The incidence of gastric cancer is very high and it has become one of the leading causes of cancer-related deaths worldwide. Changes in GPL are linked to an increased risk of gastric cancer(Choi et al. 2016). The reprogramming of cell metabolism is an important sign of cancer and is closely related to the occurrence of tumors. The characteristics of the tumor cells mainly include excessive activation of anaerobic glycolysis and aerobic respiration(Liu et al. 2019b). Epidemiological studies have shown that elevated blood glucose levels are one of the risk factors for gastric cancer(Yamagata et al. 2005). Therefore, elucidating the factors related to glycolysis is essential for the effective prevention of gastric cancer(Tennant et al. 2010). This study is consistent with our previous report that there is glycolysis in the gastric mucosa of GPL(Liu et al. 2020a). MNU induces gastric adenoma and adenocarcinoma due to inflammatory reactions to the alkylating agent(Leung et al. 2008; Tsukamoto et al. 2007; Yamachika et al. 1998), and has been utilized to induce gastric cancer in mice(Tomita et al. 2007; Yoshizawa et al. 2009).
In our study, the mouse model showed IM and Dys of gastric mucosa, and microscopic analysis showed that ischemia and hypoxia could be observed in mice receiving MNU. Thus, we proved that the GPL model was successfully established by short-term gavage of MNU in mice.
The formation of tumor is mainly governed by tissue hypoxia, which is a key molecular feature of the tumor microenvironment(Wang et al. 2020). In the response to hypoxia, angiogenesis(Jain 2014),and reprogramming, energy metabolism(Bristow and Hill 2008) is involved. Hypoxia-induced HIF-1α promotes gastric cancer cell proliferation, invasion, and migration both in vitro and in vivo(Semenza 2012; Xia et al. 2020). The key step in the occurrence and progression of cancer is angiogenesis(Hisano and Hla 2019; Jain et al. 2010). vWF, a multimeric plasma glycoprotein, acts as a marker of endothelial dysfunction(Mandorfer et al. 2018; Sadler 2013). More importantly, vWF has been widely used as a biomarker in gastric cancer(Yang et al. 2018, 2015).However, its functional role in GPL is largely unknown. Here, we report that in the gastric mucosa of mice treated with MNU, the expression levels of HIF-1α and vWF were significantly increased. These findings demonstrate the causal role of ischemic hypoxia microenvironment-derived vWF in mediating the carcinogenic characteristics of MNU and identify vWF as a new therapeutic target.
In the clinic, HIF-1α expression was correlated with aberrant P53 accumulation and cell proliferation(Zhong et al. 1999). Ki-67 is a widely used biomarker to estimate the proportion of dividing cells in order to grade tumors(Scholzen and Gerdes 2000). The presence of strong nuclear staining of P53 in the majority of cancer cells is frequently observed(Baas et al. 1994). P53 and Ki-67 immunostaining indicated that the mice treated with MNU showed more positive cells compared to the control mice. Altogether, here we reported that the ischemic hypoxic microenvironment increased the expression levels of HIF-1α and vWF, which regulated the level of P53 and promoted the expression of Ki-67.
Hypoxia-inducible miRNAs are engaged in the metabolic reprogramming process. An increasing amount of microRNAs have been found to be related to the carcinogenesis and prognosis of gastric cancer patients(Ueda et al. 2010; Yasui et al. 2004, 2009). miR-194-5p promotes gastric carcinogenesis(Sousa et al. 2016). Most of the up-regulated miRNAs have been linked to gastric cancer. For this study, though, we focused on correlations of miRNAs with GPL. In the present study, we showed that miR-194-5p expression levels increased in the GPL. We suggest that miR-194-5p upregulation is an early event in the cascade of events that lead to the conversion of GPL to cancer, and that it contributes to the establishment of an GPL expression profile through regulation of hypoxic microenvironment.
Transcription of genes encoding glycolytic enzymes are activated by HIF-1α, including stimulation of glycolysis by upregulation of LDHA, which creates an acidic tumor microenvironment(Ryan et al. 1998; Zhong et al. 1999). PCK1 is a gluconeogenic enzyme that leads to the regulation of glucose production(Troy et al. 2008). In this study, we also observed changes in gluconeogenic enzymes after MNU treatment as the level of LDHA was increased and the levels of PCK1 decreased in the stomach. In mammalian cells, FoxO3 was involved in glucose metabolism(Kops et al. 2002; Medema et al. 2000). The regulation of FoxO3a by PI3K-AKT and LKB1-AMPK may play a crucial role in controlling energy balance. The PI3K-AKT pathway controls cell survival, proliferation and tumor growth, whereas the LKB1-AMPK pathway controls cell cycle arrest and tumor suppression, and promotes longevity(Hawley et al. 2005). FoxO3a, one of the intersections between both the pathways(Biggs 3rd et al. 1999; Brunet et al. 1999), is inhibited by the PI3K-AKT pathway and activated by the LKB1-AMPK pathway (Greer et al. 2007; Shin et al. 2016). Thus, the PI3K-AKT and the LKB1-AMPK pathways may orchestrate a series of transcriptional (via FoxO3) and post-transcriptional (via mTOR) changes that allow the organism to adapt to changes in the hypoxic status. Our group has characterized the protein expression profiles of PI3K, AKT and mTOR in GPL mice(Liu et al. 2020a, b, a). In the current study, the LKB1 protein expression levels were downregulated, while the ratio of p-AMPK/AMPK and p-AKT /AKT were higher in MNU- receiving mice compared the control mice. Here, we generated the FoxO3a profile of GPL compared to normal mice. We showed evidence here that hypoxia activated miR-194/FoxO3a, inducing glycolytic metabolism reprogramming by upregulating the expression of LDHA and downregulating the expression of PCK1 in GPL (Fig. 7).
In summary, our findings identified a novel miR-194-5p, which was up-regulated in GPL. Mechanistically, miR-194-5p functioned as an oncogenic miRNA by regulation of the hypoxic microenvironment and subsequently regulated the PI3K-AKT and the LKB1-AMPK pathways via FoxO3a, inducing reprogramming of glycolytic metabolism.