Clinical values of expression signature of circCDR1AS and circHIAT1 in prostate cancer: Two circRNAs with regulatory function in androgen receptor (AR) and PI3K/AKT signaling pathways

Abstract Background Prostate cancer (PCa) is a genetically heterogeneous disease with highly molecular aberrations. It has been revealed that a newly discovered class of non‐coding RNAs called circular RNAs (circRNAs) play key roles in dictating tumor behaviors and phenotypes of the prostate tumors. In the current study, our aim was to determine the expression profiles of circHIAT1 and circCDR1AS in PCa compared with benign prostatic hyperplasia (BPH) tissues, as well as their clinicopathological relevance. Methods The 50 prostate tissues including 25 PCa tissues and 25 BPH samples were collected for analyzing the expression levels of target circRNAs by quantitative real‐time PCR (qRT‐PCR). Results The results revealed that expression of circCDR1AS was significantly elevated in PCa compared with the BPH (p < 0.05). We also observed that PCa patients over the age of 60 had a higher expression of the circCDR1AS than patients under the age of 60 (p = 0.017). Moreover, a lower expression level of circHIAT1 was found in the PCa than BPH tissues (p < 0.05), and finally, the findings indicated that the area under the curve (AUC) of circCDR1AS was 0.848, with 92% sensitivity and 76% specificity, as well as an AUC of 0.828, with the 80% sensitivity and 76% specificity for circHIAT1. Conclusion These observations suggest that the abnormal expression of circCDR1AS and circHIAT1 can be regarded as two different types of molecular pathology with potential biomarker values for PCa, although further studies are needed.


| INTRODUC TI ON
Prostate cancer (PCa) is of great clinical importance in terms of both its prevalence and mortality in men globally. 1 Despite the significant advances that have been made in the diagnosis and treatment of this cancer, it still has heavy costs on the health systems of different countries in the world. 2 The phases of formation, development, and progression of PCa are driven by heterogeneous molecular changes that phenotypically dictate the heterogeneous behaviors and pathophysiological features of prostate tumors. 3 On the contrary, there is limited evidence for the early and accurate detection of PCa. 4 Therefore, it seems that the best goal to find new and more specific and sensitive diagnostic, therapeutic and prognostic biomarkers is to better understand the molecular pathophysiology of this cancer by revealing tumor-specific genetic changes and linking them to emerging phenotypes. 3,5 Thanks to advanced expression analysis technologies, it has been revealed that one of the key such molecular changes that play important roles in the pathophysiology of cancers is the abnormal expression of a newly discovered class of non-coding RNA (ncRNAs) called circular RNAs (circRNAs); however, little is known about their roles in PCa. 6,7 In this regard, there are some clues that abnormalities in the circRNAs can manifest themselves through dysregulating the important signaling pathways involved in the development of tumorigenesis, such as androgen receptor (AR) and PI3K/AKT pathways. 8,9 The AR and PI3K/AKT are the most commonly deregulated pathways in PCa. [10][11][12] The abnormal implication of the AR signaling in PCa is executed via modulating the cell proliferation, survival, and invasion in early and late tumors. 12 In addition, intensification and activation of signaling from the PI3K/AKT pathway in PCa have been shown to lead to resistant, metastatic, and aggressive phenotypes in prostate tumors by regulating the metabolism, survival, growth, progression, and cytoskeleton reorganization of tumor cells. 13 There is also evidence of reciprocal regulatory interaction between members of these two signaling pathways, which has been shown to play an important role in PCa development and progression. 12 Accordingly, the key to these two pathways in PCa has led to the development of some potent AR and PI3K/AKT targeted drug treatments. 12 Interestingly, there is evidence that two key circRNAs, circHIAT1 and circCDR1AS, play important roles in tumor development by controlling AR and PI3K/AKT signaling pathways, respectively. 14,15 For example, published data have shown that circCDR1AS is overexpressed in gastric cancer and hepatocellular carcinoma (HCC) and through regulating some target genes in the PI3K/AKT pathway, promotes cell proliferation and invasion of the tumor cells. 15,16 In the case of PCa, only data on the circCDR1AS are available, and a new study on the PCa cell lines has revealed that this circRNA plays a prominent role in the invasion and migration tumor cells by suppressing miR-641. 17 Structurally, circCDR1AS is a highly conserved single-chain circular RNA with length of 1485 nucleotide and without 5' cap and 3' poly-A tail structure. 18,19 CDR1as shows tissue-time specific expression, and because of its regulatory strong effects on the miR-7, it is co-expressed with this miRNA in the brain. 19,20 Under physiological conditions, it has the highest expression levels in brain tissue and spinal cord, and the lowest in the lungs, muscles, and heart. 18 Pathologically, changes in its expression also occur in some diseases, including the pulmonary fibrosis, myocardial infarction, and cancers mentioned above. [21][22][23] The circHIAT1 has a spliced sequence length of 807 nucleotides that is transcribed from its host gene called hippocampus abundant transcript 1 (HIAT1). The HIAT1 gene encodes a transmembrane protein of unknown function with homology to the solute carrier protein family and may transport a solute required for spermatogenesis from the bloodstream. 24 A previous study has reported that expression of circHIAT1 is downregulated by AR in cancer cell lines and tumor tissue of clear cell renal cell carcinoma (ccRCC) cancer, and this led to increased proliferation, migration, and invasion of tumor cells in vitro and in vivo, as well as poor clinical features, including a worse overall survival of the patients. 14 Therefore, given the important involvement of AR and PI3K/AKT pathways in the development of prostate tumors and also the existence of such key clues about the regulatory roles of circHIAT1 and circCDR1AS in these pathways, we hypothesized that the expression levels of these two circRNAs could be altered in prostate tumors in relation to patients' clinical phenotypes. Accordingly, this study was aimed to determine the expression profiles of cir-  Of note, a written informed consent was gained from each study participant. This research was approved by the Ethics Committee of the Zanjan University of Medical Sciences.

| Data collection
The clinicopathological characteristics, age, BMI, DRE, PSA level before surgery, Free/total PSA ratio, PSA density, Gleason score, ISUP, bone metastasis, and Family history of PCa obtained from medical reports ( International society of urological pathology (ISUP) (n/%) Note: Data were presented as count or percentage. PCa; prostate Cancer. BPH; benign prostatic hyperplasia. p < 0.05 was considered significant (in bold). For some variables, including prostate-specific antigen (PSA) levels prior to surgery, free/total PSA ratio, PSA density, and bone scan, the data in the patients' medical records were incomplete.

| Quantitative real-time polymerase chain reaction (RT-qPCR)
The    (Table 2). Also, as shown in Table 3, no relationship was found between the study variables and the change in circHIAT1 expression.

| Potential diagnostic values of circCDR1AS and circHIAT1 for PCa
We further assessed the diagnostic effectiveness of candidate cir-  Figure 3D). Furthermore, when the circCDR1as combined with total PSA, the sensitivity and specificity were 90.48% and 82.61%, respectively (AUC of 0.961, 95% CI (0.73-0.096), Std. Error 0.57, R 2 McF = 0.6258, p < 0.0001, Figure 3E). Correspondingly, when we combined the circHIAT1 with total PSA, the sensitivity and specificity were 90% and 87%, respectively (AUC of 0.994, 95% CI (0.73-0.096), Std. Error 0.57, R 2 McF = 0.5706, p < 0.0001, Figure 3F). Therefore, in the combined model of one of the two selected circRNAs with total PSA, the sensitivity and AUC were better than total PSA alone, but not specificity. In line with these observations, the results showed that the AUC of three combined factors (circCDR1AS, circHIAT1, and total PSA) was 0.995 with the sensitivity and specificity of 100% and 95.45%, respectively (95% CI (0.98-1), Std. Error 0.005, R 2 McF = 0.9046, p < 0.0001, Figure 3G). Therefore, by combining two candidate circRNAs with total PSA, the highest biomarker values can be obtained to differentiate PCa from BPH.

| DISCUSS ION
Heterogeneous phenotypes of prostate tumors are a reflection of their heterogeneous genetic changes, which also have an important effect on its physiopathology, treatment, and diagnosis. [26][27][28][29] Thus, revealing PCa-related genetic signatures could be useful in identifying the molecular etiology of the tumors as well as introducing new diagnostic, therapeutic, and prognostic biomarkers. [26][27][28]30 In this study, we investigated the expression levels of two key circR-NAs including the circCDR1AS (hsa_circ_0001946) and circHIAT1 Another report showed that circCDR1AS is overexpressed as an oncogene in HCC and executes its role through suppressing miR-7 in a PTEN/PI3K/AKT-related signaling network and functionally stimulates proliferation and invasion of HCC cells. 36 Moreover, its expression was clinically associated with a more malignant form of HCC patients. 36 Therefore, our findings and such evidence suggests that Note: Data were presented as mean value ±standard deviation or count. p < 0.05 was considered significant (in bold).
expression of the circCDR1AS than patients ≤60 years of age. Age is a known risk factor for PCa, so as the age increases, the risk of developing this cancer increases. 37 Thus, this expression signature may be age-dependent event, and it can be thought that one of the possible molecular mechanisms behind the relationship of increasing age with increasing risk of PCa is the elevating of circCDR1AS, although this observation needs to be studied in more detail.
We furthermore found that the expression level of circHIAT1 in the tumors was reduced compared with BPH tissues. This result is important because previous data showed that this circRNA has a tumor-suppressive function and its reduced expression plays a prominent role in the development of some cancers. 14, 38 Li et al.  ROC curve analysis showed an AUC of 0.848 with 92% sensitivity and 76% specificity. Also, it was found an AUC of 0.828 with 80% sensitivity and 76% specificity for circHIAT1. However, these data for the total PSA were an AUC of 0.819 as well as the sensitivity and specificity of 96% and 33%, respectively. Notably, when the biomarker value data were combined as the circHIAT1+circCDR1AS and also in the form of three factors including circCDR1AS+cir-cHIAT1+total PSA, the higher values of AUC, sensitivity, and specificity were obtained than the total PSA alone. Therefore, these two circRNAs are expected to be potentially important biomarkers for PCa screening and diagnosis, although, this hypothesis needs further research.
In summary, the findings of current study indicated that in patients with PCa, compared with BPH individuals, the expression levels of circCDR1AS increased but the circHIAT1 decreased. Also, the expression of circCDR1AS in the PCa group showed a significant relationship with the increasing age, and finally, we indicated the potential biomarker potential of the molecular signature of these two circRNAs to differentiate PCa from BPH. Such data suggest that aberrant expressions of circCDR1AS and circHIAT1 are the two different molecular pathologies that occur in PCa and on the con-

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.