Patent Application
20230193397
The disclosure relates to the field of biotechnology, in particular to a cDNA related to glioma prognosis, an mRNA transcribed from the cDNA, a protein encoded by the mRNA, a kit for evaluating glioma prognosis and a system for evaluating glioma prognosis.
Glioma is a general name of tumors derived from neuroepithelia, accounting for 40%-50% of craniocerebral tumors, and is the most common primary intracranial tumor. Gliomas are difficult to radically treat, often relapse after the operation, and have a poor prognosis. The World Health Organization grading system divides gliomas into grades I-IV according to the histological characteristics, and the prognosis of different grades of gliomas varies.
Accurately evaluating the prognosis of glioma patients has important clinical, scientific and social values. In clinical work, accurate prognosis evaluation can guide doctors to formulate personalized examination and treatment schedules for high-risk patients and help the doctors to formulate reasonable reexamination and follow-up visit plans, thus improving the quality of medical services. In scientific research, accurately assessing the risk level of patients' prognosis can provide an important basis for developing effective treatment schedules for high-risk patients, and can become an important reference for testing the effect of new treatments. From the perspective of society, accurate evaluation of the prognosis of patients can provide scientific survival expectations for patients and their families, guide the patients to comply with treatment plans, avoid excessive medical treatment, reduce family financial pressure, and help to improve the doctor-patient relationship.
At present, the evaluation of glioma prognosis is carried out mainly according to the pathological grade of glioma, but the prognosis of different patients with glioma of a same pathological grade may also vary greatly, and the evaluation of glioma prognosis is still not accurate enough only according to the pathological grade of glioma.
The disclosure aims to provide a cDNA related to glioma prognosis, an mRNA transcribed from the cDNA, a protein encoded by the mRNA, a kit for evaluating glioma prognosis and a system for evaluating glioma prognosis, capable of further improving the accuracy of glioma prognosis evaluation.
In a first aspect, the disclosure provides a cDNA, where a sequence of the cDNA contains a fragment of SEQ ID NO: 1, or the sequence of the cDNA contains a complementary fragment of the fragment of SEQ ID NO: 1; wherein the fragment of SEQ ID NO: 1 at least contains a sequence at positions 2635-2652, 2623-2640 or 2627-2645 of SEQ ID NO: 1.
Preferably, the fragment of SEQ ID NO: 1 at least contains a sequence at positions 2588-2687 of SEQ ID NO: 1.
Optionally, the cDNA corresponds to a human MET gene, and the sequence of the cDNA does not contain an exon 19 of the human MET gene.
Optionally, the cDNA is single-stranded DNA, and the sequence of the cDNA is the sequence as shown in SEQ ID NO: 1, or the sequence of the cDNA is a complementary sequence of the sequence as shown in SEQ ID NO: 1.
Optionally, the cDNA is double-stranded DNA, and the sequence of one single strand of the cDNA is the sequence as shown in SEQ ID NO: 1, and the sequence of the other single strand of the cDNA is the complementary sequence of the sequence as shown in SEQ ID NO: 1.
In a second aspect, the disclosure provides an mRNA, where the mRNA is a reverse transcription template for any cDNA in the first aspect.
Optionally, the sequence of the mRNA contains a fragment of SEQ ID NO: 2, and the fragment of SEQ ID NO: 2 at least contains a sequence at positions 3820-3837, 3832-3849 or 3827-3845 of SEQ ID NO: 2.
Preferably, the fragment of SEQ ID NO: 2 at least contains a sequence at positions 3785-3884 of SEQ ID NO: 2.
Optionally, the sequence of the mRNA is the sequence as shown in SEQ ID NO: 2.
In a third aspect, the disclosure provides a protein, where the protein is encoded by any mRNA in the second aspect.
Optionally, the amino acid sequence of the protein contains a fragment of SEQ ID NO: 3, and the fragment of SEQ ID NO: 3 at least contains a sequence at positions 1207-1220 of SEQ ID NO: 3.
Preferably, the fragment of SEQ ID NO: 3 at least contains a sequence at positions 1175-1220 of SEQ ID NO: 3.
Optionally, the amino acid sequence of the protein is the sequence as shown in SEQ ID NO: 3.
In a fourth aspect, the disclosure provides a kit for evaluating glioma prognosis, where the kit includes a first primer pair capable of specifically amplifying any cDNA in the first aspect, and/or a first probe capable of specifically hybridizing with any cDNA in the first aspect; and/or,
the kit includes a second primer pair capable of specifically amplifying any mRNA in the second aspect, and/or a second probe capable of specifically hybridizing with any mRNA in the second aspect, and/or,
the kit includes an antibody for resisting any protein in the third aspect, and any protein in the third aspect.
Optionally, the first primer pair contains a first primer as shown in SEQ ID NO: 4 and a second primer as shown in SEQ ID NO: 5, and the sequence of the first probe contains a sequence as shown in SEQ ID NO: 6.
The second primer pair contains a third primer as shown in SEQ ID NO: 7 and a fourth primer as shown in SEQ ID NO: 8, and the sequence of the second probe contains a sequence as shown in SEQ ID NO: 9.
In a fifth aspect, the disclosure provides application of a molecular reagent in preparation of a kit for evaluating glioma prognosis, where the molecular reagent includes at least one of (1)-(8) as follows:
(1) any cDNA in the first aspect;
(2) a first primer pair capable of specifically amplifying any cDNA in the first aspect;
(3) a first probe capable of specifically hybridizing with any cDNA in the first aspect;
(4) any mRNA in the second aspect;
(5) a second primer pair capable of specifically amplifying any mRNA in the second aspect;
(6) a second probe capable of specifically hybridizing with any mRNA in the second aspect;
(7) any protein in the third aspect; and
(8) an antibody for resisting any protein in the third aspect.
In a sixth aspect, the disclosure provides a system for evaluating glioma prognosis, where the system includes an amplification device, a sequencing device, a computing device and an output device.
The amplification device includes a collection unit and an amplification unit, where the collection unit is used for collecting a template nucleic acid fragment and an amplification primer, and the amplification unit is used for amplifying the template nucleic acid fragment by using the amplification primer to obtain an amplification product.
The sequencing device is used for sequencing the nucleic acid sequence of the amplification product to obtain a sequence of the amplification product.
The computing device includes a memory and a processor, where a computer program is stored in the memory, and the processor is configured to execute the computer program stored in the memory so as to achieve the following determination:
if the sequence of the amplification product contains the sequence of any cDNA in the first aspect and/or the sequence of any mRNA in the second aspect, it is determined that the prognosis of glioma corresponding to the template nucleic acid fragment is poor.
The output device is used for outputting a determination result of the computing device.
In a seventh aspect, the disclosure provides a method for evaluating glioma prognosis, where the method includes a step of detecting whether a to-be-detected glioma sample contains the cDNA and/or the mRNA and/or the protein as described above, and if the to-be-detected glioma sample contains the cDNA and/or the mRNA and/or the protein as described above, it is indicated that the prognosis of a glioma patient corresponding to the to-be-detected glioma sample is poorer.
Through the above technical solutions, the accuracy of glioma prognosis evaluation is effectively improved in the disclosure.
Other features and advantages of the disclosure will be described in detail in the subsequent specific examples.
The drawings are used to provide a further understanding of the disclosure and constitute a part of the description, and are used to explain the disclosure together with the following specific examples, but do not constitute limitations of the disclosure. In the drawings:
The specific examples of the disclosure are described in detail below in combination with the drawings. It should be understood that the specific examples described herein are only used to illustrate and interpret the disclosure, but not to limit the disclosure.
In a first aspect, the disclosure provides a cDNA, where a sequence of the cDNA contains a fragment of SEQ ID NO: 1, or the sequence of the cDNA contains a complementary fragment of the fragment of SEQ ID NO: 1; wherein the fragment of SEQ ID NO: 1 at least contains a sequence at positions 2635-2652, 2623-2640 or 2627-2645 of SEQ ID NO: 1; preferably, the fragment of SEQ ID NO: 1 at least contains a sequence at positions 2588-2687 of SEQ ID NO: 1.
Preferably, the cDNA corresponds to a human MET gene, and the sequence of the cDNA does not contain an exon 19 of the human MET gene.
According to the disclosure, the cDNA can be single-stranded DNA, and the sequence of the cDNA can be the sequence as shown in SEQ ID NO: 1, or the sequence of the cDNA can be a complementary sequence of the sequence as shown in SEQ ID NO: 1.
According to the disclosure, the cDNA can be double-stranded DNA, and the sequence of one single strand of the cDNA can be the sequence as shown in SEQ ID NO: 1, and the sequence of the other single strand of the cDNA can be the complementary sequence of the sequence as shown in SEQ ID NO: 1.
The cDNA is cDNA corresponding to the human MET gene with the exon 19 deleted. The inventors of the disclosure have found that when the cDNA can be reversely transcribed from mRNA of glioma cells, the malignant degree of glioma is higher, the drug resistance is stronger, and the prognosis of patients is poorer. Where the fragment of SEQ ID NO: 1 contains a deletion site of the exon 19, the sequence shown in SEQ ID NO: 1 contains all exons, except the exon 19, of the human MET gene, and the sequence shown in SEQ ID NO: 1 is as follows:
Where the sequence at the positions 2635-2652 of SEQ ID NO: 1 is ccaatacagtttcttgca; the sequence at the positions 2623-2640 of SEQ ID NO: 1 is cacgccaaaggaccaata; and the sequence at the positions 2627-2645 of SEQ ID NO: 1 is ccaaaggaccaatacagtt.
In a second aspect, the disclosure provides an mRNA, where the mRNA is a reverse transcription template for any cDNA in the first aspect.
Preferably, the sequence of the mRNA contains a fragment of SEQ ID NO: 2, and the fragment of SEQ ID NO: 2 at least contains a sequence at positions 3820-3837, 3832-3849 or 3827-3845 of SEQ ID NO: 2; preferably, the fragment of SEQ ID NO: 2 at least contains a sequence at positions 3785-3884 of SEQ ID NO: 2.
Preferably, the sequence of the mRNA is a sequence as shown in SEQ ID NO: 2.
Where the sequence as shown in SEQ ID NO: 2 is an RNA sequence complementary to the sequence as shown in SEQ ID NO: 1, and the sequence as shown in SEQ ID NO: 2 is as follows:
Where the sequence at the positions 3820-3837 of SEQ ID NO: 2 is ugcaagaaacuguauugg; the sequence at the positions 3832-3849 of SEQ ID NO: 2 is uauugguccuuuggcgug; and the sequence at the positions 3827-3845 of SEQ ID NO: 2 is aacuguauugguccuuugg.
In a third aspect, the disclosure provides a protein, where the protein is encoded by any mRNA in the second aspect.
Preferably, the amino acid sequence of the protein contains a fragment of SEQ ID NO: 3, and the fragment of SEQ ID NO: 3 at least contains a sequence at positions 1207-1220 of SEQ ID NO: 3.
Preferably, the fragment of SEQ ID NO: 3 at least contains a sequence at positions 1175-1220 of SEQ ID NO: 3.
Preferably, the amino acid sequence of the protein is the sequence as shown in SEQ ID NO: 3.
Where the sequence as shown in SEQ ID NO: 3 is an amino acid sequence encoded by the RNA sequence as shown in SEQ ID NO: 2, and the amino acid sequence as shown in SEQ ID NO: 3 is as follows:
Where the sequence at the positions 1207-1220 of SEQ ID NO: 3 is ARNCIGPLACSSGS .
The sequence at the positions 1175-1220 of SEQ ID NO: 3 is NPTVKDLIGFGLQVAKGMKYLASKKFVHRDLAARNCIGPLACSSGS.
In a fourth aspect, the disclosure provides a kit for evaluating glioma prognosis, where the kit includes a first primer pair capable of specifically amplifying any cDNA in the first aspect, and/or a first probe capable of specifically hybridizing with any cDNA in the first aspect; and/or the kit includes a second primer pair capable of specifically amplifying any mRNA in the second aspect, and/or a second probe capable of specifically hybridizing with any mRNA in the second aspect; and/or the kit includes an antibody for resisting any protein in the third aspect, and any protein in the third aspect.
Where the first primer pair only needs to be able to specifically amplify any cDNA in the first aspect, and preferably, the first primer pair contains a first primer as shown in SEQ ID NO: 4 and a second primer as shown in SEQ ID NO: 5. The first probe only needs to be able to specifically hybridize with any cDNA in the first aspect, and preferably, the sequence of the first probe contains a sequence as shown in SEQ ID NO: 6. The second primer pair only needs to be able to specifically amplify any mRNA in the second aspect, and preferably, the second primer pair contains a third primer as shown in SEQ ID NO: 7 and a fourth primer as shown in SEQ ID NO: 8. The second probe only needs to be able to specifically hybridize with any mRNA in the second aspect, and preferably, the sequence of the second probe contains a sequence as shown in SEQ ID NO: 9.
Where the sequence as shown in SEQ ID NO: 4 is aatttctgaccgagggaatcat; the sequence as shown in SEQ ID NO: 5 is acgccaaaggaccaatacagtttc; the sequence as shown in SEQ ID NO: 6 is gccaaaggaccaatacagtttctt; the sequence as shown in SEQ ID NO: 7 is taatacgactcactataggggagcacgccaaaggaccaataca; the sequence as shown in SEQ ID NO: 8 is tgaaatatcttgcaagcaaaaagtt; and the sequence as shown in SEQ ID NO: 9 is agcacgccaaaggaccaatacagt.
Where the antibody for resisting any protein in the third aspect can be a monoclonal antibody and/or a polyclonal antibody.
In a fifth aspect, the disclosure provides application of a molecular reagent in preparation of a kit for evaluating glioma prognosis, where the molecular reagent includes at least one of (1)-(8) as follows:
(1) any cDNA in the first aspect; (2) a first primer pair capable of specifically amplifying any cDNA in the first aspect; (3) a first probe capable of specifically hybridizing with any cDNA in the first aspect; (4) any mRNA in the second aspect; (5) a second primer pair capable of specifically amplifying any mRNA in the second aspect; (6) a second probe capable of specifically hybridizing with any mRNA in the second aspect; (7) any protein in the third aspect; and (8) an antibody for resisting any protein in the third aspect.
In a sixth aspect, the disclosure provides a system for evaluating glioma prognosis. The system includes an amplification device, a sequencing device, a computing device and an output device, where the amplification device includes a collection unit and an amplification unit, where the collection unit is used for collecting a template nucleic acid fragment and an amplification primer, and the amplification unit is used for amplifying the template nucleic acid fragment by using the amplification primer to obtain an amplification product; the sequencing device is used for sequencing the nucleic acid sequence of the amplification product to obtain a sequence of the amplification product; the computing device includes a memory and a processor, where a computer program is stored in the memory, and the processor is configured to execute the computer program stored in the memory so as to achieve the following determination: if the sequence of the amplification product contains the sequence of any cDNA in the first aspect and/or the sequence of any mRNA in the second aspect, it is determined that the prognosis of glioma corresponding to the template nucleic acid fragment is poor; and the output device is used for outputting a determination result of the computing device.
In a seventh aspect, the disclosure provides a method for evaluating glioma prognosis, where the method includes a step of detecting whether a to-be-detected glioma sample contains the cDNA and/or the mRNA and/or the protein as described above, and if the to-be-detected glioma sample contains the cDNA and/or the mRNA and/or the protein as described above, it is indicated that the prognosis of a glioma patient corresponding to the to-be-detected glioma sample is poorer. Where the cDNA and/or mRNA can be detected by a method of PCR, NASBA, TMA, RT-RPA, nucleic acid hybridization or high-throughput sequencing, and the protein can be detected by immunoblotting.
The disclosure is further described in detail below by the examples.
This preparation example was used for obtaining glioma samples and obtaining total RNAs and total cDNAs therein.
1211 glioma samples were collected by using operations conforming to the standards of the medical ethical committee, and the pathological characteristics of each glioma sample were determined. Where for each patient from whom a sample was collected, the consent of himself or herself and his or her therapist was obtained before the sample was collected, having a written proof material. Where glioma was diagnosed by utilizing a pathological diagnosis method, the prognosis of the glioma samples was evaluated according to the overall survival of patients corresponding to the glioma samples, and the longer the overall survival of the patients is, the better the prognosis of the glioma is. The characteristics such as the gender, age, pathological grade, tumor category, and overall survival of the patients corresponding to the glioma samples are shown in Table 1.
The total RNAs of the glioma samples were extracted by using a DNA extraction kit (purchased from Qiagen) according to its operation instruction. The total RNAs were detected by an integrity analyzer, and it was confirmed that the RNA integrity number (MN) of the total RNAs was larger than 7.0. Double-stranded cDNA was synthesized by using a reverse transcription kit (purchased from Invitrogen) according to its operation instruction and by taking the total RNAs as a template.
In this example, the cDNAs of the glioma samples synthesized by the preparation example were subjected to PCR verification.
Primers used for the PCR verification were the first primer as shown in SEQ ID NO: 4 and the second primer as shown in SEQ ID NO: 5. The PCR operation was carried out according to the instructions of synthetic primers and the PCR kit. The PCR product was subjected to agarose gel nucleic acid electrophoresis to show whether an amplification band existed or not, as shown in
It can be known from
In this example, RNAs of the glioma samples collected in the preparation example were sequenced.
An RNA library was constructed from RNA of each sample by using a RNA library construction kit (purchased from Illumina), then RNA sequencing was performed on the RNA library by using a sequencing platform (Illumina HiSeq 2000), and glioma samples containing mRNA as shown in SEQ ID NO: 2 were screened from the RNA sequencing results as shown in Table 2.
It can be seen from Table 2 that 12 glioma samples containing mRNA as shown in SEQ ID NO: 2 were detected with a median survival of 284 days, and 1199 glioma samples not containing the mRNA as shown in SEQ ID NO: 2 were detected, with a median survival of 866 days, which indicated that the prognosis of glioma containing the mRNA as shown in SEQ ID NO: 2 disclosed by the disclosure was poor, so that the mRNA can be used for evaluating glioma prognosis.
Besides, in the full-grade glioma, the IDH-mutated full-grade glioma, the secondary glioblastoma and the IDH-mutated glioblastoma, the overall survival condition of the glioma samples containing the mRNA shown as the SEQ ID NO: 2 and the overall survival condition of the glioma samples not containing the mRNA shown as the SEQ ID NO: 2 were compared through a survival curve (a Kaplan-Meier curve). Results show that the glioma samples containing the mRNA as shown in SEQ ID NO: 2 were poor in overall survival, as shown in
The preferred examples of the disclosure are described in detail above in combination with the drawings, however, the disclosure is not limited to the specific details in the above examples, in the technical concept range of the disclosure, the technical solution of the disclosure can be subjected to various simple variations, and these simple variations all belong to the protection range of the disclosure.
In addition, it should be noted that the specific technical features described in the above examples can be combined in any suitable mode without contradiction.
In addition, various different examples of the disclosure can also be combined at will, and as long as the examples do not violate the idea of the disclosure, the examples also should be regarded as the contents disclosed by the disclosure.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202010318367.3 | Apr 2020 | CN | national |
This application is a national stage application under 35 U.S.C. 371 of International Application No. PCT/CN2021/088497, filed on Apr. 20, 2021 and designated the U.S., which claims priority to Chinese Patent Application No. 202010318367.3, filed on Apr. 21, 2020. The contents of each are herein incorporated by reference. The material in the ASCII text file inno_13145_20221025_Sequence_listing.txt, created on Oct. 18, 2022, having the size of 29,255 bytes, filed for the present application via EFS-web in replacement of electronic sequence listing (inno_13145_20221013_Sequence_listing.txt; Size: 28,178 bytes; and Date of Creation: Oct. 10, 2022), is incorporated by reference in the specification for all purposes. The Sequence listing includes no new matter.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/088497 | 4/20/2021 | WO |
