CN108796074B - Application of reagent for detecting circular RNA circRNF13 in preparation of tumor auxiliary diagnosis preparation and kit - Google Patents
Application of reagent for detecting circular RNA circRNF13 in preparation of tumor auxiliary diagnosis preparation and kit Download PDFInfo
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Abstract
The invention discloses application of a reagent for detecting circular RNA circRNF13 in preparing a tumor auxiliary diagnosis preparation and a kit. In particular to a preparation for assisting in diagnosing tumors by a real-time fluorescence quantitative analysis method. Research proves that the circRNF13 is up-regulated in serum of patients with nasopharyngeal carcinoma, oral cancer, thyroid cancer, ovarian cancer and lung cancer. Therefore, the expression condition of the circRNF13 is used for serological auxiliary diagnosis of tumors, and has profound clinical significance and important popularization and application prospects.
Description
Technical Field
The invention belongs to the technical field of tumor molecular biology, and particularly relates to application of a reagent for detecting circular RNA circRNF13 in preparation of a tumor auxiliary diagnosis preparation and a corresponding kit.
Background
Malignant tumors seriously harm human health, and the incidence rate of Chinese malignant tumors is on the rise in recent years. According to statistics of Chinese cancer statistical center, the number of new malignant tumors is about 380.4 ten thousand, wherein 211.4 ten thousand of male patients and 169.0 ten thousand of female patients can be diagnosed as cancer about more than 1 ten thousand times per day, and about 7 times per minute. The incidence rate of the malignant tumor is 278.07/10 ten thousand, wherein the incidence rate of the malignant tumor in men is 301.67/10 ten thousand, and the incidence rate of the malignant tumor in women is 253.29/10 ten thousand. The average rate of the incidence rate of the malignant tumor is 190.63/10 ten thousand (the population standard rate is based on the standard population structure of China in 2000), the world standard rate of the incidence rate of the malignant tumor is 186.53/10 ten thousand (the population standard rate is based on the Segi's world standard population structure), and the cumulative incidence rate of 0-74 years is about 21.58%. The mortality rate of malignant tumor is about 167.89/10 ten thousand, wherein the medium rate is about 106.98/10 ten thousand, the world standard rate is about 106.09/10 ten thousand, and the cumulative mortality rate of 0-74 years is about 12.00%. According to the statistics of the American Cancer Society (ACS), the number of new diseases of American malignant tumors is estimated to be about 173.5 ten thousands, and 4700 new diseases are all found every day. However, the incidence of malignant tumors in the united states began to steadily decline in the last 90 s of the century, with the mortality rate of malignant tumors also declining at a rate of 1.5% per year. The significant decline in the incidence of malignant tumors in the united states may be attributed to the control of smoking, the promotion of multi-level cancer screening, and the widespread use of novel cancer therapies.
It is thus clear that the reduction of the causative factors of malignant tumors, the effective early diagnosis, and the search for novel cancer therapies play a significant role in the control of cancer. Therefore, in the medical research of China, the research is dedicated to searching for novel tumor markers in order to provide a new way for early diagnosis of malignant tumors and for early, convenient and accurate diagnosis of diseases so as to be beneficial to treatment.
Circular RNA (circRNA) is a new class of RNA molecules with a configuration that is different from conventional linear RNA (containing 5 'and 3' ends), and circular RNA molecules are in a closed circular structure (i.e. 5 'and 3' ends end to end), and circular RNA is more stable than linear RNA because there are no free 5 'and 3' ends. At first, circRNA is considered as a byproduct of missplicing and is not widely concerned, in recent years, the wide application of RNA-Sequencing based on a new generation Sequencing technology promotes the rapid development of many fields of RNA, and a large amount of new circRNA is found, so that the circRNA is pushed to the stage center of malignant tumor genomics research.
As an emerging RNA, circRNA, its mechanism of action and function is attracting increasing research. As more and more circrnas were identified, some were found to be expressed at levels much higher than their associated linear genes. The circRNAs form a circular structure by covalent bonds, and most of the circRNAs are reverse splicing products, are widely expressed in organisms, sometimes have the expression level 10 times that of linear RNAs, and are mostly localized in cytoplasm. The CircRNA can be used as a molecular sponge of miRNA to adsorb miRNA, bind protein, or be used as a regulatory factor of gene transcription and protein translation, and the like to play important biological functions, is likely to become a new biomarker and a clinical treatment target of malignant tumor, and provides a new idea for diagnosis and prognosis of tumor.
Therefore, more novel circrnas need to be screened and verified as biomarkers for cancer diagnosis and prognosis and their applications, and can be well protected in the patent field as soon as possible, and can significantly improve international competitiveness of our country in the technical field.
Disclosure of Invention
The invention screens out circRNA with high expression from RNA seq data, and verifies the expression conditions of the circRNA in various cancers in sequence, and finds out new circRNA which can be used as a serum molecular marker for diagnosing malignant tumors.
Therefore, the first purpose of the invention is to provide the application of a reagent for detecting circular RNA circRNF13 in the preparation of a tumor auxiliary diagnostic preparation, wherein the sequence of the circular RNA circRNF13 is shown as SEQ No. 1. Provides a new accurate, reliable, simple and convenient detection way for the auxiliary diagnosis of the tumor.
Compared with tissue detection, the serum is used as a detection sample, and has the advantages of convenient sample acquisition mode and operation, low cost, high accuracy and ideal diagnosis for tumors. Therefore, the applicant uses serum as a sample in the research process, and searches and verifies the molecules in the serum relevant to tumor diagnosis and the relationship between the molecules and the tumor through a large number of tests. The applicant firstly discovers that the circular RNA circRNF13 in serum has positive correlation with various types of tumors, and the circular RNA circRNF13 can be conveniently used for assisting or primarily diagnosing the possibility of having the tumors by detecting the content of the circular RNA circRNF13 in the serum.
According to the invention, an experiment method of SYBR-qPCR or Taqman probe q-PCR is adopted to successively verify the expression condition of circRNF13 in nasopharyngeal carcinoma, oral cancer, thyroid cancer, ovarian cancer and lung cancer.
The invention has sufficient number of samples collected for verifying the expression condition of the circRNF13, has statistical significance, and has reasonable sample source, strict screening standard, strict test process and real and reliable result.
The tumors selected in the verification process of the invention basically comprise representative multiple tumor types appearing from human head and neck, and internal organs. And test results show that the expression of the circRNF13 in the serum of patients with nasopharyngeal carcinoma, oral cancer, thyroid cancer, ovarian cancer and lung cancer is obviously up-regulated, the consistency of the expression trend in various tumors is shown, and the circRNF13 molecule can be used as a molecular marker for tumor diagnosis.
In the application, the reagent for detecting the circular RNA circRNF13 comprises a real-time fluorescent quantitative detection reagent.
Due to the particularity of the circular RNA structure, in order to ensure the specificity of the primer and the accuracy of qPCR, the applicant strictly follows the circular RNA primer design principle and designs a primer which can accurately amplify the expression of the circular RNA circRNF13 for real-time fluorescent quantitative detection:
an upstream primer: 5'-GTCCAGGATAGACATAGAGC-3'
A downstream primer: 5'-GTGTAGACTTGTGTGGCTGA-3' are provided.
The second purpose of the invention is to provide a kit for tumor auxiliary diagnosis; provides a new accurate and reliable detection product for the tumor auxiliary diagnosis.
The kit contains a reagent capable of detecting circular RNA circRNF13 in serum.
Further, a primer capable of amplifying circular RNA circRNF 13; the sequence of the circular RNA circRNF13 is shown in SEQ No. 1.
Further, the primer capable of amplifying the circular RNA circRNF13 is preferably the following primer:
an upstream primer: 5'-GTCCAGGATAGACATAGAGC-3'
A downstream primer: 5'-GTGTAGACTTGTGTGGCTGA-3' are provided.
However, the primer capable of amplifying the circular RNA circRNF13 according to the present invention is not limited to the primers provided above.
Because human serum has no reference gene, pGL3 plasmid is preferably added as a reference to reduce errors in the RNA extraction process, such as differences in RNA adsorption efficiency and elution efficiency, and provide more reliable experimental data for research. Because pGL3 plasmid is added quantitatively, the copy number of the target gene can be calculated from the CT value obtained by q-PCR reaction, and absolute quantification can be achieved. The addition of the external reference gene reduces the system error in the experiment, thereby providing data information with higher reliability for research.
Therefore, the temperature of the molten metal is controlled,
further, the kit of the present invention further comprises: internal control pGL3 primer:
an upstream primer: 5'-TCCATCTTGCTCCAACACCC-3'
A downstream primer: 5'-TCGTCTTTCCGTGCTCCAAA-3' are provided.
However, the reference primer of the present invention is not limited to the reference pGL3 primer provided above.
The Taqman probe has high specificity, high sensitivity and good specificity. The SYBR method needs to carry out PCR reaction in different EP tubes, while the Taqman probe method can realize the simultaneous detection of a plurality of genes in one EP tube, thereby reducing the experimental error. The Taqman probe-qPCR experimental method is rigorous, has high reliability and repeatability, can simultaneously detect various samples, has good experimental repeatability, obtains more reliable data, and provides a solid basis for the evaluation of clinical application value.
Therefore, the temperature of the molten metal is controlled,
further, the kit of the present invention preferably further comprises: the Taqman probe matched with the primer for real-time fluorescent quantitative detection of circular RNA circRNF13 expression comprises the following components: 5 '-Cy 5-AGGATAGACATAGAGCTAGAAGAAACAGACTTCGT-BHQ 2-3'.
Further, the kit of the present invention preferably further comprises: a Taqman probe matched with an internal reference pGL3 primer: 5 '-HEX-ACGCAGGTGTCGCAGGTCTTCC-BHQ 1-3'.
However, the Taqman probe used in the present invention is not limited to the above-mentioned kit probe.
Further, the kit of the present invention further comprises: a serum total RNA extraction reagent, an RNA reverse transcription PCR reaction reagent and a real-time fluorescent quantitative detection reagent.
The invention extracts RNA from serum of various tumor patients and normal human serum samples, performs reverse transcription, detects the expression of circRNF13 by a real-time fluorescence quantitative method, and shows that the expression of circRNF13 is up-regulated in the serum of various tumor patients. The circular RNA molecule circRNF13 exists in serum for the first time, and has a uniform positive correlation with various types of tumors, so that the circRNF13 can be used as a molecular marker for tumor auxiliary diagnosis. The invention provides a powerful molecular biology tool for the auxiliary diagnosis of tumors, and has profound clinical significance and important popularization and application prospects.
Drawings
FIG. 1 is a circRNF13 map;
FIG. 2 is a reference plasmid pGL3 q-PCR standard curve;
FIG. 3 shows the expression of circRNF13 in the serum of nasopharyngeal carcinoma patients as detected by SYBR-qPCR;
SYBR-qPCR is used for detecting the expression level of circRNF13 in the serum of a nasopharyngeal carcinoma patient, wherein compared with a normal control group, the expression level of circRNF13 in the serum of the nasopharyngeal carcinoma patient is up-regulated, and P is less than 0.001; n represents a normal control group, T represents a nasopharyngeal carcinoma group, and N represents the number of samples. P <0.05, P <0.01, P < 0.001.
FIG. 4 is a graph showing the Taqman-qPCR assay for the expression of circRNF13 in the serum of nasopharyngeal carcinoma patients;
detecting the expression level of the circRNF13 in the serum of a nasopharyngeal carcinoma patient by Taqman-qPCR (quantitative polymerase chain reaction), wherein compared with a normal control group, the circRNF13 is up-regulated in the serum of the nasopharyngeal carcinoma patient, and P is less than 0.001; n represents a normal control group, T represents a nasopharyngeal carcinoma group, and N represents the number of samples. P <0.05, P <0.01, P < 0.001.
FIG. 5 is a ROC curve for the nasopharyngeal carcinoma group circRNF 13.
FIG. 6 is a Taqman-qPCR assay for the expression of circRNF13 in the serum of oral cancer patients;
Taqman-qPCR detects the expression level of circ RNF13 in the serum of an oral cancer patient, wherein compared with a normal control group, the expression of circ RNF13 in the serum of the oral cancer patient is up-regulated, and P is less than 0.001; n represents a normal control group, T represents an oral cancer group, and N represents the number of samples. P <0.05, P <0.01, P < 0.001.
FIG. 7 is a ROC curve for the oral cancer group circRNF 13.
FIG. 8 is a Taqman-qPCR assay for circRNF13 expression in serum from thyroid cancer patients;
detecting the expression level of circRNF13 in the serum of a thyroid cancer patient by Taqman-qPCR (quantitative polymerase chain reaction), wherein the expression of circRNF13 in the serum of the thyroid cancer patient is up-regulated compared with a normal control group, and P is less than 0.001; n represents a normal control group, T represents a thyroid cancer group, and N represents the number of samples; p <0.05, P <0.01, P < 0.001.
FIG. 9 is Taqman-qPCR detection of circRNF13 expression in serum of female thyroid cancer patients; the expression level of circRNF13 in the serum of female thyroid cancer patients is detected by Taqman-qPCR, and compared with a normal control group, the expression level of circRNF13 in the serum of female thyroid cancer patients is up-regulated, and P is less than 0.001. N represents a normal control group, T represents a thyroid cancer group, and N represents the number of samples. P <0.05, P <0.01, P < 0.001.
FIG. 10 is a ROC curve for the thyroid cancer group circRNF 13.
FIG. 11 is a ROC curve for the female thyroid cancer group circRNF 13.
FIG. 12 is a Taqman-qPCR assay for the expression level of circRNF13 in ovarian cancer patient serum, wherein circRNF13 is upregulated compared to normal controls, with P < 0.001. N represents a normal control group, T represents an ovarian cancer group, and N represents the number of samples. P <0.05, P <0.01, P < 0.001.
FIG. 13 is a ROC curve for circRNF13 in the ovarian carcinoma group.
FIG. 14 is a Taqman-qPCR assay of the expression level of circ RNF13 in serum of lung cancer patients, wherein the expression level of circ RNF13 in serum of lung cancer patients is up-regulated compared to a normal control group, and P is less than 0.001. N represents a normal control group, T represents a lung cancer group, and N represents the number of samples. P <0.05, P <0.01, P < 0.001.
FIG. 15 is a ROC curve for circRNF13 in lung cancer group.
Detailed Description
The following detailed description is intended to further illustrate the invention and is not to be construed as limiting the invention.
The normal control group and the tumor group serum samples adopted in the invention are respectively from the Hunan Yabi hospital examination center and the Hunan province tumor hospital examination center of the university in the south China. 121 cases are collected in the normal control group, and the tumor diseases, infectious diseases, serious immune diseases and other serious diseases are eliminated; tumor group serum samples included: 100 nasopharyngeal carcinoma patient serum samples, 55 oral cancer patient serum samples, 57 thyroid cancer patient serum samples, 36 ovarian cancer patient serum samples, 45 lung cancer serum samples, and a blood sample collection time interval: 3 months in 2017 to 1 month in 2018. The collected blood samples gradually complete clinical data, including patient name, sex, age, hospitalization number, pathological type, pathological stage, identity card number, treatment condition, etc. All serum samples were informed by the patient or the person who collected the serum samples, and a blood sample bank with complete clinical data was gradually established.
1. Realtime PCR primer
The primers and probes used in the invention are designed through a special Primer design website Primer 3.0. The primer synthesis work entrusted the synthesis of the department of Engineers. To ensure primer specificity and qPCR accuracy, we strictly followed the following primer design principles:
the design principle of the probe is as follows:
the primer and the Taqman probe used by the invention have the following sequences:
(1) pGL3 primer
An upstream primer: 5'-TCCATCTTGCTCCAACACCC-3', respectively;
a downstream primer: 5'-TCGTCTTTCCGTGCTCCAAA-3', respectively;
taqman probe: 5 '-HEX-ACGCAGGTGTCGCAGGTCTTCC-BHQ 1-3';
(2) circRNF13 primer
An upstream primer: 5'-GTCCAGGATAGACATAGAGC-3'
A downstream primer: 5'-GTGTAGACTTGTGTGGCTGA-3'
Taqman probe: 5 '-Cy 5-AGGATAGACATAGAGCTAGAAGAAACAGACTTCGT-BHQ 2-3';
2 method
2.1 cancer patients and Normal persons peripheral blood sample Collection
The sample collection procedure was as follows:
(1) collecting peripheral blood samples by using an EDTA (ethylene diamine tetraacetic acid) anticoagulation blood collection tube, collecting 1-2 ml of fasting venous blood, and turning the blood collection tube upside down to mix the blood samples gently and uniformly so as to avoid hemolysis;
(2) centrifuging the whole blood sample at 4 ℃ for 10min at 1600g, extracting 500-;
(3) immediately placing the plasma specimen in a refrigerator at-80 ℃ for storage after extraction, and finishing the whole process after blood separation within 4 hours;
(4) filling in 'sample information record table', collecting the information of patient and normal person's name, sex and age, and the clinical data of patient's hospitalization number, pathological type, pathological stage and received treatment.
2.2 serum Total RNA extraction
The method adopts a miRNeasy Serum/plasmaikit kit, and comprises the following specific steps:
(1) 200ul plasma was placed in 2ml EP tubes;
(2) add 5 times volume of lysis buffer (i.e. 1000ul) to EP tube, vortex or mix well with pipette;
(3) the dissolved product in the EP tube is placed for 5min at room temperature (15-25 ℃);
(4) adding radix Ginseng PGL3, and mixing (1.6 x 10)8copies/ul)；
(5) Add equal sample volume of chloroform (i.e. 200ul), vortex or shake vigorously for 15 s;
(6) standing at room temperature (15-25 deg.C) for 2-3 min;
(7) centrifuging 12000g at 4 ℃ for 15min, adjusting the temperature to room temperature after using the centrifuge, and obtaining an upper colorless water phase of RNA; the middle white layer is DNA; the lower red organic phase is protein;
(8) sucking the upper water phase into a new 2ml EP tube (600ul), adding 1.5 times of 100% alcohol (900 ul), mixing well with a pipette, adding alcohol to form precipitate without influence;
(9) placing the column into a 2ml tube, sucking 700ul of the column, closing the cover, centrifuging at room temperature (15-25 deg.C) for 15s at more than or equal to 8000g (10000rpm), and discarding the waste liquid in the collection tube;
(10) repeating (9), passing the residual sample through the column, and discarding the waste liquid in the collection tube;
(11) adding 700ul Buffer RWT to the column, closing the cover, centrifuging to more than or equal to 8000g (10000rpm) for 15s, and discarding the waste liquid in the collection tube;
(12) adding 500ul Buffer RPE to the column, closing the cover, centrifuging for 15s at a speed of more than or equal to 8000g (10000rpm), and discarding the waste liquid in the collection tube;
(13) adding 500ul 80% ethanol into the column, slightly closing the cover, centrifuging to more than or equal to 8000g (10000rpm), washing the column for 2min, and discarding the collecting tube (carefully moving the column to avoid getting on the waste liquid of the collecting tube);
(14) placing the column in a new 2ml Tube, opening the lid, centrifuging at full speed for 5min, drying, discarding the collection Tube (to avoid damaging the lid, the Tube is placed at an interval such that the lid faces upwards and the rotor turns in the opposite direction);
(15) place the column in a fresh 1.5ml Tube, add 14ul of enzyme-free water to the center of the column membrane, gently close the column, centrifuge at full speed for 1min, and elute the RNA to about 12ul in a 1.5ml Tube.
We added the pGL3 plasmid as a reference, providing more reliable experimental data for the present invention.
The reference plasmid was added at a concentration of about 2X 10 pGL3 per ml of serum8Copies were calculated to be approximately 1ng plasmid per ml serum;
a10. mu.l reaction for Real time PCR was as follows:
the pGL3 plasmid standard curve was plotted as follows: 1ng plasmid is taken and added with enzyme-free water to be constant volume to 500 mul, and then 2 mul plasmid is taken and added with enzyme-free water to be constant volume to 1ml to be used as a 1 st tube; then gradually diluting in half, i.e.: 500ul of plasmid solution is taken from the 2 nd tube and put into the 2 nd tube, 500ul of non-enzyme water is added and mixed evenly; remove 500ul plasmid solution from the 2 nd tube and put into the third tube, add 500ul without enzyme water, mix; and so on. Finally, 1ul of each tube is taken as a template of the fluorescent quantitative PCR.
The Q-PCR results were as follows:
the plotted standard graph is shown in fig. 2.
2.3 RNA reverse transcription PCR reaction
The concentration of the extracted total RNA is determined, then RNA is quantified, and cDNA is synthesized from the 3' end according to the set reverse transcription program.
1. The following reagents were added to a sterile, enzyme-free tube in order:
2. mixing gently, centrifuging instantly, and incubating at 65 deg.C for 5 min;
3. adding the following reagents into the mixed solution, wherein the final volume is 20 ul;
4. gently mix and centrifuge instantaneously, operating according to the following procedure:
the cDNA (complementary DNA) obtained after completion of the reaction was stored at-20 ℃.
2.4 real-time fluorescent quantitative PCR
SYBR method Real-time fluorescent Quantitative PCR (Quantitative Real-time PCR, q-PCR) reaction system: iTaqTMUniversal
Green Supermix：
SYBR method real-time fluorescence quantitative PCR reaction steps:
after the reaction, the amplification curve and the melting curve of the real-time fluorescent quantitative PCR were confirmed, and the expression intensity of each gene was normalized based on the CT value (threshold cycle values) and the internal reference gene (pGL3), and the P value was calculated by unpaired t-test.
The real-time fluorescent quantitative PCR reaction system of the Taqman probe method comprises the following steps:
iTaqTMUniversal Probes Supermix：
taqman probe Real-time fluorescent Quantitative PCR (Quantitative Real-time PCR, q-PCR) reaction system:
the Taqman probe real-time fluorescence quantitative PCR reaction step:
after completion of the reaction, the expression intensity of each gene was confirmed by labeling with CT value (threshold cycle values) and reference gene (pGL3), and then calculating P value by unpaired t-test.
2.5 statistical analysis
Statistical analysis was performed using SPSS 13.0 and Graphpad 5.0 software.
3 results
3.1 high expression of circRNF13 in serum of nasopharyngeal carcinoma patients
The experiment adopts 100 nasopharyngeal carcinoma patient serums and 51 normal control serums, and the experimental result shows that: as compared with the normal control group, circRNF13 was up-regulated in serum of nasopharyngeal carcinoma patients, as shown in FIG. 3.
To ensure the authenticity and reliability of experimental data and reduce errors in the experimental process, a Taqman probe of circRNF13 is designed, the expression condition of circRNF13 in the serum of a nasopharyngeal carcinoma patient is detected by adopting an experimental method of Taqman probe q-PCR, and a normal control is increased to 121 cases, as shown in figure 4. The results show that: compared with the serum of a normal control group, the expression level of the circRNF13 in the serum of a nasopharyngeal carcinoma patient is obviously increased, and the statistical significance is achieved (P is less than 0.001).
3.2 nasopharyngeal carcinoma ROC curve evaluation clinical application value
The invention verifies that the expression of the circRNF13 is up-regulated in nasopharyngeal carcinoma, and the clinical application value of the circRNF13 is evaluated by adopting an ROC curve. Particularly, the AUC (Area Under the Curve) is used for representing that the AUC value is between 0.5 and 1.0, and the closer the AUC is to 1, the better the specificity and the sensitivity are, and the higher the clinical application value is.
We performed ROC curve analysis based on the expression level of circRNF13 in nasopharyngeal carcinoma, as shown in figure 5. The AUC value for ROC curve analysis was 0.877.
Single molecule ROC curve analysis of circRNF13
Area under the curve (AUC)
Parameter analyzed as circRNF13
a. Based on nonparametric assumptions
b. Zero hypothesis true area is 0.5
3.3 high expression of circRNF13 in serum of oral cancer patients
The experiment adopts 55 cases of oral cancer patient serums and 121 cases of normal control serum, designs a Taqman probe of the circRNF13, and adopts an experiment method of Taqman probe q-PCR to detect the expression condition of the circRNF13 in the oral cancer patient serums, as shown in figure 6. The results show that: compared with the serum of a normal control group, the expression level of the circRNF13 in the serum of the oral cancer patient is obviously increased, and the statistical significance is achieved (P is less than 0.001).
3.4 oral cancer ROC Curve assessment clinical application value
The invention verifies that the expression of the circRNF13 is up-regulated in oral cancer, and the clinical application value of the circRNF13 is evaluated by adopting an ROC curve.
We performed ROC curve analysis based on the expression level of circRNF13 in oral cancer, as shown in fig. 7. The AUC value of ROC curve analysis was 0.731.
Single molecule ROC curve analysis of circRNF13
Area under the curve (AUC)
Parameter analyzed as circRNF13
a. Based on nonparametric assumptions
b. Zero hypothesis true area is 0.5
3.5 high expression of circRNF13 in serum of thyroid carcinoma patients
The expression level of circ RNF13 in the serum of thyroid cancer patients is detected by adopting an experimental method of Taqman probe q-PCR in the serum of 57 thyroid cancer patients and 121 normal control patients, and the result shows that: compared with a normal control group, the expression level of the circRNF13 in the serum of the thyroid cancer patient is obviously increased and has statistical significance (P is less than 0.001), and the result is shown in figure 8.
The incidence of thyroid cancer is higher in women than in men, and 43 of thyroid cancer patients and 14 of thyroid cancer patients collected by the inventor are women and men. In contrast, 36 women in the normal control group and 85 men in the normal control group have obvious difference in male and female proportion from the thyroid cancer group. For experimental stringency, we performed further statistics in thyroid female patients alone. We used 43 female patients with thyroid cancer and 36 women in the normal control group to verify the expression level of circRNF 13. circRNF13 was also significantly up-regulated in serum from thyroid cancer patients compared to the normal control group with statistical differences (P < 0.001) as shown in fig. 9.
3.6 thyroid cancer ROC curve evaluation clinical application value
The invention proves that the expression of the circRNF13 is up-regulated in thyroid cancer, and the clinical application value of the circRNF13 is evaluated by adopting an ROC curve according to the expression level of the circRNF13 in the thyroid cancer. As shown in fig. 10, the AUC value of ROC curve analysis was 0.798.
Single molecule ROC curve analysis of circRNF13
Area under the curve (AUC)
Parameter analyzed as circRNF13
a. Based on nonparametric assumptions
b. Zero hypothesis true area is 0.5
We performed ROC curve analysis based on circRNF13 expression levels in female patients with thyroid cancer, as shown in figure 11. The AUC value of ROC curve analysis was 0.874.
Single molecule ROC curve analysis of circRNF13
Area under the curve (AUC)
Parameter analyzed as circRNF13
a. Based on nonparametric assumptions
b. Zero hypothesis true area is 0.5
3.7 high expression of circRNF13 in the serum of patients with ovarian cancer
In the ovarian cancer group, 36 sera of ovarian cancer patients and 36 sera of female volunteers in a normal control group were tested and verified by an experimental method of Taqman probe q-PCR, and the expression level of circRNF13 is shown in FIG. 12. The results show that: compared with a normal control group, the expression level of the circRNF13 in the serum of the ovarian cancer patient is obviously increased and has statistical significance (P is less than 0.001).
3.8 evaluation of the clinical application value of the ROC Curve
The invention verifies that the expression of the circRNF13 is up-regulated in ovarian cancer, and the clinical application value of the circRNF13 is evaluated by adopting an ROC curve according to the expression level of the circRNF13 in the ovarian cancer. As shown in fig. 13, the AUC value of ROC curve analysis was 0.706.
Single molecule ROC curve analysis of circRNF13
Area under the curve (AUC)
Parameter analyzed as circRNF13
a. Based on nonparametric assumptions
b. Zero hypothesis true area is 0.5
3.9 circRNF13 high expression in Lung cancer serum
The expression of circRNF13 in the serum of lung cancer patients is detected by adopting an experimental method of Taqman probe q-PCR, wherein 45 patients with lung cancer and 121 patients with normal control are detected. The experimental results show that: compared with the normal control group, the expression level of circRNF13 in the serum of the lung cancer patient is obviously increased and has statistical significance (P is less than 0.001), as shown in figure 14.
3.10 Lung cancer clinical data analysis and ROC Curve
We performed ROC curve analysis based on circRNF13 expression levels in lung cancer patients, respectively, as shown in fig. 15. The results are as follows: circRNF13 had an AUC of 0.689.
Single molecule ROC curve analysis of circRNF13
Area under the curve (AUC)
Parameter analyzed as circRNF13
a. Based on nonparametric assumptions
b. Zero assumes that the real area is 0.5.
Sequence listing
<110> university of south-middle school
Application of reagent for detecting circular RNA circRNF13 in preparation of tumor auxiliary diagnosis preparation and kit
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 553
<212> DNA
<213> Unknown (Unknown)
<400> 1
ggaagaggaa gaacgtctga gaaataaaat tcgagctgat catgagaagg ccttggaaga 60
agcaaaagaa aaattaagaa agtcaagaga ggaaattcga gcagaaattc agacagagaa 120
aaataaggta gtccaagaaa tgaagataaa agagaacaag ccactgccac cagtccctat 180
tcccaacctt gtaggaatac gtggtggaga cccagaagat aatgacataa gagagaaaag 240
ggaaaaaatt aaagagatga tgaaacatgc ttgggataac tataggacat atgggtgggg 300
acataatgaa ctcagaccta ttgcaaggaa aggacactcc cctaacatat ttggaagttc 360
acaaatgggt gctaccatag tagatgcttt ggataccctt tatatcatgg gacttcatga 420
tgaattccta gatgggcaaa gatggattga agacaacctt gatttcagtg tgaattcaga 480
ggtgtctgtg tttgaagtca acattcgatt tattggaggc ctacttgcag catattacct 540
atcaggagag gag 553
<210> 2
<211> 20
<212> DNA
<213> Unknown (Unknown)
<400> 2
<210> 3
<211> 20
<212> DNA
<213> Unknown (Unknown)
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<210> 4
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<212> DNA
<213> Unknown (Unknown)
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<210> 5
<211> 20
<212> DNA
<213> Unknown (Unknown)
<400> 5
<210> 6
<211> 35
<212> DNA
<213> Unknown (Unknown)
<400> 6
aggatagaca tagagctaga agaaacagac ttcgt 35
<210> 7
<211> 22
<212> DNA
<213> Unknown (Unknown)
<400> 7
acgcaggtgt cgcaggtctt cc 22
Claims (3)
1. The application of a reagent for detecting circular RNA circRNF13 in preparing a tumor auxiliary diagnostic preparation, wherein the sequence of the circular RNAcircRNF13 is shown as SEQ No. 1; the reagent for detecting the circular RNA circRNF13 is a reagent for detecting the circular RNA circRNF13 in serum; tumor types include: at least one of nasopharyngeal carcinoma, oral cancer, thyroid cancer, and ovarian cancer.
2. The use of claim 1, wherein the reagent for detecting the cyclic rnacircrrnf 13 comprises a real-time fluorescent quantitative detection reagent.
3. The use of claim 2, wherein the real-time fluorescence quantitative detection reagent comprises primers for real-time fluorescence quantitative detection of circular RNA circRNF13 expression:
an upstream primer: 5'-GTCCAGGATAGACATAGAGC-3'
A downstream primer: 5'-GTGTAGACTTGTGTGGCTGA-3' are provided.
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| CN112870364B (en) * | 2021-04-12 | 2022-04-12 | 中南大学湘雅三医院 | Application of circ-RNF13-TRIM41-p53 complex as target site detection preparation |
| CN113101369B (en) * | 2021-04-12 | 2022-03-11 | 中南大学湘雅三医院 | Application of circular RNA circ-RNF13 as target site inhibitor |
| CN113999849A (en) * | 2021-11-04 | 2022-02-01 | 山东大学齐鲁医院 | Cancer biomolecule marker and application |
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| CN105441581A (en) * | 2016-01-29 | 2016-03-30 | 中南大学 | Application of reagent for detecting long-chain non-coding RNA LOC284454 expression quantity in tongue cancer tissue |
| CN105506154A (en) * | 2016-01-29 | 2016-04-20 | 中南大学 | Application of long non-coding RNA LOC284454 reagent in in-situ hybridization detection of nasopharyngeal carcinoma tissues |
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| CN105441581A (en) * | 2016-01-29 | 2016-03-30 | 中南大学 | Application of reagent for detecting long-chain non-coding RNA LOC284454 expression quantity in tongue cancer tissue |
| CN105506154A (en) * | 2016-01-29 | 2016-04-20 | 中南大学 | Application of long non-coding RNA LOC284454 reagent in in-situ hybridization detection of nasopharyngeal carcinoma tissues |
Non-Patent Citations (1)
| Title |
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| CircRNF13 regulates the invasion and metastasis in lung adenocarcinoma by targeting miR-93-5p.;Wang L.等;《Gene》;20180425;摘要，2. Materials and methods * |
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