CN115820861A - Application of marker in preparation of prostate cancer diagnosis product - Google Patents
Application of marker in preparation of prostate cancer diagnosis product Download PDFInfo
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- CN115820861A CN115820861A CN202211524730.2A CN202211524730A CN115820861A CN 115820861 A CN115820861 A CN 115820861A CN 202211524730 A CN202211524730 A CN 202211524730A CN 115820861 A CN115820861 A CN 115820861A
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Abstract
The invention belongs to the technical field of medical biology, and particularly relates to application of a marker in preparation of a prostate cancer diagnosis product, wherein the marker is LncRNAJAZF1-AS1, and the nucleotide sequence of the marker is shown in SEQ ID No. 5. The expression level of LncRNAJAZF1-AS1 in the prostate cancer cell line is obviously reduced; indicating that LncRNAJAZF1-AS1 has expression deletion in prostate cancer cell lines to different degrees.
Description
Technical Field
The invention relates to the technical field of medical biology, in particular to application of a marker in preparation of a prostate cancer diagnosis product.
Background
Prostate cancer (PCa) is an important disease that gradually affects the health of elderly men in china. According to the data of the world cancer report in 2020, the incidence rate of Chinese prostate cancer is 15.6/10 ten thousand, the number of new cases is more than 11 ten thousand, and the number of deaths is more than 5 ten thousand, so that the incidence rate and the mortality rate of the Chinese prostate cancer show obvious rising trends. The treatment mode of early-stage prostate cancer is radical operation, but because early-stage prostate cancer has no obvious symptoms and lacks specific diagnosis indexes, most patients have middle and late-stage prostate cancer when finding the early-stage prostate cancer, and screening, early-stage diagnosis and treatment of high-risk people are effective means for improving the overall survival rate of Chinese prostate cancer patients. Therefore, the search for key molecules for early accurate diagnosis and target therapy of prostate cancer is a great need for prostate cancer patients.
Long non-coding RNA (LncRNA) is a non-coding RNA molecule with transcript length larger than 200 nt. With the progress of the functional research on LncRNA, it is shown that lncRNA participates in the biological processes of proliferation, metastasis, invasion, apoptosis and the like of PCa development by regulating and controlling multiple layers of transcription, post-transcription, translation and the like. The most widely used molecular diagnostic marker for Prostate cancer is Prostate Specific Antigen (PSA), but its specificity is poor. Therefore, there is a need to find markers that are closely related to the development of prostate cancer.
Disclosure of Invention
In order to solve the technical problems, the invention provides an application of a marker in preparing a prostate cancer diagnosis product.
The application of a marker in preparing a prostate cancer diagnosis product, wherein the marker is LncRNA JAZF1-AS1, the NCBI gene ID of the marker is 100128081, and the nucleotide sequence of the marker is shown in SEQ ID NO. 5.
Preferably, the nucleotide sequence of the specific primer for detecting LncRNAJAZF1-AS1 is shown AS SEQ ID NO.1 and SEQ ID NO. 2.
Preferably, the LncRNA JAZF1-AS1 is applied to the preparation of products for diagnosing the growth of Du145 cells.
Preferably, the LncRNAJAZF1-AS1 is applied to the preparation of products for diagnosing the growth of PC-3 cells.
A kit for diagnosis of prostate cancer, comprising a reagent for detecting the expression level of LncRNAJAZF1-AS1 in a sample.
Preferably, the sample is one of a tissue, a cell, and a body fluid.
An application of a detection reagent for detecting LncRNA JAZF1-AS1 in preparing a product for prostate cancer auxiliary diagnosis.
Compared with the prior art, the invention has the beneficial effects that:
the expression conditions of LncRNA JAZF1-AS1 in the prostate cancer cell lines DU145 and PC-3 and a normal prostate stromal cell line WPMY-1 are detected through real-time fluorescent quantitative PCR, and the expression level of LncRNAJAZF1-AS1 in the prostate cancer cell lines is found to be remarkably reduced; indicating that LncRNA JAZF1-AS1 has expression deletion in prostate cancer cell lines to different degrees.
The product for detecting the expression level of the LncRNAJAZF1-AS1 gene by real-time quantitative PCR comprises a pair of specific primers for amplifying the LncRNAJAZF1-AS1 gene. Wherein, lncRNAJAZF1-AS 1F end primer (5 '-3'): TGCCCTGGAGGATAAG; r-terminal primer (5 '-3'): ccactaaactctctccgttcca.
Drawings
Fig. 1 is a comparison of expression levels of JAZF1-AS1 in each group of cells, where x represents P <0.0001.
Detailed Description
The following detailed description of specific embodiments of the invention is provided, but it should be understood that the scope of the invention is not limited to the specific embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. The experimental methods described in the examples of the present invention are all conventional methods unless otherwise specified.
The DU145 cells, PC3 cells and WPMY-1 cells used in the examples were all from the Shanghai cell Bank of the Chinese academy. MEM medium, RPMI-1640 medium and DMEM medium were obtained from gibco.
Examples
1. Extraction of Total RNA from cells
(1) Culturing in vitro prostate cancer cells, DU145 cells in MEM medium containing 10% volume fraction of fetal calf serum and 1% volume fraction of penicillin, and PC3 cells in MEM medium containing 10% volume fraction of fetal calf serumAnd mixed solution of 1% by volume of streptomycin in RPMI-1640 culture medium, culturing WPMY-1 cells in DMEM culture medium containing 10% by volume of fetal calf serum and 1% by volume of streptomycin, and culturing the three cells at 37 deg.C, saturation humidity, and 5% by volume of CO 2 Subculturing in an incubator.
(2) The cells were cultured using 6-well plates until the cell state was good, the culture solution was discarded, and washed 3 times with 1mL of PBS buffer, 500. Mu.L of Trizol reagent was added, pipetted, mixed well, and allowed to stand at room temperature for 5min.
(3) The blended liquid was transferred to a 1.5mL centrifuge tube.
(4) Chloroform (100. Mu.L) was added to each tube in a volume ratio of 1 to Trizol, the cap was closed, and the tube was shaken vigorously for 30 seconds and then allowed to stand at room temperature for 5min.
(5) After centrifugation at 12,000g for 15min at 4 ℃ the mixture was divided into three layers, RNA was dissolved in the supernatant clear liquid phase and the supernatant was transferred to a new 1.5mL centrifuge tube.
(6) Adding isopropanol (precooled at 20 ℃) with the same volume as the Tirzol into the centrifuge tube, covering the cover tightly, turning upside down and mixing evenly for several times, and then placing for 10min at room temperature.
(7) The tube was centrifuged at 12,000g at 4 ℃ for 15min to remove the supernatant.
(8) The tubes were filled with 500. Mu.L of 75% ethanol (prepared with 0.1% DEPC-treated water), capped, shaken for 15s and allowed to stand at room temperature for 5min.
(9) Centrifuging the tube at 4 deg.C and 12,000g for 15min, and removing supernatant.
(10) Placing the centrifugal tube on a clean ultra-clean workbench for air drying for 30min, and adding 10-15 mu L of 0.1% DEPC treated water into the centrifugal tube to dissolve the RNA when no water drops are evident in the centrifugal tube.
(11) And (3) detecting the concentration and purity of the RNA by using a micro spectrophotometer, wherein the quality of the RNA is better when A260/A280 is between 1.8 and 2.0. In addition, the integrity of the RNA can be detected by agarose gel electrophoresis, and if the 28S and 18S bands of the RNA are clear, the RNA integrity is better.
(12) The RNA with better quality can be directly used for the next experiment or stored at minus 80 ℃ for a long time.
2. Reverse transcription
The reverse transcription system is as follows:
(1) Removal of genomic DNA
TABLE 1 reaction solution System for removing genomic DNA
Note: the reaction mixture was prepared in advance in the amount of reaction number +2 (excluding RNA) on ice in the above amounts, and the reaction mixture was dispensed into each reaction tube, and finally an RNA sample was added.
(2) The reaction tube is placed at room temperature and reacts for 10min to obtain the first-step reaction liquid.
(3) Reverse transcription PCR reaction
TABLE 2 reverse transcription reaction System
(4) Mixing the above dosage, and performing reverse transcription in PCR instrument at 37 deg.C, 15min,85 deg.C, 5s,4 deg.C.
The above-mentioned genomic DNA removal and reverse transcription reaction are TAKARAPrimeScript TM Reagent in RT reagent Kit with gDNA Eraser Kit.
3. Fluorescent quantitative PCR
Using GAPDH AS an internal reference, the expression of LncRNA JAZF1-AS1 in the prostate cancer cell lines DU145, PC-3 and the normal prostate stromal cell line WPMY-1 was detected by PCR reaction.
The nucleotide sequences of the specific amplification primers for JAZF1-AS1 and GAPDH are shown in Table 3.
TABLE 3 JAZF1-AS1 and Interdoped sequences
(1) Reaction system
TABLE 4 PCR reaction System
(2) Reaction conditions
TABLE 5PCR reaction conditions
After the reaction, the melting curve analysis confirmed the specificity of the PCR product, the presence or absence of a band and a primer dimer. Ct value of each obtained sample is calculated by the formula
Calculating the relative expression amount of mRNA of each sample by the method, repeating the experiment for 3 times, representing the result data in a mode of mean value plus or minus standard deviation, performing statistical analysis by SPSS 18.0 statistical software, and performing t test on the difference between the two, wherein the difference is considered as P<It is statistically significant at 0.05.
The results are shown in FIG. 1. Wherein, Δ Ct = target gene Ct value-reference gene Ct value, - Δ Δ Ct = blank control group Δ Ct average value-each sample group Δ Ct value. Note:
the method is carried out under conditions that the amplification efficiencies of the target gene and the reference gene are close to 100% and that the efficiencies are within 5% of each other.
As shown in figure 1, the qRT-PCR experiment result shows that the expression level of JAZF1-AS1 in two cancer cells is obviously reduced (p value is less than 0.0001) compared with that of normal cell WPMY-1, wherein Du145 is more obvious. Therefore, the expression level of LncRNAJAZF1-AS1 is related to the occurrence of the prostate cancer, and can be used AS a molecular marker of the prostate cancer for clinical auxiliary diagnosis of the prostate cancer.
It should be noted that when the following claims refer to numerical ranges, it should be understood that both endpoints of each numerical range and any number between the endpoints are optional, and the preferred embodiments of the present invention are described in order to avoid redundancy.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (7)
1. The application of the marker in preparing a prostate cancer diagnosis product is characterized in that the marker is LncRNAJAZF1-AS1, and the nucleotide sequence of the marker is shown AS SEQ ID NO. 5.
2. The use of a marker in the preparation of a prostate cancer diagnostic product according to claim 1, wherein the nucleotide sequence of the specific primer for detecting LncRNAJAZF1-AS1 is shown in SEQ ID No.1 and SEQ ID No. 2.
3. The use of a marker in the preparation of a prostate cancer diagnostic product AS claimed in claim 1, wherein the use of LncRNA JAZF1-AS1 in the preparation of a product for diagnosing Du145 cell growth.
4. The use of a marker according to claim 1 in the preparation of a product for diagnosing prostate cancer, wherein LncRNAJAZF1-AS1 is used in the preparation of a product for diagnosing the growth of PC-3 cells.
5. A kit for diagnosis of prostate cancer, comprising a reagent for detecting the expression level of LncRNAJAZF1-AS1 in a sample.
6. The kit for prostate cancer diagnosis according to claim 5, wherein said sample is one of a tissue, a cell, and a body fluid.
7. Use of a detection reagent for detecting LncRNAJAZF1-AS1 of claim 1 in the preparation of a product for use in the adjuvant diagnosis of prostate cancer.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106367526A (en) * | 2016-11-04 | 2017-02-01 | 叶伟亮 | Product for diagnosing prostatic cancer and application thereof |
| WO2018137435A1 (en) * | 2017-01-26 | 2018-08-02 | 上海长海医院 | Prostatic cancer marker, pcdh9, and application thereof |
| CN113462780A (en) * | 2021-07-01 | 2021-10-01 | 河南省人民医院 | Marker and kit for auxiliary diagnosis of prostate cancer |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106367526A (en) * | 2016-11-04 | 2017-02-01 | 叶伟亮 | Product for diagnosing prostatic cancer and application thereof |
| WO2018137435A1 (en) * | 2017-01-26 | 2018-08-02 | 上海长海医院 | Prostatic cancer marker, pcdh9, and application thereof |
| CN113462780A (en) * | 2021-07-01 | 2021-10-01 | 河南省人民医院 | Marker and kit for auxiliary diagnosis of prostate cancer |
Non-Patent Citations (1)
| Title |
|---|
| ZHIYUAN ZHANG等: "Construction of ceRNA network reveals potential IncRNA biomarkers in rectal adenocarcinoma", ONCOLOGY REPORTS, vol. 39, pages 2101 - 2113 * |
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