Screening of differentially expressed miRNAs in tensile strain­treated HepG2 cells by miRNA microarray analysis.

Cirrhosis and portal hypertension are associated with an increased risk of developing liver cancer. However, it is unknown how changes in the cellular mechanical microenvironment induced by portal hypertension affect the occurrence and development of liver cancer. The aim of this study was to determine the effect of tensile strain on the proliferation of a human liver cancer cell line (HepG2 cells) using methods such as flow cytometry, Cell Counting Kit­8 and 5­bromodeoxyuridine assays, and to examine the changes in microRNA (miRNA/miR) expression using microarray, reverse transcription­quantitative (RT­q)PCR and bioinformatics analyses. It was demonstrated that cyclic tensile force promoted the proliferation of HepG2 cells. The most suitable research conditions were as follows: Tensile strain force loading amplitude 15%; frequency 1 Hz; and time 24 h. After loading the HepG2 cells under such conditions, the differentially expressed miRNAs were screened out using an Agilent Human miRNA Microarray, identifying seven miRNAs with significant differences (expression difference >2 times and P<0.05). A total of five were upregulated, including hsa­miR­296­5p, hsa­miR­6752­5p, hsa­miR­6794­5p, hsa­miR­6889­5p and hsa­miR­7845­5p; and two were downregulated, hsa­miR­4428 and hsa­miR­503­5p. The results of RT­qPCR also further confirmed the expression changes of these miRNAs. Gene Ontology and pathway analyses showed the involvement of these miRNAs in numerous important physiological processes. These findings may provide novel miRNA­based information, thus enhancing the understanding of the pathophysiological processes leading to liver cancer.

Identification of miRNAs associated with the mechanical response of hepatic stellate cells by miRNA microarray analysis.

It has been suggested that hepatic stellate cells (HSCs) could be used in the regulation of liver microcirculation and portal hypertension. The effects of tensile strain on the microRNA (miRNA) profile of HSCs are largely unknown. In this study, we aimed to explore the changes of miRNA expression in tensile strain-treated HSCs. The purity and activation of HSCs were determined by immunofluorescence staining with antibody against desmin and a-SMA, respectively. miRNA profile analysis was performed on HSCs with and without tensile strain treatment (n=3) using microarray analysis. We identified 6 significantly differentially expressed miRNAs (DEMs), including 1 downregulated (rno-miR-125b-2-3p) and 5 upregulated (rno-miR-1224, rho-miR-188-5p, rho-miR-211-3p, rho-miR-3584-5p and rho-miR-466b-5p), which were validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) experiments. Further analysis of the DEMs revealed that many important biological processes and signal pathways were triggered in tensile strain-treated HSCs. These include the signal transduction mechanisms associated with protein binding, apoptosis, proliferation, and the FoxO and Wnt signaling pathways. In conclusion, this study presents the specific DEMs in tensile strain-treated HSCs. Our study provide novel miRNA-based information that may enhance our understanding of the pathophysiological processes leading to portal hypertension.

Mechanical force and tensile strain activated hepatic stellate cells and inhibited retinol metabolism.

OBJECTIVES: Hepatic stellate cells (HSCs), the principal producers of extracellular matrix proteins, play a major role in the development of liver fibrosis which is accompanied with elevated sinusoidal pressure and portal hypertension. We have isolated primary rat HSCs and investigated the effect of mechanical pressure and tensile strain on retinol metabolism in the cells. RESULTS: Mechanical force and tensile strain significantly increased the expression of α-smooth muscle actin (α-SMA) and collagen I, and notably inhibited the expression of cellular retinol-binding protein I (CRBP-I), lecithin-retinol acyltransferase (LRAT), retinyl ester hydrolase (REH), retinoic acid receptor-ß (RAR-ß) and retinoid X receptor-α (RXR-α). Such effects were partially reversed by the retinoid X receptor antagonist, HX531, and the retinoic acid receptor antagonist, LE135. CONCLUSION: Mechanical force and tensile strain significantly activate HSCs by regulating the retinoid metabolic pathway. Activation of HSCs can therefore be manipulated through mechanical force and tensile strain in vitro.