Establishment and Validation of a Four-stress Granule-related Gene Signature in Hepatocellular Carcinoma

Background and Aims:Stress granules(SGs)as membrane-less cytoplasmic foci formed in response to unfavorable external stimuli could promote cancer cells to adapt to hostile environments.Hepatocellular carcinoma(HCC)is prone to be highly aggressive once diagnosed,which markedly reduces patient survival time.Therefore,it is crucial to develop valid diagnostic markers to prognosticate HCC patient prognosis,which promotes individualized precision therapeutics in HCC.Considering the pro-tumorigenic activity of SGs,it is of great potential value to construct a prognostic tool for HCC based on the expression profiles of SG-related genes(SGGs).Methods:Bioinformatic analysis was employed to establish an SGG-based prognostic signature.Western blotting and realtime polymerase chain reaction assays were used to assess the expression patterns of the related SGGs.Loss-of-function experiments were performed to analyze the effect of the SGGs on SG formation and cell survival.Results:A four-SGG signature(KPNA2,MEX3A,WDR62,and SFN)targeting HCC was established and validated to exhibit a robust performance in predicting HCC prognosis.Consistently,all four genes were further found to be highly expressed in human HCC tissues.More important,we demonstrated that individually knocking down the four SGGs significantly reduced HCC cell proliferation and metastasis by compromising the SG formation process.Conclusions:We developed an SGG-based predictive signature that can be used as an independent prognostic tool for HCC.The strong predictive power of this signature was further elucidated by the carcinogenic activity of KPNA2,MEX3A,WDR62,and SFN in HCC cells by regulating SG formation.

R158Q and G212S,novel pathogenic compound heterozygous variants in SLC12A3 of Gitelman syndrome

The dysfunction of Na^(+)-Cl^(−)cotransporter(NCC)caused by mutations in solute carrier family12,member 3 gene(SLC12A3)primarily causes Gitelman syndrome(GS).In identifying the pathogenicity of R158Q and G212S variants of SLC12A3,we evaluated the pathogenicity by bioinformatic,expression,and localization analysis of two variants from a patient in our cohort.The prediction of mutant protein showed that p.R158Q and p.G212S could alter protein’s three-dimensional structure.Western blot showed a decrease of mutant Ncc.Immunofluorescence of the two mutations revealed a diffuse positive staining below the plasma membrane.Meanwhile,we conducted a compound heterozygous model—Ncc^(R156Q/G210S)mice corresponding to human NCC R158Q/G212S.Ncc^(R156Q/G210S)mice clearly exhibited typical GS features,including hypokalemia,hypomagnesemia,and increased fractional excretion of K^(+)and Mg^(2+)with a normal blood pressure level,which made Ncc^(R156Q/G210S)mice an optimal mouse model for further study of GS.A dramatic decrease and abnormal localization of the mutant Ncc in distal convoluted tubules contributed to the phenotype.The hydrochlorothiazide test showed a loss of function of mutant Ncc in Ncc^(R156Q/G210S)mice.These findings indicated that R158Q and G212S variants of SLC12A3 were pathogenic variants of GS.

Cyclophilin D as a potential therapeutic target of liver ischemia/reperfusion injury by mediating crosstalk between apoptosis and autophagy

Background:Liver ischemia/reperfusion(I/R)injury is a complex and multifactorial pathophysiological process.It is well recognized that the membrane permeability transition pore(mPTP)opening of mitochondria plays a crucial role in cell death after I/R injury.Cyclophilin D(CypD)is a critical positive regulator of mPTP.However,the effect of CypD on the pathogenesis of liver I/R injury and whether CypD is a potential therapeutic target are still unclear.Methods:We constructed liver-specific CypD knockout and AAV8-peptidyl prolyl isomerase F(PPIF)overexpression mice.Then,a 70%liver I/R injury model was established in mice,with 90 min of ischemia and 6 h of reperfusion.The liver function was detected by the level of serum glutamic pyruvic transaminase(alanine transaminase)and glutamic oxaloacetic transaminase(aspartate aminotransferase),the liver damage score and degree of necrosis were measured by hematoxylin and eosin(H&E)staining of liver tissues.Reactive oxygen species(ROS)staining,apoptosis,and autophagy-related molecules were used to detect apoptosis and autophagy during liver I/R.Results:The liver-specific knockout of CypD alleviated necrosis and dysfunction in liver I/R injury,by reducing the excessive production of ROS,and inhibiting cell apoptosis and autophagy.On the contrary,overexpression of CypD exacerbated I/R-induced liver damage.Conclusion:We found that the downregulation of CypD expression alleviated liver I/R injury by reducing apoptosis and autophagy through caspase-3/Beclin1 crosstalk;in contrast,the upregulation of CypD expression aggravated liver I/R injury.Therefore,interfering with the expression of CypD seems to be a promising treatment for liver I/R injury.

Exosomes loaded a smart bilayer-hydrogel scaffold with ROS-scavenging and macrophage-reprogramming properties for repairing cartilage defect

Enhancing the regeneration of cartilage defects remains challenging owing to limited innate self-healing as well as acute inflammation arising from the overexpression of reactive oxygen species(ROS)in post-traumatic microenvironments.Recently,stem cell-derived exosomes(Exos)have been developed as potential cell-free therapy for cartilage regeneration.Although this approach promotes chondrogenesis,it neglects the emerging inflammatory microenvironment.In this study,a smart bilayer-hydrogel dual-loaded with sodium diclofenac(DC),an anti-inflammatory drug,and Exos from bone marrow-derived mesenchymal stem cells was developed to mitigate initial-stage inflammation and promote late-stage stem-cell recruitment and chondrogenic differentiation.First,the upper-hydrogel composed of phenylboronic-acid-crosslinked polyvinyl alcohol degrades in response to elevated levels of ROS to release DC,which mitigates oxidative stress,thus reprogramming macrophages to the pro-healing state.Subsequently,Exos are slowly released from the lower-hydrogel composed of hyaluronic acid into an optimal microenvironment for the stimulation of chondrogenesis.Both in vitro and in vivo assays confirmed that the dual-loaded bilayer-hydrogel reduced post-traumatic inflammation and enhanced cartilage regeneration by effectively scavenging ROS and reprogramming macrophages.The proposed platform provides multi-staged therapy,which allows for the optimal harnessing of Exos as a therapeutic for cartilage regeneration.