Fig 1: THOC2 or THOC5 knockdown suppresses the tumorigenicity of radioresistant TNBC in vivo. A) THOC2- or THOC5-silenced MDA-MB-231-RR cells were subcutaneously injected into the nude mice. The xenograft tumors derived from these MDA-MB-231-RR cells were photographed and compared at the end of the experiment. B) Tumor growth was monitored every 4 days for 8 weeks, and the volume was recorded till the experiment ended. C) Tumor weight was measured and compared at the end of the experiment. D) Tumor incidence in mice transplanted with MDA-MB-231-RR cells expressing sh-control, sh-THOC2, or sh-THOC5 was shown and, a limiting dilution method was used to examine the frequency of CSCs in tumors. E) The protein level of THOC2, THOC5, NANOG, SOX2, and Ki-67 in xenograft tumors was detected by IHC. Representative IHC images are shown at 200× magnification. ***P < 0.001 versus sh-control group (n = 8).
Fig 2: The expression of THOC2 and THOC5 is upregulated in TNBC cells and associated with a worse prognosis in patients. A) The protein expression of THOC2 and THOC5 in TNBC cell lines was detected by WB. GAPDH was used as the loading control. B) The box-and-whisker plots were generated using the UALCAN to describe THOC2 and THOC5 protein expression differences among human normal breast tissues and various BC subtypes. The protein expression data were obtained from the CPTAC database. C) The protein level of THOC2 and THOC5 in human TNBC tissues and paired adjacent normal breast epithelia in TMA was evaluated by IHC. Representative IHC images are displayed at 100× and 400× magnification. Brown represents target protein staining, while blue represents the nuclei. D) The protein staining of THOC2 and THOC5 by IHC between TNBC tissues and paired adjacent normal breast epithelia was scored and compared (n = 80). E) The association of THOC2 and THOC5 protein levels with OS in TMA was analyzed according to the IHC score. F) The hazard ratio plot (Forest plot) was generated with the results of multivariate analysis by Cox regression model for OS in TMA. G) The association of THOC2 and THOC5 protein expression with OS was analyzed using the dataset obtained from the work of Liu, et al. [ 23 ]a and Tang et al. [ 23 b], respectively. *P < 0.05 and ***P < 0.001; ns, nonsignificant.
Fig 3: The schematic of the THOC-mediated stemness enhancement and radioresistance in TNBC. The export of mRNA from the nucleus to the cytoplasm is a key step in protein synthesis, which is essential for all living eukaryotic cells. The THOC is a key component in the formation of cotranscription that messenger ribonucleoparticles can be transported into the cytoplasm for translation. THOC2 and THOC5, as the backbone protein and specific adaptor for the THOC, respectively, play a vital role in maintaining the protein expression of pluripotent transcription factors and the switching of embryonic stem cell differentiation and proliferation. In this study, we found that radioresistant TNBC can employ this mechanism via upregulating the protein expression of THOC2 and THOC5 to promote the THOC-mediated spliced mRNA efflux, increase the translation and protein synthesis of NANOG and SOX2, and enhance the stem-like properties of TNBC cells. The upregulation of NANOG and SOX2 gene expression also provides a prerequisite for this mechanism. The enhanced stemness thus confers TNBC cells a survival advantage upon RT-induced oxidative stress injury and apoptosis. (This figure was created with Biorender.com)
Fig 4: THOC2 knockdown decreases the stemness of radioresistant TNBC cells and caused radiosensitization. THOC2 was silenced in MDA-MB-231-RR and 436-RR cells using a lentiviral system with two different shRNAs. A) The protein expression of THOC2 in TNBC cell lines was detected by WB. GAPDH was used as the loading control. B) The proliferation rate of cells was determined using the proliferation assay. C) The clonogenic ability of cells was evaluated by colony formation assay. D) The stemness of TNBC cell lines was evaluated by mammosphere formation assay, and the formation efficiency was calculated. Representative images are shown at 100× magnification. E) The percentage of CD44+CD24-/low cells in MDA-MB-231-RR cells was detected using flow cytometry. F) The percentage of ALDH+ cells in MDA-MB-436-RR cells was detected by flow cytometry. THOC2-depleted TNBC cells were treated with 0 or 4 Gy IR. G) The intracellular ROS level was detected 24 h after IR. H) Cell apoptosis was analyzed 24 h after IR using flow cytometry, and the percentage of apoptotic cells was calculated as the percentage of cells in Q2 and Q3. I) The protein expression of cleaved PARP1, caspase-3, and caspase-9 was detected by WB 24 h after IR. GAPDH was used as the loading control. **P < 0.01 and ***P < 0.001 versus sh-control group; # P < 0.05, ## P < 0.01, and ### P < 0.001 versus sh-control + IR group (n = 3).
Fig 5: THOC2 knockdown decreases the protein expression of SOX2 and NANOG by disrupting their transcript export. A) The relative mRNA expression of OCT4, NANOG, and SOX2 in TNBC cell lines was detected by qRT-PCR. B) The enrichment of GAPDH, OCT4, NANOG, and SOX2 mRNAs against THOC2 and IgG antibodies in TNBC cell lines was analyzed by RIP assay. THOC2 was silenced in MDA-MB-231-RR and 436-RR cells using a lentiviral system with two different shRNAs. C) The protein expression of OCT4, NANOG, and SOX2 was detected by WB. GAPDH was used as the loading control. D) The total mRNA expression of OCT4, NANOG, and SOX2 was detected by qRT-PCR. E) The relative expression of polysome-associated OCT4, NANOG, and SOX2 mRNAs (fractions 7–12) was detected by qRT-PCR. F) The intracellular distribution of NANOG, SOX2, and GAPDH mRNAs was evaluated by RNA-FISH. Representative images are shown at 630× magnification. Red represents target mRNA, while blue represents the nuclei. *P < 0.05, **P < 0.01, and ***P < 0.001 versus parental cells or sh-control group (n = 3).
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