Effect of miR-451a cells in non-small cell lung cancer

A mir-451a, non-small cell lung cancer technology, applied in gene therapy, genetic material components, drug combinations, etc., can solve the problem of lack of early diagnosis methods for lung cancer

Inactive Publication Date: 2016-03-23
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

About 80% of lung cancers are non-small cell lung cancers (non-small cell lung cancer, NSCLC), due to the lack of effective ...

Method used

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  • Effect of miR-451a cells in non-small cell lung cancer
  • Effect of miR-451a cells in non-small cell lung cancer
  • Effect of miR-451a cells in non-small cell lung cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: According to the results of Solexa sequencing, it is determined that miR-451a is underexpressed in the mouse induced tumor model

[0034] The KRAS gene mutant mouse lung cancer models (L822T1, L703T2, L903T1) 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. where L822T1 is genotype KRAS + / + of benign tumors, L703T2 is genotype LKB1 - / - / KRAS + / + malignancy of L903T1 to P53 - / - / KRAS + / + of malignant tumors.

[0035]The lung tissue of normal mice and the lung tissue of induced cancer mice were sampled separately, and the total RNA in the tissue was extracted by Trizol method. The specific steps are: lyse the tissue with Trizol, place it at room temperature for 5 minutes to fully lyse the tissue, and obtain the lysate; add chloroform, vortex for 15 seconds, and centrifuge at 12000g / 4°C for 10 minutes;...

Embodiment 2

[0064] Example 2: Detection of miR-451a expression in human non-small cell lung cancer tissue clinical samples

[0065] The 41 pairs of lung cancer and paracancerous tissues involved in this example were all from Shanghai Chest Hospital.

[0066] In this example, the Trizol method in Example 1 was used to extract the total RNA of the tissue, and the OneStepPrimeScript miRNAcDNASynthesis Kit (Code No. D350A) of Takara Company was used for reverse transcription to construct the cDNA library of the above tissue, and the oligodt was used as the primer for reverse transcription. Two steps of reverse transcription were used to obtain the miRNA cDNA library required for subsequent experiments.

[0067] The study found that the tissues of 41 cases were classified according to the TNM stage. In the NOM1 group, the expression level of miR-451a decreased to 30.64% compared with the adjacent tissues, and the significance was good at Pfigure 1 b)

Embodiment 3

[0068] Example 3: Detection of miRNA expression differences in common lung cancer cell lines

[0069] Common lung cancer cell lines include: A549, H1299, Spca-1, Hcc827, 95-D, etc. Among them, A549 and Hcc827 are human non-small cell lung cancer cells, Spca-1 is human lung adenocarcinoma cells, and H1299 is non-small cell lung cancer epithelioid cells, and 95-D is human highly metastatic lung cancer cells. In this example, normal lung epithelial cells Beas-2B were used as a reference, and the above five lung cancer cell lines were cultured simultaneously. Cell culture was carried out according to the ATCC standard procedure, and they were respectively cultured in the corresponding medium containing 10% fetal bovine serum. The cell culture incubator was kept humid and maintained at 5% CO 2 In culture state, cells were cultured at 37°C.

[0070] When the cells reached about 80% density in the 6cm culture dish, use the Trizol method in Example 1 to extract cellular RNA, and ...

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Abstract

The invention relates to the effect of miR-451a cells in the non-small cell lung cancer. Migration of A549 cells can be significantly inhibited through overexpression of miR-451a in the A549 cells. By means of the bioinformatics analysis means, it is predicted that translation of the target gene mRNA is inhibited or the target gene mRNA is directly degraded by miR-451a through regional complementation with 3'-UTR of the target gene CAB39 mRNA; it is determined that the CAB39 gene is the target gene of miR-451a through double-luciferase reporter gene system analysis and Western Blot experiment verification. Finally, it is verified through the Transwell Assay and Flow Cytometry Assay experimental means that miR-451a performs the function of inhibiting migration of cells by reducing the CAB39 gene in the A549 cells, and a novel accurate treating researching and treating scheme is provided. By means of the effect, important research guidance and application value is provided for using miRNA for diagnosing and treating the non-small cell lung cancer and searching for drug targets in the fields such as tumor immunotherapy clinically.

Description

technical field [0001] The invention relates to the application of miR-451a cells in non-small cell lung cancer. Background technique [0002] Lung cancer is the leading cause of cancer death worldwide, with more than 1 million deaths and 1.2 million new cases each year. About 80% of lung cancers are non-small cell lung cancers (non-small cell lung cancer, NSCLC), due to the lack of effective early diagnosis methods for lung cancer, and NSCLC has a strong ability to metastasize, its 5-year survival rate is only 15%-30 %. Timely treatment of NSCLC patients will greatly increase the survival rate of patients and improve the health status of patients. Therefore, research on the early diagnosis of NSCLC and the mechanism of tumor metastasis is urgent. [0003] Functional regulation of microRNA (miRNA, miRNA) is an important frontier in current life sciences. miRNA is a kind of endogenous non-coding small molecule RNA with a length of about 22nt, which regulates gene expressi...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61P35/04A61P35/00
CPCA61K48/0066
Inventor 金由辛金言王强党启李雪
Owner SHANGHAI UNIV
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