A kit for early screening of prostate cancer and a preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a kit for early screening of prostate cancer, a preparation method and application thereof. The kit for early screening of prostate cancer comprises primer pairs for detecting hsa_circ_0058040, hsa_circ_0096498 and hsa_circ_0000994. The application finds that the three circRNAs have expression differences in serum of healthy people and prostate cancer patients through high-throughput sequencing and experimental verification, and the three circRNAs are applied to preparation of the kit. The content of hsa_circ_0058040, hsa_circ_0096498 and hsa_circ_0000994 in serum can be detected through real-time fluorescent quantitative PCR technology, and the kit has high diagnostic value and wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a reagent kit for early screening of prostate cancer, its preparation method, and its application. Background Technology

[0002] Prostate cancer is a common malignant tumor of the male genitourinary system and has become a leading cause of new cancer cases and cancer deaths among men worldwide. It seriously threatens men's physical and mental health and quality of life. Early symptoms of prostate cancer often include frequent urination, urgency, difficulty urinating, and hematuria, significantly impacting patients' lives. In advanced stages, the tumor is prone to metastasis, increasing the difficulty of treatment and worsening the prognosis. Studies show that once prostate cancer has spread, the five-year survival rate is significantly lower than in the localized stage. Therefore, early diagnosis is crucial for improving patient outcomes.

[0003] Currently, various potential biomarkers for prostate cancer have been reported in clinical practice and research, such as prostate-specific antigen (PSA), miRNAs, exosome markers, and DNA methylation markers. With the rapid development of high-throughput sequencing technology and bioinformatics analysis methods, circular RNAs (circRNAs) have gradually become a research hotspot due to their aberrant expression in various cancers and their involvement in tumorigenesis and development. Studies have confirmed that circRNAs possess multiple biological functions, including transcriptional regulation, competitive splicing, protein-protein interaction, acting as miRNA sponges, and regulating mRNA expression and translation. Furthermore, circRNAs exhibit high stability in both intracellular and extracellular fluids, providing a significant advantage as non-invasive or minimally invasive biomarkers for early cancer screening and prognostic assessment.

[0004] Currently, there are no reports on the combined detection of circular RNAs hsa_circ_0058040, hsa_circ_0096498, and hsa_circ_0000994 for early prostate cancer screening. Therefore, this invention aims to provide a kit for early prostate cancer screening, its preparation method, and its application. Summary of the Invention

[0005] The primary objective of this invention is to provide a kit for early screening of prostate cancer.

[0006] The second objective of this invention is to provide a method for preparing a reagent kit for early screening of prostate cancer.

[0007] The third objective of this invention is to provide a kit for early screening of prostate cancer and its application in the preparation of products for detecting prostate cancer.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A kit for early screening of prostate cancer, the kit comprising primer pairs for quantitative detection of hsa_circ_0058040, hsa_circ_0096498, and hsa_circ_0000994.

[0009] Further, the nucleotide sequence of hsa_circ_0058040 is shown in SEQ ID NO.1; the nucleotide sequence of hsa_circ_0096498 is shown in SEQ ID NO.2; and the nucleotide sequence of hsa_circ_0000994 is shown in SEQ ID NO.3.

[0010] Further, the upstream primer sequence for the quantitative detection of hsa_circ_0058040 is shown in SEQ ID NO.4, and the downstream primer sequence is shown in SEQ ID NO.5; the upstream primer sequence for the quantitative detection of hsa_circ_0096498 is shown in SEQ ID NO.6, and the downstream primer sequence is shown in SEQ ID NO.7; the upstream primer sequence for the quantitative detection of hsa_circ_0000994 is shown in SEQ ID NO.8, and the downstream primer sequence is shown in SEQ ID NO.9.

[0011] Furthermore, the prostate cancer early screening kit also includes primer pairs for amplifying the internal reference gene GAPDH and qPCR amplification reagents.

[0012] Furthermore, the upstream primer sequence for amplifying the internal reference gene GAPDH is shown in SEQ ID NO.10, and the downstream primer sequence is shown in SEQ ID NO.11.

[0013] Furthermore, the qPCR amplification reagents include SYBR Green, qPCR buffer, dNTPs, and DNA polymerase.

[0014] According to the above-described method for preparing a reagent kit for early prostate cancer screening, the preparation method specifically includes the following steps: (1) Primers were synthesized according to the sequences of SEQ ID NO.4-SEQ ID NO.11 described above, and working solutions for each primer pair were prepared with sterile water; (2) Mix SYBR Green, qPCR buffer, dNTPs and DNA polymerase in the specified proportions to prepare 2× SYBR Green qPCR premix; (3) The working solutions of each primer pair prepared in step (1) and the premixed solution prepared in step (2) are loaded into the kit to obtain the kit for early screening of prostate cancer.

[0015] The above-described reagent kit for early prostate cancer screening is used in the preparation of products for detecting prostate cancer.

[0016] Compared with the prior art, the main advantages of the present invention are as follows: This invention, through high-throughput sequencing and experimental verification, found that hsa_circ_0058040, hsa_circ_0096498, and hsa_circ_0000994 show differential expression in the serum of healthy individuals and prostate cancer patients. Furthermore, circular RNAs exhibit high stability and are not easily degraded in body fluids, making them suitable as biomarkers for prostate cancer. This technology is also applied to the preparation of a reagent kit. The kit includes primer pairs for amplifying hsa_circ_0058040, hsa_circ_0096498, hsa_circ_0000994, and the internal reference gene GAPDH. Serum mRNA is extracted and reverse transcribed into cDNA. Using the cDNA as a template, real-time quantitative PCR is used to specifically detect the expression levels of hsa_circ_0058040, hsa_circ_0096498, and hsa_circ_0000994 in serum. The combined use of these three markers provides better diagnostic efficacy for prostate cancer, enabling effective early detection of prostate cancer and improving the diagnostic accuracy of this disease, thus demonstrating broad application prospects. This reagent kit uses serum samples, and the detection method is non-invasive, convenient, and facilitates early screening. The process is fast and cost-effective. Attached Figure Description

[0017] Figure 1 To screen for differentially expressed hsa_circRNAs associated with prostate cancer; Figure 2 The relative expression level of hsa_circ_0058040 in the control group and the prostate cancer patient group; Figure 3 The relative expression level of hsa_circ_0000994 in the control group and the prostate cancer patient group; Figure 4 The relative expression levels of hsa_circ_0096498 in the control group and the prostate cancer patient group; Figure 5 ROC curve analysis was performed to assess the diagnostic value of hsa_circ_0096498, hsa_circ_0000994, and hsa_circ_0058040 alone and in combination for detecting prostate cancer. Detailed Implementation

[0018] The technical solution of the present invention will be further described below with reference to specific embodiments. However, those skilled in the art should understand that the following embodiments are only for illustrating the present invention and should not be regarded as limiting the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used are all conventional products obtained through commercial channels.

[0019] Example 1 Screening for differentially expressed hsa_circRNAs associated with prostate cancer: High-throughput sequencing was performed on the whole transcriptomes of serum samples from 50 prostate cancer patients and 50 healthy individuals to analyze the changes in the expression profiles of hsa_circRNAs in the serum of healthy individuals and prostate cancer patients, and hsa_circRNAs with significantly different fold-over expression in the serum of healthy individuals and prostate cancer patients were screened as candidate molecules.

[0020] The results are as follows Figure 1 As shown, six candidate hsa_circRNAs with significantly fold-fold differential expression associated with prostate cancer patients (hsa_circ_0000745, hsa_circ_0092339, hsa_circ_0006357, hsa_circ_0058040, hsa_circ_0096498, hsa_circ_0000994) were screened out. From these six candidate hsa_circRNAs, the candidate hsa_circ_0058040, hsa_circ_0096498, and hsa_circ_0000994 with the largest fold-fold differential expression in the serum of prostate cancer patients were further screened out as serum biomarkers for prostate cancer patients.

[0021] Example 2 Serum mRNA extraction: Serum samples were collected from 50 healthy individuals and 50 prostate cancer patients. Total RNA was extracted from the serum using a column chromatography method. The specific experimental steps are as follows: (1) Collect 250 μL of serum sample and place it in a 1.5 mL centrifuge tube. Add 750 μL of lysis buffer LB, repeatedly pipette and shake vigorously to mix. Let stand at room temperature for 10 min.

[0022] (2) Add 200 μL of chloroform, shake vigorously for 20 s to mix, let stand at room temperature for 5 min, centrifuge at 12000 rpm for 10 min to separate the sample into layers, and transfer the upper aqueous phase to a 1.5 mL centrifuge tube.

[0023] (3) Add 1.5 times the volume of anhydrous ethanol to the upper aqueous phase, mix by pipetting, transfer the mixture to an RNA purification column, centrifuge at 12000 rpm for 1 min, and discard the waste liquid.

[0024] (4) Add 700 μL of protein removal washing buffer to the RNA purification column of step (3), centrifuge at 12000 rpm for 30 s, and discard the waste liquid.

[0025] (5) Place the RNA purification column from step (4) back into the collection tube, add 500 µL of wash buffer, centrifuge at 12000 rpm at room temperature for 30 s, discard the waste liquid, and repeat this step once.

[0026] (6) Place the RNA purification column from step (5) back into the collection tube and centrifuge the empty column at 12,000 rpm for 2 min at room temperature to remove residual wash solution.

[0027] (7) Place the RNA purification column from step (6) into a new 1.5 mL centrifuge tube, add 30 µL of RNase-free H2O to the center of the RNA purification column, and incubate at room temperature for 2 min. Centrifuge at 12000 rpm for 1 min. Collect the filtrate, which is the mRNA solution. The mRNA solution can be stored at -80℃ for a long time.

[0028] Example 3 mRNA is reverse transcribed into cDNA: The mRNA solution extracted in Example 2 was reverse transcribed into cDNA using a reverse transcription kit. The reverse transcription reaction system is shown in Table 1. Using RNA as a template, 5× ABScript III RT Mix and Nuclease-free dH2O were added, gently mixed, and centrifuged. 1 μg of mRNA was reverse transcribed using a 20 μL reverse transcription system. The reverse transcription was completed by setting the reaction program on the PCR instrument. The reverse transcription reaction conditions set on the PCR instrument are shown in Table 2.

[0029] Table 1 Reverse transcription reaction system Table 2 Reverse transcription reaction conditions Example 4 Preparation of a kit for early prostate cancer screening A method for preparing a reagent kit for early prostate cancer screening, the method specifically including the following steps: (1) Primers were designed based on the nucleotide sequences of hsa_circ_0058040, hsa_circ_0096498, and hsa_circ_0000994. The nucleotide sequence of hsa_circ_0058040 is shown in SEQ ID NO.1; the nucleotide sequence of hsa_circ_0096498 is shown in SEQ ID NO.2; and the nucleotide sequence of hsa_circ_0000994 is shown in SEQ ID NO.3. The upstream primer sequence for quantitative detection of hsa_circ_0058040 is shown in SEQ ID NO.4, and the downstream primer sequence is shown in SEQ ID NO.5; the upstream primer sequence for quantitative detection of hsa_circ_0096498 is shown in SEQ ID NO.6, and the downstream primer sequence is shown in SEQ ID NO.7; the upstream primer sequence for quantitative detection of hsa_circ_0000994 is shown in SEQ ID NO.8, and the downstream primer sequence is shown in SEQ ID NO.9; the upstream primer sequence for amplifying the internal reference gene GAPDH is shown in SEQ ID NO.10, and the downstream primer sequence is shown in SEQ ID NO.11. Primers were synthesized according to the sequences SEQ ID NO.4-SEQ ID NO.11, and each primer pair was prepared to a final concentration of 0.2 μM using sterile water. Specific sequence information is as follows.

[0030] SEQ ID NO.1: ; SEQ ID NO.2: SEQ ID NO.3: Table 3 Sequence List (2) Take 100 μL of 100× SYBR Green, 1.00 mL of 10× qPCR buffer, 400 μL of 10 mMdNTPs, and 100 μL of 5 U / μL DNA polymerase, and add them to 7.30 mL of nuclease-free water. Gently invert and mix (avoid vortexing) to obtain 10 mL of 2× SYBR Green qPCR premix.

[0031] (3) Put the working solutions of each primer pair prepared in step (1) and the 2× SYBR GreenqPCR premix prepared in step (2) into the kit to obtain the early screening kit for prostate cancer.

[0032] Experimental Example 1 Real-time quantitative PCR to verify serum biomarker expression levels: Fifty cDNA samples from prostate cancer patients prepared in Example 3 were used as the control group, and 50 serum cDNA samples from healthy individuals were used as the observation group. The early prostate cancer screening kit prepared in Example 4 was used to perform real-time quantitative PCR detection on the cDNA template prepared in Example 3 to quantify the expression levels of hsa_circ_0058040, hsa_circ_0096498, and hsa_circ_0000994 in serum. The specific experimental steps are as follows: Using cDNA as a template, upstream and downstream primers for each primer pair of hsa_circ_0058040, hsa_circ_0096498, and hsa_circ_0000994 were added to the working solution, along with 2×SYBR Green qPCR premix. 20 μL of reaction solution was prepared according to the quantitative PCR reaction system shown in Table 4. The quantitative PCR reaction procedure is shown in Table 5. Each sample was divided into three replicates. GAPDH was used as an internal control. The relative expression levels of serum markers hsa_circ_0058040, hsa_circ_0096498, and hsa_circ_0000994 were calculated using the 2^(-ΔΔCt) method. Statistical analysis was performed on the quantitative PCR data. The relative expression levels of hsa_circ_0058040, hsa_circ_0096498, and hsa_circ_0000994 in serum of healthy individuals and prostate cancer patients were plotted using bar charts generated with Origin software. The results are shown below. Figure 2 , Figure 3 , Figure 4 As shown.

[0033] Table 4. Real-time PCR reaction system Table 5. Quantitative PCR reaction procedure Figure 2 , Figure 3 , Figure 4 The figures show the relative expression levels of serum biomarkers hsa_circ_0058040, hsa_circ_0000994, and hsa_circ_0096498, respectively. As the figure shows, serum biomarkers hsa_circ_0058040, hsa_circ_0096498, and hsa_circ_0000994 are expressed at low levels in the control group but at high levels in the serum of prostate cancer patients. This is consistent with high-throughput sequencing results. The expression levels of hsa_circ_0058040, hsa_circ_0096498, and hsa_circ_0000994 in serum can clearly distinguish between prostate cancer patients and healthy individuals. Therefore, hsa_circ_0058040, hsa_circ_0096498, and hsa_circ_0000994 can serve as serum biomarkers for prostate cancer patients and can provide new ideas and methods for the early prediction and diagnosis of prostate cancer.

[0034] Experimental Example 2 ROC curve assessment of the diagnostic value of serum biomarkers in prostate cancer: Based on the relative expression levels of hsa_circ_0058040, hsa_circ_0096498, and hsa_circ_0000994 obtained from Experiment 1 in prostate cancer patients and healthy individuals, statistical analysis was performed using ROC curves to evaluate the predictive ability of the selected hsa_circ_0058040, hsa_circ_0096498, and hsa_circ_0000994 closely associated with prostate cancer. ROC curves were constructed for the individual diagnosis of prostate cancer using hsa_circ_0096498, hsa_circ_0000994, and hsa_circ_0058040, as well as for the combined diagnosis of prostate cancer using hsa_circ_0096498, hsa_circ_0000994, and hsa_circ_0058040. The results are as follows: Figure 5 As shown.

[0035] Figure 5 ROC curve analysis was performed to assess the diagnostic value of hsa_circ_0096498, hsa_circ_0000994, and hsa_circ_0058040 alone and in combination for detecting prostate cancer. Figure 5 The AUC values ​​of serum biomarkers hsa_circ_0096498, hsa_circ_0000994, and hsa_circ_0058040, when used individually to detect prostate cancer, were 0.891, 0.854, and 0.813, respectively. An AUC above 0.7 indicates that the model has good classification ability and can specifically distinguish between prostate cancer patients and healthy individuals. By detecting the expression levels of hsa_circ_0058040, hsa_circ_0096498, and hsa_circ_0000994 in serum, the goal of screening for prostate cancer can be achieved. When these three biomarkers are combined to construct a diagnostic model, the combined diagnostic AUC value is significantly higher than that of any single biomarker. This indicates that combined detection can greatly improve the ability to distinguish between prostate cancer patients and healthy individuals, exhibiting superior diagnostic performance. Therefore, combined detection of these three circRNAs can serve as an effective strategy for early prostate cancer screening.

[0036] In summary, this invention used high-throughput sequencing and quantitative real-time PCR to detect and validate serum biomarkers hsa_circ_0096498, hsa_circ_0000994, and hsa_circ_0058040 in samples from prostate cancer patients. The results showed that these serum biomarkers exhibited specific high expression in prostate cancer patients, verifying a close correlation between these biomarkers and the occurrence of prostate cancer. Furthermore, the combined AUC value of these three biomarkers (hsa_circ_0096498, hsa_circ_0000994, and hsa_circ_0058040) was higher than the AUC value of any single biomarker for prostate cancer diagnosis, indicating high diagnostic value and providing important reference for the early diagnosis of prostate cancer.

[0037] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. A reagent kit for early screening of prostate cancer, characterized in that, The kit for early prostate cancer screening includes primer pairs for quantitative detection of hsa_circ_0058040, hsa_circ_0096498, and hsa_circ_0000994.

2. The reagent kit for early prostate cancer screening according to claim 1, characterized in that, The nucleotide sequence of hsa_circ_0058040 is shown in SEQ ID NO.1; the nucleotide sequence of hsa_circ_0096498 is shown in SEQ ID NO.2; and the nucleotide sequence of hsa_circ_0000994 is shown in SEQ ID NO.

3.

3. The reagent kit for early prostate cancer screening according to claim 1, characterized in that, The upstream primer sequence for the quantitative detection of hsa_circ_0058040 is shown in SEQ ID NO.4, and the downstream primer sequence is shown in SEQ ID NO.5; the upstream primer sequence for the quantitative detection of hsa_circ_0096498 is shown in SEQ ID NO.6, and the downstream primer sequence is shown in SEQ ID NO.7; the upstream primer sequence for the quantitative detection of hsa_circ_0000994 is shown in SEQ ID NO.8, and the downstream primer sequence is shown in SEQ ID NO.

9.

4. The reagent kit for early prostate cancer screening according to claim 1, characterized in that, The early prostate cancer screening kit also includes primer pairs for amplifying the internal reference gene GAPDH and 2× SYBR Green qPCR premix.

5. A reagent kit for early prostate cancer screening according to claim 4, characterized in that, The upstream primer sequence for amplifying the internal reference gene GAPDH is shown in SEQ ID NO.10, and the downstream primer sequence is shown in SEQ ID NO.

11.

6. A reagent kit for early prostate cancer screening according to claim 4, characterized in that, The 2× SYBRGreen qPCR premix includes SYBR Green, qPCR buffer, dNTPs, and DNA polymerase.

7. A method for preparing a reagent kit for early prostate cancer screening according to any one of claims 1-6, characterized in that, The preparation method specifically includes the following steps: (1) The sequence synthesis primers according to claim 3 SEQ ID NO.4-SEQ ID NO.11, and the working solutions of each primer pair were prepared with sterile water; (2) Mix SYBR Green, qPCR buffer, dNTPs and DNA polymerase in the specified proportions to prepare 2× SYBR Green qPCR premix; (3) Put the working solutions of each primer pair prepared in step (1) and the 2× SYBR Green qPCR premix prepared in step (2) into the kit to obtain the kit for early screening of prostate cancer.

8. The use of a prostate cancer early screening kit according to any one of claims 1-6 in the preparation of products for detecting prostate cancer.

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