Even with considerable progress in cancer research, GC is still one of the global health problems. Genes profiling is the most appropriate approach for establishing new diagnostic biomarkers.
The present results revealed significant down regulation of SEC13 and up regulation of SMAD7 expression in GC patients group as compared to control group. In normal and premalignant cells, TGF-β mainly functions as a tumor suppressor through promoting apoptosis, maintaining genome stability, and reducing proliferation. While cancer cells could evade TGF-β suppression effect; they use TGF-β advertising functions to acquire a growth benefit and undergo processes, like epithelial-mesenchymal transition, which facilitates their invasion, and migration17.
Under well oxygenated conditions, SMAD7 is an effective inhibitor of cancer invasion. Hypoxia, which is commonly found in solid tumors, activates SMAD7 expression in HIF- and von Hippel-Lindau protein-dependent manner. The upregulated SMAD7 expression in tumors is correlated with hypoxic gene expression where hypoxia might convert SMAD7 from invasion inhibitor to activator18. Hypoxia triggers posttranslational covalent protein alterations (hydroxylation, and phosphorylation). Provoking or preventing these alterations, hypoxia might favor further formation of permanent R-SMAD / SMAD7 complexes and SMAD7 could dephosphorylate R-SMADs then, impact SMAD-stimulated transcription. Moreover, HIF is well-known to bind SMAD3 and changes the activation of hypoxic gene19. On the contrary, it would be reasonable that HIF could modify R-SMAD-SMAD4-SMAD7 complex binding to DNA. Additionally, hypoxia has been recognized to stimulate a large number of genes, so hypoxia-caused alterations in gene expression might change responses of TGF-β to support instead of prevent invasion18.
SMADs access to their genes is exactly signal dependent, causing SMAD nuclear translocation an important step in TGF-β signal transduction into nucleus. SEC13 has specific properties for SMADs, where SEC13 can provide mechanism by which TGF-β accelerates nuclear import rate of SMADs. Furthermore, SEC13 participates in directing SMADs to their chromatin binding sites and contributes to SMAD-facilitated transcriptional control20. According to SEC13 functions, the significant down regulation of SEC13 expression in GC patients may be one of the factors that leads to aberrant TGF-β signaling. This study confirmed decrease in SEC13 expression with all cancer stages (II, III, IV); while SMAD7 tended to be especially highly expressed in GC patients with stage IV, which could explain the significant negative correlation between SEC13 and SMAD7.
The present study revealed down expression of GHRL in GC patients as compared to control group. Also, GHRL expression was not associated with GC stages. This result agreed with Pritchett et al., 2020 and Hu et al., 202121.22. Ghrelin has anti-inflammatory effects where it alleviates production of nuclear factor-κB and pro-inflammatory cytokines. Additionally, GHRL suppresses macrophage-produced inflammatory cytokines and cyclooxygenase-2 expression23. GHRL activates gastric vagus nerve which delivers the gastrointestinal tract immune information to the hypothalamus24. Therefore, ghrelin’s anti-inflammatory impacts may protect against tumorigenesis25.
AMP-activated protein kinase (AMPK) is the master sensor of cell energy level and it has been proven to control the cell critical functions, involving growth and metabolism, and constantly could be involved in initiation and development of tumor26. Also, AMPK is a negative regulator of Warburg effect to suppress GC development27. A recent study by Hu et al. 202122 recognized that overexpression of GHRL may inhibit GC cell proliferation, invasion, and support apoptosis by activating AMPK pathway. GHRL knockdown enhanced uptake of glucose and release of lactic acid, indicating that GHRL provoked the anti-Warburg effect through AMPK signaling pathway to prevent GC. Accordingly, the observed decrease in GHRL expression in GC patients led to assume that GHRL may function as the tumor suppressor.
The present results showed a significant down regulation in lncRNA GHRLOS in GC patients group as compared to control group. As well as a significant association with GC stages (II, III, IV). These findings are consistent with other research which proved that lncRNA GHRLOS may act as a tumor suppressor during colorectal carcinogenesis and its down regulation stimulates CRC progression28 through its functional and regulatory roles in ghrelin axis12.
LncRNA GHRLOS has been recognized as an overlapping gene on GHRL antisense strand and can serve as a suppressor of its overlapping gene28. Interestingly, it has been proven that ghrelin-AMPK signaling utilizes the anti-warburg effect to inhibit GC progress22. Regarding lncRNA GHRLOS, a previous study suggested that lncRNA GHRLOS can play a role in the regulation of gluco-metabolism29. Therefore, it can be suggested that lncRNA GHRLOS has comparable inhibitory effect on cancer cell as it is related to GHRL gene.
In accordance with previous studies30.31, the present results revealed that HIF-1α gene expression and protein level were significantly elevated in GC patient as compared to control group and were significantly associated with GC clinical stages (II, III, IV). Furthermore, the level of HIF-1α protein was consistent with its mRNA expression in GC patient.
In cancer cells the main feature in response to hypoxia is induction of HIF-1α as well as its downstream target to enhance blood vessel formation, and aggression32. Reactive oxygen species (ROS) induce HIF-1α transcriptional activity through stimulation of NF-κB, extracellular signal-regulated kinases (ERK1 / 2) and PI3K / Akt / m-TOR pathways. Reactive nitrogen species (RNS) induces S-nitrosation of prolyl 4-hydroxylase 2 (PHD2) to elevate HIF-1α stability and activity14.33. In addition to oxygen, HIF-1 may be regulated by other stimuli involving hormones (insulin), growth factors (platelet-derived growth factor) and vasoactive peptide (angiotensin-2).34.
The present study demonstrated that a significant elevation in CEA in GC patients that trends to be significantly increased with GC clinical stages as compared to control group. The secretion and transcription of CEA are controlled by TGF-β pathway and SMAD3-facilitated tumor growth factor35.
Hypoxia increases CEA protein level and its promoter activity directly through HIF-1α binding36 and indirectly through increase cellular pH by activation of plasma membrane Na + / H + exchanger37. Previous study has revealed that E-box represents an important site for the total activity of CEA promoter and contain potential binding sites for various transcription factors as HIF-1α38.
The correlations of all studied parameters with clinicopathological characteristics of GC patients showed some variation than other previous studies39.40 and this may be due to difference in sample numbers and methods used.
The observed correlations between all studied parameters are reasoning since under hypoxia condition TGF-β / SMAD signaling is promoted. TGF-β upregulates HIF-1α expression and induces HIF-1α DNA binding activity. TGF-β affects HIF-1α activity and accumulation via enhancing stability of HIF-1α protein41. Besides, HIF-1α plays a main role for the elevation of CEA protein level and promotes its activity36. Furthermore, SMAD7 is activated by hypoxia in HIF- and VHL-dependent manner and its inhibitory effect on invasion is totally lost18. Also, with hypoxic condition, GHRL reduces the level of HIF-1α42. Additionally, lncRNAs can act as a direct or indirect regulator of HIFs and can improve or reduce its function in cancer.43. GHRLOS LncRNA is positively correlated with GHRL and negatively correlated with HIF-1α protein. So that, lncRNA GHRLOS may exert the same inhibitory effect.
The present results indicated that the validity of using SEC13, SMAD7, GHRL, lncRNA GHRLOS, HIF-1α genes, and HIF-1α protein as diagnostic markers for GC. Additionally, HIF-1α protein was superior to lncRNA GHRLOS followed by CEA protein, SEC13, HIF-1α, SMAD7, and GHRL genes for GC diagnosis.
Conclusion: SEC13, SMAD7, GHRL, lncRNA GHRLOS, HIF-1α genes, and HIF-1α protein may be considered as promising biomarkers for the early detection of gastric cancer. SMAD7, HIF-1α gene and HIF-1α protein may be jointly involved in tumor development and prognosis (act as oncogenic factors), while SEC13, GHRL, and lncRNA GHRLOS may act as tumor suppressor factors and thus could be considered as novel therapeutic targets for gastric cancer.