Application of miR-34a gene in non-small cell lung cancer

A non-small cell lung cancer and gene technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, drug combination, etc., can solve the problems of lack of early diagnosis and treatment methods for lung cancer

Inactive Publication Date: 2013-07-31
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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

Method used

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  • Application of miR-34a gene in non-small cell lung cancer
  • Application of miR-34a gene in non-small cell lung cancer
  • Application of miR-34a gene in non-small cell lung cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: According to the results of Solexa sequencing, it is determined that miR-34a is under-expressed in non-small cell lung cancer cell lines

[0039] The k-ras gene mutation mouse lung cancer model (L703T2) and the normal lung cancer model (L1805) used in the present invention are provided by the research group of Professor Ji Hongbin from the Institute of Biochemical Cells, Chinese Academy of Sciences.

[0040] The lung tissues of normal mice and those of mice with non-small cell lung cancer were sampled. The tissues were lysed by the TRIZOL method, and chloroform was added. After the protein and nucleic acid were layered, the supernatant was removed by centrifugation and isopropanol precipitation was added. After centrifugation again, the pellet was washed with ethanol and dried to obtain total RNA. TaKaRa company’s reverse transcription kit was used to construct cDNA libraries of the two tissues, oligo dT was used as primer for reverse transcription, and the cDNA ...

Embodiment 2

[0042] Example 2: The effect of miR-34a on the proliferation of H1299 cells

[0043] Spread the H1299 cells evenly in a 96-well plate with a medium of 1640. After 24 h, pass the mimics of miR-34a through lipo2000 transfection reagent and co-incubate with serum-free medium to transfer them into H1299 cells for transfection. After 6 h, replace the cell culture medium, put in fresh culture medium, then immediately add CCK8 reagent, incubate in the incubator for 2.5 h, then transfer the solution to the microplate, and detect the absorbance value at 450 nm every 24 h Measure once.

[0044] The test found that the transfection group had a significant decrease in absorbance compared with the control group, that is, the proliferation rate of the cells transfected with miR-34a was significantly lower than that of the control group, indicating that miR-34a can inhibit the proliferation of H1299 cells ( See figure 2 ).

Embodiment 3

[0045] Example 3: The effect of miR-34a on H1299 cell apoptosis

[0046] Spread the H1299 cells evenly in a 6-well plate. After 24 hours, the miR-34a mimics were incubated with lipo2000 and serum-free medium 1640 and then transferred to H1299 cells. After 6 hours of transfection, the cell culture medium was replaced with fresh culture liquid. 48 h after transfection, the collected cells were stained with annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (Sigma-Aldrich, USA). Flow cytometry was used to detect the apoptosis of H1299 cells. The results showed that the apoptosis rate of H1299 cells transfected with miR-34a was significantly higher than that of the control group (see image 3 , Figure 4 with Figure 5 ).

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Abstract

The invention relates to an application of an miR-34a gene in non-small cell lung cancer. A target gene TGF[beta]R2 of the miR-34a in an H1299 cell line is also provided. The miR-34a is complemented with 3'-UTR of the mRNA of the target gene, such that the translation of the mRNA of the target gene is inhibited, or the mRNA of the target gene is directly degraded. The miR-34a and the TGF[beta]R2 have interaction with each other in the H1299 cell line. The interaction influences life actions such as propagation, apoptosis and the like of the H1299 cells. TGF[beta]R2 is primarily validated as the target gene of the miR-34a by software prediction, vector construction and luciferase reporter gene analysis in HEK293T cells; and then the phenomenon is further validated by a technology of expressing the miR-34a and qRT-PCR in the H1299 cells. The disclosure is the first report that the TGF[beta]R2 in the H1299 cell line is the target gene of the miR-34a. Application values in diagnosis and treatment for the non-small cell lung cancer by using the miRNA clinically and in drug target aspects are provided by the invention.

Description

technical field [0001] The invention relates to the application of miRNA gene in non-small cell lung cancer. Background technique [0002] Functional regulation of microRNA (miRNA, miRNA) is an important frontier in current life sciences. miRNA is a class of non-coding RNA molecules with a length of 21-22 nt, which regulate the activity of target genes through post-transcriptional gene silencing. Studies on the biological function of miRNA and the target genes it regulates show that miRNA plays an important role in the process of tumorigenesis, and miRNA is likely to become a new approach for cancer treatment. There is a one-to-many relationship between miRNA and the non-coding sequence (3′-UTR or 5′-UTR) of target gene mRNA. miRNA is usually transcribed by RNA polymerase II in the nucleus, the primary transcription product is pri-miRNA, and pri-miRNA is processed by nuclease RNase III Drosha and its cofactor Pasha into a hairpin precursor miRNA (pre-miRNA). miRNA is expo...

Claims

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Application Information

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IPC IPC(8): C12N15/113A61P35/00
Inventor 金由辛马中良李艳利侯品品朱绍良
Owner SHANGHAI UNIV
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