PLOD3 regulates the expression of YAP1 to affect the progression of non-small cell lung cancer via the PKCδ/CDK1/LIMD1 signaling pathway.

Procollagen-lysine, 2-oxoglutarate 5-dioxygenase (PLOD3) is a crucial oncogene in human lung cancer, whereas protein kinase C δ (PKCδ) acts as a tumor suppressor. In this study, we aimed to explore the regulation by PLOD3 on the expression of YAP1 to affect the progression of non-small cell lung cancer (NSCLC) via the PKCδ/CDK1/LIMD1 signaling pathway. We found that PLOD3, CDK1, and YAP1 were highly expressed, while LIMD1 was poorly expressed in NSCLC tissues. Mechanistic investigation demonstrated that silencing PLOD3 promoted the cleavage of PKCδ in a caspase-dependent manner to generate a catalytically active fragment cleaved PKCδ, enhanced phosphorylation levels of CDK1, and LIMD1 but suppressed nuclear translocation of YAP1. Furthermore, functional experimental results suggested that loss of PLOD3 led to increased phosphorylation levels of CDK1 and LIMD1 and downregulated YAP1, thereby suppressing the proliferation, colony formation, cell cycle entry, and resistance to apoptosis of NSCLC cells in vitro and inhibiting tumor growth in vivo. Taken together, these results show that PLOD3 silencing activates the PKCδ/CDK1/LIMD1 signaling pathway to prevent the progression of NSCLC, thus providing novel insight into molecular targets for treating NSCLC.

Intermittent hypoxia-induced downregulation of microRNA-320b promotes lung cancer tumorigenesis by increasing CDT1 via USP37.

Obstructive sleep apnea-hypopnea (OSAH) is correlated with an increased incidence of lung cancer. In our study, we explored the functional roles of microRNAs (miRNAs) in lung cancer patients that were complicated with OSAH involving the deubiquitination enzyme. The miR-320b expression pattern in lung cancer tissues and cells was determined. The interactions between ubiquitin-specific peptidase 37 (USP37) and miR-320b were evaluated by a dual-luciferase reporter gene assay, whereas USP37 and Cdc10-dependent transcript 1 (CDT1) was assessed by co-immunoprecipitation and immunofluorescence. After the induction of intermittent hypoxia (IH), a gain-of function approach was performed to investigate roles of miR-320b, USP37, and CDT1 in lung cancer cell proliferation and invasion. In addition, nude mouse xenograft models were used to study their effects on tumor growth in vivo. miR-320b was poorly expressed in lung cancer patients with OSAH. IH treatment downregulated the expression of miR-320b but promoted the proliferation and invasion capabilities of lung cancer cells, both of which were suppressed by the overexpression of miR-320b through decreasing USP37. USP37 interacted with and deubiquitinated CDT1 to protect it from proteasomal degradation. Our study uncovered that IH-induced downregulation of miR-320b promoted the tumorigenesis of lung cancer by the USP37-mediated deubiquitination of CDT1.

SOX2-Upregulated microRNA-30e Promotes the Progression of Esophageal Cancer via Regulation of the USP4/SMAD4/CK2 Axis.

Esophageal cancer (EC) is a highly aggressive disease, and its progression involves a complex gene regulation network. Transcription factor SOX2 is amplified in various cancers including EC. A pathway involving SOX2 regulation of microRNAs (miRNAs) and their target genes has been previously revealed. This study aims to delineate the ability of SOX2 to influence the EC progression, with the involvement of miR-30e/USP4/SMAD4/CK2 axis. SOX2 expression was first examined in the clinical tissue samples from 30 EC patients. Effects of SOX2 on proliferation, migration, and invasion alongside tumorigenicity of transfected cells were examined by means of gain- and loss-of-function experiments. EC tissues and cells exhibited high expression of SOX2, miR-30e, and CK2 and poor expression of USP4 and SMAD4. Mechanistically, SOX2 was positively correlated with miR-30e and upregulated the expression of miR-30e. miR-30e specifically targeted USP4, which induced deubiquitination of SMAD4 and promoted its expression. Meanwhile, SMAD4 was enriched in the CK2 promoter region and thus inhibited its expression. SOX2 stimulated EC cell proliferative, invasive, and migratory capacities in vitro and tumor growth in vivo by regulating the miR-30e/USP4/SMAD4/CK2 axis. Collectively, our work reveals a novel SOX2-mediated regulatory network in EC that may be a viable target for EC treatment.

MicroRNA let-7i Inhibits Histone Lysine Demethylase KDM5B to Halt Esophageal Cancer Progression.

Recent studies have suggested that microRNA let-7i is a tumor suppressor in human cancers, including esophageal cancer, but its underlying mechanism is not yet fully understood. We investigated the role and mechanisms of let-7i in the progression of esophageal cancer. We first showed that let-7i was downregulated in esophageal cancer tissues and cells and then linked its low expression to cancer progression. Bioinformatic analysis predicted KDM5B as a target gene of let-7i, which was confirmed by a dual-luciferase reporter assay. Loss- and gain-of function approaches were adopted to examine the interactions of let-7i, KDM5B, SOX17, and GREB1 in vitro and in vivo. Overexpression of let-7i suppressed esophageal cancer cell proliferation and invasion and promoted apoptosis. Mechanistic investigation showed that let-7i targeted and inhibited KDM5B expression, whereas KDM5B enhanced H3K4me3 at the SOX17 promoter region. Overexpression of let-7i suppressed the expression of GREB1 in esophageal cancer cells by regulating the KDM5B/SOX17 axis in vivo and in vitro. Taken together, our findings reveal the tumor-suppressive properties of let-7i in esophageal cancer in association with an apparent KDM5B-dependent SOX17/GREB1 axis. This study offers a potential prognostic marker and therapeutic target for esophageal cancer.

A new technology for reducing anastomotic fistula in the neck after esophageal cancer surgery.

BACKGROUND: To explore the risk factors and prevention methods of cervical mechanical anastomotic fistula and stenosis after the radical resection of esophageal cancer. METHODS: From March 2018 to November 2018, 128 patients undergoing mechanical anastomosis of esophageal cancer were selected from the Department of Thoracic Surgery of The First Affiliated Hospital of Zhengzhou University. All the enrolled patients were operated on using the Mckeown method, and a retrospective study was conducted. Data for preoperative and postoperative test indices, intraoperative embedding materials, postoperative complications, and preoperative and postoperative treatment were collected, and the relationship between various factors and the incidence of cervical anastomotic fistula and stenosis was analysed. Univariate analysis was conducted using t tests or Fisher's exact probability method, and multivariate analysis was conducted using logistic regression models. RESULTS: All 128 patients successfully underwent surgery without dying. The enrolled patients were evaluated using the Stooler classification, with 28 patients having grade 0, 41 patients having grade 1, 34 patients having grade 2, 21 patients having grade 3, and 4 patients having grade 4 stenosis. Patients with stenosis of grade 3 or above had obvious choking sensation, which could only be relieved by balloon dilation. Symptoms in all patients with stenosis were relieved by balloon dilation. There were no significant differences between the two groups regarding embedding materials, preoperative choking history, history of alcohol consumption, history of hypertension, history of coronary heart disease, history of diabetes, postoperative calcium concentration, average albumin concentration, average platelet concentration, body mass index, anastomotic fistula, preoperative chemotherapy, postoperative chemotherapy, or postoperative cough (P>0.05). There were significant differences in postoperative reflux (χ2=11.338, P<0.05) and scar constitution (χ2=12.497, P<0.05). The effects of embedding materials in patients with anastomotic fistula were significantly different (χ2=4.372, P<0.05). CONCLUSIONS: Postoperative reflux and scar constitution may be risk factors for postoperative anastomotic stenosis after resection of esophageal cancer. There was almost no difference in the effects on esophageal anastomotic stenosis between embedding materials and the omentum majus, but Neoveil® may have certain advantages in preventing cervical anastomotic fistula, and thus may have certain clinical application value.

LINC00184 silencing inhibits glycolysis and restores mitochondrial oxidative phosphorylation in esophageal cancer through demethylation of PTEN.

BACKGROUND: Total lesion glycolysis has been reported to be a satisfactory predictor of survival in patients with locally advanced esophageal cancer (EC). The aim of the present study is to investigate the function of long intergenic non-protein coding RNA 184 (LINC00184) on the EC cell glycolysis and mitochondrial oxidative phosphorylation (OXPHOS). METHODS: The expression of LINC00184 was determined to be highly expressed and PTEN was poorly expressed in EC tissues and cells by RT-qPCR. In order to evaluate the effects of LINC00184 on cellular process in vitro and in vivo, gain- and loss-of-function approaches were performed to alter the expression of LINC00184 and PTEN in EC cells. RESULTS: Silencing of LINC00184 was observed to inhibit the proliferation, migration, invasion, colony formation, and glycolysis of EC cells and tumour growth, while the mitochondrial OXPHOS was restored. By recruiting DNMT1, LINC00184 enhanced the promoter methylation of PTEN. Inhibition of PTEN promoter methylation suppressed EC glycolysis, whereas, improved mitochondrial OXPHOS. Mechanically, LINC00184 modulated glycolysis and mitochondrial OXPHOS in EC cells through induction of the Akt phosphorylation. After blockage of Akt signaling pathway by an Akt inhibitor, LY294002, the regulatory effects of LINC00184 on the glycolysis and mitochondrial OXPHOS of EC cells were reversed. CONCLUSION: Taken together, the LINC00184/PTEN/Akt axis mediates glycolysis and mitochondrial OXPHOS in EC cells. This study highlighted a potential intervention target for treating EC.

Genetic variation of lncRNA GAS5 contributes to the development of lung cancer.

Lung cancer remains the leading cause of cancer-related deaths throughout the world. In spite of great effort for the research of carcinogenesis, the molecular mechanisms of lung cancer remain unclear. In current study, we investigated the possible association between susceptibility of lung cancer and GAS5 rs145204276, which showed contradictory roles in carcinogenesis of colorectal cancer and hepatocellular carcinoma. We found that the del allele was significantly associated with 21% decreased risk of lung cancer (OR=0.79; 95% CI=0.66-0.93; P value = 0.006). Compared with the genotype ins/ins, both the genotype ins/del (OR=0.78; 95% CI=0.62-0.99) and del/del (OR=0.59;95% CI=0.39-0.89) showed decreased susceptibility of lung cancer. Real-time PCR analysis found that the expression levels of lncRNA GAS5 in lung cancer tissues were significantly lower than those in the corresponding normal tissues (P<0.01). Also the relative GAS5 expression level in samples with del/del genotype was significantly higher than that in samples with ins/del and ins/ins genotype (P<0.01). Taken together, our findings provided strong evidence for the hypothesis that GAS5 rs145204276 were significantly associated with the susceptibility of lung cancer, and GAS5 functions as a tumor suppressor in carcinogenesis of lung cancer.

[Analysis of Pathological Types and Clinical Epidemiology of 6,058 Patients with Lung Cancer].

BACKGROUND AND OBJECTIVE: The epidemiology of lung cancer changes with time, region, and population. The pathological and clinical characteristics of patients with primary bronchial lung cancer were retrospectively analyzed to understand the epidemic trend of lung cancer in recent years. METHODS: We reviewed the clinical data of patients with primary bronchogenic carcinoma. These patients, who are permanent residents of Henan province, were treated in the First Affiliated Hospital of Zhengzhou University from 2012 to 2014. The distributions of gender, age, urban/rural residency, smoking history, drinking history, operation history, and histological types of tumor were compared among the patients and analyzed. RESULTS: A total of 6,058 cases of lung cancer were collected, including 1,495 cases in 2012, 2,070 cases in 2013 and 2,493 cases in 2014. The proportions of male to female patients with lung cancer were 2.26:1, 2.29:1 and 2.20:1 in 2012 to 2014, respectively (χ²=0.367, P=0.832). The age of onset was 60-69 years old, which accounted for 35.72% of the patients, and exhibited statistically significant differences between male and female patients (χ²=109.848, P<0.001). Moreover, differences in the pathological types of patients with lung cancer were statistically significant in 2012-2014 (χ²=25.344, P=0.013). Lung adenocarcinoma accounted for 38.5% and 73.63% in male and female patients, respectively, and the difference was statistically significant (χ²=562.382, P<0.001). Adenocarcinoma also accounted for 60.62%, 56.59%, 49.84%, 45.15%, 47.03% and 41.25% in all ages of patients with lung cancer; the difference was statistically significant (χ²=48.886, P<0.001). Furthermore, the proportions of various pathological types in rural and urban patients were statistically significant among the five regions of Henan Province. The proportions of squamous cell carcinoma were the highest in smoking and drinking patients, accounting for 38.39% and 37.37%, respectively. About 15.4% of the patients included in the study received surgical treatment. CONCLUSIONS: The proportion of adenocarcinoma in all types of lung cancer has increased in recent years, whereas the incidence of squamous cell carcinoma has declined. Adenocarcinoma is common in male patients with lung cancer as well as in young- and middle-aged patients. Squamous cell carcinoma is associated with smoking and drinking in male patients.

Side population cells separated from A549 lung cancer cell line possess cancer stem cell-like properties and inhibition of autophagy potentiates the cytotoxic effect of cisplatin.

Recent studies have suggested that cancer stem cells (CSCs) may be responsible for tumorigenesis and contribute to resistance to chemotherapy. Side population (SP) cells are thought to be enriched for CSCs in most types of human tumors. Therefore, the aim of the present study was to sort SP cells using an A549 lung cancer cell line, identify the cancer stem cell-like properties of SP and determine the role of autophagy in the survival of SP cells of lung cancer. SP cells were isolated by fluorescence-activated cell sorter (FACS) from A549 lung cancer cells, and the CSC-like properties were verified through confocal fluorescence imaging, sphere formation assays, cell proliferation and colony formation assay, gene expression in vitro and tumor formation in vivo. The role of autophagy in the survival of SP cells was assessed by western blotting and flow cytometric analysis. A549 lung cancer cells contained 1.10% SP cells. SP cells showed higher abilities of sphere and colony formation, cell proliferation and self-renewal. Moreover, compared to non-SP, SP cells demonstrated a higher mRNA expression of stem cell markers (MDR1, ABCG2 and OCT-4). The clone formation efficiency of SP cells was significantly higher than that non-SP cells under the same conditions. Expression of autophagosomes in SP cells was markedly lower than that in non-SP cells. However, the level of autophagy in SP cells was found to be markedly increased in the presence of cisplatin. In addition, inhibition of autophagy enhanced the effects of apoptosis induced by cisplatin. SP cells from the A549 lung cancer cell line possessed the properties of CSCs and were used to investigate the further characteristics of lung CSCs. SP cells were more resistant to chemotherapy and inhibition of autophagy enhanced the effects of apoptosis induced by the chemotherapeutic agent, cisplatin. These results may provide insight into novel therapeutic targets.

Identification of potential plasma biomarkers for esophageal squamous cell carcinoma by a proteomic method.

Among malignant tumors, the mortality rate of esophageal squamous cell carcinoma (ESCC) ranks sixth in the world. Late-stage diagnosis of ESCC increases the mortality. Therefore, more effective biomarkers for early diagnosis of ESCC are necessary. Unfortunately, appropriate biomarkers for clinical diagnosis and prognosis have not been identified yet. However, recent progresses in quantitative proteomics have offered opportunities to identify plasma proteins as biomarkers for ESCC. In the present study, plasma samples were analyzed by differential in-gel electrophoresis (DIGE) and differentially expressed proteins were identified by matrix assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS). A total of 31 proteins representing 12 unique gene products were identified, in which 16 proteins were up-regulated and 15 down-regulated in tumors. The up-regulated proteins were alpha-2-HS-glycoprotein (AHSG), leucine-rich alpha-2-glycoprotein (LRG), zinc-alpha-2-glycoprotein, alpha-1-antichymotrypsin, complement factor I and complement C4-B, whereas the down-regulated proteins were serum albumin, Ig alpha-2 chain C region, alpha-1-antitrypsin, fibrinogen gamma chain, haptoglobin and hemoglobin subunit alpha. Among all the differentially expressed proteins, AHSG and LRG were validated by ELISA. The results were consistent with the data from the proteomics results, further suggesting that AHSG and LRG may be employed as potential biomarkers for the early diagnosis of ESCC. In summary, this study was the first time to use DIGE combined MALDI-TOF/TOF platform to identify the potential plasma biomarkers for ESCC. The plasma AHSG and LRG showed great potential for ESCC screening.

Proteasome inhibitor MG132 inhibits the proliferation and promotes the cisplatin-induced apoptosis of human esophageal squamous cell carcinoma cells.

Comprehensive treatment based on chemotherapy is regarded as the first-line treatment for patients with unresectable or metastatic esophageal squamous cell carcinoma (ESCC). However, chemoresistance is common among patients with ESCC. Therefore, there is a need to explore new therapeutic strategies or adjuvant drugs. One promising possibility is to use dietary agents that can increase tumor cell sensitivity to drugs. In this study, we initially investigated the antitumor activity of proteasome inhibitor MG132 in vitro and in vivo. Effects of MG132 on the enhancment of the anticancer functions of cisplatin were then investigated in human esophageal cancer EC9706 cells in relation to apoptosis and cell signaling events. Exposure of cells to MG132 resulted in a marked decrease in cell viability in a dose- and time-dependent manner. Administration of MG132 markedly inhibited tumor growth in the EC9706 xenograft model. MG132 significantly enhanced cisplatin-induced apoptosis in association with the activation of caspase-3 and -8. These events were accompanied by the downregulation of NF-κB, which plays a key role in cell apoptosis. Taken together, these findings demonstrate a novel mechanism by which proteasome inhibitor MG132 potentiates cisplatin-induced apoptosis in human ESCC and inhibitory activity of tumor growth of the EC9706 xenograft model.

Insulin enhances apoptosis induced by cisplatin in human esophageal squamous cell carcinoma EC9706 cells related to inhibition of autophagy.

BACKGROUND: Chemoresistance is common among patients with esophageal squamous cell carcinoma (ESCC). We investigated the effect and mechanism of insulin on enhancing anticancer functions of cisplatin in human esophageal cancer cell line EC9706. METHODS: The viability of EC9706 cells exposed to cisplatin was assessed using MTT assay. The times T1, when the number of living cells reached a plateau and T2, when the number of living cells reached a new plateau after the addition of insulin were found. T1 and T2 plateau cells were stained by Annexin V-FITC/PI and monodansylcadaverin (MDC). Fluorescent microscopy was used to observe the expression of apoptosis and autophagy intuitively. Apoptotic ratio and fluorescent intensity were analysed by flow cytometry (FCM) quantitatively. Western blotting analysis was used to estimate the protein expression levels of AKT, mTOR, PI3K, PTEN, autophage related indicator LC3-II and autophage related protein Beclin1 changes that occurred in the course of treatment. RESULTS: A larger number of typical autophagosomes were detected in EC9706 cells exposed to cisplatin. Insulin can increase the apoptosis induced by cisplatin. Apoptotic ratio of T1 plateau cells ((32.6 ± 4.3)%) is significantly less than T2 plateau ((47.5 ± 5.6)%). MDC fluorescent intensity at T1 plateau (104.9 ± 13.2) was significantly higher than intensity at T2 plateau (82.6 ± 10.3). After cotreatment with insulin, the expression level of LC3-II, Beclin1 and PTEN in T2 plateau cells were significantly downregulated, but AKT, mTOR and PI3K expressions significantly upregulated compared with T1 plateau. CONCLUSIONS: Insulin could enhance cisplatin-induced apoptosis in human esophageal squamous cell carcinoma EC9706 cells related to inhibition of autophagy. The activation of PI3K/Akt/mTOR signaling pathway induced by insulin resulted in the suppression of autophagy in EC9706 cells, which may be attributed to the anticancer effects of cisplatin.

Biomechanical comparison of transforaminal lumbar interbody fusion with 1 or 2 cages by finite-element analysis.

BACKGROUND: Anterior lumbar interbody fusion and posterior lumbar interbody fusion with 1 cage have been shown to have similar biomechanics compared with the use of 2 cages. However, there have been no reports on the biomechanical differences between using 1 or 2 cages in transforaminal lumbar interbody fusion (TLIF) surgery. OBJECTIVE: To determine the biomechanical differences between the use of 1 or 2 cages in TLIF by finite-element analysis. METHODS: Three validated finite-element models of the L3-L5 lumbar segment were created (intact model and single- and paired-cage TLIF models). To study the biomechanics, a compressive preload of 400 N over 7.5 N-m was applied to the superior surfaces of the L3 vertebral body to simulate flexion, extension, rotation, and lateral bending. RESULTS: There was no significant difference in the range of motion between single-cage and paired-cage TLIF models, < 1° for all loading cases. Cage stress was high in the single-cage TLIF model under all loading conditions. Bone graft stress was high in the single-cage TLIF model. Pedicle screw stress was higher in the single-cage compared with the paired-cage TLIF. CONCLUSION: Single-cage TLIF approximates biomechanical stability and increases the stress of the bone graft. The use of a single cage may simplify the standard TLIF procedure, shorten operative times, decrease cost, and provide satisfactory clinical outcomes. Thus, single-cage TLIF is a useful alternative to traditional 2-cage TLIF.