CN116024336A

CN116024336A - Detection and application of novel gastric cancer molecular marker

Info

Publication number: CN116024336A
Application number: CN202210815913.3A
Authority: CN
Inventors: 陆洪鹏; 李培飞; 徐磊; 罗守军
Original assignee: Ningbo First Hospital
Current assignee: Ningbo First Hospital
Priority date: 2022-07-12
Filing date: 2022-07-12
Publication date: 2023-04-28

Abstract

The invention designs a circular RNA molecular marker for diagnosing gastric cancer, which is hsa_circ_0001789. Stomach cancer remains one of the most common malignant tumors, with mortality rates very high worldwide. Although radical surgery increases the overall survival rate of gastric cancer patients, the 5-year survival rate is still not satisfactory. At present, early treatment is the only reliable way for reducing the death rate of gastric cancer, so that the improvement of early detection of gastric cancer has important significance. The invention provides application of hsa_circ_0001789 as a gastric cancer diagnosis and/or prognosis marker, wherein the expression level of the marker in focus tissues and blood plasma of tumor patients is obviously reduced, the marker is related to poor prognosis of gastric cancer, and the marker has important detection value for clinical diagnosis and poor prognosis prevention of gastric cancer.

Description

Detection and application of novel gastric cancer molecular marker

Technical Field

The invention belongs to the technical field of tumor markers, and particularly relates to application of hsa_circ_0001789 as a gastric cancer diagnosis and/or prognosis marker.

Background

Gastric Cancer (GC) is the third leading cause of death in cancer patients worldwide. Stomach cancer is the most common malignancy of the gastrointestinal tract in china, and mortality rates rise year by year. Although great progress has been made in the diagnosis and treatment of gastric cancer in recent years, prognosis of most gastric cancer patients is still unsatisfactory because the 5-year survival rate of such patients is rarely more than 30%. Therefore, the excavation of new molecular mechanisms and new biomarkers for gastric cancer is of great importance for improving early diagnosis of gastric cancer patients and providing new therapeutic targets.

Circular RNA (circRNA) is a novel endogenous non-coding RNA, an RNA molecule with a closed-loop structure formed by covalent bonds. In recent years, the rapid development of high throughput sequencing technology has further driven intensive research into circRNA, which is believed to be widely involved in human physiology and pathology. Studies have shown that circRNA is involved in the development and progression of a variety of malignancies, such as glioma, gastric cancer, liver cancer, colorectal cancer and pancreatic cancer. In addition, since the circRNA can avoid degradation by exonuclease, it tends to be stable in structure, so that it has great potential as a novel tumor biomarker for diagnosis/prognosis of gastric cancer.

Disclosure of Invention

In order to obtain a marker with important diagnostic and prognostic significance for gastric cancer, in the screening process, the invention is proved based on ROC curve analysis, hsa_circ_0001789 is obviously down-regulated in gastric cancer tissues and blood plasma, gastric cancer tissues and tissues beside the gastric cancer can be well distinguished, the area under the curve is 0.82, the sensitivity is 0.51, and the specificity is 0.84.

The low expression of the CircRNA is positively correlated with gastric cancer tumor invasion, differentiation, lymphatic metastasis, tumor-nodule-metastasis (TNM) stage and distant metastasis. From the above results, hsa_circ_0001789 can be used to distinguish gastric cancer from normal tissue effectively, and can be used as a diagnostic and prognostic marker in clinic.

Based on the research results, the invention provides the following technical scheme:

in a first aspect of the invention there is provided the use of hsa_circ_0001789 as a diagnostic and/or prognostic marker for gastric cancer.

Applications described in the first aspect above include, but are not limited to, any of the following:

(1) Determining whether the patient is a gastric cancer patient by detecting the expression level of hsa_circ_0001789 in a patient detection sample;

(2) Detecting the expression content of hsa_circ_0001789 in a gastric cancer patient detection sample, so as to evaluate the prognosis condition of the patient;

(3) The application of hsa_circ_0001789 detection reagent in preparing gastric cancer diagnosis and/or prognosis detection reagent.

In the application of the first aspect, the detection sample of the diagnostic marker is a clinical biological sample of a patient, including but not limited to one or more of serum, plasma, whole blood, secretion, body fluid, tissue, organ, paraffin section, and the like. In one mode of the invention where validation is feasible, the test samples are focal tissue samples and plasma samples of patients with gastric cancer.

According to the results of the study of the present invention, the expression level of hsa_circ_0001789 is lower in lesion and plasma samples of patients with gastric cancer than in paracancestral tissues and normal human plasma, and the decrease in expression level thereof is correlated with pathological parameters indicating poor prognosis, such as tumor invasion, differentiation, lymphatic metastasis, tumor-nodular-metastasis (TNM) stage and distant metastasis of gastric cancer.

Thus, in the above applications of the respective aspects, detecting a low expression level of hsa_circ_0001789 in a sample means that a subject has a greater likelihood of having gastric cancer, or that gastric cancer has a higher tendency to have a poor prognosis.

In a second aspect of the invention, there is provided a combination of reagents for the diagnosis and/or prognosis of gastric cancer for detecting the expression level of hsa_circ_0001789 in a sample.

The kind of the reagent combination according to the second aspect is determined according to the detection method of the circRNA, and the detection methods are available in the art, such as PCR. In one embodiment with good effect, the invention provides a diagnostic kit suitable for detecting hsa_circ_0001789 by a PCR method, wherein the diagnostic kit at least comprises the following diagnostic reagents:

(1) A primer;

(2) A sample tissue lysing agent;

(3) And (5) a PCR reaction system.

Further, the primer comprises a to-be-detected circRNA primer and also comprises a primer of an internal reference marker; in a specific example, the circRNA primer sequence is as follows:

Forward：5’-CTGCTCACGCAAGCTGAA-3’(SEQ ID NO.1)

Reverse：5’-TCTGTGAAGTCAACCTTTCCA-3’(SEQ ID NO.2)

the primer sequences of the internal reference markers are as follows:

Forward：5’-TCGACAGTCAGCCGCATCTTCTTT-3’(SEQ ID NO.3)

Reverse：5’-ACCAAATCCGTTGACTCCGACCTT-3’(SEQ ID NO.4)

the sample tissue lysing reagent includes, but is not limited to, TRIZOL reagent, isopropanol, ethanol, buffer reagent.

In a third aspect of the present invention, there is provided a kit for diagnosis and/or prognosis of gastric cancer, comprising the reagent combination according to the second aspect.

In a fourth aspect of the present invention, there is provided a method for determining prognosis of a gastric cancer patient, the method comprising detecting hsa_circ_0001789 content in focal tissue and plasma of the patient.

In the above determination method, the content of hsa_circ_0001789 can be detected by the kit according to the third aspect, and if the content of hsa_circ_0001789 in the focal tissue or plasma sample of the patient is lower than that of the corresponding control group, it indicates that the progression of the gastric cancer of the patient has a greater risk of exacerbation such as tumor invasion, differentiation, lymphatic metastasis, tumor-nodule-metastasis (TNM) stage and distant metastasis. The hsa_circ_0001789 critical value is technical content which can be conventionally determined by a person skilled in the art according to factors such as a kit, an expression level calculation method, a sample and the like.

The beneficial effects of the above technical scheme are:

in one scheme, the invention provides application of hsa_circ_0001789 as a gastric cancer diagnosis and prognosis judgment marker, and compared with a paracancerous tissue or normal blood plasma, the marker has good detection specificity and has important clinical application value for diagnosis and prognosis judgment of gastric cancer.

Drawings

FIG. 1 shows the expression of hsa_circ_0001789 in gastric and paracancerous tissues as described in example 1. FIGS. 1A and B show that hsa_circ_0001789 is expressed at a lower level in gastric cancer tissue than in paracancerous tissue. FIG. 1C shows the expression pattern of hsa_circ_0001789 in 108 pairs of tissue samples.

FIG. 2 shows the expression of hsa_circ_0001789 in the plasma of patients with gastric cancer and healthy controls as described in example 1. FIGS. 2A and B show that the expression level of hsa_circ_0001789 in the plasma of gastric cancer patients was lower than that of healthy control group.

FIG. 3A is an ROC curve of hsa_circle_ 0001789 described in example 1. Fig. 3B is the threshold value of hsa_circle_ 0001789.

FIG. 4 is a comparison of the potential of hsa_circ_0001789 described in example 1 to conventional biomarkers (CEA, CA199, CA125 and CA 724) in predicting malignant biological behavior of gastric cancer. FIG. 4A shows the efficacy of hsa_circ_0001789 in predicting the invasive phase (Tis & T1-T3, or T4). FIG. 4B shows the efficacy of hsa_circ_0001789 in predicting gastric cancer lymphatic metastasis (N0, or N1-N3). FIG. 4C shows the predicted efficacy of hsa_circ_0001789 for the distal transition period (M0 or M1). FIG. 4D shows the efficacy of hsa_circ_0001789 in predicting TNM stage (0 & I & II, or III & IV). And (3) injection: hsa_circle_ 0001789 is denoted by RNA.

Detailed Description

In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.

Example 1

(1) Clinical sample acquisition

108 gastric cancer patients and 24 healthy subjects were recruited from the people hospitals in northern Legend of Ningbo city and the first hospitals of Ningbo city in total from month 1 in 2016 to month 12 in 2021.

Cancer and paracancerous tissue from 108 gastric cancer patients were collected and the paracancerous tissue was evaluated by a highly experienced pathologist to ensure that no tumor cells were present in the sample. After tissue excision, all samples were immediately placed in RNA-fixer liquid nitrogen free RNA sample stock solution and stored in a refrigerator at-80 ℃.

For plasma samples, fresh peripheral blood samples of 24 gastric cancer patients and 24 healthy control groups were collected with EDTA tubes, centrifuged at 3,000g for 10 min, and the upper plasma was isolated and stored in a-80 ℃ refrigerator until further analysis.

Gastric cancer specimens were taken from patients who did not receive any chemotherapy, radiation therapy or targeted therapy prior to surgery. Clinical pathological characteristics of the patients, including gender, age, maximum tumor diameter, infiltration degree, TNM stage, differentiation grade, distal metastasis, lymphatic metastasis, etc., were also collected. All participants obtained written informed consent. The study protocol was approved by the human research ethics committee of both hospitals.

(2) RNA extraction

1) About 50mg of tissue specimens stored in a-80℃refrigerator were removed and rapidly transferred to EP tubes, 800. Mu.L of Trizol reagent was added, and the tissue was sufficiently minced with tissue scissors until it became paste-like (no visible particles were apparent). To the above lysate, 320. Mu.L of RNase-free pure water was added, and the mixture was mixed upside down, left standing at room temperature for 5 minutes, followed by centrifugation at 12,000rpm for 15 minutes at 4 ℃. The processing of the plasma samples is the same except for the shearing.

2) The centrifuge tube was removed, at which time the solution separated into an upper RNA-containing aqueous phase and a lower precipitated phase of Han protein and organelle debris, and the upper aqueous phase was carefully transferred to a new EP tube.

3) Equal volumes of isopropanol were added, mixed upside down and allowed to stand for 10 minutes.

4) Centrifuge at 12000rpm for 10 min at 4℃to give a white precipitate, and carefully discard the supernatant.

5) Add 75% ethanol 1mL to EP tube and mix the precipitate by inversion

6) Centrifuge at 12000rpm for 5 minutes at 4 ℃, carefully discard the supernatant and air dry at room temperature.

7) An appropriate amount of RNase-free pure water was added to the EP tube to dissolve the precipitate, and the RNA precipitate was sufficiently dissolved by vortexing.

8) The concentration and purity of RNA were measured. 1. Mu.L of each RNA sample was taken after equilibration with RNase-free pure water, and the concentration of RNA was measured on a micro-spectrophotometer and recorded, followed by storing the RNA in a-80℃refrigerator for use.

(3) RNA reverse transcription

And (3) carrying out reverse transcription on the RNA obtained by extraction by using a reverse transcription kit to obtain cDNA, and storing the cDNA in a refrigerator at the temperature of-20 ℃ for later use.

(4) Real-time quantitative polymerase chain reaction (qRT-PCR)

1) Preparation of the reaction System

2) Sample addition sequence: mu.L of the mixture was added to each well of the PCR plate, followed by 2. Mu.L of the corresponding cDNA template in each well. Carefully stick the seal plate membrane and briefly mix by centrifugation.

3) Parameter setting of a PCR instrument:

4) Melting curve analysis: the expression of circRNA was normalized using GAPDH as an internal control, using 2- ^△△Ct The relative quantification of hsa_circ_0001789 expression was assessed by the method.

5) Statistical analysis: statistical analysis was performed using SPSS software 22.0. The chi-square test and student t test were used to analyze group-to-group differences. The subject operating profile (ROC) was used to compare the diagnostic value of hsa_circ_0001789 to conventional tumor markers. The threshold value of hsa_circ_0001789 was analyzed by Sigma Plot 12.3 software. P <0.05 is considered statistically significant.

3. Advantageous effects

3.1 significant downregulation of hsa_circ_0001789 expression in gastric cancer compared to its paracancerous tissues

The expression level of hsa_circ_0001789 in stomach cancer tissues and paracancerous tissues was examined by qRT-PCR. The results indicate that hsa_circ_0001789 expression was significantly down-regulated in gastric cancer tissue compared to non-cancerous tissue (P <0.001, fig. 1).

3.2 plasma from gastric cancer patients hsa_circ_0001789 expression was significantly down-regulated compared to healthy subjects

The expression level of hsa_circ_0001789 in plasma samples of gastric cancer patients and normal subjects was also detected by qRT-PCR, and the results show that the expression level of hsa_circ_0001789 in plasma of gastric cancer patients is significantly lower than that of healthy control group (P <0.001, FIG. 2).

3.3 diagnostic value of differentially expressed hsa_circ_0001789 for gastric cancer

The area under the subject's working characteristics curve (AUC) was analyzed and calculated using ROC curves to determine the potential diagnostic value of hsa_circ_0001789 in gastric cancer. The results show that hsa_circ_0001789 can identify gastric and paracancerous tissues with AUC of 0.82, cut-off of 9.5, and specificity and sensitivity of 0.84 and 0.51, respectively (fig. 3).

3.4hsa_circ_0001789 expression level for predicting prognosis of gastric cancer

Chi-square test analyzes the relationship between hsa_circ_0001789 expression level and clinical pathological parameters of gastric cancer patients. As shown in table 1, hsa_circ_0001789 low expression was associated with tumor invasion (P < 0.001), differentiation (P < 0.001), lymphatic metastasis (p=0.014), TNM staging (P < 0.001) and distant metastasis (p=0.028). These factors often indicate a relatively high degree of malignancy in the tumor and a poor prognosis for the patient. This suggests that a low expression of hsa_circ_0001789 may suggest that the patient is ill-prognosticated.

3.5hsa_circ_0001789 value in predicting malignant biological characteristics of gastric cancer

As shown in Table 1, the increased expression level of hsa_circ_0001789 is closely related to the malignant characteristics of GC, such as tumor invasion, differentiation, TNM staging, distant metastasis, lymphatic metastasis, etc. On this basis, we further compared the predictive value of hesa_circ_0001789 with traditional tumor markers, such as CEA, CA199, CA724, and CA 125. The results show that for the predicted invasive phase (Tis & T1-T3, or T4), the AUC of hsa_circ_0001789 reached the second highest 0.786 (95% Confidence Interval (CI), 0.629-0.942, p=0.013). Meanwhile, the threshold, sensitivity and specificity were 10.170, 76.1% and 85.7%, respectively (fig. 4A). For the phases (N0 or N1-N3) where lymphatic metastasis was predicted, AUC for hsa_circ_0001789 reached 0.603 (95% ci,0.384-0.821, p=0.344), with thresholds, sensitivity and specificity of 9.885, 82.9% and 50%, respectively (fig. 4B). For the phases (M0 or M1) predicting distal metastasis, the AUC of hsa_circ_0001789 was 0.722 (95% CI,0.602-0.843, P=0.040; FIG. 4C), the thresholds, sensitivities and specificities were 10.655, 100% and 54.3%, respectively. In addition, the AUC of the predictive TNM stage (0 & i & ii, or III & IV; fig. 4D) was 0.786 (95% ci,0.629-0.942, p=0.013), with cut-off, sensitivity and specificity of 10.170, 76.1% and 85.7%, respectively.

Table 1.Hsa_circ_0001789 expresses a correlation with clinical pathological factors of gastric cancer patients.

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Claims

Use of hsa_circ_0001789 as a diagnostic and/or prognostic marker for gastric cancer.
2. Use of hsa_circ_0003045 as a diagnostic and/or prognostic marker for gastric cancer according to claim 1, including but not limited to any of the following:

(1) Determining whether the patient is a gastric cancer patient by detecting the expression level of hsa_circ_0001789 in a patient detection sample;

(2) Detecting the expression content of hsa_circ_0001789 in a gastric cancer patient detection sample, so as to evaluate the prognosis condition of the patient;

(3) The application of hsa_circ_0001789 detection reagent in preparing gastric cancer diagnosis and/or prognosis detection reagent.
3. The use of hsa_circ_0001789 as a diagnostic and/or prognostic marker for gastric cancer according to claim 2, wherein the diagnostic marker test sample is a clinical biological sample of a patient, including but not limited to one or more of serum, plasma, whole blood, secretions, body fluids, tissues, organs, paraffin sections and the like;

preferably, the test sample is a tumor focus tissue sample and plasma of a patient.
4. The use of hsa_circ_0001789 as a diagnostic and/or prognostic marker for gastric cancer according to claim 2, wherein in the various aspects of use, a reduced hsa_circ_0001789 content in the test sample means that there is a greater likelihood that the patient is suffering from gastric cancer, or that gastric cancer has a poor prognosis.
5. A combination of reagents for the diagnosis and/or prognosis of gastric cancer, characterized in that the combination of reagents is used for detecting the expression level of hsa_circ_0001789 in a sample.
6. The combination of reagents for the diagnosis and/or prognosis of gastric cancer according to claim 5, wherein the type of said combination of reagents is determined according to the method of detection of the circRNA, such as PCR method, which is feasible.
7. The combination of reagents for the diagnosis and/or prognosis of gastric cancer according to claim 6, wherein the diagnostic kit suitable for the detection of hsa_circ_0001789 by PCR method comprises at least the following diagnostic reagents:

(1) A primer;

(2) A sample tissue lysing agent;

(3) A PCR reaction system; preferably, the primer comprises a to-be-detected circRNA primer and also comprises a primer of an internal reference marker; in a specific example, the circRNA primer sequence is as follows:

Forward：5’-CTGCTCACGCAAGCTGAA-3’(SEQ ID NO.1)

Reverse：5’-TCTGTGAAGTCAACCTTTCCA-3’(SEQ ID NO.2)

the primer sequences of the internal reference markers are as follows:

Forward：5’-TCGACAGTCAGCCGCATCTTCTTT-3’(SEQ ID NO.3)

Reverse：5’-ACCAAATCCGTTGACTCCGACCTT-3’(SEQ ID NO.4)。
8. the combination of reagents for the diagnosis and/or prognosis of gastric cancer according to claim 6, wherein the sample tissue lysis reagent comprises, but is not limited to, TRIZOL reagent, isopropanol, ethanol, buffer reagent.
9. A kit for diagnosing and/or prognosis of gastric cancer, comprising the combination of reagents according to any one of claims 5 to 8.
10. The method for judging the prognosis of the gastric cancer patient is characterized by comprising the steps of detecting the content of hsa_circ_0001789 in a patient sample;

in the above determination method, the content of hsa_circ_0001789 can be detected by the kit of claim 9, and if the content of hsa_circ_0001789 in the patient sample is lower than that in the control group, it indicates that there is a greater risk of metastasis or exacerbation of gastric cancer development in the patient.