Crustacyanin has the function of binding astaxanthin which is the best antioxidant, and plays an important role in the body color variation of crustaceans. To investigate the causes of body color variation of the ridgetail white prawn, Exopalaemon carinicauda, the present study obtained four subtypes of crustacyanin gene: C1, C2, A1, and A2. Based on fluorescence quantitative polymerase chain reaction, lipocalin-C1 is mainly expressed in the eyestalk, lipocalin-C2 is in the ventral nerve cord, and lipocalin-A1 and lipocalin-A2 are in subcutaneous adipose tissues. Under the inhibiting effect of Cd 2+ stress, the expression of four subtypes first increases and then decreases within 24 h, and reaches the maximum at 6 or 12 h. RNA interference experiments showed a decrease in the expression of lipocalin genes in subcutaneous adipose tissue for each subtype, with the body color changing from transparent to red, and the dark red spots on the epidermis changing to bright red. Moreover, the blue protein in the subcutaneous adipose tissue largely disappeared, based on the light micrographs. In view of these findings, the crustacyanin gene appears to fulfill some function in the resistance to heavy metal stress and body color formation of E. carinicauda.
For the purpose of studying the epigenetic characteristics of the mitochondrial genome of the ridgetail white prawn, Exopalaemon carinicauda (Holthuis, 1950) under Cd2+ and Cr6+ heavy metal stress, the mitochondrial DNA methylation of E. carinicauda was analysed by bisulfite sequencing PCR (BSP). Many methylation sites were found at the 3′ end sequence of COX3 and at the starting region sequence of ND3, while only a few methylation sites were found at the 3′ end sequence of ND5. The mitochondrial genome was inferred to regulate the energy metabolism through the methylation process. In addition, under Cd2+ stress, mitochondrial DNA methylation was more common, and found during all stress periods (3, 6, 12, 24, 48, 72 and 96 h), while under Cr6+ stress, mitochondrial DNA methylation was less common, mainly occurring after 48 hours of stress. The sensitivity of the mitochondrial genome response to Cd2+ stress was inferred to be greater than that to Cr6+. This study revealed for the first time that methylation occurs in the mitochondrial genome of E. carinicauda in response to heavy metal stress.
Background
Effects of mesenchymal stem cells (MSCs) on tumors have been extensively studied in recent years, but results still remain controversial. In this study we investigated the roles of MSCs on the expression of programmed death-ligand 1 (PD-L1) in lung cancer cells. Since overexpression of PD-L1 has been found to attenuate body's immune system against tumor cells, PD-1/PD-L1 blockages have become the most effective immunotherapy to the late stage non-small cell lung cancer (NSCLC). Therefore, understanding the effects of MSCs on PD-L1 expression is critical in managing immunotherapy for NSCLC patients.
Method
PD-L1 expressions were tested in two NSCLC cell lines, A549 and H1299 using qRT-PCR and Western Blot to qualitatively measure mRNA and protein levels, respectively. MSCs were isolated from umbilical cord blood and co-cultured with A549 and H1299 cells in complete medium using Transwell filters for 24-h, 48-h and 72-h. Then both A549 and H1299 cells were collected and PD-L1 expressions were tested. MSC-Conditioned Medium (MSC-CM) was generated by culturing 80% confluence of MSCs with serum-free medium. After 48 h, the medium was collected as MSC-CM. Both A549 and H1299 were then cultured in MSC-CM for 24-h, 48-h and 72-h, and PD-L1 expressions were measured from these two cell lines separately.
Result
Both MSCs and MSC-CM significantly increased the PD-L1 mRNA levels in the two NSCLC cells, while the MSC-CM had stronger stimulation to PD-L1 expressions (P < 0.0001) comparing to MSCs treatment (P<0.01). A549 cells reached the most significant effect at 48-h, while H1299 cells attended the same effect at 24-h treatment. The similar results were observed with PD-L1 protein levels. Both cell lines showed increased PD-L1 protein expressions after treating with MSCs and MSC-CM, but the augmented scales were smaller than the mRNA levels. The PD-L1 protein expressions in A549 cells increased significantly (p < 0.05) at 48-h when treating with MSCs, while increased much higher when treating with MSC-CM (P < 0.01). H1299 cells also showed the similar increase in PD-L1 protein expressions after 24-h treatment.
Conclusion
Both MSCs and MSC-CM significantly increase PD-L1 expressions in lung cancer cells, which ultimately attenuate the immune system against tumor cells.
Citation Format: Hui Zhao, Hui Yang, Jinqiu Sun, Guoqiang Liang, Qianqian Zhao, Xiufang Zhang, Shengxian Liang, Rui Guo, Li Zhong. Mesenchymal stem cells promote PD-L1 expression in lung cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6011.
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