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Human fascin-1 gene siRNA and applications thereof

A fascin-1, gene technology, applied in the field of bioengineering, can solve the problems of fascin-1 malignant proliferation and the like

Active Publication Date: 2015-04-22
FIRST HOSPITAL OF SHANXI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In order to solve the problem of malignant proliferation, invasion and metastasis of tumor cell fascin-1, the present invention provides a kind of siRNA targeting human fascin-1 gene and its application, so as to achieve the malignant biology of inhibiting its malignant proliferation, invasion and metastasis phenotype, which may be a druggable target for tumor-targeted therapy

Method used

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  • Human fascin-1 gene siRNA and applications thereof
  • Human fascin-1 gene siRNA and applications thereof
  • Human fascin-1 gene siRNA and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Expression of fascin-1 protein in laryngeal squamous cell carcinoma tissue and corresponding normal paracancerous mucosa tissue

[0032] Paraffin-embedded tissues were sectioned serially at 4 μm, deparaffinized and hydrated; repaired with citrate buffer under high pressure, blocked with 3% hydrogen peroxide methanol solution for 10 min, washed 3 times with phosphate buffered saline (PBS); added blocking solution 10 min, incubate with primary antibody overnight at 4°C; wash 3 times with PBS Tween 20 solution, incubate with secondary antibody for 15-20 min, wash 3 times with PBS, develop color with diaminobenzidine (DAB); counterstain with hematoxylin and dehydrate to seal; PBS Instead of primary antibody as negative control, known positive samples of lung squamous cell carcinoma were used as positive control, and vascular endothelial cells as internal control; mouse anti-human monoclonal antibody fascin-1 (MS-1112) was purchased from Thermo Fisher Corporatio...

Embodiment 2

[0035] Example 2 Expression of fascin-1 mRNA and protein in laryngeal squamous carcinoma cell line Hep-2 and normal oral pharyngeal mucosal keratinocyte line HOK

[0036] 1. Cell culture

[0037]Preheat the water bath to 37°C; wipe the surface of the ultra-clean workbench exposed to ultraviolet light for 30 minutes with 75% alcohol; place sterile centrifuge tubes, straws, culture bottles, etc. in order on the ultra-clean workbench; take out the cryotube; quickly To thaw, quickly put the frozen tube into a water bath that has been preheated at 37°C to thaw quickly, and shake it constantly to make the liquid in the tube melt quickly within 2 minutes, and there is still a little unthawed liquid in the frozen tube. When the time comes, take it out; wipe the outer wall of the cryotube with an alcohol cotton ball, and then put it into the ultra-clean bench; prepare the cell suspension, transfer the cells to a 15ml centrifuge tube, gradually add the preheated medium, and centrifu...

Embodiment 3

[0066] Example 3 Transfection of fascin-1 siRNA in Hep-2 and screening of effective siRNA

[0067] 1. siRNA design:

[0068] Using the siRNA online design tool of Ambion Company (http: / / www.ambion.com / techlib / misc / siRNA_

[0069] finder.html), and based on the principles of siRNA design, siRNA sequence design was performed for the sequence of fascin-1 mRNA (NM_003088.3) in the human NCBI gene database, and the candidate target sequence was submitted to BLAST (http: / / www.ncbi.nlm .nih.gov / blast / Blast.cgi) website, after sequence homology BLAST analysis, it was proved that the designed interfering sequence had no homology with other genes, so as to ensure the specificity of the interfering target gene.

[0070] The designed sequence is:

[0071] No.1

[0072] Sense strand: 5′-CAAAGACTCCACAGGCAAAtt-3′

[0073] Antisense strand: 5′-TTTGCCTGTGGAGTCTTTGtt-3′

[0074] No.2

[0075] Sense strand: 5′-CGACTATAACAAGGTGGCCATtt-3′

[0076] Antisense strand: 5′-ATGGCCACCTTGTT...

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Abstract

The present invention discloses a human fascin-1 gene siRNA and applications thereof, and belongs to the technical field of bio-engineering, wherein problems of malignant proliferation, invasion and metastasis of tumor cells fascin-1 are solved with the human fascin-1 gene siRNA. The human fascin-1 gene siRNA comprises the following sequences: a sense strand: 5'-CGACTATAACAAGGTGGCCATtt-3'; and an antisense strand: 5'-ATGGCCACCTTGTTATAGTCGtt-3'. According to the present invention, malignant biological phenotypes such as malignant proliferation, invasion and metastasis of tumor cells can be inhibited with the expressed human fascin-1 gene siRNA; and the human fascin-1 gene siRNA can be widely used for researching inhibition on malignant phenotypes of tumor cells, and can further be used for preparing anti-tumor biological targeting agents or drugs.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to an siRNA aimed at human fascin-1 gene and its application. Background technique [0002] Laryngeal cancer is one of the most common malignant tumors of the head and neck, originating from the epithelium of the laryngeal mucosa, and has a high mortality rate. According to epidemiological studies in North America and Europe, the incidence rate is 7.0-13.2 / 100,000 people, showing an upward trend in the past 20 years. Among the biological characteristics of tumor cells, invasion and metastasis are the most critical. According to clinical statistics, more than 80% of cancer patients die of invasion and metastasis, while those without invasion and metastasis have a better prognosis. However, due to the limited anatomical structure of the laryngeal cavity and the rich lymphoid tissue, the laryngeal squamous cell carcinoma has the malignant biological behavior of local invasion an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113A61K48/00A61P35/00
Inventor 高伟王斌全张春明温树信皇甫辉陈钢钢张海利
Owner FIRST HOSPITAL OF SHANXI MEDICAL UNIV
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