Suppression of Ov-grn-1 encoding granulin of Opisthorchis viverrini inhibits proliferation of biliary epithelial cells

Abstract

Multistep processes likely underlie cholangiocarcinogenesis induced by chronic infection with the fish-borne liver fluke, Opisthorchis viverrini. One process appears to be cellular proliferation of the host bile duct epithelia driven by excretory-secretory (ES) products of this pathogen. Specifically, the secreted growth factor Ov-GRN-1, a liver fluke granulin, is a prominent component of ES and a known driver of hyper-proliferation of cultured human and mouse cells in vitro. We show potent hyper-proliferation of human cholangiocytes induced by low nanomolar levels of recombinant Ov-GRN-1 and similar growth produced by low microgram concentrations of ES products and soluble lysates of the adult worm. To further explore the influence of Ov-GRN-1 on the flukes and the host cells, expression of Ov-grn-1 was repressed using RNA interference. Expression of Ov-grn-1 was suppressed by 95% by day 3 and by ~100% by day 7. Co-culture of Ov-grn-1 suppressed flukes with human cholangiocyte (H-69) or human cholangiocarcinoma (KKU-M214) cell lines retarded cell hyper-proliferation by 25% and 92%, respectively. Intriguingly, flukes in which expression of Ov-grn-1 was repressed were less viable in culture, suggesting that Ov-GRN-1 is an essential growth factor for survival of the adult stage of O. viverrini, at least in vitro. To summarize, specific knock down of Ov-grn-1 reduced in vitro survival and capacity of ES products to drive host cell proliferation. These findings may help to contribute to a deeper understanding of liver fluke induced cholangiocarcinogenesis.

Keywords: Cellular proliferation; Cholangiocarcinoma; Granulin; Liver fluke; O. viverrini; RNA interference.

Figure 1. Proteins from Opisthorchis viverrini stimulate the proliferation of human cholangiocytes in vitro

Normalized Cell Index is the output from the xCELLigence instrument. Ov-GRN-1 is produced in E. coli expressed and refolded into an active growth factor. The lysate of O. viverrini represents a soluble extract of adult Opisthorchis viverrini worms. OvES (excreted/secreted products) is the supernatant from cultured adult O. viverrini. TRX is thioredoxin from E. coli and is an expression-matched control for Ov-GRN-1. Error bars reveal the standard error of triplicate biological replicates. **** denotes statistical significance at the level of P ≤ 0.0001.

Figure 2. Gene-specific silencing of Ov-grn-1 in Opisthorchis viverrini following delivery of dsRNA by square wave electroporation

Real-time qPCR analysis revealed the transcript levels of Ov-grn-1 normalized to levels of Ov-actin in flukes after electroporation and soaking with Ov-grn-1 or luciferase (Luc) dsRNAs. Ov-grn-1 transcript levels had decreased significantly by day 1 and were not detectable by day 7. Ov-grn-1 RT-PCR products amplified from total RNA of adult flukes after electroporation with either Ov-grn-1or Luc dsRNAs (inset). Data are represented by mean ± 1 standard deviation; *** denotes statistical significance at the level of P ≤ 0.001.

Figure 3. Survival rate of Opisthorchis viverrini after RNAi-based silencing of Ov-grn-1

Twenty adult flukes were cultured for five days. The worms were examined the viability with Trypan blue by light microscopy. Data were represented by mean ± 1 standard deviation. Significance at the level of P ≤ 0.05 level denoted with * in a comparison with flukes not exposed to dsRNA but subjected to identical electroporation (mock).

Figure 4. Proliferation of cholangiocytes repressed after exposure to Opisthorchis viverrini subjected to gene knockdown of Ov-grn-1

Growth expressed as the ratio of cell numbers per well between treatment and control groups (“no parasites” used as 100% negative control). The effect of specific repression of expression of Ov-grn-1 on growth of lines of immortalized cholangiocytes (H69 cell line) (panel A) and cholangiocarcinoma cells (KKU-M214 cells) (Panel B) resulted in significantly decreased proliferation compared to controls during cultivation for three days. Findings presented as the means of biological quadruplicates, with the standard deviation shown as bars. Asterisks indicate significant differences between mock control and Ov-grn-1 dsRNA treatments: ** and *** represent P ≤ 0.01 and 0.001, respectively.