阅读说明：本技术 一种作为诊断结直肠癌肝转移标志物的血浆外泌体circRNA (Plasma exosome circRNA as marker for diagnosing colorectal cancer liver metastasis ) 是由 张力图 刘海洲 宁淑芳 利基林 于 2020-05-06 设计创作，主要内容包括：本发明公开了一种作为诊断结直肠癌肝转移标志物的血浆外泌体circRNA。它包括人血浆外泌体中的hsa_circ_0001296和hsa_circ_0006773。本发明利用微阵列分析用于检测血浆外泌体中差异表达的circRNA,并证实这些候选circRNA确实存在差异并且可以用作潜在的结直肠癌肝转移诊断生物标记。结果发现结直肠癌患者血浆外泌体中的hsa_circ_0001296和hsa_circ_0006773表达改变可以作为诊断结直肠癌肝转移的标志物的组合,这将有助于探索这种疾病的发病机理,并为CRC的诊断提供新的方向。(The invention discloses a plasma exosome circRNA as a marker for diagnosing colorectal cancer liver metastasis. It includes hsa _ circ _0001296 and hsa _ circ _0006773 in human plasma exosomes. The invention utilizes microarray analysis for detecting circrnas differentially expressed in plasma exosomes and confirms that these candidate circrnas are indeed differential and can be used as potential colorectal cancer liver metastasis diagnostic biomarkers. As a result, the expression change of hsa _ circ _0001296 and hsa _ circ _0006773 in plasma exosome of colorectal cancer patients can be used as a combination of markers for diagnosing liver metastasis of colorectal cancer, which helps to explore the pathogenesis of the disease and provides a new direction for the diagnosis of CRC.)

1. A marker for diagnosing liver metastasis of colorectal cancer, comprising hsa _ circ _0001296 and hsa _ circ _0006773 in human plasma exosomes.

2. The marker for diagnosing colorectal cancer according to claim 1, wherein hsa _ circ _0001296 and hsa _ circ _0006773 are expressed in an increased amount in plasma exosomes of patients with liver metastases of colorectal cancer.

3. A kit for diagnosing liver metastasis of colorectal cancer, comprising hsa _ circ _0001296 and hsa _ circ _ 0006773.

Technical Field

The invention belongs to the technical field of colorectal cancer diagnosis, and particularly relates to plasma exosome circRNA serving as a marker for diagnosing colorectal cancer liver metastasis.

Background

Circular RNA (circRNA) is a novel non-coding RNA that forms a covalently closed continuous loop. The CircRNA has no 3 'and 5' ends, but has a large number of miRNA binding sites. Most circrnas are exon circrnas, which are derived from exon regions of known protein-encoding genes by reverse splicing. The CircRNA is abundant and stable in exosome and plasma, and provides a more convenient method for detection. Exosomes are nanoscale bilayer membrane vesicles, 40-100nm in diameter, usually appearing in a cup shape. Many human cells can release exosomes by extracellular secretion, and these vesicles are widely distributed in blood, saliva, urine and other body fluids. Exosomes can carry liposomes, proteins, mRNA and non-coding RNA to target tissues and can be involved in cellular communication, immune responses and tumor growth and migration.

Measuring exosome-derived RNA as a biomarker has many advantages over RNA free in blood. In one aspect, the exosome membrane can effectively protect the contents from RNase degradation, thereby maintaining the integrity and functionality of exosome RNA in circulating blood. On the other hand, cancer cells secrete exosomes into peripheral circulation fluid, and exosome RNA can more accurately reflect changes of cancer cells in the tumor evolution process. circRNA is stable and abundant in blood, saliva and exosomes, and is a promising indicator for early diagnosis and prognosis of cancer. Human serum exosomes contain large amounts of intact and stable circRNA, which has great potential as a biomarker for future tumor detection.

Disclosure of Invention

The invention aims to provide plasma exosome circRNA serving as a liver metastasis marker for diagnosing colorectal cancer.

A marker for diagnosing liver metastasis of colorectal cancer comprises hsa _ circ _0001296 and hsa _ circ _0006773 in human plasma exosomes.

The hsa _ circ _0001296 and hsa _ circ _0006773 have increased expression levels in plasma exosomes of colorectal cancer liver metastasis patients.

A kit for diagnosing liver metastasis of colorectal cancer comprises hsa _ circ _0001296 and hsa _ circ _ 0006773.

The invention has the beneficial effects that: the present invention utilizes microarray analysis for the detection of circrnas differentially expressed in plasma exosomes (DE-circrnas) and demonstrates that these candidate circrnas do differ and can be used as potential colorectal cancer liver metastasis diagnostic biomarkers. As a result, the expression changes of hsa _ circ _0001296 and hsa _ circ _0006773 in plasma exosomes of patients with colorectal cancer liver metastasis can be used as a combination of markers for diagnosing colorectal cancer liver metastasis, which can help to explore the pathogenesis of the disease and provide a new direction for diagnosing CRC.

Drawings

FIG. 1 is a TEM image (scale bar, 100nm) of exosomes.

Figure 2 is the size range of exosomes confirmed by NTA analysis.

Figure 3 is a western blot of three representative exosome-specific markers: TSG101, CD63, and CD 9.

Figure 4 is a heatmap analysis of differentially expressed circRNA.

Figure 5 is a volcano plot analysis of differentially expressed circRNA.

FIG. 6 depicts the qRT-PCR detection of hsa _ circ _0001296 and hsa _ circ _0006773 expression in plasma exosomes; all data are expressed as mean ± SD, p <0.05, p <0.01, p <0.001 compared to the control group.

FIG. 7 is ROC curves for liver metastasis detection of hsa _ circ _0001296 and hsa _ circ _0006773 colorectal cancer; all data are expressed as mean ± SD, p <0.05, p <0.01, p <0.001 compared to the control group.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

The human CRC cell lines (SW480, SW620, LoVo, HT-29) and normal NCM460 colonic epithelial cells were purchased from the national institute of bioscience, Shanghai, academy of sciences, China, in the examples described below. All cell lines were in a suitable culture environment (5% CO)₂Stored at 37 ℃ in RPMI1640 medium (Gibco, USA) supplemented with 10% FBS (Gibco).