CN117402965A - Colorectal cancer marker, primer, kit and application - Google Patents

Abstract

The invention belongs to the field of molecular biology, and particularly relates to a colorectal cancer marker, a primer, a kit and application. The colorectal cancer marker disclosed by the invention is a serum miRNA marker. The marker comprises: one or more of hsa-miR-2276-3p, hsa-miR-3915, hsa-miR-4728-5p, hsa-miR-5190 and hsa-miR-5699-3p, and the corresponding coding nucleotide sequence of each microRNA is shown in SEQ ID NO. 1-SEQ ID NO. 5. The blood microRNA provided by the invention has a noninvasive and efficient effect in detection as a colorectal cancer screening or diagnosis marker, and has a wide application prospect in preparation of colorectal cancer screening and diagnosis kits.

Description

Colorectal cancer marker, primer, kit and application

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a colorectal cancer marker, a primer, a kit and application.

Background

Colorectal cancer is currently the most common cancer at position 3 worldwide, and is also the cause of death from cancer at position 2. Over 100 thousands of colorectal cancer patients are newly increased worldwide only in 2018, and about 80 thousands of colorectal cancer death cases are newly increased. Colorectal cancer thus severely threatens human health.

Colorectal cancer is usually asymptomatic early, and early screening and accurate diagnosis thereof are important factors in improving prognosis of patients. However, the existing colorectal cancer screening and diagnosis means have a plurality of limitations and disadvantages. For example, the traditional colorectal cancer related tumor markers are not suitable for colorectal cancer screening and auxiliary diagnosis due to low sensitivity and specificity, and can only be used for postoperative recurrence monitoring; the enteroscopy screening and diagnosis of colorectal cancer has high accuracy, but the preparation process is complex, and the examination process is painful, so that the enteroscopy screening and diagnosis of colorectal cancer is not suitable for auxiliary diagnosis. Therefore, there is a great need in clinic for simple, non-invasive and high-efficiency colorectal cancer screening and diagnosis methods.

The duration of colorectal cancer patient survival is closely related to clinical staging, and the earlier the finding, the greater the chance of cure. If the early treatment is carried out, the survival rate of 5 years can reach 70-95%; mid-term treatment, five-year survival is approximately 50-60%, but late-stage treatment has a 5-year survival rate of only 5-10%. Early discovery and early treatment are therefore of paramount importance. Most cancer patients are in early stage, either feel no symptoms or the symptoms are mild and easily ignored; when the disease state is advanced to obvious symptoms and medical treatment is needed, the optimal treatment time is missed, comprehensive treatment is needed, the treatment cost is obviously increased, the treatment side effect is increased, the treatment effect is poor, and the survival rate of 5 years is obviously reduced.

Screening and early diagnosis are one of the main methods to improve prognosis in colorectal cancer patients. The development and progression of most colorectal cancers is a complex process involving multiple stages, steps, factors, genes, and the like, as a result of interactions between external environmental factors and genetic factors inherent to the body.

The ideal biological markers should satisfy the following characteristics: 1. can be obtained by a non-invasive method and can be isolated by a simple method; 2. high specificity for different diseases; 3. highly diagnostic sensitivity; 4. biological rationality of disease-related mechanisms; 4. expression is stable to allow for clinically routine use. There is increasing evidence that blood micrornas possess these characteristics and are expected to be biological markers for screening and diagnosing disease.

Micrornas (mirnas) are a class of non-coding single-stranded RNA molecules of about 19-25 nucleotides in length, encoded by endogenous genes, which are widely found in eukaryotic cells. miRNA genes account for about 1% of the entire genome, which regulate gene expression by decreasing the stability of target mRNA or inhibiting translational efficiency, thereby participating in a variety of biological processes including cell proliferation, differentiation, apoptosis, development, and stress response. The miRNA in the blood often forms a complex with proteins and the like, so that the miRNA has good RNase degradation resistance, and the expression level of the miRNA in the blood is not affected after freezing, thawing or long-term storage at room temperature. In addition, the expression level of miRNA in blood is very similar for people with different sexes and ages.

With more and more miRNAs as biomarkers and therapeutic targets of human diseases, finding a marker capable of accurately diagnosing colorectal cancer has great significance in optimizing clinical colorectal cancer diagnosis strategies.

Disclosure of Invention

In view of the above, the present invention aims to provide a colorectal cancer marker for use in colorectal cancer screening or diagnosis, and a kit and application for colorectal cancer diagnosis, the technical scheme is as follows:

in a first aspect, the present invention provides a colorectal cancer marker comprising: at least one of hsa-miR-2276-3p, hsa-miR-3915, hsa-miR-4728-5p, hsa-miR-5190 and hsa-miR-5699-3 p.

As one implementation mode, the base sequence of the hsa-miR-2276-3p is shown as SEQ ID NO.1, the base sequence of the hsa-miR-3915 is shown as SEQ ID NO.2, the base sequence of the hsa-miR-4728-5p is shown as SEQ ID NO.3, the base sequence of the hsa-miR-5190 is shown as SEQ ID NO.4, and the base sequence of the hsa-miR-5699-3p is shown as SEQ ID NO. 5.

The colorectal cancer markers listed above are serum miRNA markers, through experimental detection, the expression levels of the markers in serum tissues of colorectal cancer patients are statistically different from the expression levels of tissues corresponding to normal people, and the markers of hsa-miR-2276-3p, hsa-miR-3915, hsa-miR-4728-5p, hsa-miR-5190, hsa-miR-5699-3p and the like can be independently used for detecting and diagnosing colorectal cancer, can also be used for jointly detecting and diagnosing colorectal cancer by more than two markers, and simultaneously can timely reflect the disease states of colorectal cancer patients, are particularly suitable for early diagnosis, prognosis judgment, postoperative recurrence detection and the like of colorectal cancer, avoid complicated detection, save time and labor cost and facilitate clinicians to adopt personalized treatment schemes. Moreover, the method for detecting the microRNA markers in the serum samples is very mature, the detection process is simple and convenient and is easy to repeat, the detection investment of hospitals or detection institutions can be greatly reduced by common technicians, meanwhile, the detection samples are easy to obtain, the clinical operability is high, the traumatology is small, the stability of the serum microRNA is good, and the detection is convenient.

In a second aspect, the present invention also provides a primer for detecting the colorectal cancer marker, the primer comprising: an upstream primer and/or a reverse transcription primer;

wherein, the base sequence of the upstream primer of hsa-miR-2276-3p is shown in SEQ ID NO. 6;

the base sequence of the upstream primer of hsa-miR-3915 is shown in SEQ ID NO. 7;

the base sequence of the upstream primer of hsa-miR-4728-5p is shown in SEQ ID NO. 8;

the base sequence of the upstream primer of hsa-miR-5190 is shown in SEQ ID NO. 9;

the base sequence of the upstream primer of hsa-miR-5699-3p is shown in SEQ ID NO. 10;

the base sequence of the reverse transcription primer of hsa-miR-2276-3p is shown in SEQ ID NO. 11;

the base sequence of the reverse transcription primer of hsa-miR-3915 is shown in SEQ ID NO. 12;

the base sequence of the reverse transcription primer of hsa-miR-4728-5p is shown in SEQ ID NO. 13;

the base sequence of the reverse transcription primer of hsa-miR-5190 is shown in SEQ ID NO. 14;

the base sequence of the reverse transcription primer of hsa-miR-5699-3p is shown in SEQ ID NO. 15.

In a third aspect, the invention also provides a kit comprising: the above primer.

As one embodiment, the kit further comprises; the base sequence of the universal reverse primer of each colorectal cancer marker is shown as SEQ ID NO. 18.

As an embodiment, the kit further comprises: the base sequence of the probe is shown as SEQ ID NO. 19.

As an embodiment, the kit further comprises: an RNA extraction reagent;

the RNA extraction reagent comprises TRIzol ^TM LS、Beyozol、PureZOL、Aurum ^TM At least one of them.

The kit provided by the invention can realize detection of colorectal cancer markers in serum by utilizing a reverse transcription reaction technology and a PCR reaction technology, realizes screening or diagnosis of colorectal cancer, is mature in method, has a simple and convenient detection process, is easy to repeat, and is beneficial to realizing early diagnosis, prognosis judgment, postoperative recurrence detection and the like of colorectal cancer.

When the above kit is used for detecting colorectal cancer, the detection method thereof comprises:

1) Collecting a serum sample;

2) Extracting total RNA in a sample;

3) Synthesizing cDNA by taking the extracted RNA as a template;

4) Carrying out RT-qPCR reaction on the primer and the probe to obtain the cycle number, namely CT value, of the target primer and the external reference primer in the serum sample to be detected when the fluorescent signals reach a set threshold value;

5) According to the 2-delta Cq calculation method, the delta CT value of the target primer in the serum sample is calculated through the external reference primer.

In a fourth aspect, the invention also provides the use of the aforementioned colorectal cancer marker or the aforementioned primer or the aforementioned kit for the preparation of an anti-colorectal cancer drug.

The colorectal cancer marker, the primer or the kit provided by the invention are applied to the preparation of the anti-colorectal cancer medicament, so that the time and the labor cost are saved, and the research and the production period of the anti-colorectal cancer medicament are shortened.

Drawings

FIG. 1 is a graph showing the expression of hsa-miR-2276-3p in serum of colorectal cancer patients and healthy control groups;

FIG. 2 is a ROC curve of hsa-miR-2276-3 p;

FIG. 3 is an expression profile of hsa-miR-3915 in serum of colorectal cancer patients and healthy control groups;

FIG. 4 is an ROC curve of hsa-miR-3915;

FIG. 5 is a graph showing the expression of hsa-miR-4728-5p in serum of colorectal cancer patients and healthy control groups;

FIG. 6 is a ROC curve of hsa-miR-4728-5 p;

FIG. 7 is a graph showing the expression of hsa-miR-5190 in serum of colorectal cancer patients and healthy control groups;

FIG. 8 is an ROC curve of hsa-miR-5190;

FIG. 9 is a graph showing expression of hsa-miR-5699-3p in serum of colorectal cancer patients and healthy control groups;

FIG. 10 is a ROC curve of hsa-miR-5699-3 p.

Detailed Description

In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the invention is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

Example 1

(1) Serum sample collection and preparation

Serum from all volunteers was collected 120 cases of colorectal cancer patients with confirmed diagnosis, serum from healthy persons with 120 cases of physical examination, each with 2ml of peripheral blood, centrifuged at 4℃for 2 hours at 3600g for 10 minutes, and the upper serum was stored in a refrigerator at-80℃for further use.

(2) Extraction of RNA and Synthesis of cDNA in serum samples

200uL of serum extracted in the step (1) is taken, 0.1pM nematode cel-miR-54 is added as an external reference, 1ml of TRIzol is added according to the ratio of 1:5, and the mixture is fully and uniformly mixed, and total RNA is extracted according to the scheme provided by the TRIzol. According to the method of the reverse transcription kit, reverse transcription enzyme (Sensiscript Reverse Transcriptase), dNTP (dNTP Mix) and reaction buffer (10× Sensiscript Reverse Transcription Buffer) provided by the kit are added on a common PCR instrument by taking the extracted total RNA (Purified RNA) as a template, and the microRNA marker and the reverse transcription primer mixture (Reverse transcription Mix) of cel-miR-54 are subjected to reverse transcription into cDNA. Preserving at-20 ℃ for standby.

TABLE 1 composition of cDNA Synthesis reaction System

Table 2 reverse transcription primers used

(3) RT-qPCR detection

The cDNA of the serum sample is subjected to reverse transcription, fluorescent real-time quantitative PCR is carried out according to the method of Golden HS TaqMan fluorescent real-time quantitative PCR kit, and Ct values of all samples are read out by a light cycler480 (Roche) fluorescent quantitative PCR instrument. The qRT-RCR application instrument is LightCycler480 (Roche), and the reaction conditions are: (1) pre-denaturation, 95 ℃,5 min; (2) denaturation, 95 ℃,10 seconds; annealing at 60℃for 30 seconds for a total of 40 cycles.

According to the 2-delta Cq calculation method, calculating delta CT value of the serum sample by taking cel-miR-54 as an external reference, and according to a formula delta CT (detection) =CT (microRNA, detection) -CT (external reference, detection); Δct (calibration) =ct (colorectal cancer, calibration) -CT (control group, calibration); Δct=Δct (detection) - Δct (calibration), based on the obtained CT value(s), and carrying out relative quantitative detection on the target gene according to the 2-delta CT value.

Data are sorted by adopting a 2-delta CT method, data analysis and graphic processing are analyzed by adopting GraphPad Prism 8.3.0 software, all data are detected by adopting a Two-driven Student's test statistical method, the data are expressed by mean value +/-standard deviation, and P <0.05 is statistically significant.

TABLE 3 real-time fluorescent quantitative PCR reaction system

TABLE 4 colorectal cancer screening or diagnostic markers, upstream primers and probe sequences of this example

Example 2 comparison of hsa-miR-2276-3p expression in serum of colorectal cancer patients and normal persons

Serum from patients with confirmed colorectal cancer 120 cases and serum from healthy persons with physical examination 120 cases were collected. Quantitative analysis was performed by the method of example 1 with the exception of peripheral blood, with informed consent of all volunteers, and the results are shown in FIGS. 1-2.

As shown in fig. 1-2, hsa-miR-2276-3P expression levels in serum of colorectal Cancer patients (Cancer) were significantly down-regulated (P < 0.0001) compared to normal (Control) (fig. 1); the area under the curve corresponding to 0.7406 (95% CI:0.6794-0.8018, P < 0.0001) (FIG. 2), sensitivity 76.67% and specificity 53.33% were obtained by ROC curve.

The experimental results of this example show that hsa-miR-2276-3p in serum of colorectal cancer patients is down-regulated compared with normal human expression, and that the difference is statistically significant. ROC curve analysis of diagnostic efficacy of circulating hsa-miR-2276-3p in colorectal cancer patients showed auc= 0.7406 with a sensitivity of 76.67% and a specificity of 53.33%.

Example 3 comparison of hsa-miR-3915 expression in serum of colorectal cancer patients and normal persons

Serum from patients with confirmed colorectal cancer 120 cases and serum from healthy persons with physical examination 120 cases were collected. Quantitative analysis was performed by the method of example 1 with the exception of peripheral blood, respectively, with informed consent of all volunteers, and the results are shown in FIGS. 3 to 4.

Significant downregulation of hsa-miR-3915 expression levels in serum of colorectal Cancer patients (Cancer) compared to normal (Control) (P < 0.0001) (FIG. 3); the area under the curve corresponding to the ROC curve was 0.8452 (95% CI:0.7941-0.8963, P < 0.0001) (FIG. 4), the sensitivity was 87.5%, and the specificity was 72.5%.

The experimental results of this example show that hsa-miR-3915 in serum of colorectal cancer patients is down-regulated compared with normal human expression, and that the difference is statistically significant. ROC curve analysis of diagnostic efficacy of circulating hsa-miR-3915 in colorectal cancer patients showed auc= 0.8452, sensitivity of 87.5% and specificity of 72.5%.

Example 4 comparison of hsa-miR-4728-5p expression in serum of colorectal cancer patients and normal persons

Serum from patients with confirmed colorectal cancer 120 cases and serum from healthy persons with physical examination 120 cases were collected. Quantitative analysis was performed by the method of example 1 with the exception of peripheral blood, respectively, with informed consent of all volunteers, and the results are shown in FIGS. 5 to 6.

Significant downregulation of hsa-miR-4728-5P expression levels in serum of colorectal Cancer patients (Cancer) compared to normal (Control) (P < 0.0001) (FIG. 5); the area under the curve corresponding to the ROC curve was 0.7593 (95% CI:0.699-0.8196, P < 0.0001) (FIG. 6), sensitivity 77.5%, specificity 64.17%.

The experimental results of this example show that hsa-miR-4728-5p in serum of colorectal cancer patients is down-regulated compared with normal human group expression, and that the difference is statistically significant. ROC curve analysis of diagnostic efficacy of circulating hsa-miR-4728-5p in colorectal cancer patients showed auc= 0.7593, sensitivity of 77.5% and specificity of 64.17%.

Example 5 comparison of hsa-miR-5190 expression in serum of colorectal cancer patients and normal persons

Serum from patients with confirmed colorectal cancer 120 cases and serum from healthy persons with physical examination 120 cases were collected. Quantitative analysis was performed by the method of example 1 with the exception of peripheral blood, respectively, with informed consent of all volunteers, and the results are shown in FIGS. 7 to 8.

Significant down-regulation of hsa-miR-5190 expression levels in serum of colorectal Cancer patients (Cancer) compared to normal (Control) (P < 0.0001) (FIG. 7); the area under the curve corresponding to the ROC curve was 0.8574 (95% CI:0.8103-0.9045, P < 0.0001) (FIG. 8), the sensitivity was 83.33%, and the specificity was 70.83%.

The experimental results of this example show that hsa-miR-5190 in serum of colorectal cancer patients is down-regulated compared with normal human expression, and the difference is statistically significant. ROC curve analysis of diagnostic efficacy of circulating hsa-miR-5190 in colorectal cancer patients showed auc= 0.8574 (fig. 8), sensitivity of 83.33% and specificity of 70.83%.

Example 6 comparison of hsa-miR-5699-3p expression in serum of colorectal cancer patients and normal persons

Serum from patients with confirmed colorectal cancer 120 cases and serum from healthy persons with physical examination 120 cases were collected. Quantitative analysis was performed by the method of example 1 with the exception of peripheral blood, respectively, with informed consent of all volunteers, and the results are shown in FIGS. 9 to 10.

As shown in fig. 9-10, hsa-miR-5699-3P expression levels in serum of colorectal Cancer patients (Cancer) were significantly down-regulated (P < 0.0001) compared to normal (Control) (fig. 9); the area under the curve corresponding to the ROC curve was 0.7882 (95% CI:0.7312-0.8452, P < 0.0001) (FIG. 10), the sensitivity was 76.67%, and the specificity was 67.53%.

The experimental results of this example show that hsa-miR-5699-3p in serum of colorectal cancer patients is down-regulated compared with normal human expression, and that the difference is statistically significant. ROC curve analysis of diagnostic efficacy of circulating hsa-miR-5699-3p in colorectal cancer patients showed auc= 0.7882 (fig. 10), sensitivity of 76.67% and specificity of 67.5%.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.

Claims (8)

1. A colorectal cancer marker, comprising: at least one of hsa-miR-2276-3p, hsa-miR-3915, hsa-miR-4728-5p, hsa-miR-5190 and hsa-miR-5699-3 p.

2. The colorectal cancer marker of claim 1, wherein the base sequence of hsa-miR-2276-3p is shown in SEQ ID No.1, the base sequence of hsa-miR-3915 is shown in SEQ ID No.2, the base sequence of hsa-miR-4728-5p is shown in SEQ ID No.3, the base sequence of hsa-miR-5190 is shown in SEQ ID No.4, and the base sequence of hsa-miR-5699-3p is shown in SEQ ID No. 5.

3. A primer for detecting a colorectal cancer marker according to claim 1 or 2, characterized in that the primer comprises: an upstream primer and/or a reverse transcription primer;

wherein, the base sequence of the upstream primer of hsa-miR-2276-3p is shown in SEQ ID NO. 6;

the base sequence of the upstream primer of hsa-miR-3915 is shown in SEQ ID NO. 7;

the base sequence of the upstream primer of hsa-miR-4728-5p is shown in SEQ ID NO. 8;

the base sequence of the upstream primer of hsa-miR-5190 is shown in SEQ ID NO. 9;

the base sequence of the upstream primer of hsa-miR-5699-3p is shown in SEQ ID NO. 10;

the base sequence of the reverse transcription primer of hsa-miR-2276-3p is shown in SEQ ID NO. 11;

the base sequence of the reverse transcription primer of hsa-miR-3915 is shown in SEQ ID NO. 12;

the base sequence of the reverse transcription primer of hsa-miR-4728-5p is shown in SEQ ID NO. 13;

the base sequence of the reverse transcription primer of hsa-miR-5190 is shown in SEQ ID NO. 14;

the base sequence of the reverse transcription primer of hsa-miR-5699-3p is shown in SEQ ID NO. 15.

4. A kit, comprising: the primer according to claim 3.

5. The kit of claim 4, further comprising; the base sequence of the universal reverse primer of each colorectal cancer marker is shown as SEQ ID NO. 18.

6. The kit of claim 4, further comprising: the base sequence of the probe is shown as SEQ ID NO. 19.

7. The kit of claim 4, further comprising: an RNA extraction reagent;

the RNA extraction reagent comprises TRIzol ^TM LS、Beyozol、PureZOL、Aurum ^TM At least one of them.

8. Use of a colorectal cancer marker according to claim 1 or 2 or a primer according to claim 3 or a kit according to any one of claims 4 to 7 for the preparation of an anti-colorectal cancer medicament.