Hypoxia-induced UBE2K Promotes The Malignant Progression Of HCC

Objective: Hepatocellular carcinoma(HCC),mainly referring to liver cell cancer,is one of the common malignant tumors in China,ranking third in terms of mortality and causing approximately 900,000 cases and 830,000 deaths,posing a significant threat to the life and health of the Chinese people.Ubiquitination is one of the important post-translational modification processes in cells.Through the enzymatic cascade reaction composed of ubiquitin-activating enzyme E1,ubiquitin-conjugating enzyme E2,and ubiquitin ligase enzymes E3,it ubiquitinates protein substrates and degrades them.Multiple studies have shown that the ubiquitination process is involved in various physiological and pathological processes,including the occurrence and development of tumors.Hypoxia,which commonly exists in human solid tumors,is a key driving factor for malignant progression.Hypoxia-inducible factors-1α(HIF-1α)is upregulated in response to cellular hypoxia,and also regulate the malignant behaviors of cancer cells,such as growth and migration.Recent studies have shown that the relationship between hypoxia and ubiquitination plays an important role in the field of tumors,and various hypoxia-responsive ubiquitination enzymes have been discovered and studied.This study aims to explore the role of hypoxia in the ubiquitin-conjugating enzyme E2-K(UBE2K)in HCC,and investigate the effects of UBE2K on HCC cell proliferation and migration.These research results highlight the potential application of UBE2K as a new biomarker and therapeutic target for HCC,providing important scientific basis for the development and optimization of early diagnosis and treatment strategies for HCC in the future.Method:(1)Elevated levels of UBE2K were observed through gene microarray analysis under hypoxic conditions.(2)Western blotting(WB)was performed to determine the expression levels of UBE2K in Hep3 B and Huh7 cells under hypoxic and normoxic conditions.(3)Stable knockdown and overexpression cell lines of HIF-1α were constructed in Hep3 B and Huh7 cells.(4)WB was used to determine the expression levels of UBE2K in Hep3 B and Huh7 cells under hypoxic conditions and HIF-1αoverexpression conditions.(5)Hypoxic conditions were induced in Hep3 B and Huh7 cells with stable knockdown of HIF-1α,and changes in UBE2K expression were detected by WB.(6)The correlation between UBE2K and HIF-1α expression in HCC tissues was analyzed by immunohistochemistry(IHC)staining and bioinformatics analysis.(7)IHC staining and bioinformatics analysis revealed that UBE2K is highly expressed in HCC tissues.(8)WB was used to compare the expression levels of UBE2K in normal liver cell line MIHA and liver cancer cell lines MHCC97 H,Huh7,and Hep3 B.(9)Stable transfection cell lines with UBE2K knockdown and overexpression were constructed in Hep3 B and Huh7 cells.(10)The effects of UBE2K knockdown and overexpression on the proliferation of Huh7 and Hep3 B cells were evaluated using CCK-8 proliferation assay and colony formation assay.(11)Transwell migration assay and scratch assay were performed to investigate the effects of UBE2K knockdown and overexpression on the migration ability of Huh7 and Hep3 B cells.(12)WB,CCK-8 proliferation assay,colony formation assay,Transwell migration assay,and scratch assay revealed that UBE2K knockdown inhibited hypoxia-induced cell proliferation and migration in HCC cells.(13)WB,CCK-8 proliferation assay,colony formation assay,Transwell migration assay,and scratch assay demonstrated that increasing UBE2K levels reversed the inhibition of cell proliferation and migration caused by HIF-1α knockdown under hypoxic conditions.Results: UBE2K is a potential hypoxia-responsive gene in HCC cells.Our research suggests that hypoxia-induced HIF-1α expression promotes UBE2K expression,while knockdown of HIF-1α under hypoxic conditions decreases UBE2K expression.IHC staining and bioinformatics analysis confirmed that UBE2K is highly expressed in HCC tissues compared to adjacent normal tissues,and UBE2K expression is positively correlated with HIF-1α expression in HCC tissues.Further functional studies revealed that overexpression of UBE2K promotes proliferation and migration of Hep3 B and Huh7 cells,whereas knockdown of UBE2K inhibits proliferation and migration of these cells.Moreover,functional reversal experiments demonstrate that knockdown of UBE2K inhibits hypoxia-induced cell proliferation and migration in HCC cells,whereas overexpression of UBE2K reverses the inhibition of cell proliferation and migration caused by HIF-1α knockdown under hypoxic conditions.Conclusion:(1)UBE2K is a hypoxia-responsive gene,and its expression is positively regulated by HIF-1α under hypoxic conditions.(2)UBE2K is upregulated in HCC and positively correlates with the expression of HIF-1α.(3)UBE2K functions as an oncogene and can promote the proliferation and migration ability of HCC cells.(4)The functional HIF-1α/UBE2K axis promotes the growth and migration of HCC cells under hypoxic conditions.Significance: Our study reveals that UBE2K functions as a novel hypoxia-responsive gene with a pro-oncogenic role in HCC and its potential molecular mechanism.This finding offers a new perspective and contributes to a deeper understanding of the pathogenesis and progression of HCC.