Tielin Chen

FLNA and PGK1 are Two Potential Markers for Progression in Hepatocellular Carcinoma

Background/Aims: Hepatocellular carcinoma (HCC) is one of the most deadly diseases; metastasis and recurrence are the most important factors that affect the therapy of HCC chronically. Until now, the prognosis for the metastasis of HCC had not improved. Recently, several proteins that are related to metastasis and invasion of HCC were identified, but the effective markers still remain to be elucidated. Methods: In this study, comparative proteomics was used to study the differentially expressed proteins in two HCC cell lines MHCC97L and HCCLM9, which have low and high metastatic potentials, respectively. Results: Our findings indicated that filamin A (FLNA) and phosphoglycerate kinase 1 (PGK1) were two significantly differentially expressed proteins, with high expression in HCCLM9 cells, and may influence the metastasis of HCC cells. Conclusion: Taken together with the confirmation of expression on the mRNA level, we propose the use of FLNA and PGK1 as potential markers for the progression of HCC.

Overexpression of FoxO1 Causes Proliferation of Cultured Pancreatic β Cells Exposed to Low Nutrition

Multiple lines of evidence have shown that the functional defect of pancreatic beta cells is the root cause of type 2 diabetes. FoxO1, a key transcription factor of fundamental cellular physiology and functions, has been implicated in this process. However, the underlying molecular mechanism is still largely unknown. Here, we show that the overexpression of FoxO1 promotes the proliferation of cultured pancreatic beta cells exposed to low nutrition, while no change in apoptosis was observed compared with the control group. Moreover, by using two specific inhibitors for PI3K and MAPK signaling, we found that FoxO1 might be the downstream transcription factor of these two pathways. Furthermore, a luciferase assay demonstrated that FoxO1 could regulate the expression of Ccnd1 at the transcription level. Collectively, our findings indicated that FoxO1 modulated by both MAPK and PI3K signaling pathways was prone to cause the proliferation, but not the apoptosis, of pancreatic beta cells exposed to low nutrition, at least partially, by regulating the expression of Ccnd1 at the transcription level.

A Modified TALEN-Based Strategy for Rapidly and Efficiently Generating Knockout Mice for Kidney Development Studies

The transcription activator-like effector nucleases (TALENs) strategy has been widely used to delete and mutate genes in vitro. This strategy has begun to be used for in vivo systemic gene manipulation, but not in an organ-specific manner. In this study, we developed a modified, highly efficient TALEN strategy using a dual-fluorescence reporter. We used this modified strategy and, within 5 weeks, we successfully generated kidney proximal tubule-specific gene Ttc36 homozygous knockout mice. Unilateral nephrectomy was performed on the 6-week-old founders (F0) to identify the knockout genotype prior to the birth of the offspring. This strategy was found to have little effect on reproduction in the knockout mice and inheritability of the knockout genotypes. The modified TALEN knockout strategy in combination with unilateral nephrectomy can be readily used for studies of gene function in kidney development and diseases.

The miR-17-92 cluster regulates FOG-2 expression and inhibits proliferation of mouse embryonic cardiomyocytes

MicroRNAs (miRNAs) have gradually been recognized as regulators of embryonic development; however, relatively few miRNAs have been identified that regulate cardiac development. A series of recent papers have established an essential role for the miRNA-17-92 (miR-17-92) cluster of miRNAs in the development of the heart. Previous research has shown that the Friend of Gata-2 (FOG-2) is critical for cardiac development. To investigate the possibility that the miR-17-92 cluster regulates FOG-2 expression and inhibits proliferation in mouse embryonic cardiomyocytes we initially used bioinformatics to analyze 3′ untranslated regions (3′UTR) of FOG-2 to predict the potential of miR-17-92 to target it. We used luciferase assays to demonstrate that miR-17-5p and miR-20a of miR-17-92 interact with the predicted target sites in the 3′UTR of FOG-2. Furthermore, RT-PCR and Western blot were used to demonstrate the post-transcriptional regulation of FOG-2 by miR-17-92 in embryonic cardiomyocytes from E12.5-day pregnant C57BL/6J mice. Finally, EdU cell assays together with the FOG-2 rescue strategy were employed to evaluate the effect of proliferation on embryonic cardiomyocytes. We first found that the miR-17-5p and miR-20a of miR-17-92 directly target the 3′UTR of FOG-2 and post-transcriptionally repress the expression of FOG-2. Moreover, our findings demonstrated that over-expression of miR-17-92 may inhibit cell proliferation via post-transcriptional repression of FOG-2 in embryonic cardiomyocytes. These results indicate that the miR-17-92 cluster regulates the expression of FOG-2 protein and suggest that the miR-17-92 cluster might play an important role in heart development.

Fibroblast growth factor 21 enhances cholesterol efflux in THP-1 macrophage-derived foam cells.

Fibroblast growth factor 21 (FGF21) is a novel metabolic regulator. The present study aimed to investigate the effect of FGF21 on cholesterol efflux and the expression of ATP binding cassette (ABC) A1 and G1 in human THP-1 macrophage-derived foam cells. Furthermore, the present study aimed to investigate the role of the liver X receptor (LXR) α in this process. A model of oxidized low-density lipoprotein-induced foam cells from human THP-1 cells was established. The effect of FGF21 on cholesterol efflux was analyzed using a liquid scintillation counter. The expression of ABCA1 and ABCG1 was determined using quantitative polymerase chain reaction and western blot analyses. FGF21 was found to enhance apolipoprotein A1- and high-density lipoprotein-mediated cholesterol efflux. FGF21 was also observed to increase the mRNA and protein expression of ABCA1 and ABCG1. Furthermore, LXRα-short interfering RNA attenuated the stimulatory effects induced by FGF21. These findings suggest that FGF21 may have a protective effect against atherosclerosis by enhancing cholesterol efflux through the induction of LXRα-dependent ABCA1 and ABCG1 expression.

Overexpression of Trpp5 contributes to cell proliferation and apoptosis probably through involving calcium homeostasis

Trpp5 is one member of the polycystic kidney disease (PKD) family, which belongs to transient receptor potential (TRP) superfamily. Our previous study has shown that Trpp5 is developmentally expressed in mouse testis and overexpression of Trpp5 increases intracellular free calcium concentration in MDCK cells. However, the roles of this protein in cellular processes are largely unknown. Here, we demonstrated that Trpp5 resided in both cytoplasm and cell membrane of HEK293 cells. We found that overexpression of Trpp5 slightly increased the calcium current amplitude of HEK293 cells and shifted the reversal potential to a more negative value. Meanwhile, overexpression of Trpp5 suppressed proliferation of Hela cells via inhibiting DNA replication and induced apoptosis of Hela cells with morphological changes and accumulation of fragmented DNA. Collectively, these findings suggest that Trpp5 might involve calcium homeostasis contributing to cell proliferation and apoptosis.

Down-regulated Six2 by knockdown of neurofibromin results in apoptosis of metanephric mesenchyme cells in vitro

Embryonic Six2-positive nephron progenitor cells adjacent to ureteric bud tips ultimately give rise to nephron structures, including proximal and distal tubules, podocytes, Bowman’s capsules, and the glomeruli. This process requires an internal balance between self-renew and differentiation of the nephron progenitor cells, which is mediated by numerous molecules. Recent studies have shown that the neurofibromin (Nf1) null mutant mouse embryos have an 18- to 24-h developmental delay in metanephros manifesting retardation in its cephalad repositioning and reduction number of glomeruli. However, the underlying inter-/intracellular signaling mechanisms responsible for reducing number of glomeruli during nephrogenesis remain to be fully elucidated. Here, we originally detected the Nf1 expression in developing kidney and metanephric mesenchyme cells. Surprisingly, Nf1 knockdown by small interfering RNAs in the metanephric mesenchyme cells (mK3) resulted in a decreased expression of Six2, the key marker of renal progenitor cells, while the ratio of apoptotic cells was significantly increased. Furthermore, overexpression of Six2 in mk3 cells partially rescued apoptosis phenotype. Collectively, these results implied that knockdown of Nf1 resulted in apoptosis of mK3 cells in vitro probably through down-regulation of Six2 expression. Collectively, we demonstrated that down-regulated Six2 by knockdown of Nf1 resulted in apoptosis of mK3 cells in vitro. These results implied that inhibition of Nf1 may delay metanephros development via down-regulation of Six2.

Comparative proteomic analysis suggests that mitochondria are involved in autosomal recessive polycystic kidney disease

Autosomal recessive polycystic kidney disease (ARPKD), characterized by ectatic collecting duct, is an infantile form of PKD occurring in 1 in 20 000 births. Despite having been studied for many years, little is known about the underlying mechanisms. In the current study, we employed, for the first time, a MS‐based comparative proteomics approach to investigate the differently expressed proteins between kidney tissue samples of four ARPKD and five control individuals. Thirty two differently expressed proteins were identified and six of the identified protein encoding genes performed on an independent group (three ARPKD subjects, four control subjects) were verified by semi‐quantitative RT‐PCR, and part of them were further validated by Western blot and immunohistochemistry. Moreover, similar alteration tendency was detected after downregulation of PKHD1 by small interfering RNA in HEK293T cell. Interestingly, most of the identified proteins are associated with mitochondria. This implies that mitochondria may be implicated in ARPKD. Furthermore, the String software was utilized to investigate the biological association network, which is based on known and predicted protein interactions. In conclusion, our findings depicted a global understanding of ARPKD progression and provided a promising resource of targeting protein, and shed some light further investigation of ARPKD.

Highly Effective Ex Vivo Gene Manipulation to Study Kidney Development Using Self-Complementary Adenoassociated Viruses

Background. Ex vivo culture of intact embryonic kidney has become a powerful system for studying renal development. However, few methods have been available for gene manipulation and have impeded the identification and investigation of genes in this developmental process. Results. Here we systemically compared eight different serotypes of pseudotyped self-complementary adenoassociated viruses (scAAVs) transduction in cultured embryonic kidney with a modified culture procedure. We demonstrated that scAAV was highly effective in delivering genes into and expressing in compacted tissues. scAAV serotypes 2 and 8 exhibited higher efficiency of transduction compared to others. Expression kinetics assay revealed that scAAV can be used for gene manipulation at the study of UB branching and nephrogenesis. Repressing WT1 in cultured kidney using shRNA impairs tubule formation. We for the first time employed and validated scAAV as a gene delivery tool in cultured kidney. Conclusions. These findings are expected to expedite the use of the ex vivo embryonic kidney cultures for kidney development research. For other ex vivo cultured organ models, scAAV could also be a promising tool for organogenesis study.

Comparative proteomics and correlated signaling network of kidney in ApoE deficient mouse

Apolipoprotein E knockout (apoE−/−) mouse is one of the most popular models for cardiovascular research, especially in the study of atherosclerosis. Naturally, large amount of studies try to uncover the role of apoE in atherosclerosis, and indeed apoE plays an important role in this pathogenesis. Kidney is an organ that contains lots of capillaries and also largely expresses apoE. Moreover, a protective role of apoE in kidney as an autocrine regulator has been demonstrated previously, however, the underlying mechanism is largely unknown.