MicroRNAs play key roles in tumor proliferation and invasion. Here we show distinct expression of miR-222-3p between ERα-positive and ERα-negative endometrial carcinoma (EC) cell lines and primary tumors, and investigation of its relationship with ERα and other clinical parameters. In vitro, the function of miR-222-3p was examined in RL95-2 and AN3CA cell lines. MiR-222-3p expression was negatively correlated with ERα. Over-expressed miR-222-3p in RL95-2 cells promoted cell proliferation, enhanced invasiveness and induced a G1 to S phase shift in cell cycle. Furthermore, the miR-222-3p inhibitor decreased the activity of AN3CA cells to proliferate and invade. In vivo, down-regulated miR-222-3p of AN3CA cells inhibited EC tumor growth in a mouse xenograft model. Additionally, miR-222-3p increased raloxifene resistance through suppressing ERα expression in EC cells. In conclusion, miR-222-3p plays a significant role in the regulation of ERα expression and could be potential targets for restoring ERα expression and responding to antiestrogen therapy in a subset of ECs.

Endometrial cancer stem cells (ECSCs) are stem-like cells endowed with self-renewal and differentiation abilities, and these cells are essential for cancer progression in endometrial cancer (EC). As hallmarks of the tumour microenvironment (TME), hypoxia and hypoxia-inducing factors (HIFs) give rise to the dysregulation of tumour stemness genes, such as SOX2. Against this backdrop, we investigated the regulatory mechanisms regulated by HIFs and SOX2 in ECSCs during EC development. Here, ECSCs isolated from EC cell lines and tissues were found to express stemness genes (CD133 and aldehyde dehydrogenase, ALDH1) following the induction of their ECSC expansion. Notably, m6A methylation of RNA and HIF-1α/2α-dependent AlkB homologue 5 (ALKBH5) participate in the regulation of HIFs and SOX2 in EC, as confirmed by the observations that mRNA levels of m6A demethylases and ALKBH5 significantly increase under hypoxic conditions in ECSCs. Moreover, hypoxia and high ALKBH5 levels restore the stem-like state of differentiated ECSCs and increase the ECSC-like phenotype, whereas the knockdown of HIFs or ALKBH5 significantly reduces their tumour initiation capacity. In addition, our findings validate the role of ALKBH5 in promoting SOX2 transcription via mRNA demethylation, thereby maintaining the stem-like state and tumorigenicity potential of ECSCs. In conclusion, these observations demonstrate a critical role for m6A methylation-mediated regulation of the HIF-ALKBH5-SOX2 axis during ECSC expansion in hypoxic TMEs.

Tumor-stroma interactions contribute greatly to intratumoral estrogen biosynthesis in endometrial carcinoma, but the mechanisms involved remain largely unknown. Previous study demonstrated that intratumoral aromatase upregulation in stromal cells participated in this process, but the specific aromatase-regulators have not been reported. In the present study, we found that aromatase expression in intratumoral stroma, but not in tumor epithelium, correlated positively with interleukin 6 (IL-6) expression in cancer epithelial cells by immunohistochemistry, which was confirmed using laser capture microdissection/real-time reverse transcription-PCR. With stimulation by exogenous IL-6, aromarase expression was increased in stromal cells not but not in cancer cells. Aromatase mRNA levels in endometrial cancer cells were not influenced by cocultivation with intratumoral stromal cells. When cocultured with 17b-estradiol (E 2 )-treated cancer cells, aromatase mRNA in stromal cells was significantly elevated and increased IL-6 protein levels were detected in E 2 -treated culture medium. Next, we demonstrated that E 2 -induced IL-6 production was through cooperation between estrogen receptor a and nuclear factor-kappa B. Furthermore, an IL-6 receptor blocking antibody could attenuate the upregulation of aromatase expression in stromal cells and the E 2 concentration in coculture systems of cancer and stromal cells. The results were confirmed by an orthotopic nude endometrial carcinoma model in vivo. These studies elucidated the activation of a positive feedback loop, that is, IL-6 stimulated by E 2 in endometrial cancer cells induced aromatase expression in stromal cells, promoting enhanced intratumoral E 2 synthesis. Blocking of this tumor-stroma interaction may be a therapeutic strategy to overcome in situ estrogen biosynthesis in endometrial carcinoma.Endometrial carcinoma is the most common gynecologic malignancy, with an estimated 47,130 diagnosed cases and 8,010 deaths in 2012 in the United States. 1 Existing evidence indicates that unopposed estrogens contribute to the tumorigenesis and promotion of endometrial carcinoma. 2 The conversion of androstenedione and testosterone into estrogen is catalyzed by the cytochrome P450 aromatase enzyme. 3 Although postmenopausal women have low levels of circulating plasma estrogens, the intratumoral production of estrogens can lead to higher estrogen levels in tumors. 4 Several studies have indicated that, in normal and abnormal human endometrium, aromatase expression and activity are associated with malignancy and only aromatase expression in stromal cells, but not in epithelial cells, correlates positively with poor survival. 5,6 Additionally, an indirect coculture model confirmed that tumor-stroma communication contributed to upregulation of aromatase activity. 7 Therapeutic strategies that focus on tumor-stroma interactions require the identification of molecular targets that regulate intercellular interactions. To the best of our knowledge, however, no exact stimulators have...