CN115851729A - Marker tRF-Thr-TGT-4-M2 for detecting prostate cancer by urine and application thereof - Google Patents

Abstract

The invention provides a marker tRF-Thr-TGT-4-M2 for detecting prostate cancer by urine and application thereof, belonging to the technical field of molecular biological detection. The invention provides a marker tRF-Thr-TGT-4-M2 for detecting prostate cancer, wherein the nucleotide sequence of the marker is shown in SEQ ID NO. 1. The invention can judge the proliferation level of the prostate cancer cells by detecting the relative expression level of tRF-Thr-TGT-4-M2 in urine.

Description

Marker tRF-Thr-TGT-4-M2 for detecting prostate cancer by urine and application thereof

Technical Field

The invention relates to the technical field of molecular biology detection, in particular to a prostatic cancer marker tRF-Thr-TGT-4-M2 detected by urine and application thereof.

Background

The 2016 Nature Genetics journal reports that tRNA is a highly abundant, ubiquitous, passively involved mRNA decoder and protein translation element. tRNA significantly affects biological processes and disease progression by binding its anticodon to the codon of mRNA. Meanwhile, the high abundance of tRNA in body fluid makes tRNA become a biomarker for clinical application, and can be applied to the detection of tumor proliferation and metastasis. tRFs and TiRNAs have subsequently been reported to be small fragments of RNA of a particular size that are cleaved at the tRNA loop by specific nucleases [ e.g., dicer, angiogenin (ANG) ] in specific cells/tissues or under specific conditions such as cell stress. tRFs and tirRNAs belong to a class of small non-coding RNAs, collectively referred to as tsRNAs. tRFs are classified as tRF-1, tRF-2, tRF-3, tRF-5 and i-tRF; tiRNA is divided into 5'tiRNA and 3' tiRNA. Mature tRNA is converted into tRF and TiRNA after being sheared, and the tRF and the TiRNA play important roles in inhibiting protein synthesis, regulating gene expression, starting virus reverse transcriptase and regulating DNA damage reaction and can be regarded as a functional unit of the tRNA.

The current methods for detecting prostate cancer cell proliferation are generally judged by serum PSA detection. However, in the process of wide application, the detection method is found to have poor specificity and insufficient sensitivity, and cannot distinguish severe inflammation of prostate and prompt early prostate cancer cell proliferation. Although detection methods such as ultrasound and nuclear magnetic resonance have been used as a supplement, these methods still have the above-mentioned disadvantages. Therefore, the search for markers with high sensitivity and strong specificity to detect the proliferation of prostate cancer cells has become a hot spot of medical research.

Disclosure of Invention

The invention aims to provide a marker tRF-Thr-TGT-4-M2 for detecting prostate cancer by urine and application thereof in detecting prostate cancer cell proliferation.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a marker tRF-Thr-TGT-4-M2 for detecting prostate cancer, wherein the nucleotide sequence of the marker is shown as SEQ ID NO. 1.

The invention also provides a primer for detecting the marker, wherein the primer comprises an upstream primer shown as SEQ ID NO. 2 and a downstream primer shown as SEQ ID NO. 3.

The invention also provides application of the marker or the primer in preparation of a reagent or a kit for detecting prostate cancer.

Preferably, the detection sample of the reagent or the kit comprises a prostate cell strain, fresh prostate tissue, fresh punctured prostate tissue, fresh urine, fresh prostate massage solution or blood.

The invention also provides a kit for detecting the prostate cancer by using the urine, and the kit comprises the primer.

According to the invention, the proliferation level of the prostate cancer cells can be judged by detecting the relative expression level of tRF-Thr-TGT-4-M2 in urine. the nucleotide sequence of tRF-Thr-TGT-4-M2 is shown in SEQ ID NO 1 and belongs to the tRF-5c type.

Drawings

FIG. 1 shows the expression of tRF-Thr-TGT-4-M2 gene in prostate cancer tissue and prostate normal tissue.

Detailed Description

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

In this embodiment, fresh urine is used as the test sample. When the urine is discharged out of the body, the urine passes through the prostatic urethra, so that the urine sediment detection device has certain significance for relevant detection of the urine sediment. During the period from 10 months at 2021 to 1 month at 2022, the patients who had prostate disease clinical symptoms at the subsidiary hospital of Yangzhou university collected urine for the first urination in the morning, and immediately after collection, the following procedures were performed.

1. Homogenate

200mL of middle urine of the first urination of the examinee in the morning is taken, and then the middle urine is centrifuged at 6000 rpm at 4 ℃ for 20 minutes, and the urinary sediment is collected for later use. Repeated pipetting with TRI REAGENT REAGENT was used to lyse the cells, 1ml TRI REAGENT REAGENT per gram of sediment, and to avoid washing the cells prior to lysis with TRI REAGENT REAGENT, since washing would likely lead to degradation of mRNA.

2. Two-phase separation

After homogenization, the sample was incubated at 25 ℃ for 5 minutes to completely dissociate the nucleic acid protein complex. Then 0.2ml of chloroform (Shanghai chemical Co., ltd.) was added to 1ml of the TRI REAGENT reagent homogenate sample and the tube cap was closed. After shaking the tube vigorously by hand for 15 seconds, it was incubated at 25 ℃ for 3 minutes. Centrifuge at 12000 rpm for 15 minutes at 4 ℃. After centrifugation, the mixed liquid will be separated into a lower red phenol chloroform phase, an intermediate layer and an upper colorless aqueous phase. The RNA was partitioned in the aqueous phase in its entirety. The volume of the aqueous phase was 60% of the TRI REAGENT reagent added during homogenization.

RNA precipitation

The aqueous phase was transferred to a fresh centrifuge tube. The aqueous phase was mixed with isopropanol (Shanghai chemical Co., ltd.) to precipitate RNA therein, the amount of isopropanol added was 1ml of TRI REAGENT REAGENT added to 0.5ml of isopropanol per sample homogenization. After mixing, the mixture was incubated at 30 ℃ for 10 minutes and centrifuged at 12000 rpm at 4 ℃ for 10 minutes. At this point the invisible RNA pellet before centrifugation will form a gelatinous pellet at the bottom and on the side walls of the tube.

RNA washing

The supernatant was removed and the RNA pellet was washed with 75% ethanol (prepared with DEPC water from Shanghai Philippine industries, ltd.). The amount of 75% ethanol added was 1ml of TRI REAGENT REAGENT added to 1ml of 75% ethanol for each sample homogenization. After shaking, the mixture was centrifuged at 7500 rpm at 4 ℃ for 5 minutes.

5. Re-solubilization of RNA pellets

The ethanol solution was removed, the RNA pellet was air dried for 10 minutes, and dried by vacuum centrifugation. During the procedure care was taken that the RNA pellet was not completely dried. When RNA was dissolved, 1mL of RNase-free water was added and the mixture was repeatedly blown with a gun several times, followed by incubation at 60 ℃ for 10 minutes. The RNA solution obtained was stored at-70 ℃.

6. Extraction of control sample RNA

RNA was extracted from cells of a control sample human prostate hyperplasia cell line (BPH-1 cell line, purchased from Shanghai Bingsui Biotech, ltd.).

Add 800. Mu.l of TRI REAGENT reagent to 1mL of control sample, after sample lysis, repeat the procedure from step 2 to step 5. In addition, 10. Mu.g of RNase-free glycogen was added as an aqueous carrier before precipitating RNA with isopropanol. To reduce the solution viscosity, the samples were passed twice through a 26-gauge needle to shear the genomic DNA prior to the addition of chloroform. After separation of the two phases glycogen remains in the aqueous phase and co-precipitates with RNA. As long as the glycogen concentration is not higher than 4mg/ml, the synthesis of cDNA is not affected, and the PCR process is not affected as well.

7.RNA quality detection

By UV absorption measurement using

ND-1000 determination of RNA concentration and purity, before the measurement with dissolved RNA DEPC water 1 u l withered treatment of the surface of the measuring base, then RNA detection operation method as follows:

a) Add 1. Mu.l of RNA sample dropwise to the surface of the measurement base.

b) The liquid drops will automatically form a liquid column between the upper base and the lower base and automatically complete the determination, and various parameters of RNA concentration and quality will be automatically generated into files in a computer.

c) After one measurement, the sample liquid on the surfaces of the upper and lower bases is wiped off by using a soft lens wiping paper, and then the next sample measurement can be carried out.

d) Measurement results (EXCEL and JPEG files automatically generated by computer attached to the laboratory report folder):

and (3) concentration determination:

a reading at 260nm of 1 indicates 40ng RNA/. Mu.l. The formula for calculating the RNA concentration of the sample is as follows: a. The ₂₆₀ (reading). Times.40 ng/. Mu.l. An example of a specific calculation is as follows:

the RNA was dissolved in 20. Mu.l of DEPC water, 1. Mu.l was used for the measurement, and A was measured ₂₆₀ ＝65.003

RNA concentration =65.003 × 40 ng/. Mu.l =2600.12 ng/. Mu.l

After taking 1. Mu.l for measurement, the remaining sample RNA was 19. Mu.l, and the total amount of remaining RNA was: 19 μ l × 2600.12ng/μ l =49.4 μ g

And (3) purity detection:

a of RNA solution _260/A280 The ratio of (a) is an RNA purity detection method, and the ratio ranges from 1.8 to 2.1. Even if the ratio is outside this range, the RNA sample can be used in common experiments such as Northern hybridization, RT-PCR and RNase protection.

Example 2

RNA Pre-treatment and cDNA Synthesis

Reagent rtStar used in this example ^TM tRF&tiRNA Pretreatment Kit(Cat#AS-FS-005)、rtStar ^TM First-Strand cDNA Synthesis Kit (3 and 5' adaptor) (Cat # AS-FS-003) was purchased from Arraystar, USA; other reagents were purchased from Saimer Feishale science (China) Co.

1.3' terminal deacetylation treatment

(1) The deacetylation reaction solution was prepared according to the following table 1:

TABLE 1 configuration of deacetylation reaction solution

RNA	5μg
		Deacylation reaction buffer (5X)	3uL
SUPERase·In TM RNase inhibitors	1μL
		Nuclease-free water	Variables of
Total volume	Adding to 15 mu L

(2) Vortex and incubate at 37 ℃ for 40 min.

(3) Add 19 u L Deacylation Stop buffer, vortex mixing, room temperature incubation for 5 minutes, stop the deacetylation reaction.

2. Removal of 3'-cP and addition of 5' -P

(1) Placing the reaction solution in the previous step on ice, and sequentially adding the reagents in the following table 2:

TABLE 2 reagents

Terminal enzyme reaction buffer (10X)	5μL
		10mMATP	5μL
TerminalEnzymeMix	3U(1μL)
		Nuclease-free water	5μL
Total volume/sample	Adding to 50 μ L

(2) Vortex and incubate at 37 ℃ for 40 min.

(3) The reaction was terminated by incubation at 70 ℃ for 5 minutes.

(4) The RNA was re-extracted.

3. Demethylation treatment

(1) Preparing a demethylation reaction solution:

TABLE 3 demethylation reaction solution configuration

(2) Carrying out demethylation reaction: after incubation for 2 hours in a 37 ℃ water bath, 40. Mu.l of nuclease-free water and 10. Mu.l of Demethylation Stop buffer (5X) were added to terminate the Demethylation reaction.

(3) The RNA was re-extracted.

4. Connecting 3' joints

(1) The following reagents were added sequentially to a 200 μ L rnase-free PCR tube:

TABLE 4 reagents

Nuclease-free water	Variables of
		Sample RNA	2μL
3’Adaptor	0.5μL
		RNASpike-in	0.5μL
Total volume	Adding to 3.5 μ L

(2) Incubate at 70 ℃ for 2 minutes in a thermal cycler, then transfer the PCR tube to ice.

(3) The following reagents were added:

TABLE 5 reaction reagents

3' ligationreaction buffer	5μL
		3’LigationEnzymeMix	1.5μL
Total volume	After addition, the volume of the solution is 10 mu L

(4) Incubate for 1 hour at 25 ℃ in a thermal cycler.

5. Hybridization of Reverse Transcription Primer (Reverse Transcription Primer)

The reverse transcription primer can hybridize to the excess 3' adaptor, thereby converting the single-stranded DNA adaptor into a double-stranded DNA molecule. If the initial amount of total RNA is 100ng, reverse transcription primer is applied to non-enzyme water 1: and 2, diluting.

(1) Adding the following reagents into the PCR tube of the previous step (4):

TABLE 6 reagents

Nuclease-free water	2.3μL
		ReverseTranscriptionPrimer	0.5μL
Total volume	After addition, it became 12.8. Mu.L

(2) And sequentially incubating at 75 ℃ for 5min, at 37 ℃ for 15min and at 25 ℃ for 15min in a thermal cycler.

6. Connecting 5' joints

(1) Resuspend 5' linker in 20. Mu.L nuclease-free water. Note that: if the total RNA starting amount is 100ng, the 5' joint with nuclease free water 1:2 dilution.

(2) Add 0.6. Mu.L of 5' linker to a separate nuclease-free 200. Mu.LPCR tube. (N is the number of samples treated in the experiment) was incubated at 70 ℃ for 2 minutes in a thermocycler and immediately cooled on ice. Note that: the remaining 5' resuspension linker was stored in a-80 ℃ freezer. To avoid RNA degradation, please use the linker within 30 minutes after linker denaturation.

(3) The following reactants were sequentially added to the PCR tube in the previous step (1) and mixed well.

TABLE 7 reactants

Incubate for 1 hour at 25 ℃ in a thermal cycler.

7. Reverse transcription reaction

(1) The following reactants were added to a nuclease-free 200 μ LPCR tube:

TABLE 8 reactants

(2) The thermal cycler was incubated at 50 ℃ for 1 hour and then immediately cooled on ice, and the reaction product was directly used for PCR amplification reaction.

Example 3

EXAMPLE 2 cDNA synthesized for real-time quantitative PCR detection

Reagent 2 XPCR mastermix (AS-MR-006-5) was purchased from Arraystar, USA; primer design software is Primer 5.0; quantstudio ^TM 5ReThe al-time PCR System (Applied Biosystems) was purchased from Applied Biosystems, USA.

1. Preparation of a gradient diluted DNA template for Standard Curve drawing

(1) Selecting a cDNA template which is determined to express the marker gene and the housekeeping gene to carry out PCR reaction:

mixing the solution at the bottom of the flick tube, centrifuging for a short time at 5000 r/min, setting PCR reaction: at 95 ℃ for 10min;40 PCR cycles (95 ℃,10 sec; 60 ℃,60 sec (fluorescence collection)).

(2) The PCR product and 100bp DNAladder are subjected to 2% agarose gel electrophoresis, ethidium bromide staining is carried out, and whether the PCR product is a single specificity amplification band or not is detected.

(3) PCR products were diluted in 10-fold gradients: the concentration of PCR product was set to 1, and the dilution was 1X 10 ^-1 ，1×10 ^-2 ，1×10 ^-3 ，1×10 ^-4 ，1×10 ^-5 ，1×10 ^-6 ，1×10 ^-7 ，1×10 ^-8 ，1×10 ^-9 Several gradients of DNA.

2. Performing a Realtime PCR reaction

(1) The synthesized cDNA sample was placed in a Realtime PCR reaction system. The system is configured as follows:

the solution was mixed by flicking the bottom of the tube, centrifuged at 5000 rpm for 5 minutes.

(2) Sample application

a. Add 8. Mu.l of the mixture to each well of the 384-PCR plate.

b. The corresponding 2. Mu.l of cDNA was added.

c. A Sealing Film was carefully applied and centrifuged briefly for 5 minutes.

c. The prepared PCR plate was placed on ice before setting up the PCR program.

The 384-PCR plate was placed on a Realtime PCR machine for PCR reaction.

All the indexes were carried out according to the following procedure:

at 95 ℃ for 10min;40 PCR cycles (95 ℃,10 sec; 60 ℃,60 sec (fluorescence collection)).

In order to establish the melting curve of the PCR product, after the amplification reaction is finished, the temperature is controlled according to the formula (95 ℃,10 seconds, 60 ℃,60 seconds, 95 ℃,15 seconds); and slowly heated from 60 ℃ to 95 ℃ (instrument auto-Ramp Rate 0.075 ℃/sec).

3. Results and calculations

And respectively carrying out Realtime PCR reaction on the target gene and the housekeeping gene of the sample. According to the drawn gradient dilution DNA standard curve, the concentration results of the target genes and housekeeping genes of the sample are directly generated by a machine. The concentration of the target gene of the sample is divided by the concentration of the housekeeping gene of the sample, so that the corrected relative content of the gene of the sample is obtained.

4. Correcting gene to be detected by internal reference

In real-time quantitative PCR, the sample adding amount of a sample to be detected and a control sample is 2 mu l, however, because of the influence of RNA concentration quantitative error, RNA reverse transcription efficiency error and the like, the content of cDNA in the volume of 2 mu l of each sample is not completely the same, in order to correct the difference, a housekeeping gene U6 (the expression amount of different samples is basically constant) is used as an internal reference, the value of the gene to be detected of the sample is divided by the value of the internal reference of the sample, and the finally obtained ratio is the relative content of the gene to be detected of the sample.

5. Primer list for real-time quantitative PCR

TABLE 9 primer List for real-time quantitative PCR

6. Evaluation of sample prostate cancer proliferation results

After the detection sample and the control sample are processed by the above operation steps, the relative content of tRF-Thr-TGT-4-M2 in the tissue can be obtained, and the result is shown in FIG. 1.

As can be seen from FIG. 1, the relative content of tRF-Thr-TGT-4-M2 in the test sample is significantly higher than that in the control sample, indicating that the prostate cancer cells are in a proliferative state.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.

Claims (5)

1. A marker tRF-Thr-TGT-4-M2 for detecting prostatic cancer is characterized in that the nucleotide sequence of the marker is shown as SEQ ID NO. 1.

2. A primer for detecting the marker of claim 1, wherein the primer comprises an upstream primer shown as SEQ ID NO. 2 and a downstream primer shown as SEQ ID NO. 3.

3. Use of the marker of claim 1 or the primer of claim 2 in the preparation of a reagent or kit for detecting prostate cancer.

4. The use of claim 3, wherein the test sample of the reagent or kit comprises a prostate cell line, fresh prostate tissue, fresh punctured prostate tissue, fresh urine, fresh prostate massage solution or blood.

5. A kit for detecting prostate cancer, comprising the primer of claim 2.

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