ABSTRACT
Redirecting immune cells to the tumor cells and enhancing its anti-tumor immune response is a very promising cancer treatment strategy. AS1411 aptamers have high affinity for malignant tumors with high nucleolin expression, and cytotoxic T lymphocyte associated protein 4 (CTLA-4) aptamers can specifically bind to CTLA-4, which is expressed by T cells. In this study, a dual-affinity aptamer targeted liposome (Dat. Lipo) was constructed based on AS1411 aptamer and CTLA-4 aptamer, and its immunotherapeutic effect on T cells was studied. After the aptamer was modified with cholesterol, Dat. Lipo was prepared by instillation method; its effect of redirecting T cells was determined by confocal micrographs; its T cell immunotherapy effect was evaluated by cell counting kit-8 (CCK8) and T cell penetration was evaluated by tumor spheroids. The results showed that compared with liposomes loaded with one type aptamer, Dat. Lipo could effectively promote the redirection of T cells to tumor cells; Dat. Lipo had good biosafety and immunotherapeutic effect on MCF-7 and HepG2 cells in a concentration-dependent manner; Dat. Lipo could also promote T cells to infiltrate into the tumor spheroids and enhance the immunotherapy effect of T cells in different dimensions. In summary, Dat. Lipo can use the high affinity of aptamers to redirect T cells to tumor cells, enhance the effect of immunotherapy, and has a promising application prospect in tumor therapy. This study was approved by the Examination Committee of Cancer Hospital of Xiangya School of Medicine, Central South University, Hunan Cancer Hospital.
ABSTRACT
Objective • To generate a doxycycline (Dox)-inducible multiplexed CRISPR interference (CRISPRi) system for multiple gene inhibition in human embryonic stem cells (hESCs) to explore the function of gene families and model multigene diseases. Methods • A Dox-inducible multiplexed CRISPRi system was developed by Golden Gate assembly in hESCs. This system consisted of two plasmids, one expressing modified repressive nuclease-deactivated CRISPR-associated protein 9 (dCas9) and Krüppel-associated box (KRAB) transcriptional repressor domain under the control of Dox, the other carrying eight independent guide RNA (gRNA) expression cassettes. PCR was conducted using total genomic DNA as a template to confirm whether these two plasmids were integrated into genome. Western blotting was performed to confirm whether the expression of dCas9-KRAB could be induced by Dox treatment. Results • Using this tunable CRISPRi system, multiple genes were successfully silenced simultaneously in hESCs. The silence of genes and related to hESC self-renewal caused obvious cell differentiation in terms of changed cell morphology, decreased activity of alkaline phosphatase, and reduced expression of stage-specific embryonic antigen 4 (SSEA4), a marker of undifferentiated hESCs. Conclusion • This Dox-inducible multiplexed CRISPRi system can be used for quick and efficient silence of multiple genes in hESCs in a highly controlled manner.
ABSTRACT
Objective To study the expression of monocarboxylate transporter l(MCTl), CD147, and CD44 mRNA in non-small cell lung cancer(NSCLC), and its role in the development, progression and prognosis of NSCLC. Methods Fifty-six NSCLC specimens and the adjacent tissues (more than 5 cm from the tumors), 20 specimens of benign pulmonary diseases and 7 specimens of normal pulmonary tissues were included in the present study. The expression of MCT1, CD147, and CD44 mRNA was examined by real-time quantitative RT-PCR in the above specimens. The relationship of their expression with the clinical and pathological features of NSCLC patients was analyzed. Results (l)The expression rates of MCT1, CD147, and CD44 mRNA in NSCLC specimens were significantly higher than those in the adjacent tissues, normal pulmonary tissues, and benign pulmonary disease tissues (all P<0. 05). (2) The expression of MCT1, CD147, and CD44 in NSCLC tissue was significantly correlated with each other(P<0. 05). (3) The expression of MCT1, CD147, and CD44 mRNA in NSCLC tissue was not correlated with age, gender, smoking, site and dimension of lesion, or pTNM stage, and was correlated with the histological types (all P<0. 05) and lymph node metastasis of NSCLC(all P<0. 05). In addition, we also found that MCT1 and CD44 expression was correlated with the differentiation degree of NSCLC(all P<0. 05). Conclusion The expression of MCT1, CD147, and CD44 mRNA is up-regulated in the NSCLC specimens. MCT1, CD147 and CD44 may participate in the development and progression of NSCLC, and they may also act as markers for diagnosis and prognosis of NSCLC.