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We thank Chen et al. for their interest in our study1 identifying microRNA (miR) 19b as an inhibitor of hepatic stellate cell (HSC)-mediated fibrogenesis. We also welcome the opportunity to address reported discrepancies in miR profiles between quiescent and activated HSCs. Although Chen et al. state the same approaches were used in several studies,1-5 resulting in different miR profiles, several noticeable differences in experimental designs exist. Each of the above-mentioned studies used different platforms for miR profiling: 1) Exiqon miRCURY LNA (spotted) array v. 8.03; 2) Ncode Multi-Species miR (spotted) microarray V25; 3) Agilent Rat miR (spotted) microarray (version not specified)1; 4) Affymetrix GeneChip miR 1.0 (oligo) array2; 5) Quantitative reverse-transcription polymerase chain reaction (qRT-PCR).4 Moreover, not all miR assays interrogate the same set of miRs. Additionally, expression miR profiles should be verified by qRT-PCR. For further verification, an in vivo assessment by in situ hybridization would strengthen the findings demonstrated in our study.1 Another technical difference likely to introduce variability is methods and criteria for sample size and data and statistical analyses, which are not always reported.

In addition to methodology/technical differences, the day of HSC harvest is important, and it is clear there are variations in what is considered quiescent and activated cells. Although Chen et al. state that all studies used the same quiescent and activated HSCs, in actuality this was not the case. Among the studies described in this Letter to the Editor, variations in the state of quiescence and activation were reported. Some studies reported freshly isolated, day 2 or day 3 (culture-activated) as a quiescent cell population. Similarly, the activated state of HSCs varied as well; day 7, 10, and 14 (culture-activated) were used. We have shown previously that daily variations in gene expression exist throughout the in vitro HSC transdifferentiation/activation process.6 Our data indicated the specific day of culture activation used can significantly impact studies examining differences between selected populations of quiescent and activated cells. Thus, it is not surprising that different miR array profiles were generated among studies considering the variation in what researchers deemed quiescent and activated. Furthermore, several profiling experiments using miR arrays have been conducted comparing quiescent and in vivo activated HSCs (i.e., carbon tetrachloride [CCl4]7, 8 or bile duct ligation [BDL]9 fibrotic models). Therefore, taking into account differences observed in gene expression profiles between in vitro and in vivo activated HSCs,10 miR array analyses may also produce disparate profiles depending on the activation process selected (i.e., in vitro versus in vivo activation).

Overall, the authors should also note that while some variation does exist in the number/identity of miRs that are differently expressed in quiescent versus activated HSCs, by and large each of the referenced array studies as well as other recent publications do seem to support each other's findings (supported by your table). We hope our response has offered some clarity and insight on miR expression profile discrepancies in HSCs that exist among different research groups/studies.

References

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  • 1
    Lakner AM, Steuerwald NM, Walling TL, Ghosh S, Li T, McKillop IH, et al. Inhibitory effects of microRNA 19b in hepatic stellate cell-mediated fibrogenesis. HEPATOLOGY 2012; 56: 300310.
  • 2
    Chen C, Wu CQ, Zhang ZQ, Yao DK, Zhu L. Loss of expression of miR-335 is implicated in hepatic stellate cell migration and activation. Exp Cell Res 2011; 317: 17141725.
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    Guo CJ, Pan Q, Li DG, Sun H, Liu BW. miR-15b and miR-16 are implicated in activation of the rat hepatic stellate cell: an essential role for apoptosis. J Hepatol 2009; 50: 766778.
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    Ji J, Zhang J, Huang G, Qian J, Wang X, Mei S. Over-expressed microRNA-27a and 27b influence fat accumulation and cell proliferation during rat hepatic stellate cell activation. FEBS Lett 2009; 583: 759766.
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    Maubach G, Lim MC, Chen J, Yang H, Zhuo L. miRNA studies in in vitro and in vivo activated hepatic stellate cells. World J Gastroenterol 2011; 17: 27482773.
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    Lakner AM, Moore CC, Gulledge AA, Schrum LW. Daily genetic profiling indicates JAK/STAT signaling promotes early hepatic stellate cell transdifferentiation. World J Gastroenterol 2010; 16: 50475056.
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    Guo CJ, Pan Q, Cheng T, Jiang B, Chen GY, Li DG. Changes in microRNAs associated with hepatic stellate cell activation status identify signaling pathways. FEBS J 2009; 276: 51635176.
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    Roderburg C, Urban GW, Bettermann K, Vucur M, Zimmermann H, Schmidt S, et al. Micro-RNA profiling reveals a role for miR-29 in human and murine liver fibrosis. HEPATOLOGY 2011; 53: 209218.
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    Venugopal SK, Jiang J, Kim TH, Li Y, Wang SS, Torok NJ, et al. Liver fibrosis causes downregulation of miRNA-150 and miRNA-194 in hepatic stellate cells, and their overexpression causes decreased stellate cell activation. Am J Physiol Gastrointest Liver Physiol 2010; 298: G101106.
  • 10
    De Minicis S, Seki E, Uchinami H, Kluwe J, Zhang Y, Brenner DA, et al. Gene expression profiles during hepatic stellate cell activation in culture and in vivo. Gastroenterology 2007; 132: 19371946.

Ashley M. Lakner Ph.D.*, Nury M. Steuerwald Ph.D.*, Laura W. Schrum Ph.D.*, * Carolinas Medical Center, Charlotte, NC.