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Application of KPNA2 (karyopherin alpha 2) gene and application of siRNA (small interfering ribonucleic acid) for inhibiting expression of KPNA2 gene

A gene and expression technology, applied in the field of pharmaceutical genetic engineering, to achieve the effect of improving accuracy, improving five-year survival rate, inhibiting value-added and migration

Active Publication Date: 2017-08-08
HARBIN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, there is no literature report on the correlation between colorectal cancer and KPNA2 gene expression at home and abroad

Method used

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  • Application of KPNA2 (karyopherin alpha 2) gene and application of siRNA (small interfering ribonucleic acid) for inhibiting expression of KPNA2 gene
  • Application of KPNA2 (karyopherin alpha 2) gene and application of siRNA (small interfering ribonucleic acid) for inhibiting expression of KPNA2 gene
  • Application of KPNA2 (karyopherin alpha 2) gene and application of siRNA (small interfering ribonucleic acid) for inhibiting expression of KPNA2 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1 Patient and sample collection

[0069] First, 30 colorectal cancer patients (15 male patients and 15 female patients, covering stage I to stage IV) who underwent radical surgery in the Colorectal Surgery Department of the Second Affiliated Hospital of Harbin Medical University from January 2014 to June 2014 were collected. These patients did not receive neoadjuvant chemoradiotherapy before operation. 10 mL of venous blood was collected from the patient before the operation, and after centrifugation (1000 rpm, centrifuged for 5 minutes), the patient’s serum was collected and stored in a -80°C refrigerator for later use. Postoperative lesion samples and adjacent normal mucosal tissues were collected from 30 patients for real-time reverse transcriptase polymerase chain reaction (RT-PCR). Immediately after surgical resection of the lesion, the obtained tissue was placed in liquid nitrogen and stored in a -80°C refrigerator, soaked in 10% formalin solution for ...

Embodiment 2

[0070] Example 2 Immunohistochemical staining

[0071]Immunohistochemical staining analysis was performed by the avidin peroxidase method. After the sample tissue provided in Example 1 was embedded in paraffin, the paraffin tissue was cut into 4 micron thick sections, dewaxed and dehydrated. After soaking in methanol for 10 minutes, endogenous peroxidase activity was terminated with 0.5% hydrogen peroxide. Non-specific binding should be eliminated by soaking in phosphate buffered saline (PBS) containing 10% goat serum for 1 hour at room temperature. Then slice in PBS containing mouse anti-human KPNA2 polyclonal antibody (1:300; Wuhan Proteintech, Wuhan, China) overnight at 4°C. After that, it was incubated with biotin-labeled goat anti-mouse immunoglobulin (IgG; 1:400; ZSGB-Bio, Beijing, China) for 1 hour at room temperature. This is followed by streptomycin-peroxidase treatment. Sections were incubated for 5 minutes at room temperature containing 0.1% 3,3-diaminobenzidine...

Embodiment 3

[0079] Example 3 Real-time reverse transcription polymerase chain reaction

[0080] Total RNA from 30 cases of colorectal cancer and adjacent normal tissues provided in Example 1 was extracted with Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the instructions. Follow the instructions to adopt RevertAid TM H Minus First Strand cDNASynthesis Kit (Fermentas, Burlington, Ontario, Canada) was used for reverse transcription. Primers for KPNA2 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (TaKaRa Biotechnology, Dalian, China) are:

[0081]

[0082] PCR using SYBR Green dye method. Use the ABI PRISM(R) 7500 real-time quantitative PCR instrument to perform the reaction according to the corresponding procedures in the instructions. In order to prove the specificity of the amplification products, the primers were subjected to melting curve analysis. The PCR cycling conditions were as follows:

[0083]

[0084] The results were analyzed by relative quantit...

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Abstract

The invention provides application of a KPNA2 (karyopherin alpha 2) gene and application of siRNA (small interfering ribonucleic acid) for inhibiting expression of the KPNA2 gene, and relates to the technical field of medicine gene engineering. The application of the KPNA2 gene as a colorectal cancer biomarker provided by the invention can be used for effectively instructing the clinical stages of the colorectal cancer, and accurately judging the prognosis of a colorectal cancer patient. The siRNA for inhibiting the expression of the KPNA2 gene can effectively inhibit the proliferation and transfer of colorectal cancer cells. The invention also provides application of the siRNA in preparing a KPNA2 gene silent colorectal cancer cell line. The cell line can be used for studying the correlation and mechanism between KPNA2 and the colorectal cancer, and providing a reliable study foundation for deeply studying the possible mechanism of generation and development between the KPNA2 and the colorectal cancer, thereby providing an effective therapeutic target for the target therapy of the colorectal cancer.

Description

technical field [0001] The invention relates to the technical field of medical genetic engineering, in particular to the application of a KPNA2 gene and the application of siRNA for inhibiting the expression of the KPNA2 gene. Background technique [0002] Colorectal cancer is the third most common malignant tumor in the four countries. The annual incidence is about 1.2 million, and the median age of diagnosis is 70 years old, but according to epidemiological statistics, the incidence tends to be younger. In some western countries, the incidence of colorectal cancer shows a downward trend, but in some countries, especially developing countries, the incidence of colorectal cancer still shows an obvious upward trend. With the development of my country's economy and the change of people's lifestyle and diet structure, the incidence of colorectal cancer in our country shows a trend of increasing year by year. The prognosis of patients with colorectal cancer is related to tumor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/113A61P35/00A61P35/04
CPCC12N15/113C12N2310/14C12Q1/6886C12Q2600/112C12Q2600/118C12Q2600/158
Inventor 郁雷高丽吕延杰
Owner HARBIN MEDICAL UNIVERSITY
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