[Expression of hepatocyte nuclear factor 4γ in gastric carcinoma and its role in cell proliferation and stemness].

Objective: To explore the role and molecular mechanism of hepatocyte nuclear factor 4γ (HNF4γ) in proliferation and stemness of gastric cancer. Methods: A total of 102 cases of paraffin-embedded gastric cancer tissues and matched adjacent gastric tissues and 42 cases of fresh-frozen tissues derived from gastric patients who received radical gastrectomy were collected from the First Affiliated Hospital of Zhengzhou University between 2012 to 2015. The expression of HNF4γ was tested by immunohistochemical staining, quantitative real-time polymerase chain reaction (qRT-PCR). HNF4γ overexpressed (AGS-HNF4γ) and shRNA silenced (HGC27-shHNF4γ) gastric cell lines were established. The effects of HNF4γ on cell proliferation and stemness were verified by XTT, clone formation and sphere formation assay. The expression of CD44 was detected by western blot. Results: The mRNA expression level of HNF4γ in fresh-frozen gastric cancer tissue was (12.43±2.702), which was significantly higher than (3.639±1.109) in normal tissue (P<0.001). The high protein expression rate of HNF4γ in paraffin-embedded gastric cancer tissues was 41.2% (42/102), which was significantly higher than 8.8% (9/102) in normal gastric mucosa tissue (P< 0.001). The protein expression of HNF4γ was closely related to the tumor differentiation, infiltration depth, lymph node metastasis and tumor stage (P<0.05). The median survival interval of patients with HNF4γ high expression was 25 months, the 3-year survival rate was 4.8% (2/42), significantly lower than 38 months and 51.7% (31/60) of patients with normal HNF4γ expression (P<0.001). The proliferation and CD44 protein expression of AGS-HNF4γ cells were significantly higher than those of the AGS-Vector cells. The number of clone formation, sphere formation rate of AGS-HNF4γ cells were 243.5±24.5 and (83.5±3.9)%, significantly higher than 81.0±16.0 and (21.8±5.6)% of AGS-Vector cells (P=0.030 and P=0.010, respectively). The proliferation and CD44 protein expression of HGC27-shHNF4 cells were significantly lower than those of the HGC27-vector cells. The number of clone formation, sphere formation rate of HGC27-shHNF4 cells were 26.0±1.0 and (20.8±8.4)%, significantly higher than 83.5±4.5 and (72.5±4.8)% of HGC27-vector cells (P=0.006 and P=0.030, respectively). Conclusions: HNF4γ is upregulated in the gastric cancer tissues and related with the poor prognosis of patients with gastric cancer. Overexpression of HNF4γ promotes the proliferation and remains the stemness of gastric cancer cells by upregulating the expression of CD44.

[Significance of hydroxymethylglutaryl-CoA synthase 2 on prognosis of esophageal squamous cell carcinoma].

Objective: To explore the roll and function of hydroxymethylglutaryl-CoA synthase 2 (HMGCS2) in the development and progression of human esophageal squamous cell carcimoma(ESCC). Method: Using immunohistochemistry, the expression of HMGCS2 was determined in 150 primary ESCC patients from July 2002 to December 2005 in the People's Hospital of Linzhou City, Henan Province. And HMGCS2 over-expression ESCC cell lines were established to verify HMGCS2 gene function. Result: In 150 cases of ESCCs, the expression rate of HMGCS2 was 58% (87/150), which was lower than 72% (108/150) in paired normal tissues, the difference was statistically significant (P=0.013). HMGCS2 down-regulated expression was associated with tumor cell differentiation (P=0.022), pT status (P=0.036), pN status (P=0.017) and TNM stage(P=0.012). The 5-years disease-specific survival (DSS) in down HMGCS2 expression group (14 months) was poorer than those in normal expression group (20 months; P=0.002). In addition, multivariate Cox regression analysis showed that HMGCS2 expression (Wald=7.136, P=0.008) was an independent risk factor for DSS. Furthermore, functional studies demonstrated that HMGCS2 gene could suppress the tumorigenic ability of ESCC cells (OD: 0.79±0.04 vs 1.25±0.68; P=0.01), the formation of colone (number of colones: 30±10 vs 189±15, P=0.002), and cell motility (number of cells: 27±14 vs 222±40, P=0.009). Conclusion: HMGCS2 can inhibit the proliferation and migration of ESCC cells, and could be an important candidate tumor suppressor gene for ESCC.

MicroRNA-377 suppresses initiation and progression of esophageal cancer by inhibiting CD133 and VEGF.

Esophageal cancer is one of the most lethal cancers worldwide with poor survival and limited therapeutic options. The discovery of microRNAs created a new milestone in cancer research. miR-377 is located in chromosome region 14q32, which is frequently deleted in esophageal squamous cell carcinoma (ESCC), but the biological functions, clinical significance and therapeutic implication of miR-377 in ESCC are largely unknown. In this study, we found that miR-377 expression was significantly downregulated in tumor tissue and serum of patients with ESCC. Both tumor tissue and serum miR-377 expression levels were positively correlated with patient survival. Higher serum miR-377 expression was inversely associated with pathologic tumor stage, distant metastasis, residual tumor status and chemoradiotherapy resistance. The roles of miR-377 in suppressing tumor initiation and progression, and the underlying molecular mechanisms were investigated. Results of in vitro and in vivo experiments showed that miR-377 overexpression inhibited the initiation, growth and angiogenesis of ESCC tumors as well as metastatic colonization of ESCC cells, whereas silencing of miR-377 had opposite effects. Mechanistically, miR-377 regulated CD133 and VEGF by directly binding to their 3' untranslated region. Moreover, systemic delivery of formulated miR-377 mimic not only suppressed tumor growth in nude mice but also blocked tumor angiogenesis and metastasis of ESCC cells to the lungs without overt toxicity to mice. Collectively, our study established that miR-377 plays a functional and significant role in suppressing tumor initiation and progression, and may represent a promising non-invasive diagnostic and prognostic biomarker and therapeutic strategy for patients with ESCC.

Overexpression of matrix metalloproteinase 10 is associated with poor survival in patients with early stage of esophageal squamous cell carcinoma.

Matrix metallopeptidase 10 (MMP10) is frequently expressed and correlates closely with metastasis and poor prognosis in various human cancers. However, the significance of MMP10 expression in esophageal squamous cell carcinoma (ESCC) and its role in ESCC progression remains unclear. In this report, upregulation of MMP10 mRNA was detected in 39/60 (65.0%) of primary ESCC tissues compared with their paired nontumor esophageal tissues. Tissue microarray (TMA) study found protein overexpression of MMP10 in 188/239 (78.7%) of primary ESCC tissues but not in their corresponding nontumor esophageal tissues, suggesting that overexpression of MMP10 may play important roles in ESCC development and progression. Although the overexpression of MMP10 was not significantly associated with disease-specific survival rate (P= 0.182) for all tested ESCCs, it was significantly associated with poorer disease-specific survival (P= 0.001) in early stage of ESCCs (I-IIA). In addition, multivariate analysis found that MMP10 expression in tumor tissues was evaluated as a potential independent prognostic factor for early stage ESCC patients. These findings suggest that MMP10 plays an important role in ESCC progression in the early stage, and overexpression of MMP10 in tumor tissues could be used as a potential prognostic marker for patients with early clinical stage of ESCC.

Directional differentiation of chicken primordial germ cells into adipocytes, neuron-like cells, and osteoblasts.

Primordial germ cells (PGCs) are useful for producing transgenic chickens and preserving genetic material in avian species. In this study, we investigated the in vitro differentiation potential of chicken PGCs into different cell types. For differentiation into adipocytes, chicken PGCs were cultured for 21 days in induction media containing dexamethasone, insulin and/or 3-isobutyl-1-methylxanthine (IBMX), and differentiation rates ranging from 74% to 91% were identified by oil red-O and alkaline phosphatase (ALP) staining. For differentiation into neuron-like cells, chicken PGCs were cultured for 3 or 7 days in the induction media containing retinoic acid (RA) and IBMX, and differentiation rates ranging from 71% to 87% were identified by toluidine blue staining and immunohistochemical staining. For differentiation into osteoblasts, chicken PGCs were cultured for 15 or 21 days in the induction media containing desamethasone, beta-glycerol phosphate and/or vitamin C, and differentiation rates ranging from 47% to 79% were confirmed by Von Kossa, cytochemical and immunohistochemical staining. These data suggest that, like mammalian PGCs, chicken PGCs can differentiate into different cell types in vitro.

Characterization of tumor suppressive function of P300/CBP-associated factor at frequently deleted region 3p24 in esophageal squamous cell carcinoma.

Deletion of 3p is one of the most frequent genetic alterations in many tumors, including esophageal squamous cell carcinoma (ESCC). In our recent study, deletion of 3p24 was frequently detected in ESCC and one candidate tumor suppressor gene (TSG), p300/CBP-associated factor (PCAF), was identified within the region. In this study, downregulation of PCAF was detected in 23/40 (57.5%) of primary ESCCs and 4/9 (44.4%) of the ESCC cell lines. A further study found that downregulation of PCAF was also associated with hypermethylation of the promoter region of PCAF gene. Methylation-specific PCR found that promoter methylation was detected in 28/40 (70%) of primary ESCCs and 5/9 (55.6%) of ESCC cell lines. In addition, the expression of PCAF could be reactivated in ESCC cell line KYSE510 after demethylation treatment with 5-aza-dC. Functional studies showed that PCAF was able to suppress tumorigenicity of ESCC cells both in vitro and in vivo, including foci formation, colony formation in soft agar and tumor formation in nude mice. Molecular study found that the tumor suppressive mechanism of PCAF was associated with its role in cell cycle arrest at the G1/S checkpoint by the downregulation of CDK2 and upregulation of p21(waf1/Cip1), Smad4, Rb and p27(Kip1). In conclusion, PCAF might be the target TSG responsible for the 3p24 deletion event, which has an important role in the development and progression of ESCC.

Behavior differentiation between wild Japanese quail, domestic quail, and their first filial generation.

The number of wild quail has dramatically reduced in China and reached a state of endangerment with the deterioration of the environment in recent years. In this study, we examined the ecological behaviors of quails in the cage to determine the differentiation level between wild Japanese quail and domestic quail, to detect the relationship between quail behavior and evolutionary differentiation and to analyze the possibility of restoring effective size of wild population. With the on-the-spot observations and measurements, the behaviors of 3 categories of quail, namely wild Japanese quail from the Weishan Lake area in China, domestic quail, and their first filial generation (F(1)) were studied. Domestic quail differed from wild Japanese quail in morphological pattern and ecological behaviors, including some indexes of figure type and egg, vocalization, aggression and fighting, and mating, but wild Japanese quail and domestic quail could succeed in mating and reproducing fertile hybrid offspring. There were significant differences between domestic quail and wild Japanese quail in reproductive traits, involved mating times, fertility rate, hatching rate, and hatching rate of fertilized eggs (P < 0.05). The first filial generation presented significant difference from the wild Japanese quail in vocalization, aggression and fighting, mating, hatching rate, hatching rate of fertilized eggs, and some egg indexes (P < 0.05) and significantly differ from the domestic quail in vocalization, hatching rate, and hatching rate of fertilized eggs (P < 0.05). Evolutionary differentiation between wild quail and domestic quail was still at a relatively low level because no reproductive isolation existed. The advantages of the F(1) hybrids in reproductive capacity, fertilization, and hatching recommend that releasing hybrids instead of domestic quails to the wild would be a more effective way to restore the effective size of wild quail population if necessary.

Uptake and accumulation of arsenic by 11 Pteris taxa from southern China.

A field survey was conducted at a deserted arsenic (As) mine in Guangxi Province, China to explore new potential As hyperaccumulators. In addition, young plants of 11 Pteris taxa were grown in glasshouse conditions for 12 weeks on As-amended soils with 0, 50 and 200 mg As kg(-1). Results of the field survey showed that the fern Pteris fauriei accumulated over 1000 mg As kg(-1) in its fronds. Of the 11 Pteris taxa, Pteris aspericaulis, Pteris cretica var. nervosa, P. fauriei, Pteris multifida, P. multifida f. serrulata, and Pteris oshimensis were all found to hyperaccumulate As in addition to P. cretica 'Albo-Lineata' and Pteris vittata (already reported as As hyperaccumulators). However, Pteris ensiformis, Pteris semipinnata and Pteris setuloso-costulata showed no evidence of As hyperaccumulation. Results also revealed a constitutive property of As hyperaccumulation in different populations of P. cretica var. nervosa, P. multifida, P. oshimensis and P. vittata.

Comparative genomic hybridization: comparison between esophageal squamous cell carcinoma and gastric cardia adenocarcinoma from a high-incidence area for both cancers in Henan, northern China.

Esophageal squamous cell carcinoma (SCC) remains the leading cause of cancer related deaths in Linzhou (formerly Linxian), the highest incidence area for esophageal cancer (EC) in Henan, northern China. In China, gastric cardia adenocarcinoma (GCA) shares very similar geographic distribution with SCC, suggesting the possibility of similar risk factors involved in SCC and GCA carcinogenesis in these areas. However, the underlying genetic alterations for esophageal and gastric cardia carcinogenesis, especially for the molecular difference between SCC and GCA, are largely unknown. The present study was thus undertaken to determine the difference in chromosomal aberrations in SCC (n = 37) and GCA (n = 31) using the comparative genomic hybridization method (CGH). All the patients were from Linzhou, Henan, a high-risk geographic region for both SCC and GCA. CGH results showed that chromosomal aberrations with different degrees were identified both in SCC and GCA. In SCC, chromosomal profile of DNA copy number was characterized by most frequently detected gains at 8q (29/37, 78%), 3q (24/37, 65%) and 5p (19/37, 51%); and frequently detected losses at 3p (21/37, 57%), 8p and 9q (14/37, 38%). In GCA, the frequently detected gains were identified at 20q (13/31, 42%), 6q (12/31, 39%) and 8q (11/31, 35%); the DNA copy number losses in GCA occurred frequently at 17p (17/31, 55%), 19p (15/31, 48%) and 1p (14/31, 45%). Statistically, there were evident differences between SCC and GCA in DNA copy number gains at 8q, 3q, 5p and 20q (P < 0.05) and in losses at 3p, 8p, 5q, 17p and 18q (P < 0.05). Gains at 8q were frequently observed in both SCC and GCA. Gains at 3q and 8p were frequently observed in TNM stage III of both SCC and GCA. The present CGH results provide candidate regions that may contain specific related genes involved in SCC and GCA in the Linzhou population. Gains at 8q, 3q and 5p and losses at 3p, 8p and 9q were specifically implicated in SCC; gains at 20q, 6q and 8q and losses at 17p, 19p and 1p were specifically implicated in GCA; gains at 8q were implicated in both SCC and GCA.