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Transcription factors (TFs) regulate the expression of genes responsible for cell growth, differentiation, and responses to environmental factors. In this study, we demonstrated that signal-induced proliferation-associated 1 (SIPA1), known as a Rap-GTPase-activating protein, bound DNA and served as a TF. Importin β1 was found to interact with SIPA1 upon fibronectin treatment. A TGAGTCAB motif was recognized and bound by DNA-binding region (DBR) of SIPA1, which was confirmed by electrophoretic mobility shift assay. SIPA1 regulated the transcription of multiple genes responsible for signal transduction, DNA synthesis, cell adhesion, cell migration, and so on. Transcription of fibronectin 1, which is crucial for cell junction and migration of triple-negative breast cancer (TNBC) cells, was regulated by SIPA1 in a DBR-dependent manner both in vivo and in vitro. Furthermore, single-cell transcriptome sequencing analysis of specimens from a metastatic TNBC patient revealed that SIPA1 was highly expressed in metastatic TNBC. Hence, this study demonstrated that SIPA1 served as a TF, promoting TNBC migration, invasion, and metastasis.

•LNCRNA SHANK3 is highly expressed in gastric cancer and is associated with poor prognosis•LNCRNA SHANK3 sponge adsorbs miR-4530 to inhibit its expression, while mine-4530 inhibits its expression by binding to 3 'UTR of MNX1.•LNCRNA SHANK3 regulates the proliferation, migration, and invasion of gastric cancer cells by regulating miR-4530/MNX1•This study has the hope of providing possible diagnostic and therapeutic targets and theoretical support for the design and development of GC preventive drugs.

ABSTRACTAs gene delivery tools, lentiviral vectors (LV) have broad applications in chimeric antigen receptor therapy (CAR-T). Large-scale production of functional LV is limited by the adherent, serum-dependent nature of HEK293T cells used in the manufacturing. HEK293T adherent cells were adapted to suspension cells in a serum-free medium to establish large-scale processes for functional LV production in a stirred bioreactor without micro-carriers. The results showed that 293 T suspension was successfully cultivated in F media (293 CD05 medium and SMM293-TII with 1:1 volume ratio), and the cells retained the capacity for LV production. After cultivation in a 5.5 L bioreactor for 4 days, the cells produced 1.5 ± 0.3 × 107TU/mL raw LV, and the lentiviral transduction efficiency was 48.6 ± 2.8% in T Cells. The yield of LV equaled to the previous shake flask. The critical process steps were completed to enable a large-scale LV production process. Besides, a cryopreservation solution was developed to reduce protein involvement, avoid cell grafting and reduce process cost. The process is cost-effective and easy to scale up production, which is expected to be highly competitive.

Papillary thyroid cancer (PTC) remains the most common endocrine malignancy, despite marked achieves in recent decades, and the mechanisms underlying the pathogenesis and progression for PTC are incompletely elucidated. Accumulating evidence show that γ-glutamylcyclotransferase (GGCT), an enzyme participating in glutathione homeostasis and is elevated in multiple types of tumors, represents an attractive therapeutic target. Using bioinformatics, immunohistochemistry, qRT-PCR, and Western blot assays, we found that GGCT expression was upregulated in PTC and correlated with more aggressive clinicopathological characteristics and worse prognosis. GGCT knockdown inhibited the growth and metastasis ability of PTC cells both in vitro and in vivo and reduced the expression of mesenchymal markers (N-cadherin, CD44, MMP2, and MMP9) while increasing epithelial marker (E-cadherin) in PTC cells. We confirmed binding of microRNA-205-5p (miR-205-5p) on the 3′-UTR regions of GGCT by dual-luciferase reporter assay and RNA-RNA pull-down assay. Delivery of miR-205-5p reversed the pro-malignant capacity of GGCT both in vitro and in vivo. Lastly, we found that GGCT interacted with and stabilized CD44 in PTC cells by co-immunoprecipitation and immunohistochemistry assays. Our findings illustrate a novel signaling pathway, miR-205-5p/GGCT/CD44, that involves in the carcinogenesis and progression of PTC. Development of miR-205-mimics or GGCT inhibitors as potential therapeutics for PTC may have remarkable applications.

PCNA (proliferating cell nuclear antigen) is a protein related to tumor development, which has been used extensively in breast cancer diagnosis and prognosis. PCNA has proven to be a useful marker to evaluate cell proliferation and prognosis when combined with other breast cancer markers. Construction of PCNA promoter luciferase reporter plasmid will provide the theory basis for researching the effect of other transcription factors on regulating PCNA transcription. In this study, a human PCNA promoter luciferase reporter construct was generated by PCR amplification of PCNA promoter. The PCR fragment was digested and cloned into pGL3 vector. The promoter sequence was verified by sequencing. The results showed that luciferase reporter plasmids of PCNA promoter were successfully constructed. Then the effects of some key transcription factors, which play important roles in breast cancer cell proliferation, were investigated by luciferase reporter assays in MCF-7 cells. The results showed that ERα can enhance transcriptional activity of PCNA. Furthermore, 17-β-estradiol (E2) also shows an obvious impact in activating PCNA transcription. Our data illuminated that E2 enhances ERα-induced proliferation potential of MCF-7 cells by stimulating the transcriptional activity of PCNA. Our research will provide a model to screen some novel factors in regulating proliferation marker transcription.

Based on observations that lactic acid bacteria has proved to be beneficial in the treatment of viral-and antibiotic-associated diarrhea but the mechanisms of action remain unknown. Some reports indicated that nitric oxide (NO) might be involved in the protective mechanisms in the gastrointestinal tract and may contribute to some of the beneficial effects of probiotics. To confirm the effect of a probiotic Lactobacillus acidophilus (L. acidophilus) strain TCCC 11036 on the NO synthesis, in the present study, HepG2 human liver cells were cultured and treated with the supernatant or precipitate of L. acidophilus TCCC 11036 cell hydrolysates, then NO synthesis and its regulation were measured with the methods of NO assay, luciferase reporter assay and RT-PCR. The results showed that the production of nitric oxide was increased after treated with precipitates of the bacterium, and its transcription was enhanced as well. These works might provide a foundation for the following study of L. acidophilus and its probiotics functions, and contribute to the development of functional foods or drugs that provide health benefits to patients suffering from inflammation, cardiovascular disease, or some other diseases correlated with NO.

Abstract: Objective: To observe the effect of all-trans retinoic acid (ATRA) on inducing human glioma MO59K cells differentiation and further explore the underlying molecular mechanisms. Methods: The expression of glial fibrillary acidic protein (GFAP) was detected by immunocytochemistry staining. The mRNA levels of GFAP, retinoid X receptor α (RXRα), p21 were examined by semi-quantitative RT-PCR analysis. Luciferase activity assay was performed in the COS-7, MO59K cells to measure p21 promoter transcription activity. Results: ATRA could significantly enhance the expression and mRNA level of GFAP by immunostaining and RT-PCR (P < 0.05). Simultaneously, the mRNA levels of RXRα and p21 were remarkably increased in dose-dependent manner by RT-PCR (P < 0.05). Furthermore, luciferase assay confirmed that ATRA and RXRα could transactivate p21 promoter in COS-7 and glioma cells (P < 0.05). Conclusion: ATRA can induce differentiation of human glioma cells. The RXRα and p21 were activated during ATRA-induced differentiation process. This effect may be caused by directly RXRα-induced p21 gene transactivation. Our findings provide novel evidence for the future studies to explore the molecular mechanism of transcriptional regulation for glioma cell differentiation and cellular therapeutic approaches for glioblastoma.