Peroxisome proliferator-activated receptor alpha regulates a microRNA-mediated signaling cascade responsible for hepatocellular proliferation

Activation of peroxisome proliferator-activated receptor alpha (PPARalpha) leads to hepatocellular proliferation and liver carcinomas. The early events mediating these effects are unknown. A novel mechanism by which PPARalpha regulates gene expression and hepatocellular proliferation was uncovered. MicroRNA (miRNA) expression profiling demonstrated that activated PPARalpha was a major regulator of hepatic miRNA expression. Of particular interest, let-7C, an miRNA important in cell growth, was inhibited following 4-h treatment and 2-week and 11-month sustained treatment with the potent PPARalpha agonist Wy-14,643 in wild-type mice. let-7C was shown to target c-myc via direct interaction with the 3' untranslated region of c-myc. The PPARalpha-mediated induction of c-myc via let-7C subsequently increased expression of the oncogenic mir-17-92 cluster; these events did not occur in Pparalpha-null mice. Overexpression of let-7C decreased c-myc and mir-17 and suppressed the growth of Hepa-1 cells. Furthermore, using the human PPARalpha-expressing mouse model, which is responsive to Wy-14,643 effects on beta-oxidation and serum triglycerides but resistant to hepatocellular proliferation and tumorigenesis, we demonstrated a critical role for let-7C in liver oncogenesis. Wy-14,643 treatment did not inhibit let-7C or induce c-myc and mir-17 expression. These observations reveal a let-7C signaling cascade critical for PPARalpha agonist-induced liver proliferation and tumorigenesis.     

MicroRNA let-7c Inhibits Cell Proliferation and Induces Cell Cycle Arrest by Targeting CDC25A in Human Hepatocellular Carcinoma

Down-regulation of the microRNA let-7c plays an important role in the pathogenesis of human hepatocellular carcinoma (HCC). The aim of the present study was to determine whether the cell cycle regulator CDC25A is involved in the antitumor effect of let-7c in HCC. The expression levels of let-7c in HCC cell lines were examined by quantitative real-time PCR, and a let-7c agomir was transfected into HCC cells to overexpress let-7c. The effects of let-7c on HCC proliferation, apoptosis and cell cycle were analyzed. The in vivo tumor-inhibitory efficacy of let-7c was evaluated in a xenograft mouse model of HCC. Luciferase reporter assays and western blotting were conducted to identify the targets of let-7c and to determine the effects of let-7c on CDC25A, CyclinD1, CDK6, pRb and E2F2 expression. The results showed that the expression levels of let-7c were significantly decreased in HCC cell lines. Overexpression of let-7c repressed cell growth, induced cell apoptosis, led to G1 cell cycle arrest in vitro, and suppressed tumor growth in a HepG2 xenograft model in vivo. The luciferase reporter assay showed that CDC25A was a direct target of let-7c, and that let-7c inhibited the expression of CDC25A protein by directly targeting its 3ʹ UTR. Restoration of CDC25A induced a let-7c-mediated G1-to-S phase transition. Western blot analysis demonstrated that overexpression of let-7c decreased CyclinD1, CDK6, pRb and E2F2 protein levels. In conclusion, this study indicates that let-7c suppresses HCC progression, possibly by directly targeting the cell cycle regulator CDC25A and indirectly affecting its downstream target molecules. Let-7c may therefore be an effective therapeutic target for HCC.     

Let-7a下调k-Ras和c-Myc癌基因的表达抑制肾癌细胞增殖

目的：MicroRNA 是近年发现的一类单链小分子RNA,对它的研究已成为一个新的热点。最近的研究发现,1et-7a 在细胞内影响着基因的表达调控,在疾病发生中起着及极重要的作用,尤其是在肿瘤的发展过程中,let-7a扮演着不可替代的角色。本文主要研究let-7a 在肾癌细胞株中的表达情况及其调控的靶基因、抑制细胞增殖的机制,对探索肾癌的致病基因,寻求肾癌新的治疗途径有重要意义。方法：应用化学合成的let-7a 模拟物（mimics）用脂质体Lipofectamine 2000 在体外瞬时转染786-O 和Caki-1肾癌细胞株,转染48 小时后采用荧光定量RT-PCR的方法检测let-7a 及c-Myc、k-Ras mRNA的表达情况,Western blot 检测这两株肾癌细胞转染了let-7a mimics 后c-Myc 及k-Ras 蛋白的表达变化;转染let-7a mimics 后分别在24、48、72 小时三个时间点用CCK-8 试剂盒检测对肾癌细胞株增殖的影响。结果：786-O 和Caki-1 肾癌细胞株中let-7a 的表达量明显低于正常肾小管上皮细胞株HK-2（P〈0.05）; 转染了let-7amimics的786-O 和Caki-1 肾癌细胞株,RT-PCR 及Western blot 结果显示c-Myc、k-Ras 在基因及蛋白的表达水平明显下调（P〈0.05）;CCK-8 检测结果显示转染了let-7a mimics的肾癌细胞株细胞增殖能力明显明显受到抑制,与阴性对照组比较差异有统计学意义（P〈0.05）。结论：Let-7a 在在肿瘤细胞与正常细胞中存在明显差异,let-7a 通过调控c-Myc、k-Ras的表达能抑制肾癌细胞增殖。Let-7a mimics 可以抑制肾癌细胞的增殖,因此上调Let-7a 的表达有可能成为肾癌基因治疗的一种有效治疗手段。     

Downregulation of microRNA let-7f mediated the Adriamycin resistance in leukemia cell line

Multidrug resistance (MDR) has become the major cause of failure chemotherapy for leukemia and high mortality of leukemia. The study aimed to investigate whether the let-7f mediate the Adriamycin (ADR) resistance of leukemia, and to explore the potential molecular mechanism. Cell proliferation was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and the soft agar clone formation assay. Flow cytometry was performed to detected cell cycle and apoptosis. The targeted regulationship was analyzed by dual-luciferase assay. Real-time polymerase chain reaction and Western blot were used to measure the expressions of let-7f, ABCC5, ABCC10, cell cycle-related proteins, and apoptosis-related proteins. The xenograft mouse model was used to conduct the tumor formation assay in vivo. The results demonstrated that the expression of let-7f was lower in multidrug-resistant K562/A02 cell lines compared to that in K562, while ABCC5 and ABCC10 were upregulated. Overexpression of let-7f in K562/A02 cell lines downregulated the ABCC5 and ABCC10 expression, enhanced cell sensitivity to ADR, promoted cell apoptosis, and inhibited cell proliferation. let-7f was proved to negatively regulate ABCC5 and ABCC10. Tumor formation assay further determined that let-7f overexpression increased sensitivity to ADR. Taken together, the let-7f downregulation induced the ADR resistance of leukemia by upregulating ABCC5 and ABCC10 expression. Our study provided a novel perspective to study the mechanism of MDR and a new target for the reversal of MDR.     

Jumonji/ARID1 B (JARID1B) protein promotes breast tumor cell cycle progression through epigenetic repression of microRNA let-7e

MicroRNAs (miRs) function as tumor suppressors or oncogenes in multiple tumor types. Although miR expression is tightly regulated, the molecular basis of miR regulation is poorly understood. Here, we investigated the influence of the histone demethylase Jumonji/ARID1 B (JARID1B) on miR regulation in breast tumor cells. In MCF-7 cells with stable RNAi-mediated suppression of JARID1B expression we identified altered regulation of multiple miRs including let-7e, a member of the let-7 family of tumor suppressor miRs. Chromatin immunoprecipitation analysis demonstrated JARID1B binding to the let-7e promoter region as well as removal of the of H3K4me3 histone mark associated with active gene expression. These results suggest that JARID1B epigenetically represses let-7e expression. JARID1B stimulates tumor cell proliferation by promoting the G(1) to S transition. As predicted, suppression of JARID1B resulted in an accumulation of MCF-7 cells in G(1). We confirmed that cyclin D1, which also promotes G(1) progression, is a direct target of let-7e, and we show that cyclin D1 expression is suppressed in JARID1B knockdown cells. Cyclin D1 expression and cell cycle progression were restored following inhibition of let-7e, suggesting that JARID1B repression of let-7e contributes to cyclin D1 expression and JARID1B-mediated cell cycle progression. Our results indicate that the JARID1B demethylase contributes to tumor cell proliferation through the epigenetic repression of a tumor suppressor miR.     

MicroRNA Let-7a Inhibits Proliferation of Human Prostate Cancer Cells In Vitro and In Vivo by Targeting E2F2 and CCND2

Background

Previous work has shown reduced expression levels of let-7 in lung tumors. But little is known about the expression or mechanisms of let-7a in prostate cancer. In this study, we used in vitro and in vivo approaches to investigate whether E2F2 and CCND2 are direct targets of let-7a, and if let-7a acts as a tumor suppressor in prostate cancer by down-regulating E2F2 and CCND2.

Methodology/Principal

Findings Real-time RT-PCR demonstrated that decreased levels of let-7a are present in resected prostate cancer samples and prostate cancer cell lines. Cellular proliferation was inhibited in PC3 cells and LNCaP cells after transfection with let-7a. Cell cycle analysis showed that let-7a induced cell cycle arrest at the G1/S phase. A dual-luciferase reporter assay demonstrated that the 3′UTR of E2F2 and CCND2 were directly bound to let-7a and western blotting analysis further indicated that let-7a down-regulated the expression of E2F2 and CCND2. Our xenograft models of prostate cancer confirmed the capability of let-7a to inhibit prostate tumor development in vivo.

Conclusions/Significance

These findings help to unravel the anti-proliferative mechanisms of let-7a in prostate cancer. Let-7a may also be novel therapeutic candidate for prostate cancer given its ability to induce cell-cycle arrest and inhibit cell growth, especially in hormone-refractory prostate cancer.     

microRNA-505 negatively regulates HMGB1 to suppress cell proliferation in renal cell carcinoma

microRNAs have been recognized to regulate a wide range of biology of renal cell carcinoma (RCC). Although miR-505 has been reported to play as a suppressor in several human tumors, the physiological function of miR-505 in RCC still remain unknown. Therefore, the role of miR-505 and relevant regulatory mechanisms were investigated in RCC in this study. Quantitative real-time polymerase chain reaction was conducted to detect the expression of miR-505 and high mobility group box 1 (HMGB1) in both RCC tissues and cell lines. Immunohistochemical staining was used to assess the correlation between HMGB1 expression and PCNA expression in RCC tissues. Subsequently, the effects of miR-505 on proliferation were determined in vitro using cell counting kit-8 proliferation assays and 5-ethynyl-2′-deoxyuridine incorporation. The molecular mechanism underlying the relevance between miR-505 and HMGB1 was confirmed by luciferase assay. Xenograft tumor formation was used to reflect the proliferative capacity of miR-505 in vivo experiments. Overall, a relatively lower miR-505 and higher HMGB1 expression in RCC specimens and cell lines were found. HMGB1 was verified as a direct target of miR-505 by luciferase assay. In vitro, overexpression of miR-505 negatively regulates HMGB1 to suppress the proliferation in Caki-1; meanwhile, knock-down of miR-505 negatively regulates HMGB1 to promote the proliferation in 769P. In addition, in vivo overexpression of miR-505 could inhibit tumor cell proliferation in RCC by xenograft tumor formation. Therefore, miR-505, as a tumor suppressor, negatively regulated HMGB1 to suppress the proliferation in RCC, and might serve as a novel therapeutic target for RCC clinical treatment.     

Expression of apoptosis and cell cycle related genes in proliferating and colcemid arrested cells of divergent lineage.

Progression through the cell cycle and redirection of cells towards programmed cell death (apoptosis) are tightly inter-related processes. However the requirement for tissue and cell type specificity suggests that a wide variety of mechanisms are used to achieve the same purpose. To examine this issue, we investigated cell cycle (c-myc, p53, p21/WAF) and apoptosis related (bcl-2, bcl-X(L), bax-alpha) gene expression in two cell lines of very different origin under proliferating and apoptosis-inducing conditions. Transformed human osteosarcoma cells (MG63) and non-transformed human kidney embryonal fibroblasts (293-0) were kept in culture in medium containing 10% FCS and growth arrest was induced by the addition of 50 ng/ml colcemid. Colcemid treatment caused growth arrest and elevated expression of cyclin B1 protein in both cell lines. Apoptosis was significantly elevated in both cell lines after colcemid exposure for at least one cell cycle. However the pattern of expression of cell cycle and apoptosis related genes, determined by RT-PCR, was quite different between the two cell lines during exponential growth and cell cycle arrest. Colcemid treatment did not markedly influence c-myc, p53 and p21/WAF expression in MG63 cells but did suppress c-myc and increase p21/WAF in 293-0 cells. Furthermore colcemid treated MG63 cells exhibited elevated bcl-2 and bax-alpha expression while similar treatment of 293-0 cells resulted in decreased bcl-X(L) and slightly increased bax-alpha expression. While growth arrest and apoptosis were induced in both MG63 and 293 cells following colcemid treatment, the differences in gene expression suggest that the mechanism by which these cells determine cell fate is quite different and may determine the sensitivity of different cell populations to anti-neoplastic drug therapy. The distinct patterns of gene expression should be carefully defined before mechanisms of apoptotic cell death are studied.     

Downregulation of c-myc RNA is not a prerequisite for reduced cell proliferation, but is associated with G1 arrest in B-lymphoid cell lines

We related the effects of c-myc expression on the ability of growth inhibitors to block the cells in the G0/G1 phase of the cell cycle. In two different B-cell lines, there was an association between the accumulation of cells in the middle to late G1 phase of the cell cycle and a rapid transient downregulation of c-myc mRNA levels. The phorbol ester TPA and the adenylate cyclase activator forskolin reduced the c-myc RNA, levels and after 3 days of treatment a proportion of the cells accumulated in G1. In contrast, neither interferon-gamma, tumor necrosis factor-alpha nor the monoclonal antibody 33-1 against DQ major histocompatibility antigens changed the cell-cycle distribution or regulated the c-myc RNA levels. Yet, all five growth inhibitors reduced the proliferation to approximately the same extent. The growth reduction was not accompanied by definite differentiation, as judged by the absence of the B-cell differentiation marker B1 (CD20).     

Measurement of c-myc protein content and cell cycle kinetics of normal and spontaneously transformed murine mastocytes by bivariate flow cytometry

Progressive in vitro culturing of interleukin-3 (IL-3) dependent normal murine mastocytes (PB-3) resulted in a variant cell line (PB-1) able to grow without exogenous IL-3 and which was tumorogenic in syngenic mice. Bivariate flow cytometry was used to evaluate the c-myc protein and DNA content of PB-3 and PB-1 cells. The c-myc protein was detected by specific monoclonal antibodies. Kinetic characteristics of PB-3 and PB-1 cell lines, namely, the duration of the G1, S and G2 + M cell cycle phases were also evaluated using the bromodeoxyuridine (BrdU) pulse-chase method and BrdU/DNA flow cytometry. Levels of c-myc protein in PB-1 cells were about two-fold higher than those of PB-3 cells in all cell cycle phases. Mean duration of the cell cycle (Tc) was 15.3 h for PB-3 cells and 12.4 h for PB-1 cells. Shortening in Tc for the transformed cells was due to a decrease of nearly 30% in mean duration of the G1 phase (from 8 h to 5.7 h). No significant differences were found in the duration of the S and G2 + M phases. These results indicate that acquired IL-3 independency in vitro and tumorogenicity of PB-1 cells were accompanied by a doubling of c-myc protein level and by a parallel shortening, or bypass, of the regulatory events within the G1 phase of the cell cycle.     

Role of ribosomal protein RPS2 in controlling let-7a expression in human prostate cancer

We discovered that an inverse relationship exists in the expression of ras/c-myc and ribosomal protein RPS2 with pre-let-7a-1/let-7a/let-7f miRNA and prostate tumor cell malignancy. Nonmalignant IBC-10a cells expressed low levels of ras/RPS2 and elevated pre-let-7a-1/let-7a/let-7f miRNA, whereas the reverse occurred in malignant PCa-20a and PC-3ML cells. Stable transfection of IBC-10a cells with pBABE.ras and pBABE.RPS2 induced ras, c-myc, and RPS2 expression, whereas the levels of let-7a/let-7f miRNA dropped to near zero. Conversely, in pBABE.pre-let-7a-1 transfected PCa-20a and PC-3ML clones, let-7a/let-7f increased whereas ras, RPS2, and c-myc dropped greater than 5-fold. Electrophoretic mobility shift assays, antibody "supershift" assays and immunoprecipitation assays revealed that RPS2 specifically binds pre-let-7a-1 to block RNA processing. Immunoflourescent studies and Northern blots confirmed that RPS2 complexes with pre-let-7a-1 (i.e., in episomal structures) to block processing to let-7a/let-7f, indicating RPS2 may prevent let-7a miRNA expression to indirectly promote oncogene expression. Functional studies further showed that the colony-forming ability (CFA) and invasive activities of IBC-10a cells were significantly enhanced in pBABE-ras.IBC-10a and pBABE-RPS2-IBC-10a clones. Conversely, with the "knockdown" of ras and RPS2 in malignant PC-3ML cells (i.e., in pLKO.TRC.shRNA.ras.PC3-ML, pLKO.TRC.shRNA.RPS2.PC-3ML transfected cells), there was both a loss of these functions and a loss of tumorigenesis in SCID mice. Likewise, with the overexpression of let-7a/let-7f in pBABE.pre-let-7a-1.PC-3ML clones (and PCa-20a clones), CFAs, invasive activities in vitro, and tumorigenesis in vivo were significantly reduced. These results show for the first time that RPS2 blocks pre-let-7a-1 processing to enable ras and c-myc expression and the transformation of primary tumor cells.     

Circular RNA circ-EGLN3 promotes renal cell carcinoma proliferation and aggressiveness via miR-1299-mediated IRF7 activation

Renal cell carcinoma (RCC) is a highly lethal cancer with increasing incidence worldwide. The purpose of the present study was to investigate the functions and molecular mechanisms of circular RNA (circRNA), circ-EGLN3, in RCC progression. The expression levels of circ-EGLN3 were assessed by a quantitative real-time polymerase chain reaction. Kaplan-Meier analysis was applied to uncover the prognostic role of circ-EGLN3 in patients with RCC. Cell viability was analyzed using cell counting kit-8 and cell apoptosis was assessed using flow cytometric experiment. Cell migratory and invasive abilities were determined by wound scratch and transwell experiments. Subcellular distribution detection was utilized to investigate the location of circ-EGLN3. Dual-luciferase reporter test was utilized for identifying the molecular mechanism of circ-EGLN3. The results indicated that circ-EGLN3 was elevated in RCC tissues and cell lines and predicted unfavorable prognosis for the patients with RCC. Silenced circ-EGLN3 hindered cell proliferation, migration, and invasion but facilitated apoptosis of RCC cells. Ectopic expressed circ-EGLN3 induced the opposite effects mentioned above. Mechanistically, circ-EGLN3 was mainly located at the cytoplasm. Circ-EGLN3 acted as a competing endogenous RNA (ceRNA) to enhance the IRF7 level via sponging miR-1299. Moreover, circ-EGLN3 mediated elevation of IRF7 is responsible for RCC cell proliferation and aggressiveness. Collectively, our study suggested that circ-EGLN3 knockdown suppressed RCC progression through acting as a ceRNA to regulate the IRF7 expression by targeting miR-1299. Circ-EGLN3 might be a potential therapeutic target for RCC management.     

Targeted Expression of Suicide Gene by Tissue-Specific Promoter and MicroRNA Regulation for Cancer Gene Therapy

In order to realise the full potential of cancer suicide gene therapy that allows the precise expression of suicide gene in cancer cells, we used a tissue specific Epithelial cell adhesion molecule (EpCAM) promoter (EGP-2) that directs transgene Herpes simplex virus–thymidine kinase (HSV-TK) expression preferentially in EpCAM over expressing cancer cells. EpCAM levels are considerably higher in retinoblastoma (RB), a childhood eye cancer with limited expression in normal cells. Use of miRNA regulation, adjacent to the use of the tissue-specific promoter, would provide the second layer of control to the transgene expression only in the tumor cells while sparing the normal cells. To test this hypothesis we cloned let-7b miRNA targets in the 3’UTR region of HSV-TK suicide gene driven by EpCAM promoter because let-7 family miRNAs, including let-7b, were found to be down regulated in the RB tumors and cell lines. We used EpCAM over expressing and let-7 down regulated RB cell lines Y79, WERI-Rb1 (EpCAM ^+ve/let-7b^{down-regulated}), EpCAM down regulated, let-7 over expressing normal retinal Müller glial cell line MIO-M1(EpCAM ^−ve/let-7b^up-regulated), and EpCAM up regulated, let-7b up-regulated normal thyroid cell line N-Thy-Ori-3.1(EpCAM ^+ve/let-7b^up-regulated) in the study. The cell proliferation was measured by MTT assay, apoptosis was measured by probing cleaved Caspase3, EpCAM and TK expression were quantified by Western blot. Our results showed that the EGP2-promoter HSV-TK (EGP2-TK) construct with 2 or 4 copies of let-7b miRNA targets expressed TK gene only in Y79, WERI-Rb-1, while the TK gene did not express in MIO-M1. In summary, we have developed a tissue-specific, miRNA-regulated dual control vector, which selectively expresses the suicide gene in EpCAM over expressing cells.     

Circular RNA circMTO1 suppressed the progression of renal cell carcinoma progression by sponging miR-211 and miR-204

Despite considerable improvements in renal cell carcinoma (RCC) diagnostic and therapeutic strategy, the clinical prognosis of patients is far from satisfactory due to its recurrence and metastasis. Here, we attempted to explore the role of circMTO1 in RCC progression, and the underlying mechanism was further elucidated. We first detected the expression of circMTO1 in 90 pairs of RCC tissues and adjacent normal tissues using qRT-PCR. Besides, circMTO1, miR-211, miR-204 and KLF6 expression levels in RCC cells were also measured using qRT-PCR. MTT assay, cell migration, flow cytometry analysis, qRT-PCR and western blotting analysis were applied to evaluating the effect of circMTO1 in RCC cells. The bioinformatics analysis and the rescue experiment were devoted to the underlying mechanism. The results demonstrated CircMTO1 expression was significantly down-regulated in RCC tissues and cell lines. Besides, CircMTO1 inhibited cell proliferation, migration and invasion, induced apoptosis in RCC cells. In addition, CircMTO1 serves as a sponge for miR-211 and miR-204, KLF6 is a direct target of miR-211 and miR-204. Furthermore, circMTO1 and KLF6 overexpression rescued the suppression of miR-211/204 in RCC cell proliferation. In short, circMTO1 repressed RCC progression by regulating KLF6 via sponging miR-211 and miR-204, which may provide new idea of diagnosis and treatment in renal cell carcinoma.

     

Low expression of PDK1 inhibits renal cell carcinoma cell proliferation,migration, invasion and epithelial mesenchymal transition through inhibition of the PI3K-PDK1-Akt pathway

As the most commonly occurring form of primary renal tumor, renal cell carcinoma (RCC) is a malignancy accompanied by a high mortality rate. 3-phosphoinositide-dependent protein kinase 1 (PDK1) has been established as a protein target and generated considerable interest in both the pharmaceutical and academia industry. The aim of the current study was to investigate the effect of si-PDK1 on the RCC cell apoptosis, proliferation, migration, invasion and epithelial mesenchymal transition (EMT) in connection with the PI3K-PDK1-Akt pathway. Microarray analysis from the GEO database was adopted to identify differentially expressed genes (DEGs) related to RCC, after which the positive expression of the PDK1 protein in tissue was determined accordingly. The optimal silencing si-RNA was subsequently selected and RCC cell lines 786-O and A498 were selected and transfected with either a si-PDK1 or activator of the PI3K-PDK1-Akt pathway for grouping purposes. The mRNA and protein expressions of PDK1, the PI3K-PDK1-Akt pathway-, EMT- and apoptosis-related genes were then evaluated. The effect of si-PDK1 on cell proliferation, apoptosis, invasion and migration was then analyzed. Through microarray analysis of GSE6344, GSE53757, GSE14762 and GSE781, PDK1 was examined. PDK1 was determined to be highly expressed in RCC tissues. Si-PDK1 exhibited marked reductions in relation to the mRNA and protein expression of PDK1, PI3K, AKT as well as Vimentin while elevated mRNA and protein expressions of E-cadherin were detected, which ultimately suggested that cell migration, proliferation and invasion had been inhibited coupled with enhanced levels of cell apoptosis. While a notable observation was made highlighting that the PI3K-PDK1-Akt pathway antagonized the effect of PDK1 silencing. Taken together, the key observations of this study provide evidence suggesting that high expressions of PDK1 are found in RCC, while highlighting that silencing PDK1 could inhibit RCC cell proliferation, migration, invasion and EMT by repressing the PI3K-PDK1-Akt pathway.     

Circ_FBLN1 promotes the proliferation and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells by regulating let-7i-5p/FZD4 axis and Wnt/β-catenin pathway

Background

Recently, more and more circular RNAs (circRNAs) have been identified in osteogenesis. In this study, we aimed to explore the effect of circ_FBLN1 on the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs).

Methods

The protein levels of osteogenesis-related genes, let-7i-5p, frizzled class receptor 4 (FZD4), Ki67, Wnt6 and β-catenin were measured by western blot assay. The levels of circ_FBLN1, FBLN1 mRNA and FZD4 mRNA were determined by quantitative real-time polymerase chain reaction (qRT-PCR) assay. The feature of circ_FBLN1 was investigated by RNase R and Actinomycin D assays. Cell proliferation ability was evaluated by colony formation assay and 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The targeting relationship between let-7i-5p and circ_FBLN1 or FZD4 was verified by dual-luciferase reporter assay.

Results

Circ_FBLN1 level was enhanced during the osteogenic differentiation of hBMSCs. Silencing of circ_FBLN1 repressed cell proliferation and osteogenic differentiation in hBMSCs. For mechanism analysis, circ_FBLN1 was found to act as a sponge for let-7i-5p and FZD4 served as a direct target gene of let-7i-5p. Let-7i-5p was downregulated during the osteogenic differentiation of hBMSCs and let-7i-5p inhibition restored the effects of circ_FBLN1 knockdown on the proliferation and osteogenesis of hBMSCs. Moreover, let-7i-5p overexpression suppressed cell proliferation and osteogenesis in hBMSCs through targeting FZD4. In addition, circ_FBLN1 knockdown reduced the levels of Wnt6 and β-catenin in hBMSCs, indicating the inactivation of Wnt/β-catenin pathway.

Conclusion

Knockdown of circ_FBLN1 inhibited the proliferation and osteogenesis of hBMSCs by regulating let-7i-5p/FZD4 axis and repressing Wnt/β-catenin pathway.

     

A polymorphism in the splice donor site of ZNF419 results in the novel renal cell carcinoma-associated minor histocompatibility antigen ZAPHIR

Nonmyeloablative allogeneic stem cell transplantation (SCT) can induce remission in patients with renal cell carcinoma (RCC), but this graft-versus-tumor (GVT) effect is often accompanied by graft-versus-host disease (GVHD). Here, we evaluated minor histocompatibility antigen (MiHA)-specific T cell responses in two patients with metastatic RCC who were treated with reduced-intensity conditioning SCT followed by donor lymphocyte infusion (DLI). One patient had stable disease and emergence of SMCY.A2-specific CD8+ T cells was observed after DLI with the potential of targeting SMCY-expressing RCC tumor cells. The second patient experienced partial regression of lung metastases from whom we isolated a MiHA-specific CTL clone with the capability of targeting RCC cell lines. Whole genome association scanning revealed that this CTL recognizes a novel HLA-B7-restricted MiHA, designated ZAPHIR, resulting from a polymorphism in the splice donor site of the ZNF419 gene. Tetramer analysis showed that emergence of ZAPHIR-specific CD8+ T cells in peripheral blood occurred in the absence of GVHD. Furthermore, the expression of ZAPHIR in solid tumor cell lines indicates the involvement of ZAPHIR-specific CD8+ T cell responses in selective GVT immunity. These findings illustrate that the ZNF419-encoded MiHA ZAPHIR is an attractive target for specific immunotherapy after allogeneic SCT.