Expression status of circ-SMARCA5, circ-NOL10, circ-LDLRAD3, and circ-RHOT1 in patients with colorectal cancer

Colorectal cancer (CRC) poses a significant burden on both the healthcare systems as well as individuals. The high mortality rate of CRC may be attributed to its metastatic potential, heterogeneity, and delayed diagnosis. CircRNAs are an essential class of regulatory RNAs that play significant roles in cancers. This study aimed to detect the expression status of circ-SMARCA5, circ-NOL10, circ-LDLRAD3, and circ-RHOT1 in patients with CRC. This study included 50 CRC patients, 30 individuals with colorectal diseases (non-cancer), and 20 healthy volunteers. By using real-time PCR, the relative expression of circ-SMARCA5, circ-NOL10, circ-LDLRAD3, and circ-RHOT1 was determined in the collected blood samples. In addition, ECLIA was used to quantify carcinoembryonic antigen (CEA) level. All circRNAs expression and CEA levels were significantly up-regulated in cancer patients (CRC, colon, rectum) as compared to healthy controls, except circ-SMARCA5. Moreover, there was a significant up-regulation of circRNAs in most non-cancer patients (UC, polyp, piles). Insignificant upregulation was observed in circRNAs and CEA when comparing cancer with non-cancer patients. No correlations were found between the studied parameters and most clinicopathological characteristics of cancer and non-cancer patients. Circ-SMARCA5, circ-NOL10, circ-LDLRAD3, and circ-RHOT1 were differentially expressed in patients with CRC as well as in non-cancer patients. Circ-SMARCA5 and circ-NOL10 may act as tumor suppressors, while circ-LDLRAD3 and circ-RHOT1 may be oncogenes. Circ-SMARCA5, circ-NOL10, circ-LDLRAD3, and circ-RHOT1 could be promising markers for the early detection of CRC.

The parameters that showed insignificant differences in cancer or non-cancer patients as compared to the control group or CRC patients respectively could not be utilized as diagnostic markers.

Discussion
Both the individuals and healthcare systems are significantly burdened by CRC.The heterogeneity, propensity for metastasis, and/or delayed detection of CRC may all contribute to its high mortality rate.CircRNAs have recently been found to play significant roles in several types of cancer.CircRNAs have been shown to control the development of disease because they can bind to miRNAs to control their target genes 10 .
The present results revealed that the expression of all studied circRNAs and CEA level were upregulated in both cancer and non-cancer patients as compared to healthy control except circ-SMARCA5 in non-cancer patients.On the other hand, insignificant upregulation was observed when compared cancer with non-cancer patients.
Up-regulation of circ-SMARCA5 expression was sufficient to impede colon cancer cell viability and change of proteins associated with proliferation (p53, p21, and cyclinD1) and apoptosis (Bax and caspase-3), suggesting that circ-SMARCA5 could suppress the growth of colon cancer.In colon cancer circ-SMARCA5 sponge miR-552, where upregulation of miR-552 partially prevented colon cancer cells from growing and invading.These changes could be linked to the stimulation of signaling pathways in cancer cells.The upregulation of circ-SMARCA5 in colon cancer cells resulted in the inhibition of the Wnt and YAP1 pathways, while miR-552 overexpression restored Wnt and YAP1 pathway activities, suggesting that circ-SMARCA5 had anti-cancer properties 11 .
Furthermore, circ-SMARCA5 could sponge miR-181b-5p and miR-17-3p, which were identified to be onco-miRNAs with proliferative, invasive, and metastatic potential in cancer cells.www.nature.com/scientificreports/metalloproteinases 3 (TIMP3) is a tumor-suppressing substance that, if eliminated, will accelerate tumor invasion and metastasis.MiR-181b-5p or miR-17-3p diminished the inhibitory impact of circ-SMARCA5 on tumor cell, confirming that circ-SMARCA5 could sponge miR-181b-5p, as well as miR-17-3p, to induce TIMP3 and suppress malignant behaviors 12,13 .Circ-NOL10 had an anticancer impact in many cancer tissues.In lung cancer, circ-NOL10 increases the expression of transcription factor sex comb on midleg-like 1 (SCML1) by preventing transcription factor ubiquitination and consequently alters the regulation of the humanin polypeptide family by SCML1.Through controlling the humanin polypeptide family and other signaling pathways, Circ-NOL10 also influences mitochondrial activity.As a result, it promotes apoptosis and reduces the growth of cancer cells 5 .Moreover, circ-NOL10 sponges miR-767-5p in breast cancer (BC) to promote the expression of suppressors of cytokine signaling 2 (SOCS2) and attenuate janus kinase 2/signal transducer and activator of transcription 5 (JAK2/STAT5) signaling, therefore preventing cancer progression 14 .
As a tumor suppressor, MiR-137 stimulates NSCLC apoptosis and prevents its growth and invasion.The downstream target of circ-LDLRAD3 and miR-137 was confirmed to be glutamine transporter solute carrier family A1 member 5 (SLC1A5).SLC1A5 is important for the metabolism of L-glutamine, which necessary for developing cancer cells.MiR-137 served as a post-transcriptional regulator to silence SLC1A5 by targeting its 3ʹ untranslated regions.Circ-LDLRAD3 positively regulated SLC1A5 by sponging miR-137 18 .
Down expression of miR-137-3p might induces KDM1A (also known as lysine-specific demethylase 1) and promote the EMT pathway, which is deeply linked to CRC metastasis 19 .KDM1A expression is frequently increased in various malignancies, and its upregulation is thought to induce cancer cell proliferation, and migration 20 .Since miRNA-491-5P and miR-137-3P are expressed in CRC, circ-LDLRAD3 may be regulate CRC progression by sponging these miRNAs.
Circ-RHOT1 controls the proliferation of pancreatic cells by downregulating the expression of miR-125a-3p to increase E2F3 expression.PANC-1 cells had lower levels of miR-125a-3p, and its overexpression had a negative impact on PANC-1 growth 21 .E2F3 is a transcription factor that belongs to the E2F family and is elevated in several malignancies.It may play a significant role in tumor growth 22 .MiR-125a-3p/E2F3 axis is crucial for PANC-1 cell invasion 21 .
The expression of miR-125a-5p was significantly reduced in CRC tissues and cell lines.In vitro, miR-125a-5p inhibition accelerated the development of CRC.Vascular endothelial growth factor-A (VEGFA) may be a viable target gene of miR-125a-5p, where it could bind to its receptor VEGFR2 and be strongly related to tumor growth.MiR-125a-5p prevented the progression of CRC by VEGFA/VEGFR2 signaling pathway.Mechanisms by which miR-125 controls CRC form a complicated network, miR-125 could negatively modulate B cell lymphoma-2 like protein-12 (BCL2L12), B cell lymphoma-2 (BCL2), and myeloid cell leukemia-1 (Mcl-1), and down-regulated miR-125 promoted colon cancer cell proliferation and suppressed apoptosis 23,24 .www.nature.com/scientificreports/www.nature.com/scientificreports/growth.Circ-RHOT1 may act as a target for miR-326 to indirectly control PDK2 expression and therefore affect cancer development, whereas circ-RHOT1 positively controlled PDK2 expression by sponging miR-326 27 .
In CRC, miR-326 has ability to impede cancer growth by directly inhibiting E2F1.Up-regulation of E2F1 can promote CRC proliferation and prevent apoptosis 28 .E2F1 is a crucial transcription factor that involved in S-phase specific expression of ribonucleotide reductase small subunit M2 (RRM2) under physiological situations.On the other hand, in response to DNA damage, E2F1 also triggered RRM2 transcription via ATM/ATR-CHK1 signal pathway.E2F1 regulates the transcription of several genes involved in DNA synthesis, namely thymidine kinase, dihydrofolate reductase, and thymidylate synthase 29 .In CRC cell lines, overexpression of E2F1 upregulated RRM2 expression through enhancing its transcriptional activation 30 .
Circ-RHOT1 acts as a molecular sponge for miR-3666.Overexpression of miR-3666 may inhibit proliferative activity, and natural killer cell sensitivity of BC.MiR-3666 may target SMAD5 and negatively regulate SMAD5 expression in BC cells, indicating that SMAD5 is necessary for circ-RHOT1 to exhibit its functional role in the progression of BC 31 .MiR-3666 suppresses the growth and metastasis of CRC cells, and its direct target gene is SATB2.Via the Wnt pathway, the transcription factor SATB2 promoted the development of CRC.MiR-3666 and SATB2 in CRC were negatively correlated 32 .
Since circ-RHOT-1 sponge miR-125a, miR-326 and miR-3666 and these miRNAs are expressed in CRC.So, circ-RHOT-1 may regulate the proliferation and invasion of CRC by sponging these miRNAs.www.nature.com/scientificreports/ The elevated level of CEA in CRC patients may be associated with the extent and severity of active inflammation of the colonic mucosa.CEA is expressed in the inflamed mucosa related to hemorrhoidal disease (piles) and in ulcerative colitis patients, but not in healthy mucosa.Furthermore, most adenocarcinomas of the colon, rectum, pancreas, and stomach, as well as breast cancers and NSLC, have overexpressed CEA 33 .CEA proteins are bound to a cell membrane through a glycosylphosphatidylinositol (GPI) anchor linkage.The detected elevation of CEA may be caused by phospholipase enzymes that interfere with the breakdown of GPI binding and the removal of CEA from the cell surface.In addition, during colon cancer progresses, activation of oncogenic c-Ki-ras proteins causes an increase in CEA level and a disturbance of basolateral polarity 34 .
Gastrointestinal tract is affected by inflammatory bowel disease (IBD), a chronic inflammatory disorder that is linked to several pathogenic causes, including immunological, microbial, genetic, and environmental variables.Inflammatory factors have been found to interact with genetic components, like mRNAs and microRNAs, to cause the progression of IBD.CircRNAs exhibit variable expression in UC, and several of them exhibit severe dysregulation.CircRNAs also react to different pro-inflammatory stimuli.Due to their capacity to control gene expression, circRNAs may have changed in concentration 35 .Furthermore, circRNAs are involved in the intestinal epithelial barrier and immune homeostasis disruption, which is a key mechanism to develop IBD 36 .
The microbial communities within polyps and the infiltrating T lymphocytes of polyps regulate intra-polyp inflammatory reactions, which are the primary cause of polyposis.CircRNAs are linked to the abnormal development of inflammatory polyps 37 .
Piles are prevalent disorders that impair the normal functions of the anus and rectum.The vascular wall and the connective tissue around the hemorrhoids suffer from a significant inflammatory response that gradually results in mucosal ischemia, ulceration, and thrombosis.In this region, tissue injury triggers an inflammatory response that emerges as the invasion of neutrophils, macrophages, T lymphocytes, monocytes, and dendritic cells.Both pro-and anti-inflammatory cytokines are secreted by these cells.But because pro-inflammatory cytokines are produced in excess, the pro-inflammatory response eventually dominates the anti-inflammatory response 38 .Upregulated circRNAs were prevalent in anorectal malformations (ARMs) and may have helped ARMs develop, where they have an impact on cellular, regulatory, and metabolic processes 39 .
Lack of significant correlation between studied parameters and most clinicopathological data may be due to the small number of available samples for patients (cancer and non-cancer).These observed results are in line with previous studies which indicated that no significant correlation between circ-SMARCA5 and gender, age, smoking, tumor size, TNM system 40 and between circ-NOL10 and age, gender, smoking, tumor location 41 as well as between circ-RHOT1 and tumor size, stage 21 .
In CRC, CEA, circ-RHOT1, circ-NOL10 and circ-LDLRAD3 could be used as diagnostic markers where CEA was superior to the expression of circ-RHOT1 followed by circ-NOL10 and circ-LDLRAD3.To be differentiated from CRC, in UC patients both circ-SMARCA5 and CEA could be used as diagnostic markers where circ-SMARCA5 superior to CEA; in polyp patients CEA could be a diagnostic marker only, and in piles patients circ-SMARCA5 superior to CEA followed by circ-RHOT1 for prediction of piles.

Conclusion
This study provides new insights into the possible implication of circ-SMARCA5, circ-NOL10, circ-LDLRAD3, circ-RHOT1 in CRC progression, where they are expressed differentially in CRC patients as well as non-cancer patients (UC, polyp, and piles).Since circ-SMARCA5 and circ-NOL10 are tumor suppressor genes, whereas circ-LDLRAD3 and circ-RHOT1 are oncogenes.
Due to the dynamic variation of the expressions of circ-SMARCA5, circ-NOL10, circ-LDLRAD3 and circ-RHOT in cancer, and non-cancer patients, these circRNAs might be prospective candidates for early diagnosis of CRC.However, to validate such a role of the studied circRNAs, large clinical cohort studies are highly recommended.

Table 1 .
Characteristics of control and cancer subjects (CRC, colon, and rectum).n, number of subjects.