HSPA4 regulated glioma progression via activation of AKT signaling pathway.

Glioma is still an incurable disease with high invasiveness. Heat shock 70 kDa protein 4 (HSPA4) is a member of the HSP110 family, and is associated with the development and progression of various cancers. In the current study, we assessed the expression of HSPA4 in clinical samples, and found that HSPA4 was up-regulated in glioma tissues and correlated with tumor recurrence and grade. Survival analyses demonstrated that glioma patients with high HSPA4 expression had lower overall survival and disease-free survival times. In vitro knockdown of HSPA4 inhibited glioma cell proliferation, mediated cell cycle arrest at G2 phase and apoptosis, and reduced the migration ability. In vivo, the growth of HSPA4-knockdown xenografts was markedly suppressed compared to the tumors formed by HSPA4-positive control cells. Additionally, Gene set enrichment analyses disclosed that HSPA4 was associated with the PI3K/Akt signaling pathway. The regulatory effect of the AKT activator SC79 on cell proliferation and apoptosis was suppressed by HSPA4 knockdown, indicating that HSPA4 is capable of promoting glioma development. In summary, these data showed that HSPA4 is likely to play a pivotal role in the progression of glioma, and consequently may be a promising therapeutic target for glioma therapy.

HSPA4 Is a Biomarker of Placenta Accreta and Enhances the Angiogenesis Ability of Vessel Endothelial Cells.

Placenta accreta spectrum (PAS) accounts for 7% of maternal mortality and is associated with intraoperative and postoperative morbidity caused by massive blood loss, infection, and adjacent organ damage. The aims of this study were to identify the protein biomarkers of PAS and to further explore their pathogenetic roles in PAS. For this purpose, we collected five placentas from pregnant subjects with PAS complications and another five placentas from normal pregnancy (NP) cases. Then, we enriched protein samples by specifically isolating the trophoblast villous, deeply invading into the uterine muscle layer in the PAS patients. Next, fluorescence-based two-dimensional difference gel electrophoresis (2D-DIGE) and MALDI-TOF/MS were used to identify the proteins differentially abundant between PAS and NP placenta tissues. As a result, nineteen spots were determined as differentially abundant proteins, ten and nine of which were more abundant in PAS and NP placenta tissues, respectively. Then, specific validation with western blot assay and immunohisto/cytochemistry (IHC) assay confirmed that heat shock 70 kDa protein 4 (HSPA4) and chorionic somatomammotropin hormone (CSH) were PAS protein biomarkers. Further tube formation assays demonstrated that HSPA4 promoted the in vitro angiogenesis ability of vessel endothelial cells, which is consistent with the in vivo scenario of PAS complications. In this study, we not only identified PAS protein biomarkers but also connected the promoted angiogenesis with placenta invasion, investigating the pathogenetic mechanism of PAS.

Functional diversity between HSP70 paralogs caused by variable interactions with specific co-chaperones.

Heat shock protein 70 (HSP70) chaperones play a central role in protein quality control and are crucial for many cellular processes, including protein folding, degradation, and disaggregation. Human HSP70s compose a family of 13 members that carry out their functions with the aid of even larger families of co-chaperones. A delicate interplay between HSP70s and co-chaperone recruitment is thought to determine substrate fate, yet it has been generally assumed that all Hsp70 paralogs have similar activities and are largely functionally redundant. However, here we found that when expressed in human cells, two highly homologous HSP70s, HSPA1A and HSPA1L, have opposing effects on cellular handling of various substrates. For example, HSPA1A reduced aggregation of the amyotrophic lateral sclerosis-associated protein variant superoxide dismutase 1 (SOD1)-A4V, whereas HSPA1L enhanced its aggregation. Intriguingly, variations in the substrate-binding domain of these HSP70s did not play a role in this difference. Instead, we observed that substrate fate is determined by differential interactions of the HSP70s with co-chaperones. Whereas most co-chaperones bound equally well to these two HSP70s, Hsp70/Hsp90-organizing protein (HOP) preferentially bound to HSPA1L, and the Hsp110 nucleotide-exchange factor HSPH2 preferred HSPA1A. The role of HSPH2 was especially crucial for the HSPA1A-mediated reduction in SOD1-A4V aggregation. These findings reveal a remarkable functional diversity at the level of the cellular HSP70s and indicate that this diversity is defined by their affinities for specific co-chaperones such as HSPH2.

The expression profiles and prognostic values of HSP70s in hepatocellular carcinoma.

BACKGROUND: The HSP70 family of heat shock protein plays a critical role in protein synthesis and transport to maintain protein homeostasis. Several studies have indicated that HSP70s are related to the development and occurrence of various cancers. METHODS: The relationship between the overall survival rate of hepatocellular carcinoma patients and the expression of 14 HSP70s from multiple databases, such as TCGA, ONCOMINE, cBioPortal was investigated. Western Blot and PCR were used to evaluate HSPA4 and HSPA14 expressions in various HCC cells to identify suitable cell lines for further experiments .Wound-healing assays, Transwell assays and EdU assays were used to verify the effects of HSPA4 and HSPA14 on the function of hepatocellular carcinoma cells, and statistical analysis was performed. RESULTS: Hepatocellular carcinoma tissues significantly expressed the 14 HSP70s compared to the normal samples. Besides, the high HSPA1A, HSPA1B, HSPA4, HSPA5, HSPA8, HSPA13, and HSPA14 expressions were inversely associated with the overall survival rate of patients, tumor grade, and cancer stage. A PPI regulatory network was constructed using the 14 HSP70s proteins with HSPA5 and HSPA8 at the network center. Univariate and multivariate analyses showed that HSPA4 and HSPA14 could be independent risk factors for the prognosis of hepatocellular carcinoma patients. Cell experiments have also confirmed that reducing HSPA4 and HSPA14 expressions can inhibit the invasion, metastasis, and proliferation of hepatocellular carcinoma cells. CONCLUSIONS: Therefore, the HSP70s significantly influence the occurrence and development of hepatocellular carcinoma. For instance, HSPA4 and HSPA14 can be novel therapeutic targets and prognostic biomarkers for hepatocellular carcinoma.

miR-434 and miR-242 have a potential role in heat stress response in rainbow trout (Oncorhynchus mykiss).

MicroRNAs (miRNAs) are being extensively studied as they function as key metabolic regulators which play a role in the heat stress response. However, the role of miRNAs in heat stress remains uncertain and many new miRNAs have not yet been discovered. In a previous study, we performed high-throughput sequencing of differentially expressed miRNAs identified on exposing rainbow trout (Oncorhynchus mykiss) to heat stress (18 vs. 24°C), which led to the identification of two novel miRNAs, temporarily named novel miR-434 and -242. The differential expression level of these miRNAs was extremely significant (P < 0.01); we analysed target gene mRNA transcripts by bioinformatics software (miRanda). We found novel miR-434 and -242 were predicted to regulate the transcripts of heat shock 70-kDa protein 4-like (HSPA4L) and calreticulin (CRT), respectively, by bioinformatics software. Here our core objective was to validate if HSPA4L and CRT are indeed the target genes of novel miR-434 and -242, respectively, and for this purpose we used the dual-luciferase reporter assay system. Target gene sequences were synthesized and cloned into a dual-luciferase vector. To better understand the function of the target genes, we combined the previous sequencing results of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. We found that novel miR-434 regulated HSPA4L expression by binding to a putative binding site in the 3'-UTR of HSPA4L, and luciferase activity inhibition was observed. In contrast, novel miR-242 was not involved in regulating CRT expression. To conclude, we believe our results should serve as a foundation for future studies aiming to comprehensively understand the mechanisms used by rainbow trout to cope with heat stress.

Exome-wide Association Study Identifies GREB1L Mutations in Congenital Kidney Malformations.

Renal agenesis and hypodysplasia (RHD) are major causes of pediatric chronic kidney disease and are highly genetically heterogeneous. We conducted whole-exome sequencing in 202 case subjects with RHD and identified diagnostic mutations in genes known to be associated with RHD in 7/202 case subjects. In an additional affected individual with RHD and a congenital heart defect, we found a homozygous loss-of-function (LOF) variant in SLIT3, recapitulating phenotypes reported with Slit3 inactivation in the mouse. To identify genes associated with RHD, we performed an exome-wide association study with 195 unresolved case subjects and 6,905 control subjects. The top signal resided in GREB1L, a gene implicated previously in Hoxb1 and Shha signaling in zebrafish. The significance of the association, which was p = 2.0 × 10-5 for novel LOF, increased to p = 4.1 × 10-6 for LOF and deleterious missense variants combined, and augmented further after accounting for segregation and de novo inheritance of rare variants (joint p = 2.3 × 10-7). Finally, CRISPR/Cas9 disruption or knockdown of greb1l in zebrafish caused specific pronephric defects, which were rescued by wild-type human GREB1L mRNA, but not mRNA containing alleles identified in case subjects. Together, our study provides insight into the genetic landscape of kidney malformations in humans, presents multiple candidates, and identifies SLIT3 and GREB1L as genes implicated in the pathogenesis of RHD.

Decreased expression of heat shock protein A4L in spermatozoa is positively related to poor human sperm quality.

Heat shock protein A4L (HSPA4L), which is highly expressed in the testis, is correlated with male fertility. However, the relationship between HSPA4L expression and sperm quality remains unknown. In the present study, a systematic characterization of HSPA4L expression on spermatozoa was performed. HSPA4L is highly expressed in the human testis, characterized by abundant localization in testicular spermatocytes and round spermatids. Compared with the testis from young adults (aged 27-36 years old), downregulated expression of HSPA4L in the testis from elderly adults (aged 78-82 years old) was observed. Immunofluorescence quantification demonstrated the localization of HSPA4L in the middle piece of sperm. Compared with mature spermatozoa, a similar lower intensity and localization percentage of HSPA4L in immature and asthenozoospermic spermatozoa were observed, and the consistently decreased expression of HSPA4L in immature and asthenozoospermic spermatozoa was validated by western blot analysis. Functional analysis revealed a correlation between HSPA4L and sperm motility by Spearman correlation analysis and its involvement in sperm-oocyte penetration by the human sperm-hamster egg penetration test. The current study demonstrates that HSPA4L is a promising marker for the assessment of sperm quality and provides clues for exploring biomarkers for the molecular diagnosis and treatment of male infertility.

[Expression of miR-429 and Its Target Gene HSPA4L in Sperms from Asthenospermia Patients.]

OBJECTIVES: To investigate the expression of miR-429 and its target gene heat shock protein A4L (HSPA4L) in sperms from asthenospermia patients. METHODS: Twenty semen samples from healthy and fertile adults and 20 semen samples from asthenospermia patients were collected,and normal sperm parameters were defined according to World Health Organization criteria.The expression levels of miR-429 and HSPA4L mRNA were determined by qRT-PCR,and the bioinformatics tool (Targetscan) was used to predict the target of miR-429.Luciferase reporter assay and transfection study were performed to confirm target gene of miR-429.The expression levels of HSPA4L mRNA and protein were further determined by qRT-CPR and Western blot,respectively. RESULTS: The motility and viability of sperms from asthenospermia patients were lower than that in control group,and miR-429 was up-regulated in sperms from asthenospermia patients.Bioinformatics analysis revealed that HSPA4L was a target of miR-429.Luciferase reporter assay and transfection study further confirmed that miR-429 suppresses the expressions of HSPA4L mRNA and protein via directly targeting HSPA4L 3'UTR.Results from clinical samples also demonstrated that HSPA4L mRNA and protein were down-regulated in sperms from asthenospermia patients and the expression level of miR-429 was inversely correlated with the expression level of HSPA4L mRNA (r=-0.725, P<0.05). CONCLUSIONS: miR-429 is up-regulated in sperms from asthenospermia patients,and it may modulate the motility and viability of sperms via suppressing the expression of HSPA4L.

Upregulation of heat shock proteins (HSPA12A, HSP90B1, HSPA4, HSPA5 and HSPA6) in tumour tissues is associated with poor outcomes from HBV-related early-stage hepatocellular carcinoma.

BACKGROUND: Heat shock proteins (HSPs) are overexpressed in human hepatocellular carcinoma (HCC) tissue and correlate with aggressiveness and prognosis of HCC. METHODS: Using the GSE14520 microarray expression profile from Gene Expression Omnibus, we compared HSP gene expression between tumour and non-tumour tissues and correlated this with outcomes in HCC patients. RESULTS: We analysed 220 hepatitis B virus (HBV)-related HCC patients and 25 HSPs in this study. With the exception of HSPA4L, HSPA12A and HSPB8, members of the HSP family, including HSPH1, HSPBP1, HSPA1A, HSPA1B, HSPA1L, HSPA2, HSPA4, HSPA5, HSPA8, HSPA9, HSPAA1, HSPAB1, HSPA14, HSPB11, HSPA13, HSP90B1 and HSPBAP1, were all overexpressed in tumour tissues (all P < 0.001). In contrast, HSPB6, HSPB7, HSPA6, HSPB2 and HSPB3 were upregulated in non-tumour tissues (all P < 0.001). Multivariate analysis showed that cirrhosis (HR = 5.282, 95% CI = 1.294-21.555, P = 0.02), Barcelona Clinic liver cancer (BCLC) staging (HR = 2.151, 95% CI = 1.682-2.750, P < 0.001), HSPA12A (HR = 1.042, 95% CI = 1.003-1.082, P = 0.033) and HSP90B1 (HR = 1.001, 95% CI = 1.000-1.001, P = 0.011) were negatively associated with survival of HBV-related HCC patients. Furthermore, advanced BCLC staging (HR = 1.797, 95% CI = 1.439-2.244, P < 0.001) was also associated with earlier recurrence of HCC. The high expression of HSPA4 (HR = 1.002, 95% CI = 1.000-1.004, P = 0.019), HSPA5 (HR = 1.0, 95% CI = 1.0-1.0, P = 0.046) and HSPA6 (HR = 1.008, 95% CI = 1.001-1.015, P = 0.021) was similarly associated with HCC recurrence. CONCLUSIONS: The expression of most HSPs was higher in tumour tissues than in non-tumour tissues. High BCLC staging scores, advanced cirrhosis and the overexpression of HSPA12A and HSP90B1 might be associated with poor survival from HCC, whereas high levels of HSPA4, HSPA5 and HSPA6 might be associated with earlier recurrence of HCC.

Unveiling the potential of HSPA4: a comprehensive pan-cancer analysis of HSPA4 in diagnosis, prognosis, and immunotherapy.

With the global rise in cancer incidence and mortality rates, research on the topic has become increasingly urgent. Among the significant players in this field are heat shock proteins (HSPs), particularly HSPA4 from the HSP70 subfamily, which has recently garnered considerable interest for its role in cancer progression. However, despite numerous studies on HSPA4 in specific cancer types, a comprehensive analysis across all cancer types is lacking. This study employs various bioinformatics techniques to delve into the role of HSPA4 in pan-cancer. Our objective is to assess its potential in clinical diagnosis, prognosis, and as a future molecular target for therapy. The research findings reveal significant differences in HSPA4 expression across different cancer types, suggesting its diagnostic value and close association with cancer staging and patient survival rates. Furthermore, genetic variations and methylation status of HSPA4 play critical roles in tumorigenesis. Lastly, the interaction of HSPA4 with immune cells is linked to the tumor microenvironment (TME) and immunotherapy. In summary, HSPA4 emerges as a promising cancer biomarker and a vital member of the HSPs family, holding potential applications in diagnosis, prognosis, and immunotherapy.

HSPA4 upregulation induces immune evasion via ALKBH5/CD58 axis in gastric cancer.

INTRODUCTION: Gastric cancer (GC) is one of the leading causes of cancer-related death worldwide. Recently, targeted therapies including PD1 (programmed cell death 1) antibodies have been used in advanced GC patients. However, identifying new biomarker for immunotherapy is still urgently needed. The objective of this study is to unveil the immune evasion mechanism of GC cells and identify new biomarkers for immune checkpoint blockade therapy in patients with GC. METHODS: Coimmunoprecipitation and meRIP were performed to investigate the mechanism of immune evasion of GC cells. Cocuture system was established to evaluate the cytotoxicity of cocultured CD8+ T cells. The clinical significance of HSPA4 upregulation was analyzed by multiplex fluorescent immunohistochemistry staining in GC tumor tissues. RESULTS: Histone acetylation causes HSPA4 upregulation in GC tumor tissues. HSPA4 upregulation increases the protein stability of m6A demethylase ALKBH5. ALKBH5 decreases CD58 in GC cells through m6A methylation regulation. The cytotoxicity of CD8+ T cells are impaired and PD1/PDL1 axis is activated when CD8+ T cells are cocultured with HSPA4 overexpressed GC cells. HSPA4 upregulation is associated with worse 5-year overall survival of GC patients receiving only surgery. It is an independent prognosis factor for worse survival of GC patients. In GC patients receiving the combined chemotherapy with anti-PD1 immunotherapy, HSPA4 upregulation is observed in responders compared with non-responders. CONCLUSION: HSPA4 upregulation causes the decrease of CD58 in GC cells via HSPA4/ALKBH5/CD58 axis, followed by PD1/PDL1 activation and impairment of CD8+ T cell's cytotoxicity, finally induces immune evasion of GC cells. HSPA4 upregulation is associated with worse overall survival of GC patients with only surgery. Meanwhile, HSPA4 upregulation predicts for better response in GC patients receiving the combined immunotherapy.

Proteomic analysis for testis of mice exposed to carbon ion radiation.

This paper investigates the mechanism of action of heavy ion radiation (HIR) on mouse testes. The testes of male mice subjected to whole body irradiation with carbon ion beam (0.5 and 4Gy) were analyzed at 7days after irradiation. A two-dimensional gel electrophoresis approach was employed to investigate the alteration of protein expression in the testes. Spot detection and matching were performed using the PDQuest 8.0 software. A difference of more than threefold in protein quantity (normalized spot volume) is the standard for detecting differentially expressed protein spots. A total of 11 differentially expressed proteins were found. Protein identification was performed using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF-TOF). Nine specific proteins were identified by searching the protein sequence database of the National Center for Biotechnology Information. These proteins were found involved in molecular chaperones, metabolic enzymes, oxidative stress, sperm function, and spermatogenic cell proliferation. HIR decreased glutathione activity and increased malondialdehyde content in the testes. Given that Pin1 is related to the cell cycle and that proliferation is affected by spermatogenesis, we analyzed testicular histological changes and Pin1 protein expression through immunoblotting and immunofluorescence. Alterations of multiple pathways may be associated with HIR toxicity to the testes. Our findings are essential for studies on the development, biology, and pathology of mouse testes after HIR in space or radiotherapy.

HSP70 Expression Signature in Renal Cell Carcinoma: A Clinical and Bioinformatic Analysis Approach.

Heat shock proteins (HSPs) are cytoprotective against stressful conditions, as in the case of cancer cell metabolism. Scientists proposed that HSP70 might be implicated in increased cancer cell survival. This study aimed to investigate the HSP70 (HSPA4) gene expression signature in patients with renal cell carcinoma (RCC) in correlation to cancer subtype, stage, grade, and recurrence, combining both clinicopathological and in silico analysis approaches. One hundred and thirty archived formalin-fixed paraffin-embedded samples, including 65 RCC tissue specimens and their paired non-cancerous tissues, were included in the study. Total RNA was extracted from each sample and analyzed using TaqMan quantitative Real-Time Polymerase Chain Reaction. Correlation and validation to the available clinicopathological data and results were executed. Upregulated HSP70 (HSPA4) gene expression was evident in RCC compared to non-cancer tissues in the studied cohort and was validated by in silico analysis. Furthermore, HSP70 expression levels showed significant positive correlations with cancer size, grade, and capsule infiltration, as well as recurrence in RCC patients. The expression levels negatively correlated with the overall survival (r = -0.87, p < 0.001). Kaplan-Meier curves showed lower survival rates in high HSP70 expressor group compared to the low expressors. In conclusion, the HSP70 expression levels are associated with poor RCC prognosis in terms of advanced grade, capsule infiltration, recurrence, and short survival.

Identification of glutamyl-prolyl-tRNA synthetase as a new therapeutic target in hepatocellular carcinoma via a novel bioinformatic approach.

Background: Hepatocellular carcinoma (HCC) has a high incidence, and current treatments are ineffective. We aimed to explore potential diagnostic and prognostic biomarkers for HCC by conducting bioinformatics analysis on genomic and proteomic data. Methods: Genome and proteome data were downloaded from The Cancer Genome Atlas (TCGA) and ProteomeXchange databases, respectively. Differentially expressed genes was determined using limma package. Functional enrichment analysis was conducted by Database for Annotation, Visualization, and Integrated Discovery (DAVID). Protein-protein analysis was established by STRING dataset. Using Cytoscope for network visualization and CytoHubba for hub gene identification. The gene mRNA and protein levels were validated using GEPIA and HPA, as well as RT-qPCR and Western blot. Results: A total of 127 up-regulated and 80 down-regulated common DEGPs were identified between the genomic and proteomic data, Mining 10 key genes/proteins(ACLY, ACACB, EPRS, CAD, HSPA4, ACACA, MTHFD1, DMGDH, ALDH2, and GLDC) through protein interaction networks. in addition, Glutamyl-prolyl-tRNA synthetase (EPRS) was highlighted as an HCC biomarker that is negatively correlated with survival. Differential EPRS expression analysis in HCC and paracancerous tissues showed that EPRS expression was elevated in HCC. RT-qPCR and Western blot analysis results showed that EPRS expression was upregulated in HCC cells. Conclusions: Our results suggest that EPRS is a potential therapeutic target for inhibiting HCC tumorigenesis and progression.

SIRT1 attenuates neuroinflammation by deacetylating HSPA4 in a mouse model of Parkinson's disease.

As a deacetylase, SIRT1 plays essential roles in various physiological events, from development to lifespan regulation. SIRT1 has been shown neuroprotective effects in neurodegeneration disorders such as Parkinson's disease (PD). However, the underlying molecular mechanisms are still not well understood. Here, we generated transgenic mice with increased expression of Sirt1 in the brain and examined the potential roles of SIRT1 in PD. Our data showed that SIRT1 repressed proinflammatory cytokine expression both in microglia and astrocytes. In MPTP induced PD model mice, lower levels of microglia and astrocyte activation were observed in SIRT1 transgenic mice. Moreover, the tyrosine hydroxylase (TH) loss in the substantia nigra pars compacta (SNpc) and striatum induced by MPTP was also attenuated by SIRT1. As a consequence, the behavioral defects induced by MPTP were largely prevented in SIRT1 transgenic mice. Mechanistically, SIRT1 interacts with heat shock 70 kDa protein 4 (HSPA4) and deacetylates it at 305, 351 and 605 lysine residues. This deacetylation modification induces the nuclear translocation of HSPA4 and thus to repress proinflammatory cytokine expression. On the contrary, mutated HSPA4, in which 305/351/605 lysine residues were replaced with arginine, was mainly localized in the cytoplasm and losses its repression on proinflammatory cytokine expression. Taken together, our data indicate that SIRT1 plays beneficial roles in PD model mice, which is likely due to, at least in part, its anti-inflammation activity in glial cells by deacetylating HSPA4. Furthermore, HSPA4 might be a druggable target for developing novel agents for treating neuroinflammation associated disorders such as PD.

Inhibition of endoplasmic reticulum stress alleviates triple-negative breast cancer cell viability, migration, and invasion by Syntenin/SOX4/Wnt/ß-catenin pathway via regulation of heat shock protein A4.

Endoplasmic reticulum stress (ER stress) is a double-edged sword in the occurrence and development of malignant cancer. The aim of this study was to explore the roles of ER stress in metastasis and epithelial-mesenchymal transitionin triple-negative breast cancer (TNBC) and potential mechanisms. In this study, 4-PBA was administrated to inhibit the ER stress. Cell viability was evaluated using a cell counting kit-8 assay. Cell migration and invasion were identified by wound healing and transwell assay, respectively. Levels of MMP2 and MMP9 were measured by enzyme-linked immunosorbent assay and immunohistochemical staining. Western blot assay was used to assess the levels of ER stress-related proteins, Syndecan-1 (SDC-1)/Syntenin-1 (SDCBP-1)/SRY-related HMG-box 4 (SOX4) signaling and Wnt/ß-catenin signaling. Moreover, a xenograft mice model was conducted to confirm the role of ER stress in TNBC. The data indicate that the ability of viability and metastasis of breast cancer cells were stronger than normal mammary epithelial cells. More aggressiveness was manifested in TNBC cells than that in non-TNBC cells. 4-PBA significantly suppressed the viability, migration, and invasion in BC cells and inhibited the SDC/SDCBP/SOX4 axis and Wnt/ß-catenin signaling. Furthermore, heat shock protein A4 (HSPA4) overexpression stimulated ER stress and activated the SDC-1/SDCBP-1/SOX4 pathway and Wnt/ß-catenin signaling. Animal experiments showed similar results that 4-PBA repressed tumor growth and inactivated the two pathways, while HSPA4 overexpression reversed the effects of 4-PBA. In summary, inhibition of ER stress inhibited TNBC viability, migration, and invasion by Syntenin/SOX4/Wnt/ß-catenin pathway via regulation of HSPA4 in vivo and in vitro.

HSPA4 Knockdown Retarded Progression and Development of Colorectal Cancer.

PURPOSE: Colorectal cancer (CRC) is a common malignancy associated with high morbidity and mortality. Heat shock 70 kDa protein 4 (HSPA4) has been shown to exert regulatory roles during tumor progression in different cancer types. Here, we investigated the expression and cellular functions of HSPA4 in CRC. MATERIALS AND METHODS: Expression of HSPA4 in CRC tissues and paracancerous tissues was analyzed by RT-qPCR and immunohistochemistry IHC staining. The functional roles of HSPA4 were explored using shRNA-mediated knockdown in HCT116 and RKO CRC cell lines, both in vitro and in tumor xenograft studies. RESULTS: HSPA4 expression was significantly increased at the RNA and protein levels in CRC tissues compared with noncancerous tissues. Moreover, HSPA4 expression was positively associated with tumor stage and its high expression of HSPA4 indicated poor patient prognosis. In vitro studies established that HSPA4 knockdown inhibited proliferation and migration, causing arrest in the G2-phase of the cell cycle along with increased levels of apoptosis. This phenotype was recapitulated in vivo where HSPA4 knockdown suppressed xenograft growth. Mechanistic investigations showed silencing of HSPA4 reduced activation of the PI3K, Akt signaling axis while also downregulating the cell cycle progression markers, CCND1 and CDK6. Similarly, there was altered expression of apoptosis-related proteins consistent with the increase in apoptosis. CONCLUSION: Our findings demonstrate clinical significance for HSPA4 in CRC, further showing that HSPA4 contributes to CRC tumorigenesis through effects on proliferation, migration and survival. Thus, HSPA4 represents a novel prognostic indicator as well as a promising therapeutic target in CRC.

lncRNA RMRP Prevents Mitochondrial Dysfunction and Cardiomyocyte Apoptosis via the miR-1-5p/hsp70 Axis in LPS-Induced Sepsis Mice.

Both long non-coding RNA (lncRNA) RMRP and heat shock protein (HSP) 70 have been known to play crucial roles in inflammation. The present study investigated the roles of lncRNA RMRP and HSP70 protein 4 (HSPA4) in lipopolysaccharide (LPS)-induced sepsis. The C57BL/6 mice were treated with LPS, following which the cardiomyocytes were isolated for in vitro experiments. Further, a cardiac muscle cell line, HL-1 was transfected with plasmids expressing RMRP and HSPA4, si-NC, si-HSPA4, miR-1-5p mimic, and controls in vitro. Cell apoptosis, mitochondrial membrane potential (MMP), and levels of intracellular reactive oxygen species (ROS), mRNAs, and proteins were detected in the transfected mice tissues and cells. The LPS treatment significantly reduced the expression levels of RMRP, MMP, and mitochondrial cytochrome C. Moreover, it enhanced the cardiomyocyte apoptosis, intracellular ROS levels, cytoplasm cytochrome C levels, and the expression of caspase-3 and caspase-9 and nuclear factor κB (NF-κB) p65 subunit. The predicted RMRP-miR-1-5p-HSPA4 network was validated by co-transfection experiments in vitro in HL-1 cells. The transfection of miR-1-5p-treated cells with pcDNA-RMRP enhanced the levels of the protein HSPA4; however, no change at the mRNA level was observed. Moreover, miR-1-5p mimic attenuated the protective effect of pcDNA-HSPA4 against LPS-induced mitochondrial damage and apoptosis. In addition, we observed that silencing of HSPA4 increased the expression of nuclear p65; however, this effect could be reversed by co-transfection with pcDNA-RMRP. The lncRNA RMRP axis acts as a sponge for miR-1-5p. RMRP inhibits LPS-induced apoptosis of cardiomyocytes and mitochondrial damage by suppressing the post-transcriptional regulatory function of miR-1-5p on HSPA4. We believe that RMRP exhibits therapeutic potential for LPS-induced myocardial dysfunction both in vitro and in vivo.

Overexpression of heat shock protein HSP90AA1 and translocase of the outer mitochondrial membrane TOM34 in HCV-induced hepatocellular carcinoma: A pilot study.

OBJECTIVE: Identification of new molecular markers to enhance early diagnosis, prognosis and/or treatment of hepatocellular carcinoma (HCC) is a need. TOM34 (34 kDa-translocase of the outer mitochondrial membrane) protein expression deregulation has demonstrated to be involved in the growth of many cancers. Here, we aimed at evaluating serum TOM34 and some heat shock proteins (HSPA4, HSPA1B, and HSP90AA1) expressions in hepatitis C virus (HCV)-related cirrhosis and HCV-induced HCC relative to controls and correlating these expressions to the clinicopathological data. METHODS: Serum specimens were collected from 90 patients with HCV associated complications (30 cirrhotic, 30 early HCC and 30 late HCC) and 60 controls. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed for relative quantification of the four target genes using the Livak method. In silico network analysis was also executed to explore the contribution of the genes in liver cancer. RESULTS: The serum TOM34 and HSP90AA1 transcripts were significantly upregulated in HCC patients compared to cirrhotic ones with more up-regulation in late HCC patients. Receiver operating characteristic analysis showed the optimum cutoff value of 0.625 corresponding to 71.7% sensitivity and 56.7% specificity, and an area under the curve (AUC) of 0.705 to discriminate HCC from cirrhotic groups (P = .002). In multivariate analysis, ordination plot showed obvious demarcation between the study groups caused by the higher levels of TOM34 among other variables. CONCLUSIONS: TOM34 and its partner HSP90AA1 might be used as a potential biomarker for monitoring HCV-induced HCC progression in the Egyptian population. Future large-scale validation studies are warranted.

Frameshift Mutations of HSPA4 and MED13 in Gastric and Colorectal Cancers.

Frameshift mutation of genes containing mononucleotide repeats is a feature of gastric (GC) and colorectal cancers (CRC) with microsatellite instability (MSI). In the public genome database, we found that human HSPA4 gene encoding a heats hock protein 70 protein (HSP70-4) and MED13 gene had mononucleotide repeats in the coding sequences that could be targets for frameshift mutation in cancers with MSI. HSP70-4 is a member of HSP70 that is known to play a role in cell survival. MED13 is a member of MED genome-wide transcription regulators that function as a regulator for diverse biological processes. In this study, we analyzed the mutations in 79 GCs and 124 CRCs including high MSI (MSI-H) and microsatellite stable/low MSI (MSS/MSI-L) cases by single-strand conformation polymorphism analysis and DNA sequencing. We found frameshift mutations of HSPA4 gene in two cancers (one GC and one CRC) and MED13 gene in the other two cancers (one GC and one CRC). The frameshift mutations were deletions of one base (c.2396delA (p.Asn799MetfsX50)) in HSPA4 and (c.2175delA (p.Lys725AsnfsX4)) in MED13. Each of HSPA4 and MED13 mutations were detected in GC with MSI-H (1/34: 2.9 %) and CRC with MSI-H (1/79: 1.3 %), but not in those with MSS. Our data show that unconventional HSPA4 and MED13 genes harbored frameshift mutations in GC and CRC with MSI. These mutations might possibly inactivate their functions and could be a feature of GC and CRC with MSI-H.