Association of sonic hedgehog signaling pathway genes IHH, BOC, RAB23a and MIR195-5p, MIR509-3-5p, MIR6738-3p with gastric cancer stage

Gastric cancer is the leading cause of cancer-related mortality worldwide. Given the importance of gastric cancer in public health, identifying biomarkers associated with disease onset is an important part of precision medicine. The hedgehog signaling pathway is considered as one of the most significant widespread pathways of intracellular signaling in the early events of embryonic development. This pathway contributes also to the maintenance of pluripotency of cancer stem cells pluripotency. In this study, we analyzed the expression levels of sonic hedgehog (Shh) signaling pathway genes IHH, BOC, RAB23a and their regulatory miRNAs including MIR-195-5p, MIR-509-3-5p, MIR-6738-3p in gastric cancer patients. In addition, the impact of infection status on the expression level of those genes and their regulatory miRNAs was investigated. One hundred samples taken from 50 gastric cancer patients (50 tumoral tissues and their adjacent non-tumoral counterparts) were included in this study. There was a significant difference in all studied genes and miRNAs in tumoral tissues in comparison with their adjacent non-tumoral counterparts. The lower expression of IHH, BOC, RAB23, miR-195-5p, and miR-6738-3p was significantly associated with more advanced cancer stage. Additionally, IHH upregulation was significantly associated with CMV infection (P < 0.001). Also, receiver operating characteristic (ROC) curve analysis indicated that mir-195 was significantly related to several clinicopathological features including tumor stage, grade, age, gender, and infection status of gastric cancer and can be considered as a potential diagnostic biomarker for gastric cancer. This study confirms the important role of Shh signaling pathway genes in gastric cancer tumorigenesis and their potential as novel molecular biomarkers and therapeutic targets.


Scientific Reports
| (2021) 11:7471 | https://doi.org/10.1038/s41598-021-86946-0 www.nature.com/scientificreports/ sex and age of gastric cancer patients (P = 0.0325). Tumors were mainly located at proximal position of the stomach (cardia, fundus, and body). Overall, 45.5% of tumors were located in the cardia region, and body and antrum regions of the stomach were the second and third most common sites, with 29.5% and 22.7% abundance, respectively. Overall, 74% of tumors were in stages I/II, and 26% in stages III/IV.

Expression levels of Shh signaling pathway genes and their regulatory miRNAs.
Expression levels of Shh signaling pathway genes (IHH, BOC, and RAB23) and their regulatory miRNAs (miR-195-5p, miR-6738-3p, and miR-509-3-5p) were evaluated in 50 gastric cancer patients using comparative relative real time PCR, and by comparing the expression in tumor tissues with their paired normal counterpart tissues. The results indicated that IHH, BOC, and RAB23 mRNA expression were significantly downregulated (Fig. 1). Similarly, miR-195-5p, miR-6738-3p, and miR-509-3-5p expression were also decreased significantly in gastric cancer tissues. The mean tumoral tissues expression levels for IHH, BOC, and RAB23 were 0.71, 0.68, and 0.57 respectively. Also, the expression levels for miR-195-5p, miR-6738-3p, and miR-509-3-5p in tumoral tissues were 0.46, 0.7, and 0.57 respectively. Scatter plot analysis indicated that IHH, BOC, and RAB23 were significantly downregulated in 52%, 58%, and 50% of tumoral tissues in comparison with their adjacent non-tumoral counterparts, respectively. Also, out of 50 GC patients, 70%, 54%, and 58% showed statistically significant downregulation of Table 1. Sequences of primers used for evaluation of SHH signaling genes and their regulatory microRNAs. The target specific portion of mRNA and micro-RNA is showed in red bold. Stem-loop sequence is underlined. Tm of the micro-RNA forward primer was increased by adding a tail (green sequences) to the 5′-end of the sequences.  TGG AGT CCA CTG GCG TCT TCAC   RT-PCR primer   GTC GTA TCC AGT GCT GCG ACC GTA TGG ATG TGT CTG CGG CGT TTT ATC ATG CAC TGG ATA  CGA CAG GCA TTG  Association of clinicopathological features and Shh signaling pathway genes and their regulatory miRNAs. Association between the expression of Shh signaling pathway genes and their regulatory miRNAs with clinicopathological features in gastric cancer patients is illustrated in Fig. 4. A significantly associated trend was observed between the expression of studied genes and their regulatory miRNAs with TNM stage. The expression of IHH, BOC, RAB23, miR-195-5p, and miR-6738-3p decreased significantly with more advanced cancer stage, and miR-509-3-5p expression was significantly decreased during early stages in gastric cancer patients (P < 0.05). Also, IHH expression level was associated with histological type, as it was significantly  www.nature.com/scientificreports/ lower in well differentiated gastric adenocarcinomas in comparison with moderately or poorly differentiated adenocarcinoma (P < 0.01). Furthermore, miR-6738-3p and miR-509-3-5p were significantly downregulated in poorly differentiated and moderately/poorly differentiated tumors, respectively (P < 0.05) (Fig. 4). Moreover, association study between Shh signaling pathway genes and their regulatory miRNAs according to age groups indicated that IHH and miR-6738-3p expression was significantly (P < 0.05) decreased in gastric cancer patients aged less than 65 years while miR-195-5p was significantly P < 0.05) down-regulated in gastric cancer patients older than 65 years. Moreover, a statistically significant decreased expression of miR-6738-3p was observed in male gastric cancer patients (P < 0.05) (Fig. 5).

Association of the infection status and Shh signaling pathway genes and their regulatory miR-NAs expression.
Furthermore, we investigate the effects of infections on the expression level Shh signaling pathway genes and their regulatory miRNAs in gastric cancer. As shown in Fig. 6, in HCMV positive patients, IHH expression was significantly increased (P < 0.001). Also, patients with no H. pylori infection showed lesser BOC and RAB23 expression in gastric tumoral tissues in comparison with H. pylori positive patients (P < 0.05). EBV and HHV6 infections had no significant effect on Shh signaling pathway genes and their regulatory miR-NAs expression in gastric cancer tissues.
Furthermore, miR-195-5p expressions were significantly decreased in gastric cancer tissues of EBV positive patients (P < 0.001). In addition, there was a significant difference for miR-509-3-5p expression level in H. pylori positive gastric cancer patients in comparison with H. pylori negative patients (P < 0.01). HCMV infections had no significant effect on Shh signaling pathway regulatory miRNAs expression (Fig. 7).
Receiver operating characteristic (ROC) curve analysis. ROC curve analysis used to reveal whether studied genes and their regulatory miRNAs can serve as diagnostic biomarkers (Fig. 8) 35 . As it is evident, total area under the curves (AUCs) of RAB23 (AUC = 0.63, sensitivity 71% and specificity 51%, P = 0.02) and miR-195-5p (AUC = 0.68, sensitivity 80% and specificity 54%, P = 0.002) were > 60%, suggesting that RAB23 and miR-195-5p can serve as diagnostic biomarkers for distinguishing patients with gastric cancer from healthy   www.nature.com/scientificreports/ Discussion. Gastric cancer is among the five most frequently diagnosed cancers, and is highly heterogeneous. Accumulating evidence strongly indicate that aberrant activation of multiple signaling pathways can contribute to gastric cancer development. Consequently, cancer stem cells (CSCs) are key driving cells for growth and metastasis of this tumor type. It has been demonstrated that Shh signaling pathway is implicated in maintaining the pluripotency of CSCs, and aberrant activation of this pathway is associated with the development and progression of various types of cancer. In this study, we investigated the clinicopathological features of gastric carcinoma as well as expression levels of Shh signaling pathway genes and their regulatory miRNAs in gastric cancer patients. Although gastric cancer is common in both sexes, its incidence is higher in males, and is more frequently observed in younger female patients [36][37][38] . Our demographic findings are consistent with these reports. We also investigated the expression level of Shh signaling genes including IHH, BOC, and RAB23 in gastric cancer patients. Remarkably, we observed that IHH expression was decreased in tumoral tissues in comparison with adjacent non-tumoral tissues. Also, IHH expression strongly correlated with the stage and grade of malignancy as well as with CMV infection. IHH is one of the three protein ligands in the mammalian hedgehog signaling pathway, and plays an essential role in bone growth and differentiation. The expression level of IHH was found to be upregulated in certain tumors such as basal cell carcinoma, pancreatic cancer, and medulloblastomas 39 . Also, immunohistochemical study indicated that IHH expression was increased in pancreatic ductal adenocarcinoma in comparison with paracancer tissue and benign lesions, and this expression was associated with tumor grade, lymph node metastasis, tumor invasion, and poor overall survival 40 . In contrast, loss of IHH expression can promote the development of dysplasia in colon carcinogenesis via Wnt signaling pathway 41 . Also, epidermal deletion of IHH can promote squamous skin tumor formation and increased malignant tumor progression and metastasis as well as prolonged loss of IHH expression leads to inflammation and mucosal damage 42 . In addition, stromal activation of Hh signaling pathways by IHH suppresses tumor growth and metastases through angiogenesis and reduction of reactive oxygen species (ROS) activity. However, the tumor suppressor or oncogenic role of IHH in cancer is controversial. Relatively, our data demonstrated a decrease in BOC mRNA levels in tumoral tissues, and this expression was associated with the tumor's stage and H.pylori infection. BOC is a co-receptor in Shh signaling pathway and a component of cell-surface receptor complex that mediates cell-cell interactions. However, its relative contribution to cancer risk is currently unknown. The hedgehog co-receptors including GAS1, CDON, and BOC modulate the levels of HH responsiveness in pancreatic fibroblasts, and loss of BOC and GAS1 was shown to reduce HH activity while promoting pancreatic tumor growth through the induction of angiogenic factors 43 . In addition, BOC may induce DNA damage and promote progression of early medulloblastoma to advanced tumors via increasing the incidence of loss of heterozygosity (LOH) of its corecep- www.nature.com/scientificreports/ tor PTCH1 44 . Moreover, our results indicated that RAB23 was downregulated in 42% of gastric cancer tissues while it was overexpressed in 34%of cases. Also, the expression of RAB23 was significantly associated with more advanced cancer stage and H. pylori infection. RAB23 is recognized as a negative regulator of the Shh signaling pathway, and also as the target of many proteins involved in cancer development. However, it remains controversial whether RAB23 acts as an oncogene or tumor suppressor. Although more and more studies have introduced RAB23 as an oncogene in variety of human cancers, there is some evidence indicating that RAB23 plays a tumor suppressive role during carcinogenesis. RAB23 through interaction with SUFU can inhibit GLI transcriptional activities and its nuclear localization. In addition, overexpression of RAB23 inhibits breast cancer cells viability and proliferation, and induces cell apoptosis 45 . Moreover, transient overexpression of miR-367 in medulloblastoma cells caused decreased RAB23 expression resulting in increased medulloblastoma cell proliferation 46 .
It is now established that both genetic and epigenetic alterations contribute to gastric cancer development. Changes in miRNAs expression, as epigenetic modulators, via regulation of cancer-related genes have a primary role in cancer onset and progression. We studied the Shh signaling pathway regulatory miRNAs by in silico analysis, and experimentally validated the expression levels of these miRNAs in gastric cancer patients. Through in silico analysis, we identified three miRNAs, including miR-195-5p, miR-6738-3p, and miR-509-3-5p, which could bind to the 3′ UTRs of Shh signaling genes and modulate the hedgehog pathway. While there is no evidence supporting a role for miR-6738-3p in cancer development, our findings showed that miR-6738-3p expression was decreased in tumoral tissues in comparison with adjacent non-tumoral tissues. Also, its expression was associated with the clinicopathological features of gastric cancer patients including stage, grade, gender, and age. Our in silico analysis predicted that mir-195-5p binding site was located in the 3′ UTR of IHH, and experimental data indicated that mir-195-5p was significantly down-regulated in tumor samples in comparison with their adjacent non-tumoral tissues. Also, mir-195-5p expression was strongly associated with the advanced cancer stage and age of gastric cancer patients, and correlated with EBV infection. Mir-195-5p is one of the well-studied miRNA that is strongly connected with various types of cancer including those of digestive system, respiratory system, urinary system, reproductive system, bone, brain, head and neck, skin, and endocrine cancer. Nevertheless, despite its strong tumor suppressive effects, there is evidence that miR-195 has an oncogenic role in some cancers. Therefore, whether mir-195-5p functions as a tumor suppressor or oncogene is still under debate. It has been reported that miR-195-5p had significant effect on oncogenicity in various types of cancer through binding to complementary sequences in crucial genes of signaling pathways. In stomach cancer, evidence had   Mir-509 is one of the anti-oncogene miRNAs that was reported to be downregulated in many prevalent human cancers. Mir-509 functions as a tumor suppressor in pancreatic and breast cancer via targeting MDM2 proto-oncogene (MDM2) and superoxide dismutase 2 (SOD2) 50,51 . Also, miR-509 as epithelial-mesenchymal transition miRNA was shown to induce the expression of E-cadherin, and inhibit cell motility and invasion 52 . In addition, miR-509 can inhibit cell motility and invasion through targeting tribbles pseudokinase 2 (TRIB2) in osteosarcoma 53 . Our findings showed that miR-509 was significantly down-regulated in tumor tissues in comparison with their adjacent non-tumoral tissues, and lower miR-509 expression in tumoral tissues was associated with high tumor grade, stage, and H. pylori infection. In line with our results, a similar study demonstrated that lower miR-509-3-5P expression was associated with advanced tumor stage and poor differentiation in gastric cancer tissues. Additionally, this lower expression promoted the migration and invasion abilities of gastric cancer cells by targeting podocalyxin like (PODXL) gene 54 . However, our finding is in disagreement with another study indicated miR-509 upregulation in H. pylori-negative gastric cancer 55 . www.nature.com/scientificreports/ Gastric cancer can be considered a complex disease that is influenced by multiple genes, miRNAs, and environmental factors. Finding valuable biomarkers for the diagnosis and prognosis of gastric cancer is difficult. Therefore, using deep convolutional neural network learning method is a very promising way to predict disease risk based on biomarkers 56 . Our ROC curve analysis indicated that mir-195 can be considered a good biomarker as it was significantly related to several clinicopathological features of gastric cancer including stage, grade, age, gender, and infection status. In conclusion, the present study demonstrated that the expression level of Shh signaling pathway genes and their regulatory miRNAs were significantly associated with gastric cancer. Also, the expression level of these genes was significantly associated with clinicopathological features including tumor stage, grade, age, gender, and infection status in gastric cancer patients. Although Hh signaling inhibitors have been developed, and two inhibitors, vismodegib and sonidegib, have been approved by the U.S. Food and Drug Administration (FDA) to treat basal cell carcinoma and medulloblastoma, none of them have been approved for gastric cancer. Thus, more researches to elucidate factors regulating Shh signaling pathway as well as their detailed mechanism of action in gastric cancer are necessary. Also, it is well known that immune regulations may affect gastric cancer stage development, and patients with common variable immunodeficiency (CVID) have a high risk of gastric cancer 57 . Recently, in vivo study indicated that immunodeficiency can promote adaptive alterations of host gut or tissue-based microbiome 58 . Therefore, additional studies on alterations of gastric mucosal immunity and microbiota and effects on signaling pathways are needed to better understand the gastric carcinogenesis.