CN108148911B - Application of the miR-582 in diagnosis, prognosis kit and the drug for preparing prostate cancer with osseous metastasis - Google Patents

Abstract

The invention discloses the application of miR-582 in the preparation of diagnostic and prognostic kits and medicines for prostate cancer bone metastasis. The inventors unexpectedly found that miR‑582‑3p and miR‑582‑5p are underexpressed in prostate cancer tissues with bone metastases, and that miR‑582‑3p or miR‑582‑5p low expression predicts lower bone metastasis-free survival In addition, both in vitro and in vivo experiments have confirmed that overexpression of pri‑miR‑583 can upregulate miR‑582‑3p and miR‑582‑5p, and inhibit prostate cancer bone metastasis through TGF‑β signaling pathway, and prostate cancer bone metastasis cell lines migration and invasion. Therefore, based on the above findings, miR‑582 can be applied to the preparation of diagnostic and prognostic kits for bone metastasis of prostate cancer, and it is expected to develop drugs that inhibit bone metastasis of prostate cancer.

Description

miR-582 in the preparation of prostate cancer bone metastasis diagnosis, prognosis kit and medicine Applications

technical field

The invention belongs to the field of biomedicine, and more specifically relates to the application of miR-582 in the preparation of diagnostic and prognostic kits and medicines for bone metastasis of prostate cancer.

Background technique

Worldwide, prostate cancer (Prostate Cancer, PCa) is one of the most common malignant tumors in men and the second leading cause of cancer death, second only to lung cancer. The main cause of death of prostate cancer is distant metastasis of tumor, especially bone metastasis. Although primary prostate cancer can be effectively controlled by surgery, hormone therapy, radiotherapy and other means, prostate cancer with bone metastasis is still incurable. The rate is extremely low. Therefore, the early diagnosis of bone metastases is very critical for the prognosis and diagnosis and treatment of prostate cancer. At the same time, the development of therapeutic drugs for bone metastasis of prostate cancer is also very helpful to improve the survival rate of patients.

In recent years, the discovery of specific miRNAs associated with bone metastasis of prostate cancer has opened up new prospects for its diagnosis and treatment. miRNAs can inhibit the translation of mRNA or promote mRNA translation by binding to the 3' untranslated region (3'-UTR) of mRNA. degradation, thereby regulating gene expression. More and more evidence shows that miRNA plays an important role in tumor metastasis. For specific cancer types, some miRNA can inhibit tumor metastasis, and some miRNA can promote tumor metastasis. The expression profile of miRNA in malignant tumors Therefore, excavating and exploring the molecular mechanism of new miRNAs in bone metastasis of prostate cancer will provide new ideas for the prevention and targeted treatment of bone metastasis of prostate cancer.

Contents of the invention

The purpose of the present invention is to provide the application of miR-582 in the preparation of diagnostic and prognostic kits and medicines for bone metastasis of prostate cancer.

The technical scheme that the present invention takes is:

Application of a reagent for detecting miR-582 and/or its coding gene in the preparation of a prostate cancer metastasis diagnostic kit.

Preferably, the reagent is selected from one or more of primers, probes and chips.

Preferably, the miR-582 is selected from one or more of pri-miR-582, pre-miR-582, miR-582-3p, miR-582-5p and fragments or variants thereof.

Preferably, the metastasis includes bone metastasis, brain metastasis and lymph node metastasis.

Application of a reagent for detecting miR-582 and/or its coding gene in the preparation of a prognostic kit for prostate cancer.

Preferably, the miR-582 is selected from one or more of pri-miR-582, pre-miR-582, miR-582-3p, miR-582-5p and fragments or variants thereof.

Preferably, the prognostic indicator is metastasis-free survival time/survival rate, wherein metastasis includes bone metastasis, brain metastasis and lymph node metastasis.

Application of one or more of the following substances in the preparation of drugs for inhibiting prostate cancer bone metastasis: 1) substances that can up-regulate the expression of miR-582; 2) substances that can enhance the activity of miR-582; 3) substances that can increase miR- 582 substances with effective action time; 4) substances that can enhance the stability of miR-582.

Preferably, the substance capable of up-regulating the expression of miR-582 is selected from: 1) miR-582 molecules; 2) miR-582 modifiers; 3) miR-582 mimics; 4) DNA encoding miR-582; 5) Expression of miR-582 vector or virus.

Preferably, the miR-582 is selected from one or more of pri-miR-582, pre-miR-582, miR-582-3p, miR-582-5p and fragments or variants thereof.

The beneficial effects of the present invention are:

The inventors unexpectedly found that miR-582-3p and miR-582-5p were underexpressed in prostate cancer tissues with bone metastases, and that miR-582-3p or miR-582-5p underexpression predicted lower bone metastasis-free survival In addition, both in vitro and in vivo experiments have confirmed that the overexpression of pri-miR-583 can up-regulate miR-582-3p and miR-582-5p, and inhibit prostate cancer bone metastasis through TGF-β signaling pathway, and prostate cancer bone metastasis cell lines migration and invasion. Therefore, based on the above findings, miR-582 can be applied to the preparation of diagnostic and prognostic kits for bone metastasis of prostate cancer, and it is expected to develop drugs that inhibit bone metastasis of prostate cancer.

Description of drawings

Figure 1: The expression of miR-582-3p and miR-582-5p; Among them, panels A and B are the expressions of miR-582-3p and miR-582-5p in normal tissues and prostate cancer tissues in the TCGA database, respectively Figure C and Figure D are the expressions of miR-582-3p and miR-582-5p in prostate cancer bone metastasis tissue and non-bone metastasis tissue respectively; Figure E and Figure F are miR-582-3p and miR- Statistical chart of 582-5p expression level and bone metastasis status. In the figure, the group marker band L is the low expression group, band H is the high expression group, nBM means no bone metastasis, BM means bone metastasis, and ANT means normal tissue , Tumor means tumor tissue;

Figure 2: Relative expression of miR-582-3p and miR-582-5p in prostate cancer cell lines;

Figure 3: Analysis of the expression levels of miR-582-3p and miR-582-5p and the survival of prostate cancer, in which Figures A and B are the bone metastasis-free survival curves of prostate cancer, and Figures C and D are the total prostate cancer Survival rate curve; in the figure, the group mark with L is the low expression group, and the band H is the high expression group;

Figure 4: The expression levels of miR-582-3p and miR-582-5p in the three groups of prostate cancer cell lines transfected with pri-miR-582 overexpression vector, in the group marker, vector indicates that the control plasmid was transfected, pri -miR-582 means transfection of pri-miR-582 overexpression vector;

Figure 5: Overexpression of pri-miR-582 inhibits prostate cancer bone metastasis in vivo; among them, Figure A is a schematic diagram of BLI imaging, Figure B is a bone X-ray image, Figure C is a bone HE staining image, and Figure D is a bone metastasis score Results; Figure E is the BLI signal result graph; Figure F is the overall survival curve, and Figure G is the bone metastasis-free survival curve. 582 indicates inoculation of PC-3 cells transfected with pri-miR-582 overexpression vector;

Figure 6: Effects of miR-582-3p mimics and miR-582-5p mimics on the migration and invasion of three groups of prostate cancer cell lines, among which, Figure A is miR-582-3p mimics and miR-582-5p The expression levels of miR-582-3p or miR-582-5p by mimics in three groups of prostate cancer cell lines; Figure B and Figure C are the expression of miR-582-3p mimics and miR-582-5p mimics respectively. Inhibition of migration and invasion ability of prostate cancer cell lines in group group, in group markers, vector means transfected with control plasmid, miR-582-3p means transfected with miR-582-3p mimic, miR-582-5p means Transfected with miR-582-5p mimic;

Figure 7: Effects of miR-582-3p, miR-582-5p, and miR-582 on the transcriptional activity of TGF-β. Among the group markers, vector means transfected with a luciferase-labeled control plasmid, and miR-582 means transfected Transfected with luciferase-tagged miR-582 expression vector, miR-582-3p means transfected with luciferase-tagged miR-582-3p expression vector, miR-582-5p means transfected with luciferase-tagged miR -582-5p expression vector, TGF-β means transfected TGF-β/Smad luciferase reporter plasmid;

Figure 8: Bioinformatics targeting prediction results of miR-582-3p and miR-582-5p;

Figure 9: Targeting results of miR-582-3p, miR-582-5p, and miR-582 verified by luciferase;

Figure 10: TGF-β signaling pathway releases the migration and invasion ability of prostate cancer cells inhibited by pri-miR-582, where + indicates that the substance is added, - indicates that the substance is not added.

Detailed ways

The present invention will be further described below in conjunction with experiments, but is not limited thereto. The experimental methods or experimental conditions used in the following experimental examples, if no special instructions, are carried out according to conventional methods or manufacturer's instructions, the materials, reagents, etc. used in the following experimental examples, if no special instructions, can be obtained from commercial sources get.

sample source

The cell lines used in the experimental examples include human prostate cancer cell lines (22Rv1, PC-3, VCaP, DU145, LNCaP) and normal prostate epithelial cells RWPE-1, all obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences; human prostate cancer cell C4-2B was purchased from from MD Anderson Cancer Center; wherein, RWPE-1 cells were cultured with Defined Keratinocyte-SFM (1X) (Invitrogen Company), PC-3, LNCaP, and 22Rv1 cells were cultured with penicillin G (100 U/ml), streptomycin (100 mg/ml) ml) and RPMI-1640 medium (Life Technologies Company) of 10% fetal bovine serum (Life Technologies Company) were cultivated, and DU145 and VCaP cells were cultivated with DMEM medium (Invitrogen Company) containing 10% fetal bovine serum; C4-2B The cells were cultured with T medium (Invitrogen Company) containing 10% fetal bovine serum, and the above cells were all cultured under the condition of 5% CO ₂ and 37°C.

The tissue samples used in the experimental example were provided by Guangzhou First People’s Hospital, including 157 prostate cancer tissue samples obtained by surgery or needle biopsy (among them, 94 cases of non-bone metastasis prostate cancer tissues, 63 cases of bone metastasis prostate cancer tissues), the above All tissue samples have corresponding clinicopathological information.

experimental method

1. RNA extraction, reverse transcription and real-time quantitative PCR

The RNA extraction kit (Qiagen Company) was used to extract the total RNA of the tissue or cells to be tested, and the mRNA and miRNA were reverse-transcribed according to the instructions of the Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Company), using iQ SYBR Green (BIO- RAD Company) amplified and quantified the cDNA on the CFX96 system (BIO-RAD Company); U6 was used as an internal reference, and the 2-ddCt relative quantification method was selected for fluorescence quantitative detection, and miR-582-3p and miR-582-5p were calculated Among them, the primers for detecting U6, miR-582-3p and miR-582-5p were synthesized and purified by Guangzhou Ruibo Biotechnology Co., Ltd.

2. Western blotting

Use the cell separation kit (Cell Signaling Technology Company) to carry out nuclei and plasma separation of the cells to be tested, use RIPA buffer (Cell Signaling Technology Company) to extract the whole cell lysate, Westernblotting is operated according to conventional conditions, wherein, SMAD2 antibody (Cat#3195) ,, pSMAD3 antibody (Cat#4664), SMAD4 antibody (Cat#5741), TGFBRI antibody (Cat#4706), TGFBRII antibody (Cat#3950) were purchased from Cell Signaling Technology Company, PDLIM7 antibody (Cat#: SAB1406807) was purchased from Sigma-Aldrich Company, α-tubulin was used as an internal reference, and its antibody was purchased from Sigma-Aldrich Company.

3. Plasmid and transfection

Human pri-miR-582 gene was amplified from genomic DNA and cloned into pMSCV-puro retroviral vector (Clontech Company), (CAGAC)12/pGL3TGF-β/Smad luciferase reporter plasmid and control plasmid were purchased From Clontech Company, used to evaluate the transcriptional activity of TGF signaling pathway components, the 3'-UTR region of SMAD2, SMAD4, TGFBRI, TGFBRII was amplified from genomic DNA, and cloned into pmirGLO plasmid (Promega Company), provided by Guangzhou City Ribo Biotechnology Co., Ltd. synthesized and purified miR-582-3p mimics, miR-582-5p mimics, TGFBRI overexpression plasmids, SMAD2 overexpression plasmids and control vectors, and transfected with Lipofectamine 3000 (Life Technologies). 4. Luciferase detection

4×10 ⁴ cells were inoculated into 24-well plates and cultured for 24 hours, and the luciferase assay was carried out according to the conventional method. Transfection with SMAD2-3′UTR luciferase plasmid, or pmirGLO-SMAD4-3′UTR luciferase plasmid, or pmirGLO-TGFBRI-3′UTR luciferase plasmid, or pmirGLO–TGFBRII-3′UTR luciferase plasmid At the time of transfection, 5ng pRL-TK vector plasmid (Promega Company) was added at the same time, the transfection reagent was Lipofectamine 3000 (Invitrogen), and the fluorescent signal was detected 36 hours after transfection.

5. Invasion and migration experiments

Invasion and migration assays were performed using the transwell chamber method. In brief, the transwell chamber was coated with or without Matrigel (BD Biosciences), the cells were trypsinized and suspended in serum-free medium, and the upper chamber was supplemented with 1.5 ×10 ⁵ cells, the lower chamber was a culture medium containing 10% fetal bovine serum, after incubation for 24-48 hours, fixed with 4% paraformaldehyde, stained with hematoxylin, and counted under a microscope (100×).

6. Animal experiments

5-6 weeks old, 18-20 g BALB/c-nu nude mice were used for the experiment, anesthetized before the experiment, and 1×10 ⁵ (100 μL PBS) PC-3 cells were inoculated into the left ventricle. The progress of bone metastasis was monitored with a bioluminescence imaging system (BLI), and the transmission damage of osteolytic lesions was monitored with X-rays. The area of osteolytic lesions was measured with Metamorph image analysis system and software, and the degree of bone destruction in each animal was expressed as a square Bone metastases were scored according to the following criteria: 0: no metastasis; 1: bone damage covered less than 1/4 of the bone width; 2: bone damage covered 1/4 to 1/2 of the bone width; 3: bone damage covered the bone width 1/2～3/4; 4: Bone damage covers more than 3/4 of the bone width; the bone metastases score of each animal comes from the comprehensive score of the four limbs, and the signs of the animal are monitored every day, when there is 10% weight loss, paralysis, head tilt, etc. Choose euthanasia when suffering symptoms.

Experimental Example 1. Low expression of miR-582-3p and miR-582-5p in bone metastatic prostate cancer tissue

The TCGA database was used to analyze the miRNA sequences in prostate cancer patients in the early stage. The results are shown in Figure 1. Compared with normal tissues, miR-582-3p and miR-582-5p were highly expressed in prostate cancer tissues (both P<0.05, refer to Figure 1A and Figure 1B).

Further research unexpectedly found that among 149 prostate cancer tissues, miR-582-3p and miR-582-5p were significantly down-regulated in 63 prostate cancer tissues with bone metastases compared to 94 prostate cancer tissues without bone metastasis (eg 1C and 1D ), about 0.5 to 0.6 times that of prostate cancer tissue without bone metastasis.

According to the median expression of miR-582-3p and miR-582-5p in prostate cancer tissues, miR-582-3p and miR-582-5p were stratified into high expression (H) and low expression (L) , counting the cases of high and low expression of miR-582-3p and miR-582-5p in prostate cancer tissues without bone metastasis and bone metastasis, as shown in Figure 1E and Figure 1F, in prostate cancer tissues with bone metastasis, miR The low expression rates of -582-3p and miR-582-5p were significantly higher than those in prostate cancer tissues without bone metastasis. The above results proved that miR-582-3p and miR-582-5p were low expressed in prostate cancer tissues with bone metastasis.

Experimental Example 2. Low expression of miR-582-3p and miR-582-5p in prostate cancer cell lines with bone metastases

Taking prostate cancer cell lines as the research object, normal human prostate epithelial cells RWPE-1 and human prostate cancer cell lines DU145, LNCaP, 22Rv1, PC-3, VCaP, C4-2B were cultured, and miR-582-3p and miR -582-5p expression, the results are shown in Figure 2, compared with the normal human prostate epithelial cell line RWPE-1, miR-582-3p in bone metastatic prostate cancer cell lines PC-3, C4-2B, VCaP Both miR-582-5p and miR-582-5p were low-expressed, and cell experiments proved that miR-582-3p and miR-582-5p were low-expressed in bone-metastatic prostate cancer cell lines.

Experimental example 3, the low expression of miR-582-3p and miR-582-5p predicts the low bone metastasis-free survival rate of prostate cancer patients

Combined with the clinicopathological data of prostate cancer tissues, miR-582-3p and miR-582-5p were stratified into high expression levels ( H) and low expression level (L), Kaplan-Meier survival analysis was performed on the cases in each stratification, the results are shown in Figure 3, the expression levels of miR-582-3p and miR-582-5p were correlated with the total prostate cancer patients Survival was not related, however, low expression of miR-582-3p and miR-582-5p both predicted lower bone metastasis-free survival.

Experimental Example 4. Overexpression of pri-miR-582 inhibits bone metastasis of prostate cancer cells in vivo

The pri-miR-582 construct vector containing miR-582-3p and miR-582-5p sequences was transfected into three groups of bone metastasis prostate cancer cell lines (PC-3, C4-2B, VCaP), as shown in Figure 4 , compared with the control vector, the expressions of miR-582-3p and miR-582-5p were significantly upregulated in three groups of prostate cancer cell lines with bone metastases.

Further, the luciferase-labeled control vector or pri-miR-582 overexpressed PC-3 cells were inoculated into the left ventricle of nude mice, and the bone metastasis status was shown in Figure 5, wherein BLI monitored the progress of bone metastasis, X X-ray monitoring of osteolytic lesions, compared with the control vector group, the overexpression of pri-miR-582 can inhibit the bone metastasis ability of prostate cancer cells (refer to Figure 5A and 5B), and staining of tibial tumor sections showed that pri-miR-582 was overexpressed. Expression can reduce the tumor burden in bone (refer to Figure 5C), and compared with the control vector group, nude mice transfected with pri-miR-582 overexpressed PC-3 cells had lower bone metastasis score, less Osteolytic destruction, higher overall survival rate and bone metastasis-free survival rate (refer to Figure 5D-5G).

Experimental example 5, the up-regulated expression of miR-582-3p or miR-582-5p inhibits the migration and invasion ability of prostate cancer cells

The miR-582-3p mimic and miR-582-5p mimic were individually transfected into three groups of prostate cancer cell lines with bone metastases (PC-3, C4-2B, VCaP) for the evaluation of miR-582-3p or miR-582-3p The effect of miR-582-5p on the migration and invasion of prostate cancer cells, the results are shown in Figure 6, up-regulation of miR-582-3p or miR-582-5p can weaken the invasion and migration of prostate cancer cells, the above results are sufficient to prove miR-582-3p or miR-582-5p can inhibit the invasion and migration of prostate cancer cells.

Experimental example 6, the mechanism of miR-582-3p and/or miR-582-5p involved in bone metastasis of prostate cancer

In order to explore the molecular mechanism of miR-582-3p and/or miR-582-5p involved in bone metastasis of prostate cancer, we found that upregulation of miR-582-3p and/or miR-582-5p can inhibit TGF-β/Smad luciferin The transcriptional activity of the enzyme reporter plasmid (refer to FIG. 7 ), so we predict that miR-582-3p and/or miR-582-5p are involved in the TGF-β signaling pathway.

Combined with bioinformatics to predict the target sites, as shown in Figure 8, it is predicted that miR-582-3p targets the 3'-UTR of SMAD2, SMAD4, and TGFBRI; while miR-582-5p targets SMAD2, SMAD4, TGFBRI, and TGFBRII 3'-UTR.

Further verified by luciferase, the results are shown in Figure 9: Up-regulation of miR-582-3p inhibits the 3'-UTR of SMAD2, SMAD4, and TGFBRI; while up-regulation of miR-582-5p inhibits the 3'-UTR of SMAD2, TGFBRI, and TGFBRII; Simultaneous up-regulation of miR-582-3p and miR-582-5p inhibited the 3'-UTR of SMAD2, SMAD4, TGFBRI, and TGFBRII.

In addition, since overexpression of miR-582-3p and miR-582-5p can inhibit migration and invasion, we further investigated whether the TGF-β signaling pathway or its components (SMAD2 or TGFBRI) can relieve the above-mentioned inhibited migration and invasiveness. The results are shown in Figure 10. In the absence of TGF-β, neither SMAD2 nor TGFBRI can relieve the inhibition of the migration and invasion ability of prostate cancer cells. In the case of TGF-β, the migration and invasion ability of prostate cancer cells Significantly improved, the migration and invasion abilities inhibited by the overexpression of miR-582-3p and miR-582-5p were restored. The above results prove that miR-582-3p and miR-582-5p indeed inhibit the invasion and migration of prostate cancer cells through the TGF-β signaling pathway.

Summarizing the above experimental examples, it can be concluded that: (1) miR-582 can be applied to the preparation of a diagnostic kit for prostate cancer metastasis; The amount is 0.5-0.6 times that of prostate cancer tissue without bone metastasis, so it can be used as a diagnostic standard to judge whether prostate cancer metastasis occurs. 2. miR-582 can be applied to the preparation of a prostate cancer prognosis kit; since the bone metastasis-free survival rate of prostate cancer is related to miR-582-3p and miR-582-5p, the bone metastasis-free survival rate can be used as a prognostic indicator, which can be used for Prognosis. 3. Both in vivo and in vitro experiments have confirmed that the up-regulated expression of pri-miR-582 can inhibit prostate cancer bone metastasis, and inhibit the migration and invasion ability of prostate cancer bone metastasis cell lines. According to common knowledge and the above conclusions, it can be expected that any of the following All substances are expected to be used in the preparation of drugs for inhibiting prostate cancer bone metastasis: 1) substances that can up-regulate the expression of miR-582; 2) substances that can enhance the activity of miR-582; 3) substances that can increase the effective time of miR-582; 4) A substance that can enhance the stability of miR-582; wherein, the substance that can up-regulate the expression of miR-582 is selected from: 1) miR-582 molecules; 2) miR-582 modifiers; 3) miR-582 mimics; 4 ) DNA encoding miR-582; 5) vector or virus expressing miR-582.

The above is only for explaining the present invention, and it should be pointed out that for those of ordinary skill in the art, some improvements and modifications made without departing from the principles of the present invention should also be considered as protection scope of the present invention.

Claims (4)

1. the reagent for detecting miR-582 is selected from preparing the application in prostate cancer with osseous metastasis diagnostic kit, the miR-582 One or more of miR-582-3p, miR-582-5p.

2. application according to claim 1, it is characterised in that: the reagent, which is selected from, to be specific to the primer of miR-582, visits One of needle, chip are a variety of.

3. detecting application of the reagent of miR-582 in preparation prostate cancer prognosis kit, the index of the prognosis is bone-free Life span/survival rate is shifted, the miR-582 is selected from one or more of miR-582-3p, miR-582-5p.

One of substance below 4. or a variety of applications inhibited in prostate cancer with osseous metastasis drug in preparation:

1) miR-582 molecule；

2) miR-582 modifier；

3) miR-582 analogies；

4) DNA of miR-582 is encoded；

5) miR-582 carrier or virus are expressed；

The miR-582 is selected from one or more of miR-582-3p, miR-582-5p.