HDAC3 Controls Liver Homeostasis More by Facilitating Deoxyribonucleic Acid Damage Repair than by Regulating Transcription in Hepatocytes.

By controlling DNA damage repair and regulating gene transcription, the critical epigenetic regulator histone deacetylase 3 (HDAC3) plays pivotal roles in liver cancer and liver regeneration; however, the role of HDAC3 in liver homeostasis has not been fully elucidated. In this study, we found that HDAC3-deficient livers developed a defective morphology and metabolism with an increasing degree of DNA damage in the hepatocytes along the portal-central axis of the lobule. Most strikingly, in the Alb-CreERT:Hdac3-/- mice, it was demonstrated that HDAC3 ablation did not impair liver homeostasis in terms of histologic characteristics, function, proliferation, or gene profiles prior to the profound accumulation of DNA damage. Next, we identified that the hepatocytes in the portal area, which carried less DNA damage than those in the central area, repopulated the hepatic lobule by active regeneration and movement toward the center. As a result, the liver became more viable after each surgery. Furthermore, in vivo tracing of keratin-19-expressing hepatic progenitor cells, which lacked HDAC3, showed that the hepatic progenitor cells gave rise to newly generated periportal hepatocytes. In hepatocellular carcinoma, HDAC3 deficiency impaired DNA damage response and enhanced radiotherapy sensitivity in vitro and in vivo. Taken together, we demonstrated that HDAC3 deficiency interferes with liver homeostasis, which is more dependent on the accumulation of DNA damage in hepatocytes than on transcriptional dysregulation. Our findings support the hypothesis that selective HDAC3 inhibition has the potential to augment the effect of chemoradiotherapy aimed at inducing DNA damage in cancer therapy.

Increased Small Ubiquitin-like Modifier-Activating Enzyme SAE1 Promotes Hepatocellular Carcinoma by Enhancing mTOR SUMOylation.

SUMOylation, one of the most important posttranslational modifications of proteins, plays an essential role in various biological processes; however, enzymes that control SUMOylation in hepatocellular carcinoma (HCC) are still unclear. Comprehensive exploration of the expression and clinical significance of SUMO enzymes in HCC would be of great value. Here, we obtained the gene expression profile of each small ubiquitin-like modifier (SUMO) protein and the corresponding clinical information from The Cancer Genome Atlas. We found that all SUMO enzymes were significantly increased in HCC tissues compared with that in adjacent nontumorous tissues. We identified a 6-gene prognostic signature, including SAE1, PIAS2, PIAS3, SENP3, SENP5, and UBC9, that could effectively predict the overall survival in patients with HCC. Specifically, SAE1 was the most valuable prognostic indicator. In 282 clinical samples, we found that SAE1 was closely related to the clinicopathologic parameters and prognosis of patients with HCC. In vitro and in vivo studies showed that SAE1 knockdown inhibits the proliferation, migration, and invasion of HCC cells. Mechanistically, we confirmed that SAE1 plays a role in driving HCC progression, which is largely dependent on the SUMOylation of mTOR signaling. In conclusion, our study revealed that the expression of SUMO enzymes, especially SAE1, is highly associated with HCC development and acts as a promising prognostic predictor.

Hepatocyte-specific HDAC3 ablation promotes hepatocellular carcinoma in females by suppressing Foxa1/2.

BACKGROUND: Hepatocellular carcinoma (HCC), the most common primary liver cancer, prevails mainly in males and has long been attributed to androgens and higher circumstantial levels of interleukin-6 (IL-6) produced by resident hepatic macrophages. METHODS: Constitutively hepatocyte-specific histone deacetylase 3 (HDAC3)-deficient (HDAC3LCKO) mice and constitutively hepatocyte-specific HDAC3 knockout and systemic IL-6 simultaneously ablated (HDAC3LCKO& IL-6-/-) mice were used in our study to explore the causes of sex differences in HCC. Additionally, we performed human HCC tissues with an IHC score. Correlation analysis and linear regression plots were constructed to reveal the association between HDAC3 and its candidate genes. To further elucidate that HDAC3 controls the expression of Foxa1/2, we knocked down HDAC3 in HUH7 liver cancer cells. RESULTS: We observed a contrary sex disparity, with an earlier onset and higher incidence of HCC in female mice when HDAC3 was selectively ablated in the liver. Loss of HDAC3 led to constant liver injury and the spontaneous development of HCC. Unlike the significant elevation of IL-6 in male mice at a very early age, female mice exhibit stable IL-6 levels, and IL-6 ablation did not eliminate the sex disparity in hepatocarcinogenesis in HDAC3-deficient mice. Oestrogen often protects the liver when combined with oestrogen receptor alpha (ERα); however, ovariectomy in HDAC3-ablated female mice significantly delayed tumourigenesis. The oestrogen-ERα axis can also play a role in tumour promotion in the absence of Foxa1 and Foxa2 in the receptor complex. Loss of HDAC3 profoundly reduced the expression of both Foxa1 and Foxa2 and impaired the binding between Foxa1/2 and ERα. Furthermore, a more frequent HDAC3 decrease accompanied by the simultaneous Foxa1/2 decline was found in female HCC compared to that in male HCC. CONCLUSION: In summary, we reported that loss of HDAC3 reduces Foxa1/2 and thus promotes HCC development in females in an oestrogen-dependent manner.

Brahma-Related Gene 1 Inhibition Prevents Liver Fibrosis and Cholangiocarcinoma by Attenuating Progenitor Expansion.

BACKGROUND AND AIMS: Intrahepatic cholangiocarcinoma (iCCA) is closely correlated with hepatic progenitor cell (HPC) expansion and liver fibrosis. Brahma-related gene 1 (Brg1), an enzymatic subunit of the switch/sucrose nonfermentable complex that is critical in stem cell maintenance and tumor promotion, is prominently up-regulated in both HPCs and iCCA; however, its role in this correlation remains undefined. APPROACH AND RESULTS: A retrospective cohort study indicated that high Brg1 expression suggests poor prognosis in patients with iCCA. In chronically injured livers induced by a 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet or bile duct ligation surgery, HPCs were dramatically activated, as indicated by their enhanced expression of Brg1 and a subset of stem cell markers; however, Brg1 ablation in HPCs strongly suppressed HPC expansion and liver fibrosis. Furthermore, in a chemically induced iCCA model, inhibition of Brg1 by a specific inhibitor or inducible gene ablation markedly improved histology and suppressed iCCA growth. Mechanistically, in addition to transcriptionally promoting both Wnt receptor genes and target genes, Brg1 was found to bind to the ß-catenin/transcription factor 4 transcription complex, suggesting a possible approach for regulation of Wnt/ß-catenin signaling. CONCLUSIONS: We have demonstrated the function of Brg1 in promoting HPC expansion, liver cirrhosis, and, ultimately, iCCA development in chronically injured livers, which is largely dependent on Wnt/ß-catenin signaling. Our data suggest that therapies targeting Brg1-expressing HPCs are promising for the treatment of liver cirrhosis and iCCA.

Pathophysiological Changes in Rhesus Monkeys with Paraquat-Induced Pulmonary Fibrosis.

PURPOSE: Pulmonary fibrosis is a life-threatening lung disorder. A comprehensive understanding of the pathophysiological changes in the development of pulmonary fibrosis will lead to new insights into its treatment. METHODS: We used a paraquat (PQ)-induced rhesus monkey model of pulmonary fibrosis to comprehensively investigate the process of pulmonary fibrosis development. Rhesus monkeys were orally administered PQ at concentrations of 25 mg/kg, 40 mg/kg, and 80 mg/kg. The dose was given once. Behavior and clinical data, such as PQ concentration, arterial oxygen saturation, biochemical evaluation, lung histopathology, and medical imaging, were continuously observed. RESULTS: Paraquat-exposed monkeys developed pulmonary fibrosis following an expected time course, especially at 25 mg/kg. CT images showed ground-glass lesions in the lung after 4 weeks, and pulmonary fibrosis persisted until the end of follow-up. Using pathological examination, the lung sustained collagen deposition and slight inflammatory cell infiltration. All rhesus monkeys had obvious inflammatory infiltration within 1 week according to the immunohistochemical results and the number of leukocytes in the blood. The CT results showed that pulmonary fibrosis had not formed, indicating that drugs with powerful anti-inflammatory ability are potential candidates for early pulmonary fibrosis treatment. CONCLUSION: Our study describes the dynamic process of paraquat-induced pulmonary fibrosis in rhesus monkeys and provided a pathophysiological basis for the treatment of pulmonary fibrosis.

Mitochondrial miR-181a-5p promotes glucose metabolism reprogramming in liver cancer by regulating the electron transport chain.

Liver cancer and other malignant tumor cells rely on the glycolytic pathway to obtain energy (i.e. the Warburg effect); however, the underlying mechanism is unclear. Mitochondria are sites of oxidative phosphorylation and adenosine triphosphate (ATP) production. The 13 constituent respiratory chain proteins encoded by the mitochondrial genome (namely, mtDNA) play essential roles. We found that in human hepatocellular carcinoma (HCC) tissues, 11 out of the 13 mtDNA-encoded genes exhibited decreased mRNA levels and 5 genes displayed decreased protein levels, including the cytochrome B (mt-CYB) and cytochrome C oxidase II (mt-CO2) genes. Mitochondrial gene sequencing revealed abnormalities in the levels of a large number of mitochondrial miRNAs (mitomiRs). MicroRNA-181a-5p (mir-181a-5p), which potentially targets genes encoding mt-CYB and mt-CO2 protein, was screened out from 549 downregulated mitomiRs via bioinformatic analysis. After overexpression of mitomiR-181a-5p, mt-CYB and mt-CO2 levels were reduced in HCC cells, and the mitochondrial membrane potential (MMP) maintained by the electron transport chain (ETC) was decreased. Furthermore, the expression of hexokinase 2 (HK2) and glucose transporter type 1 (GLUT1) was upregulated, accompanied by elevated glucose, lactic acid release, and activity of lactate dehydrogenase (LDH). In vivo experiments confirmed that constitutive mitomiR-181a-5p expression caused reprogramming of glucose metabolism and promoted tumor growth and early lung metastasis in liver cancer. In summary, the present study reveals the important role of mitomiRs in glucose metabolism reprogramming in liver cancer, which is of considerable value in exploring new therapeutic targets for HCC.

Decreased expression of HDAC8 indicates poor prognosis in patients with intrahepatic cholangiocarcinoma.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) is a highly malignant primary tumor in the liver, and the rates of incidence and mortality are rapidly increasing globally. Histone deacetylase 8 (HDAC8) is a transcriptional regulator and is associated with tumorigenesis of several tumor types. This study aimed to evaluate the correlation between HDAC8 expression and clinicopathological parameters in ICC patients. METHODS: ICC tissues and corresponding nonmalignant bile duct tissues were obtained from 60 patients. HDAC8 and Ki-67 expression were evaluated by immunohistochemistry staining. HDAC8 expression and the clinicopathological features and prognosis of the patients were analyzed. The mRNA level of HDAC8 in ICC was further analyzed using data from The Cancer Genome Atlas (TCGA). RESULTS: The expression of HDAC8 were lower in ICC tissues (39/60, 65%) than in the corresponding nonmalignant bile duct tissues (54/60, 90%) (P = 0.001). Low HDAC8 expression in ICC was significantly associated with lymph node metastases (47.6% vs. 17.9%, P = 0.015). In addition, the positive cells rate of HDAC8 was statistically and negatively correlated with the Ki-67 index in ICC lesions (r = -0.7660, P < 0.001). Importantly, the overall survival rate and recurrence-free survival rate in ICC patients with low HDAC8 expression were lower than those with high HDAC8 expression (P = 0.008 and P = 0.011, respectively). CONCLUSIONS: Decreased HDAC8 expression in ICC is related to poor prognosis, and HDAC8 may be an independent prognostic indicator of ICC patients after curative resection.

Multi-targeting inulin-based nanoparticles with cannabidiol for effective prevention of ulcerative colitis.

The pathogenesis of ulcerative colitis (UC) is closely related to severe inflammation, damaged colonic mucosal barrier, increased oxidative stress and intestinal ecological imbalance. However, due to the nonspecific distribution and poor bioavailability of drugs, UC treatment is still a serious challenge. Here, a mitochondria/colon dual targeted nanoparticles based on redox response was developed to effectively alleviate UC. Cannabidiol nanoparticles (CBD NPs) with a particle size of 143.2 ± 3.11 nm were prepared by self-assembly using polymers (TPP-IN-LA) obtained by modifying inulin with (5-carboxypentyl) triphenyl phosphonium bromide (TPP) and α-lipoic acid (α-LA). Excitingly, the constructed CBD NPs showed excellent mitochondrial targeting, with a Pearson correlation coefficient of 0.76 at 12 h. The results of animal imaging in vivo showed that CBD NPs could be effectively accumulated in colon tissue. Not only that, CBD showed significant glutathione stimulated release in the presence of 10 mM glutathione at pH 7.4. The results of in vivo animal experiments showed that CBD NPs significantly ameliorated DSS-induced colonic inflammation by modulating the TLR4-NF-κB signaling pathway. Moreover, CBD NPs significantly improved the histological damage of colon in UC mice, increased the expression level of tight junction protein ZO-1, and effectively restored the intestinal mucosal barrier function and intestinal mucosal permeability. More importantly, CBD NPs significantly improved the species composition, abundance and amount of short chain fatty acids of intestinal flora in UC mice, thus effectively maintaining the balance of intestinal flora. The dual-targeted and glutathione-responsive nanoparticles prepared in this study provide a promising idea for achieving targeted delivery of CBD for effective treatment of UC.

Identification and Characterization of Novel ACE Inhibitory and Antioxidant Peptides from Sardina pilchardus Hydrolysate.

Sardina pilchardus is a valuable source of bioactive peptides with potential applications in functional foods. In this study, we investigated the angiotensin-converting enzyme (ACE) inhibitory activity of Sardina pilchardus protein hydrolysate (SPH) produced using dispase and alkaline protease. Our results showed that the low molecular mass fractions (<3 kDa) obtained through ultrafiltration exhibited more effective ACE inhibition, as indicated by screening with ACE inhibitory activity. We further identified the low molecular mass fractions (<3 kDa) using an LC-MS/MS rapid screening strategy. A total of 37 peptides with potential ACE inhibitory activity were identified based on high biological activity scores, non-toxicity, good solubility, and novelty. Molecular docking was used to screen for peptides with ACE inhibitory activity, resulting in the identification of 11 peptides with higher -CDOCKER ENERGY and -CDOCKER INTERACTION ENERGY scores than lisinopril. The sequences FIGR, FILR, FQRL, FRAL, KFL, and KLF were obtained by synthesizing and validating these 11 peptides in vitro, all of which had ACE inhibitory activity, as well as zinc-chelating capacity. All six peptides were found to bind to the three active pockets (S1, S2, and S1') of ACE during molecular docking, indicating that their inhibition patterns were competitive. Further analysis of the structural characteristics of these peptides indicated that all six peptides contain phenylalanine, which suggests that they may possess antioxidant activities. After experimental verification, it was found that all six of these peptides have antioxidant activities, and we also found that the SPH and ultrafiltration fractions of SPH had antioxidant activities. These findings suggest that Sardina pilchardus may be a potential source of natural antioxidants and ACE inhibitors for the development of functional foods, and using LC-MS/MS in combination with an online database and molecular docking represents a promising, effective, and accurate approach for the discovery of novel ACE inhibitory peptides.

Glutamine synthetase-negative hepatocellular carcinoma has better prognosis and response to sorafenib treatment after hepatectomy.

BACKGROUND: Glutamine synthetase (GS) and arginase 1 (Arg1) are widely used pathological markers that discriminate hepatocellular carcinoma (HCC) from intrahepatic cholangiocarcinoma; however, their clinical significance in HCC remains unclear. METHODS: We retrospectively analyzed 431 HCC patients: 251 received hepatectomy alone, and the other 180 received sorafenib as adjuvant treatment after hepatectomy. Expression of GS and Arg1 in tumor specimens was evaluated using immunostaining. mRNA sequencing and immunostaining to detect progenitor markers (cytokeratin 19 [CK19] and epithelial cell adhesion molecule [EpCAM]) and mutant TP53 were also conducted. RESULTS: Up to 72.4% (312/431) of HCC tumors were GS positive (GS+). Of the patients receiving hepatectomy alone, GS negative (GS-) patients had significantly better overall survival (OS) and recurrence-free survival (RFS) than GS+ patients; negative expression of Arg1, which is exclusively expressed in GS- hepatocytes in the healthy liver, had a negative effect on prognosis. Of the patients with a high risk of recurrence who received additional sorafenib treatment, GS- patients tended to have better RFS than GS+ patients, regardless of the expression status of Arg1. GS+ HCC tumors exhibit many features of the established proliferation molecular stratification subtype, including poor differentiation, high alpha-fetoprotein levels, increased progenitor tumor cells, TP53 mutation, and upregulation of multiple tumor-related signaling pathways. CONCLUSIONS: GS- HCC patients have a better prognosis and are more likely to benefit from sorafenib treatment after hepatectomy. Immunostaining of GS may provide a simple and applicable approach for HCC molecular stratification to predict prognosis and guide targeted therapy.

Congenital biliary atresia is correlated with disrupted cell junctions and polarity caused by Cdc42 insufficiency in the liver.

Rationale: Congenital biliary atresia (BA) is a destructive obliterative cholangiopathy of neonates that affects both intrahepatic and extrahepatic bile ducts. However, the cause of BA is largely unknown. Methods: We explored the cell junctions and polarity complexes in early biopsy BA livers by immunofluorescence staining and western blot. Cdc42, as a key cell junction and polarity regulator, was found dramatically decreased in BA livers. Therefore, in order to investigate the role of Cdc42 in BA development, we constructed liver-specific and tamoxifen induced cholangiocyte-specific Cdc42 deleted transgenic mice. We further evaluated the role of bile acid in aggravating biliary damage in Cdc42 insufficient mouse liver. Results: We found a dramatic defect in the assembly of cell junctions and polarity complexes in both cholangiocytes and hepatocytes in BA livers. This defect was characterized by the disordered location of cell junction proteins, including ZO1, ß-catenin, E-cadherin and claudin-3. Cdc42 and its active form, Cdc42-GTP, which serves as a small Rho GTPase to orchestrate the assembly of polarity complexes with Par6/Par3/αPKC, were substantially reduced in BA livers. Selective Cdc42 deficiency in fetal mouse cholangiocytes resulted in histological changes similar to those found in human BA livers, including obstruction in both the intra- and extrahepatic bile ducts, epithelial atrophy, and the disruption of cell junction and polarity complexes. A reduction in bile acids notably improved the histology and serological indices in Cdc42-mutant mice. Conclusion: Our results illustrate that BA is closely correlated with the impaired assembly of cell junction and polarity complexes in liver cells, which is likely caused by Cdc42 insufficiency and aggravated by bile acid corrosion.

Loss of Numb promotes hepatic progenitor expansion and intrahepatic cholangiocarcinoma by enhancing Notch signaling.

Numb, a stem cell fate determinant, acts as a tumor suppressor and is closely related to a wide variety of malignancies. Intrahepatic cholangiocarcinoma (iCCA) originates from hepatic progenitors (HPCs); however, the role of Numb in HPC malignant transformation and iCCA development is still unclear. A retrospective cohort study indicated that Numb was frequently decreased in tumor tissues and suggests poor prognosis in iCCA patients. Consistently, in a chemically induced iCCA mouse model, Numb was downregulated in tumor cells compared to normal cholangiocytes. In diet-induced chronic liver injury mouse models, Numb ablation significantly promoted histological impairment, HPC expansion, and tumorigenesis. Similarly, Numb silencing in cultured iCCA cells enhanced cell spheroid growth, invasion, metastasis, and the expression of stem cell markers. Mechanistically, Numb was found to bind to the Notch intracellular domain (NICD), and Numb ablation promoted Notch signaling; this effect was reversed when Notch signaling was blocked by γ-secretase inhibitor treatment. Our results suggested that loss of Numb plays an important role in promoting HPC expansion, HPC malignant transformation, and, ultimately, iCCA development in chronically injured livers. Therapies targeting suppressed Numb are promising for the treatment of iCCA.

Mettl14-Mediated m6A Modification Facilitates Liver Regeneration by Maintaining Endoplasmic Reticulum Homeostasis.

BACKGROUND & AIMS: N6-methyladenosine (m6A), the most prevalent and dynamic posttranscriptional methylation modification of mammalian mRNA, is involved in various biological processes, but its role in liver regeneration has not been characterized. METHODS: We first conducted transcriptome-wide m6A mRNA sequencing and characterized the expression pattern of m6A in regenerating mouse liver. Next, we generated hepatocyte-specific Mettl3- or Mettl14-deficient mice and investigated their role in liver regeneration. A series of biochemical experiments in vitro and in vivo was further performed to investigate potential mechanisms. RESULTS: We identified an overwhelming proportion of m6A-modified genes with initially up-regulated and subsequently down-regulated m6A levels as liver regeneration progressed. Loss of Mettl14 but not of Mettl3 resulted in markedly disrupted liver regeneration, and Mettl14-ablated hepatocytes were arrested in the G1 phase of the cell cycle. Most strikingly, the Mettl14-ablated regenerating liver exhibited extensive parenchymal necrosis. mRNA transcripts, such as Hsp90b1, Erp29, Stt3a, P4hb, and Lman1, encoding proteins involved in polypeptide processing and the endoplasmic reticulum (ER) stress response, were m6A-hypomethylated, and their mRNA and protein levels were subsequently decreased, resulting in unresolved ER stress, hepatocyte death, and inhibited proliferation. CONCLUSIONS: We demonstrate the essential role of Mettl14 in facilitating liver regeneration by modulating polypeptide-processing proteins in the ER in an m6A-dependent manner.

Exosomes derived from human umbilical cord mesenchymal stem cells ameliorate IL-6-induced acute liver injury through miR-455-3p.

BACKGROUND: Using a toxin-induced nonhuman primate model of acute liver failure (ALF), we previously reported that peripheral infusion of human umbilical cord mesenchymal stem cells (hUC-MSCs) strongly suppresses the activation of circulating monocytes and interleukin-6 (IL-6) production, thereby disrupting the development of a cytokine storm and improving the prognosis of monkeys. MSCs are considered to play a therapeutic role under different stresses by adaptively producing specific factors, prompting us to investigate the factors that hUC-MSCs produce in response to high serum levels of IL-6, which plays a critical role in initiating and accelerating ALF. METHODS: We stimulated hUC-MSCs with IL-6, and the hUC-MSC-derived exosomes were deeply sequenced. The miRNAs in the exosomes that have potential to suppress IL-6-associated signaling pathway were screened, and the role of one of the most possible miRNAs was tested in the mouse model of inflammatory liver injury. RESULT: We determined that miR-455-3p, which is secreted through exosomes and potentially targets PI3K signaling, was highly produced by hUC-MSCs with IL-6 stimulation. The miR-455-3p-enriched exosomes could inhibit the activation and cytokine production of macrophages challenged with lipopolysaccharide (LPS) both in vivo and in vitro. In a chemical liver injury mouse model, enforced expression of miR-455-3p could attenuate macrophage infiltration and local liver damage and reduce the serum levels of inflammatory factors, thereby improving liver histology and systemic disorder. CONCLUSIONS: miR-455-3p-enriched exosomes derived from hUC-MSCs are a promising therapy for acute inflammatory liver injury.

Histone deacetylase 3 promotes liver regeneration and liver cancer cells proliferation through signal transducer and activator of transcription 3 signaling pathway.

Histone deacetylase 3 (HDAC3) plays pivotal roles in cell cycle regulation and is often aberrantly expressed in various cancers including hepatocellular carcinoma (HCC), but little is known about its role in liver regeneration and liver cancer cells proliferation. Using an inducible hepatocyte-selective HDAC3 knockout mouse, we find that lack of HDAC3 dramatically impaired liver regeneration and blocked hepatocyte proliferation in the G1 phase entry. HDAC3 inactivation robustly disrupted the signal transducer and activator of transcription 3 (STAT3) cascade. HDAC3 silencing impaired the ac-STAT3-to-p-STAT3 transition in the cytoplasm, leading to the subsequent breakdown of STAT3 signaling. Furthermore, overexpressed HDAC3 was further associated with increased tumor growth and a poor prognosis in HCC patients. Inhibition of HDAC3 expression reduced liver cancer cells growth and inhibited xenograft tumor growth. Our results suggest that HDAC3 is an important regulator of STAT3-dependent cell proliferation in liver regeneration and cancer. These findings provide novel insights into the HDAC3-STAT3 pathway in liver pathophysiological processes.

Systematic characterization of hypothetical proteins in Synechocystis sp. PCC 6803 reveals proteins functionally relevant to stress responses.

We described here a global detection and functional inference of hypothetical proteins involved in stress response in Synechocystis sp. PCC 6803. In the study, we first applied an iTRAQ-LC-MS/MS based quantitative proteomics to the Synechocystis cells grown under five stress conditions. The analysis detected a total of 807 hypothetical proteins with high confidence. Among them, 480 were differentially regulated. We then applied a Weighted Gene Co-expression Network Analysis approach to construct transcriptional networks for Synechocystis under nutrient limitation and osmotic stress conditions using transcriptome datasets. The analysis showed that 305 and 467 coding genes of hypothetical proteins were functionally relevant to nutrient limitation and osmotic stress, respectively. A comparison of responsive hypothetical proteins to all stress conditions allowed identification of 22 hypothetical proteins commonly responsive to all stresses, suggesting they may be part of the core stress responses in Synechocystis. Finally, functional inference of these core stress responsive proteins using both sequence similarity and non-similarity approaches was conducted. The study provided new insights into the stress response networks in Synechocystis, and also demonstrated that a combination of experimental "OMICS" and bioinformatics methodologies could improve functional annotation for hypothetical proteins.