Regulation of UDP-glucuronosyltransferases by microRNAs

Author: Dhilushi Wijayakumara

Wijayakumara, Dhilushi, 2021 Regulation of UDP-glucuronosyltransferases by microRNAs, Flinders University, College of Medicine and Public Health

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The glucuronidation of lipophilic compounds results in products that are generally more water soluble, thus promoting their detoxification and clearance (Mackenzie et al., 1997). There are four human UDP-glucuronosyltransferase (UGT) families (Mackenzie et al., 2005); however, glucuronidation is primarily carried out by members of the UGT1A and UGT2B subfamilies whose substrates for these enzymes include endogenous bioactive molecules (e.g., bilirubin, steroid hormones, bile acids, retinoids, fatty acids), carcinogens, environmental toxins, and therapeutic drugs (Guillemette, 2003, Mackenzie et al., 2005).

In humans, glucuronidation of androgens (e.g., testosterone and dihydrotestosterone) and their metabolites is carried out mainly by UGT2B15, and UGT2B17 (Turgeon et al., 2001). The androgen/androgen receptor (AR) signaling pathway plays a pivotal role in prostate growth and function; however, excessive androgen signaling contributes to prostate cancer development and progression (Kaarbo et al., 2007). UGT2B15 and UGT2B17 are expressed in the prostate and their glucuronidation of androgens effectively terminates androgen signaling (Chouinard et al., 2008). They are also expressed in liver where they reduce circulating androgen levels. Consistent with their important functions in androgen signaling, altered UGT2B15 and UGT2B17 activity is related to prostate cancer risk and progression (Turgeon et al., 2001, Heinlein and Chang, 2004, Chouinard et al., 2007).

The liver is the primary site of glucuronidation; thus, the control of hepatic UGT expression and activity is of significant biologic and pharmacological interest (Hu et al., 2014b). UGT2B4 and UGT2B7 are both highly expressed in the liver (Congiu et al., 2002, Court, 2010) as well as in extra hepatic tissues where they play important roles in drug metabolism and homeostasis of endogenous bioactive molecules (Hu et al., 2014b, Ohno and Nakajin, 2009). UGT2B7 has high activity toward various endogenous compounds including bile acids, retinoids and steroids (Hu et al., 2014b), as well as exogenous compounds including carcinogens and drugs. Around 35% of the therapeutic drugs that are glucuronidated are UGT2B7 substrates (Williams et al., 2004). UGT2B4 glucuronidates various endogenous compounds as well as a subset of drugs and toxins; however, UGT2B4 is generally considered to play a more minor role in overall drug metabolism than UGT2B7.

The transcriptional regulation of UGT2B15, UGT2B17, UGT2B7 and UGT2B4 (in the prostate and hepatic models respectively) have been well investigated. However, little is known about the mechanisms controlling these UGTs at the post-transcriptional level. MicroRNAs (miRNAs/miRs) are noncoding small RNAs that mediate post-transcriptional gene regulation through translational repression and/or mRNA degradation (Macfarlane and Murphy, 2010). This project investigated the potential for miRNA to regulate UGT2B15 and UGT2B17 in prostate cancer cells and UGT2B4 and UGT2B7 in liver cancer cells.

Putative target sites for miR-376c, miR-376b, miR-222 and miR-331-5p were identified in the 3’-untranslated regions (UTRs) of the UGT2B15 and/or UGT2B17 mRNAs. In prostate-derived LNCaP cells, miR-376c mimics reduced both UGT2B15 and UGT2B17 mRNA and protein levels and inhibited glucuronidation of the UGT2B15/UGT2B17 substrates testosterone, 4-methylumbelliferone, and androsterone. MiR-376c repressed the activity of luciferase reporters containing UGT2B15 or UGT2B17 3’-UTRs, and this was abrogated by mutating the predicted miR-376c binding sites. Consistent with the idea that miR-376c negatively regulates UGT2B15 and UGT2B17 expression, the level of this miRNA was inversely correlated with UGT2B15/UGT2B17 mRNA levels in normal prostate, primary and metastatic prostate cancer tissues, and in prostate cancer cell lines. Two other miRNAs, miR-222 and miR-376b, also reduced UGT2B15 mRNA levels in LNCaP cells and repressed the UGT2B15 3’-UTR, the latter was abrogated by mutating the relevant miRNA binding sites in the 3’ UTR. Levels of miR-222 were inversely correlated with UGT2B15/UGT2B17 mRNAs levels in normal prostate, and primary and metastatic prostate cancers. Endogenous UGT2B15 expression and activity was also repressed by miR-331-5p; however, this miRNA did not alter UGT2B17 expression. Two miR-331-5p binding sites, a canonical target site and a novel non-canonical site, were identified in the UGT2B15 3’UTR and shown to function additively. UGT2B15 and miR-331-5p levels were inversely correlated in a panel of normal human tissues, and in a liver cancer data set, but not in a prostate cancer data set. Overall, these data suggest that miR-376c and miR-331-5p (and possibly miR-222, miR-376b) are important regulators of UGT2B15 and/or UGT2B17 levels, but may play varying roles in different tissues. Moreover, miR-331-5p mediates differential regulation of the UGT2B15 and UGT2B17 mRNAs. This represents the first evidence for post-transcriptional regulation of UGT2B15 and UGT2B17 by miRNAs in prostate cancer cells and may have importance in regulating AR signaling.

Putative target sites were identified for miR-3664-3p in the UGT2B7 3’-UTR and for miR-135a-5p and miR-410-3p in the UGT2B4 3’-UTR. Transfection of miR-3664-3p mimics in HepG2 liver cancer cells significantly reduced UGT2B7 mRNA and protein levels leading to reduced enzymatic activity. Transfection of miR-135a-5p or miR-410-3p mimics significantly decreased UGT2B4 mRNA levels in Huh7 liver cancer cells. MiR-3664-3p, miR-135a-5p and miR-410-3p directly repressed the UGT2B7 and UGT2B4 3’-UTRs respectively as shown using reporter assays. This repression was abrogated by mutating the relevant miRNA binding sites in the 3’-UTR reporter constructs. The expression levels of miR-410-3p were inversely correlated with UGT2B4 mRNA levels in a cohort of liver hepatocellular carcinoma (371 specimens) and a panel of normal human tissues. Similarly, there was an inverse correlation between miR-135a and UGT2B4 mRNA levels in a panel of 18 normal human liver tissues. Together, these data suggest that miR-135a and miR- 410 control UGT2B4 and that miR-3664 controls UGT2B7 expression in liver cancer and/or normal liver cells.

Keywords: UGT, miRNA, microRNA, miR, UGT2B15, UGT2B17, UGT2B4, UGT2B7, regulation, post-transcriptional regulation, prostate cancer, PCa, glucuronidation, drug metabolism, miR and cancer, gene regulation, posttranscriptional regulation, 2B15, 2B17, 2B4, 2B7, miR-376c, miR-331-5p, miR-3664, miR-135a-5p, miR-410-3p, miR-222, miR-376b

Subject: Medical Science thesis

Thesis type: Doctor of Philosophy
Completed: 2021
School: College of Medicine and Public Health
Supervisor: Prof. Peter Mackenzie