H19 is a paternally imprinted gene encoding a noncoding RNA . As one of the first imprinted genes discovered, H19 was initially identified as a tumor suppressor because embryo-derived tumor cells overexpressing H19 exhibited growth retardation, morphological changes and abrogated clonogenicity in soft agar, as well as suppressed tumorigenicity in nude mice . Downregulation of H19 gene expression was identified as an early event in the formation of several tumor types [3–8]. Recently it was demonstrated that mice lacking H19 showed an overgrowth phenotype, while transgenic mice overexpressing H19 showed postnatal growth reduction .
In contrast, overexpression or loss of imprinting (LOI) of H19 has been observed in a wide variety of tumors, including bladder carcinoma [10, 11], epithelial ovarian cancer , esophageal cancer , lung cancer , breast adenocarcinoma [15, 16], endometrial cancer , and invasive cervical carcinoma . H19 knockdown significantly decreased the clonogenicity and anchorage-independent growth property of several breast and lung cancer cell lines . A link between H19 and several tumorigenesis-related genes, such as c-Myc , thioredoxin  and E2F1 , has been well established. Thus, it remains controversial whether H19 functions as a tumor promotor or a tumor suppressor, and it is possible that H19 plays differential roles depending on tissue type and/or developmental stage .
H19 is highly expressed in trophoblast tissue, predominantly but not exclusively from the maternal allele [22, 23]. Complete hydatidiform moles (CHM) showed high-level expression of H19 from the paternal allele, while choriocarcinomas developing from CHMs had reduced numbers of H19-positive cells . H19 downregulation was also observed in gestational trophoblastic tumors . These observations appear to support the notion that H19 functions as a tumor suppressor in trophoblast tissue. However, Ariel I et al. found that H19 expression was found to be abundant, in a decreasing order, in the intermediate trophoblast (villous and interstitial), the cytotrophoblast, and the syncytiotrophoblast. And prominent expression of H19 was found in placental site trophoblastic tumor and gestational choriocarcinoma . After choriocarcinoma-derived JEG-3 cells were subcutaneously injected into nude mice, a five-fold increase in H19 RNA level was detected in the resulting tumors, and cells highly expressing H19 were more tumorigenic . Therefore, the exact role of H19 in the trophoblast is yet undetermined.
In previous studies, we demonstrated that DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine could demethylate the promoter region of the H19 gene, upregulate the expression of H19 transcript, and reduce the proliferation, migration and invasion properties of choriocarcinoma-derived JEG-3 cells [27, 28]. As the demethylating effects of 5-aza-2′-deoxycytidine are unspecific, other genes may be demethylated in tandem and complicate the biological effects. To further clarify our understanding of the function of the H19 gene in choriocarcinoma, we developed lentiviral vectors expressing H19-specific small interfering RNA (siRNA) for use in human choriocarcinoma cells. Considering that the human choriocarcinoma cell line JAR is more easily infected by lentiviruses than the cell line JEG-3 (our unpublished observation), JAR cells were used in the present study. Using the siRNA technology, we investigated the impact of H19 knockdown on the proliferation, invasion, and apoptosis of JAR cells. Furthermore, we examined the effects of H19 knockdown on the expression of the insulin-like growth factor 2 (IGF2), hairy and enhancer of split homologue-1 (HES-1), and dual-specific phosphatase 5 (DUSP5) genes, which were chosen based on the previous evidence that H19 is a negative regulator of IGF2  and our unpublished observation that the expression of HES-1 and DUSP5 genes was altered in JEG-3 cells transfected with a eukaryotic expression vector carrying the full-length H19 cDNA.