Somatic embryogenesis (SE) is a major regeneration approach for in vitro cultured tissues of plants, including citrus. However, SE capability is difficult to maintain, and recalcitrance to SE has become a major obstacle to plant biotechnology. We previously reported that miR156-SPL modules regulate SE in citrus callus. However, the downstream regulatory pathway of the miR156-SPL module in SE remains unclear. In this study, we found that the transcription factors (TFs) CsAGL15 and CsFUS3 bind to the CsMIR156A promoter and activate its expression. Suppression of csi-miR156a function leads to up-regulation of four target genes CsSPLs and reduction of SE efficiency. In the STTM-miR156a overexpressed callus (MIM156), the number of amyloplasts and starch content were significantly reduced, and genes involved in starch synthesis and transport were down-regulated; csi-miR172d was down-regulated, whereas the target genes CsTOE1.1 and CsTOE1.2, which inhibit the expression of starch biosynthesis genes, were up-regulated. In our working model, CsAGL15 and CsFUS3 activate csi-miR156a, which represses CsSPLs and further regulates csi-miR172d and CsTOEs, thus to alter starch accumulation level in callus cells and regulate SE in citrus. This study elucidates the pathway of miR156-SPLs and miR172-TOEs mediated regulation of SE, and provides clues to enhancing SE capability in citrus.
Anaplastic thyroid cancer is an extremely lethal malignancy without reliable treatment. BRAFV600E point mutation is common in ATCs, which leads to MAPK signaling activation and is regarded as a therapeutic target. Resveratrol inhibits ATC cell growth, while its impact on BRAF-MAPK signaling remains unknown. This study aims to address this issue by elucidating the statuses of BRAF-MAPK and STAT3 signaling activities in resveratrol-treated THJ-11T, THJ-16T, and THJ-21T ATC cells and Nthyori 3-1 thyroid epithelial cells. RT-PCR and Sanger sequencing revealed MKRN1-BRAF fusion mutation in THJ-16T, BRAF V600E point mutation in THJ-21T, and wild-type BRAF genes in THJ-11T and Nthyori 3-1 cells. Western blotting and immunocytochemical staining showed elevated pBRAF, pMEK, and pERK levels in THJ-16T and THJ-21T, but not in THJ-11T or Nthyori 3-1 cells. Calcein/PI, EdU, and TUNEL assays showed that compared with docetaxel and doxorubicin and MAPK-targeting dabrafenib and trametinib, resveratrol exerted more powerful inhibitory effects on mutant BRAF-harboring THJ-16T and THJ-21T cells, accompanied by reduced levels of MAPK pathway-associated proteins and pSTAT3. Trametinib- and dabrafenib-enhanced STAT3 activation was efficiently suppressed by resveratrol. In conclusion, resveratrol acts as dual BRAF-MAPK and STAT3 signaling inhibitor and a promising agent against ATCs with BRAF mutation.
Osteosarcoma (OS) is the most common bone malignancy without a reliable therapeutic target. Glypican‐3 (GPC3) mutation and upregulation have been detected in multidrug resistant OS, and anti‐GPC3 immunotherapy can effectively suppress the growth of organoids. Further profiling of GPC3 mutations and expression patterns in OS is of clinical significance. To address these issues, fresh OS specimens were collected from 24 patients for cancer‐targeted next‐generation sequencing (NGS) and three‐dimensional patient‐derived organoid (PDO) culture. A tumor microarray was prepared using 37 archived OS specimens. Immunohistochemical (IHC) staining was performed on OS specimens and microarrays to profile GPC3 and CD133 expression as well as intratumoral distribution patterns. RT‐PCR was conducted to semiquantify GPC3 and CD133 expression levels in the OS tissues. Anti‐GPC3 immunotherapy was performed on OS organoids with or without GPC3 expression and its efficacy was analyzed using multiple experimental approaches. No OS cases with GPC3 mutations were found, except for the positive control (OS‐08). IHC staining revealed GPC3 expression in 73.77% (45/61) of OSs in weak (+; 29/45), moderate (++; 8/45), and strong (+++; 8/45) immunolabeling densities. The intratumoral distribution of GPC3‐positive cells was variable in the focal (+; 10%–30%; 8/45), partial (++; 31%–70%; 22/45), and the most positive patterns (+++; >71%; 15/45), which coincided with CD133 immunolabeling (P = 9.89 × 10−10). The anti‐GPC3 antibody efficiently inhibits Wnt/β‐catenin signaling and induces apoptosis in GPC3‐positive PDOs and PDXs, as opposed to GPC3‐negative PDOs and PDXs. The high frequency of GPC3 and CD133 co‐expression and the effectiveness of anti‐wild‐type GPC3‐Ab therapy in GPC3‐positive OS models suggest that GPC3 is a novel prognostic parameter and a promising therapeutic target for osteosarcoma.
The lack of reliable drugs is a therapeutic challenge of advanced breast cancers (ABCs). Resveratrol (Res) exerts inhibitory effects on breast cancer cell lines and animal models, while its efficacy against individual breast cancer cases remains unknown. This study aims to use ABC‐derived organoids (ABCOs) as the ex vivo therapeutic platform to clarify the effectiveness of resveratrol against different ABC subtypes. Immunohistochemical staining confirmed that the ABCOs maintained their original tumors' ER, PR, HER2, and Ki67 expression patterns. ABCO proliferation and viability tests showed >50% cell death rates in 79.2% (19/24) of Res‐treated, 28.6% (2/7) fulvestrant‐treated, 66.7% (4/6) paclitaxel‐treated, and 66.7% (6/9) gemcitabine‐treated ABCOs. pSTAT3 nuclear translocation was more frequent in Res‐sensitive (17/19; 89.47%) than that (1/5; 20%) of Res‐insensitive ABCOs, which were suppressed upon Res treatment. Statistical analysis revealed a close correlation of STAT3 activation with the efficacy of Res, but not related to tumor receptor expression patterns (ER, PR, HER2) and pathological classification. We demonstrate for the first time the higher efficacy and broader spectrum of Res against different subtypes of ABCOs in comparison with that of conventional antibreast cancer drugs, providing an alternative approach for better management of ABCs.
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