LncRNA HOTAIR regulates anoikis-resistance capacity and spheroid formation of ovarian cancer cells by recruiting EZH2 and influencing H3K27 methylation.

This study aims to investigate the role of the long non-coding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR) in the regulation of anoikis resistance of ovarian cancer cells, a prerequisite for metastasis and chemoresistance in ovarian cancer cells. Ovarian cancer SKOV3 cells were cultured in an ultra-low attachment system to establish an anoikis model. The relationship between cellular anoikis capability and HOTAIR expression level was studied by flow cytometry and RT-PCR. The ability of spheroid formation, migration, and invasion of the suspended cells was assessed following the knockdown of HOTAIR expression. The expression of EZH2, H3K27me3, representative targets of EZH2, and anoikis-related biomarkers was also detected. An increase in the duration of suspension culture time rendered the SKOV3 cells anoikis-resistant with a significantly lower apoptotic rate compared to the adherent cells. HOTAIR expression in the suspension cells increased significantly, while that in the adherent cells did not. Following small interfering RNA (siRNA)-mediated knockdown of HOTAIR expression, the abilities of anoikis resistance, migration, and invasion decreased in the suspension cells. Knockdown of HOTAIR levels also reduced the spheroid forming ability of the tumor cells in continuous suspension cultures. Moreover, EZH2 expression correlated with HOTAIR expression, thus regulating the expression of miR-193a and DOK2 via introducing H3K27me3. Western blot analysis of anoikis-related markers showed that N-cadherin, ZEB1, and TWIST1 were downregulated following inhibition of HOTAIR, while E-cadherin and ErbB3 were upregulated. In conclusion, HOTAIR enhances the anoikis resistance and spheroid forming ability of ovarian cancer cells by recruiting EZH2 and influencing H3K27 methylation that may contribute to migration, invasion, and chemoresistance of ovarian cancer cells.

[Relationship of NOR-1 with the regulation of inflammation via liver X receptor alpha in Kupffer cells].

OBJECTIVE: To investigate the relationship of NOR-1 with the inhibition of inflammatory reaction in mice Kupffer cells (KCs) induced by lipopolysaccharide (LPS) via liver X receptor alpha (LXR alpha). METHODS: KCs from male KM mice were isolated by density gradient centrifugation, incubated and then randomly assigned to three groups: control group, LPS treated group and LPS+T0901317 treated group. RESULTS: The mRNA and protein expressions of LXR alpha and NOR-1 in each group were determined by RT-PCR, immunofluorescent assay and western blot, respectively. The densities of TNF alpha and IL-10 in supernatants were evaluated by enzyme linked immunosorbent assay (ELISA). The mRNA and protein expression levels of LXR alpha in LPS + T0901317 group were the highest as compared to the other two groups (0.748+/-0.072 and 1.217+/-0.133 respectively), The mRNA and protein expression levels of NOR-1 in LPS+ T0901317 group were the highest as compared to the other two groups (2.726+/-0.065 and 0.842+/-0.058 respectively). The densities of supernatant TNF alpha in LPS group and IL-10 in LPS+T0901317 group were the highest [(450.89+/-78.52) ng/L and (537.41+/-36.41) ng/L respectively]. CONCLUSIONS: Promoting the expression of LXR alpha in KCs can elevate the NOR-1 expression and then inhibit inflammatory reaction.

Association between liver X receptor-α and neuron-derived orphan nuclear receptor-1 in Kupffer cells of C57BL/6 mice during inflammation.

The liver X receptor (LXR) isoform LXR­α has a significant role in lipid metabolism and innate immunity. Overexpression of neuron­derived orphan nuclear receptor­1 (NOR­1) in macrophages reduces the synthesis of inflammatory cytokines and chemokines. However, to date, the mechanisms via which NOR­1 inhibits lipopolysaccharide (LPS)­induced inflammation in Kupffer cells (KCs) via LXR­α have not been elucidated. T0901317 is the most potent LXR­α ligand, leading to its activation. In the present study, KCs were isolated from C57BL/6 mice and randomly divided into five groups: Control, T0901317, LPS, LPS + T0901317 and LPS + T0901317 + NOR­1 small hairpin (sh)RNA groups. In order to investigate the role of NOR­1 in inflammation, shRNA targeting NOR­1 was used to specifically knock down NOR­1 mRNA in KCs. The expression levels of LXR­α and NOR­1 in KCs were determined by reverse transcription quantitative polymerase chain reaction and western blot analyses. The protein levels of tumor necrosis factor (TNF)­α and interleukin (IL)­10 in the supernatant of KCs were evaluated by ELISA. The results revealed that LXR­α expression in the T0901317 group was higher than that in the control group; furthermore, LXR­α expression was higher in KCs treated with LPS + T0901317 compared with that in KCs treated with LPS only. The expression levels of NOR­1 in each group showed a similar trend. shRNA targeting of NOR­1 suppressed the mRNA expression of NOR­1, but had no influence on LXR­α mRNA expression. NOR­1 protein expression was augmented in the LPS + T0901317 group compared with that in the LPS + T09 + shRNA group. In the supernatant of KCs, the TNF­α levels in the LPS + T0901317 group were lower than those in the LPS group, whereas the IL­10 levels were higher in the LPS + T0901317 group compared with those in the LPS group. The results of the present study suggested that ligand T0901317 promotes LXR­α expression, which consequently suppresses LPS­induced inflammation by elevating NOR­1 expression in KCs.