Application of miR-451 in preparing medicine for treating non-small cell lung cancer

A non-small cell lung cancer, 1.mir-451 technology, applied in the field of genetic engineering, can solve the problems of low survival rate, easy development of drug resistance in patients, and loss of surgical opportunities.

Inactive Publication Date: 2010-08-18
NANJING MEDICAL UNIV +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many lung cancer patients have metastasized at the time of diagnosis, and often lose the opportunity for surgery. The chemotherapy regimens for lung cancer are also ineffectiv

Method used

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  • Application of miR-451 in preparing medicine for treating non-small cell lung cancer
  • Application of miR-451 in preparing medicine for treating non-small cell lung cancer
  • Application of miR-451 in preparing medicine for treating non-small cell lung cancer

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] 1. Amplify Pre-miR-451: Pre-miR-451, its RT and PCR primers were synthesized by Invitrogen Company, and the primer sequences are shown in the following RT-PCR. RT-PCR amplified Pre-miR-451, and the purified product was shown by agarose gel electrophoresis, and a band with the expected size was amplified at about 72 bp.

[0078] RT-PCR

[0079] Primers for miR-451 and U6 (as an internal reference) were designed and synthesized by Invitrogen.

[0080] 1) Primer sequences and reaction conditions

[0081] RT primers for MiR-451:

[0082] 5'-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAAACTCAG-3'

[0083] The RT primer of GAPDH is Oligo(dT) 15

[0084] Table 1. PCR primer sequences and reaction conditions

[0085]

[0086] 2) Synthesis of cDNA

[0087] 5X RT Buffer 4μl

[0088] dNTP 2μl

[0089] Inhibitor 1μl

[0090] Primer 1μl

[0091] RNA 1μl (mass less than 1μg)

[0092] RT ace 1 μl

[0093] h 2 O(RNase Free) 10μl

[0094] ...

Embodiment 2

[0120] MTT assay for cell proliferation:

[0121] 1) Cell inoculation: Digest monolayer cultured cells with 0.25% trypsin, prepare a single cell suspension with RPMIt604 culture medium containing 10% fetal bovine serum, inoculate 100-1000 cells per well in a 96-well culture plate, The volume of each well is 200ul. Divided into four experimental groups: empty vector group, Pre-miR-451 group, DDP group, Pre-miR-451+DDP group.

[0122] 2) Cultivate cells; move the culture plate into a COz incubator, and culture it for 3-5 days at 37° C., 5% CO 2 : and humidity.

[0123]3) Color development: After the 2nd to 5th day of culture, add 20ul of MTT solution (5mg / m1) to each well, continue to incubate at 37°C for 4h, terminate the culture, and carefully aspirate and discard the culture supernatant in the well. Add 150 μl DMSO (dimethyl sulfoxide) and shake for 10 min to fully dissolve the crystals.

[0124] 4) Colorimetry: The optical density (OD) was measured at a wavelength of 490n...

Embodiment 3

[0127] Apoptosis assay (determined by western blot)

[0128] 1. Extraction of total cell protein

[0129] 2. SDS-PAGE electrophoresis

[0130] 1) Clean and install glass plates and ceramic sheets.

[0131] 2) Prepare perfusion separation gel and let it stand at 37°C for 25 minutes.

[0132]

[0133] 3) Prepare perfusion stacking gel and let it stand at room temperature for 20 minutes.

[0134]

[0135] 4) Add protein samples and molecular weight markers, and the amount of protein loaded in each period is 40 μg.

[0136] 5) Constant voltage electrophoresis (80V for stacking gel, 160V for separating gel).

[0137] 3. Semi-dry transfer film

[0138] 1) After the electrophoresis is over, wear gloves, cut the gel (cut corner marker) according to the molecular weight range of the target protein, and cut one piece of nitrocellulose membrane (cut corner marker) and two filter papers of the same size.

[0139] 2) Soak the gel, membrane and filter paper in the transfer buffe...

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Abstract

The invention belongs to the field of genetic engineering, in particular to the application of miR-451 in preparing a medicine for treating non-small cell lung cancer. Since the recombinant of Pre-miR-451 has influence on propagation, apoptosis, migration and drug resistance of non-small cell lung cancer cells, it is proved that the miR-451 prohibits the propagation, migration and drug resistance of the non-small cell lung cancer cells and promotes the apoptosis of the non-small cell lung cancer cells; and living body level also indicates that the miR-451 has excellent anti-cancer effect.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and particularly relates to the application of miR-451 in the preparation of drugs for treating non-small cell lung cancer. Background technique [0002] RNA is one of the most important material bases in organisms, and together with DNA and proteins, it constitutes the framework of life. But for a long time, RNA was thought to be just a "transition" between DNA and protein, from which it obtained its sequence and then converted genetic information into protein. However, a series of studies have shown that these small RNAs actually manipulate many cellular functions, and they can turn off or regulate gene expression by reacting against DNA through the binding of complementary sequences. A recently discovered non-protein-coding RNA family, microRNA (miRNA, microRNA), can regulate gene expression by binding to specific mRNA or regulating the protein translation process of specific mRNA. At pres...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61P11/00A61P35/00
Inventor 德伟王朝霞陈龙邦潘旋袁栎王锐杨劲松边海波
Owner NANJING MEDICAL UNIV
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