Application of new Lnc-FLJ in the treatment of castration-resistant prostate cancer

Abstract

The present invention belongs to the field of biomedicine technology, and relates to the application of the Lnc-FLJ gene in the treatment of castration-resistant prostate cancer. The application of the Lnc-FLJ gene inhibitor in the preparation of prostate cancer drugs. The present invention discovered for the first time that Lnc-FLJ can inhibit the proliferation and autophagy of castration-resistant prostate cancer. Lnc-FLJ is more highly expressed in castration-resistant prostate cancer cells and prostate cancer tissues with higher malignancy. Inhibiting Lnc-FLJ can inhibit the occurrence and development of castration-resistant prostate cancer. The preparation of Lnc-FLJ small molecule inhibitor shRNA is used to treat castration-resistant prostate cancer and cancer insensitive to enzalutamide, thereby inhibiting the AR signaling pathway. The present invention provides new ideas for further studying the pathogenesis of castration-resistant prostate cancer and the function of the Lnc-FLJ gene, and provides a new direction for the development of castration-resistant prostate cancer drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of Lnc-FLJ in the treatment of castration-resistant prostate cancer. Background Art

[0002] Prostate cancer is a common genitourinary malignancy in men. According to the CancerStatistics 2022 report, prostate cancer accounts for 27% of new cancer cases in men and is second only to lung cancer in deaths. The development and progression of prostate cancer is closely linked to androgens. Targeted androgen suppression, known as androgen deprivation therapy (ADA), is the most effective treatment for early-stage androgen-dependent prostate cancer. Initially, it demonstrates excellent anti-tumor effects, such as relief of cancer-related symptoms, tumor shrinkage, and decreased tumor markers. However, the vast majority of patients become insensitive to ADA within one to two years, gradually developing androgen-independent castration-resistant prostate cancer (CRPC). This is the leading cause of clinical treatment failure, tumor recurrence, metastasis, and ultimately, death. Currently, a consensus suggests that castration resistance is closely linked to the androgen receptor (AR). Reactivation of the AR signaling pathway is also a primary cause of decreased sensitivity to ADA treatment. Therefore, identifying promising therapeutic targets for CRPC is crucial.

[0003] In recent years, gene transcriptional and post-transcriptional regulatory mechanisms have been shown to play a key role in prostate cancer progression, with long noncoding RNAs (lncRNAs) playing a crucial role. lncRNAs are transcripts longer than 200 nucleotides that lack protein-coding capacity and are highly tissue- and tumor-specific. Some lncRNAs have been shown to influence prostate cancer cell biological functions, such as proliferation, apoptosis, autophagy, and invasion, playing either tumor suppressor or tumor-promoting roles. Furthermore, lncRNAs have been shown to be associated with tumor drug resistance and prognosis, potentially representing key therapeutic targets for assessing prognosis and reversing multidrug resistance in prostate cancer. Based on this, our group conducted transcriptome sequencing of hormone-sensitive and castration-resistant prostate cancer and identified a novel lncRNA, FLJ20021, associated with castration resistance in prostate cancer. We named it Lnc-FLJ. To date, the role and molecular mechanisms of Lnc-FLJ in castration resistance in prostate cancer have not been reported. Summary of the Invention

[0004] The present invention aims to address the deficiencies of the prior art by, on the one hand, providing the use of Lnc-FLJ as a molecular marker for the diagnosis of prostate cancer. On the other hand, it provides an inhibitory effect on Lnc-FLJ prepared by targeting Lnc-FLJ and using it to prepare a therapeutic drug for castration-resistant prostate cancer.

[0005] The present invention provides the following technical solutions:

[0006] On the one hand, it is to provide a gene specifically expressed in prostate cancer to mark the occurrence and development of prostate cancer, and to provide the use of Lnc-FLJ as a molecular marker for the diagnosis of prostate cancer.

[0007] Another aspect is to provide a Lnc-FLJ inhibitor, which inhibits the proliferation and autophagy of castration-resistant prostate cancer and provides a use for inhibiting castration-resistant prostate cancer from being insensitive to enzalutamide.

[0008] Further provided is a Lnc-FLJ inhibitor, which inhibits the androgen receptor (AR) signaling pathway and provides application as a drug for inhibiting the AR signaling pathway.

[0009] Preferably, the Lnc-FLJ has a nucleotide sequence.

[0010] Preferably, the Lnc-FLJ inhibitor can inhibit the shRNA sequence expressed by Lnc-FLJ, and the sense strand and antisense strand of the shRNA are respectively:

[0011] Chain of Justice:

[0012] 5'-CCGGGCTCCTTCCAGTCATTCTACACTCGAGTGTAGAATGACTGGAAGGAGCTTTTTTG-3' (SEQ ID NO. 1);

[0013] Antisense strand:

[0014] 5'-aattcaaaaaaGCTCCTTCCAGTCATTCTACACTCGAGTGTAGAATGACTGGAAGGAGC-3' (SEQ ID NO. 2).

[0015] Furthermore, the inhibitor includes but is not limited to: nucleic acid molecules, carbohydrates, lipids, small molecules, chemical drugs, antibody drugs, polypeptides, proteins or interfering lentiviruses.

[0016] Furthermore, the inhibitor is in any therapeutically acceptable dose.

[0017] Through public database analysis, the present invention discovered and verified that Lnc-FLJ is highly expressed in prostate cancer and is highly correlated with the malignancy of prostate cancer. By constructing Lnc-FLJ shRNA, the expression of Lnc-FLJ in castration-resistant prostate cancer cells was inhibited. Lnc-FLJ targeted inhibitors were used to inhibit the proliferation and autophagy of castration-resistant prostate cancer cells, further inhibiting the AR signaling pathway.

[0018] Compared with the prior art, the present invention has the following beneficial effects.

[0019] This study, published in the journal Nature Communications, reveals for the first time that Lnc-FLJ is highly expressed in prostate cancer and has prostate cancer-specific expression across a wide range of cancer types. The inhibitor provided by this study significantly inhibits the proliferation and autophagy of castration-resistant prostate cancer cells while simultaneously downregulating the AR signaling pathway. Therefore, Lnc-FLJ could serve as a new therapeutic target for castration-resistant prostate cancer. This study provides new insights into the pathogenesis of castration-resistant prostate cancer and the functions of Lnc-FLJ, and offers new directions for the development of drugs for the treatment of castration-resistant prostate cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : Figure A shows the expression of Lnc-FLJ in normal prostate tissues and prostate cancer tissues in the TCGA database; Figure B shows the expression of Lnc-FLJ in pan-cancer tissues in the TCGA database;

[0021] Figure 2 Figure A shows the expression of Lnc-FLJ in benign prostatic hyperplasia and prostate cancer tissues in paraffin tissue sections; Figure B shows the mRNA expression of Lnc-FLJ in normal prostate cells (RWPE-1), benign prostatic hyperplasia cells (BPH-1), hormone-sensitive prostate cancer cells (LNCaP), castration-resistant prostate cancer cells (22Rv1, C4-2, C4-2B), and prostate cancer cells that do not express AR (PC3, DU145); Figures C and D show the nuclear and cytoplasmic expression of Lnc-FLJ in 22Rv1 cells;

[0022] Figure 3 : Figures AB show the transfection effect of Lnc-FLJ inhibitors and their inhibitory effects on Lnc-FLJ;

[0023] Figure 4Figures AB show the changes in cell proliferation ability after Lnc-FLJ inhibition under normal culture conditions or androgen-deprived culture medium conditions (cs-FBS); Figure C shows the changes in cell colony formation ability after Lnc-FLJ inhibition under normal culture conditions or androgen-deprived culture medium conditions (cs-FBS); Figures DF show the changes in cell autophagy ability after Lnc-FLJ inhibition under normal culture conditions or androgen-deprived culture medium conditions (cs-FBS);

[0024] Figure 5 : Figures AB show the expression of AR and AR pathway-related proteins after inhibition of Lnc-FLJ; Figures CD show the expression of AR and autophagy marker protein LC3B after addition of activator DHT and inhibitor ENZA. DETAILED DESCRIPTION

[0025] The embodiments of the present invention are described below with reference to the accompanying drawings.

[0026] In the description of the embodiments of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms, "connection", and "installation" should be understood in a broad sense. For example, "connection" can be a detachable connection or a non-detachable connection; it can be a direct connection or an indirect connection through an intermediate medium. In addition, "communication" can be a direct connection or an indirect connection through an intermediate medium. Here, "fixed" means that the two are connected to each other and the relative position relationship after connection remains unchanged. The directional terms mentioned in the embodiments of the present invention, such as "inside", "outside", "top", "bottom", etc., are only reference to the directions of the accompanying drawings. Therefore, the directional terms used are for better and clearer explanation and understanding of the embodiments of the present invention, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the embodiments of the present invention.

[0027] In the embodiments of the present invention, the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of the features.

[0028] In the embodiments of the present invention, "and / or" is simply a description of the association relationship between associated objects, indicating that three possible relationships exist. For example, "A and / or B" can represent three situations: A exists alone, A and B exist simultaneously, and B exists alone. Furthermore, the character " / " in this document generally indicates that the associated objects are in an "or" relationship.

[0029] References to "one embodiment" or "some embodiments" in this specification mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in one or more embodiments of the present invention. Thus, phrases such as "in one embodiment," "in some embodiments," "in other embodiments," and "in yet other embodiments" appearing in various places in this specification do not necessarily refer to the same embodiment, but rather mean "one or more, but not all, embodiments," unless otherwise specifically emphasized. The terms "including," "comprising," "having," and variations thereof mean "including but not limited to," unless otherwise specifically emphasized.

[0030] The present invention will be further described below with reference to specific embodiments. The present invention may also be implemented or applied through other different specific implementation methods. The details in this specification may also be modified or changed in various ways based on different viewpoints and applications without departing from the present invention.

[0031] The use of Lnc-FLJ in castration-resistant prostate cancer. Through extensive and in-depth research, the present invention found that Lnc-FLJ is highly expressed in prostate cancer tissues and cells. Then, the present invention found that inhibiting Lnc-FLJ inhibited the proliferation and autophagy of prostate cancer cells. Furthermore, the present invention found that inhibiting Lnc-FLJ can downregulate the AR signaling pathway.

[0032] The experimental methods in the following examples where specific conditions are not specified are generally carried out under conventional conditions or the conditions recommended by the respective manufacturers. The reagents and drugs involved in the examples are all common commercially available products unless otherwise specified.

[0033] Example 1:

[0034] The TCGA database was used to explore the expression level of Lnc-FLJ in prostate cancer.

[0035] The expression of Lnc-FLJ in prostate cancer tissues in TCGA was queried through the UALCAN cancer database. Figure 1 As shown in A.

[0036] The expression of Lnc-FLJ in pan-cancer tissues in TCGA was queried through the GEPIA database. Figure 1 As shown in B.

[0037] The results showed that Lnc-FLJ was highly expressed in prostate cancer and had prostate cancer-specific high expression in pan-cancer.

[0038] Example 2. Prostate cancer tissue and cell verification.

[0039] Benign prostatic hyperplasia and prostate cancer tissue specimens were collected and paraffin-sectioned, and the expression of Lnc-FLJ in the tissue sections was detected by lncRNA fluorescence in situ hybridization. Figure 2 As shown in A.

[0040] RNA was extracted from normal prostate cells, benign prostatic hyperplasia cells and prostate cancer cell lines, and the expression level of Lnc-FLJ in cells was detected by real-time fluorescence quantitative PCR. Figure 2 As shown in B.

[0041] 1. lncRNA fluorescence in situ hybridization

[0042] (1) Place a cell slide at the bottom of a 24-well plate and culture an appropriate number of cells. When the confluence reaches 70%, use the Ribo™ Fluorescent In Situ Hybridization Kit (R11060.7) for cell fixation and permeabilization, probe detection, and DNA staining. After sealing, observe and photograph under a confocal microscope.

[0043] (2) Hybridization conditions are as follows: Protect from light, add 100 μl of probe hybridization solution containing the probe, and incubate at 37° overnight (14-16 hours) to ensure that the cells are fully exposed to the hybridization solution and there is no dryness of the slide.

[0044] 2. Real-time Fluorescence Quantitative PCR

[0045] (1) The primer sequences are as follows:

[0046] Lnc-FLJ upstream primer: 5'-CTCCTTCCAGTCATTCTAC-3'

[0047] Lnc-FLJ downstream primer: 5'-CTCCTCTACTTTCCTTCC-3'

[0048] β-Actin upstream primer: 5'-GGGACCTGACTGACTACCTC-3'

[0049] β-Actin downstream primer: 5'-ACGAGACCACCTTCAACTCCAC-3'

[0050] (2) Conventional cell culture, after the confluence reaches 90%, total RNA of the cells is extracted using the Trizol method, reverse transcribed into cDNA, and then subjected to real-time fluorescence quantitative PCR using SYBR Green (using β-Actin as an internal reference);

[0051] (3) The reaction conditions were as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 10 s; annealing at 59°C for 30 s; and extension at 72°C for 20 s, for a total of 39 cycles.

[0052] The results showed that Lnc-FLJ was highly expressed in tissues of prostate cancer patients, lowly expressed in normal prostate cells RWPE-1, benign prostatic hyperplasia cells BPH-1, and prostate cells PC3 and DU145 that did not express AR, and expressed in prostate cancer cells LNCaP, 22Rv1, C4-2, and C4-2B, with the highest expression in 22Rv1 cells.

[0053] Example 3. Preparation of Lnc-FLJ inhibitors.

[0054] 1. Based on the nucleotide sequence of the CDS region of Lnc-FLJ, shRNA of Lnc-FLJ was designed, as shown in SEQ ID NO.1 and SEQ ID NO.2.

[0055] 2. Lentiviral packaging of Lnc-FLJ shRNA:

[0056] (1) 293ft cells were routinely cultured in six-well plates. When the confluency reached 80%, they were packaged using the following system. Fresh medium was replaced after 24 hours, and viral supernatant was collected after 48 hours. The supernatant was sterilized with a 0.45 μm filter and stored in a -80°C refrigerator.

[0057] (2) The packaging system is as follows: target plasmid (1.8ug), auxiliary plasmid PSPAX2 (1.36ug), PMD.2G (0.44ug), Opti-MEM (200ul), Lipo2000 (8ul).

[0058] The results showed that the sh-FLJ virus suspension transfection effect was good, and the expression of Lnc-FLJ was inhibited by 30%. Figure 3 As shown in AB.

[0059] Example 4. Application of Lnc-FLJ inhibitors.

[0060] The CCK8 assay detected the effect of knocking down Lnc-FLJ on the proliferation of castration-resistant prostate cancer cells 22Rv1 under normal culture conditions or androgen-deprived culture medium (cs-FBS). Figure 4 As shown in AB.

[0061] Colony formation assay was performed to detect the effect of knockdown of Lnc-FLJ on the colony formation ability of castration-resistant prostate cancer cell 22Rv1 under normal culture conditions or androgen-deprived culture medium (cs-FBS). Figure 4 As shown in C.

[0062] Western blot experiments were performed to detect the effect of knockdown of Lnc-FLJ on the expression of autophagy-related proteins in castration-resistant prostate cancer cells 22Rv1. Figure 4 As shown in D.

[0063] Immunofluorescence experiments were used to detect the effect of knockdown of Lnc-FLJ on the activity of the autophagy marker protein LC3B in castration-resistant prostate cancer cells 22Rv1. Figure 4 As shown in E.

[0064] Transmission electron microscopy experiments were performed to observe the effect of knockdown of Lnc-FLJ on the autophagosome formation of castration-resistant prostate cancer cells 22Rv1 under normal culture conditions or androgen-deprived culture medium (cs-FBS). Figure 4 As shown in F.

[0065] 1.CCK8 Experiment

[0066] (1) Prepare single cell suspension, passage into 96-well plates, and culture in a cell culture incubator;

[0067] (2) After the cells adhered to the wall (cell confluence 50%), 10 μL of CCK-8 solution was added to each well at 0 h, 24 h, 48 h, 72 h, and 96 h respectively;

[0068] (3) Place the culture plate in an incubator and incubate for 1-4 hours;

[0069] (4) Measure the absorbance at 450 nm using an enzyme-labeled instrument.

[0070] 2. Colony Formation Assay

[0071] (1) Prepare single cell suspension, subculture into 6-well plates, and culture in a cell culture incubator;

[0072] (2) After the cells adhered (cell confluence 80%), they were manually scratched (cross) using a sterilized medium-sized pipette tip;

[0073] (3) Discard the culture medium in the wells; wash twice with PBS and discard the suspended cells;

[0074] (4) Add 2 mL of serum-free or 1% serum culture medium;

[0075] (5) Observe the scratching effect under a microscope and collect white light images at different time points (0 h, 48 h);

[0076] 3. Western blot experiment

[0077] (1) Lysis of tissues and cells;

[0078] (2) BCA protein quantification to determine the sample loading amount and prepare protein samples;

[0079] (3) SDS-polyacrylamide gel electrophoresis and membrane transfer;

[0080] (4) Closed;

[0081] (5) Incubate with primary antibody at 4°C overnight;

[0082] (6) Incubate with secondary antibody at room temperature for 1 h;

[0083] (7) ECL luminescence detection.

[0084] 4. Immunofluorescence Experiment

[0085] (1) Add 500 μl of culture medium to a 24-well plate, place the slide, and inoculate cells;

[0086] (2) After cells adhered (cell confluence 50%), wash with PBS three times;

[0087] (3) Fix with 4% pre-cooled paraformaldehyde for 15 min, wash three times with PBS, 5 min each time, on a shaking platform;

[0088] (4) Permeabilize the membrane with 0.5% Triton X-100 (in PBS) for 15 min, then wash three times with PBS, each time for 5 min, on a shaking platform;

[0089] (5) 5% BSA (bovine serum albumin, PBS) blocking for 60 min;

[0090] (6) Add primary antibody (5% BSA) and incubate at 4°C overnight;

[0091] (7) Collect the primary antibody and wash with PBS three times, 5 min each time, on a shaking table;

[0092] (8) Add secondary antibody Goat anti-rabbit IgG Ifluor 647 (1:1000) and incubate at room temperature for 60 min;

[0093] (9) Recover the secondary antibody and wash with PBS three times, 5 min each time, on a shaking table;

[0094] (10) Protect from light and stain nuclei with 0.5 μg / mL DAPI (5% BSA, 2 drops / mL) for 15 min;

[0095] (11) Wash with PBS three times, 5 min each time, on a shaking table;

[0096] (12) Take a glass slide, add 10 μL of anti-fluorescence quenching mounting medium, and cover the cell surface of the slide on the mounting medium;

[0097] (13) Observe under a confocal microscope and take pictures.

[0098] 5. Transmission Electron Microscopy Experiment

[0099] (1) Collect cells (10 cm dish) by conventional centrifugation into a centrifuge tube and discard the supernatant;

[0100] (2) Use a pipette to add a 1:5 diluted fixative (3% glutaraldehyde: 0.1 mol / L PBS buffer) along the tube wall to resuspend the cells and let stand at 4°C for 5 min;

[0101] (3) Transfer the cell suspension to a 1.5 mL EP tube and centrifuge at high speed (12000 rpm for 10 min). Gently discard the supernatant and retain the pellet.

[0102] (4) Slowly add 3% glutaraldehyde fixative along the tube wall with a pipette and place the sample in a 4°C environment;

[0103] (5) The samples were pre-fixed with 3% glutaraldehyde, re-fixed with 1% osmium tetroxide, dehydrated in acetone, embedded in Ep812, and semi-thinly sectioned and stained with toluidine blue for optical positioning. Ultra-thin sections were made with a diamond knife, stained with uranyl acetate and lead citrate, and observed and filmed using a JEM-1400 FLASH transmission electron microscope.

[0104] The results showed that the constructed Lnc-FLJ stable knockdown cell line proved that knocking down Lnc-FLJ inhibited the proliferation and autophagy activity of castration-resistant prostate cancer cells. The inhibition of proliferation activity was more significant under castration conditions, while there was no effect on autophagy activity.

[0105] Example 5. Effect of Lnc-FLJ inhibitors on AR signaling pathway.

[0106] Western blot analysis revealed changes in the expression of AR and AR signaling pathway-related proteins after knockdown of Lnc-FLJ. Figure 5 As shown in A.

[0107] Immunofluorescence detection of the changes in AR protein expression activity after knockdown of Lnc-FLJ, such as Figure 5 As shown in B.

[0108] Western blot analysis revealed changes in the expression of AR protein and autophagy marker protein LC3B after the addition of AR activator DHT and inhibitor ENZA. Figure 5 As shown in C.

[0109] Immunofluorescence detection of changes in the expression activity of the autophagy marker protein LC3B after the addition of AR activator DHT and inhibitor ENZA, such as Figure 5 As shown in D.

[0110] The results showed that the constructed Lnc-FLJ stable knockdown cell line proved that after knocking down Lnc-FLJ, the AR signaling pathway was significantly inhibited, and the regulation of autophagy depended on the AR signaling pathway.

[0111] The above are only specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. The embodiments of the present invention and the features therein can be combined with each other unless there is a conflict. Therefore, the scope of protection of the present invention shall be based on the scope of protection of the claims.

Claims

1. Use of a Lnc-FLJ gene inhibitor in the preparation of a drug for treating castration-resistant prostate cancer, characterized in that: The inhibitor is shRNA, and its sense strand and antisense strand are: Sense strand: 5′-CCGGGCTCCTTCCAGTCATTCTACACTCGAGTGTAGAATGACTGGAAGGAGCTTTTTTG-3′ (SEQ ID NO. 1); Antisense strand: 5′-aattcaaaaaaGCTCCTTCCAGTCATTCTACACTCGAGTGTAGAATGACTGGAAGGAGC-3′ (SEQ ID NO. 2).

2. The use according to claim 1, characterized in that The drug works by inhibiting the androgen receptor (AR) signaling pathway.

3. The use according to claim 1, wherein The drug works by inhibiting the proliferation and autophagy of castration-resistant prostate cancer cells.

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