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Kit for detecting cancer radiosensitivity and application of kit

A technology for detecting reagents and kits, which is applied in the field of compositions for predicting radiotherapy sensitivity before treatment, and can solve problems such as detection without kits

Active Publication Date: 2019-05-21
SUN YAT SEN UNIV CANCER CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is a lack of key tumor molecular markers clinically used to detect the radiotherapy sensitivity of locally advanced rectal cancer, and there is no corresponding kit for detection

Method used

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  • Kit for detecting cancer radiosensitivity and application of kit
  • Kit for detecting cancer radiosensitivity and application of kit
  • Kit for detecting cancer radiosensitivity and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Example 1 Screening process of genome-wide shRNA library in colorectal cancer cells and construction of radiotherapy-resistant cells

[0094] The screening process is as figure 1 As shown, specifically, the entire genome shRNA library (including 75,000 shRNAs, covering about 15,000 genes) was transfected into the colorectal cancer cell line HCT116 / HT29 in the form of lentiviral vector (transfection efficiency, MOI: 0.4), After being stably screened by puromycin, the cells were divided into irradiation treatment group and control group, cultured for 7 days after treatment, DNA was extracted and amplified, and then the shRNA copy number change was analyzed by next-generation sequencing. Significantly down-regulated genes were selected as candidate genes.

[0095] Next, if figure 2 As shown, the colorectal cancer cell line HCT116 was induced by long-term step-by-step radiation to construct a stable cell line with a radiation dose of 6Gy for further screening.

Embodiment 2

[0096]Example 2 Quantitative PCR detection of mRNA levels of candidate genes in radiation-resistant and sensitive cells

[0097] The total RNA in the cells was extracted by the Trizol method, and 2 μg of the total RNA was reverse-transcribed into cDNA. Then perform reverse transcription reaction according to the requirements of the reverse transcription system to obtain cDNA. By querying the gene database of NCBI, the cDNA sequences of RFC4 and GAPDH (internal reference) were obtained, and PCR primers were designed according to the sequences as follows:

[0098] RFC4:

[0099] The upstream primer sequence is shown in SEQ ID NO: 1,

[0100] The downstream primer sequence is shown in SEQ ID NO:2.

[0101] GAPDH (internal reference):

[0102] Upstream primer sequence: AGAAGGCTGGGGCTCATTTG,

[0103] Downstream primer sequence: AGGGGCCATCCACAGTCTTC.

[0104] Real Time PCR: According to Instructions for Premix Ex TaqTM II (TaKaRa), the formula for preparing the PCR reaction ...

Embodiment 3

[0109] Example 3 Changes in protein expression level of RFC4 before and after radiotherapy

[0110] Protein immunoassays were used to detect changes in RFC4 protein content at the cellular level. The main steps included: extraction of total protein in cells, preparation of protein loading buffer, SDS-PAGE electrophoresis, electrotransfer to PVDF membrane, and western blotting (anti-RFC4( PA5-21538) antibody (1:1000) was incubated with anti-β-actin (8H10D10, American Saixintong Company) antibody (1:1000)) and so on.

[0111] The result is as Figure 4 As shown, with the gradual increase of radiation dose, the protein level of RFC4 is also up-regulated. Combined with the results in Example 2, it shows that RFC4 is the most credible radiotherapy tolerance factor among the candidate genes.

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Abstract

The invention relates to a kit for detecting cancer radiosensitivity and application of the kit to curative effect prediction. According to the kit of the invention, the screening of molecular markersis completely based on whole-genome RNAi library screening which is a more practical and effective screening mode in international research level. Identification is carried out step by step in vitroand vivo based on a radiotherapy tolerance induction process; it is finally found that RFC4 is a gene highly correlated with radiotherapy tolerance of colorectal cancer; clinical retrospective analysis and in-vitro and in-vivo functional analysis are performed on the identified RFC4 gene, it is revealed that the high expression levels of the RFC4 gene can be positively correlated with tumor regression bad performance, and the RFC4 gene can be used to predict radiotherapy tolerance / sensitivity. Based on the above technical schemes of the invention, the kit used for predicting the efficacy of local advanced neoadjuvant radiotherapy is obtained, and is of great realistic significance for solving the problem of differences between individuals in clinical efficacies and the prediction blanks ofregression / prognosis effects, and better achieving accurate treatment.

Description

technical field [0001] The invention belongs to the field of molecular diagnosis and relates to a method capable of predicting the curative effect of radiotherapy for locally advanced rectal cancer, in particular to a composition developed based on the radiotherapy resistance gene RFC4 of colorectal cancer and applied to predict radiotherapy sensitivity before treatment . Background technique [0002] In recent years, the incidence of rectal cancer in my country has been increasing year by year, seriously threatening people's health. Especially for middle and low locally advanced rectal cancer, surgical resection is more difficult, the risk of local recurrence is high, and the prognosis is poor. At present, domestic and foreign guidelines recommend neoadjuvant chemoradiotherapy combined with surgical resection as the standard treatment mode for locally advanced rectal cancer. [0003] Neoadjuvant chemotherapy and radiotherapy can improve the local control rate, surgical re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574G01N33/533G01N33/535
Inventor 刘然义岳欣王雪涔黄文林
Owner SUN YAT SEN UNIV CANCER CENT
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