Hyperpolyploidization of hepatocyte initiates preneoplastic lesion formation in the liver

Hepatocellular carcinoma (HCC) is the most predominant primary malignancy in the liver. Genotoxic and genetic models have revealed that HCC cells are derived from hepatocytes, but where the critical region for tumor foci emergence is and how this transformation occurs are still unclear. Here, hyperpolyploidization of hepatocytes around the centrilobular (CL) region is demonstrated to be closely linked with the development of HCC cells after diethylnitrosamine treatment. We identify the CL region as a dominant lobule for accumulation of hyperpolyploid hepatocytes and preneoplastic tumor foci formation. We also demonstrate that upregulation of Aurkb plays a critical role in promoting hyperpolyploidization. Increase of AURKB phosphorylation is detected on the midbody during cytokinesis, causing abscission failure and hyperpolyploidization. Pharmacological inhibition of AURKB dramatically reduces nucleus size and tumor foci number surrounding the CL region in diethylnitrosamine-treated liver. Our work reveals an intimate molecular link between pathological hyperpolyploidy of CL hepatocytes and transformation into HCC cells.


Statistics
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Software and code
Policy information about availability of computer code Data collection Quantitative PCR was performed by Lightcycler 480 system (Roche).

Data analysis
GraphPad Prism version 8.4.0 (671) was used for statistic analysis. Images were analyzed by using ImageJ 1.51j8.
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Sample size
The sample size was determined based on the magnitude and consistency of measurable differences between groups (n = (2sigma^2)(Z_1beta + Z_1-alpha/2)^2 / delta^2), and at least the minimal application size of relative statistical methods was used.
Data exclusions For the hepatocyte primary culture, cells with inappropriate density, high mortality, and uneven distribution after seeding were excluded before experimental processing. For the animal experiments, mice with unsuitable situations, such as huge change of body weight, mobility problem, distressed behavior, and fight wounds, were precluded. For the tissue slices with uneven staining were removed.

Replication
All attempts of replication were gave similar results. At least 5 to 10 mice were used for in vivo studies for each group. Primary culture experiments were done at least triplicate, and all attempts at replication were successful.
Randomization Mice and cultured hepatocytes were randomly assigned for time-course study and drugs treatment. Imaging fields were stochastically selected during image acquisition.

Blinding
Investigators were not blinded to drugs treatment during experiments. However, the investigators were blinded to group allocation during data collection, and the persons performing data analysis were unaware of the sample identity.

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