CN112813166A - Prostate cancer marker and therapeutic drug thereof - Google Patents
Prostate cancer marker and therapeutic drug thereof Download PDFInfo
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Abstract
The invention provides a prostate cancer marker and a therapeutic drug thereof, belonging to the technical field of tumor biology. According to the invention, through detecting the expression quantity difference of Lnc-RP5-952N6.1 in the prostate cancer tissue and the tissue beside the cancer, the Lnc-RP5-952N6.1 is highly expressed in the prostate cancer tissue, and the expression difference of Lnc-RP5-952N6.1 in the prostate cancer tissue and the tissue beside the cancer has excellent specificity and sensitivity, so that the Lnc-RP5-952N6.1 can be used as a marker of the prostate cancer and can be used for auxiliary diagnosis of the prostate cancer.
Description
Technical Field
The invention belongs to the technical field of tumor biology, and particularly relates to a prostate cancer marker and a therapeutic drug thereof.
Background
Prostate cancer is a common urinary system malignancy in elderly men. With changes in diet and lifestyle and aging of the population, the number of prostate cancer patients is increasing, seriously threatening the health of middle-aged and elderly men. At the same time, prostate cancer also shows a tendency to be younger. Early clinical symptoms are not evident in prostate cancer patients, and patients often have an advanced visit, thereby losing the opportunity for surgical treatment. Therefore, if early diagnosis and early intervention on the progression of prostate cancer can be achieved, it would help to reduce the risk of prostate cancer to the patient and improve the therapeutic effect of the patient. However, the pathogenesis of prostate cancer is very complex, so the pathogenesis of prostate cancer in the process of occurrence and progression is not completely understood. Therefore, the research on the occurrence and development mechanism of the prostate cancer can help better diagnose and treat the prostate cancer.
Disclosure of Invention
The invention aims to provide a prostate cancer marker and a therapeutic drug thereof.
In order to achieve the purpose, the invention provides the following technical scheme:
in one aspect, the invention provides an application of a gene in prostate cancer diagnosis, wherein the gene is Lnc-RP5-952N 6.1.
Secondly, the invention provides application of a reagent in preparing a product for diagnosing prostatic cancer, wherein the reagent is a reagent capable of specifically detecting the expression product level of Lnc-RP5-952N6.1 gene.
Preferably, the reagent comprises a specific primer for detecting the expression of Lnc-RP5-952N6.1 gene.
Preferably, the upstream primer sequence of the specific primer is 5'-CCAGCTCTGGGATCAACTGG-3'; the downstream primer sequence of the specific primer is 5'-CGTTCACGTTGAGGCTGAAC-3'.
Thirdly, the invention provides application of the Lnc-RP5-952N6.1 inhibitor in preparing a prostate cancer medicament.
Preferably, the Lnc-RP5-952N6.1 inhibitor is shRNA, and the sense strand of the shRNA is:
5'-CACCGCGGCTTCCTCTAGAATAAAGCGAACTTTATTCTAGAGGAAGCCGC -3'；
the antisense strand of the shRNA is:
5'-AAAAGCGGCTTCCTCTAGAATAAAGTTCGCTTTATTCTAGAGGAAGCCGC -3'。
the invention also provides a prostate cancer medicament, which comprises an effective dose of Lnc-RP5-952N6.1 inhibitor, wherein the Lnc-RP5-952N6.1 inhibitor is shRNA, and a sense chain of the shRNA is as follows:
5'-CACCGCGGCTTCCTCTAGAATAAAGCGAACTTTATTCTAGAGGAAGCCGC -3'；
the antisense strand of the shRNA is:
5'-AAAAGCGGCTTCCTCTAGAATAAAGTTCGCTTTATTCTAGAGGAAGCCGC -3'。
8. application of Lnc-RP5-952N6.1 inhibitor in preparing prostate cancer cell proliferation inhibitor.
9. Application of Lnc-RP5-952N6.1 inhibitor in preparing prostate cancer cell metastasis inhibitor.
The invention also provides a method for screening potential treatment drugs for the prostate cancer, which comprises the following steps:
(1) treating prostate cancer cells with a drug to be screened;
(2) determining the expression of Lnc-RP5-952N6.1 in prostate cancer cells;
wherein, if the drug to be screened can reduce the expression of Lnc-RP5-952N6.1, the drug to be screened is a potential prostate cancer treatment drug.
The invention has the beneficial effects that:
the invention discovers that Lnc-RP5-952N6.1 is highly expressed in prostate cancer tissues and prostate cancer cells, and the expression difference of Lnc-RP5-952N6.1 in the prostate cancer tissues and paracarcinoma tissues has excellent AUC value, so that Lnc-RP5-952N6.1 can be used as a marker for diagnosing prostate cancer and can be used for screening candidate therapeutic drugs for the prostate cancer.
Secondly, experiments show that the Lnc-RP5-952N6.1 inhibitor can effectively inhibit the proliferation and metastasis of prostate cancer cells, so that the Lnc-RP5-952N6.1 inhibitor can be used for preparing a prostate cancer treatment drug.
Drawings
FIG. 1 expression difference between Lnc-RP5-952N6.1 in prostate cancer tissue and paracarcinoma tissue;
FIG. 2 ROC curves showing differences in Lnc-RP5-952N6.1 expression in prostate cancer tissues and paracarcinoma tissues;
FIG. 3 expression difference of Lnc-RP5-952N6.1 in different prostate cancer cells;
FIG. 4 shows the knock-down effect of sh-Lnc-RP5-952N 6.1;
FIG. 5 detection of cell proliferation by CCK-8 assay;
FIG. 6 detection of cell proliferation by EDU assay;
FIG. 7 detection of cell migration by scratch assay;
FIG. 8 results of detection of cell invasion by Transwell assay.
Detailed Description
In order to clearly illustrate the technical features of the present solution, the present solution is explained below by way of specific embodiments.
Specimen collection
30 prostate cancer tissues diagnosed in Shandong province Hospital and corresponding tissues beside the prostate cancer are collected, prostate patients collected by the method are patients diagnosed with prostate cancer through B-ultrasonic positioning prostate puncture, and the prostate cancer is pathologically confirmed after operation.
Example 1
Tissue total RNA extraction
(1) Before starting the experiment, soaking the equipment required by the experiment in 0.1 percent DEPC water overnight, and putting the equipment into an oven to be dried for later use;
(2) taking about 50mg of fresh tumor tissue and tissue beside cancer, cutting the tissue into pieces by using an ophthalmic scissors, putting the cut tissue pieces into a precooled mortar, pouring liquid nitrogen into the mortar, quickly grinding and crushing the tissue, and transferring the tissue into a new 1.5ml EP tube without RNase after grinding;
(3) adding 1ml Trizol lysate, standing at room temperature for 5min, adding 200ul chloroform, vortexing with a vortex device for 30s, and standing on ice for 10 min;
(4) placing the EP tube into a 4 ℃ centrifuge, centrifuging for 15min at 12000g, and after the centrifugation is finished, carefully transferring the supernatant into a new 1.5ml EP tube;
(5) adding isopropanol with the same volume, mixing, standing on ice for 10min, placing the EP tube into a 4 deg.C centrifuge, centrifuging at 12000g for 10min, and discarding the supernatant;
(6) adding 1ml 75% ethanol (DEPC water preparation), washing the precipitate, placing the EP tube into a 4 deg.C centrifuge, centrifuging at 7500g for 3 min;
(7) absorbing the ethanol solution, placing the opening of the centrifugal tube at room temperature, adding 50ul of RNase-free water to dissolve the RNA after the RNA precipitate is dried, and measuring the concentration and the purity of the RNA.
Total RNA reverse transcription reaction
(1) Thermal denaturation of RNA
TABLE 1 reaction System for thermal denaturation of RNA
| Reagent | Amount of the composition used |
| Total RNA | 1ul |
| Oligo(dT)20 | 1ul |
| RNase Free H2O | 10ul |
| Total Volume | 12ul |
Reaction conditions are as follows: and (3) placing the mixed solution into a PCR instrument, treating for 5min at 65 ℃, and cooling on ice.
(2) Reverse transcription reaction of RNA
TABLE 2 reverse transcription reaction System
| Reagent | Amount of the composition used |
| Reaction solution in step (1) | 12ul |
| 5×RT Buffer | 4ul |
| dNTP Mixture | 2ul |
| RNase Inhibitor | 1ul |
| ReverTra Ace | 1ul |
| Total Volume | 20ul |
Reaction conditions
At 42 ℃ for 20 min; 5min at 99 ℃; 5min at 4 ℃; storing at-20 deg.C.
Fluorescent quantitative PCR reaction detection of expression of Lnc-RP5-952N6.1
(1) Primer sequences of Lnc-RP5-952N6.1 and beta-actin
(2) Fluorescent quantitative PCR reaction system
| Reagent | Amount of the composition used |
| cDNA | 2ul |
| Forward Primer | 1ul |
| Reverse Primer | 1ul |
| SYBR qPCR Mix | 10ul |
| ROX reference dye | 0.4ul |
| RNase Free H2O | 5.6ul |
| Total Volume | 20ul |
Reaction conditions are as follows: pre-denaturation: 60s at 95 ℃; denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 60s for 40 cycles; further extension was carried out at 72 ℃ for 5 min.
According to 2-∆∆CtCalculating the relative expression amount of Lnc-RP5-952N6.1 in prostatic cancer tissues.
Wherein, the relative expression amount of Lnc-RP5-952N6.1 in the prostatic cancer tissue is 2.581 +/-0.958, and meanwhile, a plotted ROC curve shows that the AUC value of the difference expression of Lnc-RP5-952N6.1 in the prostatic cancer tissue and the paracarcinoma tissue is 0.9144, the 95% confidence interval is 0.8420 to 0.9869, and Std. Error is 0.03695.
The above results indicate that the relative expression level of Lnc-RP5-952N6.1 in prostate cancer tissues is significantly higher than that in paraneoplastic tissues, and the difference has excellent sensitivity and specificity, so that the diagnosis of prostate cancer can be assisted by comparing the expression level of Lnc-RP5-952N6.1 in prostate cancer tissues and paraneoplastic tissues.
Example 2
Detection of expression differences of Lnc-RP5-952N6.1 in prostate cancer cells
(1) RNA was extracted from BPH-1 cells, DU-145 cells, LnCap cells, PC3 cells, VCaP cells according to the method of example 1;
(2) the difference in expression of Lnc-RP5-952N6.1 in BPH-1 cells, DU-145 cells, LnCap cells, PC3 cells, VCaP cells was examined according to the method of example 1.
Wherein the relative expression quantity of Lnc-RP5-952N6.1 in DU-145 cells is 3.606 +/-0.243;
the relative expression amount of Lnc-RP5-952N6.1 in LnCap cells is 2.613 +/-0.199;
the relative expression quantity of Lnc-RP5-952N6.1 in PC3 cells is 2.332 +/-0.452;
the relative expression amount of Lnc-RP5-952N6.1 in VCaP cells is 3.023 +/-0.310;
the results show that the relative expression level of Lnc-RP5-952N6.1 in prostate cancer cells is remarkably improved compared with that of human prostate hyperplasia cells BPH-1, wherein the expression level of Lnc-RP5-952N6.1 in DU-145 cells is the highest, so that DU-145 cells are selected for subsequent research experiments.
Example 3
Design shRNA sh-Lnc-RP5-952N6.1 of Lnc-RP5-952N6.1
(1) shRNA was designed according to the sequence SEQ ID NO.5 of Lnc-RP5-952N6.1 and constructed on pENTR/U6 plasmid as follows:
Top Strand：
5'-CACCGCGGCTTCCTCTAGAATAAAGCGAACTTTATTCTAGAGGAAGCCGC -3'；SEQ ID NO.6;
Bottom Strand
5'-AAAAGCGGCTTCCTCTAGAATAAAGTTCGCTTTATTCTAGAGGAAGCCGC -3'；SEQ ID NO.7;
(2) sh-Lnc-RP5-952N6.1 was transfected into DU-145 cells to test the inhibitory effect of sh-Lnc-RP5-952N 6.1.
Wherein, in the DU-145 cell transfected with sh-Lnc-RP5-952N6.1, the relative expression quantity of Lnc-RP5-952N6.1 is 0.198 +/-0.031, which indicates that sh-Lnc-RP5-952N6.1 can effectively inhibit the expression of Lnc-RP5-952N 6.1.
Example 4
CCK-8 was used to examine the effect of sh-Lnc-RP5-952N6.1 on the proliferation of DU-145 cells
(1) DU-145 cells were inoculated in a 96-well plate one day before transfection, and after overnight culture, sh-NC and sh-Lnc-RP5-952N6.1 were transfected using Lipofectamine 2000;
(2) the OD value of 450nm is detected by using CCK-8 after 0h, 24h,48h and 72h of transfection respectively.
Wherein the OD value of sh-NC is 0.354 +/-0.024 at 24h, the OD value of sh-Lnc-RP5-952N6.1 is 0.263 +/-0.023, and P is less than 0.05;
the OD value of sh-NC at 48h is 0.533 plus or minus 0.034, the OD value of sh-Lnc-RP5-952N6.1 is 0.372 plus or minus 0.023, and P is less than 0.05;
the OD value of sh-NC at 72h is 0.789 +/-0.034, the OD value of sh-Lnc-RP5-952N6.1 is 0.590 +/-0.032, and P is less than 0.05;
the above results indicate that sh-Lnc-RP5-952N6.1 can effectively inhibit the proliferation of DU-145 cells.
Example 5
Further examination of the effect of sh-Lnc-RP5-952N6.1 on the proliferation of DU-145 cells using EDU
(1) DU-145 cells were inoculated in a 96-well plate one day before transfection, and after overnight culture, sh-NC and sh-Lnc-RP5-952N6.1 were transfected using Lipofectamine 2000;
(2) after transfection for 48h, 100ul EDU was added for incubation for 2h, after incubation, EDU was removed, and cells were washed with PBS for 2 times;
(3) adding 4% paraformaldehyde for fixing for 30min, removing paraformaldehyde, adding 100ul 0.2% glycine, incubating for 5min, and washing cells with PBS for 2 times;
(4) adding 100ul 0.5% Triton-100, treating for 10min, washing with PBS, adding 100ul Apollo, treating for 10min, and washing with PBS;
(5) adding 100ul 0.5% Triton-100 again, processing for 10min, adding 100ul methanol, cleaning for 2 times, and cleaning with PBS;
(6) DAPI staining was added for 5min, washed with PBS and photographed under a fluorescence microscope.
As shown in FIG. 6, it can be seen that EDU-staining positive cells were significantly reduced in the cells transfected with sh-Lnc-RP5-952N6.1 compared to the control group, and the results further demonstrate the inhibitory effect of sh-Lnc-RP5-952N6.1 on the proliferation of prostate cancer cells.
Example 6
Cell migration ability detection by cell scratching experiment
(1) DU-145 cells were inoculated in a 6-well plate and cultured overnight, and sh-NC and sh-Lnc-RP5-952N6.1 were transfected using Lipofectamine 2000;
(2) when the cell confluence reaches 90%, drawing a straight line at the midpoint by using a 200ul pipette tip;
(3) the exfoliated cells were washed away with PBS and after 24h, photographed and counted under an inverted microscope.
The experimental results are shown in FIG. 7, and it can be seen from the figure that sh-Lnc-RP5-952N6.1 can effectively inhibit the migration ability of prostate cancer cells.
Example 7
Transwell chamber experiment for detecting cell invasion capacity
(1) Melting the Matrigel at 4 ℃ overnight, and diluting with a precooled serum-free culture medium according to a ratio of 1: 3;
(2) adding 30ul of diluted matrigel into a chamber, slightly shaking until the matrigel is flatly paved at the bottom of the small chamber, and placing the small chamber into a cell culture box for 4 hours;
(3) preparing DU-145 cells transfected with sh-NC and sh-Lnc-RP5-952N6.1 into cell suspension, and adjusting the cell density to 5X 104Per ml, 200ul of cell suspension is added into a chamber above the chamber;
(4) adding 500ul of DMEM medium containing FBS into a 24-well plate to form a lower chamber, and then placing a Transwe chamber into the 24-well plate;
(5) culturing for 48h in a cell culture box, taking out the Transwell chamber, cleaning by using PBS, and then gently wiping off cells in the microporous membrane by using a cotton swab;
(6) fixing with paraformaldehyde, and dyeing with crystal violet after fixing;
(7) photographs were taken under an inverted microscope and the number of cells passing through the chamber was counted.
As shown in FIG. 8, it can be seen that sh-Lnc-RP5-952N6.1 can effectively inhibit the invasion ability of prostate cancer cells.
The technical features of the present invention which are not described in the above embodiments may be implemented by or using the prior art, and are not described herein again, of course, the above description is not intended to limit the present invention, and the present invention is not limited to the above examples, and variations, modifications, additions or substitutions which may be made by those skilled in the art within the spirit and scope of the present invention should also fall within the protection scope of the present invention.
Sequence listing
<110> Shandong first medical university affiliated provincial Hospital (Shandong provincial Hospital)
<120> a prostate cancer marker and therapeutic drug thereof
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cattttagct ttgccatccg cggacggctt gagtgttaac cagctcggtg gaccgtttgc 60
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tgaaactcga gaatagttat gtaaaagaga aaaaacaaag tctatcgttc aaaggctact 180
gaaccaggaa acaggcagcc cgcgttaaaa tcccagctct gggatcaact ggttagatta 240
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gaacctttac agtgttccag gtactgtttt aagagatttt ctatactcat ataacatttt 360
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gaagaccttt atgataatcc actttcactt agtgaatcgg gattggactg ctgcattcta 480
aaaataatgg tgagaagaat gaataaatta gagtttacta tgtaataaaa ccttctaaaa 540
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aaatcaacgc acaaacataa ttggtcaaag gaagaaagct acaaaaccaa ttcaataaca 840
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aacaggataa aaatggttgc attgagagtg agctgcattt tcaccaggca gagcaaactg 960
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agaaaagacc cccactggac agatctacaa agaacaacta aagagtctca tggtagaaca 1200
ggtcagctct ggacatcatt caaatccagg ggagcctctt atactggatt gcaattgtgt 1260
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Claims (10)
1. The application of a gene in prostate cancer diagnosis, wherein the gene is Lnc-RP5-952N 6.1.
2. The application of the reagent in preparing products for diagnosing the prostatic cancer is characterized in that the reagent is a reagent capable of specifically detecting the expression product level of Lnc-RP5-952N6.1 gene.
3. The use of claim 2, wherein the reagents comprise specific primers for detecting the expression of the Lnc-RP5-952N6.1 gene.
4. The use of claim 3, wherein the upstream primer sequence of the specific primer is 5'-CCAGCTCTGGGATCAACTGG-3'; the downstream primer sequence of the specific primer is 5'-CGTTCACGTTGAGGCTGAAC-3'.
5. Application of Lnc-RP5-952N6.1 inhibitor in preparing medicament for treating prostatic cancer.
6. The use of claim 5, wherein the Lnc-RP5-952N6.1 inhibitor is an shRNA, and the sense strand of the shRNA is:
5'-CACCGCGGCTTCCTCTAGAATAAAGCGAACTTTATTCTAGAGGAAGCCGC -3'；
the antisense strand of the shRNA is:
5'-AAAAGCGGCTTCCTCTAGAATAAAGTTCGCTTTATTCTAGAGGAAGCCGC -3'。
7. the prostate cancer drug is characterized by comprising an effective dose of Lnc-RP5-952N6.1 inhibitor, wherein the Lnc-RP5-952N6.1 inhibitor is shRNA, and a sense strand of the shRNA is as follows:
5'-CACCGCGGCTTCCTCTAGAATAAAGCGAACTTTATTCTAGAGGAAGCCGC -3'；
the antisense strand of the shRNA is:
5'-AAAAGCGGCTTCCTCTAGAATAAAGTTCGCTTTATTCTAGAGGAAGCCGC -3'。
8. application of Lnc-RP5-952N6.1 inhibitor in preparing prostate cancer cell proliferation inhibitor.
9. Application of Lnc-RP5-952N6.1 inhibitor in preparing prostate cancer cell metastasis inhibitor.
10. A method for screening a potential therapeutic agent for prostate cancer, comprising the steps of:
(1) treating prostate cancer cells with a drug to be screened;
(2) determining the expression of Lnc-RP5-952N6.1 in prostate cancer cells;
wherein, if the drug to be screened can reduce the expression of Lnc-RP5-952N6.1, the drug to be screened is a potential prostate cancer treatment drug.
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| CN107699564A (en) * | 2017-10-23 | 2018-02-16 | 中国科学院苏州生物医学工程技术研究所 | Long-chain non-coding RNA sequence and its application are used in human prostata cancer early diagnosis |
| CN111420058A (en) * | 2020-04-23 | 2020-07-17 | 侯本国 | Gene inhibitor for treating prostatic cancer |
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| CN105316341B (en) * | 2015-12-08 | 2018-07-06 | 浙江理工大学 | A kind of LncRNA and its application in marker or prostate cancer prognosis recurrence marker is detected as prostate cancer |
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| CN107699564A (en) * | 2017-10-23 | 2018-02-16 | 中国科学院苏州生物医学工程技术研究所 | Long-chain non-coding RNA sequence and its application are used in human prostata cancer early diagnosis |
| CN111420058A (en) * | 2020-04-23 | 2020-07-17 | 侯本国 | Gene inhibitor for treating prostatic cancer |
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| KIMURA K,ET AL: ""Homo sapiens long intergenic non-protein coding RNA 1788 (LINC01788), NR_125938，2101bp RNA linear"", 《NCBIGENBANK》 * |
| MARIA A SMOLLE，ET AL: ""Current Insights into Long Non-Coding RNAs (LncRNAs) in Prostate Cancer"", 《INT J MOL SCI》 * |
| YIN-JIE SU,ET AL: ""Circulating long noncoding RNA as a potential target for prostate cancer"", 《INT J MOL SCI》 * |
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| 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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