Mechanisms For Hypoxia-mediated Repression Of Organic Cation Transporter 2 And Decitabine Resistance In Renal Cell Carcinoma

Renal cell carcinoma(RCC)is the most lethal type of urinary tract cancers,accounting for approximately 3.8%incidence rate and 2.5%mortality rate of adult malignancies.RCC is insensitive to radiotherapy,with merely 10%-15%efficiency of various chemotherapy regimens for metastatic renal cell carcinoma(mRCC).Human organic cation transporter 2(OCT2,encoded by SLC22A2)is the most abundant and important uptake transporter in the process of cationic drugs excretion by renal.Abnormal hypermethylation-mediated silencing of OCT2 results in resistance of renal cancer cells to oxaliplatin and platinum chemotherapeutic agents.Previous studies have shown that decitabine(DAC),a DNA methyltransferase(DNMTs)inhibitor,in combination with the traditional chemotherapy drug oxaliplatin can obtaine a satisfactory anticancer ef-fects both in vitro and vivo experiments by reversing the low-expression level of OCT2 in renal cancer cells.However,the imaging findings of the drug regimen in the course of clinical treatment showed that although it could inhibit the growth of tumors,the size of tumors did not shrink significantly.Evidence has accumulated showing that up to 50%-60%of locally advanced solid tumors may exhibit hypoxic and anoxic tissue areas that are heterogeneously distributed within the tumor mass.As a solid tumor,RCC also exhibits a hypoxic tumor microenvironment with the rapid proliferation of cancer cells.Based on the clinical phenomena mentioned above and considering the hypoxic characteristic of RCC.we try to explore the effects of hypoxia on the demethylation function of DAC in RCC cells.The mRNA/protein expression level and methylation/histone H3K4me3 modification in the promoter of OCT2 were detected in renal cancer cells after 72 h treatment of DAC under normoxia(20%oxygen concentration)and hypoxia(1%oxygen concentration)respectively.Our findings demonstrated that DAC is incapable of upregulating the expression of OCT2 under hypoxia,with hypermethylation and low H3K4me3 modification(transcriptional repression signal)in the promoter region of OCT2.We further investigated the specific mechanism of hypoxia-mediated DAC resistance in RCC cells by detecting the expression of key factors in the process of DNA dynamic methylation and DAC metabolic pathway under hypoxia.Results showed hypoxia-mediated repression of human equilibrative nucleoside transporter 1(hENTl)results in decreased cellular accumulation of DAC.In addition,qPCR,western blot and immunohistochemistry assays were performed in RCC tumor tissues and paired normal tissues,showing that the expression of ENT1 was exceptionally suppressed in RCC.Further study indicated that hypoxia downregulated the expression of ENT1 at transcriptional and post-transcr:iptional levels by hypoxia-inducible factor(HIF-la),proteasome and lysosome-dependent mechanism.Hypoxia plays a significant role in the transcriptional and post-transcriptional regulation of gene expression by affecting epigenetic modifications.We also examined hypoxia-mediated modification of histone acetylation and miRNA expression for the regulation of OCT2 expression.We found that hypoxia caused the abnormally upregulation of histone acetylate HDAC9,which impairs the enrichment of H3K27ac modification in the promoter of OCT2,and leads to transcriptional repression of OCT2.In addition,miR-489-3p,which is abnormally upregulated in RCC can repress OCT2 expression by sequence-dependent targeting of OCT2 3’-UTR and then lower the cytotoxicity of oxaliplatin.The expression of the precursor and initial transcript of miR-489-3p was also increased under hypoxia.In summary,this study elucidate the mechanism of hypoxia-mediated DAC resistance in RCC,revealed the regulation mechanism of hypoxia on OCT2 expression from multiple perspectives,and proposed the feasibility of miR-489-3p as a potential diagnostic biomarker for RCC.It highlighted the potential of a optimized combinational chemotherapy in clinical RCC therapy.