CN110229899B - Plasma marker combinations for early diagnosis or prognosis prediction of colorectal cancer - Google Patents

Abstract

The present invention discloses a combination of plasma markers for early diagnosis or prognosis prediction of colorectal cancer. Specifically, the invention obtains miRNAs which are obviously and abnormally expressed in colorectal cancer plasma, namely miR-224-5p, miR-301a-3p, miR-145-5p, miR-193b-3p and miR-1183. The invention discovers that the plasma miRNA combination can be used as a marker for early diagnosis or prognosis of colorectal cancer by researching the expression difference of the plasma miRNAs in colorectal cancer patients, colorectal adenoma patients and healthy people in each stage.

Description

Plasma marker combinations for early diagnosis or prognosis prediction of colorectal cancer

Technical Field

The present invention is in the field of biotechnology and medicine, in particular, it relates to plasma marker combinations for early diagnosis or prognosis prediction of colorectal cancer.

Background

Colorectal cancer (colorectal cancer, CRC) is one of the most common malignancies, with CRC incidence being second in females and third in males and overall CRC mortality being second in the global view. It is estimated that 180 ten thousand new colorectal cancer cases and 881,000 deaths will occur in 2018, accounting for about 1% of cancer cases and deaths. By 2030, the global incidence of CRC is expected to increase by 60%. The statistics of Chinese cancers in 2015 show that the incidence rate of CRC of men is fifth, the incidence rate of CRC of women is fourth, the total incidence rate and the mortality rate are both fifth, and the total incidence rate of digestive tract tumors rises year by year. According to literature statistics, after early CRC standard treatment, the 5-year survival time can reach 90%, but 15% -20% of CRC is found to be liver metastasis at the time of diagnosis, and the other 60% also found to be recurrence or metastasis in the subsequent treatment process. The key to CRC treatment is early screening, early diagnosis and early treatment, and CRC screening out and timely treatment at the adenoma stage are effective ways to improve survival rate of CRC patients. 80% of colorectal cancers develop from adenomatous polyps, 10-15 years of benign tumor development occurs before canceration, a time window which provides powerful opportunities for colorectal cancer prevention and early diagnosis, and the characteristic also makes colorectal cancers a few malignant tumors which can reduce morbidity and mortality through early screening. Identifying and resecting adenomas through colorectal cancer screening can reduce incidence of colorectal cancer by up to 53%. At this stage, the surgical method can almost achieve the effect of curing the disease, grasp the time of malignant changes of adenoma, and identify early lesions is the key first step of diagnosis and treatment of colorectal cancer. However, only about 59% receive colonoscopy after age 50. In addition, 60% -70% of colorectal cancer patients are found to be stage II or late. Adverse effects and poor prognosis associated with treatment lead to CRC that is prone to relapse and metastasis. Chemotherapy resistance is a major obstacle to advanced CRC treatment, while distant metastasis and local recurrence are the major causes of postoperative death in CRC patients. Knowing the risk factors associated with CRC survival facilitates screening for high risk patients who are prone to relapse and metastasis. The tumor marker can be used for screening early tumor patients, reflects the curative effect of patients after receiving surgery, radiotherapy and chemotherapy or biological targeting treatment, evaluates the recurrence and distant metastasis conditions, and has important significance for clinical treatment, recurrence and metastasis and prognosis evaluation of CRC.

The current early screening means for colorectal cancer are: fecal occult blood detection (chemical and immunological), colonoscopy, multi-target fecal detection, questionnaire risk assessment, CT mimicking colonoscopy, fecal PKM2 protein detection, plasma ctDNA Sept99 methylation detection, etc. The fecal occult blood detection chemical method is poor in sensitivity, diet control is required during sampling, the results are interfered by multiple factors, the Brenner and the like analyze nearly 4 ten thousand screening people, and compared with the colonoscope, the fecal detection rate of the chemical method is up to 90% and 75% in adenoma and colorectal cancer in the progressive stage respectively. So it is no longer proposed to use it; immunochemistry FIT has a sensitivity of 79% and a specificity of 94% for colorectal cancer screening. The colloidal gold test paper is mainly used most widely in screening. In practical application, the screening purpose can be achieved by the immunochemistry method FIT which can be carried out routinely 1 time per year. However, the screening has the defect of low sensitivity to adenomas in the progressive stage, which is only 20-30%. In addition, the reject rate is high due to the problem of stool sampling. The most sensitive means of colonoscopy intestinal tract lesion examination can intuitively observe the condition of the whole intestinal wall, besides flat lesions and rare 'intermittent cancers', most polyp lesions are difficult to diagnose, the lesion detection rate of a general asymptomatic colonoscopy screening crowd can reach 0.5-1.8% in colorectal cancer, 14.8% in advanced adenoma, and the general detection rate of intestinal polyps can be higher than 1/3. However, the colonoscopy is a first choice tool for early screening of colorectal tumors due to strict and painful preparation of intestinal tracts, high technical requirements, high risk of wound and other factors, and the colonoscope is still carefully considered as a primary screen especially in the current situation of medical resource deficiency in China. The multi-target fecal detection utilizes DNA molecular detection of tumor shedding cells in the fecal to combine with an immunoassay FIT, so that the screening sensitivity of intestinal cancer is further improved, and the screening sensitivity of progressive adenoma can be enhanced to 54%. The united states has been used for early colorectal tumor screening in asymptomatic individuals, with screening cycles recommended to be 1/1 year or 3 years. The main disadvantage of this approach is the relatively high price. Questionnaire risk assessment screening, i.e., querying and assessing the presence of high risk factors, was proposed by the university of Zhejiang Zheng Shu professor team, and was incorporated into the national defense commission standard regimen for early diagnosis and early treatment of cancer in 2007. The method has the greatest advantages of being simple and easy to implement, and improving the screening compliance rate of mass groups. However, the risk influence degree of the evaluable factors in the questionnaire is weak, and the screening sensitivity and specificity of the questionnaire on colorectal tumors in the progressive stage are not high. Other means for early colorectal tumor screening are not widely recommended because of the presence of various defects.

In conclusion, early discovery and timely intervention of high risk groups of colorectal cancer are achieved, and more accurate early warning means or biological indexes are not yet available clinically.

Summary of The Invention

The object of the present invention is to provide a plasma marker combination for early diagnosis or prognosis prediction of colorectal cancer.

In a first aspect of the invention, there is provided the use of a miRNA or detection reagent thereof for the preparation of a kit for early diagnosis or prognosis prediction of colorectal cancer, said miRNA being selected from the group consisting of: miR-224-5p, miR-301a-3p, miR-145-5p, miR-193b-3p or a combination thereof.

In another preferred embodiment, the miRNAs comprise miR-224-5p, miR-301a-3p and miR-193b-3p.

In another preferred embodiment, the miRNA comprises miR-224-5p, miR-301a-3p, miR-145-5p, miR-193b-3p; preferably, the miRNA further comprises miR-1183.

In another preferred embodiment, the kit contains miR-224-5p, miR-301a-3p, miR-145-5p, miR-193b-3p, miR-1183 or a combination thereof as a positive control.

In another preferred embodiment, the test sample of the test kit comprises blood (plasma) or tissue.

In another preferred embodiment, the detection reagent is selected from the group consisting of: antibodies, primers, probes, sequencing libraries, nucleic acid chips, or combinations thereof.

In another preferred embodiment, the detection reagent comprises a primer, an antisense nucleic acid, a probe or a chip.

In a second aspect of the present invention, there is provided a miRNA chip comprising:

a solid phase carrier; and

an oligonucleotide probe orderly immobilized on the solid support, the oligonucleotide probe specifically corresponding to a target miRNA selected from the group consisting of: miR-224-5p, miR-301a-3p, miR-145-5p, miR-193b-3p, or a combination thereof.

In another preferred embodiment, the oligonucleotide probe comprises:

a complementary binding region; and/or

A junction region attached to the solid support.

In another preferred embodiment, the miRNA of interest comprises miR-224-5p, miR-301a-3p, and miR-193b-3p.

In another preferred embodiment, the miRNA of interest comprises miR-224-5p, miR-301a-3p, miR-145-5p, miR-193b-3p, and miR-1183.

In a third aspect of the invention, there is provided a kit comprising a miRNA or detection reagent thereof, said miRNA being selected from the group consisting of: miR-224-5p, miR-301a-3p, miR-145-5p, miR-193b-3p or a combination thereof; and, the kit further comprises instructions, wherein the instructions state that the kit is used for early diagnosis or prognosis prediction of colorectal cancer.

In another preferred embodiment, the miRNAs comprise miR-224-5p, miR-301a-3p and miR-193b-3p.

In another preferred embodiment, the miRNAs include miR-224-5p, miR-301a-3p, miR-145-5p, miR-193b-3p, and miR-1183.

In another preferred embodiment, the description refers to the following:

when the measured levels of miR-224-5p, miR-301a-3p and/or miR-193b-3p in the sample are significantly different from those of the healthy sample, the detection sample is indicated to be a colorectal cancer high-risk patient sample.

In another preferred embodiment, the description refers to the following:

in the sample being assayed, the sample is, compared to a healthy sample,

the miR-224-5p level is obviously improved;

the miR-301a-3p level is obviously reduced; and/or

miR-193b-3p levels are significantly increased;

the test sample is prompted to be a colorectal cancer high risk patient sample.

In another preferred embodiment, the instructions describe a regression model, establishing a logic (P) = -1.052+86.495× (miR-193 b-3P) +30.897 × (miR-224-5P) -24.876 × (miR-301 a-3P), wherein the expression levels of miR-193b-3P, miR-224-5P, and miR-301a-3P are measured with miR-30a-5P as an internal reference, and wherein the logic (P) value in the model indicates that there is a significant difference between the test sample and the healthy sample, and the test sample is indicated to be a colorectal cancer high risk patient sample.

In another preferred embodiment, the description refers to the following:

when the measured levels of miR-224-5p, miR-301a-3p, miR-145-5p, miR-193b-3p and/or miR-1183 in the sample are significantly different from those of the healthy sample, the detection sample is indicated to be a colorectal tumor high-risk patient sample.

In another preferred embodiment, when the sample is measured, the sample is measured for a specific time, compared to a healthy sample,

the miR-224-5p level is obviously improved;

the miR-301a-3p level is obviously reduced;

miR-193b-3p levels are significantly increased;

miR-145-5p levels were significantly reduced; and/or

miR-1183 levels are significantly reduced or increased;

the test sample is prompted to be a colorectal tumor high risk patient sample.

In another preferred embodiment, the sample is a sample which, when compared to a colorectal adenoma sample,

the miR-224-5p level is obviously improved;

the miR-301a-3p level is obviously reduced;

miR-193b-3p levels are significantly increased;

miR-145-5p levels were significantly reduced; and/or

The miR-1183 level is obviously improved;

the test sample is prompted to be a colorectal cancer high risk patient sample.

In another preferred embodiment, the instructions describe a regression model, establishing a regression model with a logic (P) = -2.338+7.166× (miR-145-5P) +173.161 × (miR-193 b-3P) -27.658 × (miR-1183) +46.071 × (miR-224-5P) -58.807 × (miR-301 a-3P), wherein the expression levels of miR-224-5P, miR-301a-3P, miR-145-5P, miR-193b-3P, and miR-1183 are measured with miR-30a-5P as an internal reference, and wherein the logic (P) value in the model indicates that the test sample is significantly different from the colorectal adenoma sample, indicating that the test sample is a high risk patient sample for colorectal cancer.

In a fourth aspect of the invention, there is provided an isolated miRNA selected from the group consisting of:

(i) miR-224-5p, miR-125b-5p, miR-301a-3p, miR-145-5p, miR-193b-3p, or a combination thereof, or

(i i) miRNA complementary to miR-224-5p, miR-125b-5p, miR-301a-3p, miR-145-5p, or miR-193b-3p, or a combination thereof.

In another preferred embodiment, the miRNAs comprise miR-224-5p, miR-301a-3p and miR-193b-3p.

In another preferred embodiment, the miRNAs include miR-224-5p, miR-301a-3p, miR-145-5p, miR-193b-3p, and miR-1183.

In another preferred embodiment, the miRNAs include miR-375-3p, miR-135b-5p, miR-224-5p, miR-125b-5p, miR-301a-3p, miR-145-5p, miR-193b-3p, and miR-1183.

In another preferred embodiment, the miRNA is selected from the group consisting of:

(a) SEQ ID NO. 1-8;

(b) Any one of 8 complementary sequences complementary to any one of the sequences set forth in SEQ ID NO. 1-8; or (b)

(c) A combination from (a) or (b), and the sequence from (a) and the complementary sequence from (b) are not complementary to each other.

In a fifth aspect of the invention, there is provided an isolated or artificially constructed precursor miRNA that is capable of cleaving and expressing in a human cell a miRNA according to the fourth aspect of the invention.

In a sixth aspect of the invention, there is provided an isolated polynucleotide capable of being transcribed by a human cell into a precursor miRNA which is sheared within the human cell and expressed as a miRNA according to the fourth aspect of the invention.

In another preferred embodiment, the polynucleotide has the structure shown in formula I:

Seq _{forward direction} -X-Seq _{Reverse direction}

I is a kind of

In the formula I, the compound (I),

Seq _{forward direction} To be in human cellsThe nucleotide sequence of the miRNA is expressed;

Seq _{reverse direction} Is equal to Seq _{Forward direction} A substantially complementary or fully complementary nucleotide sequence;

x is at Seq _{Forward direction} And Seq _{Reverse direction} A spacer sequence therebetween, and said spacer sequence is identical to Seq _{Forward direction} And Seq _{Reverse direction} Are not complementary;

and the structure shown in formula I forms a secondary structure shown in formula II after being transferred into human cells:

in formula II, seq _{Forward direction} 、Seq _{Reverse direction} And X is as defined above,

the expression is shown in Seq _{Forward direction} And Seq _{Reverse direction} Complementary base pairing relationship formed between them.

In another preferred embodiment, the assay is performed by real-timeepcr.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

Fig. 1: expression of each miRNA in 60 plasma samples, figure:

(1) Represents P <0.05; * Represents P <0.005; * Represents P <0.001

(2) The columns are marked as each group compared to the normal group; black lines represent two sets of comparisons; gray lines represent three or four sets of comparisons

(3) N (20): normal group 20 cases; ad (20): colorectal adenoma group 20; CRC (20): colorectal cancer group 20; 0-II (10): 10 cases of early stage cancer group 0-II; III-IV (10): group of early stage cancer of stage III-IV 10.

Fig. 2 shows the expression verification of each miRNA in 158 plasma samples, in which:

(1) Represents P <0.05; * Represents P <0.005; * Represents P <0.001

(2) The columns are marked as each group compared to the normal group; black lines represent two sets of comparisons; gray lines represent three or four sets of comparisons

(3) N (50): normal group 50 cases; ad (50): colorectal adenoma group 50; CRC (50): colorectal cancer group 50; 0-II (28): 28 cases of early stage cancer group 0-II; III-IV (30): 30 cases of stage III-IV early stage carcinoma group.

Fig. 3a roc curve analysis of plasma mirnas diagnostic potential for colorectal cancer.

Fig. 3b roc curve analysis of plasma mirnas diagnostic potential for colorectal adenoma.

Fig. 3c roc curve analysis of plasma mirnas for identification potential for colorectal and adenoma.

Fig. 3d roc curve analysis of plasma mirnas for identification potential for colorectal early and late cancers.

Fig. 4: ROC curve analysis of two combined models.

Fig. 5: logit equations and combined diagnostic efficacy, in the figure:

n is normal, M is colorectal adenoma, T is colorectal carcinoma.

Detailed Description

Through extensive and intensive research, miRNA with obvious abnormal expression and constant expression in colorectal cancer plasma samples is determined as a preliminary research object, and the expression level of the colorectal cancer early diagnosis and prognosis prediction markers is detected by taking 30a-5p and 30e-5p as internal references, so that miRNA closely related to colorectal cancer morbidity is found out. Finally, miRNAs which are obviously and abnormally expressed in colorectal cancer plasma, namely miR-224-5p, miR-301a-3p, miR-145-5p, miR-193b-3p and miR-1183 are obtained. The invention discovers that the plasma miRNA combination can be used as an early diagnosis marker of colorectal cancer by researching the expression difference of the plasma miRNAs in colorectal cancer patients, colorectal adenoma patients and healthy people in each stage. The present invention has been completed on the basis of this finding.

Specifically, the invention uses a real-time fluorescence quantitative PCR method to verify the plasma of 78 CRC patients, 70 colorectal adenoma patients and 70 healthy volunteers in each period, detects the expression level of the 9 candidate targets, observes the difference of expression profiles in plasma samples with different clinical characteristics, and analyzes the value of the plasma specific miRNA combination in early diagnosis and prognosis prediction of colorectal cancer. After verification, the invention obtains specific plasma 3-miRNA combinations, namely miR-224-5p, miR-301a-3p and miR-193b-3p, the sensitivity of colorectal cancer detection by the biomarker combination is 0.655, the specificity is 0.920, and the area under the curve (area under the curve, AUC) capable of generating the maximum subject working characteristic curve (receiver operator characteristic curve, ROC) is 0.856. Specific plasma 5-miRNA combinations, namely miR-224-5p, miR-301a-3p, miR-145-5p, miR-193b-3p and miR-1183, were obtained, and the sensitivity of the biomarker combination to distinguish colorectal adenoma from colorectal carcinoma was 0.720, the specificity was 0.680, and the producible AUC was 0.930. Colorectal tumors herein include benign rectal adenoma, and malignant colorectal carcinoma. The progress of colorectal tumors is generally normal-adenoma-early cancer-late cancer, so that plasma miR-224-5p, miR-301a-3p, miR-145-5p, miR-193b-3p and miR-1183 can serve as potential tumor markers of colorectal tumors, colorectal cancer, colorectal adenoma and healthy controls can be accurately distinguished by the built miRNA combination, and the novel effective supplementation is performed on the existing laboratory evidence of miRNA serving as potential early colorectal cancer diagnosis biomarkers.

The invention aims to provide a colorectal cancer early diagnosis and prognosis prediction marker and develop a related kit.

The colorectal cancer early diagnosis and prognosis prediction marker provided by the invention consists of a plurality of nucleic acid molecules, wherein the plurality of nucleic acid molecules comprise nucleic acid molecules for encoding plasma miR-375-3p, miR-135b-5p, miR-224-5p, miR-125b-5p, miR-301a-3p, miR-145-5p, miR-193b-3p and miR-1183; the nucleic acid molecules are differentially expressed in at least one target plasma versus at least one control plasma. The colorectal cancer early diagnosis and prognosis prediction kit comprises the colorectal cancer early diagnosis and prognosis prediction marker.

The present invention provides a kit for distinguishing colorectal early lesions/colorectal cancer patients from healthy individuals with at least one plasma miRNA. Kits are provided for predicting the risk of metastasis or recurrence or the response to chemotherapy in colorectal cancer patients with at least one plasma miRNA.

The invention determines miRNA with obvious and constant abnormal expression in colorectal cancer plasma samples as a preliminary study object, and the expression level of the colorectal cancer early diagnosis and prognosis prediction marker is detected by taking miR-30a-5p or miR-30e-5p as an internal reference, preferably miR-30a-5p as the internal reference. miRNAs closely related to colorectal cancer onset are found. The invention selects 8 miRNAs which can be obviously abnormally expressed in colorectal cancer plasma, namely miR-375-3p, miR-135b-5p, miR-224-5p, miR-125b-5p, miR-301a-3p, miR-145-5p, miR-193b-3p and miR-1183. By studying the expression difference of the miRNAs in the blood plasma in colorectal cancer patients, colorectal adenoma patients and healthy people, the miRNA combination in the blood plasma can be used as an early diagnosis marker of colorectal cancer. Meanwhile, clinical relevant analysis is carried out by combining clinical pathological data of colorectal cancer patients, and the group of plasma miRNAs are found to be relevant to indexes such as clinical stage, differentiation degree, lymph node metastasis, distant metastasis, serum CEA expression condition and the like, so that the group of plasma miRNAs can be found to be used as a colorectal cancer prognosis prediction marker.

miRNA and precursor thereof

Small ribonucleic acid (microRNA, miRNA) is a class of non-coding single stranded small RNAs consisting of about 19-25 nt. With the continued intensive research of miRNAs, extracellular miRNAs (cfmiRNAs) was found to have good stability in various body fluids, mainly blood, but also urine, saliva, sweat, cerebrospinal fluid, etc. Based on the rapid development of high throughput techniques (such as microarrays and sequencing), comprehensive expression profiles of cfmiRNAs in a variety of cancers (mainly circulating miRNAs) are revealed, and more new miRNAs with early diagnostic and prognostic potential are discovered. However, no plasma miRNA-related kit is yet marketed in colorectal cancer.

The present invention provides a novel class of mirnas found in humans. As used herein, the term "miRNA" refers to an RNA molecule that is processed from transcripts that can form a precursor of the miRNA. Mature mirnas typically have 18-26 nucleotides (nt) (more particularly about 19-22 nt), nor are miRNA molecules with other numbers of nucleotides excluded. mirnas are generally detectable by Northern blotting.

Mirnas of human origin can be isolated from human cells. As used herein, "isolated" refers to a substance that is separated from its original environment (i.e., the natural environment if it is a natural substance). If the naturally occurring polynucleotide and polypeptide are not isolated or purified in vivo, the same polynucleotide or polypeptide is isolated or purified from other naturally occurring substances.

mirnas may be processed from precursor mirnas (Pre-mirnas) that can be folded into a stable stem-loop (hairpin) structure, typically between 50-100bp in length. The precursor miRNA can be folded into a stable stem-loop structure, and the two sides of the stem-loop structure comprise two sequences which are basically complementary. The precursor miRNA can be natural or synthetic.

The precursor miRNA may be sheared to generate a miRNA that may be substantially complementary to at least a portion of the sequence of the mRNA encoding the gene. As used herein, "substantially complementary" means that the sequences of nucleotides are sufficiently complementary to interact in a predictable manner, such as to form a secondary structure (e.g., a stem-loop structure). Typically, two "substantially complementary" nucleotide sequences are at least 70% complementary to each other; preferably, at least 80% of the nucleotides are complementary; more preferably, at least 90% of the nucleotides are complementary; further preferably, at least 95% of the nucleotides are complementary; such as 98%, 99% or 100%. Typically, there may be up to 40 mismatched nucleotides between two sufficiently complementary molecules; preferably, there are up to 30 mismatched nucleotides; more preferably, there are up to 20 mismatched nucleotides; it is further preferred to have at most 10 mismatched nucleotides, such as having 1, 2, 3, 4, 5, 8, 9 mismatched nucleotides.

As used herein, a "stem-loop" structure, also referred to as a "hairpin" structure, refers to a nucleotide molecule that can form a secondary structure that includes a double-stranded region (stem) formed by two regions of the nucleotide molecule (on the same molecule) that are flanked by double-stranded portions; it also includes at least one "loop" structure comprising a non-complementary nucleotide molecule, i.e., a single-stranded region. The double-stranded portion of the nucleotide can remain double-stranded even if the two regions of the nucleotide molecule are not fully complementary. For example, insertions, deletions, substitutions, etc. may result in the non-complementation of a small region or the small region itself forming a stem-loop structure or other form of secondary structure, however, the two regions may still be substantially complementary and interact in a predictable manner to form a double-stranded region of the stem-loop structure. The stem-loop structure is well known to those skilled in the art, and usually after obtaining a nucleic acid having a nucleotide sequence of primary structure, the skilled person is able to determine whether the nucleic acid is capable of forming a stem-loop structure.

The miRNA has a sequence shown as SEQ ID NO. 1-8. To improve the stability or other properties of the miRNA, at least one protective base, such as "TT" or the like, may also be added to at least one end of the miRNA.

Antisense oligonucleotides

According to the miRNA sequence provided by the invention, antisense oligonucleotides can be designed, and the antisense oligonucleotides can down regulate the expression of corresponding miRNA in vivo. As used herein, "antisense oligonucleotides (AS-Ons or ASO)" is also referred to AS "antisense nucleotides" and refers to DNA molecules or RNA molecules or analogs thereof that are about 18-26nt in length (more particularly about 19-22 nt).

In the present invention, the "antisense oligonucleotide" also includes modified antisense nucleotides obtained by means such as nucleic acid lock-based or nucleic acid strand backbone modification techniques, wherein the modification does not substantially alter the activity of the antisense oligonucleotide, and more preferably, the modification increases the stability, activity or therapeutic effect of the antisense oligonucleotide. Nucleic acid Locks (LNAs) generally refer to modification techniques in which the 2 'oxygen atom and 4' carbon atom of ribose are linked by a methylene bridge. LNA can prolong serum half-life of miRNA, improve affinity to target, and reduce scope and degree of off-target effect. The antisense medicine developed based on the modification technology of nucleic acid chain skeleton has greatly improved solubility, nuclease degradation resistance and other aspects, and is easy to synthesize in great amount. There are various methods for backbone modification of oligonucleotides, including thio methods, such as thio modification of a deoxynucleotide chain to a thio deoxynucleotide chain. The method is to replace oxygen atoms of phosphate bonds on the DNA skeleton with sulfur atoms, and can resist nuclease degradation. It is to be understood that any modification capable of retaining most or all of the activity of the antisense oligonucleotide is encompassed by the present invention.

As a preferred mode of the invention, the antisense oligonucleotide is subjected to nucleic acid lock modification; more preferably also thio-modifications.

After the antisense oligonucleotides are transferred into human body, they can obviously reduce the expression of miRNA.

Polynucleotide constructs

According to the miRNA sequences provided herein, polynucleotide constructs can be designed that, after being introduced, can be processed into mirnas that can affect the expression of the corresponding mRNA, i.e., the amount of the corresponding miRNA that the polynucleotide construct is capable of up-regulating in vivo. Thus, the present invention provides an isolated polynucleotide (construct) that can be transcribed by a human cell into a precursor miRNA that can be sheared by the human cell and expressed as the miRNA.

As a preferred embodiment of the present invention, the polynucleotide construct comprises a structure represented by formula I:

Seq _{forward direction} -X-Seq _{Reverse direction}

I is a kind of

In the formula I, the compound (I),

Seq _{forward direction} To express the nucleotide sequence of the miRNA in cells, seq _{Reverse direction} Is equal to Seq _{Forward direction} A substantially complementary nucleotide sequence; alternatively, seq _{Reverse direction} To express the nucleotide sequence of the miRNA in cells, seq _{Forward direction} Is equal to Seq _{Reverse direction} A substantially complementary nucleotide sequence;

x is at Seq _{Forward direction} And Seq _{Reverse direction} A spacer sequence therebetween, and said spacer sequence is identical to Seq _{Forward direction} And Seq _{Reverse direction} Are not complementary;

after the structure shown in the formula I is transferred into cells, a secondary structure shown in the formula II is formed:

in formula II, seq _{Forward direction} 、Seq _{Reverse direction} And X is as defined above;

the expression is shown in Seq _{Forward direction} And Seq _{Reverse direction} Complementary base pairing relationship formed between them.

Typically, the polynucleotide construct is located on an expression vector.

Thus, the invention also includes a vector comprising said miRNA, or said polynucleotide construct. The expression vector typically also contains a promoter, origin of replication, and/or marker gene, etc. Methods well known to those skilled in the art can be used to construct the expression vectors required for the present invention. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The expression vector preferably comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as calicheamicin, gentamicin, hygromycin, ampicillin resistance.

Chip

The microRNA expression profile chip usually contains up to several hundred probes, covers various microRNAs, and detects the content of various microRNAs in a sample on the whole genome level by utilizing the principle of DNA double-strand homologous complementation. Therefore, the transcription level of microRNA in the whole genome range in the sample to be detected can be detected at the same time.

The miRNA sequence can be used for preparing corresponding miRNA chips, so that the expression profile and the regulation mode of miRNAs can be studied.

In another aspect, the invention also provides a chip for analyzing miRNA expression profiles, which can be used to distinguish colorectal cancer samples from normal samples.

The miRNA chip comprises a solid phase carrier and oligonucleotide probes orderly fixed on the solid phase carrier.

Specifically, suitable probes can be designed according to the miRNAs of the present invention and immobilized on a solid support to form an "oligonucleotide array". By "oligonucleotide array" is meant an array having addressable locations (i.e., locations characterized by distinct, accessible addresses), each addressable location containing a characteristic oligonucleotide attached thereto. The oligonucleotide array may be divided into a plurality of subarrays, as desired.

The solid phase carrier can be made of various common materials in the field of gene chips, such as but not limited to nylon membranes, glass slides or silicon wafers modified by active groups (such as aldehyde groups, amino groups and the like), unmodified glass slides, plastic sheets and the like.

The preparation of the miRNA chip can be carried out by adopting a conventional manufacturing method of a biochip known in the art. For example, if a modified slide or a silicon wafer is used as the solid phase carrier, and the 5' -end of the probe contains an amino-modified poly dT string, the oligonucleotide probe can be prepared into a solution, then spotted on the modified slide or the silicon wafer by a spotting instrument, arranged into a predetermined sequence or array, and then fixed by standing overnight, so that the miRNA chip of the invention can be obtained. If the nucleic acid does not contain amino modifications, the preparation method can also be referred to as:

Wang Shenwu, ind. Infinite Instructions on Gene diagnosis technology-nonradioactive Manual; J.L.erisi, V.R.Iyer, P.O.BROWN.Exploringthemetabolicandgeneticcontrolofg eneexpressionnogenomic scale.science,1997;278:680 and Ma Liren, jiang Zhonghua Ji, biochip: chemical industry Press 2000,1-130.

In another aspect, the present invention also provides a method for detecting a miRNA expression profile in human tissue by a miRNA chip, comprising the steps of:

(1) Providing an RNA sample isolated from human tissue, and providing a marker on said RNA;

(2) Contacting the RNA of (1) with the chip to cause hybridization between the RNA and the oligonucleotide probe on the solid support, thereby forming an "oligonucleotide probe-RNA" binary complex on the solid support;

(3) Detecting the markers of the binary complex formed in (2), thereby determining the expression profile of the corresponding miRNA in human tissue.

Methods for extracting RNA from human tissue are well known to those skilled in the art and include the Trizol method.

More preferably, in step (1), after isolation of the RNA sample from human tissue, the RNA sample is suitably treated to enrich for RNA having a length, typically between 10 and 100 (small fragment RNA). After the treatment, the small fragment RNAs are used for subsequent hybridization, so that the accuracy of capturing miRNA by the chip can be improved. The person skilled in the art can conveniently isolate RNA having a certain fragment length, for example by gel electrophoresis.

Labelling of RNA is also a well known method to the person skilled in the art and can be achieved by adding a label, such as a labelling group, which specifically binds to RNA during hybridization. Such labeling groups include, but are not limited to: digoxin molecules (DIG), biotin molecules (Bio), fluorescein and its derivative biomolecules (FITC, etc.), other fluorescent molecules (e.g., cy3, cy5, etc.), alkaline Phosphatase (AP), horseradish peroxidase (HRP), etc. These labels and their labeling methods are all well known in the art.

When hybridizing the RNA with the miRNA chip, the miRNA chip and a prehybridization buffer solution can be prehybridized.

The solid phase hybridization between RNA and miRNA chips according to the present invention is performed according to classical methods in the art, and the person skilled in the art can easily determine the optimum conditions for buffer, probe and sample concentrations, prehybridization temperature, hybridization temperature, time, etc., according to experience. Or may be as described in the guidelines for molecular cloning experiments.

And then obtaining information to be detected according to the position, the intensity and other information of the marking signal on the miRNA chip. If the amplified product is marked by a fluorescent group, a fluorescence detection device (such as a laser confocal scanner Scanarray 3000) can be directly used for obtaining the information to be detected.

Detection kit

The invention also provides a kit, which contains the miRNA or the detection reagent (such as a chip) thereof. The kit can be used for detecting the expression profile of miRNA; or for diagnosing rectal cancer, preferably, the kit further comprises a marker for marking the RNA sample and a substrate corresponding to the marker.

In addition, various reagents required for extracting RNA, PCR, hybridization, color development, etc. can be included in the kit, including but not limited to: extract, amplification solution, hybridization solution, enzyme, control solution, color development solution, washing solution, antibody, etc.

In addition, the kit can also comprise instructions for use and/or chip image analysis software.

The present invention will be described in further detail with reference to the following examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The following examples are not to be construed as limiting the details of the experimental procedure, and are generally carried out under conventional conditions such as those described in the guidelines for molecular cloning laboratory, sambrook.J.et al, (Huang Peitang et al, beijing: scientific Press, 2002), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated. The experimental materials and reagents used in the following examples were obtained from commercial sources unless otherwise specified.

Examples

Sampling flow

Strictly obeys the technical rules of collection, arrangement and preservation of fresh organizations of Chinese human genetic resource diseases, which are issued by the national institute of health in 1998, and 2005. Collecting peripheral venous blood of 78 colorectal cancer patients, 70 colorectal adenoma patients and 70 healthy controls of a third affiliated hospital of Guangzhou medical university, gastrointestinal surgery, endoscope center, gastroenterology and physical examination center during 2017, 01-2019 and 02, wherein the colorectal cancer patients are diagnosed by pathological examination, and diagnosis of other neoplastic diseases or serious and chronic diseases and the like possibly affecting the research targets is not included, the patients are not subjected to any radiotherapy and chemotherapy before sample collection, and all the patients have complete clinical and pathological data. Male 43, female 35; age 25-90 years, average 65 years; TNM stage was performed in accordance with TNM stage standard of NCCN guideline, 38 cases of stage 0-II, 40 cases of stage III-IV. Colorectal adenomatous patients were included as standard for pathologically diagnosing definitive adenomatous polyps or tubular/villous/serrated adenomas or high/low grade intraepithelial neoplasias, and excluding neoplastic diseases or major, chronic diseases, etc. may affect the diagnosis of the targets of the study. 39 men and 31 women; age 22-88 years, mean 59 years. Healthy subjects were included as criteria to rule out a history of neoplastic disease, a history of significant or chronic disease, and other diseases that may affect the targets of the study. Of these, 34 men and 36 women; age 19-68 years, average 41 years. The study was approved by the review by the third affiliated hospital medical ethics committee of the university of guangzhou medical science, and all specimens were collected with informed consent from the patient.

Experimental main materials

1. Main instrument

Gene amplification instrument: TC-96/G/H (b), hangzhou Bo.

Fluorescent quantitative PCR instrument: lightCycler480II, roche.

2. Main reagent

Serum/plasma miRNA extraction and separation kit, DP503 and radix Caesalpiniae cristae.

All-in-OneTM miRNA First-Strand cDNA Synthesis Kit：QP014，GeneCopoeia。

HieffTM qPCR SYBR Green Master Mix (No Rox): 11201ES08, assist in the holy life.

Primer: and (3) biosynthesizing.

Experimental procedure

1. RNA extraction

200uL of plasma sample was taken and the procedure was followed as described, with a final elution volume of 35uL.

1) Sample treatment: mu.l of lysate MZA is added into 200 mu.l of serum or plasma, and the mixture is mixed for 30sec to completion by shaking with a shaker

Homogenizing, and mixing the materials upside down. If an external reference (CR 100-01) is required, the external reference is added after complete homogenization and before reverse homogenization

(1. Mu.M concentration, 1. Mu.l) was added.

2) And standing at room temperature for 5min to completely separate the nucleic acid protein complex.

3) 200 μl chloroform was added, the tube was capped, vigorously shaken for 15sec, and left at room temperature for 5min.

4) 12,000rpm (-13,400 Xg), 4℃and centrifugation for 15min, the sample will be divided into three layers: yellow organic phase, white

The middle layer and the colorless aqueous phase, the RNA being mainly in the aqueous phase, the aqueous phase is transferred to a new tube for the next operation.

5) The volume of the transfer fluid is measured, and absolute ethanol 2 times of the volume of the transfer fluid is slowly added (for example: 500 μl of transfer solution was added with 1ml of anhydrous

Ethanol), and mixing well (precipitation may occur at this time). Transferring the obtained solution and precipitate into an adsorption column mirinlite, and a chamber

Standing for 2min, centrifuging at room temperature at 12,000rpm (13,400Xg) for 30sec, centrifuging, discarding effluent, and retaining adsorption

Column mirinlite.

6) Adding 700 μl deproteinized MRD (please check whether ethanol has been added) into the adsorption column mirinlite, standing at room temperature

Centrifuge at 12,000rpm (13,400 Xg) for 30sec at room temperature for 2min, discard the waste liquid.

7) 500. Mu.l of rinse solution RW (please check whether ethanol has been added) was added to the column mirilute, allowed to stand at room temperature for 2min,

centrifuge at 12,000rpm (13,400 Xg) for 30sec at room temperature, discard the waste liquid.

8) The step 7 is repeated once.

9) Centrifuge at 12,000rpm (13,400Xg) for 2min and discard the collection tube.

2. cDNA Synthesis

1) Thawing the reagent of miRNA qRT-PCR Detection Kit, gently reversing the top and bottom, uniformly mixing, and standing on ice for later use after short centrifugation.

2) Preparing miRNA reverse transcription reaction liquid on ice, adding the following components into a precooled RNase-free reaction tube to a total volume of 25 mu L

Composition of the components	Addition amount of	Final concentration
			Total RNA		50ng
2.5U/μl PolyA Polymerase	1μL
			RTase Mix	1μL
5×Reaction Buffer	5μL	1×
			DEPC treated water	to 25μL

3) Reverse transcription reaction: mix well and incubate for 1 hour at 37℃after brief centrifugation.

4) Terminating the reaction: inactivating at 85 deg.C for 5min, diluting with sterilized water for 8 times, and preserving the reverse transcription product at-20deg.C.

3. Quantitative PCR detection

1) Mix was thawed at 4 ℃, gently mixed upside down and centrifuged briefly.

2) The reaction solutions in the following table were prepared on ice

3) The reaction tube was centrifuged briefly to ensure that all reaction solution was at the bottom of the reaction well.

4) The following procedure was used for the reaction:

4. primer sequences

5. Statistical analysis

Statistical analysis was performed using Primer Premier 5.0 software and SPSS 16.0. When the data of the plasma miRNA are normally distributed, adopting t-test analysis to compare the two groups of data; when the data is distributed in a biased state or uneven in variance, the plasma samples are compared between two groups and the correlation analysis of clinical pathological parameters is carried out by using a Mann-Whitney U test, and the plasma samples are compared between 3 groups by using a Kruskal-Wallis test. And (5) carrying out multi-factor analysis by using logistic binary regression and establishing a logistic equation. The diagnostic efficacy of plasma mirnas for colorectal and adenomas was analyzed with a subject working profile (receiver operator characteristic, ROC) curve. The difference of P <0.05 is statistically significant.

Experimental results

1. Clinical case profile

Clinical pathology data from 218 (78 colorectal cancers, 70 colorectal adenomas and 70 healthy controls) sample sources were collected and collated (table 1).

TABLE 1.218 clinical case characterization data for sample sources

2. Relation between expression of plasma miRNA and clinical pathological characteristics

The correlation of miRNA expression levels in the plasma of 78 colorectal cancer patients and clinical pathological parameters is analyzed by adopting t-test, and the result shows that the expression level of the plasma miR-1183 of the colorectal cancer patients is obviously correlated with pathological stage (P=0.015) and N stage (P=0.018) of the patients; plasma miR-301a-3P expression levels were significantly correlated with patient M-staging (p=0.000); plasma miR-193b-3P expression levels were significantly correlated with patient serum CEA expression levels (p=0.028); plasma miR-145-5P expression levels were significantly correlated with patient M-staging (p=0.001) (table 2). No significant differences were seen in other correlation analyses. Mirnas that indicate differences may be involved in malignant progression of colorectal cancer, with potential as colorectal tumor prognostic biomarkers.

The correlation of the expression level of miRNA in the plasma of 70 colorectal adenoma patients with sex, age and serum CEA expression is analyzed by adopting t test, and the result shows that the expression level of the plasma miR-301a-3P of the colorectal adenoma patients is obviously correlated with the sex (P=0.038) and age (P=0.004) of the patients; plasma miR-224-5P (p=0.045) and miR-145-5P (p=0.043) expression levels were significantly correlated with patient age (table 3). No significant differences were seen in other correlation analyses. It was shown that plasma miR-301a-3p, plasma miR-224-5p and miR-145-5p expression could be affected by the sex and age distribution of the patient. The correlation of miRNA expression levels in plasma of 70 healthy volunteers with gender and age was also analyzed using t-test, and the results showed that the measured plasma miRNA expression levels were not significantly correlated with the gender and age of the patients (table 4).

TABLE 2 correlation of expression levels of miRNAs in colorectal cancer plasma with clinical pathological parameters

TABLE 2 continuous process

TABLE 3 correlation of expression levels of miRNAs in colorectal adenoma plasma with clinical pathological parameters

TABLE 3 continuous process

TABLE 4 correlation of expression levels of miRNAs in plasma of healthy controls with clinical pathological parameters

TABLE 4 continuous process

3. Detection of expression levels of 7 targets in a round of 60 plasma samples

The expression levels of miR-301a-3p, miR-145-5p, miR-193b-3p, miR-125b-5p, miR-224-5p, miR-375-3p and 135b-5p in 60 plasma samples (20 cases of colorectal cancer, colorectal adenoma and healthy controls) are detected by real-time fluorescent quantitative PCR. The results show that miR-301a-3p, miR-145-5p, miR-193b-3p, miR-125b-5p and miR-224-5p have obvious statistical differences among groups, and the differences of miR-375-3p and miR-135 b-5p are not obvious. Five targets with significant differences can enter two rounds of large sample validation. (see FIG. 1 for partial results).

4. Verification of expression levels of 6 targets in two rounds of 158 plasma samples

Five targets from the first round of results, namely miR-224-5p, miR-125b-5p, miR-301a-3p, miR-145-5p and miR-193b-3p, are screened to enter two rounds of verification, and miR-1183 with significant significance is added to form 6 targets to enter two rounds of large sample verification. Real-time fluorescent quantitative PCR detects expression of 6 targets in 158 plasma samples (58 colorectal cancers, 50 colorectal adenomas and 50 healthy controls). The results show that the miR-224-5p, miR-301a-3p, miR-145-5p and miR-1183 have obvious differences. (see FIG. 2 for partial results).

5. ROC curve analysis of diagnostic value of single mirnas for colorectal and adenoma

Two rounds of data were pooled and analyzed for diagnostic value of miR-224-5p, miR-125b-5p, miR-301a-3p, miR-145-5p, miR-193b-3p and miR-1183 alone for colorectal cancer and adenoma. ROC curve analysis showed that plasma miR-224-5p, miR-301a-3p, and miR-145-5p levels were able to identify colorectal cancer patients and healthy subjects (figure 3A). Plasma miR-1183, miR-224-5p and miR-301a-3p levels are capable of identifying colorectal adenoma patients and healthy subjects (FIG. 3B). Plasma miR-1183, miR-224-5p and miR-301a-3p levels were able to identify colorectal and adenomatous subjects (FIG. 3C). Plasma miR-1183 levels were able to identify early and late colorectal cancer (figure 3D). The area under the curve AUC, 95% confidence interval CI, sensitivity, specificity and p-value statistics of the above ROC curves are shown in table 1.

FIG. 3A ROC curve analysis of diagnostic potential of plasma miRNAs for colorectal cancer

FIG. 3B ROC curve analysis of diagnostic potential of plasma miRNAs for colorectal adenomas

FIG. 3C ROC curve analysis of the differential potential of plasma miRNAs for colorectal and adenomas

Figure 3D ROC curve analysis of the differential potential of plasma mirnas for colorectal early and late cancers

TABLE 5 diagnostic value of individual miRNAs for colorectal and adenomas

Note that: the cut-off value takes the maximum value of the reduction log index.

6. multifactor analysis of miRNA, combined model establishment and diagnostic potential detection

After the differential expression detection of miRNA targets in various plasma samples, all meaningful targets are put into logistic binary regression, and targets which can independently influence colorectal adenoma and cancer are screened. And establishing a logic equation, and analyzing the ROC curve after combination. The diagnostic efficacy of the logistic equation and combination is summarized in fig. 5 below. The results show that the first equation represents that miR-224-5p, miR-301a-3p and miR-193b-3p can serve as independent predictors of CRC occurrence, the area under the curve for diagnosing colorectal cancer by the combination of the three miRNAs is 0.856, 95% CI is 0.781-0.930, and sensitivity and specificity are 0.655 and 0.92 (A in FIG. 4); the second equation represents that miR-145-5p, miR-224-5p, miR-301a-3p, miR-193B-3p and miR-1183 can identify adenomas and cancers, and that the area under the curve for identifying adenomas and cancers by a combination of five miRNAs is 0.930, 95% CI is 0.881-0.979, and sensitivity and specificity are 0.72 and 0.68, respectively (B in FIG. 4). These data show that these two combined models have higher diagnostic value for colorectal tumors and higher detection efficacy than current early screening and auxiliary diagnostic means. From the group comparison analysis, miR-224-5p, miR-301a-3p and miR-193b-3p can have obvious sign significance in cancer groups, and can participate in occurrence and development of colorectal from normal-adenoma-cancer stage.

Figure 5 diagnostic efficacy of Logit equations and combinations

Detection verification

100 detection samples (comprising 30 colorectal cancer samples, 30 colorectal adenoma samples and 40 healthy samples) are subjected to detection of the expression level of the miRNA markers according to the method, and the category of the sample is judged according to the expression level of the miRNA markers shown in the invention, so that double-blind verification is performed.

The results show that the combination of the three miRNAs can effectively identify colorectal cancer samples, the sensitivity of the colorectal cancer samples can reach 70% (21/30), the specificity of the colorectal cancer samples can reach 92.9% (65/70), and the colorectal cancer samples have good repeatability and stability. The combination comprising the five miRNAs can effectively distinguish rectal adenoma samples from colorectal carcinoma samples, the sensitivity of the five miRNAs can reach 76.7 percent (23/30), the specificity of the five miRNAs can reach 73.3 percent (22/30), and the repeatability and the stability are good.

The invention relates to a combination of a plurality of plasma miRNAs as a colorectal cancer early diagnosis and prognosis judgment marker and a related kit. Compared with the traditional fecal occult blood detection, the sensitivity is improved, particularly the sensitivity to adenoma in the progressive stage is obviously improved, and the sampling has no strict diet control, so that the reject rate is greatly reduced. Compared with colonoscopy, the kit does not need to be subjected to strict and painful intestinal preparation before screening, has obviously reduced technical requirements, very small risk of wound and low popularity of colonoscopy as a first screen, and can greatly improve the detection rate of large intestine lesions if the kit can be used for carrying out high-sensitivity preliminary screening and then carrying out diagnosis under the colonoscopy. Compared with multi-target fecal detection newly incorporated into a screening guide, the invention has the advantages of low price, simple and convenient operation, easy implementation and the like under the condition of equivalent sensitivity and specificity. Compared with a questionnaire risk assessment screening method, the plasma miRNA molecule provided by the invention has the advantages that the influence degree of the risk can be assessed, and the defects of low screening sensitivity and specificity of colorectal tumors in the progressive stage are overcome. In conclusion, early discovery and timely intervention of high risk groups of colorectal cancer are achieved, and more accurate early warning means or biological indexes are not yet available clinically. The kit is a detection means which has good sensitivity, low cost, simple and convenient operation and easy implementation, has value for early screening/diagnosis of CRC and has better significance for prognosis judgment of CRC.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Sequence listing

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DA AN GENE CO., LTD. OF SUN YAT-SEN University

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Claims (2)

1. The application of the miRNA detection reagent in the blood plasma is characterized in that the reagent kit for identifying colorectal adenoma and colorectal carcinoma is prepared, and the miRNA consists of miR-224-5p, miR-301a-3p, miR-145-5p, miR-193b-3p and miR-1183;

in the samples assayed, the sample is, compared to colorectal adenoma samples,

the miR-224-5p level is obviously improved;

the miR-301a-3p level is obviously reduced;

miR-193b-3p levels are significantly increased;

miR-145-5p levels were significantly reduced;

the miR-1183 level is obviously improved;

the test sample is prompted to be a colorectal cancer high risk patient sample.

2. The use of claim 1, wherein the regression model is determined as a regression model of logic (P) = -2.338+7.166× (miR-145-5P) +173.161 × (miR-193 b-3P) -27.658 × (miR-1183) +46.071 × (miR-224-5P) -58.807 × (miR-301 a-3P), wherein expression levels of miR-224-5P, miR-301a-3P, miR-145-5P, miR-193b-3P, and miR-1183 are measured with miR-30a-5P as an internal reference, and wherein a logic (P) value in the model indicates that the test sample is significantly different from the colorectal adenoma sample, indicating that the test sample is a high risk patient sample for colorectal cancer.