TWI485252B - A method of detecting the possibility of crc by specific gene profile from stool samples - Google Patents

Description

Method for detecting colorectal cancer possibility by cell gene kit in feces

The invention utilizes the gene expression difference between the fecal gene composition of the colorectal cancer patient and the non-colorectal cancer subject, performs the specificity detection of the gene in the feces, and develops a rapid and high-sensitivity non-invasive colorectal cancer detecting molecule. The imprinting kit is used to detect the fecal gene group by non-invasive methods and improve the detection rate of colorectal cancer patients.

The current related technologies are: immunochemical faecal occult blood test (iFOBT), endoscopic Exam, and barium Enema.

Moreover, in February 2009, the inventors have obtained two patents for the purification of expression genes from human feces (Republic of China Invention No. I306474 and Invention No. I306475), and purified total RNA from human feces for profit. The human differentially expressed gene clusters of human colorectal cancer patients and normal humans in the feces were screened from human whole-genome oligonuclear microarray wafers, and some genes have been obtained by reverse transcription PCR in tissues or blood of patients with colorectal cancer. It is used as a tumor marker for screening and diagnosing colorectal cancer.

The prior art fecal immune occult blood method must pay attention to the quantitative recovery and storage of the specimen, which is very inconvenient; and because the immunological method is extremely sensitive, the incorrect sample volume will reduce the correctness of the result. In the prior art, the endoscopy or colorectal X-ray examination is required to go to a professional medical institution, and the enema and the large intestine should be cleaned. Otherwise, the differential diagnosis will be affected, which may cause inconvenience and incompatibility of the subject. Influence the willingness to check.

1. As shown in the "first image", use whole-genome microarray wafer screening in normal people and large Intestinal cancer patients have genes with different performance, and initially found about 4,000 genes with significant differences. Further use of custom microarray wafers and gene annotation confirmed the differences in the performance of these genes in normal and colorectal cancer patients, and found 47 genes that showed significant differences in performance and were labeled with specific functions.

2. Further use of custom microarray wafers and gene annotation to confirm the differences in the performance of these genes in normal and colorectal cancer patients, found that there are 47 genes with significant differences in performance and labeled with specific functions.

3. Using 14 to 28 fecal samples to confirm the gene expression by real-time quantitative polymerase chain reaction, 8 genes with significant differences and confirmed by real-time quantitative polymerase chain reaction were confirmed.

4. For these 8 genes, the number of samples was further enlarged to 54 stool samples (18 non-cancer patients, and 36 colorectal cancer patients), and the 54 fecal samples were reconfirmed to show differences in the expression of the genes. A total of 6 genes were confirmed to have significant differences (UBE2N, IMPDH1, SLC15A4, DYNC1LI1, HRASLS2, and STK17B) (as shown in the "second graph").

5. In the 54 fecal samples, a total of 63 of the 6 genes (C6 takes 1+C6, 2+C6 takes 3+C6, 4+C6 takes 5+C6 takes 6=63) and the prediction model is combined. And the sensitivity and accuracy of 63 predictive models for colorectal cancer prediction were tested with independent 22 fecal samples. According to the known technical sensitivity, the sensitivity is higher than 90%, which can be used as a method to detect colorectal cancer, but sensitive. The higher the degree of detection, the lower the accuracy, and the two are inversely proportional. Therefore, the inventors selected the model with sensitivity higher than 90% among the detection results of 63 prediction models, and then selected the highest accuracy (>85%). The two models, namely the two prediction models with the highest sensitivity and accuracy, "UBE2N, SLC15A4, HRASLS2, STK17B" and "SLC15A4, HRASLS2, STK17B (as shown in the "third figure").

6. The predictive ability of the two predictive models for colorectal cancer was tested by 11 independent stool samples. It was found that the predictive model consisting of "UBE2N, SLC15A4, HRASLS2, and STK17B" can effectively predict colorectal cancer. Sample.

7. In summary, the present invention finds that a specific expression gene group combination (UBE2N, SLC15A4, HRASLS2 and STK17B) can be summarized in the stool of a colorectal cancer patient, and the gene components of the gene combination are not related to each other, but through biological information. According to the analysis and statistics, these gene groups will become specific molecular markers for screening or diagnosing colorectal cancer.

(1) UBE2N (Homo sapiens ubiquitin-conjugating enzyme E2N (UBC13 homolog, yeast), gene number NM_003348.3)

(2) SLC15A4 (Homo sapiens solute carrier family 15, member 4, gene number NM_145648.3)

(3) HRASLS2 (Homo sapiens HRAS-like suppressor 2, gene number NM_017878.1)

(4) STK17B (Homo sapiens serine/threonine kinase 17b, gene number NM_004226.2)

1. Compared with the fecal immune occult blood method of the prior art, the present invention does not need to pay attention to the preservation of the quantitative feces and the specimen, and increases the convenience of operation.

2. Compared with the prior art intestinal endoscopy or colorectal X-ray examination, the present invention does not need to go to a professional medical institution, nor does it require the enema and the large intestine to clean, which can exempt the subject from inconvenience and incompatibility. The side effects of intestinal endoscopy or colorectal X-ray examination can also be avoided, thereby increasing the willingness to examine.

3. The effective rate of detecting colorectal cancer is more than 90%. This data is significantly higher than the occult blood method that uses feces as the specimen.

First figure: Schematic diagram of the process of the invention.

Figure 2: Schematic diagram of the differences between UBE2N, IMPDH1, SLC15A4, DYNC1LI1, HRASLS2, and STK17B in colorectal cancer patients and non-large colorectal cancer subjects.

Figure 3: Schematic diagram of "predictive model with sensitivity greater than 90% and accuracy greater than 85%".

The four genes UBE2N, SLC15A4, HRASLS2 and STK17B are significantly different between colorectal cancer patients and non-colorectal cancer patients, so the four genes can be used as a detection tool for screening colorectal cancer.

Claims (2)

A method for detecting the possibility of colorectal cancer by a cell gene kit in feces, comprising: (1) obtaining feces of a subject, and (2) analyzing and detecting UBE2N, SLC15A4, HRASLS2 and STK17B genes in feces. Performance, to determine whether there is a possibility of colorectal cancer. According to the method of claim 1, the step (2) should include: (1) quantitative detection of UBE2N, SLC15A4, HRASLS2 and STK17B gene expression, and UBE2N, SLC15A4 and HRASLS2 gene expression, colorectal rectum Cancer patients have higher performance than non-rectal colorectal cancer subjects. In STK17B gene expression, patients with colorectal cancer have lower performance than those with non-colorectal cancer, and (2) the gene expression is clinically Judging criteria for the possibility of colorectal cancer.