OBJECTIVE: Frizzled 7 (FZD7) is abnormally expressed and activated in a variety of cancers. In ovarian cancer, overexpression of FZD7 reduces the sensitivity of platinum-resistant ovarian cancer cells to ferroptosis, thereby allowing cancer cells to survive. However, whether FZD7 inhibits ferroptosis in ovarian cancer cells and its mechanisms are remain unclear. This study aims to explore the effects of FZD7 and its upstream regulator miR-1-3p on ferroptosis in ovarian cancer cells are evaluated to clarify the molecular mechanism for miR-1-3p and FZD7\'s involvement in ferroptosis in ovarian cancer cells.
METHODS: Human ovarian cancer cell lines HO8910 and SKOV3 were used as the research subjects. In the first part of the experiment, human ovarian cancer cells were transfected with blank plasmid and FZD7 overexpression plasmid, respectively; in the second and third parts, human ovarian cancer cells were transfected with miR-1-3p mimics negative control, miR-1-3p mimics, miR-1-3p inhibitors negative control, and miR-1-3p inhibitors, respectively; in the fourth part of the experiment, human ovarian cancer cells were transfected with miR-1-3p mimics and miR-1-3p mimics+FZD7 overexpression plasmid, respectively, and normal cultured cells were set as the control group. The human ovarian cancer cell ferroptosis model was established by incubating human ovarian cancer cells with different treatments with ferroptosis inducer Erastin or RSL3. Real-time RT-PCR was used to detect the mRNA expression levels of FZD7 and miR-1-3p; Western blotting was used to detect the protein expression levels of FZD7; CCK-8 assay was used to detect the cell viability; lipid peroxidation colorimetric assay kit was used to detect the level of intracellular MDA; and iron assay kit was used to detect the level of intracellular Fe2+. Dual-luciferase assay was used to detect the targeting relationship between miR-1-3p and FZD7.
RESULTS: Overexpression of FZD7 increased the cell viability of human ovarian cancer cell lines HO8910 or SKOV3 (P<0.05, P<0.01, or P<0.001) and decreased the intracellular MDA levels (P<0.01) in Erastin-treated or RSL3-treated ovarian cancer cells. FZD7 was a direct target of miR-1-3p, which inhibited the expression of FZD7 (P<0.01) by binding to the 3\'-untranslated region (3\'UTR) site of FZD7. MiR-1-3p mimics decreased the cell viability of human ovarian cancer cell lines HO8910 or SKOV3 (P<0.05, P<0.01, or P<0.001) and increased the intracellular MDA levels (P<0.01) in Erastin-treated or RSL3-treated ovarian cancer cells; while miR-1-3p inhibitors significantly increased the cell viability of human ovarian cancer cell lines HO8910 or SKOV3 (P<0.05, P<0.01, or P<0.001) and decreased the intracellular MDA levels (P<0.01) in Erastin-treated or RSL3-treated ovarian cancer cells. The effect of miR-1-3p mimics on enhancing the sensitivity of human ovarian cancer cells to Erastin-induced or RSL3-induced ferroptosis was abrogated by overexpression of FZD7(P<0.05 or P<0.01).
CONCLUSIONS: MiR-1-3p enhances the sensitivity of ovarian cancer cells to ferroptosis by targeting FZD7.
目的: 卷曲蛋白7(Frizzled 7，FZD7)在多种癌症中异常表达和激活。在卵巢癌中，FZD7的过表达可降低铂耐药性卵巢癌细胞对铁死亡的敏感性，从而使癌细胞存活，但FZD7是否抑制卵巢癌细胞铁死亡及其相关机制尚未被阐明。本研究通过探究FZD7及其上游调控因子miR-1-3p对卵巢癌细胞铁死亡的影响，旨在明确miR-1-3p及FZD7参与卵巢癌细胞铁死亡的分子机制。方法: 以人卵巢癌细胞系HO8910和SKOV3为研究对象，第1部分实验将空白质粒和FZD7过表达质粒分别转染人卵巢癌细胞；第2，3部分实验将miR-1-3p模拟物阴性对照、miR-1-3p模拟物、miR-1-3p抑制剂阴性对照和miR-1-3p抑制剂分别转染人卵巢癌细胞；第4部分实验将miR-1-3p模拟物、miR-1-3p模拟物+FZD7过表达质粒分别转染人卵巢癌细胞，另设正常培养的对照组。采用铁死亡诱导剂Erastin或RSL3分别孵育经不同处理后的人卵巢癌细胞构建人卵巢癌细胞铁死亡模型。使用real-time RT-PCR检测FZD7和miR-1-3p的mRNA表达水平，蛋白质印迹法检测FZD7的蛋白质表达水平，CCK-8实验检测细胞活力，脂质过氧化比色测定试剂盒检测细胞内MDA水平，铁检测试剂盒检测细胞内Fe2+水平，双荧光素酶实验检测miR-1-3p和FZD7的靶向关系。结果: FZD7的过表达提高经Erastin或RSL3处理的人卵巢癌细胞系HO8910或SKOV3细胞的活力(P<0.05、P<0.01或P<0.001)，并降低细胞内MDA的水平(P<0.01)。FZD7是miR-1-3p的直接靶点，miR-1-3p通过与FZD7的3\'非翻译区(3\'-untranslated region，3\'UTR)位点结合，抑制FZD7的表达(P<0.01)。MiR-1-3p模拟物降低经Erastin或RSL3处理的人卵巢癌细胞系HO8910或SKOV3细胞的活力(P<0.05、P<0.01或P<0.001)，提高细胞内MDA的水平(P<0.01)，而miR-1-3p抑制剂则显著提高经Erastin或RSL3处理的人卵巢癌细胞系HO8910或SKOV3细胞的活力(P<0.05、P<0.01或P<0.001)，降低细胞内MDA的水平(P<0.01)。MiR-1-3p模拟物增强人卵巢癌细胞对Erastin或RSL3诱导细胞铁死亡敏感性的作用可以被FZD7的过表达所取消(P<0.05或P<0.01)。结论: MiR-1-3p靶向FZD7增强卵巢癌细胞对铁死亡的敏感性。.

Mesenchymal-like stemness is characterized by epithelial-mesenchymal transition (EMT). Breast cancer (BC) cell mesenchymal-like stemness is responsible for distal lung metastasis. Interrogation of databases showed that Fzd7 was closely associated with a panel of mesenchymal-related genes and a panel of stemness-related genes. Fzd7 knockdown in mesenchymal-like MDA-MB-231 and Hs578T cells reduced expression of Vimentin, Slug and Zeb1, induced an epithelial-like morphology, inhibited cell motility, impaired mammosphere formation and decreased Lgr5+ subpopulation. In contrast, Fzd7 overexpression in MCF7 cells resulted in opposite changes. Fzd7 knockdown delayed xenograft tumor formation, suppressed tumor growth, and impaired lung metastasis. Mechanistically, Fzd7 combined with Wnt5a/b and modulated expression of phosphorylated Stat3 (p-STAT3), Smad3 and Yes-associated protein 1 (Yap1). Moreover, Fzd7-Wnt5b modulated expression of collagen, type VI, alpha 1 (Col6a1). Both Wnt5b knockdown and Col6a1 knockdown disrupted BC cell mesenchymal phenotype and stemness. Taken together, Fzd7 contributes to BC cell EMT and stemness, inducing tumorigenesis and metastasis, mainly through a non-canonical Wnt5b pathway. Col6a1 is implicated in Fzd7-Wnt5b signaling, and mediates Fzd7-Wnt5b -induced mesenchymal-like stemness. Video Abstract.

Wnts are secreted signaling molecules that are implicated in a variety of growth-related processes. Frizzled proteins have been identified as receptors for Wnt ligands in vertebrates and invertebrates, but a functional role for dioecious flatworm Frizzleds has not been determined. To evaluate the endogenous role of Frizzled proteins during development, we have identified and characterized a Schistosoma japonicum frizzled gene (Sjfz7). We found that Sjfz7 encodes a 698 amino acid protein with typical characteristics of Frizzled proteins. The immunohistochemical localization pattern showed that Sjfz7 protein was extensively distributed in almost all tissues of S. japonicum, including subtegumental muscle cells, parenchymal cells, intestinal epithelial cells and male and female germ cells. This indicated that Sjfz7-mediated Wnt signaling might be associated with the development of musculature, intestinal tract and reproductive organs in schistosome. Comparing mRNA levels between frizzled family members showed that Sjfz7 mRNA was consistently higher in the developmental stages analyzed, suggesting that Sjfz7 may be responsible for more functional tasks than other frizzled family members. Comparing frizzled mRNA levels between not fully developed and normal worms suggested that Wnt signaling might be abnormal in not fully developed worms.