Upregulation of the oestrogen target gene SIX1 is associated with higher growth speed and decreased survival in HCV-positive women with hepatocellular carcinoma

Rosina Maria Critelli, Fabiola Milosa, Adriana Romanzi, Simone Lasagni, Gemma Marcelli, Lorenza Di Marco, Alessandra Pivetti, Filippo Schepis, Dante Romagnoli, Serena Mancarella, Francesco Dituri, Maria-Luz Martinez-Chantar, Gianluigi Giannelli, Erica Villa, Rosina Maria Critelli, Fabiola Milosa, Adriana Romanzi, Simone Lasagni, Gemma Marcelli, Lorenza Di Marco, Alessandra Pivetti, Filippo Schepis, Dante Romagnoli, Serena Mancarella, Francesco Dituri, Maria-Luz Martinez-Chantar, Gianluigi Giannelli, Erica Villa

Abstract

The male/female ratio of patients with hepatocellular carcinoma (HCC) is often unbalanced towards the male sex, indicating a sex predisposition for HCC development. A possible explanation may be attributed to different hormonal statuses, including the pro-inflammatory action of androgens in men and the protective effects of oestrogen against excessive inflammation in women. Although several studies have studied gene expression in patients with HCC, very few have attempted to identify features that could be distinctive between male and female patients. The present study aimed to identify distinctive signalling mechanisms between men and women that may be associated with HCC progression. The present study analysed a detailed microarray database that was obtained from the prospective study of 78 patients with HCC to study gene expression according to sex. In addition, the present study aimed to evaluate whether the differentially expressed genes were known oestrogen targets. Moreover, RNAs from the HCC cohort were evaluated for microRNA (miRNA/miR) expression, and a relationship between miRNA and gene expression according to sex was investigated. One gene, sineoculis homeobox homolog 1 (SIX1), which is known to be an oestrogen target gene, was revealed to be highly upregulated in hepatitis virus C (HCV)-positive female patients with HCC but not in HCV-positive male patients. In addition, SIX1 upregulation had a significant relationship with tumour growth speed (assessed as tumour doubling time in two CTs performed 6 weeks apart) and survival (P=0.009 and P=0.042, respectively) in female patients only. Furthermore, SIX1 upregulation was related with miR-421 and miR-9-5p only in male patients; however, in female patients, SIX1 upregulation had a direct relationship with miR-181b, miR-503-5p and miR-125b (miRNAs with potential oncogenic capacity), and an inverse correlation with miR139-5p, miR-26b, let7c-3p and let7c-5p (putatively oncosuppressive microRNAs). These data suggested a distinctive model for liver carcinogenesis in HCV-positive women, with downregulation of protective mechanisms against tumour progression and the activation of potential oncogenes, in relation to the oestrogen target gene SIX1. (IRB10/08_CE_UniRer; ClinicalTrials ID: NCT01657695).

Keywords: TGF-β; biologic aggressiveness; hepatocellular carcinoma; microRNA; sex; sineoculis homeobox homolog 1.

Conflict of interest statement

The authors declare that they have no competing interests.

Copyright: © Critelli et al.

Figures

Figure 1.
Figure 1.
Transcriptomic and proteomic levels of SIX1 in nontumor and tumour tissue according to sex. (A) mRNA levels were evaluated by microarray analysis (Agilent Whole Human Genome Oligo Microarrays). Expression levels in non-tumor tissue were non-significantly different between males and females. In the tumour tissue, SIX1 levels only moderately increased in males while in females a sharp increase was observed. Comparisons were performed by paired t-test between T and NT in males, and T and NT in females. T-test for independent groups was used to compared SIX1 in tumour tissue of males vs. females, as well as in non-tumor tissue of males vs. females. (*P

Figure 2.

Evaluation of growth speed (evaluated…

Figure 2.

Evaluation of growth speed (evaluated by the estimate of the doubling time) and…

Figure 2.
Evaluation of growth speed (evaluated by the estimate of the doubling time) and of survival in HCV-positive females and males in relation to low or high SIX1 expression. (A and B) Probability of having elevated growth speed in relation to SIX1 median values in (A) females and (B) males. HCC in females who had elevated SIX1 median values was characterised by significantly higher growth speed (P=0.003 by log-rank test). (C) HCV-positive females with higher median SIX1 levels had significantly lower survival in comparison with those with lower median levels (P=0.020, log-rank test). (D) No difference was present in males in relation to SIX1 levels. (E and F) Survival analysis was performed according to SIX1 proteomic expression in the tumour tissue of (E) females and (F) males. Data confirm survival analysis according to transcriptomic data. The probability of having elevated speed growth and survival was evaluated by the Kaplan-Meier method. For panel 2C and 2E two-stage hazard rate comparison method was used. DT, doubling time.
Figure 2.
Figure 2.
Evaluation of growth speed (evaluated by the estimate of the doubling time) and of survival in HCV-positive females and males in relation to low or high SIX1 expression. (A and B) Probability of having elevated growth speed in relation to SIX1 median values in (A) females and (B) males. HCC in females who had elevated SIX1 median values was characterised by significantly higher growth speed (P=0.003 by log-rank test). (C) HCV-positive females with higher median SIX1 levels had significantly lower survival in comparison with those with lower median levels (P=0.020, log-rank test). (D) No difference was present in males in relation to SIX1 levels. (E and F) Survival analysis was performed according to SIX1 proteomic expression in the tumour tissue of (E) females and (F) males. Data confirm survival analysis according to transcriptomic data. The probability of having elevated speed growth and survival was evaluated by the Kaplan-Meier method. For panel 2C and 2E two-stage hazard rate comparison method was used. DT, doubling time.

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