Regulation of microRNA expression by p73-isoforms in hepatocellular carcinoma

Question:

p53 is one of the most mutated genes in cancer cells. Besides p53, p63 and p73 belong to the p53-family of transcription factors which respond to cellular stress signals. p63 and p73 share lot of functions with p53, but can also exert independent effects. Depending on their splice variants – with transactivation domain (TA)/dominant negative (DN) – and the characteristics of the particular binding site p53 proteins activate or inhibit specific target genes. Micro-RNAs are small, non-coding RNA-molecules with a length of about 22 nucleotides, which also play an important role in gene regulation. Their expression profiles can in turn be controlled by the p53 family. Little is known about p73-dependent microRNA profiles in hepatocellular carcinoma (HCC). The aim of this study was therefore to identify microRNAs in HCC which are regulated by p73 and its isoforms.

Methods:

Hep3B-cells were transfected with rAd-TAp73, rAd-ΔNp73 or rAd-GFP. Tap73- and ΔNp73-dependent microRNA expression was determined by qPCR. To evaluate effects of HCC-relevant therapeutics on TA/ΔNp73-dependent microRNA regulation cells were treated with doxorubicin, bleomycin, regorafenib, sorafenib or tivantinib for up to 72 hours.

Results:

72 hours after transfection, overexpression of TAp73 induced a strong induction of microRNA 34a by 28.1-fold, microRNA 145 by 21.2-fold and microRNA 149 by 11.3-fold. While treatment with doxorubicin led to an only slight enhancement of microRNA 34a to a 29-fold expression, tivantinib had synergistic effects, leading to a 34.3-fold increased expression in combination with TAp73 overexpression. Interestingly, ΔNp73 overexpression showed contrary effects: expression levels of microRNAs 34a, 145 and 149 were only raised by 2.4- to 3.2-fold 72 hours after rAd-ΔNp73 transfection. In combination with drug treatment their expression levels were unchanged or even reduced compared to untreated cells.

Conclusion:

p73 isoforms divergently affect microRNA profiles in HCC. We demonstrate that TAp73 is an important regulator of microRNAs 34a, 145 and 149 and provide evidence for a synergistic action of Tap73 overexpression and chemotherapeutics and targeted therapies in vitro. In contrast, ΔNp73 downregulated microRNA expression levels. We hypothesize that regulation of tumorsuppressive microRNAs represents an effector mechanism by which Tap73 exerts its role in tumor development and treatment response. Synergistic effects with HCC-relevant therapeutics might provide new options for the development of therapeutic and prognostic measures in HCC.