim 4 or 5, wherein the pharmaceutical formulation is an injectable formulation or an oral formulation.
- 7. Use of a pharmaceutical formulation according to any one of claims 4 to 6 in the manufacture of a medicament for the prophylaxis or treatment of ovarian cancer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710657458.8A CN107354159B (en) | 2017-08-03 | 2017-08-03 | Application of siRNA of long-chain non-coding RNA SMAD5-AS1 in ovarian cancer treatment |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710657458.8A CN107354159B (en) | 2017-08-03 | 2017-08-03 | Application of siRNA of long-chain non-coding RNA SMAD5-AS1 in ovarian cancer treatment |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107354159A CN107354159A (en) | 2017-11-17 |
| CN107354159B true CN107354159B (en) | 2021-05-25 |
Family
ID=60288211
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710657458.8A Expired - Fee Related CN107354159B (en) | 2017-08-03 | 2017-08-03 | Application of siRNA of long-chain non-coding RNA SMAD5-AS1 in ovarian cancer treatment |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107354159B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112826826B (en) * | 2021-02-03 | 2023-03-17 | 上海兰天生物医药科技有限公司 | Application of siRNA sequence in preparation of medicine for treating ovarian cancer |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105085648A (en) * | 2015-05-04 | 2015-11-25 | 北京市肿瘤防治研究所 | Corresponding antigen of anti-ovarian cancer monoclonal antibody COC166-9 and application thereof |
| CN105779452A (en) * | 2016-03-10 | 2016-07-20 | 上海吉玛制药技术有限公司 | Oligonucleotide capable of inhibiting tumor growth and application of oligonucleotide |
| CN106676110A (en) * | 2017-02-20 | 2017-05-17 | 中国医科大学附属第医院 | Small interfering ribonucleic acid (siRNA) of long noncoding RNA (lncRNA) ABHD11-AS1 related to ovarian cancer and endometrial cancer, and application of siRNA |
-
2017
- 2017-08-03 CN CN201710657458.8A patent/CN107354159B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105085648A (en) * | 2015-05-04 | 2015-11-25 | 北京市肿瘤防治研究所 | Corresponding antigen of anti-ovarian cancer monoclonal antibody COC166-9 and application thereof |
| CN105779452A (en) * | 2016-03-10 | 2016-07-20 | 上海吉玛制药技术有限公司 | Oligonucleotide capable of inhibiting tumor growth and application of oligonucleotide |
| CN106676110A (en) * | 2017-02-20 | 2017-05-17 | 中国医科大学附属第医院 | Small interfering ribonucleic acid (siRNA) of long noncoding RNA (lncRNA) ABHD11-AS1 related to ovarian cancer and endometrial cancer, and application of siRNA |
Non-Patent Citations (3)
| Title |
|---|
| "Carboplatin-docetaxel-induced activity against ovarian cancer is dependent on up-regulated lncRNA PVT1";Enling Liu等;《International journal of clinical and experimental pathology》;20150415;第8卷(第4期);摘要，3804页，3806页 * |
| "Distinct Sets of lncRNAs are Differentially Modulated after Exposure to High and Low Doses of X Rays";Mariona等;《Radiation Research》;20161114;第186卷(第6期);第7页 * |
| "Homo sapiens DAMS mRNA, complete cds";NCBI;《NCBI_GeneBank》;19990325;序列信息 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107354159A (en) | 2017-11-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhu et al. | Gut microbiota regulate tumor metastasis via circRNA/miRNA networks | |
| Su et al. | MicroRNA-101, down-regulated in hepatocellular carcinoma, promotes apoptosis and suppresses tumorigenicity | |
| Wang et al. | Human tumor microRNA signatures derived from large‐scale oligonucleotide microarray datasets | |
| Gong et al. | LncRNA HAND2‐AS1 represses cervical cancer progression by interaction with transcription factor E2F4 at the promoter of C16orf74 | |
| Dong et al. | LncRNA COL1A1-014 is involved in the progression of gastric cancer via regulating CXCL12-CXCR4 axis | |
| Chong et al. | Overexpression of microRNA-196b accelerates invasiveness of cancer cells in recurrent epithelial ovarian cancer through regulation of homeobox A9 | |
| Wu et al. | MicroRNA-128 suppresses cell growth and metastasis in colorectal carcinoma by targeting IRS1 Retraction in/10.3892/or. 2021.8140 | |
| Shukla et al. | Enumeration of deregulated miRNAs in liquid and tissue biopsies of cervical cancer | |
| CN111778338A (en) | Application of circular RNA biomarker | |
| Mao et al. | Circ-XPR1 promotes osteosarcoma proliferation through regulating the miR-214-5p/DDX5 axis | |
| Zhang et al. | MiR-212-3p suppresses high-grade serous ovarian cancer progression by directly targeting MAP3K3 | |
| Yan et al. | Long non-coding RNA GAS5 regulates the growth and metastasis of human cervical cancer cells via induction of apoptosis and cell cycle arrest | |
| Ling et al. | Expression and prognostic significance of microRNA-451 in human epithelial ovarian cancer | |
| Tuo et al. | miR-532–3p suppresses proliferation and invasion of ovarian cancer cells via GPNMB/HIF-1α/HK2 axis | |
| Balatti et al. | MicroRNA profiling of salivary duct carcinoma versus Her2/Neu overexpressing breast carcinoma identify miR-10a as a putative breast related oncogene | |
| Wu et al. | AUF1-induced circular RNA hsa_circ_0010467 promotes platinum resistance of ovarian cancer through miR-637/LIF/STAT3 axis | |
| Feng et al. | Seed targeting with tiny anti-miR-155 inhibits malignant progression of multiple myeloma cells | |
| Fang et al. | PUS1 is a novel biomarker for predicting poor outcomes and triple-negative status in breast cancer | |
| CN107354159B (en) | Application of siRNA of long-chain non-coding RNA SMAD5-AS1 in ovarian cancer treatment | |
| Li et al. | MiR-218 affects the invasion and metastasis of cervical cancer cells by inhibiting the expression of SFMBT1 and DCUNIDI | |
| Qiao et al. | CD44 occurring alternative splicing promotes cisplatin resistance and evokes tumor immune response in oral squamous cell carcinoma cells | |
| CN110923324A (en) | Breast cancer miRNA marker and application thereof | |
| Wu et al. | G‐quadruplex‐enhanced circular single‐stranded DNA (G4‐CSSD) adsorption of miRNA to inhibit colon cancer progression | |
| Zhang et al. | MiRNA-8073 targets ZnT1 to inhibit malignant progression of ovarian cancer. | |
| Fang et al. | MIIP inhibits malignant progression of hepatocellular carcinoma through regulating AKT. |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20210525 |