Molecular marker miR-504 for regulating radiation resistance of nasopharyngeal carcinoma

A technology of mir-504 and molecular markers, applied in the field of new molecular markers, can solve the problems of unclear molecular biological mechanism, abnormal structure and function of tumor cell mitochondria, etc., and achieve easy mass screening, convenient sampling, and high specificity Effects on Sex and Sensitivity

Inactive Publication Date: 2015-12-23
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This phenomenon is related to the abnormal structure and function of tumor cell mitochondria, and its molecular biological mechanism is still unclear

Method used

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  • Molecular marker miR-504 for regulating radiation resistance of nasopharyngeal carcinoma
  • Molecular marker miR-504 for regulating radiation resistance of nasopharyngeal carcinoma
  • Molecular marker miR-504 for regulating radiation resistance of nasopharyngeal carcinoma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1. Identification of radioresistant phenotypes of nasopharyngeal carcinoma radioresistant cell lines.

[0044] Plate colony formation assay was used to investigate whether the radioresistant nasopharyngeal carcinoma cell lines were radioresistant. Disperse the monolayer cultured cells in the logarithmic growth phase into a single cell suspension by general passage method, and count them. Dilute the cell suspension in gradient multiples, inoculate in a culture dish or a culture plate at an appropriate cell density according to the purpose of the experiment, and then perform radiation treatment with different doses (0, 2, 4, 6Gy), place at 37°C, 5% CO 2 Continue culturing in the incubator for 12-14 days. When colonies visible to the naked eye appeared in the Petri dish, the culture was terminated. Discard the culture medium, wash carefully with PBS or DHanks solution 2-3 times, add methanol 5ml to fix for 15min, stain with 0.1% crystal violet or Giemsa staining...

Embodiment 2

[0046] Example 2. The expression of miR-504 is up-regulated in nasopharyngeal carcinoma radioresistant cell lines.

[0047] The expression of miR-504 was detected by Taqman fluorescent quantitative PCR method. The TaqMan probe method is a highly specific quantitative PCR technology, and its core is to use the 3′→5′ exonuclease activity of Taq enzyme to cut off the probe and generate a fluorescent signal. Since the probe is specifically bound to the template, the strength of the fluorescent signal represents the amount of the template. In the quantitative PCR reaction system of the TaqMan probe method, a pair of PCR primers and a probe are included. The probe only specifically binds to the template, and its binding site is between the two primers. The 5' end of the probe is labeled with a reporter group (Reporter, R), such as FAM, VIC, etc., and the 3' end is labeled with a fluorescent quencher group (Quencher, Q), such as TAMRA, etc. When the probe is intact, the fluorescen...

Embodiment 3

[0049] Example 3. miR-504 promotes the proliferation of nasopharyngeal carcinoma cell lines and their resistance to radiation, and inhibits the apoptosis of nasopharyngeal carcinoma cell lines.

[0050] MTS method was used to detect the effect of miR-504 on the proliferation of nasopharyngeal carcinoma cell lines. MTS indirectly reflects the proliferation ability of cells by detecting cell viability. MTS is an improved reagent based on MTT, which contains a new type of tetrazole compound (MTS) and an electron coupling agent (phenazineethosulfate, PES). PES has enhanced chemical stability, which allows it to be mixed with MTS to form a stable solution. MTS is bioreduced by cells into a colored formazan product, which can be directly dissolved in the culture medium. This conversion is accomplished by the action of NADPH or NADH produced by dehydrogenases in metabolically active cells. During detection, read the absorbance value at a wavelength of 490nm on a microplate reader ...

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Abstract

The invention discloses a molecular marker miR-504 for regulating the radiation resistance of nasopharyngeal carcinoma. Substantial up-regulation of the miR-504 in a nasopharyngeal carcinoma radiation resistance cell line is found, and the miR-504 influences expression of the mitochondrial energy metabolism related molecule TFAM and the oxidative phosphorylation complexes I, III and IV of the downstream of the miR-504 though direct targeting inhibition of NRF1, so the resistance of nasopharyngeal carcinoma cells to radiation is regulated through interfering the mitochondrial energy metabolism. The expression level of the miR-504 in serum of a nasopharyngeal carcinoma radiotherapy patient is positively correlated with the total tumor volume, so the miR-504 provides a new detection index for radiotherapy reaction control in the radiotherapy process of the nasopharyngeal carcinoma patient, and provides a new target for nasopharyngeal carcinoma radiotherapy.

Description

technical field [0001] The present invention relates to an action mechanism of microRNA in regulating tumor radioresistance, in particular to the molecular mechanism of miR-504 regulating nasopharyngeal carcinoma radioresistance and its use as a novel molecular marker. Background technique [0002] microRNA (miRNA) is a family of small non-coding RNAs consisting of 21-25 nucleotides (approximately 22 nucleotides), which recognize the 3'-UTR of the target gene through the "seed sequence" of its own 5' region region, and bind to the 3'-UTR region of the target gene in a complete or incomplete complementary pairing manner, thereby degrading or inhibiting the expression of the target gene at the post-transcriptional level, affecting the translation of the target gene and the expression of the protein level. The regulation of miRNAs on target genes is multi-level and multi-step. One miRNA can regulate the expression of multiple target genes, and one target gene can be regulated b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 曹亚赵璐晴孙仑泉
Owner CENT SOUTH UNIV
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