High glucose facilitates hepatocellular carcinoma cell proliferation and invasion via WTAP-mediated HK2 mRNA stability.

Abstract

Hepatocellular carcinoma (HCC) is one of the most common cancers, and diabetes is a risk factor for hepatocarcinogenesis. N6-methyladenosine (m6A) methyltransferase WT1-associated protein (WTAP) is highly expressed in HCC and contributes to tumor progression. However, its role in high glucose-driven HCC progression remains unclear. The m6A quantitative assay was used to detect the m6A modification level. The levels of mRNAs and proteins were detected by qPCR, Western blot, and immunohistochemistry. CCK-8, colony formation, EdU, and transwell assays were used to detect HCC cell proliferation, invasion, and migration. Immunoprecipitation and CHX assays were used to reveal the regulatory effect of high glucose on WTAP. An RNA degradation experiment was used to explore WTAP's regulation of HK2 mRNA. To demonstrate the effect of high glucose on HCC growth in vivo, a diabetic mouse model was constructed, and HCC cells were subcutaneously injected. High glucose prominently increased the global level of m6A in HCC cells. Interestingly, high glucose upregulated WTAP protein rather than mRNA expression. We found that WTAP expression was significantly upregulated in HCC tissues, especially in tumor tissues of diabetic patients. WTAP knockdown markedly attenuated high glucose-induced abilities of HCC cell proliferation, colony formation, migration, and invasion. Meanwhile, WTAP overexpression significantly enhanced the malignant behaviors of HCC cells under low glucose conditions. High glucose reduced the ubiquitination of WTAP, thereby inhibiting its proteasomal and lysosomal degradation. Phosphorylated ERK (p-ERK) was required for high glucose-mediated WTAP stability. WTAP knockdown prominently abrogated high glucose-induced global m6A levels and HK2 expression in HCC cells. WTAP positively regulated HK2 expression by increasing mRNA stability. HK2 overexpression remarkably reversed the suppressive effects of WTAP knockdown on HCC cells. HK2 knockdown prominently abolished the promoting role of WTAP in HCC cells. Importantly, the growth of HCC cells in diabetic mice was significantly faster than that in control mice, which was prominently attenuated by WTAP knockdown. Our study demonstrated that high glucose decreased WTAP degradation and maintained its protein level by activating ERK phosphorylation. WTAP promoted HCC cell proliferation, colony formation, migration, and invasion by stabilizing HK2 mRNA.