CN117535414A - Prostate cancer marker, probe, kit and application thereof - Google Patents
Prostate cancer marker, probe, kit and application thereof Download PDFInfo
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- CN117535414A CN117535414A CN202311498811.4A CN202311498811A CN117535414A CN 117535414 A CN117535414 A CN 117535414A CN 202311498811 A CN202311498811 A CN 202311498811A CN 117535414 A CN117535414 A CN 117535414A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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Abstract
The invention relates to a prostate cancer marker, a probe, a kit and application thereof, belonging to the technical field of molecular biology detection. The nucleotide sequence of the prostate cancer marker is shown as SEQ ID NO.1, can be used for detecting prostate cancer, solves the problem of insufficient accuracy and stability of the marker for detecting prostate cancer in the prior art, and has the characteristics of high accuracy and strong stability.
Description
Technical Field
The invention relates to the technical field of molecular biological detection, in particular to a prostate cancer marker, a probe, a kit and application thereof.
Background
tRNA is a passively involved mRNA decoder and protein translation element that is abundant and widely present in the body, and significantly affects biological processes and disease progression through anticodon binding to mRNA codons. the high abundance of tRNA in body fluid makes it a biomarker for clinical application, and is widely used for detecting proliferation and metastasis of tumor or cancer cells.
the tRNA is cleaved by a specific nuclease to form novel non-coding small RNAs (i.e., tsRNAs) such as tFRs and tRNAs. Where tRFs can be categorized as tRFs-1, tRFs-2, tRFs-3, tRFs-5 and i-tRFs, and the tRNAs can be categorized as 5'tRNA and 3' tRNA. tRF and tiRNA play important roles in inhibiting protein synthesis, regulating gene expression, initiating viral reverse transcriptase, and regulating DNA damage response, and can be considered as functional units of trnas.
Argonaute proteins (AGOs) are a large family of proteins, including AGO1, AGO2, AGO3 and AGO4.AGO proteins mainly comprise four domains, N, PAZ, MID and PIWI, wherein the N domain is responsible for charging tRNA, the PAZ domain is capable of binding to the 3 'end of tRNA, the MID domain is capable of binding to the 5' end of tRNA, and the PIWI domain has ribonuclease H (RNase H) activity.
Although tRNA plays an important role in the involvement in the proliferation and metastasis of tumor or cancer cells, its use in biology, especially medicine, is limited by its inadequate in vivo stability. Although the Argonaute protein is capable of significantly increasing the stability of the tRF or the tiRNA by binding to the tRF or the tiRNA to form a corresponding complex, there is no related study or application of Argonaute protein-tRNA fragment complexes as markers in the detection of tumor or cancer cell proliferation and metastasis.
Disclosure of Invention
The invention aims to provide a prostate cancer marker, a probe, a kit and application thereof, so as to solve the problems of insufficient accuracy and stability of the marker for detecting prostate cancer in the prior art.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a prostate cancer marker, and the nucleotide sequence of the prostate cancer marker is shown as SEQ ID NO. 1.
The invention also provides application of the prostate cancer marker in preparing products for detecting prostate cancer.
The invention also provides a kit for detecting the prostate cancer, which comprises a reagent for detecting the nucleotide sequence of the prostate cancer marker.
The invention also provides a probe for detecting the prostate cancer, and the nucleotide sequence of the probe is shown as SEQ ID NO. 2;
preferably, the nucleotide sequence of the probe is the 27 th to 47 th sites of the nucleotide sequence of the prostate cancer marker.
The invention also provides application of the probe in preparation of a product for detecting the prostate cancer marker.
The invention also provides a kit for detecting the prostate cancer marker, which comprises the probe.
The invention also provides application of the probe in preparation of products for detecting prostate cancer.
The invention also provides a kit for detecting the prostate cancer, which comprises the probe.
The invention has the following technical effects and advantages:
(1) The accuracy is high: the development condition of the prostate cancer can be accurately judged through the dynamic change of the prostate cancer markers;
(2) The stability is strong: in blood, the prostate cancer marker is less prone to degradation than 5' tRF-mtMetCAT, and can reflect biological effects.
Drawings
FIG. 1 is a tRNAargonaute protein complex in blood of healthy volunteers;
FIG. 2 shows the tRNAargonaute protein complex in blood of a prostate cancer patient;
FIG. 3 shows the differential expression of the tRNAargonaute protein complex in blood of healthy volunteers and in blood of prostate cancer patients.
Detailed Description
The invention provides a prostate cancer marker, and the nucleotide sequence of the prostate cancer marker is shown as SEQ ID NO. 1.
In the present invention, the nucleotide sequence of the prostate cancer marker is a nucleotide fragment sequence of the junction of 5' tRF-mtMetCAT and Argonaute protein.
The invention also provides application of the prostate cancer marker in preparing products for detecting prostate cancer.
The invention also provides a kit for detecting the prostate cancer, which comprises a reagent for detecting the nucleotide sequence of the prostate cancer marker.
The invention also provides a probe for detecting the prostate cancer, and the nucleotide sequence of the probe is shown as SEQ ID NO. 2.
In the present invention, the nucleotide sequence of the probe is the 27 th to 47 th sites of the nucleotide sequence of the prostate cancer marker.
The invention also provides application of the probe in preparation of a product for detecting the prostate cancer marker.
The invention also provides a kit for detecting the prostate cancer marker, which comprises the probe.
The invention also provides application of the probe in preparation of products for detecting prostate cancer.
The invention also provides a kit for detecting the prostate cancer, which comprises the probe.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1: screening for prostate cancer markers
10mL of each of 1 patient diagnosed with prostate cancer and 1 healthy volunteer from a medical examination of the attached hospitals at Yangzhou university was taken and sent to a digital spectrum (Shanghai) Biotechnology Co., ltd for detection of tRNAargonaute protein complex (contract number 2304008) in blood, and the detection results are shown in FIGS. 1 to 2.
By performing differential expression analysis on 926 tRNAargonaute protein complexes obtained through detection, 508 tRNAargonaute protein complexes are up-regulated and 418 tRNAargonaute protein complexes are down-regulated, and the differential expression analysis result is shown in FIG. 3, and the tRNAargonaute protein complex with the largest difference is obtained through screening from the tRNAargonaute protein complexes with up-regulated expression, namely, the 5' tRNAt-mtMetCAT and the Argonaute protein complex.
Example 2: method for detecting prostate cancer markers
1. Detection reagent
RIPA lysate (cat No. 89900), PBS buffer from sammer femto; 5 Xloading solution and lysate were purchased from Bao Ri doctor materials technology (Beijing) Co., ltd; NETbuffer fluid is available from Shanghai such as Ji Biotechnology development Co., ltd; decolorized solution was purchased from Shanghai ze Biotechnology Co., ltd; 2 XSSC buffer, 0.1% NP-40/2 XSSC solution, DAPI counterstain from Shanghai Charpy technologies Co., ltd; coomassie brilliant blue staining kit was purchased from Shanghai department biotechnology limited.
2. Detection method
Step 1: plasma collection and lysis
Blood of 1 healthy volunteer and 1 prostate cancer patient are respectively extracted for 10mL, centrifuged for 20min at 8000rpm at 4 ℃, the sediment is collected and added with 1mL RIPA lysate to be fully and evenly mixed, ice bath ultrasound is carried out for 5min until the solution is clarified, and then centrifugation is carried out for 20min at 8000rpm at 4 ℃, and the supernatant is the blood plasma.
Step 2: argonaute protein preparation
Taking 50 mu L of the plasma prepared in the step 1, rotating at 4 ℃ for incubation for 3 hours, washing with NETbuffer solution for 4 times, then washing with PBS buffer solution for 1 time, and mixing the mixture upside down after each washing. Re-suspending with 1mL PBS buffer to obtain re-suspension, extracting RNA from 700 μL re-suspension, reverse transcription, centrifuging 300 μL re-suspension at 8000rpm for 20min at 4deg.C, collecting precipitate, adding 30 μL RIPA lysate and 15 μL 5×loading solution, incubating at 95deg.C for 10min, and performing agarose gel electrophoresis for 30min.
Step 3: coomassie brilliant blue staining and decolorizing
And (3) carrying out coomassie brilliant blue staining on the protein glue prepared in the step (2), wherein the staining step refers to the instruction book of the kit. Cutting the dyed albumin glue, immersing the albumin glue in the decoloring liquid, vibrating for 30min, replacing the decoloring liquid, and repeating the operation until the adhesive tape is decolored into a colorless adhesive tape.
Step 4: reduction and alkylation of Argonaute proteins
The colorless tape obtained in the step 3 is placed in 500 mu L of acetonitrile for soaking for 10min, after the colorless tape is contracted and whitened, the colorless tape is centrifuged at 8000rpm for 20min at 27 ℃, 10 mu L of 5mmol/LTECP is added to the precipitate, and the solution is incubated for 10min at 55 ℃ to reduce disulfide bonds in Argonaute protein. After the temperature was lowered to 27 ℃, 10. Mu.L of 10mmol/L indoleacetic acid was added and reacted for 20min away from light to carry out alkylation. Centrifuging at 8000rpm at 27deg.C for 20min, collecting precipitate, adding 500 μl acetonitrile, reacting for 10min, centrifuging at 8000rpm at 27deg.C for 20min after colorless adhesive tape contracts, and collecting precipitate to obtain alkylated adhesive tape.
Step 5: trypsin digestion
1. Mu.g trypsin was mixed with 100. Mu.LABC buffer to give a trypsin solution.
70 mu L of trypsin solution is added into the alkylation adhesive tape prepared in the step 4, the mixture is kept stand at 4 ℃ for 2 hours until the alkylation adhesive tape is fully expanded, 20 mu LABC buffer solution is added for enzymolysis at 37 ℃ overnight, and then the mixture is centrifuged at 8000rpm at 4 ℃ for 30 minutes, and the precipitate is collected.
Step 6: preparation of cell sections
And (3) placing the precipitate collected in the step (5) in 95% ethanol for fixing for 24 hours, and preparing the cell wax block through a standardized dehydration and wax dipping process. The cell wax block was cut into cell wax pieces having a thickness of 3. Mu.m, and immediately after slicing by a microtome, the pieces were baked at 65℃for 2 hours, and conventionally dewaxed to obtain cell slices.
Step 7: probe preparation
The nucleotide segment (shown as SEQ ID NO. 1) at the joint of the 5' tRF-mtMetCAT and the Argonaute protein is used as a template, the 27 th to 47 th nucleotide core sequences are selected as probes (shown as SEQ ID NO. 2) and sent to the Bodhisattva bioengineering Co.
Step 8: pretreatment of cell sections
Immersing the cell slice prepared in the step 7 into absolute ethyl alcohol for 5min and repeating for 1 time, then immersing in 80% ethanol, 75% ethanol and deionized water for 3min respectively, immersing in a boiled penetrating agent for 30min, and immersing in pepsin working solution at 37 ℃ for enzymolysis for 10min; taking out the cell slice, rinsing in 2 XSSC buffer solution for 2 times and 5min each time, soaking in 70% ethanol, 85% ethanol and absolute ethanol for 3min respectively, and air drying to obtain pretreated cell slice.
Step 9: fluorescence in situ hybridization
Taking 10 mu L of probe, standing for 5min at 27 ℃, mixing the probe upside down, centrifuging at 8000rpm for 2min, dripping the probe onto the pretreated cell slice prepared in the step 8, immediately covering a cover glass, and sealing edges. The slide glass was placed on a hybridization apparatus, denatured at 85℃for 5min, and hybridized overnight at 37℃to obtain a hybridized cell slice.
Step 10: washing, counterstaining and observation
Taking out the hybridized cell slice obtained in the step 9, soaking in 2 XSSC buffer solution for 5 seconds, soaking in 2 XSSC buffer solution at 37 ℃ for 40 minutes, soaking in 2 XSSC buffer solution at 60 ℃ for 10 minutes, soaking in 0.1% NP-40/2 XSSC solution for 5 minutes, soaking in 75% ethanol for 3 minutes, then dripping 10 mu L of DAPI counterstain on the hybridized cell slice for counterstaining, standing for 10 minutes in dark environment, and observing by using a fluorescence microscope.
Example 3: determination of results for markers of prostate cancer
Because the complex of the 5 'tRCA-mtMetCAT and the Argonaute protein has the effect of promoting proliferation of the prostate cancer cells, the fluorescence in situ hybridization detection value is reduced when the detection is carried out for the second time and above based on the fluorescence in situ hybridization detection value in the first blood, which indicates that the complex of the 5' tRCA-mtMetCAT and the Argonaute protein is increased and the proliferation level of the prostate cancer cells is improved; the increase in fluorescence in situ hybridization assay values upon the second and subsequent assays indicated a decrease in the 5' tRF-mtMetCAT and Argonaute protein complex and a decrease in the proliferation level of prostate cancer cells.
From the above examples, the present invention provides a prostate cancer marker, a probe, a kit and applications thereof. The prostate cancer marker, the probe and the kit detect the prostate cancer, solve the problems of insufficient accuracy and stability of the marker for detecting the prostate cancer in the prior art, and have the advantages of high accuracy, strong stability, high sensitivity, strong specificity and the like.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (8)
1. The prostate cancer marker is characterized in that the nucleotide sequence of the prostate cancer marker is shown as SEQ ID NO. 1.
2. Use of the prostate cancer marker of claim 1 in the manufacture of a product for detecting prostate cancer.
3. A kit for detecting prostate cancer, comprising reagents for detecting the nucleotide sequence of the prostate cancer marker of claim 1.
4.A probe for detecting prostate cancer, which is characterized in that the nucleotide sequence of the probe is shown as SEQ ID NO. 2;
the nucleotide sequence of the probe is 27 th to 47 th sites of the nucleotide sequence of the prostate cancer marker according to claim 1.
5. Use of the probe of claim 4 for the preparation of a product for detecting a marker for prostate cancer.
6. A kit for detecting a marker of prostate cancer, comprising the probe of claim 4.
7. Use of the probe of claim 4 for the preparation of a product for detecting prostate cancer.
8. A kit for detecting prostate cancer, comprising the probe of claim 4.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311498811.4A CN117535414A (en) | 2023-11-10 | 2023-11-10 | Prostate cancer marker, probe, kit and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311498811.4A CN117535414A (en) | 2023-11-10 | 2023-11-10 | Prostate cancer marker, probe, kit and application thereof |
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| Publication Number | Publication Date |
|---|---|
| CN117535414A true CN117535414A (en) | 2024-02-09 |
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| CN202311498811.4A Pending CN117535414A (en) | 2023-11-10 | 2023-11-10 | Prostate cancer marker, probe, kit and application thereof |
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