Colorectal cancer biomarker

A biomarker and colorectal cancer technology, applied in the field of biomedicine, can solve problems such as genome instability

Pending Publication Date: 2017-08-04
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Overexpression of HERVs can lead to genome instability, and abnor...

Method used

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  • Colorectal cancer biomarker
  • Colorectal cancer biomarker
  • Colorectal cancer biomarker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Detection of IFITM1 expression in colorectal cancer and hESCs ( figure 1 )

[0029] Materials and Methods:

[0030] 1. Randomly select patients with colorectal cancer, and take frozen tissue sections (including tumor and normal parts) from patients with colorectal cancer for immunofluorescence staining.

[0031] 2. Randomly select patients with colorectal cancer, extract RNA, and detect the mRNA level of IFITM1 by quantitative PCR; and take RNA from hESCs cell lines (WA26 and RUES2) to detect the expression level of IFITM1.

[0032]3. Results: The expression of IFITM1 in the tumor site of colorectal cancer was significantly higher than that in normal tissues, and its expression was high in hESCs cell lines (with HEF cells as the control).

Embodiment 2

[0034] Using CRISPR / Cas9 technology to construct IFITM1 knockout hESCs cell line ( figure 2 )

[0035] 1. Cell culture: using Essential 8 (invitrogen) culture medium at 37°C, 5% CO 2 To culture hESCs cells under certain conditions, the medium needs to be changed every day.

[0036] 2. Plasmid construction: Design primers according to the CDS region sequence of the IFITM1 protein published by NCBI, and insert it into the px459 plasmid through amplification and ligation. After positive clone PCR identification, sequencing, and BLAST comparison, the results showed that px459-IFITM1 was successfully constructed .

[0037] 3. Cell transfection and identification: hESCs cell lines were stored in Essential 8 medium at 37°C, 5% CO 2 cultured in an incubator. The plasmid is transferred into cells by nuclear transfer technology, and then the cells are cultured. IFITM1 knockout cells were obtained through drug screening and monoclonal cell selection, and then verified by sequencing...

Embodiment 3

[0039] To verify the effect of IFITM1 knockout on hESCs ( figure 2 ,3,4)

[0040] 1. There was no change in cell pluripotency after IFITM1 knockout was detected by western blot; telomerase was not changed after IFITM1 knockout was detected by quantitative PCR and TRAP experiments; detected by quantitative PCR (T / S ratio) There was no change in cellular telomere length.

[0041] 2. Through PCR experiments, we detected that after IFITM1 was knocked out, the cells were harvested to extract RNA, and the detection showed that HERVs in the cells increased. The primers used for detection are as follows:

[0042]

[0043]

[0044] 3. Through ChIP-qPCR experiments (using 2 primers), we found that after IFITM1 was knocked out, the enrichment of H3K9me3 at HERVs sites decreased, because H3K9me3 can inhibit the expression of HERVs, thus leading to an increase in the expression of HERVs.

[0045] 4. Collect the cells of the same generation in 3 for immunofluorescence (5mC and 5hm...

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Abstract

The invention discloses a colorectal cancer biomarker, relating to application of interferon-induced transmembrane protein 1 in inhibition of endogenous retrovirus in colorectal cancer. The hESCs of IFITM1-KO is constructed by using CRISPR/Cas9 technology so as to study the functions of IFITM1. Studies find that the IFITM1-deleted human embryonic stem cell (hESCs) is identical with IFITM1-WT in the aspects of cell proliferation, cell pluripotency, telomere length, telomerase activity and the like. The expression of human endogenous retrovirus in hESCs of IFITM1-KO increases, and expression in para-carcinoma tissue is higher than that in colorectal carcinoma tissue. ChIP-qPCR detection finds that the enrichment of H3K9me3 in hESCs of IFITM1-KO is reduced at the HERVs site. Data shows that the expression level of IFITM1 in hESCs and colorectal carcinoma is in negative correlation to HERVs expression, and the IFITM1 can inhibit the expression of HERVs by regulating epigenetic inheritance.

Description

[0001] Technical field: [0002] The invention belongs to the technical field of biomedicine, and specifically relates to a method for diagnosing colorectal cancer based on detecting the expression levels of IFITM1 and HERVs-related genes regulated by it. [0003] Background technique: [0004] 1. In recent years, a number of studies have shown that IFITM1 is highly expressed in various malignant tumors. For example, IFITM1 is highly expressed in cervical cancer, esophageal cancer, ovarian cancer, and brain cancer, and it is upregulated in colorectal cancer and is considered to be Colorectal cancer molecular markers. More and more people pay attention to its role in the occurrence and development of malignant tumors and the early invasion of tumors. In colorectal cancer, IFITM1 is only expressed in tumor tissue, and the activation of Wnt / β-catenin signaling pathway can induce the expression of IFITM1 in the process of intestinal tumor formation; in chronic myeloid leukemia, it...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N33/68G01N33/574
CPCC12Q1/6886C12Q2600/158G01N33/57419G01N33/57484G01N33/68
Inventor 付雨栋王华龚鹏周忠诚戚峰刘林
Owner NANKAI UNIV
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