BackgroundThe transcription factor forkhead box P3 (Foxp3) is a master regulatory gene necessary for the development and function of CD4+CD25+ regulatory T cells (Tregs). Mesenchymal stem cells (MSC) have recently emerged as promising candidates for cell-based immunosuppression/tolerance induction protocols. Thus, we hypothesized that MSC-based Foxp3 gene therapy would improve immunosuppressive capacity of MSC and induce donor-specific allograft tolerance in rat’s liver allograft model.MethodsThe present study utilized a lentivirus vector to overexpress the therapeutic gene Foxp3 on MSC. In vivo, Injections of 2 × 106 MSC, FUGW-MSC or Foxp3-MSC into the portal vein were carried out immediately after liver transplantation.ResultsSuccessful gene transfer of Foxp3 in MSC was achieved by lentivirus carrying Foxp3 and Foxp3-MSC engraftment in liver allograft was confirmed by fluorescence microscopy. Foxp3-MSC treatment significantly inhibited the proliferation of allogeneic ACI CD4+ T cells to splenocytes (SC) from the same donor strain or third-party BN rat compared with MSC. Foxp3-MSC suppressive effect on the proliferation of CD4+ T cells is contact dependent and associated with Programmed death ligand 1(PD-L1) upregulation in MSC. Co-culture of CD4+ T cells with Foxp3-MSC results in a shift towards a Tregs phenotype. More importantly, Foxp3-MSC monotherapy achieved donor-specific liver allograft tolerance and generated a state of CD4+CD25+Foxp3+ Tregs-dependent tolerance.ConclusionFoxp3-engineered MSC therapy seems to be a promising and attractive cell therapy approach for inducing immunosuppression or transplant tolerance.
Hepatocellular carcinoma (HCC) is known for its high mortality rate worldwide. Based on intensive studies, microRNA (miRNA) expression functions in tumor suppression. Therefore, we aimed to evaluate the contribution of miR-146a-5p to radiosensitivity in HCC through the activation of the DNA damage repair pathway by binding to replication protein A3 (RPA3). First, the limma package of R was performed to differentially analyze HCC expression chip, and regulative miRNA of RPA3 was predicted. Expression of miR-146a-5p, RPA3, and DNA damage repair pathway-related factors in tissues and cells was determined. The effects of radiotherapy on the expression of miR-146a-5p and RPA3 as well as on cell radiosensitivity, proliferation, cell cycle, and apoptosis were also assessed. The results showed that there exists a close correlation between miR-146a and the radiotherapy effect on HCC progression through regulation of RPA3 and the DNA repair pathway. The positive rate of ATM, pCHK2, and Rad51 in HCC tissues was higher when compared with that of the paracancerous tissues. SMMC-7721 and HepG2 cell proliferation were significantly inhibited following 8 Gy 6Mv dose. MiR-146a-5p restrained the expression of RPA3 and promoted the expression of relative genes associated with the DNA repair pathway. In addition, miR-146a-5p overexpression suppresses cell proliferation and enhances radiosensitivity and cell apoptosis in HCC cells. In conclusion, the present study revealed that miR-146a-5p could lead to the restriction of proliferation and the promotion of radiosensitivity and apoptosis in HCC cells through activation of DNA repair pathway and inhibition of RPA3.
Axis inhibition protein 1 (AXIN1) is a negative regulator of Wnt/beta-catenin signaling via regulating the level of beta-catenin. However, the role of AXIN1 in the tumorigenesis and progression of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) is less clear. PCR sequence analysis, immunohistochemistry, and Western blot were performed on 22 HBV-related HCC samples and corresponding nontumor liver tissues to detect variants in AXIN1 gene and the expression level of AXIN1. Human hepatoma cell lines SNU475 and SNU423 were transfected with pCDNA3.1-AXIN1-myc or AXIN1 G425S-myc mutant. The growth curve and apoptosis rate of cell lines, phosphorylation of beta-catenin, and cell cycle regulatory proteins depending on beta-catenin transcriptional activity were detected. We identified four mutations of AXIN1 in 22 primary HBV-related HCCs and demonstrated a lower expression of AXIN1 in HBV-related HCC tissues than that in paired adjacent nontumor tissues. Overexpression of AXIN1 wild-type but not AXIN1 mutant inhibited the growth of HCC cell lines, accelerated their apoptosis, and negatively regulated beta-catenin-dependent transcriptional activity. Our study revealed that alterations of AXIN1 were involved in HBV-related HCC. Overexpression of AXIN1 but not AXIN1 mutant negatively regulated beta-catenin-dependent transcriptional activity and downregulated the level of cell cycle regulatory proteins, suggesting that AXIN1 may be a potential target for gene therapy of primary HCC.
Long non-coding RNAs (lncRNAs) have a considerable regulatory influence on multiple biological processes. Nevertheless, the role of TMEM220-AS1 in hepatocellular carcinoma (HCC) remains unclear. We used The Cancer Genome Atlas (TCGA) database to analyze the differentially expressed lncRNAs. qRT-PCR was used to verify the results for a large population. The in vitro effects of TMEM220-AS1 on HCC cells were determined using Cell Counting Kit-8 (CCK-8), 5-ethynyl-2’-deoxyuridine (EdU), flow cytometry, and Transwell assays in HCC cells. We used qRT-PCR and western blotting to identify the epithelial-mesenchymal transition (EMT). Moreover, we performed bioinformatics analysis, western blotting, dual luciferase reporter gene assay, RNA pull-down, and RNA binding protein immunoprecipitation (RIP) to investigate the underlying molecular mechanisms of TMEM220-AS1 function. Finally, the function of TMEM220-AS1 was verified in vivo. The results showed that TMEM220-AS1 was expressed at considerably low levels in HCC. It was demonstrated that malignant phenotypes and EMT of HCC cells were promoted by the knock down of TMEM220-AS1 both in vivo and in vitro. TMEM220-AS1, which was detected primarily in the cytoplasm, functioned as an miRNA sponge to bind miR-484 and promote the level of membrane-associated guanylate kinase, WW, and PDZ domain containing 1 (MAGI1), thereby curbing the malignant phenotypes of HCC cells. In conclusion, low levels of TMEM220-AS1 promote proliferation and metastasis through the miR-484/MAGI1 axis in HCC.
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