GKN1–miR-185–DNMT1 Axis Suppresses Gastric Carcinogenesis through Regulation of Epigenetic Alteration and Cell Cycle

Abstract

Purpose: Gastrokine 1 (GKN1) functions to protect the gastric antral mucosa and promotes healing by facilitating restoration and proliferation after injury. GKN1 is downregulated in Helicobacter pylori-infected gastric epithelial cells and loss of GKN1 expression is closely associated with gastric carcinogenesis, but underlying mechanisms of the tumor-suppressing effects of GKN1 remain largely unknown.Experimental Design: AGS, MKN1, MKN28 gastric cancer cells and HFE-145 immortalized nonneoplastic gastric mucosal cells were transfected with GKN1 or shGKN1. We conducted molecular and functional studies of GKN1 and miR-185 and investigated the mechanisms of alteration. We also analyzed epigenetic alterations in 80 gastric cancer tissues.Results: Restoration of GKN1 protein suppressed gastric cancer cell growth by inducing endogenous miR-185 that directly targets epigenetic effectors DNMT1 and EZH2 in gastric cancer cells. In addition, ectopic expression of GKN1 upregulated Tip60 and downregulated HDAC1 in an miR-185-independent manner, thereby inducing cell-cycle arrest by regulating cell-cycle proteins in gastric cancer cells. Notably, GKN1 expression was inversely correlated with DNMT1 and EZH2 expression in a subset of 80 gastric cancer tissues and various gastric cancer cell lines. Interestingly, it was found that GKN1 exerted a synergistic anticancerous effect with 5-fluorouracil on tumor cell growth, which suggests a possible therapeutic intervention method for gastric cancer.Conclusion: Our results show that GKN1 has an miR-185-dependent and -independent mechanism for chromatic and DNA epigenetic modification, thereby regulating the cell cycle. Thus, the loss of GKN1 function contributes to malignant transformation and proliferation of gastric epithelial cells in gastric carcinogenesis.

Gastrokine 1 inhibits gastric cancer cell migration and invasion by downregulating RhoA expression

“…We had previously found that GKN1 induces miR-185 expression in gastric epithelial cells and that miR-185 plays an inhibitory role in cell proliferation [11]. In the present study, we have shown that GKN1 induces miR-185 expression in AGS and MKN1 gastric cancer cells (Fig.…”

“…4f). c-Myc inhibits miR-185 expression by binding to a promoter of miR-185 [11], and as such it is likely that c-Myc expression, induced by miR-34a silencing, leads to the inhibition of miR-185 expression.…”

“…To investigate whether GKN1 directly regulates miR-34a and miR-185 expression, expression of miR-34a and miR-185 was also quantified by qPCR and normalized to the level of human U6 small nuclear RNA. Data are reported as relative quantities according to an internal calibrator using the 2 -44Ct method [11]. The primer sequences are shown in Table 1.…”

“…Since we have previously reported that GKN1 induced miR-185 expression in gastric cancer cells [11], and miR-185 is a negative regulator of RhoA by directly binding to the 3 0 untranslated region (UTR) of RhoA [20], we investigated whether GKN1 inhibits RhoA expression in a miR-185-dependent manner. A significant increase in the expression of miR-185 was confirmed in AGS and MKN1 cells transfected with GKN1 (Fig.…”

“…GKN1 is involved in the maturation and integrity of gastric mucosal epithelial cells [10]. Previously, we reported that GKN1 possesses miR-185-dependent and miR-185-independent mechanisms, for chromatic and DNA epigenetic modifications, thereby regulating the cell cycle [11]. In addition, GKN1 inhibits EMT by downregulating reactive oxygen species production and the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway, as well as by inactivating nuclear factor jB dependent matrix metalloproteinases [12,13].…”