CN112592978A

CN112592978A - Application of substance for detecting genetic marker in preparation of risk early warning and colorectal cancer early diagnosis kit

Info

Publication number: CN112592978A
Application number: CN202011641948.7A
Authority: CN
Inventors: 杨进; 陈尔飞; 燕变变
Original assignee: Northwestern University
Current assignee: Northwestern University
Priority date: 2020-12-31
Filing date: 2020-12-31
Publication date: 2021-04-02

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to application of a substance for detecting a genetic marker in preparation of a kit for risk early warning and early diagnosis of rectal cancer. The invention utilizes exome sequencing technology to sequence and analyze tumor tissue samples of 3 Chinese Han nationality sCRC patients, and finally determines the functional genetic markers of new candidate genes BMP5, RASSF6, DDI2 and SARDH for early sCRC risk early warning by genotyping large sample tumor tissues and self normal control tissues aiming at the specific low-frequency function deletion mutation of the screened tumor tissues.

Description

Application of substance for detecting genetic marker in preparation of risk early warning and colorectal cancer early diagnosis kit

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to application of a substance for detecting a genetic marker in preparation of a kit for risk early warning and early diagnosis of rectal cancer.

Background

Sporadic Colorectal cancer (CRC), a type of malignant tumor without obvious family genetic predisposition and family history and originating from the large intestine mucosal epithelium, accounts for approximately 70% of Colorectal cancer (CRC). According to the annual book of Chinese health department of 2010 Chinese health statistics, malignant tumors leap the top of the mortality rate of main diseases in China, wherein sCRC is one of five kinds of malignant tumors with the greatest harm in China, and the mortality rate of urban residents is as high as 9.78/10 ten thousand. With the development of economy and the change of life style of people in China, the morbidity and mortality of sCRC tend to rise year by year, and the sCRC becomes another main factor threatening the health of people.

The sCRC is caused by the change of the expression level of cancer suppressor genes or oncogenes caused by gene mutation, epigenetic change and the like accumulated in colorectal tissues under the stimulation of adverse environmental factors. Therefore, researches between genomics, proteomics, related gene expression regulation and control and the like in cancer tissues and cancer phenotypes can be deeply carried out, and the system screening of specific functional mutation sites of sCRC tissues, differences of related gene expression and changes of epigenetics in an omics range can be helped, so that the understanding of the pathogenesis of sCRC molecules is promoted, and reliable evidence is provided for accurate early warning and diagnosis and targeted intervention of the sCRC stage. On the basis, the precise early warning and early diagnosis (precise medical treatment) of the individualized sCRC are realized by combining the sCRC related adverse environment, living habits and the like.

Aiming at early accurate early warning and intervention of sCRC, not only can repeatable laboratory data be effectively integrated and combined with external adverse risk factor analysis, but also personalized health management suggestions under gene guidance are given according to differences of individual living habits. With the development of various omics and multidisciplinary applications, the molecular regulatory network of complex diseases and the generation and development processes thereof are systematically explained from multiple layers, which is a necessary trend of early accurate early warning of sCRC.

The prior art has the problems that treatment is delayed due to missed diagnosis and misdiagnosis because early symptoms of sCRC are not obvious; meanwhile, compared with early prevention and treatment of hereditary colorectal cancer, some definite genetic markers directly used for early warning and early diagnosis of disease risk are lacked at present.

Disclosure of Invention

In order to solve the technical problems, the invention provides an application of a substance for detecting a genetic marker in preparing a kit for risk early warning and early diagnosis of rectal cancer.

The invention aims to provide application of a substance for detecting genetic markers in preparing a risk early warning and early colorectal cancer diagnosis kit, wherein the genetic markers are genes BMP5, DDI2, RASSF6 and SARDH.

Preferably, the BMP5, DDI2, RASSF6 and SARDH are involved in the generation of colorectal cancer.

Preferably, in the application of the substance for detecting the genetic marker in preparing a kit for risk early warning and early diagnosis of rectal cancer, the expression level of the BMP5 gene in HT-29, Colo320, SW480 and LoVo cell lines is reduced.

Preferably, the gene BMP5 is inserted into the pEF-Bos vector to obtain the pEF-Bos-BMP5 recombinant vector in the application of the substance for detecting the genetic marker in the preparation of a kit for risk early warning and early diagnosis of rectal cancer.

Preferably, the substance for detecting the genetic marker is used for preparing a kit for risk early warning and early diagnosis of rectal cancer, and after the pEF-Bos-BMP5 recombinant vector is transfected, the proliferation capacity and the migration capacity of the rectal cancer cell line HT-29 are reduced.

Compared with the prior art, the invention has the following beneficial effects:

1. in order to meet the urgent need of sCRC early prevention at the present stage in China, tumor tissue samples of 3 Chinese Han nationality sCRC patients are sequenced and analyzed by using exome sequencing technology, and the functional genetic markers of new candidate genes BMP5, RASSF6, DDI2 and SARDH for early sCRC risk early warning are finally determined by genotyping large sample tumor tissues and self normal control tissues aiming at the specific low-frequency function deletion mutation of the screened tumor tissues.

2. The invention screens and determines new candidate genes and tissue specific functional genetic markers of Chinese Han nationality population sCRC from a whole genome angle system by a whole exome sequencing technology for the first time.

3. The invention researches the molecular action mechanism of BMP5 and other cancer suppressor genes and their functional genetic markers participating in and influencing sCRC generation and development for the first time.

4. The invention firstly determines the multi-type tissue specific functional genetic markers related to the genes such as BMP5 and the like which can be used for sCRC early warning or diagnosis through independent large samples and in-vivo and in-vitro function experimental research.

Drawings

FIG. 1 shows the sequencing depth distribution of case sample 1 in the exoscope region according to the present invention;

FIG. 2 shows the sequencing depth distribution of case sample 2 in the exoscope region according to the present invention;

FIG. 3 is a graph showing the sequencing depth distribution of case sample 3 in the exoscope region according to the present invention;

FIG. 4 shows the results of a functional test of BMP5 of the present invention;

a is the mRNA expression level of BMP5, B is the protein expression level of BMP 5;

FIG. 5 shows experimental results of proliferation and migration abilities of cells according to the present invention;

a is the cell proliferation assay result and B is the cell migration assay result.

Detailed Description

In order that those skilled in the art will better understand the technical solutions of the present invention to be implemented, the present invention will be further described with reference to the following specific embodiments and accompanying drawings.

Example 1

(1) Clinical sample collection and preparation

According to the clinical diagnosis and physical examination results, tumor tissues and self normal tissues of 150 patients with sCRC of Chinese Han nationality are collected, wherein 3 frozen fresh tissues and self blood samples are used for sequencing early stage exons, and the rest are paraffin-embedded samples which are used for researching later stage independent samples, and the clinical and pathological information of the samples are recorded in detail. Disease group selection criteria: the mutation type detection result of MLH1 and MSH2 genes which are confirmed to be sCRC through primary immunohistochemical detection (necessarily containing pathological diagnosis), have no obvious family medical history and have diagnostic significance on the confirmed hereditary CRC of Chinese Han people is negative; the tumor tissue is subjected to HE staining and the proportion of the tumor cells is determined to be more than 80 percent; the normal tissue refers to the tissue which has no tumor cells and is more than 2cm away from the tumor edge in pathological detection; at the same time, we have addressed the study objectives of this study to each sample provider, and have signed informed consent provided by the ethical committee of northwest university. Extracting whole genome DNA of Blood by using standard operation steps of a PAXgene Blood DNA kit; paraffin embedded Tissue whole genome DNA was extracted using standard procedures of Qiagen QIAamp DNA FFPE Tissue Kit. The DNA of the genome is quantified and the quality is evaluated by an ultraviolet spectrophotometry and then used for experiments, wherein the DNA information of 21 cases is shown in a table 1, and the genetic CRC diagnostic gene mutation sites of Chinese Han nationality population are shown in a table 2.

Pathology and DNA sample information for 121 sCRC cases in table

TABLE 2 genetic CRC diagnostic gene mutation sites for Chinese Han nationality population

(2) Whole exome sequencing of 3 sCRC cancer tissue samples

Exon capture was performed by the Solexa Turseq exon capture platform and exon sequencing was performed by the Hiseq2000 next generation sequencing platform. Reads were aligned to Reference Sequences of the thousand human genomes (human _ g1k _ v37.fasta) with default parameters of BWA software, mapping files were processed with default parameters of samtools and picard-tools software to obtain mapping rates of Reads, sequencing multipliers of exons, etc., and the results are shown in table 3. The data size of 3 cases sequenced was above 58M reads (5.8G Bases data), the mapping efficiency between reads and Reference Sequences was about 98%, the exon capture efficiency was about 42%, the effective average sequencing depth of the exon regions was about 40 ×, and the sequencing depth distribution of DNA samples of 3 cases in the exon regions is shown in FIGS. 1-3, respectively.

TABLE 33 cases mapping rates of reads, exon sequencing multipliers, etc

(3) Determination of novel LoF site in cancer tissue

a) sCRC raw data result analysis of 3 cases

All loss of function (LoF) variation sites were screened in 3 case samples. Including nonsense mutation (STOP _ GAINED), frameshift mutation (FRAME _ SHIFT), and splicing mutation (SPLICE _ SITE) SITEs. The number of the screened LoF loci is basically consistent with the LoF number determined by the whole genome sequencing of the Asian normal population, the results are shown in the table 4, about 460 LoF variant loci are provided for each person, and the accuracy of the project based on exon sequencing is proved to meet the standard.

LoF site statistics for Table 43 case samples

Variant Type	Sample	1	Sample 2	Sample 3
					Stop Gained	107	113	111
Frame_Shift	210	225	256
				Splice Site	88	92	95
Total	405	430	462

b) Deleting the existing mutation sites in the dbSNP database, and reserving the low-frequency new mutation sites.

The sequencing quality value >100 was tightly controlled according to the relevant bioinformatics knowledge, with sequencing depths between 10 x and 200 x. The number of new LoF sites in

case samples

1, 2, and 3 was determined to be 38, 56, and 67, respectively, for a total of 161 new sites, and the results are shown in table 5.

Statistics of Low frequency LoF sites in 53 samples

variant type	Sample 1	Sample 2	Sample 3
				Stop	8	13	12
Splice	4	8	12
				frameshift indel	26	35	43
Total	38	56	67

c) Determination of sCRC tissue specific novel low-frequency LoF mutation site

The LoF variant sites found in 161 tissues were subjected to first-generation re-sequencing in sCRC cancer tissues and blood of the original 3 cases, the consistency of the second-generation sequencing and the first-generation sequencing is verified, 51 new LoF mutant sites specific to sCRC tumor tissues are determined (the sites which are difficult to detect due to high repeated sequences and genes which are reported to be related to sCRC are deleted), and the results are shown in Table 6.

Table 63 samples sCRC tissue-specific LoF mutation sites

variant type	Sample	1	Sample 2	Sample 3
					Stop_Gained	1	8	6
Splice_Site	0	3	17
				frameshift indel	2	12	2
Total	3	23	25

Through the literature search of a Pubmed database, candidate genes reported by CRC are deleted, and 4 new genes which are possibly used as cancer suppressor genes and involved in carcinogenesis are screened: BMP5, DDI2, RASSF6, and SARDH. Around the possible candidate genes, in order to determine whether the candidate genes participate in the generation and development of CRC, deep sequencing of a gene coding region is carried out in an independent large sample, and subsequent in vivo and in vitro function experiments are carried out on the high-frequency mutant genes.

(4) Functional test of candidate gene BMP5

BMP5 gene expression in colorectal cancer cell lines and normal intestinal cell lines. The cell lines used were NCM460, HT-29, Colo320, SW480 and LoVo, respectively, all of which are commercially available. Fig. 4A shows the mRNA expression level of BMP5, fig. 4B shows the protein expression level of BMP5, and compared to NCM460 normal gut cells, p <0.05 represents statistically different values (× p <0.001, × p <0.01, × p <0.05), and from the results in fig. 4, it can be seen that the BMP5 gene is expressed in reduced amounts in HT-29, Colo320, SW480 and LoVo cell lines.

(5) Construction of recombinant vector pEF-Bos-BMP5 Using commercial pEF-Bos vector

Inserting a target gene BMP5 into a pEF-Bos vector to obtain a pEF-Bos-BMP5 recombinant vector, then transfecting the recombinant vector to HT-29 cells, and observing the proliferation and migration capabilities of the HT-29 cells, wherein the result is shown in figure 5, the figure 5A is a cell proliferation experiment result, the upper line in the figure A is an HT-29 cell control, and the lower line is an HT-29 cell transfected with the pEF-Bos-BMP5 recombinant vector; FIG. 5B shows the results of a cell migration experiment, control HT-29 cell control, BMP 5-transfectad. The results show that after the pEF-Bos-BMP5 recombinant vector is transfected, the proliferation capacity and the migration capacity of the rectal cancer cell line HT-29 are reduced.

It should be noted that, when the present invention relates to a numerical range, it should be understood that two endpoints of each numerical range and any value between the two endpoints can be selected, and since the steps and methods adopted are the same as those in the embodiment, in order to prevent redundancy, the present invention describes a preferred embodiment. While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims

1. Use of a substance for detecting a genetic marker in the preparation of a kit for risk early warning and early diagnosis of colorectal cancer is characterized in that the genetic marker is genes BMP5, DDI2, RASSF6 and SARDH.

2. Use of the substance for detecting genetic markers for the preparation of a kit for risk pre-warning and early diagnosis of colorectal cancer according to claim 1, wherein BMP5, DDI2, RASSF6 and SARDH are involved in the development of colorectal cancer.

3. Use of the substance for detecting genetic markers for the preparation of a kit for risk pre-warning and early diagnosis of rectal cancer according to claim 1, wherein the BMP5 gene is expressed in reduced amounts in HT-29, Colo320, SW480 and LoVo cell lines.

4. The use of the substance for detecting genetic markers for the preparation of a kit for risk pre-warning and early diagnosis of rectal cancer according to claim 1, wherein the gene BMP5 is inserted into the pEF-Bos vector to obtain the pEF-Bos-BMP5 recombinant vector.

5. The use of the material for detecting genetic markers in the preparation of a kit for risk pre-warning and early diagnosis of rectal cancer according to claim 4, wherein the proliferation and migration capabilities of the rectal cancer cell line HT-29 are reduced after transfection of the pEF-Bos-BMP5 recombinant vector.