CN111793694A

CN111793694A - Use of circ CDK13 in prostate cancer bone metastasis

Info

Publication number: CN111793694A
Application number: CN202010844605.4A
Authority: CN
Inventors: 黄帅; 林卓远; 梁宝霞; 瓦庆德; 唐欲博; 谢尚延
Original assignee: Second Affiliated Hospital of Guangzhou Medical University
Current assignee: Second Affiliated Hospital of Guangzhou Medical University
Priority date: 2020-08-20
Filing date: 2020-08-20
Publication date: 2020-10-20
Anticipated expiration: 2040-08-20
Also published as: CN111793694B

Abstract

The invention finds that the circ CDK13 is remarkably and highly expressed in prostatic cancer bone metastasis tissues and cells, the circ CDK13 is up-regulated to promote the proliferation of prostatic cancer bone metastasis cells, and the circ CDK13 targets miR-505-3p, so that the invention provides the application of the circ CDK13 as a molecular marker and a drug target of prostatic cancer bone metastasis.

Description

Use of circ CDK13 in prostate cancer bone metastasis

Technical Field

The invention belongs to the field of biomedicine, and more particularly relates to application of circ CDK13 in prostate cancer bone metastasis.

Background

Prostate cancer (PCa) is one of the most prone tumors to bone metastasis, and accounts for the first malignancy in american men, second only to lung cancer. In China, along with the change of the nutritional structure and the aging trend, the morbidity and mortality of PCa have a remarkable increasing trend. After bone metastasis occurs in tumors, bone-related complications such as pain, pathological fractures, hypercalcemia, spinal cord compression, loss of motor function and the like are accompanied, and finally, the death of the patient is caused. At present, the treatment aiming at the bone metastasis is palliative, and no matter the treatment is surgery, radiotherapy and drug treatment, the corresponding symptoms are only relieved and improved, and the survival expectation of patients cannot be improved. Therefore, once bone metastasis occurs, not only the quality of life and the life span of a patient are seriously reduced, but also great difficulty is brought to clinical treatment. Different from developed countries in the western world, the stage of prostate cancer is late in the discovery of the prostate cancer in China, the tumor is locally or remotely transferred, and the low-differentiation tumor has high occupation ratio. There is no definite molecular mechanism to elucidate prostate cancer metastasis, nor is there a relevant target to effectively predict prostate cancer metastasis. Therefore, the potential target of the PCa bone metastasis is searched, so that a new specific prediction factor and a potential therapeutic target are provided for the bone metastasis of the PCa patient, and the method has important practical significance and clinical significance.

Circular RNA (circular RNA) was first reported abroad in the 70's 20 th century, circular RNA (circular RNA), or circular RNA, is a recently identified class of specific non-coding RNA (ncRNA) molecules, and is usually produced by a specific alternative splicing mechanism and comprises more than one exon which is circularly formed to form circular RNA, which exists in almost all species and in a large amount in the cytoplasm of eukaryotic cells. circRNA plays an important regulatory role in disease, and it is currently most recognized that circular RNA regulates the expression level of miRNA targets by binding to miRNA to block the inhibitory effect of the latter on target RNA. Research has shown that circular RNA plays an important role in the development and progression of atherosclerosis, nervous system diseases, diabetes and tumors. There are no reports on the relationship between circ CDK13 and PCa bone metastasis.

Disclosure of Invention

The invention aims to provide the application of circ CDK13 in prostate cancer bone metastasis.

The technical scheme adopted by the invention is as follows:

use of a reagent for detecting a molecular marker which is circ CDK13 in the manufacture of a diagnostic tool for bone metastases from prostate cancer.

Preferably, wherein the reagents comprise primers or probes for detecting circ CDK 13.

Preferably, the reagent is used for detecting prostate cancer tissue samples.

Use of an agent which down-regulates the expression of circ CDK13 in the manufacture of a medicament for the prevention or treatment of prostate cancer bone metastases.

Preferably, wherein the agent comprises an siRNA against circ CDK13, an antisense oligonucleotide or an agent that knocks out or knocks out a circCDK13 gene.

Preferably, wherein the agent is capable of inhibiting the proliferation of bone metastases from prostate cancer.

Use of an agent that blocks the binding of circ CDK13 to miR-505-3p in the preparation of a medicament for preventing or treating prostate cancer bone metastasis.

The invention has the beneficial effects that:

Drawings

FIG. 1 shows the expression of circ CDK13 in prostate cancer bone metastasis (PCa/BM) and bone metastasis free (PCa/nBM) tissues;

FIG. 2 is a graph of circ CDK13 expression in a plurality of prostate cancer cells;

FIG. 3 is a graph of the proliferation of prostate cancer cells overexpressing circ CDK 13;

FIG. 4 shows the results of the analysis of the gene chip;

significant statistical differences are indicated in the above figures.

Detailed Description

The invention will now be further illustrated, but is not limited, by the following specific examples.

The experimental methods and experimental conditions used in the following examples were carried out by a conventional method or according to manufacturer's instructions unless otherwise specified, and materials, reagents and the like used in the following experiments were commercially available unless otherwise specified.

The term "non-bone metastasis" or "bone metastasis free" in the medical field means that no bone metastasis occurs, and generally, a collected tumor sample is analyzed and classified as a bone metastasis free tumor sample if the tumor sample does not have bone metastasis, and it is understood by those skilled in the art that the bone metastasis free tumor sample may be a tumor sample in which no metastasis occurs, and may also be a tumor sample in which other non-bone metastasis (e.g., liver, brain, lung) occurs.

Example 1, circ CDK13 was relatively highly expressed in PCa bone metastasis tissue

The samples used in this example were from the second hospital affiliated to Guangzhou university of medical science, and included 31 prostate cancer tissues (of which, 17 non-bone metastatic prostate cancer tissues and 14 bone metastatic prostate cancer tissues).

Cell lines used in this example 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 Chinese academy; human prostate cancer cell C4-2B was purchased from MD anderson cancer center; among them, RWPE-1 cells were cultured with refined Keratinocyte-SFM (1X) (Invitrogen Co.), PC-3, LNCaP, 22Rv1 cells were cultured with RPMI-1640 medium (Life Technologies Co.) containing penicillin G (100U/ml), streptomycin (100mg/ml) and 10% fetal bovine serum (Life Technologies Co.), and DU145, VCaP cells were cultured with DMEM medium (Invitrogen Co.) containing 10% fetal bovine serum; C4-2B cells were cultured in T medium (Invitrogen) containing 10% fetal bovine serum, all at 5% CO₂And culturing at 37 ℃.

RNA was extracted using Trizol reagent, 2. mu.g of RNA was subjected to reverse transcription, and after completion of the reaction, cDNA was stored at-20 ℃ for future use. SYBR Green mix (Roche) required for DNA amplification and related primers were prepared according to the kit instructions. The amplification reaction conditions were set according to the kit instructions in a Bio-rad real-time quantitative PCR apparatus, and 40 cycles of reactions were performed. The U6 gene was used as an internal control, and at least 3 auxiliary wells were set for all samples. The relative expression quantity of the target gene DNA is obtained by Schmitgen and Livak2^-ΔΔCtAnd calculating by using a formula.Primers for detection of circ CDK13 (see hsa _ circ _0079928 for molecular details) and U6 were designed, synthesized and purified by lebo biotechnology limited, guangzhou.

As shown in fig. 1, circ CDK13 was significantly highly expressed in the tissues with bone metastasis compared to the tissues without bone metastasis, and the results in fig. 2 showed significantly high expression of circ CDK13 in prostate cancer cells, with the highest expression of PC-3 cells.

In conclusion, the circ CDK13 can be used as a molecular marker for diagnosing bone metastasis of the prostate cancer and used for guiding the development of diagnostic reagents for the bone metastasis of the prostate cancer.

Example 2 circ CDK13 promotion of proliferation of PCa bone metastasis cells

The human circ CDK13 gene was cloned into pMSCV-puro retroviral vector (Clontech Co.) to obtain a circ CDK13 expression plasmid, and the negative control plasmid or the circ CDK13 expression plasmid, respectively, was transfected into PC-3 or C4-2B cells using Lipofectamine 3000(Life Technologies Co.).

Cell proliferation assay was performed on constructed PC-3 cells and C4-2B cells, and after digesting the cells with 0.25% trypsin, the cell concentration was 3X 10⁴Each cell/mL was inoculated uniformly in a 96-well plate at 100. mu.L per well for 0 day, 3 days, and 5 days by adding 10. mu.L of MTT reagent, incubating at 37 ℃ in a thermostat for 4 hours, adding 150ul of dimethyl sulfoxide per well, and measuring the absorbance A (490nm) of each well with a multifunctional microplate reader to observe the cell proliferation with time.

The results are shown in figure 3, where high expression of circ CDK13 promotes proliferation of PCa bone metastasis cells.

Example 3 targeting of circ CDK13 miR-505-3p

Our previous studies have confirmed the relationship between miR-505-3p and bone metastasis of prostate cancer (Downalignment of miR-505-3p precursors boiler metastasis-free clearance in pro state cancer), and the present invention further found that circ CDK13 targets miR-505-3p by gene chip analysis (FIG. 3).

Taken together with the results of examples 2 and 3, it can be seen that circ CDK13 plays a role in promoting bone metastasis of prostate cancer by targeting miR-505-3p, and therefore, a substance that down-regulates circ CDK13 or blocks the binding of circ CDK13 to miR-505-3p can conceivably play a role in preventing or treating prostate cancer or its metastasis, for guiding the development of an agent for preventing or treating bone metastasis of prostate cancer.

Claims

1. Use of a reagent for detecting a molecular marker which is circ CDK13 in the manufacture of a diagnostic tool for bone metastases from prostate cancer.

2. Use according to claim 1 wherein the reagent comprises a primer or probe for detecting circ CDK 13.

3. Use according to claim 1, wherein the reagent is for detecting prostate cancer tissue samples.

4. Use of an agent which down-regulates the expression of circ CDK13 in the manufacture of a medicament for the prevention or treatment of prostate cancer bone metastases.

5. Use according to claim 4 wherein the formulation comprises an siRNA, antisense oligonucleotide or knock-out formulation of a circ CDK13 gene directed against circ CDK 13.

6. The use of claim 4, wherein the agent is capable of inhibiting the proliferation of bone metastases from prostate cancer.

7. Use of an agent that blocks the binding of circ CDK13 to miR-505-3p in the preparation of a medicament for preventing or treating prostate cancer bone metastasis.