CN110894528A - CircRNA marker for diagnosing gastric poorly differentiated adenocarcinoma and application thereof - Google Patents
CircRNA marker for diagnosing gastric poorly differentiated adenocarcinoma and application thereof Download PDFInfo
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Abstract
A set of circRNA markers for diagnosing the gastric poorly differentiated adenocarcinoma comprises five circRNAs, namely hsa _ circ _0102434 shown in SEQ ID NO:1, hsa _ circ _0005505 shown in SEQ ID NO:2, hsa _ circ _0077837 shown in SEQ ID NO:3, hsa _ circ _0072309 shown in SEQ ID NO:4 and hsa _ circ _0070033 shown in SEQ ID NO: 5.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a group of circRNA markers for diagnosing gastric poorly differentiated adenocarcinoma and application thereof.
Background
Generally, adenocarcinomas with a small number of glandular structures in the histological range are diagnosed as poorly differentiated adenocarcinomas (Poorlydifferenced adenocaricinomas). Among Gastric cancers (Gastric cancer), poorly differentiated adenocarcinomas occur mainly in women and young patients, including signet ring cell carcinoma, poorly adherent adenocarcinomas, and the like. Current standard treatments for poorly differentiated adenocarcinoma of the stomach include surgical resection, chemotherapy, and targeted therapies. The gastric poorly differentiated adenocarcinoma has high malignancy and poor prognosis. Although the 5-year survival rate of patients with gastric poorly differentiated adenocarcinomas is still low with progressive improvement in the level of diagnosis. Therefore, the identification of novel biomarkers and targeted treatment sites has great significance for gastric poorly differentiated adenocarcinomas.
Circular RNA is a special class of non-coding RNA formed by the special splicing machinery of the reverse splicing reaction (back-splicing), having a closed loop structure, and abundantly present in eukaryotic transcriptomes. Compared with linear non-coding RNA, the circRNA molecule has a closed ring structure, is not influenced by RNA exonuclease and has more stable expression. Research shows that the circRNA can regulate the expression of genes on multiple layers of transcription, post-transcription and the like, plays an important role in regulating and controlling multiple physiological and pathological processes of organisms, recent research also discovers that the circRNA participates in the generation and development of tumors, and currently, some circRNA markers with diagnostic potential are discovered in gastric cancer successively, but the circRNA markers with diagnostic potential in gastric poorly differentiated adenocarcinoma are not reported yet.
The invention content is as follows:
the technical problem to be solved is as follows: the invention provides a circRNA marker for diagnosing gastric poorly differentiated adenocarcinoma and application thereof, and provides a reference basis for diagnosis of gastric cancer. The circRNA biomarker provided by the invention has high sensitivity and specificity on gastric cancer, and can be used as a novel biomarker for detecting gastric cancer.
The invention can be realized by the following technical scheme: a set of circRNA markers for diagnosing gastric poorly differentiated adenocarcinoma comprises five circRNAs shown in SEQ ID NO:1, hsa _ circ _0102434, SEQ ID NO:2, hsa _ circ _0005505, SEQ ID NO:3, hsa _ circ _0077837, SEQ ID NO:4, and hsa _ circ _0070033, SEQ ID NO: 5.
Preferably, the method comprises the following steps: the primer combination comprises: specific primers for hsa _ circ _0102434, specific primers for hsa _ circ _0005505, specific primers for hsa _ circ _0077837, specific primers for hsa _ circ _0072309, specific primers for hsa _ circ _ 0070033.
Preferably, the method comprises the following steps: the specific primers aiming at hsa _ circ _0102434 comprise an upstream primer and a downstream primer aiming at
The specific primers for hsa _ circ _0005505 included an upstream primer and a downstream primer, the specific primers for hsa _ circ _0077837 included an upstream primer and a downstream primer, the specific primers for hsa _ circ _0072309 included an upstream primer and a downstream primer, and the specific primers for hsa _ circ _0070033 included an upstream primer and a downstream primer.
The marker is applied to the preparation of a kit for diagnosing the gastric poorly differentiated adenocarcinoma.
The primer combination is applied to the preparation of a kit for diagnosing the gastric poorly differentiated adenocarcinoma.
The primer combination is applied to screening or preparing gastric poorly differentiated adenocarcinoma targeted drugs.
A diagnostic kit for gastric poorly differentiated adenocarcinoma comprises the primer combination.
The method adopts real-time fluorescence PCR (RT-PCR) to detect a group of circRNAs and provides reference value for discovery and diagnosis of gastric poorly differentiated adenocarcinoma. (2) The method has strong specificity, high sensitivity and stable result, and the provided circRNA biomarker for diagnosing the gastric poorly differentiated adenocarcinoma can be used for diagnosing the gastric poorly differentiated adenocarcinoma, and has wide clinical application prospect and great commercial value.
Description of the drawings:
FIG. 1 is a graph of the expression levels of hsa _ circ _0102434 and hsa _ circ _0005505 in normal stomach tissue versus high, medium and poorly differentiated adenocarcinomas
FIG. 2 is a graph showing the expression levels of hsa _ circ _0077837, hsa _ circ _0072309, and hsa _ circ _0070033 in normal stomach tissue control high-medium poorly differentiated adenocarcinomas
FIG. 3 is a ROC plot of hsa _ circ _0102434 for diagnosis of poorly differentiated adenocarcinoma of the stomach.
FIG. 4 is a ROC plot of hsa _ circ _0005505 for diagnosis of poorly differentiated adenocarcinoma of the stomach.
FIG. 5 is a ROC plot of hsa _ circ _0077837 for diagnosis of poorly differentiated adenocarcinoma of the stomach.
FIG. 6 is a ROC plot of hsa _ circ _0072309 for diagnosis of poorly differentiated adenocarcinoma of the stomach.
FIG. 7 is a ROC plot of hsa _ circ _0070033 for diagnosis of poorly differentiated adenocarcinoma of the stomach.
Detailed Description
The embodiments of the present invention will be described below with reference to specific embodiments.
Example 1
1. Clinical specimen collection
Case population and clinical specimens: 40 cases of clinical information from patients with radical gastric cancer therapy performed in the hospital of people in Zhejiang province from 2017 to 2018 at 12 months, corresponding surgical specimens of gastric cancer and matched paracancer surgical specimens, all tissue specimens were taken out from the body and washed with PBS to remove blood, then stored in a liquid nitrogen tank, and then stored in a biological specimen bank at-80 ℃ until use, paracancer tissues were taken from 5cm away from the edge of cancer tissues and had no obvious tumor cells,
2. total RNA was extracted and quantified, and total tissue RNA was extracted from gastric cancer and para-carcinoma tissues using TRIzol Reagent kit. The total RNA extracted was then quantified using a ultramicro UV-Vis spectrophotometer (NanoDrop ND 2000).
The 5 groups hsa _ circ _0102434, hsa _ circ _0005505, hsa _ circ _0077837, hsa _ circ _0072309 and hsa _ circ _0070033 provided by the invention are screened from the detection result of the circRNA chip of the gastric cancer patient, and the primer sequences are shown as follows:
3. reverse transcription:
cDNA was synthesized using the PrimeScript RT Reagent Kit with gDNA Eraser (Takara, RR047A, Japan) Kit. The reverse transcription process Primer is a Random Primer (Random Primer) in the kit.
3.1 genomic DNA Elimination reaction
Preparing a master mix of other components than the RNA sample in a volume sufficient to accommodate 2 times the number of reactions, placing an appropriate amount of the master mix in a microtube, and then adding the RNA sample, specific reagents are as shown in Table 2:
composition (I) | Dosage of |
5X gDNA Eraser Buffer | 2.0μL |
gDNAEraser | 1.0μL |
total RNA | *1 |
RNase Free dH2O | |
Total | 10.0μL |
The temperature is as follows: storing at 42 deg.C for 2 min (5 min at room temperature) at 4 deg.C
3.2 reverse transcription Process:
preparation of reverse transcription reaction solution on ice surface
A sufficient reaction amount of the master mix solution, which should contain all the components except the genomic DNA elimination reaction solution, was prepared, and 10. mu.L of the mixed mother solution was added from step 1, and then gently mixed to immediately perform the reverse transcription reaction, and specific reagents were as shown in Table 3:
15min at 37 ℃ for 5 seconds at 85 DEG C
3.3 real-time fluorescent quantitative PCR:
Real-Time polymerase chain reaction (RT-qPCR) was achieved using the CFX96 Real Time PCR system using TB Green Premix Ex Taq (Takara, RR420A, Japan). The amplification has _ circ _0006401 used a diverging Primer (Divergent Primer) and the amplification of internal control GAPDH used a more general converging Primer (Convergent Primer). Primers for has _ circ _0006401 and GAPDH were synthesized by the manufacturer (Sangon Biotech, Shanghai, China). The reaction system is carried out according to the following reaction system, wherein the reaction system is 25 mu l/hole): TB GreenPremix Ex Taq 12.5. mu.l, cDNA 2.0. mu.l, upstream and downstream primers (10. mu.M) 0.5. mu.l each, and RNase Free water 9.5. mu.l. Reaction conditions are as follows: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 30s, 55 cycles, and finally adding a melting curve to detect the specificity of the PCR amplification product. Setting 3 duplicate wells for each well, taking the average of the Ct values of the three duplicate wells as the Ct value of the sample, taking GAPDH as an internal reference, and the primer sequences of has _ circ _0006401 are as follows: 5'-gctctcactgaaacagaaatg-3' (sense) and 5'-ggcttcacagatggctgatt-3' (antisense). The primer sequences for GAPDH are as follows: 5'-CTCTCTGCTCCTCCTGTTCGAC-3' (sense) and 5'-TGAGCGATGTGGCTCGGCT-3' (antisense). And analyzing the RT-qPCR data by using a delta Ct method and a 2-delta Ct method, wherein the delta Ct value is the expression quantity of the sample, and the 2-delta Ct value is the relative expression quantity of the sample. The experimental results are expressed as mean ± standard deviation.
The specific reaction system is shown in Table 4:
composition (I) | Dosage of |
SYBR Premix Ex Taq II | 12.5μL |
PCR Forward Primer(10μM) | 1.0μL |
PCR Reverse Primer(10μM) | 1.0μL |
RT reaction solution(CDNA) | 2.0μL |
dH2O(sterillized distilled water) | 8.5μL |
Tatal | 25.0μL |
4. Analysis of circRNA expression level
Differences in circRNA expression in cancer tissues versus normal tissues were analyzed using unpaired sample t test, with bilateral P < 0.05 considered statistically different.
5. circRNA diagnostic value analysis
ROC curves were plotted and the area under the curve, AUC, was calculated to evaluate the sensitivity and specificity of these two circrnas for cancer tissue diagnosis, respectively, and for cancer tissue diagnosis in combination. AUC < 0.5, indicating no diagnosis; when AUC is 0.5-0.7, the diagnosis accuracy is low; when AUC is 0.7-0.9, the diagnosis accuracy is moderate; when AUC > 0.9, the diagnosis accuracy is high.
5. Results
5.1 expression levels of hsa _ circ _0102434 and hsa _ circ _0005505 in normal stomach tissue control poorly differentiated adenocarcinomas in 40 normal stomach tissue samples and 40 normal stomach tissue control samples, the expression levels of hsa _ circ _0102434 and hsa _ circ _0005505 in poorly differentiated adenocarcinomas were significantly up-regulated, the expression levels of hsa _ circ _0077837, hsa _ circ _0072309 and hsa _ circ _0070033 in poorly differentiated adenocarcinomas were significantly down-regulated, with P values < 0.0001, respectively (FIG. 1);
5.2 diagnostic value of hsa _ circ _0102434, hsa _ circ _0005505, hsa _ circ _0077837, hsa _ circ _0072309, hsa _ circ _0070033 alone on poorly differentiated adenocarcinomas of the stomach
The analysis of the ROC curve is as follows:
as shown in fig. 3: hsa _ circ _0102434 has an area under the curve of 0.743 (95% CI-0.629-0.875), sensitivity of 57.9%, specificity of 100%;
as shown in fig. 4: hsa _ circ _0005505 had an area under the curve of 0.763 (95% CI-0.645-0.880), a sensitivity of 57.9%, and a specificity of 94.7%;
as shown in fig. 5: hsa _ circ _0077837 had an area under the curve of 0.875 (95% CI: 0.902-1.000), sensitivity of 89.5%, specificity of 94.7%;
as shown in fig. 6: hsa _ circ _0072309 had an area under the curve of 0.858 (95% CI: 0.731-1.000), a sensitivity of 78.9%, and a specificity of 89.5%;
as shown in fig. 7: hsa _ circ _0070033 had an area under the curve of 0.761 (95% CI-0.590-0.932), a sensitivity of 100.0%, and a specificity of 52.9%;
in conclusion, hsa _ circ _0102434, hsa _ circ _0005505, hsa _ circ _0077837, hsa _ circ _0072309 and hsa _ circ _0070033 all have certain diagnostic value for diagnosing the gastric poorly differentiated adenocarcinoma, wherein the area under the AUC curve of hsa _ circ _0077837 diagnosis is the largest, and the sensitivity and specificity are 89.5% and 94.7% respectively, so that the specific biomarker is reliable and is a specific biomarker for diagnosing the gastric poorly differentiated adenocarcinoma.
It should be understood that after reading the above description of the present invention, those skilled in the art can make various modifications and embellishments to the present invention, however, all of them are related to the detection of the SNP site described in the present invention, or are applied to various forms of the site, and these modifications and embellishments are within the scope of the present invention.
Claims (5)
1. A set of circRNA markers for diagnosing gastric poorly differentiated adenocarcinoma, characterized in that the markers include five circRNAs shown in SEQ ID NO:1, hsa _ circ _0102434, SEQ ID NO:2, hsa _ circ _0005505, SEQ ID NO:3, hsa _ circ _0077837, SEQ ID NO:4, hsa _ circ _0072309, and SEQ ID NO:5, hsa _ circ _ 0070033.
2. The primer combination for a set of circRNA markers according to claim 1, characterized in that the primer combination comprises: specific primers for hsa _ circ _0102434, specific primers for hsa _ circ _0005505, specific primers for hsa _ circ _0077837, specific primers for hsa _ circ _0072309, specific primers for hsa _ circ _ 0070033.
3. The primer combination of a set of circRNA markers in claim 2, wherein the specific primers for hsa _ circ _0102434 comprise an upstream primer and a downstream primer, the specific primers for hsa _ circ _0005505 comprise an upstream primer and a downstream primer, the specific primers for hsa _ circ _0077837 comprise an upstream primer and a downstream primer, the specific primers for hsa _ circ _0072309 comprise an upstream primer and a downstream primer, and the specific primers for hsa _ circ _0070033 comprise an upstream primer and a downstream primer.
4. Use of the primer combination of claim 2 or 3 for the preparation of a kit for diagnosing gastric poorly differentiated adenocarcinoma.
5. A diagnostic kit for poorly differentiated adenocarcinoma of the stomach, comprising the primer combination according to claim 3.
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