Diverse Roles and Targets of miRNA in the Pathogenesis of Testicular Germ Cell Tumour.

Testicular germ cell tumour (TGCT) is the most common cancer type among young adults in many parts of the world. Although the pathogenesis of TGCT is not well understood, the involvement of heritable components is evident, and the risk is polygenic. Genome-wide association studies have so far found 78 susceptibility loci for TGCT, and many of the loci are in non-coding regions indicating the involvement of non-coding RNAs in TGCT pathogenesis. MicroRNAs (miRNAs), a class of non-coding RNAs, have emerged as important gene regulators at the post-transcriptional level. They are crucial in controlling many cellular processes, such as proliferation, differentiation, and apoptosis, and an aberrant miRNA expression may contribute to the pathogenesis of several cancers, including TGCT. In support of this notion, several studies reported differential expression of miRNAs in TGCTs. We previously demonstrated that miRNAs were the most common group of small non-coding RNAs in TGCTs, and several functional studies of miRNAs in TGCTs suggest that they may act as either oncogene or tumour suppressors. Moreover, individual miRNA targets and downstream pathways in the context of TGCT development have been explored. In this review, we will focus on the diverse roles and targets of miRNAs in TGCT pathogenesis.

Transcriptomic and functional studies reveal miR-431-5p as a tumour suppressor in pancreatic ductal adenocarcinoma cells.

The lactate receptor HCAR1 (hydroxycarboxylic acid receptor 1) is highly expressed in pancreatic ductal adenocarcinoma (PDAC), where it regulates lactate transport between the cancer cells. Little is known about the underlying cause of high HCAR1 expression in PDAC, and in the present study, we investigated whether HCAR1 could be a target of miRNA regulation. By searching for predicted miRNA candidates in silico, we identified miR-431-5p as a possible regulator of HCAR1. We found miR-431-5p to repress HCAR1 expression through direct binding to the 3' UTR. The endogenous expression of miR-431-5p and HCAR1 was found to be negatively related in the PDAC cell lines BxPC-3, Capan-2, and PANC-1. Overexpression of miR-431-5p inhibited cell proliferation in all the cell lines, and a shift in cell cycle progression and induction of apoptosis were found in the BxPC-3 cells. Transcriptomic analysis of mRNA from BxPC-3 cells revealed numerous differentially expressed genes (DEGs), including HCAR1, in response to miR-431-5p overexpression. A significant proportion of these DEGs was enriched in cancer-related processes and signalling pathways. Among the most significantly repressed DEGs was ATP6V0E1, encoding the integral subunit e of vacuolar ATPase (V-ATPase), an enzyme that is important for cancer cell survival. Small interfering RNA (siRNA)-mediated knockdown of HCAR1 and ATP6V0E1 showed that only knockdown of ATP6V0E1 mimicked the effect of miR-431-5p overexpression on cell viability. Our findings indicate that miR-431-5p acts as a tumour suppressor in PDAC cells, with BxPC-3 cells being most responsive.

L-lactate induces neurogenesis in the mouse ventricular-subventricular zone via the lactate receptor HCA1.

AIM: Adult neurogenesis occurs in two major niches in the brain: the subgranular zone of the hippocampal formation and the ventricular-subventricular zone. Neurogenesis in both niches is reduced in ageing and neurological disease involving dementia. Exercise can rescue memory by enhancing hippocampal neurogenesis, but whether exercise affects adult neurogenesis in the ventricular-subventricular zone remains unresolved. Previously, we reported that exercise induces angiogenesis through activation of the lactate receptor HCA1. The aim of the present study is to investigate HCA1 -dependent effects on neurogenesis in the two main neurogenic niches. METHODS: Wild-type and HCA1 knock-out mice received high intensity interval exercise, subcutaneous injections of L-lactate, or saline injections, five days per week for seven weeks. Well-established markers for proliferating cells (Ki-67) and immature neurons (doublecortin), were used to investigate neurogenesis in the subgranular zone and the ventricular-subventricular zone. RESULTS: We demonstrated that neurogenesis in the ventricular-subventricular zone is enhanced by HCA1 activation: Treatment with exercise or lactate resulted in increased neurogenesis in wild-type, but not in HCA1 knock-out mice. In the subgranular zone, neurogenesis was induced by exercise in both genotypes, but unaffected by lactate treatment. CONCLUSION: Our study demonstrates that neurogenesis in the two main neurogenic niches in the brain is regulated differently: Neurogenesis in both niches was induced by exercise, but only in the ventricular-subventricular zone was neurogenesis induced by lactate through HCA1 activation. This opens for a role of HCA1 in the physiological control of neurogenesis, and potentially in counteracting age-related cognitive decline.

Blood lactate dynamics in awake and anaesthetized mice after intraperitoneal and subcutaneous injections of lactate-sex matters.

Lactate treatment has shown a therapeutic potential for several neurological diseases, including Alzheimer's disease. In order to optimize the administration of lactate for studies in mouse models, we compared blood lactate dynamics after intraperitoneal (IP) and subcutaneous (SC) injections. We used the 5xFAD mouse model for familial Alzheimer's disease and performed the experiments in both awake and anaesthetized mice. Blood glucose was used as an indication of the hepatic conversion of lactate. In awake mice, both injection routes resulted in high blood lactate levels, mimicking levels reached during high-intensity training. In anaesthetized mice, SC injections resulted in significantly lower lactate levels compared to IP injections. Interestingly, we observed that awake males had significantly higher lactate levels than awake females, while the opposite sex difference was observed during anaesthesia. We did not find any significant difference between transgenic and wild-type mice and therefore believe that our results can be generalized to other mouse models. These results should be considered when planning experiments using lactate treatment in mice.

Knockdown of SPRY4 and SPRY4-IT1 inhibits cell growth and phosphorylation of Akt in human testicular germ cell tumours.

Testicular germ cell tumour (TGCT) is the most common cancer in young men in large parts of the world, but the aetiology is mainly unknown. Genome-wide association studies have so far identified about 50 susceptibility loci associated with TGCT, including SPRY4. SPRY4 has shown tumour suppressor activity in several cancer cells, such as lung and prostate, while it was found to act as an oncogene in ovarian cancer. An intronic region within the SPRY4 gene produces a long non-coding RNA, SPRY4-IT1, which has been reported to act as an oncogene in melanoma, breast cancer, and colorectal cancer, and as a tumour suppressor in lung cancer. The roles of SPRY4 and SPRY4-IT1 in TGCT development are yet unknown. We found higher expression levels of SPRY4, both mRNA and protein, and of SPRY4-IT1 in human TGCT than in normal adult testis. Small-interfering RNA (siRNA)-mediated transient knockdown of SPRY4 and SPRY4-IT1 in two TGCT cell lines 833 K and NT2-D1 resulted in decreased cell growth, migration, and invasion. Knockdown of SPRY4 and SPRY4-IT1 also led to a significant reduction in the phosphorylation of Akt. Our findings indicate that SPRY4 and SPRY4-IT1 may act as oncogenes in TGCTs via activation of the PI3K / Akt signalling pathway.

Exercise induces cerebral VEGF and angiogenesis via the lactate receptor HCAR1.

Physical exercise can improve brain function and delay neurodegeneration; however, the initial signal from muscle to brain is unknown. Here we show that the lactate receptor (HCAR1) is highly enriched in pial fibroblast-like cells that line the vessels supplying blood to the brain, and in pericyte-like cells along intracerebral microvessels. Activation of HCAR1 enhances cerebral vascular endothelial growth factor A (VEGFA) and cerebral angiogenesis. High-intensity interval exercise (5 days weekly for 7 weeks), as well as L-lactate subcutaneous injection that leads to an increase in blood lactate levels similar to exercise, increases brain VEGFA protein and capillary density in wild-type mice, but not in knockout mice lacking HCAR1. In contrast, skeletal muscle shows no vascular HCAR1 expression and no HCAR1-dependent change in vascularization induced by exercise or lactate. Thus, we demonstrate that a substance released by exercising skeletal muscle induces supportive effects in brain through an identified receptor.

Impaired dynamics and function of mitochondria caused by mtDNA toxicity leads to heart failure.

Cardiac mitochondrial dysfunction has been implicated in heart failure of diverse etiologies. Generalized mitochondrial disease also leads to cardiomyopathy with various clinical manifestations. Impaired mitochondrial homeostasis may over time, such as in the aging heart, lead to cardiac dysfunction. Mitochondrial DNA (mtDNA), close to the electron transport chain and unprotected by histones, may be a primary pathogenetic site, but this is not known. Here, we test the hypothesis that cumulative damage of cardiomyocyte mtDNA leads to cardiomyopathy and heart failure. Transgenic mice with Tet-on inducible, cardiomyocyte-specific expression of a mutant uracil-DNA glycosylase 1 (mutUNG1) were generated. The mutUNG1 is known to remove thymine in addition to uracil from the mitochondrial genome, generating apyrimidinic sites, which obstruct mtDNA function. Following induction of mutUNG1 in cardiac myocytes by administering doxycycline, the mice developed hypertrophic cardiomyopathy, leading to congestive heart failure and premature death after ∼2 mo. The heart showed reduced mtDNA replication, severely diminished mtDNA transcription, and suppressed mitochondrial respiration with increased Pgc-1α, mitochondrial mass, and antioxidative defense enzymes, and finally failing mitochondrial fission/fusion dynamics and deteriorating myocardial contractility as the mechanism of heart failure. The approach provides a model with induced cardiac-restricted mtDNA damage for investigation of mtDNA-based heart disease.