Long non-coding RNA related to melanoma and application thereof

A melanoma, non-coding technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, microbial measurement / inspection, etc., to achieve the effect of improving the survival rate

Inactive Publication Date: 2020-04-07
刘秀玲
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on melanoma mostly focuses on traditional protein-coding genes and protein signal transduction, while the research on non-coding RNA is less

Method used

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  • Long non-coding RNA related to melanoma and application thereof
  • Long non-coding RNA related to melanoma and application thereof
  • Long non-coding RNA related to melanoma and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Detection of the expression of LncRNA-FA2H-2 in melanoma cancer tissues and adjacent tissues

[0025] 1. Research object

[0026] 20 cases of melanoma cancer tissues and corresponding paracancerous tissues were selected for the experiment.

[0027] 2. Tissue RNA Extraction

[0028] (1) Put about 50 mg of tissue into a mortar, add a small amount of liquid nitrogen, and grind quickly. After the tissue becomes soft, add a small amount of liquid nitrogen for grinding again, and repeat three times;

[0029] (2) Add 1ml Trizol, use an electric homogenizer to homogenize for 2 minutes, transfer to a centrifuge tube after homogenization, and place at room temperature for 5 minutes;

[0030] (3) Centrifuge at 12000rpm for 5 minutes, take the supernatant, and remove the precipitate;

[0031] (4) Add 200ul of chloroform, shake and mix, and let stand at room temperature for 15 minutes;

[0032] (5) Centrifuge at 12000rpm at 4°C for 15min;

[0033] (6) Take the supernatant (be c...

Embodiment 2

[0069] Knockdown of LncRNA-FA2H-2 Gene

[0070] 1. siRNA design

[0071] si-RNA-1

[0072] 5'-AUUCUUCUGACAUGUUUCCUG-3'

[0073] 5'-GGAAACAUGUCAGAAGAAUGC-3'

[0074] si-RNA-2

[0075] 5'-UCACAUUCAAUAUGUACAGCU-3'

[0076] 5'-CUGUACAUAUUGAAUGUGAAU-3'

[0077] si-NC

[0078] 5'-ACAGAGCCUCGCCUUUGCCGAU-3'

[0079] 5'-CUUGCACAUGCCGGAGCCGUU-3'

[0080] 2. Cell Culture

[0081] Human melanoma cell line A357 was cultured in high-glucose DMEM medium (10% fetal bovine serum) at 37°C, 5% CO 2 .

[0082] 3. siRNA transfection

[0083] (1) The day before transfection, 2×10 5 A357 cells were seeded onto 6-well culture plates at 37 °C, 5% CO 2 After culturing in the incubator for 24 hours, transfect according to the instructions of Lipofectamine 2000;

[0084] (2) The experimental groups were divided into si-NC group, si-RNA-1 and si-RNA-2.

[0085] 4. Real-time quantitative PCR detection of LncRNA-FA2H-2 gene expression after knockdown

[0086] (1) Extraction of total cellular...

Embodiment 3

[0094] 1. Cell Proliferation Experiment

[0095] (1) Inoculate A357 in a 96-well plate at a cell density of 5000 cells / ml, 200 μl per well, set up 3 replicate wells for each group, place at 37°C, 5% CO 2 Static culture in the incubator;

[0096] (2) A357 cells were transfected with si-NC, si-RNA-1, and si-RNA-2 respectively. 4 hours before the end of the culture, 20 μl of 5 mg / ml MTT solution was added to each well, and the culture was continued at 37°C and 5% CO2 Stand in the box and cultivate until the end;

[0097] (3) Aspirate and discard the culture medium in the wells, add 150 μl DMSO to each well, shake horizontally for 10 min, detect the absorbance at 492 nm with a microplate reader, and draw the growth curve.

[0098] (4) SPSS19.0 statistical software was used to establish a database and conduct statistical analysis. The measurement data were expressed as mean ± standard deviation. The comparison of the means of the two groups was carried out by independent sample t...

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Abstract

The invention discloses a long non-coding RNA related to melanoma and application thereof. The long non-coding RNA is LncRNA-FA2H-2. Researches prove that the expression of the LncRNA-FA2H-2 in tissues of a melanoma patient is up-regulated; the proliferation capacity of melanoma cells A357 can be remarkably reduced by knocking down the LncRNA-FA2H-2 through the si-RNA; and thus the result indicates that the LncRNA-FA2H-2 can be used as a molecular marker and a target point of melanoma for diagnosis and treatment of the melanoma. The long non-coding RNA related to melanoma has the beneficial effects that the biomarker LncRNA-FA2H-2 is provided for the diagnosis of melanoma, and the early diagnosis of melanoma can be realized by detecting the expression of the gene; the invention also provides application of siRNA of LncRNA-FA2H-2 in preparation of a melanoma pharmaceutical composition; and the long non-coding RNA has important clinical significance and broad application prospects.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a long non-coding RNA for detecting melanoma and its application. Background technique [0002] Melanoma is the most malignant primary skin tumor in the skin. It originates from melanoma cells and is the most aggressive malignant tumor among all skin tumors. The prognosis of patients with melanoma is poor, and the survival time of patients is short. Although melanoma is less common than other skin cancers such as squamous cell carcinoma and basal cell carcinoma, it accounts for approximately 75% of deaths from skin malignancies. Because the early symptoms of melanoma patients are not obvious, the patients are usually in the middle and advanced stages when they are diagnosed. Therefore, it is of great value to study the specific mechanism of the occurrence and development of melanoma to improve the early diagnosis and prognosis of patients. At present, the resea...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/158C12Q2600/178
Inventor 刘秀玲
Owner 刘秀玲
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