Application of miR-486-5p in non-small cell lung carcinoma cell line

A non-small cell lung cancer, cell technology, applied in the field of application of miR-486-5p in non-small cell lung cancer cell lines

Inactive Publication Date: 2014-10-01
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Lung adenocarcinoma (lung adenocarcinoma) is the most common cancer, and about 80% of lung cancers are non-small c...

Method used

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  • Application of miR-486-5p in non-small cell lung carcinoma cell line
  • Application of miR-486-5p in non-small cell lung carcinoma cell line
  • Application of miR-486-5p in non-small cell lung carcinoma cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: According to Solexa sequencing results, it is determined that miR-486-5p is lowly expressed in non-small cell lung cancer cell lines

[0038] The k-ras gene mutant mouse lung cancer model (L703T2) and normal lung cancer model (L1805) used in the present invention were provided by the research group of Professor Ji Hongbin, Institute of Biochemical Cells, Chinese Academy of Sciences.

[0039] The lung tissue of normal mice and the lung tissue of mice with non-small cell lung cancer were sampled, the tissue was lysed by TRIZOL method, chloroform was added, and the protein and nucleic acid were separated. After centrifugation again, the precipitate was washed with ethanol and dried to obtain total RNA. The cDNA libraries of the two tissues were constructed using the reverse transcription kit from TaKaRa Company, reverse transcription was performed with oligo dT as the primer, and the cDNA for subsequent experiments was obtained through two steps of denaturatio...

Embodiment 2

[0041] Example 2: Effect of miR-486-5p on the proliferation of H1299 cells

[0042] Spread the H1299 cells evenly in a 96-well plate, and the medium used is 1640. After 24 h, the mimics of miR-486-5p were transferred into the H1299 cells through lipo2000 transfection reagent and co-incubation in serum-free medium. After 6 hours of transfection, replace the cell culture medium, put in fresh culture medium, then immediately add CCK8 reagent, incubate in the incubator for 2.5 hours, then transfer the solution to a microtiter plate, and detect the absorbance value at 450 nm. Measured every 24 hours.

[0043] The test found that compared with the control group, the absorbance of the transfection group decreased significantly, that is, the proliferation rate of the cells transfected with miR-486-5p was significantly lower than that of the control group, indicating that miR-486-5p has the ability to inhibit the growth of H1299 cells. role in proliferation (see image 3 ).

Embodiment 3

[0044] Example 3: Effect of miR-486-5p on H1299 cell cycle

[0045] H1299 cells were evenly plated in 6-well plates, and each sample was replicated three times. MiR-146a-5p mimics were transfected 24 h later. Cells were collected after 48 h. Wash twice with PBS and fix overnight at 4°C with 70% ethanol. Wash once with PBS, digest with 50 mg / ml RNase for 1 h, room temperature. Stain with 50 mg / ml PI for 30 min at 4°C and detect with flow cytometry. (see Figure 4 ).

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Abstract

The invention relates to application of miR-486-5p in a non-small cell lung carcinoma cell line. The miR-486-5p performs specific low-expression in a non-small cell lung carcinoma cell and a series of lung carcinoma tissue clinical specimen; in an H1299 cell, the over-expressed miR-486a-5p can inhibit cell proliferation of H1299 and inhibit G1/S conversion to block the cell in the G1 phase. Experiments based on bioinformatics analysis discovers that the miR-486a-5p is complementary with 3'-UTR () of a target gene mRNA (messenger ribonucleic acid), so that translation of the mRNA of the target gene can be inhibited or the mRNA of the target gene can be directly degraded; furthermore, western blotting and other experiments prove that the CDK4 gene is the target gene of the miR-486a-5p. The experiment first proves that the CDK4 gene is the garget gene of miR-486a-5p, and the miRNA is utilized to diagnose and treat non-small cell lung carcinoma in clinical application, and a certain application value is created for providing the aspect of drug targets.

Description

technical field [0001] The present invention relates to the application of miR-486-5p in non-small cell lung cancer cell lines. Background technique [0002] MicroRNA ( microRNA , miRNA ) function regulation is an important frontier of current life sciences. MicroRNA (microRNA, miRNA) is a kind of non-coding small molecule RNA of about 22 nt, which widely exists in higher eukaryotes, and most of them have high conservation. miRNAs act on specific mRNAs to degrade them or inhibit their translation, and negatively regulate gene expression at the post-transcriptional level, while some miRNAs can activate the transcription of target genes. The 2nd to 7th bases at the 5′ end of mature miRNA are called “seed sequence”, which is the key to effective regulation of mRNA. The binding site of miRNA can be not only the 3′-UTR of the target gene, but also the target gene 5′-UTR or CDS region of the In mammals, about 30% of protein-coding genes are regulated by miRNAs. [0003] Lung c...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61P35/00
Inventor 金由辛马中良申雨晴袁天蔚张冰洁李艳利韦嘉励
Owner SHANGHAI UNIV
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