Ovarian cancer is a malignant tumor originating from the ovary, characterized by its high mortality rate and propensity for recurrence. In some patients, especially those with recurrent cancer, conventional treatments such as surgical resection or standard chemotherapy yield suboptimal results. Consequently, there is an urgent need for novel anti-cancer therapeutic strategies. Ferroptosis is a distinct form of cell death separate from apoptosis. Ferroptosis inducers have demonstrated promising potential in the treatment of ovarian cancer, with evidence indicating their ability to enhance ovarian cancer cell sensitivity to cisplatin. However, resistance of cancer cells to ferroptosis still remains an inevitable challenge. Here, we analyzed genome-scale CRISPR-Cas9 loss-of function screens and identified PAX8 as a ferroptosis resistance protein in ovarian cancer. We identified PAX8 as a susceptibility gene in GPX4-dependent ovarian cancer. Depletion of PAX8 rendered GPX4-dependent ovarian cancer cells significantly more sensitive to GPX4 inhibitors. Additionally, we found that PAX8 inhibited ferroptosis in ovarian cancer cells. Combined treatment with a PAX8 inhibitor and RSL3 suppressed ovarian cancer cell growth, induced ferroptosis, and was validated in a xenograft mouse model. Further exploration of the molecular mechanisms underlying PAX8 inhibition of ferroptosis mutations revealed upregulation of glutamate-cysteine ligase catalytic subunit (GCLC) expression. GCLC mediated the ferroptosis resistance induced by PAX8 in ovarian cancer. In conclusion, our study underscores the pivotal role of PAX8 as a therapeutic target in GPX4-dependent ovarian cancer. The combination of PAX8 inhibitors such as losartan and captopril with ferroptosis inducers represents a promising new approach for ovarian cancer therapy.

UNASSIGNED: Small cell lung cancer (SCLC) shows poor prognosis since it metastasizes widely at early stage. Paired box gene (PAX) 8 is a transcriptional factor of PAX family, of which the expression in lung cancer is a controversial issue, and its prognostic value of PAX8 in SCLC is still unclear.
UNASSIGNED: Overall, 184 subjects who were pathologically diagnosed with SCLC were enrolled in the study. Immunohistochemical analysis of PAX8 and Ki-67 were performed. The correlations between PAX8 expression and clinical features or Ki-67 index were further analyzed. Subsequently, an analysis of the association between PAX8, stage, Ki-67 status, and overall survival (OS) were performed in 169 subjects with follow-up information.
UNASSIGNED: PAX8 was positive in 53.8% (99/184) SCLC specimens. The positive rate is significantly higher in extensive-stage specimens (61.0%) than in limited-stage specimens (45.24%). PAX8 expression is positively correlated with Ki-67 index (P = 0.001) while negatively correlated with OS (HR = 3.725, 95% CI 1.943-7.139, P＜0.001). In combination groups, the PAX8 negative and limited stage group had the most promising OS.
UNASSIGNED: PAX8 expression rate in SCLC specimens is not low. It has prognostic value in small cell lung cancer.

Ribbon synapses in the cochlear hair cells are subject to extensive pruning and maturation processes before hearing onset. Previous studies have highlighted the pivotal role of thyroid hormone (TH) in this developmental process, yet the detailed mechanisms are largely unknown. In this study, we found that the thyroid hormone receptor α (Thrα) is expressed in both sensory epithelium and spiral ganglion neurons in mice. Hypothyroidism, induced by Pax8 gene knockout, significantly delays the synaptic pruning during postnatal development in mice. Detailed spatiotemporal analysis of ribbon synapse distribution reveals that synaptic maturation involves not only ribbon pruning but also their migration, both of which are notably delayed in the cochlea of Pax8 knockout mice. Intriguingly, postnatal hyperthyroidism, induced by intraperitoneal injections of liothyronine sodium (T3), accelerates the pruning of ribbon synapses to the mature state without affecting the auditory functions. Our findings suggest that thyroid hormone does not play a deterministic role but rather controls the timing of cochlear ribbon synapse maturation.

Primary congenital hypothyroidism (CH) is a common neonatal endocrine disorder characterized by elevated concentrations of thyroid stimulating hormone (TSH) and low concentrations of free thyroxine (FT4). PAX8 and NKX2-1 are important transcription factors involved in thyroid development. In this study, we detected three novel variants in PAX8 (c.149A > C and c.329G > A) and NKX2-1 (c.706A > G) by whole exome sequencing (WES) in three unrelated CH patients with variable phenotypes. The results of Western blot and immunofluorescence analysis showed that the three variants had no effect on protein expression and subcellular localization. However, the results of the electrophoretic mobility shift assay (EMSA) and dual-luciferase reporter assay suggested that the three variants in PAX8 and NKX2-1 both affected their DNA-binding ability and reduced their transactivation capacity. Moreover, a dominant-negative effect in K236E−NKX2-1 was identified by dual-luciferase reporter assay. To sum up, our findings extend our knowledge of the current mutation spectrum of PAX8 and NKX2-1 and provide important information for diagnosing, treating, and preventing CH in these families.

Nocardia rubra cell-wall skeleton (Nr-CWS) is reported as an external immunotherapeutic enhancer with the advantage of antitumor effect on human cancers. However, the immune regulatory role of Nr-CWS is not fully illustrated. We studied mouse CD4+ T lymphocytes isolated from mice spleen were induced by Nr-CWS and observed that the differentiation of Th1 CD4+ T cells and the cytokines of IL-2, TNF-α, IFN-γ were all enhanced by Nr-CWS. Furthermore, RNA sequencing was conducted to investigate the different mRNA profiling induced by Nr-CWS. We observed that paired box 8 (PAX8) was significantly up-regulated in Nr-CWS-treated Th1 cells compared to control. As a transcription factor, chromatin immunoprecipitation sequencing was carried out to study the genome-wide distribution of PAX8. Interestingly, we found that the binding domain of PAX8 was elevated by Nr-CWS, and the target genes associated with these binding sites showed a positive correlation between their transcription and PAX8 binding strength. Finally, we determined that Nr-CWS could enhance the activity of the PI3 K/Akt signaling pathway. Akt agonist could mimic the effect of Nr-CWS for PAX8 up-regulation, while Akt inhibitor compromised the expression of PAX8. Taken together, we determined a novel role of Nr-CWS in boosting the activity of Th1 maturation via the PI3 K/Akt/PAX8 axis.

Preeclampsia (PE), a pregnancy disorder that affects 5-7% of pregnant women, is among the primary causes for maternal and perinatal mortality. PE is believed to be associated with insufficient invasion of villous and extravillous trophoblasts (EVTs), which hampers uterine spiral artery remodeling and finally induces PE. But the mechanism responsible for reduction of trophoblast invasion remains unclear. In this study, placental tissues taken from healthy donors and PE patients were used to evaluate the miR-326 expression; CCK8 and colony formation assays were used to confirm the effect of miR-326 on cell proliferation; transwell assay was used to demonstrate the effect of miR-326 on cell invasion capability; western blot was used to investigate the underlying mechanism; and luciferase assay was used to detect the effect of miR-326 on YAP/TAZ-mediated transcription activity. It was revealed the miR-326 expression was higher in placentas from PE patients than from healthy donors. After transfection of miR-326 mimics, trophoblast proliferation and invasion were impaired. Using TargetScan, we speculated that PAX8 was a target of miR-326, which was later confirmed by western blot. The YAP/TAZ expression was also downregulated after transfection with miR-326. Luciferase assay demonstrated that overexpression of miR-326 suppressed YAP/TAZ-mediated transcription activity by targeting PAX8. Overexpression of PAX8 could partly rescue miR-326-induced suppression of trophoblast proliferation and invasion. Taken together, our result indicated that miR-326 suppresses trophoblast growth, invasion, and migration by means of targeting PAX8 via the Hippo pathway.

Our previous studies have shown that the Adipose-derived mesenchymal stem cells (ADSCs) can regulate metastasis and development of ovarian cancer. However, its specific mechanism has yet to be fully revealed. In this study, an RNA-seq approach was adopted to compare the differences in mRNA levels in ovarian cancer cells being given or not given ADSCs. The mRNA level of paired box 8 (PAX8) changed significantly and was confirmed as an important factor in tumour-inducing effect of ADSCs. In comparison with the ovarian cancer cells cultured in the common growth medium, those cultured in the medium supplemented with ADSCs showed a significant increase of the PAX8 level. Moreover, the cancer cell growth could be restricted, even in the ADSC-treated group (P < .05), by inhibiting PAX8. In addition, an overexpression of PAX8 could elevate the proliferation of ovarian cancer cells. Moreover, Co-IP assays in ovarian cancer cells revealed that an interaction existed between endogenous PAX8 and TAZ. And the PAX8 levels regulated the degradation of TAZ. The bioluminescence images captured in vivo manifested that the proliferation and the PAX8 expression level in ovarian cancers increased in the ADMSC-treated group, and the effect of ADSCs in promoting tumours was weakened through inhibiting PAX8. Our findings indicate that the PAX8 expression increment could contribute a role in promoting the ADSC-induced ovarian cancer cell proliferation through TAZ stability regulation.

Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) is associated with congenital absence of the uterus, cervix, and the upper part of the vagina; it is a sex-limited trait. Disrupted development of the Müllerian ducts (MD)/Wölffian ducts (WD) through multifactorial mechanisms has been proposed to underlie MRKHS. In this study, exome sequencing (ES) was performed on a Chinese discovery cohort (442 affected subjects and 941 female control subjects) and a replication MRKHS cohort (150 affected subjects of mixed ethnicity from North America, South America, and Europe). Phenotypic follow-up of the female reproductive system was performed on an additional cohort of PAX8-associated congenital hypothyroidism (CH) (n = 5, Chinese). By analyzing 19 candidate genes essential for MD/WD development, we identified 12 likely gene-disrupting (LGD) variants in 7 genes: PAX8 (n = 4), BMP4 (n = 2), BMP7 (n = 2), TBX6 (n = 1), HOXA10 (n = 1), EMX2 (n = 1), and WNT9B (n = 1), while LGD variants in these genes were not detected in control samples (p = 1.27E-06). Interestingly, a sex-limited penetrance with paternal inheritance was observed in multiple families. One additional PAX8 LGD variant from the replication cohort and two missense variants from both cohorts were revealed to cause loss-of-function of the protein. From the PAX8-associated CH cohort, we identified one individual presenting a syndromic condition characterized by CH and MRKHS (CH-MRKHS). Our study demonstrates the comprehensive utilization of knowledge from developmental biology toward elucidating genetic perturbations, i.e., rare pathogenic alleles involving the same loci, contributing to human birth defects.

Upper urinary tract urothelial carcinomas (UUT-UCs) are defined as malignant neoplasms of the urothelium from the upper urinary tract, including renal calyces, the renal pelvis and the distal ureter. The natural attributes of UUT-UCs differ from those of bladder cancer. The aim of the present study was to investigate PAX8 expression in the normal urothelium and in urothelial carcinomas (UCs) of the upper urinary tract. Immunohistochemistry was conducted in 35 cases of renal pelvic and 30 cases of ureteral papillary UCs and the adjacent normal urothelium respectively. PAX8 mRNA expression was evaluated by RT-PCR in a different set of normal urothelial mucosa of the urinary tract and UUT-UCs. In immunohistochemical studies, the positive rates of PAX8 staining in UCs of the renal pelvis and ureter were 17% and 6.6% respectively, presenting focally positive in most cases, while the positive rates in the adjacent normal epithelia of the pelvis and ureter were 100% and 93% respectively. PAX8 mRNA was detected in all of the tumors and adjacent normal urothelial mucosa specimens of the upper urinary tract. 4 types of PAX8 isoforms, PAX8a, PAX8b, PAX8c and PAX8e, were detected in UUT-UCs in this study. As in bladder cancer, PAX8 expression was highly heterogeneous in terms of the splicing mRNA isoforms, with the different isoforms differentially expressed in the UUT-UCs. Among the 4 types of PAX8 isoforms, the PAX8e isoform was found in almost all UUT-UCs tumor tissues, but the PAX8d isoform was not detected in UUT-UCs that were different from the transcriptional splicing patterns of PAX8 in bladder cancer reported in the literature. In addition, the above 4 types of PAX8 splicing isoforms were simultaneously detected in almost all of the normal mucosal epithelia of the upper urinary tract, which was very different from that of bladder mucosa. Further studies are suggested to reveal whether or not the differences in natural attributes between UCs of the upper and lower urinary tracts are related to their PAX8 transcriptional splicing patterns.

Long noncoding RNAs play vital roles in several biological processes, including cell growth and embryonic development. We showed that MACC1-AS1 was overexpressed in hepatocellular carcinoma (HCC) cells and tissues. The MACC1-AS1 expression level was dramatically upregulated in HCC samples compared to adjacent normal samples, and 77.5% (31 of 40) of HCC samples showed overexpression of MACC1-AS1. Ectopic MACC1-AS1 expression enhanced cell proliferation and cyclin D1 expression in both SMMC7721 and MHCC-97H cells. Ectopic expression of MACC1-AS1 promoted vimentin, N-cadherin and snail expression and decreased E-cadherin expression in both SMMC7721 and MHCC-97H cells. MACC1-AS1 overexpression also induced cell invasion in the same two cell lines. Furthermore, MACC1-AS1 overexpression enhanced PAX8 expression in HCC cells. The PAX8 level was dramatically increased in HCC samples compared to adjacent normal samples, and 75% (30 of 40) of HCC samples showed overexpression of PAX8. PAX8 expression was positively correlated with MACC1-AS1 expression in HCC samples. MACC1-AS1 overexpression promoted HCC cell proliferation, EMT and invasion through regulating PAX8. These results suggest that MACC1-AS1 acts as an oncogene in the development of HCC.