An lncRNA ENST00000510619 for the treatment of prostate cancer and its applications

Abstract

The present invention belongs to the field of biomedicine, and particularly relates to an lncRNA ENST00000510619 for the treatment of prostate cancer and its application. The cDNA sequence of lncRNA ENST00000510619 is shown as SEQ ID NO.1. The present invention discovers that the expression of lncRNA ENST00000510619 in mCRPC and mHSPC prostate cancer tissues is significantly higher than that in normal prostate tissues, and the expression of lncRNA ENST00000510619 in mCRPC prostate cancer tissues is significantly higher than that in mHSPC prostate cancer tissues; inhibiting / knocking out lncRNA ENST00000510619 can inhibit the proliferation, migration and invasion of prostate cancer cells, and enhance the sensitivity of docetaxel-induced apoptosis of prostate cancer cells. Therefore, lncRNA ENST00000510619 can be used as a therapeutic target for prostate cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to an lncRNA ENST00000510619 for the treatment of prostate cancer and its application. Background Art

[0002] Prostate cancer (PCa) ranks second in the incidence rate among men globally. Although the incidence rate of prostate cancer in China is relatively lower than that in developed countries, with the aging of the population, changes in lifestyle and eating habits, the incidence rate and mortality rate of prostate cancer in China show an increasing trend year by year, and it has become the urinary system malignant tumor with the highest incidence rate in many regions. For early-stage localized prostate cancer, radical prostatectomy is considered the most effective treatment method. However, due to the lack of obvious symptoms in early-stage prostate cancer patients, most patients are already in the advanced stage at the time of diagnosis, missing the best opportunity for surgery. The first choice for the treatment of advanced prostate cancer is endocrine therapy. However, after a period of treatment, most prostate cancers will still progress to castration-resistant prostate cancer (CRPC), with rapid progression and ultimately leading to the death of patients. Currently, the treatment methods for CRPC are limited and costly, and may even be accompanied by obvious adverse reactions. Therefore, there is an urgent clinical need to find new treatment targets and drugs for CRPC.

[0003] Long non-coding RNA (lncRNA) is a transcriptional product with a length exceeding 200 nucleotides. LncRNA does not encode proteins, but can play roles through various mechanisms, including interacting with proteins, reverse complementarity with DNA, binding to various RNAs, and playing roles such as transcriptional interference of gene clusters, epigenetic silencing, or post-transcriptional changes of target proteins. LncRNA is closely related to the occurrence and development of tumors. Research shows that lncRNA is widely involved in various cell biological functions, including cell proliferation, cell cycle, cell differentiation, apoptosis, and metabolism. However, the mechanism of action of lncRNA is complex and diverse, and the research on the exact molecular mechanism of regulating CRPC treatment is still in its infancy. Therefore, in-depth research on lncRNA may provide new ideas for the treatment of advanced prostate cancer and has important clinical significance. Summary of the Invention

[0004] The purpose of the present invention is to provide an lncRNA ENST00000510619 for the treatment of prostate cancer and its application.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions:

[0006] An lncRNA ENST00000510619 for the treatment of prostate cancer, the cDNA sequence of the lncRNA ENST00000510619 is shown in SEQ ID NO.1.

[0007] Use of a reagent for inhibiting lncRNA ENST00000510619 in at least one of the following A1)-A5):

[0008] A1) Preparing a drug for treating prostate cancer;

[0009] A2) Preparing a drug for inhibiting the proliferation of prostate cancer cells;

[0010] A3) Preparing a drug for reducing the migration ability of prostate cancer cells;

[0011] A4) Preparing a drug for reducing the invasion ability of prostate cancer cells;

[0012] A5) Preparing a drug for enhancing the sensitivity of docetaxel-induced apoptosis of prostate cancer cells;

[0013] The reagent includes siRNA for inhibiting the expression level of lncRNA ENST00000510619;

[0014] The sequence of the siRNA is:

[0015] Sense strand: 5′-GUUCUGCUCUCAUUUAUUATT-3′,

[0016] Antisense strand: 5′-UAAUAAAUGAGAGCAGAACTT-3′.

[0017] Use of a reagent for knocking out lncRNA ENST00000510619 in at least one of the following A1)-A5):

[0018] A1) Preparing a drug for treating prostate cancer;

[0019] A2) Preparing a drug for inhibiting the proliferation of prostate cancer cells;

[0020] A3) Preparing a drug for reducing the migration ability of prostate cancer cells;

[0021] A4) Preparing a drug for reducing the invasion ability of prostate cancer cells;

[0022] A5) Preparing a drug for enhancing the sensitivity of docetaxel-induced apoptosis of prostate cancer cells;

[0023] The reagent includes gRNA targeting lncRNA ENST00000510619

[0024] The target sequence of the gRNA is as follows:

[0025] gRNA-A1: 5′-TTACGCGTTGTCATTGAAAG-3′

[0026] gRNA-A2: 5′-GGAGGGCTGGAATCGAACTC-3′.

[0027] The remarkable advantages of the present invention are as follows:

[0028] LncRNA plays an important role in prostate cancer. The research of the present invention finds that lncRNA ENST00000510619 is closely related to prostate cancer. The expression of lncRNA ENST00000510619 in mHSPC and mCRPC prostate cancer tissues is significantly higher than that in normal prostate tissues, and the expression of lncRNA ENST00000510619 in mCRPC prostate cancer tissues is significantly higher than that in mHSPC prostate cancer tissues; artificially inhibiting / knocking out lncRNA ENST00000510619 can inhibit the proliferation, migration and invasion of prostate cancer cells, and enhance the sensitivity of docetaxel-induced apoptosis of prostate cancer cells. Therefore, lncRNA ENST00000510619 can be used as a therapeutic target for prostate cancer. Description of the Drawings

[0029] Figure 1 : Expression levels of lncRNA ENST00000510619 in prostate cancer tissues and normal prostate tissues. Control represents normal prostate tissues, mHSPC represents metastatic hormone-sensitive prostate cancer tissues, and mCRPC represents metastatic castration-resistant prostate cancer tissues. ** indicates P < 0.01 compared with the Control group; # indicates P < 0.05 compared with the mHSPC group.

[0030] Figure 2 : Real-time fluorescence quantitative PCR verification of si-lncRNA ENST00000510619 transfected into PC3 cells. si-NC PC3 represents PC3 cells transfected with si-NC, and si-lncRNA PC3 represents PC3 cells transfected with si-lncRNA ENST00000510619. ** indicates P < 0.01 compared with the si-NC PC3 group.

[0031] Figure 3: PCR and sequencing identification of the knockout of lncRNA ENST00000510619 in prostate cancer DU145 cells by CRISPR / Cas9 gene editing technology. A: PCR identification sites: E1 is exon 1, and E2 is exon 2. B: PCR identification results: 1 (monoclonal 1) is homozygous, 2 (monoclonal 2) is heterozygous, and WT is wild-type DU145 cells. Region1: WT 658bp, heterozygous 658bp, homozygous 0bp; Region 2: WT 164bp, heterozygous 164bp, homozygous 0bp; Region 3: WT has no band, heterozygous has no band, homozygous 359bp. C: Sequencing identification results: Compared with the original gene of wild-type DU145 cells, the gene of the knockout cells lacks a 7137bp fragment.

[0032] Figure 4 : CCK-8 assay to detect the effect of downregulating the expression of lncRNA ENST00000510619 on the proliferation ability of prostate cancer PC3 cells (A) and DU145 cells (B). A: si-NC PC3 represents PC3 cells transfected with si-NC, si-lncRNA PC3 represents PC3 cells transfected with si-lncRNA ENST00000510619, ** indicates P < 0.01 compared with the si-NC PC3 group. B: DU145-KO represents DU145 cells with lncRNA ENST00000510619 knocked out, ** indicates P < 0.01 compared with the DU145 group.

[0033] Figure 5 : Transwell assay to detect the effect of downregulating the expression of lncRNA ENST00000510619 on the invasion ability of prostate cancer PC3 cells (A) and DU145 cells (B). A: si-NC PC3 represents PC3 cells transfected with si-NC, si-lncRNAPC3 represents PC3 cells transfected with si-lncRNAENST00000510619, * indicates P < 0.05 compared with the si-NC PC3 group. B: DU145-KO represents DU145 cells with lncRNA ENST00000510619 knocked out, ** indicates P < 0.01 compared with the DU145 group.

[0034] Figure 6: Scratch assay was used to detect the effect of down - regulating the expression of lncRNA ENST00000510619 on the migration ability of prostate cancer PC3 cells (A) and DU145 cells (B). A: si - NC PC3 represents PC3 cells transfected with si - NC, si - lncRNA PC3 represents PC3 cells transfected with si - lncRNA ENST00000510619, ** indicates P < 0.01 compared with the si - NC PC3 group. B: DU145 - KO represents DU145 cells with lncRNA ENST00000510619 knocked out, * indicates P < 0.05 compared with the DU145 group.

[0035] Figure 7 : To detect the effect of knocking out lncRNA ENST00000510619 on the apoptosis of docetaxel - induced DU145 cells. DU145 - KO represents DU145 cells with lncRNA ENST00000510619 knocked out, * indicates P < 0.05 compared with the DU145 group. Detailed implementation manners

[0036] To make the content of the present invention easier to understand, the technical solutions of the present invention are further described below in conjunction with specific implementation manners, but the present invention is not limited thereto.

[0037] The cDNA sequence of lncRNA ENST00000510619 of the present invention is shown in SEQ ID NO.1.

[0038] Example 1:

[0039] 1. Tissue collection

[0040] From September 2020 to September 2022, 60 cases of prostate tissue specimens were collected, including 20 cases of metastatic hormone - sensitive prostate cancer (mHSPC), 20 cases of metastatic castration - resistant prostate cancer (mCRPC), and 20 cases of normal prostate tissue (Control). All prostate tissue specimens were pathologically confirmed. After the fresh specimens were taken out, they were immediately stored in liquid nitrogen for later use.

[0041] 2. Extraction and quality analysis of RNA samples

[0042] (1) Grind the tissue specimens after freezing in liquid nitrogen and add TransZol Up (TransGen Biotech).

[0043] (2) Transfer the lysate to a centrifuge tube, add 0.2 ml of RNA Extraction Agent, and pipette up and down repeatedly until there is no obvious precipitate in the lysate.

[0044] (3) Vortex the vortexer at room temperature for 5 min.

[0045] (4) Centrifuge at 10000×g and 4 °C for 15 min, transfer the upper colorless aqueous phase to a new centrifuge tube, add an equal volume of absolute ethanol (precipitation may occur at this time), and gently invert and mix well.

[0046] (5) Add the obtained solution and precipitate together into the centrifugal column, centrifuge at 12000×g at room temperature for 30 s, and discard the effluent.

[0047] (6) Add 500 μl of CB9, centrifuge at 12000×g at room temperature for 30 s, and discard the effluent.

[0048] (7) Repeat step (6) once.

[0049] (8) Add 500 μl of WB9, centrifuge at 12000×g at room temperature for 30 s, and discard the effluent.

[0050] (9) Repeat step (8) once.

[0051] (10) Centrifuge at 12000×g at room temperature for 2 min to completely remove the residual ethanol, and air dry for 5 min.

[0052] (11) Place the centrifugal column into an RNase-free Tube, add 40 μl of RNase-free water, let stand at room temperature for 1 min, and centrifuge at 12000×g for 1 min to elute the RNA.

[0053] (12) Repeat step (11) for a second elution.

[0054] (13) Detect the RNA concentration and identify the quality and purity of the RNA.

[0055] 3. Reverse Transcription

[0056] (1) Prepare the following reaction system in an RNase-free centrifuge tube: 600 ng of template RNA, 4 μl of 4×gDNA wiperMix, and make up to 16 μl with RNase-free ddH2O. Gently pipette and mix well, place in a thermostatic instrument, and incubate at 42 °C for 2 min.

[0057] (2) Add 4 μl of 5×HiScript II qRT SuperMix II (Vazyme Biotech) to the reaction tube in step (1). Gently pipette and mix well.

[0058] (3) Perform the reverse transcription reaction according to the following program: 50 °C for 15 min, 85 °C for 5 s.

[0059] 4. Real-time fluorescence quantitative PCR

[0060] (1) Primer design

[0061] Design real-time fluorescence quantitative PCR primers according to the lncRNA ENST00000510619 and β-actin gene sequences. The sequences are as follows:

[0062] lncRNA ENST00000510619:

[0063] Forward primer: 5’-ACATGCTTGGCCTCATTTCTCTG-3’,

[0064] Reverse primer: 5’-CTAAGGAGTGTAAGGGGCTTTGTC-3’;

[0065] β-actin:

[0066] Forward primer: 5’-GTTGTCGACGACGAGCG-3’;

[0067] Reverse primer: 5’-GCACAGAGCCTCGCCTT-3’,

[0068] (2) Prepare the following reaction system in each well of a 96-well plate: Hieff Universal Blue qPCR SYBR Green Master Mix 10 μl, forward primer 0.5 μl, reverse primer 0.5 μl, cDNA template 2 μl, make up to 20 μl with sterile ultrapure water.

[0069] (3) Place the 96-well plate in a fluorescence quantitative PCR instrument and perform real-time fluorescence quantitative PCR reaction according to the following program: 95°C for 2 min; 95°C for 10 sec, 60°C for 30 sec, 40 cycles; 95°C for 15 s, 60°C for 1 min. Set 3 replicates for each sample, and repeat all amplification reactions three times to ensure the reliability of the results. Use the 2 -ΔΔCt method to calculate the relative expression level of lncRNA ENST00000510619.

[0070] 5. Detection results

[0071] As Figure 1As shown, the expression of lncRNA ENST00000510619 in both the mHSPC group and the mCRPC group was significantly higher than that in the Control group (both P < 0.01), and there was also a significant difference in the expression of lncRNA ENST00000510619 between the mHSPC group and the mCRPC group (P < 0.05). This indicates that the high expression of lncRNA ENST00000510619 is significantly correlated with prostate cancer.

[0072] Example 2:

[0073] 1. Cell culture

[0074] Both PC3 cells (human prostate cancer cells, derived from bone metastases of prostate adenocarcinoma patients) and DU145 cells (human prostate cancer cells, derived from brain metastases of prostate adenocarcinoma patients) were gifted by the Prostate Disease Center of the Naval Medical University. PC3 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, and DU145 cells were cultured in MEM medium containing 10% fetal bovine serum. The culture conditions were: 37°C, 5% CO2. The growth of the cells was observed every day, and the medium was changed every other day.

[0075] 2. Down-regulating the expression of lncRNA ENST00000510619 in PC3 cells by siRNA technology

[0076] (1) Cell transfection

[0077] ① Design siRNA specifically targeting lncRNA ENST00000510619, namely si-lncRNA ENST00000510619, and entrust Sangon Biotech (Shanghai) Co., Ltd. to synthesize it. The control was si-NC. The sequences of si-lncRNA ENST00000510619 and si-NC are shown as follows:

[0078] si-lncRNA ENST00000510619:

[0079] Sense strand: 5'-GUUCUGCUCUCAUUUAUUATT-3',

[0080] Antisense strand: 5'-UAAUAAAUGAGAGCAGAACTT-3';

[0081] si-NC:

[0082] Sense strand: 5'-UUCUCCGAACGUGUCACGUTT-3',

[0083] Antisense strand: 5'-ACGUGACACGUUCGGAGAATT-3'.

[0084] ② The PC3 cells were divided into a negative control group (si-NC) and an experimental group (si-lncRNA ENST00000510619). According to the instructions of Hieff Liposomal Nucleic Acid Transfection Reagent (Yeasen Biotechnology), transfection was carried out. The specific steps are as follows:

[0085] 1) 24 h before transfection, observe that the PC3 cells grow and fuse to 80 - 90%. After trypsin digestion, transfer the cells into a centrifuge tube, centrifuge at 1500 rpm at room temperature for 3 min, collect the cell pellet, and resuspend it in complete medium to 1×10 5 cells / ml, and inoculate them into a 6-well plate to make the density 70 - 95% at the time of transfection.

[0086] 2) Prepare the DNA-Hieff Liposomal Nucleic Acid Transfection Reagent complex: For each well of cells, dilute 5 μl of Hieff Liposomal Nucleic Acid Transfection Reagent with 250 μl of serum-free MEM medium, and incubate at room temperature for 5 min. Additionally, dilute 100 pmol of siRNA with 250 μl of serum-free MEM medium and mix well.

[0087] 3) Mix the diluted siRNA and Liposomal Nucleic Acid Transfection Reagent in equal volumes (total volume 500 μL), gently mix, and incubate at room temperature for 20 min to form the DNA-liposome complex.

[0088] 4) Remove the growth medium from each well, replace it with 2 mL of serum-free medium, add 500 μL of the DNA-liposome complex to each well, shake the culture plate, and gently mix.

[0089] 5) Replace with growth medium 4 - 6 h after transfection.

[0090] 6) Incubate the cells in an incubator at 37 °C and 5% CO2 for 48 h.

[0091] (2) Verification of the transfection efficiency of si-lncRNA ENST00000510619

[0092] ① Total RNA extraction

[0093] 1) 48 h after transfection of prostate cancer cells in each group, add 1 ml of TransZolUp (TransGen Biotech) to each well of cells in the 6-well plate, place it horizontally for 2 min to evenly distribute the lysate on the cell surface and lyse the cells, and then use a pipette to blow and beat the cells to make them detach.

[0094] 2) Transfer the cell lysate into a centrifuge tube, add 0.2 ml of RNA Extraction Agent, and pipette up and down repeatedly until there is no obvious precipitate in the lysate.

[0095] 3) Vortex at room temperature for 5 min.

[0096] 4) Centrifuge at 10000×g for 15 min at 4 °C. Transfer the upper colorless aqueous phase to a new centrifuge tube, add an equal volume of absolute ethanol (precipitation may occur at this time), and gently invert and mix well.

[0097] 5) Add the obtained solution and precipitate together into the centrifugal column, centrifuge at 12000×g for 30 s at room temperature, and discard the effluent.

[0098] 6) Add 500 μl of CB9, centrifuge at 12000×g for 30 s at room temperature, and discard the effluent.

[0099] 7) Repeat step 6) once.

[0100] 8) Add 500 μl of WB9, centrifuge at 12000×g for 30 s at room temperature, and discard the effluent.

[0101] 9) Repeat step 8) once.

[0102] 10) Centrifuge at 12000×g for 2 min at room temperature to completely remove the residual ethanol. Air dry for 5 min.

[0103] 11) Place the centrifugal column into an RNase-free Tube, add 40 μl of RNase-free water, let stand at room temperature for 1 min, and centrifuge at 12000×g for 1 min to elute the RNA.

[0104] 12) Repeat step 11) for secondary elution.

[0105] 13) Detect the RNA concentration and identify the yield and purity of the RNA.

[0106] ② Reverse transcription and real-time fluorescence quantitative PCR: The implementation steps and conditions are the same as those in Example 1.

[0107] 3. Construction of prostate cancer DU145 cells with lncRNA ENST00000510619 knockout by CRISPR / Cas9 gene editing technology

[0108] (1) Target design

[0109] Using lncRNA ENST00000510619 as the transcript, design 1 target each upstream and downstream of exon1 and exon2 (select the guide RNA with fewer off-targets as the target):

[0110] gRNA-A1: 5'-TTACGCGTTGTCATTGAAAG-3',

[0111] gRNA-A2: 5'-GGAGGGCTGGAATCGAACTC-3'.

[0112] Design primers to sequence the genomic sequences near the gRNA targeting sites to confirm the genotype of prostate cancer DU145 cells.

[0113] (2) Preparation of knockout materials

[0114] Synthesize gRNA materials and Cas9 protein according to the design scheme.

[0115] (3) Cell transfection

[0116] 1) The instrument used for electroporation is Neon TM Transfection instrument (MPK5000), and the experimental operation is carried out strictly according to the official website instructions.

[0117] 2) Take the target cells in the logarithmic growth phase with good state, digest, centrifuge to remove the supernatant, resuspend with PBS, count, and take 1×10 6 cells into an EP tube, and centrifuge to obtain cell pellets.

[0118] 3) Incubate and combine gRNA and Cas9 protein to form an RNP complex, and resuspend the cells as a suspension.

[0119] 4) Input the optimal electroporation parameters, perform electroporation on the above mixture, and perform electroporation on the EGFP control group under the same parameters.

[0120] 5) Transfer the electroporated cells in each group to the corresponding culture vessels respectively, place them in an incubator for culture, observe the cell state after 24 h, and judge the electroporation efficiency of this time according to the situation of the EGFP control group.

[0121] (4) Monoclonalization

[0122] 1) Digest the cells, count, and inoculate monoclonal cells.

[0123] 2) Observe the growth state of the clones during this period and change the medium in time.

[0124] 3) Wait until the clones grow to a sufficient number, backup and send for PCR testing after that.

[0125] (5) PCR identification

[0126] Extract the genomic DNA of the clone samples for PCR detection to identify the gRNA targeting sites and knockout bands. The identified sites are as Figure 3As shown in A. According to the two exons of lncRNA ENST00000510619, primers at three sites were designed respectively. The upstream primer of Region 1 is before the knockout region, and the downstream primer is within the knockout region; the upstream primer of Region 2 is within the knockout region, and the downstream primer is after the knockout region; the upstream primer of Region 3 is before the knockout region, and the downstream primer is after the knockout region. The primer sequences are shown as follows:

[0127]

[0128] Preliminarily identify the monoclonal cell lines with successful knockout and send them for sequencing.

[0129] (6) Sequencing identification

[0130] After the sequencing results were compared by Snapgene software, the monoclonal cell lines with successful knockout were determined.

[0131] (7) Positive clone amplification

[0132] Amplify and culture the monoclonal cell lines with successful knockout.

[0133] 4. Experimental results

[0134] (1) Down-regulation of lncRNA ENST00000510619 expression in PC3 cells by siRNA technology and identification: The results of real-time fluorescence quantitative PCR showed ( Figure 2 ), compared with si-NC PC3 cells, the mRNA expression level of lncRNA ENST00000510619 in si-lncRNA PC3 cells was significantly decreased (P<0.01), indicating that in this example, PC3 cells with down-regulated lncRNA ENST00000510619 expression were successfully constructed by transfecting si-RNA ENST00000510619.

[0135] (2) Knockout of lncRNA ENST00000510619 in prostate cancer DU145 cells by CRISPR / Cas9 gene editing technology and identification: The PCR identification results are as Figure 3 shown in B. Since the Region 1 primer and Region 2 are primers designed across the knockout region, some sequences in the homozygote (knockout cells) were knocked out, so they could not be amplified and there was no band; the Region 3 primer spanned the entire knockout region (7137bp). Because the knockout fragment was deleted in the homozygote, it could be amplified successfully (359bp), while the wild type had a complete sequence (7478bp), and this fragment was too large to be amplified successfully, so there was no band. The sequencing results of the preliminarily identified monoclonal cell lines with successful knockout showed ( Figure 3C). After knocking out, compared with the wild-type DU145 cells, the gene sequence was missing 7137 bp. Combining the results of PCR identification and sequencing identification, it was determined that the lncRNA ENST00000510619 was successfully knocked out.

[0136] Example 3:

[0137] 1. Detection of cell proliferation ability by CCK-8 method

[0138] (1) Respectively, the control group PC3 cells (si-NC PC3), PC3 cells transfected with si-lncRNA ENST00000510619 (si-lncRNA PC3), control group DU145 cells (DU145), and DU145 cells with lncRNA ENST00000510619 knocked out (DU145-KO) were digested and centrifuged sufficiently, and then resuspended and counted with 1 ml of cell culture medium containing 10% fetal bovine serum. 100 μl of cell suspension was inoculated into 96-well plates, about 5×10 3 cells per well (each group of cells was inoculated in 3 replicate wells).

[0139] (2) Add 100 μl of PBS to the other wells without cells in the periphery of the 96-well plate.

[0140] (3) Incubate in an incubator at 37 °C and 5% CO2 for 48 h.

[0141] (4) Take out the 96-well plate and add 10 μl of CCK-8 reagent (ApexBio Technology) to each well.

[0142] (5) Incubate the culture plate in the incubator for 1 h.

[0143] (6) Gently mix on a shaker. Then use an enzyme-linked immunosorbent assay (ELISA) reader (BioTek, VT, USA) to measure the absorbance at 450 nm. The cell survival rate (%) = [(absorbance of experimental wells - absorbance of blank wells) / (absorbance of control wells - absorbance of blank wells)] × 100% was calculated.

[0144] 2. Experimental results

[0145] As Figure 4 shown, compared with the control group PC3 cells, the cell viability of the si-lncRNA PC3 group decreased significantly; compared with the control group DU145 cells, the cell viability of the DU145-KO group decreased significantly. It was shown that inhibiting the expression of lncRNA ENST00000510619 or knocking out lncRNA ENST00000510619 could both inhibit the proliferation of prostate cancer cells.

[0146] Example 4:

[0147] 1. Detection of the effect of down-regulating the expression of lncRNA ENST00000510619 on the invasiveness of prostate cancer cells by Transwell assay (1) Dilute Matrigel (MedChemExpress) with serum-free medium at a ratio of 1:8. Take 100 μL and spread it on the inside of the insert, covering the bottom of the insert completely. Incubate it in an incubator at 37 °C and 5% CO2 for 4 h.

[0148] (2) After digesting and centrifuging the control group PC3 cells (si-NC PC3), PC3 cells transfected with si-lncRNA ENST00000510619 (si-lncRNA PC3), control group DU145 cells (DU145), and DU145 cells with lncRNA ENST00000510619 knocked out (DU145-KO) sufficiently, resuspend them with 1 ml of serum-free cell culture medium and count. Adjust the cell concentration to 5×10 5 / ml.

[0149] (3) Take 200 μL of the cell suspension and inoculate it into the upper chamber of an 8-μm pore size Transwell insert (Corning, NY, USA). Add 500 μL of cell culture medium containing 10% fetal bovine serum to the lower chamber of a 24-well plate, then place the upper chamber into the well plate and incubate it in an incubator at 37 °C and 5% CO2 for 24 h.

[0150] (4) Discard the medium of the non-migrated cells in the upper layer, wash it twice with PBS, add methanol for fixation for 15 min to fix the cells.

[0151] (5) Discard the liquid in the lower chamber, wash it twice with PBS, add 0.1% crystal violet for staining for 10 min.

[0152] (6) Wash the insert, gently wipe the upper chamber with a cotton swab to remove the crystal violet not bound to the cells.

[0153] (7) Randomly select 5 fields of view under the microscope to observe and count the cells.

[0154] 2. Experimental results

[0155] As Figure 5 shown, compared with the control group PC3 cells, the number of cells passing through the membrane in the si-lncRNA PC3 group was significantly reduced; compared with the control group DU145 cells, the number of cells passing through the membrane in the DU145-KO group was also significantly reduced. It indicates that inhibiting the expression of lncRNA ENST00000510619 or knocking out lncRNA ENST00000510619 can both inhibit the invasive ability of prostate cancer cells.

[0156] Example 5:

[0157] 1. Detect the effect of down-regulating the expression of lncRNA ENST00000510619 on the migration ability of prostate cancer cells by scratch test. (1) After fully digesting and centrifuging the control group PC3 cells (si-NC PC3), PC3 cells transfected with si-lncRNA ENST00000510619 (si-lncRNA PC3), control group DU145 cells (DU145), and DU145 cells with lncRNA ENST00000510619 knocked out (DU145-KO), resuspend them with 1 ml of complete cell culture medium and count, and adjust the cell concentration to 5×10 5 / ml.

[0158] (2) Seed the cells on a 6-well plate and culture them in an incubator at 37°C and 5% CO2 for 24 h;

[0159] (3) Use a 200 μL pipette tip against the lid of the 6-well plate or a ruler to make a vertical scratch;

[0160] (4) Wash the cells 3 times with PBS to remove the scratched cells, and add serum-free medium;

[0161] (5) Observe and take pictures under an inverted microscope at 0 h and 12 h respectively, ensure that the scratch is centered and vertical, and pay attention to the consistent background.

[0162] (6) After opening the picture with Image J software, randomly draw 6 horizontal lines and calculate the average value of the cell-to-cell distance.

[0163] (7) Calculate the cell migration rate. Cell migration rate = (initial scratch width - cell-to-cell distance after 12 hours) / initial scratch width × 100%.

[0164] 2. Experimental results

[0165] As Figure 6 shown, compared with the control group PC3 cells, the cell migration rate in the si-lncRNA PC3 group decreased significantly; compared with the control group DU145 cells, the cell migration rate in the DU145-KO group also decreased significantly. It indicates that inhibiting the expression of lncRNA ENST00000510619 or knocking out lncRNA ENST00000510619 can both inhibit the migration ability of prostate cancer cells.

[0166] Example 6:

[0167] 1. Detect the effect of knocking out lncRNA ENST00000510619 on docetaxel-induced apoptosis of prostate cancer cells by flow cytometry

[0168] (1) DU145 cells (control group) and DU145 cells with lncRNA ENST00000510619 knocked out (DU145-KO group) were respectively seeded in 6-well plates and cultured in an incubator at 37 °C and 5% CO2 for 24 h;

[0169] (2) Docetaxel with a final concentration of 10 μmol / L was added to the two groups of cells and cultured for another 48 h;

[0170] (3) After digesting the two groups of cells with trypsin, 10 5 cells were collected, centrifuged at 2000 rpm for 3 min, the supernatant was discarded, the cells were resuspended and washed with pre-cooled PBS, centrifuged at 2000 rpm for 3 min, the supernatant was discarded, and the precipitate was retained.

[0171] (4) 195 μl of Binding Buffer was added to resuspend the cells;

[0172] (5) 5 μl of Annexin V-FITC was added and mixed evenly;

[0173] (6) 10 μl of PI Stain was added;

[0174] (7) Incubate in the dark at room temperature for 15 min, and invert several times during incubation to enhance the staining effect;

[0175] (8) Detect by flow cytometry. Set three groups of controls: unstained group, PI single-stained group and Annexin V-FITC single-stained group.

[0176] 2. Experimental results

[0177] As Figure 7 shown, compared with the control group of DU145 cells, the apoptosis rate (early apoptosis + late apoptosis) of DU145-KO group cells increased significantly [(24.01 ± 3.16)% vs (17.04 ± 1.82)%, t = 3.313, P = 0.03], and the difference was statistically significant, indicating that knocking out lncRNA ENST00000510619 can enhance the sensitivity of prostate cancer cells to docetaxel-induced apoptosis.

[0178] The above are only the preferred embodiments of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

Claims

1. Use of a reagent for inhibiting lncRNA ENST00000510619 in the preparation of a medicament for treating prostate cancer, characterized in that: The cDNA sequence of the lncRNA ENST00000510619 is shown as SEQ ID NO.1; the reagent includes siRNA that inhibits the expression level of lncRNA ENST00000510619; the sequence of the siRNA is: sense strand: 5′-GUUCUGCUCUCAUUUAUUATT-3′, antisense strand: 5′-UAAUAAAUGAGAGCAGAACTT-3′.

2. The application according to claim 1, wherein: The drug is used for inhibiting the proliferation of prostate cancer cells, reducing the migration ability of prostate cancer cells, reducing the invasion ability of prostate cancer cells, or enhancing the sensitivity of docetaxel-induced apoptosis of prostate cancer cells.

3. Use of a reagent for knocking out lncRNA ENST00000510619 in the preparation of a drug for treating prostate cancer, characterized in that: The cDNA sequence of the lncRNA ENST00000510619 is shown as SEQ ID NO.1; the reagent includes gRNA targeting lncRNA ENST00000510619 and Cas9 protein; the target sequences of the gRNA are: gRNA-A1: 5′-TTACGCGTTGTCATTGAAAG-3′ and gRNA-A2: 5′-GGAGGGCTGGAATCGAACTC-3′.

4. The application according to claim 3, characterized in that: The drug is used for inhibiting the proliferation of prostate cancer cells, reducing the migration ability of prostate cancer cells, reducing the invasion ability of prostate cancer cells, or enhancing the sensitivity of docetaxel-induced apoptosis of prostate cancer cells.

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