CN107164378B - siRNA for specifically inhibiting SPIB gene expression and recombinant vector and application thereof - Google Patents
siRNA for specifically inhibiting SPIB gene expression and recombinant vector and application thereof Download PDFInfo
- Publication number
- CN107164378B CN107164378B CN201710470667.1A CN201710470667A CN107164378B CN 107164378 B CN107164378 B CN 107164378B CN 201710470667 A CN201710470667 A CN 201710470667A CN 107164378 B CN107164378 B CN 107164378B
- Authority
- CN
- China
- Prior art keywords
- spib
- sirna
- taxol
- cells
- ovarian cancer
- 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
- 108020004459 Small interfering RNA Proteins 0.000 title claims abstract description 57
- 230000014509 gene expression Effects 0.000 title claims abstract description 49
- 239000013598 vector Substances 0.000 title claims abstract description 28
- 101150113537 Spib gene Proteins 0.000 title claims abstract description 23
- 230000002401 inhibitory effect Effects 0.000 title claims abstract description 12
- 229930012538 Paclitaxel Natural products 0.000 claims abstract description 78
- 229960001592 paclitaxel Drugs 0.000 claims abstract description 78
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 claims abstract description 78
- 206010033128 Ovarian cancer Diseases 0.000 claims abstract description 33
- 206010061535 Ovarian neoplasm Diseases 0.000 claims abstract description 33
- 230000000692 anti-sense effect Effects 0.000 claims abstract description 18
- 206010059866 Drug resistance Diseases 0.000 claims abstract description 15
- 239000003814 drug Substances 0.000 claims abstract description 15
- 108091081021 Sense strand Proteins 0.000 claims abstract description 13
- 229940079593 drug Drugs 0.000 claims abstract description 11
- 238000002360 preparation method Methods 0.000 claims abstract description 7
- 239000002773 nucleotide Substances 0.000 claims description 5
- 125000003729 nucleotide group Chemical group 0.000 claims description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 3
- 108020004999 messenger RNA Proteins 0.000 abstract description 13
- 108090000623 proteins and genes Proteins 0.000 abstract description 10
- 230000012292 cell migration Effects 0.000 abstract description 9
- 230000006907 apoptotic process Effects 0.000 abstract description 8
- 230000004663 cell proliferation Effects 0.000 abstract description 7
- 230000002441 reversible effect Effects 0.000 abstract description 4
- 208000000102 Squamous Cell Carcinoma of Head and Neck Diseases 0.000 abstract description 3
- 230000004709 cell invasion Effects 0.000 abstract description 3
- 201000000459 head and neck squamous cell carcinoma Diseases 0.000 abstract description 3
- 102000004169 proteins and genes Human genes 0.000 abstract description 3
- 206010009944 Colon cancer Diseases 0.000 abstract description 2
- 208000001333 Colorectal Neoplasms Diseases 0.000 abstract description 2
- 208000005718 Stomach Neoplasms Diseases 0.000 abstract description 2
- 206010017758 gastric cancer Diseases 0.000 abstract description 2
- 201000011549 stomach cancer Diseases 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 76
- 239000004055 small Interfering RNA Substances 0.000 description 49
- 238000001890 transfection Methods 0.000 description 44
- 101000825182 Homo sapiens Transcription factor Spi-B Proteins 0.000 description 38
- 102100022281 Transcription factor Spi-B Human genes 0.000 description 37
- 239000013642 negative control Substances 0.000 description 17
- 230000000694 effects Effects 0.000 description 15
- 108020004414 DNA Proteins 0.000 description 13
- 239000013612 plasmid Substances 0.000 description 13
- 238000001514 detection method Methods 0.000 description 12
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 11
- 238000006243 chemical reaction Methods 0.000 description 10
- 238000000034 method Methods 0.000 description 9
- 238000011160 research Methods 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 239000012634 fragment Substances 0.000 description 8
- 230000002829 reductive effect Effects 0.000 description 8
- 210000004881 tumor cell Anatomy 0.000 description 8
- 206010028980 Neoplasm Diseases 0.000 description 7
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 230000009368 gene silencing by RNA Effects 0.000 description 7
- 239000012528 membrane Substances 0.000 description 7
- 238000011282 treatment Methods 0.000 description 7
- 238000005406 washing Methods 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 230000035945 sensitivity Effects 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 238000001262 western blot Methods 0.000 description 6
- 230000009471 action Effects 0.000 description 5
- 230000000903 blocking effect Effects 0.000 description 5
- 230000009545 invasion Effects 0.000 description 5
- 230000005012 migration Effects 0.000 description 5
- 238000013508 migration Methods 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- WOVKYSAHUYNSMH-RRKCRQDMSA-N 5-bromodeoxyuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(Br)=C1 WOVKYSAHUYNSMH-RRKCRQDMSA-N 0.000 description 4
- 241000283707 Capra Species 0.000 description 4
- 102000003952 Caspase 3 Human genes 0.000 description 4
- 108090000397 Caspase 3 Proteins 0.000 description 4
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 4
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 4
- 108091027967 Small hairpin RNA Proteins 0.000 description 4
- 238000000137 annealing Methods 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 238000001976 enzyme digestion Methods 0.000 description 4
- 239000013604 expression vector Substances 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
- 230000008506 pathogenesis Effects 0.000 description 4
- 230000035755 proliferation Effects 0.000 description 4
- 238000010839 reverse transcription Methods 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 102000003960 Ligases Human genes 0.000 description 3
- 108090000364 Ligases Proteins 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 description 3
- 229910052794 bromium Inorganic materials 0.000 description 3
- 239000011248 coating agent Substances 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000030279 gene silencing Effects 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 230000002452 interceptive effect Effects 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 108010082117 matrigel Proteins 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 229940122498 Gene expression inhibitor Drugs 0.000 description 2
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 239000002033 PVDF binder Substances 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000002512 chemotherapy Methods 0.000 description 2
- 238000002247 constant time method Methods 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000037440 gene silencing effect Effects 0.000 description 2
- 238000012226 gene silencing method Methods 0.000 description 2
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 210000003519 mature b lymphocyte Anatomy 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000001543 one-way ANOVA Methods 0.000 description 2
- 230000000149 penetrating effect Effects 0.000 description 2
- 210000005134 plasmacytoid dendritic cell Anatomy 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- 238000006748 scratching Methods 0.000 description 2
- 230000002393 scratching effect Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- CZVCGJBESNRLEQ-UHFFFAOYSA-N 7h-purine;pyrimidine Chemical compound C1=CN=CN=C1.C1=NC=C2NC=NC2=N1 CZVCGJBESNRLEQ-UHFFFAOYSA-N 0.000 description 1
- 238000012935 Averaging Methods 0.000 description 1
- 241000244203 Caenorhabditis elegans Species 0.000 description 1
- 102000053642 Catalytic RNA Human genes 0.000 description 1
- 108090000994 Catalytic RNA Proteins 0.000 description 1
- 238000007808 Cell invasion assay Methods 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 208000009849 Female Genital Neoplasms Diseases 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 108010018070 Proto-Oncogene Proteins c-ets Proteins 0.000 description 1
- 102000004053 Proto-Oncogene Proteins c-ets Human genes 0.000 description 1
- 108091030071 RNAI Proteins 0.000 description 1
- 208000034331 Squamous cell carcinoma of the stomach Diseases 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 239000006180 TBST buffer Substances 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 101150063416 add gene Proteins 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 108010078837 aspartyl-glutamyl-valyl-aspartyl-p-nitroanilide Proteins 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000003560 cancer drug Substances 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 208000016496 colorectal squamous cell carcinoma Diseases 0.000 description 1
- 238000009096 combination chemotherapy Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000011500 cytoreductive surgery Methods 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000012938 design process Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 239000000890 drug combination Substances 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 230000001516 effect on protein Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 201000000496 gastric squamous cell carcinoma Diseases 0.000 description 1
- 238000012224 gene deletion Methods 0.000 description 1
- 210000001280 germinal center Anatomy 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 238000010832 independent-sample T-test Methods 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 238000010232 migration assay Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000002980 postoperative effect Effects 0.000 description 1
- 230000032361 posttranscriptional gene silencing Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000003531 protein hydrolysate Substances 0.000 description 1
- 239000012474 protein marker Substances 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000000611 regression analysis Methods 0.000 description 1
- 230000009933 reproductive health Effects 0.000 description 1
- 201000011453 reproductive organ cancer Diseases 0.000 description 1
- 208000028466 reproductive system neoplasm Diseases 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 238000010583 slow cooling Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000011272 standard treatment Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
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
-
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- 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.
Landscapes
- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Plant Pathology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses siRNA for specifically inhibiting SPIB gene expression, a recombinant vector thereof and application of the siRNA in preparation of drugs for reversing drug resistance of ovarian cancer taxol, and belongs to the technical field of molecular biology and biomedicine. The siRNA comprises a sense strand and an antisense strand, wherein the sense strand: 5'-AAUCAGGGUAGCUGGAAUGCU-3', respectively; antisense strand: 5'-CAUUCCAGCUACCCUGAUUCA-3' are provided. The siRNA provided by the invention can specifically and efficiently inhibit the mRNA and protein expression of the SPIB gene, reduce cell proliferation, increase cell apoptosis, reduce cell migration and invasion capacity, and effectively reverse the drug resistance of ovarian cancer cells to paclitaxel. The invention also provides application of the siRNA and the recombinant vector thereof in preparing medicines for treating ovarian cancer, gastric cancer, colorectal cancer, head and neck squamous cell carcinoma or reversing drug resistance of ovarian cancer.
Description
Technical Field
The invention relates to the technical field of molecular biology and biomedicine, in particular to siRNA for specifically inhibiting SPIB gene expression, a recombinant vector and application thereof.
Background
RNA interference (RNAi) is a widely occurring sequence-specific post-transcriptional gene silencing mechanism in animals and plants. In 1998, Andrew Fire, an American scientist, found for the first time that the dsRNA produced gene silencing effect is at least 10 times of the inhibition effect of antisense nucleotide in C.elegans of caenorhabditis elegans, and can induce the same gene inhibition phenomenon in offspring. The mechanism research of RNAi phenomenon shows that the key molecule capable of degrading homologous RNA efficiently and specifically to result in sequence specific gene silencing is small double-stranded oligonucleotide with 21-23 base length, also called small interfering RNA (siRNA). Further research shows that the double-stranded RNA shorter than 21bp or longer than 25bp can not effectively start RNAi, and the gene silencing effect is obviously reduced or even eliminated as long as one base is mismatched, so that the specificity of the siRNA effect is fully embodied.
siRNA has attracted attention as a significant finding in recent biotechnology because it has a gene suppression function of specifically and efficiently blocking all homologous genes and is a powerful tool for gene function studies. The method of using small interfering RNA (siRNA) as gene silencing is widely used for the research of malignant tumor mechanism, and thus the development process of tumor specific high-efficiency targeted therapy strategy based on the technology is promoted.
Ovarian cancer is the leading cause of death in reproductive tumors, has occult disease, lacks early symptoms and effective early diagnosis means, and has rapid malignant progression. The standard treatment regimen is tumor cytoreductive surgery with post-operative platinum/taxol drug combination chemotherapy, and most recurred within 18 months, although complete remission can occur in 70% of patients. Even if the ovarian cancer after relapse is treated by chemotherapeutics with completely different action mechanisms, the ovarian cancer can generate drug resistance, so that the survival rate after tumor treatment cannot be obviously improved. The five-year survival rate of advanced ovarian cancer is consistently around 30%. High recurrence rate and drug resistance are important reasons for poor prognosis of ovarian cancer. How to improve the treatment effect of ovarian cancer and search a solution of chemotherapy resistance of ovarian cancer is an important subject of gynecological tumor research.
The combined application of molecular targeted drugs and chemotherapy is the most promising treatment method for improving the survival rate of malignant tumor patients at present. SPIB (Spi-B transcription factor) is a member of the Ets transcription factor family, and the gene is located on chromosome 19q, is normally expressed only on mature B cells, T cell progenitors, and plasmacytoid dendritic cells, plays an important role in the differentiation process of mature B cells into plasmacytoid cells and plasmacytoid dendritic cells, plays a central role in the germinal center reaction during embryonic development, and is slowly developed in mice with SPIB gene deletion, and a large number of apoptotic cells are produced in vivo as compared with wild-type mice. Recent studies found that SPIB is highly expressed in the solid tumors gastric, colorectal and head and neck squamous cell carcinoma. Our previous studies showed that SPIB is also highly expressed in ovarian cancer cells, and abnormal increase in the paclitaxel-resistant cell line a2780/Taxol in ovarian cancer is found, but whether the gene is related to the onset of ovarian cancer remains to be further studied. Therefore, the interference of the expression of the SPIB gene in the ovarian cancer drug-resistant cell strain by adopting the RNAi technology is an important supplement to the pathogenesis of the ovarian cancer and an important exploration and application to the ovarian cancer and the drug-resistant treatment thereof.
Disclosure of Invention
The invention aims to provide siRNA for specifically inhibiting the expression of an SPIB gene, which is used for researching the pathogenesis of ovarian cancer. The invention also aims to provide the application of the siRNA in preparing a medicine for reversing the drug resistance of the ovarian cancer taxol.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention designs and synthesizes 3 pairs of siRNA for specifically inhibiting the expression of the SPIB gene, and the siRNA is transfected into an ovarian cancer Taxol resistant cell strain A2780/Taxol, and the result shows that the interference effect of S2 for inhibiting the expression of the SPIB gene is most obvious.
The invention provides siRNA (S2) for specifically inhibiting the expression of an SPIB gene, which comprises a sense strand and an antisense strand,
the sense strand: 5'-AAUCAGGGUAGCUGGAAUGCU-3' (SEQ ID NO. 1);
the antisense strand: 5'-CAUUCCAGCUACCCUGAUUCA-3' (SEQ ID NO. 2).
Preferably, the sense and antisense strands are 2 ' -methoxy modified at the 5 ' and 3 ' ends of the 3 bases. The research of the invention proves that the stability of the siRNA (S2) modified by the 2' -methoxy group is increased, the capability of resisting the hydrolysis of ribozyme in vivo can be improved, the immune stimulation reaction is reduced, the action time of siRNA interfering the expression of gene to be reduced is prolonged, and the action has high efficiency and specificity.
The invention provides an RNA interference kit comprising a DNA sequence encoding the siRNA. The kit comprises a DNA plasmid vector cloned with the siRNA, and when the kit is applied, the plasmid vector transcribes and expresses the required siRNA in eukaryotic cells, so that the expression of silencing the SPIB gene is achieved.
The invention provides a recombinant vector containing a DNA sequence for encoding the siRNA. Preferably, the original vector used is the lentiviral vector plko.1puro.
The invention also provides a construction method of the recombinant vector, which comprises the following steps:
(1) synthesizing an SPIB-S2 fragment, selecting two enzyme cutting sites of Age I and EcoR I, and designing shRNA sequence according to the sequence of S2, wherein the sequence is as follows:
sense strand:
5’-CCGGTGAATCAGGGTAGCTGGAATGCTTTCAAGAGAGACATTCCAGCTACCCTGATTCATTTTTTGGTACC-3’(SEQ ID NO.7)；
antisense strand:
5’-AATTGGTACCAAAAAATGAATCAGGGTAGCTGGAATGTCTCTCTTGAAAGCATTCCAGCTACCCTGATTCA-3’(SEQ ID NO.8)；
(2) annealing to obtain a DNA fragment of the SPIB-S2;
(3) pLKO.1-SPIB-sh2 recombinant vector was constructed using lentiviral vector pLKO.1puro.
The siRNA provided by the invention can efficiently and specifically inhibit the expression of the SPIB gene of ovarian cancer cells, reduce cell proliferation, increase apoptosis and reduce cell migration and invasion capacity, so that the siRNA and the recombinant vector serving as the SPIB gene expression inhibitor can be applied to the research of pathogenesis of tumor diseases.
The invention provides application of the siRNA and the recombinant vector in preparation of an SPIB gene expression inhibitor.
The invention provides application of the siRNA and the recombinant vector in preparing a medicament for treating ovarian cancer, gastric cancer, colorectal cancer or head and neck squamous cell carcinoma.
The research of the invention shows that after the siRNA is transfected by the paclitaxel resistant strain A2780/Taxol, the sensitivity of the cell strain to paclitaxel is obviously improved, and the reversal index is 19.51, which shows that the reversal effect of the siRNA provided by the invention on the paclitaxel resistant strain A2780/Taxol is very obvious, therefore, the siRNA has potential application value in the treatment of reversing the paclitaxel resistance of ovarian cancer.
The invention provides application of the siRNA and the recombinant vector in preparation of a medicine for reversing drug resistance of ovarian cancer taxol.
The invention has the following beneficial effects:
the siRNA provided by the invention can specifically and efficiently inhibit the expression of mRNA and protein of the SPIB gene, reduce the proliferation of tumor cells, increase the apoptosis, reduce the migration and invasion capacity of the tumor cells, and effectively reverse the drug resistance of ovarian cancer cells to paclitaxel, and has important significance in the research of tumor pathogenesis and the preparation of drugs for tumor treatment and drug resistance treatment of the reversed ovarian cancer.
Drawings
FIG. 1 shows the expression of SPIB mRNA in A2780 cells and A2780/Taxol cells detected by qRT-PCR.
FIG. 2 shows the detection of the expression of SPIB protein in A2780 cells and A2780/Taxol cells by Western Blotting.
FIG. 3 shows the expression of SPIB mRNA in A2780/Taxol cells 48h after transfection with S1, S2, S3 by qRT-PCR.
FIG. 4 shows the expression of SPIB protein in A2780/Taxol cells after transfection with S1, S2 and S3 for 72h by Western Blotting.
FIG. 5 is a schematic diagram of pLKO.1-SPIB-sh2 recombinant plasmid and inserted restriction sites.
FIG. 6 is a schematic diagram of a small hairpin shRNA. The U6 promoter directs the transcription of downstream small hairpin shRNA; comprises 23S 2 sense strand bases and 23S 2 antisense strand bases.
FIG. 7 shows the detection of the expression of SPIB protein in A2780/Taxol cells after pLKO.1-SPIB-sh2 transfection by Western Blotting.
FIG. 8 shows the number and morphology of A2780/Taxol cells after pLKO.1-SPIB-sh2 transfection under phase contrast microscope observation.
FIG. 9 shows the proliferation of A2780/Taxol cells detected by bromine labeling method after transfection of pLKO.1-SPIB-sh 2.
FIG. 10 shows the detection of Caspase3 activity in apoptosis of A2780/Taxol cells after pLKO.1-SPIB-sh2 transfection.
FIG. 11 shows the migration ability of A2780/Taxol cells after pLKO.1-SPIB-sh2 transfection in cell scratch assay.
FIG. 12 shows the migration ability (A) and invasion ability (B) of A2780/Taxol cells after transfection of pLKO.1-SPIB-sh2 by Transwell assay.
FIG. 13 shows the reversal of paclitaxel resistance in A2780/Taxol cells after pLKO.1-SPIB-sh2 transfection.
Detailed Description
The present invention will be further described with reference to the following examples. The methods used in the following examples are intended to better understand the invention but are not intended to limit it. Unless otherwise specified, the experimental methods mentioned in the examples are all conventional methods, and the experimental materials used are all purchased from conventional reagents companies.
The SPSS 18.0 statistical analysis software is adopted, and the data of each sample are averaged to be +/-standard deviationAs shown, the differences between the two groups were measured by T Test (Independent-Sample T Test), and the differences between the groups were measured by One-Way analysis of variance (One-Way ANOVA), IC50Using probit regression analysis, P < 0.05 was statistically different.
Ovarian cancer cell strain A2780 and ovarian cancer Taxol resistant cell strain A2780/Taxol are preserved in a cell bank of a key laboratory of female reproductive health research in Zhejiang province;
rabbit anti-human SPIB primary antibody (Cat.15768-1-AP), mouse anti-human GAPDH primary antibody (Cat.60004-1-Ig), horseradish peroxidase-labeled goat anti-mouse IgG (H + L) secondary antibody (Cat.SA00001-1), and horseradish peroxidase-labeled goat anti-rabbit IgG (H + L) secondary antibody (Cat.SA00001-2) were purchased from Proteintech;
western Blotting Luminol Reagent kit (Cat. sc-2048) was purchased from Santa Cruz;
reverse transcription of cDNAKit PrimeScriptTMRT Master Mix (Cat. RR036A), fluorescent quantitative PCR detection kit SYBR Premix Ex Taq (perfect Real time, Cat. DRR041A) purchased from TaKaRa company; lipofectamine3000 transfection kit (cat. l3000008) was purchased from Invitrogen;
the eukaryotic expression vector selects a conventional RNAi vector pLKO.1puro, and is originated from a global scientist plasmid sharing non-profit tissue Addgene;
restriction enzymes Age I (Cat. R0552S), EcoR I (Cat. R0101S), Kpn I (Cat. R0142S) were purchased from NEB; t4 ligase (Cat.2011A), a DNA fragment purification kit (Cat.9761), a DNA gel recovery kit (Cat.9762) and a plasmid DNA small purification kit (Cat.9760) are all purchased from TaKaRa company;
siRNA was synthesized by TaKaRa; PCR primers and DNA for cloning were synthesized by Shanghai Bioengineering Co., Ltd;
prestained protein Marker (cat.26616) available from Fermentas corporation;
cell Proliferation ELISA, BrdU (colorimetric, Cat.11647229001) was purchased from Roche;
CaspACE Assay System (colorimetric, Cat. G7351) available from Promega;
cell migration, invasion model Transwell Permeable Supports (Cat.3428) was purchased from Corning;
Lab-Tek II Chamber Slide System-Lab-Tek Chamber Slide System was purchased from Nunc corporation (Cat.154526);
BD MatrigelTMbasic Membrane matrix membranes (cat.356234) available from BD;
siRNA Negative Control AllStars Negative Control SiRNA (Cat.1027281) was purchased from QIAGEN;
SDS-PAGE gel preparation kit (cat. cw0022m) purchased from kazai century corporation;
0.45um PVDF membrane (Cat. IPVH00010) was purchased from Millipore;
paclitaxel (cat. P106868) was purchased from Aladdin.
Example 1 study of the differences in expression of SPIB in ovarian cancer cell line A2780 and paclitaxel-resistant cell line A2780/Taxol thereof
First, real-time fluorescent quantitative RT-PCR (qRT-PCR) is used for detecting mRNA expression of SPIB gene
After 48h incubation, the medium in the 6-well plate was aspirated, washed twice with PBS, and total RNA was extracted with Trizol, and the RNA concentration was determined with Thermo Nano Drop2000 spectrophotometer, according to SYBR Premix Ex Taq (perfect reaction time) kit instructions. First step RNA denaturation. Reaction system: RNA0.5ug, adding DEPC water to 6.8 ul; reaction conditions are as follows: incubate at 70 ℃ for 10min and place on ice. And a second step of reverse transcription. Reaction system: reverse transcription was performed according to PrimeScript RTMaster Mix kit instructions; reaction conditions are as follows: incubating at 42 deg.C for 60min, inactivating at 85 deg.C for 5min, and storing at-20 deg.C.
Taking 1ul of reverse transcription product to perform fluorescent quantitative PCR reaction. PCR primer sequences:
5’-AAGAAGCTGCGCCTGTACC-3’；
5'-TCTTGGCGTAGTTTCGGAGG-3', product length: 211 bp;
reaction conditions are as follows: 10s at 95 ℃, 5s at 95 ℃ and 30s at 6 ℃ for 40 cycles.
The amount of expression of SPIB mRNA in each group of samples was calculated by the 2-DELTA CT method.
As a result: as shown in FIG. 1, the expression of SPIB mRNA was increased by 88.19% (P < 0.05) in A2780/Taxol cells compared to its parental cell, A2780, indicating a significant increase in SPIB mRNA expression in paclitaxel-resistant cell lines.
Second, Western Blotting detection of SPIB protein expression
After culturing for 72h, absorbing and removing the culture medium in the 6-well plate, washing with PBS for 3 times, adding RIPA protein lysate (100 ul/well), blowing and beating for several times, incubating on ice for 5min to fully crack, centrifuging at 4 ℃, 12000 rpm for 5min, collecting supernatant, and subpackaging at-20 ℃ for storage; denaturing each sample at 95 deg.c for 5min, taking 10ul sample, 8% SDS-PAGE electrophoresis at 200V for 10 min; 100V for 100 min; turning to a PVDF membrane: 110V for 120 min; sealing with TBS sealing solution containing 5% skimmed milk powder for 60 min; primary antibody incubation: incubating SPIB primary antibody (1: 2000) and GAPDH primary antibody (1: 5000) at room temperature for 2 h; washing membrane with TBS for 10min × 3 times; and (3) secondary antibody incubation: incubating a goat anti-mouse IgG (H + L) secondary antibody (1: 10000) marked by horseradish peroxidase and a goat anti-rabbit IgG (H + L) secondary antibody (1: 10000) marked by horseradish peroxidase for 1H; washing membranes with TBST for 10min × 3 times and TBS for 10min × 1 time; after ECL development, the images were scanned with ImageQuantLAS4000 mini (ge healthcare).
As a result: as shown in FIG. 2, the expression of the SPIB protein was also significantly increased in A2780/Taxol cells compared to its parent cell, A2780 (P < 0.05).
Example 2 SPIB siRNA design Synthesis
The mRNA sequence of the SPIB gene (NM-003121.4) was obtained from Genebank, and 3 pairs of siRNA sequences (shown in SEQ ID NO.1-SEQ ID NO.6) were obtained by on-line design using siDirect Ver2.0 software (http:// sidirect2.rnai. jp /). In the design process, a sequence simultaneously meeting three algorithms (Ui-Tei x Reynolds x Amarzguioui) reported in the literature is selected, a 23nt length segment with the highest siRNA action specificity is selected, the design can avoid the generation of an interferon-like immune response in future in-vivo experiments, 100nt after an initiation codon is selected, 5 'and 3' end UTR regions are avoided, and the GC content is controlled to be 30-70%. 3 pairs of siRNA with the length of 23nt are selected as experimental screening interference fragments, the structural characteristics are that the 3' ends of the sense strand and the antisense strand are respectively externally hung with two basic groups, and the structural characteristics are as follows
Followed by BLASTN(https://blast.ncbi.nlm.nih.gov/Blast.cgi)Homology search is carried out on line, sequences with homology are excluded, and the influence of nonspecific fragments on the specific action effect of siRNA is avoided as much as possible.
And finally, carrying out 2 '-OMe (2' -methoxyl) modification on 3 continuous purine (pyrimidine) bases at the 5 'end and the 3' end of the sense strand and the antisense strand during chemical synthesis, so that the chemical stability of the siRNA molecule in cells is increased, and the time and effect of siRNA interfering gene expression down regulation are prolonged. The final sequences and modifications are shown in Table 1 and formula (I) below:
TABLE 1
Example 3 detection and screening of three pairs of SPIB siRNAs on the interfering Effect of SPIB genes in Taxol-resistant Strain A2780/Taxol for ovarian cancer
Firstly, experimental grouping:
A2780/Taxol normal group (no siRNA transfection), hereinafter referred to as AR;
A2780/Taxol negative control group (transfection negative control siRNA), hereinafter referred to as AR-N;
A2780/Taxol panel (transfection S1), hereinafter referred to as AR-S1;
A2780/Taxol panel (transfection S2), hereinafter referred to as AR-S2;
A2780/Taxol panel (transfection S3), hereinafter referred to as AR-S3.
Two, grouped transfection
To ensure transfection efficiency and reduce cytotoxicity, we used Lipofectamine3000 transfection reagent for siRNA transfection. One day before transfection, cells were trypsinized and counted, and cells were plated in six-well plates at a density of 0.5X 10 on the day of transfection6Per ml, cells were fused to 70-90%. Each well was diluted with 125. mu.l of serum-free OPTI-MEM medium to 5ul of Lipofectamine3000 reagent and mixed well; preparing siRNA premix, diluting siRNA with 125 mul of serum-free OPTI-MEM culture medium to a final concentration of 50nM, and fully mixing; adding siRNA premix (1:1) into diluted Lipofectamine3000 reagent, and incubating for 5min at room temperature; finally, the siRNA-liposome complex was added to the cells at 37 ℃ with 5% CO2The culture is continued. After 48h, SPIB mRNA expression was detected, and after 72h, SPIB protein expression was detected.
Third, real-time fluorescent quantitative RT-PCR (qRT-PCR) is used for detecting mRNA expression of the SPIB gene
The detection steps are the same as the previous steps, and the expression quantity of the SPIB mRNA in each group of samples is calculated by adopting a 2-delta CT method.
As a result: as shown in FIG. 3, after the A2780/Taxol cells were transfected with S1, S2 and S3, the expression of the SPIB mRNA was significantly reduced, wherein the S2 interference effect was 83.07% lower than that of the negative control group, and the difference was significant (P < 0.05%) compared with S1 (62.05%) and S3 (55.75%). The results show that S2 has the best interference effect on SPIB.
Fourth, Western Blotting detection of SPIB protein expression
The detection procedure was as before, after ECL development, the Image was scanned with Image Quant LAS4000 mini (GE healthcare).
As a result: as shown in FIG. 4, after transfection with S2, there was a significant decrease in SPIB protein expression in A2780/Taxol cells (P < 0.05) compared to the negative control, and a significant difference (P < 0.05) compared to S1 and S3. The results show that S2 has the best interference effect on protein expression of SPIB in the paclitaxel resistant strain A2780/Taxol. Therefore, S2 was selected as siRNA for subsequent studies.
Example 4 construction of eukaryotic vector pLKO.1-SPIB-sh2 and detection of interference effect on expression of SPIB Gene
Firstly, experimental grouping:
A2780/Taxol normal group (without transfection of any vector), hereinafter referred to as AR;
A2780/Taxol negative control group (pLKO.1puro empty vector transfected), hereinafter referred to as AR-N;
A2780/Taxol experimental group 1 (pLKO.1-SPIB-sh 2 transfection), hereinafter referred to as AR-sh 2;
II, synthesizing the fragment SPIB-S2
Two enzyme cutting sites of Age I and EcoR I are selected, and shRNA sequence is designed according to the sequence of S2 and is constructed into a eukaryotic expression vector pLKO.1puro. The sequence is as follows:
sense strand:
5’-CCGGTGAATCAGGGTAGCTGGAATGCTTTCAAGAGAGACATTCCAGCTACCCTGATTCATTTTTTGGTACC-3’(SEQ ID NO.7)；
antisense strand:
5’-AATTGGTACCAAAAAATGAATCAGGGTAGCTGGAATGTCTCTCTTGAAAGCATTCCAGCTACCCTGATTCA-3’(SEQ ID NO.8)；
thirdly, construction of eukaryotic vector pLKO.1-SPIB-sh2
pLKO.1-SPIB-sh2 recombinant expression vector was constructed using eukaryotic expression vector pLKO.1puro (FIGS. 5 and 6), and the specific method was described in molecular cloning, a laboratory Manual, of Cold spring harbor Press, USA.
The sense and antisense strands of SPIB-S2 were annealed (denaturation at 95 ℃ for 2 min; annealing by slow cooling to 25 ℃) and stored at 4 ℃. Completely digesting the vector pLKO.1puro by Age I and EcoR I, and standing overnight at 37 ℃; and recovering DNA from the enzyme digestion product by using a DNA gel recovery kit. The annealing product and the carrier enzyme digestion recovery fragment are subjected to a connection reaction, and the reaction system (10 ul): 1ul of T4 ligase, 1ul of T4 ligase buffer solution, and carrying out enzyme digestion on the annealing product and a carrier to recover a fragment mixture (the molar ratio is 3: 1); supplementing deionized water to 10ul, and connecting overnight at 16 ℃; placing 5ul of the ligation product in 100ul of JM109 competent bacteria, carrying out ice bath for 30min, carrying out heat shock at 42 ℃ for 90s, carrying out ice bath for 5min, adding 1000ul of LB culture medium, carrying out shake culture at 37 ℃ for 30min, centrifuging at 5000rpm for 5min, discarding the supernatant, uniformly coating the bacteria on an LB plate (containing 50ug/ml ampicillin), and carrying out inverted culture at 37 ℃ for overnight; selecting a plurality of independent colonies, inoculating the colonies into an LB culture medium containing corresponding resistance, and oscillating the colonies overnight at 37 ℃ for strain expansion; collecting bacteria, extracting by using a plasmid DNA purification kit to obtain plasmid DNA, carrying out enzyme digestion identification by Kpn I to obtain pLKO.1-SPIB-sh2 recombinant plasmid.
Fourth, pLKO.1-SPIB-sh2 recombinant plasmid is used for observing interference effect after transfecting cells
pLKO.1-SPIB-sh2 was transfected into A2780/Taxol cells in logarithmic growth phase. Transfection 5ul Lipofectamine3000 reagent was diluted in 125. mu.l serum-free OPTI-MEM medium per well and mixed well according to Lipofectamine3000 protocol; adding 5ug of recombinant plasmid DNA into 125 mul of serum-free OPTI-MEM culture medium, adding 10ul of P3000 reagent, and mixing to obtain recombinant plasmid premix; the diluted Lipofectamine3000 reagent was added to the recombinant plasmid premix (1:1) and incubated at room temperature for 5 mm; finally, 250ul of the recombinant plasmid-liposome complex was added to the cells at 37 ℃ with 5% CO2The culture is continued. SPIB protein expression was detected after 72 h.
As shown in FIG. 7, after the pLKO.1-SPIB-sh2 recombinant plasmid is transfected, the expression of the SPIB protein in A2780/Taxol cells is remarkably reduced (P < 0.05) compared with that of a negative control (transfection of an empty plasmid), and the result shows that the pLKO.1-SPIB-sh2 recombinant plasmid is transfected to effectively interfere the protein expression of the SPIB.
Example 5 Effect of pLKO.1-SPIB-sh2 transfection on proliferation, apoptosis, migration and invasion of tumor cells after specific blocking of SPIB expression
Firstly, experimental grouping:
A2780/Taxol normal group (without transfection of any vector), hereinafter referred to as AR;
A2780/Taxol negative control group (pLKO.1puro empty vector transfected), hereinafter referred to as AR-N;
A2780/Taxol experimental group (pLKO.1-SPIB-sh 2 transfection), hereinafter referred to as AR-sh 2.
Two, grouped transfection
The transfection procedure was as before, and cells were cultured after transfection for detection of proliferation, apoptosis, migration and invasion of cells.
Third, cell proliferation assay
Cells are transfected into a 96-well plate pLKO.1-SPIB-sh2 and then cultured for 72h, 10ul BrdU labeling solution is added into each well until the final BrdU concentration is 10uM, and the cells are incubated for 2h at 37 ℃; absorbing BrdU labeling solution, adding 200ul FixDenat into each well, and incubating at 20 deg.C for 30 min; removing FixDenat by suction, adding 100ul anti-BrdU-POD into each well, and incubating at 20 deg.C for 90 min; washing 3 times with 200ul washing Solution per well; adding 100 ul/well substrate solution, incubating at 20 deg.C for 20min, detecting absorbance (A) at wavelength of 370nm (reference wavelength of 492nm), and measuring cell proliferation ability with AExperimental group/AControl groupAnd (4) showing.
As shown in FIGS. 8 and 9, in A2780/Taxol cells, pLKO.1-SPIB-sh2 is transfected, and the observation under a phase contrast microscope shows that the number of cells in an experimental group is obviously reduced, the number of suspended cells is increased, and the cell debris is increased; the results of cell proliferation tested by the bromine standard method also show that: compared with a negative control, the cell proliferation capacity of the experimental group is reduced by 71.22%, and the significant difference is realized (P is less than 0.05). Indicating that the tumor cell proliferation can be inhibited after the expression of the SPIB is specifically blocked.
Fourthly, detecting apoptosis by Caspase3 activity
Collecting cells after transfection for 72h, adjusting cell density to 1 × 10 with lysate8-/ml, split on ice for 15min, 15000g × 20min, collect supernatant. According to CaspACE Assay System (colorimetric)) The instructions prepare positive and negative control samples simultaneously, and determine and adjust the same concentration of each histone. Caspace Assay Buffer32ul, DMSO 2ul, 100nM DTT 10ul, deionized water adjusted to 98ul volume, 2ul DEVD-pNA substrate was added to each well of 96-well plate, incubated at 37 ℃ for 4h, the absorbance was measured at 405nM wavelength, and Caspase3 activity was calculated for each group of samples by the Δ A method.
The result is shown in FIG. 10, after pLKO.1-SPIB-sh2 is transfected by A2780/Taxol cells, Caspase3 activity is increased by 4.19 times, and compared with a negative control, the significant difference (P is less than 0.05) is generated, which indicates that tumor cell apoptosis can be promoted after the expression of the specific blocking SPIB.
Fifth, cell migration ability test
And uniformly marking transverse lines at the back of the six-hole plate by using a straight ruler, wherein one transverse line is marked by about 0.5cm and penetrates through the holes, and each hole is provided with at least 6 transverse lines. After cell transfection pLKO.1-SPIB-sh2, when the cells are continuously cultured for 24h and fused into a single layer state, vertically scratching the selected area in a six-hole plate by using a 200ul pipette tip, washing by PBS for 3 times to remove the scratched cells, and adding a serum-free culture medium for continuous culture. Photographing is carried out at the time points of 0h, 24h and 48h, 6 horizontal lines are randomly selected, and the average value of the distances between cells is calculated.
The result is shown in fig. 11, after cells are transfected by pLKO.1-SPIB-sh2 for 24h and 48h, the distance between A2780 and Taxol cells is obviously greater than that of a negative control group, the healing capacity of the cells after scratching is obviously reduced, and the tumor cell migration can be inhibited after the expression of the specific blocking SPIB.
Sixth, Transwell test for detecting cell migration ability
48h after cell transfection, the cells were collected by trypsinization, resuspended in serum-free medium and adjusted to a cell density of 5X 105Adding 2ml of cell suspension into an upper chamber, adding 2ml of 10% FBS complete culture medium into a lower chamber, continuously culturing for 24h, taking out a small chamber, washing with PBS for 3 times, carefully removing cells on the surface of the upper layer of the upper chamber by using a cotton swab, inverting and airing, fixing with 95% ethanol for 25min, staining with hematoxylin, observing under a microscope, counting and taking a picture. 10 fields were counted per chamber, averaged and analyzed for changes in cell migration capacity.
The results of the cell migration experiments are shown in FIG. 12A, after pLKO.1-SPIB-sh2 transfection, the cell numbers of the A2780/Taxol experimental group and the negative control group penetrating the chamber are 46 + -15 and 279 + -27 respectively, and the two groups have statistical difference (P < 0.05); the results indicate that the specific blocking of SPIB expression inhibits tumor cell migration.
Seventhly, cell invasion capacity detection by Transwell test
Liquefying matrigel stored at-20 deg.C at 4 deg.C, mixing matrigel with OPTI-MEM at a ratio of 1:6 ice, diluting, coating the upper surface of the bottom membrane of the chamber, solidifying at 37 deg.C for 30min, and removing the liquid separated out from the chamber. The remaining steps after matrigel coating were as above, 10 fields were counted per chamber, and changes in cell invasiveness were counted and analyzed by averaging.
The results of the cell invasion assay are shown in FIG. 12B, the numbers of cells penetrating the chamber of the A2780/Taxol test group and the negative control group are 36 + -8 and 177 + -28, respectively, and the two groups have statistical difference (P < 0.05); the results show that tumor cell infiltration can be inhibited after the expression of the SPIB is specifically blocked.
Example 6 pLKO.1-SPIB-sh2 reversal of drug resistance in ovarian cancer after specific blockade of SPIB expression
Firstly, experimental grouping:
a2780 normal group (without transfection of any vector), hereinafter referred to as a;
A2780/Taxol normal group (without transfection of any vector), hereinafter referred to as AR;
A2780/Taxol negative control group (pLKO.1puro empty vector transfected), hereinafter referred to as AR-N;
A2780/Taxol experimental group (pLKO.1-SPIB-sh 2 transfection), hereinafter referred to as AR-sh 2.
Two, grouped transfection
The transfection procedure was as before, and cells were cultured for 24h after transfection.
Thirdly, detecting the sensitivity of the cells to the paclitaxel after pLKO.1-SPIB-sh2 transfection
Taking cells in logarithmic growth phase from each group, re-suspending the cells after trypsinization, counting the cells and adjusting the density of the cell suspension to 1 × 105And/ml, inoculating into a 96-well plate and continuing culturing for 24 hours. The next day, paclitaxel was added to each group,setting concentration gradient at 200ug/ml, 100ug/ml, 50ug/ml, 25ug/ml, 12.5ug/ml, 6.25ug/ml, 3.125ug/ml and 0ug/ml, respectively, reacting for 24h, measuring absorbance (A) at wavelength of 370nm (reference wavelength of 492nm) by bromine standard method, and calculating the inhibition rate of paclitaxel on each group of cellsExperimental group/ANegative control group.3 replicate wells were set for each concentration and the average was taken.
The drug concentration at 50% inhibition was half the Inhibitory Concentration (IC)50)；
IC of drug-resistant strain A2780/Taxol50IC with its parent cell line A278050The ratio of (A) to (B) is the Resistance Fold (RF);
IC of drug-resistant strain A2780/Taxol50IC after transfection with pLKO.1-SPIB-sh2 (Reversant)50The ratio of (A) to (B) is the resistance Reversal Index (RI).
The results are shown in Table 2, Table 3 and FIG. 13, IC of A2780/Taxol vs. paclitaxel50(42.65 + -4.88 ug/ml) is significantly higher than the IC of parent A2780 for paclitaxel50(1.36 +/-0.29 ug/ml), the drug resistance times are as high as 31.36, which indicates that the sensitivity of A2780/Taxol to paclitaxel is obviously lower than that of a parent cell A2780, and the drug resistance is high. And after the A2780/Taxol is transfected with pLKO.1-SPIB-sh2, the sensitivity to paclitaxel is obviously improved (2.12 +/-0.47 ug/ml), and the reversal effect of the transfected pLKO.1-SPIB-sh2 to the drug resistance of the A2780/Taxol is very obvious, and the reversal index is 19.51.
TABLE 2 drug sensitivity of A2780 and A2780/Taxol to paclitaxel
TABLE 3 reversion of the sensitivity of A2780/Taxol to paclitaxel drugs after pLKO.1-SPIB-sh2 transfection
SEQUENCE LISTING
<110> Zhejiang university
<120> siRNA for specifically inhibiting SPIB gene expression, recombinant vector and application thereof
<130>
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> RNA
<213> Artificial sequence
<400> 1
aaucagggua gcuggaaugc u 21
<210> 2
<211> 21
<212> RNA
<213> Artificial sequence
<400> 2
cauuccagcu acccugauuc a 21
<210> 3
<211> 21
<212> RNA
<213> Artificial sequence
<400> 3
ucauagaaga cgccaucugg g 21
<210> 4
<211> 21
<212> RNA
<213> Artificial sequence
<400> 4
cagauggcgu cuucuaugac c 21
<210> 5
<211> 21
<212> RNA
<213> Artificial sequence
<400> 5
ucugauagca cagggcuggg g 21
<210> 6
<211> 21
<212> RNA
<213> Artificial sequence
<400> 6
ccagcccugu gcuaucagag g 21
<210> 7
<211> 71
<212> DNA
<213> Artificial sequence
<400> 7
ccggtgaatc agggtagctg gaatgctttc aagagagaca ttccagctac cctgattcat 60
tttttggtac c 71
<210> 8
<211> 71
<212> DNA
<213> Artificial sequence
<400> 8
aattggtacc aaaaaatgaa tcagggtagc tggaatgtct ctcttgaaag cattccagct 60
accctgattc a 71
Claims (4)
1. The application of the siRNA for specifically inhibiting the expression of the SPIB gene in the preparation of the medicine for reversing the drug resistance of the ovarian cancer taxol is characterized in that the siRNA comprises a sense strand and an antisense strand, wherein the nucleotide sequence of the sense strand is shown as SEQ ID NO.1, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 2.
2. The use of claim 1, wherein 3 bases at the 5 ' and 3 ' ends of the sense and antisense strands are 2 ' -methoxy modified.
3. The application of a recombinant vector containing a DNA sequence of siRNA for specifically inhibiting the expression of SPIB gene in the preparation of drugs for reversing the drug resistance of ovarian cancer taxol is characterized in that the siRNA comprises a sense strand and an antisense strand, wherein the nucleotide sequence of the sense strand is shown as SEQ ID NO.1, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 2.
4. The use of claim 3, wherein 3 bases at the 5 ' and 3 ' ends of the sense and antisense strands are 2 ' -methoxy modified.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710470667.1A CN107164378B (en) | 2017-06-20 | 2017-06-20 | siRNA for specifically inhibiting SPIB gene expression and recombinant vector and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710470667.1A CN107164378B (en) | 2017-06-20 | 2017-06-20 | siRNA for specifically inhibiting SPIB gene expression and recombinant vector and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107164378A CN107164378A (en) | 2017-09-15 |
| CN107164378B true CN107164378B (en) | 2019-12-31 |
Family
ID=59820250
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710470667.1A Expired - Fee Related CN107164378B (en) | 2017-06-20 | 2017-06-20 | siRNA for specifically inhibiting SPIB gene expression and recombinant vector and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107164378B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR102672646B1 (en) * | 2020-07-02 | 2024-06-07 | 연세대학교 산학협력단 | A Method for Inhibiting Cancer Metastasis by Modulating Anchorage-Dependency of Cancer Cells |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101701217A (en) * | 2009-11-18 | 2010-05-05 | 王理伟 | SiRNA restraining gene expression of transcription factor Spl and application thereof |
| WO2015097210A1 (en) * | 2013-12-23 | 2015-07-02 | Issazadeh-Navikas Shohreh | Immunosuppressive foxa1-expressing t cells |
| CN106609300A (en) * | 2015-10-23 | 2017-05-03 | 北京乐普基因科技股份有限公司 | Coronary artery disease risk assessment kit and risk assessment method |
-
2017
- 2017-06-20 CN CN201710470667.1A patent/CN107164378B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101701217A (en) * | 2009-11-18 | 2010-05-05 | 王理伟 | SiRNA restraining gene expression of transcription factor Spl and application thereof |
| WO2015097210A1 (en) * | 2013-12-23 | 2015-07-02 | Issazadeh-Navikas Shohreh | Immunosuppressive foxa1-expressing t cells |
| CN106609300A (en) * | 2015-10-23 | 2017-05-03 | 北京乐普基因科技股份有限公司 | Coronary artery disease risk assessment kit and risk assessment method |
Non-Patent Citations (2)
| Title |
|---|
| The ETST ranscription Factor Spi-B Is Required for Human Plasmacytoid Dendritic Cell Development;Remko Schotte等;《The Journal of Experimental Medicine》;20041206;第200卷(第11期);第1504页左栏第3段，摘要 * |
| 血细胞特异性转录因子SPIB在肺癌转移中的功能研究;牛卿;《万方学位论文数据库》;20141231;1-63 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107164378A (en) | 2017-09-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Chen et al. | RETRACTED: LINC01234/MicroRNA-31-5p/MAGEA3 Axis Mediates the Proliferation and Chemoresistance of Hepatocellular Carcinoma Cells | |
| Gong et al. | LncRNA HAND2‐AS1 represses cervical cancer progression by interaction with transcription factor E2F4 at the promoter of C16orf74 | |
| JP4658936B2 (en) | Compositions and methods for treating lung cancer | |
| Wang et al. | Hsa-let-7e-5p inhibits the proliferation and metastasis of head and neck squamous cell carcinoma cells by targeting chemokine receptor 7 | |
| Wang et al. | Gankyrin promotes epithelial-mesenchymal transition and metastasis in NSCLC through forming a closed circle with IL-6/STAT3 and TGF-β/SMAD3 signaling pathway | |
| He et al. | LncRNA DLEU2 promotes cervical cancer cell proliferation by regulating cell cycle and NOTCH pathway | |
| Wu et al. | LncRNA SLCO4A1-AS1 modulates colon cancer stem cell properties by binding to miR-150-3p and positively regulating SLCO4A1 | |
| Xiao et al. | Long noncoding RNA LINC01123 promotes the proliferation and invasion of hepatocellular carcinoma cells by modulating the miR-34a-5p/TUFT1 axis | |
| Wang et al. | Long non-coding RNA LOC283070 mediates the transition of LNCaP cells into androgen-independent cells possibly via CAMK1D | |
| Wang et al. | Long non-coding RNA FOXD1-AS1 promotes the progression and glycolysis of nasopharyngeal carcinoma by sustaining FOXD1 expression | |
| Xue et al. | LncRNA loc339803 acts as CeRNA of miR-30a-5p to promote the migration and invasion of hepatocellular carcinoma cells | |
| Huang et al. | LncRNA AC098934 promotes proliferation and invasion in lung adenocarcinoma cells by combining METTL3 and m6A modifications | |
| Zhang et al. | HnRNPU-AS1 inhibits the proliferation, migration and invasion of HCC cells and induces autophagy through miR-556-3p/miR-580-3p/SOCS6 axis | |
| Zhang et al. | circ_SMAD2 regulate colorectal cancer cells proliferation through targeting miR-1258/RPN2 signaling pathway | |
| CN107354157B (en) | siRNA for specifically inhibiting LAMB1 gene expression, recombinant vector and application thereof | |
| Cai et al. | The let-7c/HoxB7 axis regulates the cell proliferation, migration and apoptosis in hepatocellular carcinoma | |
| EP1599586B1 (en) | Rna-interference for znfn3a1-gene as a method for inhibiting cancer cell growth | |
| Zhang et al. | GR silencing impedes the progression of castration-resistant prostate cancer through the JAG1/NOTCH2 pathway via up-regulation of microRNA-143-3p | |
| CN107164378B (en) | siRNA for specifically inhibiting SPIB gene expression and recombinant vector and application thereof | |
| Ye et al. | Circ-0036602 acts as a sponge of MiR-34a-5p and MiR-431-5p to promote cervical cancer proliferation and invasion | |
| CN107541514B (en) | siRNA for specifically inhibiting CO L12A 1 gene expression, recombinant vector and application thereof | |
| CN107287200B (en) | The siRNA and its recombinant vector of specificity inhibition Eya2 gene expression and application | |
| CN107541515B (en) | siRNA for specifically inhibiting PRV1 gene expression and recombinant vector and application thereof | |
| CN107236736B (en) | The siRNA and its recombinant vector of specificity inhibition MSI-1 gene expression and application | |
| Zhao et al. | Effects of EZH2 gene on the growth and migration of hepatocellular carcinoma HepG2 cells |
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: 20191231 |