Background
PC (short for prostate cancer) is a popular malignant tumor in men [
1,
2]. Its mortality rate ranks second in cancer in Europe and the United States. Its incidence is also the second among all malignant tumors in men worldwide [
1,
3]. For the past few years, the occurrence rate of prostate cancer in males has increased year by year, which is closely related to the continuous growth of life expectancy, the aging of the population, the change of diet structure and the continuous improvement of diagnostic techniques [
4‐
6]. Prostate cancer poses a heavily threat to the health and life of men, particularly in the middle and late stages. After endocrine therapy, most of the patients eventually progress to CRPC (castration-resistant prostate cancer), which is insensitive to radiotherapy and chemotherapy. So far, there is no effective treatment, which is a worldwide problem. At present, there are many methods for treating prostate cancer, including surgical treatment, hormone therapy, and radiation therapy [
7‐
9]. Although these treatment methods are widely used in clinical practice, there are many limitations in terms of efficacy or prognosis. Therefore, the current research hotspot is to find a biomarker with sensitivity and specificity for early diagnosis and cure of prostate cancer.
The development and progression of prostate cancer is complicated, including changes in molecular genetics and epigenetics. Research on the pathogenesis of prostate cancer has never stopped, from proteomics to genomics, from DNA to RNA, from encoding RNA to non-coding RNA [
10,
11]. Although researchers have put a lot of effort into the study of prostate cancer, the mechanism of action for the pathogenesis of prostate cancer has not been unveiled. With the advancement of science and technology, non-coding RNAs that cannot encode proteins after transcription gradually enter the field of view.
Recent studies have shown that circRNAs are a kind of single-stranded closed circular RNA, which are characterized by stable structure, tissue-specific and conservative evolution [
12,
13]. Recent studies have gradually revealed the function of circRNAs, for example, circCDR1as has about 70 miR-7 binding sites, which can regulate EGFR expression by adsorbing miR-7 [
14]. Circ_001569 can promote the invasion and proliferation of colorectal tumor cells by adsorbing mi R-145 to regulate the expression level of target E2F5 gene [
15]. In general, in recent years, it has been found that circRNAs mainly function as competitive endogenous RNA (ce RNA) or MI RNA sponge. In addition, CIRC RNAs also play a role in regulating selective splicing and gene transcription, regulating parental gene expression and transcription translation [
16,
17]. Cir-ITCH is located on human chromosome 20, 20q11.22. It is homologous to the RNA sequence of ITCH, a protein-coding gene. It usually spans 1–5 exons [
18]. Cir-ITCH was first found to be low-expressed in esophageal cancer, then in colon cancer, hepatocellular carcinoma and lung cancer [
19,
20]. At present, the mechanism of action of cir-ITCH in the progression and development of prostate cancer is still under investigation. To solve these problems, this study was set to study the effect of cir-ITCH gene on the apoptosis and proliferation of prostate cancer by detecting the expression of cir-ITCH in PC cell lines and tissues, and further interpret the functional role of cir-ITCH in regulating the apoptosis and proliferation of prostate cancer cells. To explain the pathogenesis of PC maybe offer an effective and precise therapeutic target.
Methods
Patient organization
The study was authorized by the Research Ethics Committee of The Jintan Hospital Affiliated with Jiangsu University. Informed consent was gained from all the patients. PC tissues and matched nearby normal tissues were obtained from 52 patients who received surgical resection of PC between Feb. 2014 to Apr. 2018. After excision of the prostate tissue, all tissue samples were stored in liquid nitrogen. Histopathological results validated the diagnosis of prostate cancer.
Cell culture and cell transfection
Human PC cell lines (C4-2, LNCaP, DU145, 22Rv1, PC-3 and VcaP) and human prostate epithelial cell line (RWPE-1) were obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China). Human prostate cancer cell lines were maintained in RPMI-1640 medium. Human prostate epithelial cell line (RWPE-1) was maintained in Eagle medium with 10% FBS (fetal bovine serum) (Invitrogen).
The fragment of 873 bp cDNA was inserted into a PLCDH-cir plasmid vector (Ribobio, China) to construct a circ-ITCH overexpression vector. A lentivirus was packaged by Hanbio Company (Shanghai, China). Transfection procedures were performed as described previously. The si-HOXB13, miR-17-5p mimetic and the responding vector controls (GenePharma, China) were transfected into PC cells with Lipofectamine 2000 reagents (Invitrogen, USA). The sequence of si-HOXB13 was as follows: 5′-TAAGAAAGCATGTGTGAACTA-3′.
Quantitative reverse transcription PCR (qRT-PCR)
qRT-PCR measure was applied with SYBR Premix Ex Taq reagents (TaKaRa, China). The expression levels of each gene was detected and normalized to the U6 snRNA or GAPDH using the Ct method of 2
−ΔΔ. Total RNA was enriched from PC tissues or cells lines with TRIzol reagent. cDNA (Complementary DNA) was generated according to total RNA with SuperScript III. The gene expression of circRNA/mRNA and miRNA was determined by LightCyclerTM 480 system. The internal controls of miRNA and mRNA/circRNA were U6 and GAPDH, respectively. The oligonucleotides are as shown in Table
1.
Table 1
The oligonucleotides
CircITCH | 5′-GTTGGTCAATCGCCTGCTA-3′ | 5′-GGTCCCGCACAGGGGTA-3′ |
ITCH | 5′-GGTGAGTGCTGGACGTTG-3 | 5′-TGGAAATCAGGTCTTCGTT-3′ |
miR-17-5p | 5′-CGTTTCTGTACCAAGGGGCTG-3′ | 5′-CAACGGCGGATGGGGGAAGCC-3′ |
HOXB13 | 5′-AAAGTGGCCTATGCTATCGA-3 | 5′-GCGACTTGCCAATAAGCAC-3 |
GAPDH | 5′-CGGCAGAGCGATAAGACCT-3′ | 5′-CGGCAGAGCGATAAGACCT-3′ |
U6 | 5′-ATGATGGCACTGTACTGGGCC-3′ | 5′-GATTGGCAGCGATTATACACC-3′ |
Animal research
A total of 24 Female BALB/c nude mice, 6 weeks old, were randomly grouped into 4 groups, 6 mice in each group. Prostate cells were with a transfection of control vector and circ-ITCH. Prostate cancer cells with a density of 1 × 107 tumor cells per mice were subcutaneously injected into the lateral flank of female athymic BALB/C nude mice. The size of tumor was detected twice every a week using a caliper and the formula V = (width2 × length)/2 was used. One month after the subcutaneous injection, the nude mice were sacrificed and the last total weight of tumor was detected. Animal studies were applied in strictly comply with the institutional ethics guidelines for animal experiments approved by the The Jintan Hospital Affiliated with Jiangsu University Animal Management Committee.
IHC staining: Frozen sections from mouse xenografts tumor were stained with Ki67 primary antibodies. Protein level was detected by an I-View Detection chemistry system and a NexES automated stainer (Ventana Medical Systems, AZ).
CCK-8 method
Transfected cells were placed into wells of 96-well plates at a cell density of 1 × 103 cells/well. Three wells were replicated in each group and 10 μL/well of CCK8 was added to each well of cells for the last 2 h of incubation. Finally, the OD value of the cell liquid was measured by an enzyme-linked immunosorbent assay.
The transfected cells (1 × 103 cells per well) were seeded into a 6-well plate to culture for 24 h. After the cells were exposed to ADR for 24 h, each well was washed, the culture medium was exchanged, and the cells were maintained for 8 days. Finally, colonies were counted after fixation for 10 min with 10% formaldehyde, and incubated with crystal violet (0.1%) for 10 min. Cell viability was analyzed using GraphPad Prism 6 (GraphPad Sofware, USA).
BrdU incorporation labeling
The transfected cells (1 × 103 cells per well) were seeded in a 6-well plate to adhere within 24 h. After transfection for 48 h, the cell proliferation assay was conducted with the BrdU cell growth assay kit.
Apoptosis detection
The transfected cells were adhered to the wall and washed two times with cooled PBS, then mixed up in 100 μL volume of working buffer. The assay kit of Annexin V-FITC-PI (Roche Applied Science, Germany) was utilized for cell staining following the vendor guidance. Apoptosis results were run by a FACS cytometer (Can II, BD Biosciences, USA).
Luciferase assay
The pmirGLO miRNA target expression vector containing dual luciferase was used in the 3′-untranslated region (UTR) luciferase reporter gene assay (Promega, USA). Cells were placed into a 24-well plate at a cell density of 0.4 million cells per mL, after that cells were cotransfected by Lipofectamine 2000 (Invitrogen) with HOXB13 and circITCH mimics and wild type or mutant sequences. Cells were collected 48 h after transfection, after that, a dual luciferase reporter assay system from premega was used to assess firefly or Renilla luciferase activities and calculated as ratio of firefly to Renilla luciferase activity.
Western blot
After the total protein was cleaved using a lysis buffer containing 1% PMSF, the concentration of protein was measured with a BCA Protein Detection Kit (Sigma, USA). The lysate was applied to a 12% polyacrylamide gel for electrophoresis experiments, and after electrophoresis, it was transferred to a PVDF membrane. Membranes were blocked using 5% milk and incubated with anti-HOXB13 antibody (Proteintech, Chicago, IL, USA) over a night at 4–8 °C. After that, it was incubated with anti-rabbit IgG antibody (diluted in 1/5000 n; GE, Little) for 2–3 h at 37 °C temperature. Commerical GAPDH antibody reagent (diluted in 1/2500; Bioworld) was applied as an internalcontrol standard. The kit of ECL Western blotting (Amersham, UK) was used to measure all protein bands.
Statistical method
The monitoring data were analyzed by SPSS19.0 statistical software. The data analysis data was showed as mean ± SD (mean ± standard deviation). The data analysis between the 2 groups was applied by t test. The data analysis between 2 more groups was applied with ANOVA method (one-way variance analysis), and the LSD test was conducted for the next analysis. Kaplan–Meier plot was utilized to analyze the survival curves. P < 0.05 means that there was statistically significant difference.
Discussion
Cancer of prostate is a usual tumor of the male genitourinary system, and its incidence rate is not only the highest in Europe and the United States, but also shows a significant upward trend [
21,
22]. Early hormone-dependent prostate cancer can achieve the purpose of reducing tumors and lowering blood PSA through endocrine therapy, but gradually evolves into hormone-independent prostate cancer after treatment, leading to the death of patients eventually from hormone-insensitive cells [
23,
24]. The molecular mechanism of the progression and development of hormone-independent prostate tumor has not comprehension. Therefore, it is very necessary to study the molecular mechanism of hormone-independent prostate tumorigenesis and provide a gist for clinical protection and treatment of hormone-independent prostate tumor.
Non-coding RNA (ncRNA) is a biomolecule that is ubiquitous in organisms and plays an important role in regulating life activities [
25,
26]. Circular RNA (circRNA) is a type of endogenous ncRNA molecule that is widely and diversely present in a variety of biological cells and regulates gene expression [
27,
28]. CircRNA has a key function role in gene regulation, such as miRNA adsorption as miRNA sponge, regulation of related gene expression, expression of peptides, and so on [
29,
30]. As a new regulatory endogenous RNA, circRNA has attracted more and more attention in the development of tumors and has become a new research hotspot [
31,
32]. It has been reported that many circRNAs are abnormally expressed in different tumors types such as gastric cancer, colon cancer and esophageal cancer, and exert an significant role in tumor growth, invasion and migration [
33,
34]. Studies have found that the expression levels of circ MYLK in PCA samples and PCA cells are significantly higher than that in the normal tissues and normal prostate cells. Overexpression of circRNA MYLK promotes proliferation, invasion and migration of PCA cells, while is circRNA MYLK accelerates apoptosis of PCA cells. In addition, the study has also found that si circRNA MYLK affects the function of PCA cells by targeting mir-29a [
35]. In esophageal, colon and lung cancer, cir-ITCH binds to MI R-7/mi R-17/mi R-214 through molecular sponge to form cir-ITCH-mi RNA-7/mi R-17/mi R-214-ITCH regulatory signal axis, up-regulates the expression of ITCH, promotes the ubiquitination degradation of DVL2, inhibits the classical Wnt/β-catenin signaling pathway, and thus inhibiting the growth of cancer cells [
19,
20]. Other articles have showed that the expression of cir-ITCH in bladder cancer is also down-regulated. Overexpression of cir-ITCH can suppress the growth, invasion and migration of bladder cancer cells, and further confirm that cir-ITCH targets miR-17 and miR-224, up-regulates the target genes p21 and PTEN of mi R-17 and mi R-224, thus suppressing the growth, invasion, migration and metastasis of cancer cells [
36]. In this study, it was found that the expression of cir-ITCH was reduced in breast cancer cell lines and tissues, which was consistent with the above reported results, and confirmed that its expression level was significantly correlated with preoperative PSA, Gleason score and tumor stage. Furthermore, in vivo experiments showed that circITCH overexpression inhibited tumor growth rate and tumor volume. In vitro experiments showed that up-regulation of circITCH significantly suppressed the growth, invasion and migration of prostate cancer cells and induced apoptosis. These data suggested that overexpression of circITCH can effectively delay the progression of prostate cancer.
The study found that circRNA contains a large number of MERs that act as competitive endogenous RNA (ceRNA) and become a new member of competitive endogenous RNA [
37]. How to form a regulatory network between circRNAs and miRNAs to maintain balance and regulate life activities of the organism needs further research and exploration. Some study found that using a TaqMan-based reverse transcriptase polymerase chain reaction assay, the relationship between cir-ITCH and ESCC was analyzed in a total of 684 ESCC and paired adjacent non-tumor tissue samples from eastern and southern China. We found that cir-ITCH expression was usually low in ESCC compared to the peritumoral tissue. The functional relevance of cir-ITCH was further examined by biochemical assays. As sponge of miR-7, miR-17, and miR-214, cir-ITCH might increase the level of ITCH [
20]. In recent years, the relationship between miRNAs and tumors has become a research hotspot. In general, the decline or loss of prostate cancer gene expression is considered to be a tumor inhibitor gene. The expression of microRNA17 is generally increased in cancer cells, and it is highly expressed in all detected solid tumors. It is currently believed that miRNA-17-5p is widely involved in tumor proliferation, migration, apoptosis and growth [
38]. Studies have found that patients with higher miR-17-5p expression level have lower survival rates overall and there is a highly negative relation between p130 and mir-17-5p expression [
39]. This study suggested that circITCH can bind to target miR-17-5p, and the expression level of miR-17-5p was significantly elevated in PC tissues. And a high negative relationship was found between the expression of miR-17-5p and circITCH in PC tissues. This indicated that circITCH may exert its biological function through spongy miR-17-5p, which was consistent with the literature reports.
CircITCH can act on multiple target genes like p63, p73, Notch1, Wnt/pathway [
19,
36]. A study has shown that ITCH degrades the phosphorylated form of disheveled (Dvl) via the proteasome pathway, thus inhibiting the action of the canonical Wnt pathway [
20]. The expression of HOXB13 is mostly in the posterior structure of the embryo and plays an important role in the development of the spinal cord, digestive tract and genitourinary system [
40]. HOXB13 is a recently discovered gene closely related to the growth and development of normal prostate tissue and the occurrence and progression of prostate cancer. HOXB13 also plays a variety of functions during the progression of prostate cancer [
41,
42]. On the one hand, it is believed that the high expression of HOXB13 can inhibit the activation of AR, regulate the cell cycle and inhibit the growth of prostate cancer, on the other hand, HOXB13 is thought to promote the invasion and metastasis of prostate cancer [
43]. This study indicated that miR-17-5p-overexpression reversed the increase in protein and mRNA expression yeilds of HOXB13 induced by circITCH overexpression. Furthermore, circITCH expression was positively correlated with HOXB13 expression in prostate cancer tissues. It was shown that circITCH enhanced the expression of the tumor suppressor HOXB13 by acting as a sponge of miR-17-5p in prostate cancer.
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