Blockade of ONECUT2 expression in ovarian cancer inhibited tumor cell proliferation, migration, invasion and angiogenesis.

One cut homeobox 2 (ONECUT2 or OC-2) is a newly discovered transcription factor. Aberrant expression of OC-2 is closely related to cell proliferation, migration, invasion, and angiogenesis. In this study, we found that OC-2 expression was upregulated in ovarian adenocarcinoma cells, by Western blot analysis. The results of immunohistochemistry showed that the expression of OC-2 was also increased in malignant ovarian cancer tissue. In order to explore the role of OC-2 in the development of ovarian cancer, siRNAs that specifically targets OC-2 were designed. The siRNA targeting OC-2 could effectively inhibit the vascular endothelial growth factor A (VEGFA) expression, but silence and overexpression of VEGFA did not affect OC-2 expression. In addition, OC2-siRNA could block the proliferation, migration, and invasion, and inhibit epithelial-mesenchymal transition and the AKT/ERK signaling pathway, of human ovarian cancer cells in vitro. In a mouse model of ovarian cancer xenograft tumors, OC2-siRNA could significantly inhibit tumor cell growth and the tumor inhibition rate reached approximately 73%. The results of immunohistochemistry showed that the densities of microvessels stained with CD31, the expression of OC-2 and VEGFA were significantly decreased in tumors. These data indicated that OC-2 was an upstream regulator of VEGFA and silencing OC-2 could inhibit ovarian cancer angiogenesis and tumor growth.

YTHDC1 gene polymorphisms and neuroblastoma susceptibility in Chinese children.

Neuroblastoma (NB) is the most common extracranial tumor in children. YTHDC1, a member of RNA methylation modification binding proteins, plays critical roles in tumor occurrence and metastasis. However, it is unclear whether YTHDC1 gene polymorphisms are related to NB susceptibility. Herein, we aimed to evaluate the association between YTHDC1 gene polymorphisms (rs2293596 T>C, rs2293595 T>C, rs3813832 T>C) and susceptibility of NB by logistic regression models. In this eight-center case-control study, 898 patients with NB and 1734 healthy controls were genotyped by TaqMan assay. The results showed that rs3813832 TC genotype could significantly reduce the susceptibility of NB compared with the TT genotype [adjusted odds ratio (AOR) = 0.81, 95% confidence interval (CI) = 0.68-0.96, P = 0.018]. Combined genotype analysis revealed that individuals with 3 protective genotypes had a prominently lower NB risk than those with 0-2 protective genotypes (AOR = 0.80, 95% CI = 0.68-0.94, P = 0.006). The stratified analysis also demonstrated the protective effect of rs3813832 TC/CC and 3 protective genotypes in certain subgroups. Further functional experiments revealed that YTHDC1 siRNA-554, targeting the area near the rs3813832 T>C polymorphism site, could observably inhibit the proliferation and migration of NB cells. In conclusion, our findings highlight the involvement of YTHDC1 gene and its genetic variants in the etiology of NB.

Knockout of EGFL6 by CRISPR/Cas9 Mediated Inhibition of Tumor Angiogenesis in Ovarian Cancer.

Tumor angiogenesis plays an important role in the progression and metastasis of ovarian cancer. EGFL6 protein is highly expressed in ovarian cancer and has been proposed to play an important role in promoting tumor angiogenesis. Here, a CRISPR/Cas9 system was used to knockout the EGFL6 gene in the ovarian cancer cell line SKOV3, using specific guide RNA targeting the exons of EGFL6. The knockout of EGFL6 markedly inhibited the proliferation, migration, and invasion of SKOV3 cells, as well as promoted apoptosis of tumor cells. In the nude mouse model of ovarian cancer, knockout of EGFL6 remarkably inhibited tumor growth and angiogenesis. The transcript profile assays detected 4,220 differentially expressed genes in the knockout cells, including 87 genes that were correlated to proliferation, migration, invasion, and angiogenesis. Moreover, Western blotting confirmed that EGFL6 knockout downregulated the FGF-2/PDGFB signaling pathway. Thus, the results of this study indicated that EGFL6 could regulate cell proliferation, migration, and angiogenesis in ovarian cancer cells by regulating the FGF-2/PDGFB signaling pathway.

CRISPR/Cas9-Mediated OC-2 Editing Inhibits the Tumor Growth and Angiogenesis of Ovarian Cancer.

Ovarian cancer is the leading cancer-related cause of death in women worldwide. It is of great relevance to understand the mechanism responsible for tumor progression and identify unique oncogenesis markers for a higher chance of preventing this malignant disease. The high-expression OC-2 gene has been shown to be a potential candidate for regulating oncogenesis and angiogenesis in ovarian cancer. Hence, we wished to investigate the impact of OC-2 gene on ovarian cancer aggressiveness. CRISPR/Cas9, a gene editing tool, allows for direct ablation of OC-2 at the genomic level, and we successfully generated OC-2 KO cell lines from SKOV3 and CAOV3 cells. In an apoptosis assay, OC-2 KO induced the apoptosis activation of tumor cells, with the up-regulation of Bax/Caspase-8 and the down-regulation of Bcl-2. Consequently, the proliferation, migration, and invasion of OC-2 KO cell lines were significantly inhibited. Assays of qRT-PCR and Western blotting showed that the expression levels of pro-angiogenic growth factors VEGFA, FGF2, HGF, and HIF-1α and the activation of Akt/ERK pathways were significantly down-regulated at the loss of OC-2. In the xenograft model, OC-2 KO potently suppressed the subcutaneous tumor growth, with the inhibition exceeding 56%. The down-regulation of CD31 and relevant pro-angiogenic growth factors were observed in OC-2 KO tumor tissues. Taken together, OC-2 depletion negatively regulated the ovarian cancer progression possibly by apoptosis activation and angiogenesis inhibition. This work revealed a pivotal regulator of apoptosis and angiogenesis networks in ovarian cancer, and we applied the CRISPR/Cas9 system to the transcription factor pathway for developing a broad-acting anti-tumor gene therapy.

miR-618 rs2682818 C>A polymorphism decreases Hirschsprung disease risk in Chinese children.

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that play an important role in the development of many malignant tumors. In addition, recent studies have reported that single nucleotide polymorphisms (SNPs) located in the miRNA functional region was inextricably linked to tumor susceptibility. In the present study, we investigated the susceptibility between miR-618 rs2682818 C>A and Hirschsprung disease (HSCR) in the Southern Chinese population (1470 patients and 1473 controls). Odds ratios (ORs) and 95% confidence intervals (CIs) were used for estimating the strength of interrelation between them. We found that the CA/AA genotypes of miR-618 rs2682818 were associated with a decreased risk of HSCR when compared with the CC genotype (OR = 0.84, 95% CI = 0.72-0.99, P=0.032). Based on the stratified analysis of HSCR subtypes, the rs2682818 CA/AA genotypes were able to significantly lessen the risk of HSCR compared with CC genotype in patients with long-segment HSCR (adjusted OR = 0.70, 95% CI = 0.52-0.93, P=0.013). In conclusion, our results indicated that the miR-618 rs2682818 C>A polymorphism was associated with a reduced risk of HSCR in Chinese children, especially in patients with long-segment HSCR (L-HSCR) subtype.

miR-6086 inhibits ovarian cancer angiogenesis by downregulating the OC2/VEGFA/EGFL6 axis.

miRNAs have emerged as a pivotal component of gene regulatory networks, mediating cytokines secretion, cell cycle, and differentiation regulation. However, how miRNAs collaborate with transcription factors and downstream effector proteins that determine the fate of ovarian cancer cells remains to be understood, especially regarding to mechanism of tumor angiogenesis regulation. Based on the qRT-PCR and IHC analysis, we found that miR-6086 was maintained a very low level both in ovarian cancer cell lines and tissues. Further, we identified OC2 and EGFL6 as the direct targets of miR-6086 by luciferase assay and we observed an inverse relationship between the expression of miR-6086 and the OC2/VEGFA/EGFL6 axis. The Western blotting analysis suggested that OC2 could directly upregulate VEGFA and indirectly up-regulate EGFL6 through VEGFA. Moreover, miR-6086 could indirectly downregulate VEGFA through OC2. In addition, miR-6086, siOC2 and siEGFL6 could negatively regulate the tumor growth and angiogenesis of ovarian cancer (Skov3) in the animal studies, with the inhibition rates of 77.07%, 69.89%, and 73.62%, respectively (**p < 0.01). Moreover, the tumor cell proliferation, migration, and invasion of ovarian cancer cell lines (Caov3 and Skov3) and vascular formation (HUVECs) were significantly suppressed in vitro, by decreasing the AKT/MAPK pathways (*p < 0.05). Taken together, our results reveal that miR-6086 can suppress the angiogenesis networks in ovarian cancer by down-regulating the OC2/VEGFA/EGFL6 axis, directly or indirectly, which may provide potential targets for tumor therapeutics.

AURKA rs8173 G>C Polymorphism Decreases Wilms Tumor Risk in Chinese Children.

Wilms tumor is the most common type of renal malignancy in children. Previous studies have demonstrated that single nucleotide polymorphisms (SNPs) in the AURKA gene could predispose to several human malignancies. We recruited 145 cases and 531 cancer-free controls to investigate whether AURKA gene variants modify Wilms tumor susceptibility. Three AURKA SNPs (rs1047972 C>T, rs2273535 T>A, and rs8173 G>C) were genotyped by the Taqman methodology. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the strength of association between AURKA SNPs and Wilms tumor risk. We found that only the rs8173 G>C polymorphism was significantly associated with Wilms tumor risk (GC vs. GG: adjusted OR (AOR) = 0.50, 95% CI = 0.35-0.73, P=0.0002; GC/CC vs. GG: AOR = 0.60, 95% CI = 0.42-0.88, P=0.008). Stratification analysis revealed that rs8173 GC/CC genotypes were associated with Wilms tumor risk among children aged >18 months (AOR = 0.56, 95% CI = 0.34-0.93, P=0.024), male children (AOR = 0.54, 95% CI = 0.33-0.90, P=0.017), and children with clinical stage III + IV diseases (AOR = 0.56, 95% CI = 0.35-0.90, P=0.017). Haplotype analysis indicated that the CAG haplotype was significantly associated with increased Wilms tumor risk. In conclusion, our findings indicated that the AURKA rs8173 G>C polymorphism was associated with decreased Wilms tumor risk in Chinese children.

Association of miR-34b/c rs4938723 and TP53 Arg72Pro Polymorphisms with Neuroblastoma Susceptibility: Evidence from Seven Centers.

Neuroblastoma is a pediatric malignancy arising from the developing peripheral nervous system. p53 and downstream effector miR-34b/c have critical tumor suppressing functions. TP53 Arg72Pro (rs1042522 C > G) and miR-34b/c rs4938723 (T > C) polymorphisms have been known to modify cancer susceptibility. This study was performed to validate the association of these two polymorphisms and neuroblastoma risk with 819 cases and 1780 controls. Odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were used to assess the strength of the associations. False positive report possibility analysis was adopted to dissect out real significant associations from chance findings. We found that both TP53 Arg72Pro (CG/GG vs. CC: adjusted OR = 0.82, 95% CI = 0.69-0.98) and miR-34b/c rs4938723 (TC/CC vs. TT: adjusted OR = 0.64, 95% CI = 0.54-0.75) were associated with decreased neuroblastoma susceptibility. Stratify analyses further confirmed the protective effect among some subgroups. Moreover, subjects with variant alleles of both polymorphisms were associated with more significantly decreased neuroblastoma risk (CG/TC vs. CC/TT: adjusted OR = 0.38, 95% CI = 0.28-0.50; GG/TC vs. CC/TT: adjusted OR = 0.43, 95% CI = 0.30-0.63) than those carrying variant allele of either one polymorphism (CC/TC vs. CC/TT: adjusted OR = 0.51, 95% CI = 0.37-0.69; CG/TT vs. CC/TT: adjusted OR = 0.71, 95% CI = 0.55-0.92), suggesting cumulative effects of the polymorphisms. False positive report possibility analysis further verified that our findings are noteworthy. Overall, we confirmed that miR-34b/c rs4938723 and TP53 Arg72Pro conferred decreased neuroblastoma risk and two polymorphisms exerted stronger protective effects against neuroblastoma than either one alone.