MicroRNA-5195-3p mediated malignant biological behaviour of insulin-resistant liver cancer cells via SOX9 and TPM4.

BACKGROUND: Primary liver cancer is a malignant tumour of the digestive system, ranking second in cancer mortality in China. In different types of cancer, such as liver cancer, microRNAs (miRNAs) have been shown to be dysregulated. However, little is known about the role of miR-5195-3p in insulin-resistant liver cancer. METHODS AND RESULTS: In this study, in vitro and in vivo experiments were conducted to identify the altered biological behaviour of insulin-resistant hepatoma cells (HepG2/IR), and we proved that HepG2/IR cells had stronger malignant biological behaviour. Functional experiments showed that enhanced expression of miR-5195-3p could inhibit the proliferation, migration, invasion, epithelial-mesenchymal transition (EMT) and chemoresistance of HepG2/IR cells, while impaired expression of miR-5195-3p in HepG2 cells resulted in the opposite effects. Bioinformatics prediction and dual luciferase reporter gene assays proved that SOX9 and TPM4 were the target genes of miR-5195-3p in hepatoma cells. CONCLUSIONS: In conclusion, our study demonstrated that miR-5195-3p plays a critical role in insulin-resistant hepatoma cells and might be a potential therapeutic target for liver cancer.

iTRAQ-Based Proteome Profiling of Differentially Expressed Proteins in Insulin-Resistant Human Hepatocellular Carcinoma.

Recently, the incidences of insulin resistance (IR) and IR-related complications have increased throughout the world, which also associate with poor prognosis in hepatocellular carcinoma (HCC). Numerous studies had been focused on the role of IR in tumorigenesis and prognosis of HCC. The proteomic analysis of IR related hepatocellular carcinoma had not been reported by now. In the present study, 196 differentially expressed proteins (DEPs) were identified between insulin resistant HepG2 cells and their parental cells, of which 109 proteins were downregulated and 87 proteins were upregulated. Bioinformatics analysis indicated that these DEPs were highly enriched in process of tumorigenesis and tumor progression. PPI network analysis showed that SOX9, YAP1 and GSK3ß as the key nodes, were involved in Wnt and Hippo signaling pathways. Survival analysis revealed that high expression of SOX9 and PRKD3 were strongly associated with reduced patient survival rate. parallel reaction monitoring (PRM) and Western blot analysis were applied to verify the protein level of these four key nodes mentioned above, which showed the same trend as quantified by isobaric tags for relative and absolute quantitation (iTRAQ) and confirmed the reliability of our Proteome Profiling analysis. Our results indicated that IR related dysregulation of protein expression might participated in tumorigenesis and malignant phenotype of hepatocarcinoma cells.

Value of biomarkers in epithelial-mesenchymal transition models of liver cancer under different interventions: a meta-analysis.

Aim: A meta-analysis was conducted to evaluate the effectiveness of crucial biomarkers in HepG2 cells during epithelial-mesenchymal transformation induced by multiple interventions. Methods: PubMed, Web of Science, Embase, China National Knowledge Infrastructure, Chinese Biomedical Literature Database, Wan Fang Data and VIP databases were systematically searched from inception to 14 June 2020, by two independent reviewers. Results: A total of 58 studies were included in the meta-analysis. E-cadherin, N-cadherin and vimentin performed well under medicinal interventions. E-cadherin worked well under genetic interventions. E-cadherin and N-cadherin also performed significantly well under tumor microenvironment interventions. Under ncRNA interventions, the expression of E-cadherin significantly changed. Conclusion: Different sets of biomarkers should be selected under various interventions based on their performance.

Global Analysis of miRNA Signature Differentially Expressed in Insulin-resistant Human Hepatocellular Carcinoma Cell Line.

Chemoresistance mediated by insulin resistance (IR) in HCC has already been validated. However, the underlying mechanism, especially the involvement of microRNAs (miRNAs) was unelucidated. In this study, miRNA microarrays and bioinformatics methods were employed to determine the dysregulation of miRNA by IR in HCC cells, and quantitative RT-PCR (qRT-PCR) was applied to valid the miRNA array data. Of all the 2006 miRNAs screened, 32 miRNAs were found up or down regulated between the HepG2/IR cells and its parental cells. Further literature mining revealed that some of these miRNAs may function as oncogenes or tumor suppressors that contribute to tumor progression, recurrence, and metastasis which eventually lead to chemotherapeutic resistance. Interestingly, bioinformatics analysis by Gene Ontology (GO) enrichment pathway indicating that function of the predicted target genes of these dysregulated miRNAs were significantly enriched in the processes related with biosynthesis, catabolism, modification etc., and Kyoto Encyclopedia of Genes and Genomes (KEGG) mapping showed that the biological regulatory mechanisms were integrated in cancer-related pathways. Moreover, we also constructed a network which connected the differentially expressed miRNAs to target genes, GO enrichments and KEGG pathways to reveal the hub miRNAs, genes and pathways. Collectively, our present study demonstrated the possible miRNAs and predicted target genes involving in the pathophysiology of insulin resistant HCC, providing novel insights into the molecular mechanisms of multidrug resistance in the insulin resistant HepG2 cells.

A polysaccharide from the dried rhizome of Drynaria fortunei (Kunze) J. Sm. prevents ovariectomized (OVX)-induced osteoporosis in rats.

In the present study, a homogenous polysaccharide (DFPW) was isolated and purified from the dried rhizome of Drynaria fortunei, and its protective effect against osteoporosis was investigated in ovariectomized (OVX) rats. Histological analysis indicated that oral administration of DFPW (100 and 400 mg/kg) for 12 weeks significantly improved trabecular bone mass, as demonstrated by the increase in trabecular area, trabecular thickness and its number in OVX rats. Furthermore, the decline of bone mineral density and bone mineral content including Ca, P and Mg induced by OVX was reversed by the DFPW administration. This function was achieved by the decreased levels of the bone turnover markers, such as serum ALP, urinary deoxypyridinoline (DPD), Ca and P excretions. Besides, DFPW improved biomechanical parameters (maximum load, energy, Young's, modulus and maximum stress) to strengthen the hardness and strength femoral diaphysis in OVX rats. These results strongly suggested that DFPW might be a hopeful alternative therapeutics to treat postmenopausal osteoporosis.

Environmental inhibitors of the expression of cytochrome P450 17A1 in mammals.

Cytochrome P450 17A1 (CYP17A1; EC: 1.14.14.19) is a critically important bifunctional enzyme with nicotinamide adenine dinucleotide phosphate (NADPH) as its cofactor that catalyzes the formation of all endogenous androgens. Its hydroxylase activity catalyzes the 17α-hydroxylation of pregnenolone (PREG)/progesterone (P4) to 17α-OH-pregnenolone/17α-OH-progesterone, and its 17,20-lyase activity converts 17α-OH-pregnenolone/17α-OH-progesterone to dehydroepiandrosterone/androstenedione. Androgens are required for male reproductive development, so androgen deficiency resulting from CYP17A1 inhibition may lead to reproductive disorders. There has been some advances on the study of environmental chemicals inhibiting mammalian CYP17A1 expression but no related review was available so we think it now necessary to review their characteristics and inhibiting properties.

Protective effects of Dipsacus asper polysaccharide on osteoporosis in vivo by regulating RANKL/RANK/OPG/VEGF and PI3K/Akt/eNOS pathway.

A homogenous polysaccharide (DAP), with a molecular weight of 2.61 × 104 Da, was isolated from the roots of Dipsacus asper Wall. Gas chromatography (GC) indicated that DAP was composed of galactose and mannose with a molar ratio of 1:1. The purpose of this study was to evaluate the effect of DAP on the progress of bone loss in the ovariectomized (OVX) rat model of osteoporosis. Administration of DAP (50 and 200 mg/kg/body wt. day) for 12 weeks significantly prevented OVX-induced bone loss, biomechanical reduction, the body weight gain, the loss of the uterus weight, as well as increased U-Ca/Cr, U-P/Cr, ALP, TRAP, OC and DPD/Cr levels in rats, which was further supported by the histopathological examinations. Furthermore, we found that the mechanism by which DAP elicited anti-osteoporotic effects was mediated by up-regulation of VEGF and OPG, but down-regulation of RANK and RANKL in both protein and mRNA expression in OVX rats, as well as the activation of PI3K/Akt/eNOS signaling pathway, indicating that DAP can be clinically used as a potential alternative medicine for the prevention and treatment of postmenopausal osteoporosis.

Knockdown of lncRNA XIST suppresses osteosarcoma progression by inactivating AKT/mTOR signaling pathway by sponging miR-375-3p.

BACKGROUND: Osteosarcoma (OS) is one of the most common bone tumors in adolescents and young adults. Emerging evidence suggested ncRNA (lncRNA and miRNA) are closely associated with cell progression, apoptosis and autophagy. However, the role of regulatory network between ncRNA and mRNA in OS has not been fully verified. METHODS: lncRNA XIST, miRNA expression were detected by qRT-PCR. The protein expression of LC3, p62, AKT, p-AKT, mTOR and p-mTOR was measured by western blot. MTT assay and flow cytometry were applied to measure cell proliferation and apoptosis. Luciferase assay was used to ensure the relationship between lncRNA, miRNA and mRNA. GFP-LC3 cells were observed using fluorescence microscope. RESULTS: XIST expression was up-regulated but miR-375-3p was down-regulated in OS tissues and cells. Luciferase assay results demonstrated that miR-375-3p was a target of XIST and mTOR was a target mRNA of miR-375-3p. In addition, knockdown of XIST and mTOR inhibited OS cell proliferation and autophagy, but induced apoptosis. Knockdown of XIST could reverse the effect of miR-375-3p inhibitor on OS cells. The effects of si-mTOR of OS cells could be reversed by silencing miR-375-3p. Moreover, knockdown of XIST inhibited AKT/mTOR signaling pathway via sponging miR-375-3p. CONCLUSION: Knockdown of XIST inhibited cell growth and autophagy but induced cell apoptosis by suppressing the AKT/mTOR signaling pathway by sponging miR-375-3p.

Suppressive oligodeoxynucleotide-induced dendritic cells rein the aggravation of osteoarthritis in mice.

OBJECTIVE: To explore the effect of suppressive oligodeoxynucleotide-induced dendritic cells (S-DCs) in the osteoarthritis (OA) therapy. METHODS: S-DCs were prepared from splenic CD11c + cells by in vitro culture with suppressive oligodeoxynucleotide. The function and phenotypes of S-DCs were measured by ELISA and flow cytometry. The innate immune signaling pathways were detected by western blotting in the non-treated DCs and S-DCs upon stimulation. In vivo, we employed an iodoacetate-induced OA mice model. S-DCs were transferred by intravenous route. The weight bearing of mice was evaluated and pro-inflammatory factors in OA joint were measured by real-time PCR. Treg cell ratio and CD4 + IL10+ cells in spleen were detected by flow cytometry at day 5 post OA induction. RESULTS: The S-DCs showed less inflammatory phenotypes upon stimulation. The expression of pro-inflammatory cytokines and mature makers in the S-DCs were blunt, due to the impaired innate immune signal transduction. In an iodoacetate-induced OA model, transfer of S-DCs significantly controlled the process of OA. Restricted inflammatory responses were observed in the joint of S-DC recipients. Moreover, after S-DC transfer, Tregs and CD4 + IL10+ cells were mounted in the spleen. CONCLUSION: Transfer of suppressive oligodeoxynucleotides-induced autologous DCs may represent a potential agent to control the aggravation of OA in patients.

Clindamycin Administration Increases the Incidence of Collagen-Induced Arthritis in Mice Through the Prolonged Impact of Gut Immunity.

The profound influence of gut flora on host immune system and its link with autoimmune disorders have been established. However, the role of certain antibiotic in progression of autoimmune disorder is still confusing. Here, we employed a collagen-induced arthritis (CIA) model to explore the role of clindamycin administration in different scenarios. In the first scenario, mice treated with antibiotics for 4 weeks were performed with the induction of CIA immediately. The results showed that clindamycin administration promoted the incidence and severity of CIA, while the recipients of vancomycin showed completed tolerance. We also found that increased gut-associated Th1 and Th17 cells might be related to the subsequent expansion of collagen-specific immune response. In the second scenario, mice treated with antibiotics for 4 weeks were performed with CIA induction 4 weeks later. Notably, clindamycin administration showed a prolonged impact on the incidence and severity of CIA, as well as the gut immunity as compared to vancomycin administration. In addition, antibody depletion of integrin α4ß7 systemically resulted in an impaired CIA response, underlining the influence of gut immunity. In the mice that received clindamycin, the abundance of anaerobic bacteria was significantly decreased and showed little recovery at 4 weeks later. Our observations highlighted the different characteristics of antibiotic administration on the development of autoimmune disorders and indicated its link with gut immunity.

A multivariate analysis of prognostic determinants for stages II and III colorectal cancer in 141 patients.

BACKGROUND: Previous prognosis analyses of colorectal cancer (CRC) patients with stage II and III disease were done as separate categories. The purpose of this study was to analyze prognostic factors associated with survival in a group of patients who underwent radical resection of stages II and III CRC. METHODS: A retrospective review was performed for 141 consecutive stages II and III patients who had undergone radical resection of colorectal adenocarcinoma between May 2003 and November 2003. Univariate and multivariate analyses were performed to assess the effect of record variables on disease free survival and overall survival. RESULTS: The median follow-up time was 59 months, and the 3- and 5-year survival rates were 76% and 68%, respectively. Four factors were independently associated with a worse disease-free survival: diabetes (hazard ratio (HR) 2.338; 95% confidence interval (CI) 1.011 - 5.407), expression of cyclooxygenase-2 (Cox-2) (HR 0.335; 95%CI 0.126 - 0.888), expression of matrix metalloproteinases 2 (MMP-2) (HR 0.233; 95%CI 0.101 - 0.541), expression of vascular endothelial growth factor (VEGF) (HR 0.295; 95%CI 0.088 - 0.996). Four factors were independently associated with a worse overall survival: lymph nodes metastasis (HR 1.67; 95%CI 1.29 - 2.14), Cox-2 positive (HR 0.056; 95%CI 0.247 - 0.731), MMP-2 positive (HR 0.398; 95%CI 0.190 - 0.836), VEGF (HR 0.364; 95%CI 0.090 - 0.716). CONCLUSIONS: Diabetes, expression of Cox-2, MMP-2 and VEGF were independently associated with a worse disease- free survival. Lymph nodes metastasis, expression of Cox-2, MMP-2 and high level of VEGF predicted a poor overall survival.

Expression of COX-2, MMP-2 and VEGF in stage II and III colorectal cancer and the clinical significance.

OBJECTIVES: To investigate the expression of COX-2, MMP-2 and VEGF in colorectal cancer and the clinical/pathological significance. METHODS: Stage II and III colorectal cancer patients (149 cases) that received radical resection between May 2003 and November 2008 and who had complete clinical and pathological data, were recruited in this study. Expression of COX-2, MMP-2 and VEGF were detected by immunohistochemistry. RESULTS: The positive rate of COX-2, MMP-2, and VEGF expression was 60.4%, 50.3% and 69.1%, respectively. COX-2 correlated with stage, lymph node metastasis, postoperative recurrence and metastasis, and survival rate; MMP-2 correlated with intestinal wall invasion, stage, number of lymph node metastasis, postoperative recurrence and metastasis, and survival rate; VEGF correlated with preoperative serum levels of CEA and CA199, postoperative recurrence and metastasis, and survival rate; the positive rate of COX-2, MMP-2 and VEGF co-expression was 32.9%, which correlated with stage, number of lymph node metastasis, preoperative serum level of CEA, postoperative recurrence and metastasis, and survival rate. CONCLUSION: The expression of COX-2, MMP-2 and VEGF in colorectal cancer plays a synergistic promoting effect on the malignant biological behavior of tumors, which could be used as a marker to determine the malignant progression, invasion and metastasis, and prognosis of the tumor.

[Prognostic analysis of 443 cases of stage II colorectal cancer and the value of adjuvant chemotherapy].

BACKGROUND AND OBJECTIVE: Prognosis of stage II colorectal cancer varies. Whether or not to perform adjuvant chemotherapy on patients with stage II colorectal cancer is controversial. This study was to explore the prognostic factors for the patients with stage II colorectal cancer and evaluate the effect and the necessity of adjuvant chemotherapy. METHODS: Between January 2000 and January 2005, 443 patients with stage II colorectal cancer receiving radical surgery at Sun Yat-sen University Cancer Center were retrospectively analyzed. The overall survival rate and survival curve were analyzed using the Kaplan-Meier method and the log-rank test. The univariate and multivariate prognostic analyses were performed by the Cox regression model. Patients with or without chemotherapy (Xelox/Folfox regimen) with high-risk factors were analyzed respectively. RESULTS: The median follow-up time was 59 months, and the 3-and 5-year survival rates were 88.4% and 82.5%, respectively. Univariate analysis showed that intestinal obstruction or perforation, diabetes mellitus, inadequate surgical margin, and the number of sampled nodes < 9 were poor prognostic factors. Patients with intestinal obstruction or perforation, the number of sampled nodes < 9 achieved higher 5-year survival (80% and 86%) undergoing adjuvant chemotherapy than those receiving surgery alone (67% and 64%). CONCLUSIONS: The prognosis of colorectal cancer patients with intestinal obstruction or perforation, diabetes mellitus, inadequate surgical margin, and the number of sampled nodes < 9 are relatively poor. Adjuvant chemotherapy is recommended to patients with intestinal obstruction, perforation or sampled nodes < 9.