Regulation of the PD-1/PD-L1 Axis and NK Cell Dysfunction by Exosomal miR-552-5p in Gastric Cancer.

OBJECTIVE: NK cells play a vital role in tumor immune resistance. Various factors affect NK cell activity. While NK cell dysfunction has been observed in numerous malignancies, the underlying mechanisms in gastric cancer remain unclear. METHOD: Flow cytometry was used to identify the phenotypic distribution and expression of activated receptors on NK cells. ELISA was used to determine the expression of cytokines. We examined the expression of NK cell-related genes and explored their association with survival and prognosis. Additionally, we conducted PCR detection of miR-552-5p expression levels in plasma exosomes of patients and investigated its correlation with phenotypic distribution and activated receptors. We used flow cytometry and ELISA to verify the role of miR-552-5p in NK cell dysfunction. Furthermore, we investigated the potential role of PD-1/PD-L1 in regulating NK cell dysfunction in patients' cells. RESULTS: We observed a significant decrease in the percentage of NKG2D and NKp30 and IFN-γ and TNF-α in patients than in healthy volunteers. Patients with low levels of CD56, CD16, NKG2D, and NKP46 exhibited poorer survival prognoses. Moreover, increased expression levels of plasma exosomal miR-552-5p in patients were negatively associated with NK cell phenotypic distribution and activated receptor expression. MiR-552-5p downregulated the secretion of perforin, granzyme, and IFN-γ as well as the expression of NKp30, NKp46, and NKG2D. Additionally, it suppressed the cytotoxicity of NK cells. The inhibitory effect of miR-552-5p, on NK cell function was reversed when anti-PD-L1 antibodies were used. CONCLUSION: Exosomal miR-552-5p targets the PD-1/PD-L1 axis, leading to impaired NK cell function.

Downregulation of the enhancer of zeste homolog 1 transcriptional factor predicts poor prognosis of triple-negative breast cancer patients.

Background: Triple-negative breast cancer (TNBC) is the most malignant subtype of breast cancer and lacks effective biomarkers. This study seeks to unravel the expression status and the prospective transcriptional mechanisms of EZH1/EZH2 in TNBC tissue samples. Moreover, another objective of this study is to reveal the prognostic molecular signatures for risk stratification in TNBC patients. Methods: To determine the expression status of EZH1/EZH2 in TNBC tissue samples, microarray analysis and immunohistochemistry were performed on in house breast cancer tissue samples. External mRNA expression matrices were used to verify its expression patterns. Furthermore, the prospective transcriptional mechanisms of EZH1/EZH2 in TNBC were explored by performing differential expression analysis, co-expression analysis, and chromatin immunoprecipitation sequencing analysis. Kaplan-Meier survival analysis and univariate Cox regression analysis were utilized to detect the prognostic molecular signatures in TNBC patients. Nomogram and time-dependent receiver operating characteristic curves were plotted to predict the risk stratification ability of the prognostic-signatures-based Cox model. Results: In-house TMAs (66 TNBC vs. 106 non-TNBC) and external gene microarrays, as well as RNA-seq datasets (1,135 TNBC vs. 6,198 non-TNBC) results, confirmed the downregulation of EZH1 at both the protein and mRNA levels (SMD = -0.59 [-0.80, -0.37]), as is opposite to that of EZH2 (SMD = 0.74 [0.40, 1.08]). The upregulated transcriptional target genes of EZH1 were significantly aggregated in the cell cycle pathway, where CCNA2, CCNB1, MAD2L1, and PKMYT1 were determined as key transcriptional targets. Additionally, the downregulated transcriptional targets of EZH2 were enriched in response to the hormone, where ESR1 was identified as the hub gene. The six-signature-based prognostic model produced an impressive performance in this study, with a training AUC of 0.753, 0.981, and 0.977 at 3-, 5-, and 10-year survival probability, respectively. Conclusion: EZH1 downregulation may be a key modulator in the progression of TNBC through negative transcriptional regulation by targeting CCNA2, CCNB1, MAD2L1, and PKMYT1.

Fas-associated factor 1 inhibits tumor growth by suppressing Helicobacter pylori-induced activation of NF-κB signaling in human gastric carcinoma.

Loss of Fas-associated factor 1 (FAF1) may act as a pro-survival signal in diseased cells, but whether this is true in gastric carcinoma remains unclear. Here we report that FAF1 was expressed at low levels in gastric carcinoma tissues and cell lines, and its expression correlated with larger tumors, higher histology grade, higher TNM stage, tumor infiltration, and lymph node metastasis. Univariate analysis and survival curve analysis identified low FAF1 expression as a predictor of poor prognosis. FAF1 overexpression in HGC-27 gastric cancer cells induced cell apoptosis and inhibited cell proliferation and growth. It also reduced colony formation in vitro and tumor growth in mice. We found that Helicobacter pylori, a risk factor for gastric cancer, down-regulated FAF1 expression via NF-κB signaling. Knock-down of IKKß or p65 expression in gastric cancer cells reversed H. pylori-induced down-regulation of FAF1 expression and partially blocked H. pylori-induced secretion of inflammatory cytokines TNF-α and IL-8. Our results suggest that loss of FAF1 contributes to human gastric carcinogenesis by allowing H. pylori to activate NF-κB signaling.

The Relationship between Telomerase Activity and Clinicopathological Parameters in Colorectal Cancer: A Meta-Analysis.

BACKGROUND: Recently, accumulated research has found that the expression of telomerase activity (TA) was associated with colorectal cancer (CRC) advancement, whereas the TA prognostic effect in CRC patients is still controversial. AIMS: To investigate relationships between TA and CRC clinicopathological parameters. STUDY DESIGN: Meta-analysis study. METHODS: We searched published studies in databases, such as EMBASE, the Cochrane Library, PubMed, and Ovid databases (last search updated to October 2014) by meeting specified search criteria. The quality of the included studies was usually evaluated and a meta-analysis was implemented by Stata 12.0 software. We used an odds ratio (OR) with a 95% confidence interval (CI) to evaluate relationship strengths between TA and CRC clinicopathological parameters. RESULTS: In total, 11 studies (715 patients) were included to assess the relation between TA and metastasis-related parameters in CRC patients. The results indicate that a senior TA expression was connected with the existence of lymph node metastasis (180 patients; OR=2.85, 95% CI=1.40-5.81, p=0.004), and tumor site (522 patients; OR=2.93, 95% CI=1.29-6.67, p=0.010). However, a senior TA expression was not connected with tumor size (137 patients; OR=1.57, 95% CI=0.71-3.47, p=0.267), histological differentiation (570 patients; OR=1.28, 95% CI=0.78-2.09, p=0.332), depth of invasion (57 patients; OR=3.76, 95% CI=0.61-23.04, p=0.152), distant metastasis (123 patients; OR=1.76, 95% CI=0.54-5.74, p=0.346), and clinical stage of the cancer (543 patients; OR=1.59, 95% CI=0.74-3.38, p=0.232). CONCLUSION: This meta-analysis suggests that a positive TA was correlated with lymph node metastasis progression and tumor site of the CRC but did not correlate with other important clinicopathological parameters. TA can play a useful part in the prognosis and treatment of CRC patients, but further studies are required to confirm this.

Screening and preliminary validation of miRNAs with the regulation of hTERT in colorectal cancer.

The overexpression of human telomerase reverse transcriptase (hTERT) has been associated with the invasion and metastasis of colorectal cancer (CRC) and has received extensive attention, although the underlying mechanism involved remains unclear. The aim of the present study was to screen and preliminarily validate new tumor­suppressor microRNAs (miRNAs) that potentially inhibit hTERT expression and to assess its clinical significance. Screening for downregulated miRNAs in CRC tissues was performed by retrieving and analysing microRNA microarray data. miRNA candidates were then filtered by bioinformatics analysis. The expression of miRNAs candidates was verified by quantitative polymerase chain reaction in the CRC and corresponding normal tissues. Immunohistochemistry (IHC) was used for the detection of hTERT protein expression. Spearman's correlation coefficient between miRNA candidates and hTERT protein expression was calculated (r) to identify hTERT-targeting miRNAs. A survival analysis was performed to assess the prognostic significance of hTERT-targeting miRNAs in CRC. Eight miRNAs with the potential to interact with hTERT were predicted: miR­29c-3p, miR­124-3p, miR­133a-3p, miR­133b, miR-138-5p, miR-150-5p, miR-378a-3p and miR-422a, respectively. Following detection of the miRNAs using RT-qPCR, miR-29c-3p was excluded. miR-138-5p and miR-422a were observed to potentially interact with hTERT (r=-0.362, P=0.001; r=-0.306, P=0.005, respectively). The Kaplan-Meier survival curves demonstrating high- vs. low-expression group of miR­422a showed a highly significant difference in CRC patients (P=0.024), which suggests that the downregulation of miR-422a was associated with a poorer prognosis. The results indicated that miR-138-5p and miR-422a potentially inhibited hTERT expression in CRC, and suggest a potential application of miR­422a in prognosis prediction and CRC treatment.

RNAi-mediated knockdown of the c-jun gene sensitizes radioresistant human nasopharyngeal carcinoma cell line CNE-2R to radiation.

This study aimed to investigate the effect of RNA interference (RNAi)-mediated downregulation of the expression of the c-jun gene (a proto-oncogene) on the radiosensitivity of a radioresistant human nasopharyngeal carcinoma cell line (CNE-2R) and to validate its potential as an anticancer target. A lentiviral vector with c-jun small hairpin RNA (shRNA) was constructed and transfected into CNE-2R cells. The gene silencing efficiency of these recombinants was confirmed by RT-PCR and western blotting. Radiosensitivity, cell proliferation, cell cycle profile and apoptosis were assessed using colony formation assay, CCK-8 assay and flow cytometry, respectively. The lentiviral shRNA efficiently knocked down the expression of c-jun at both the mRNA and protein levels (P<0.05). c-jun-downregulated CNE-2R cells exhibited significantly decreased cell proliferation and enhanced radiosensitivity compared to the control group (P<0.05), and the effects were likely due to G2/M phase arrest and enhanced cell apoptosis. These data provide evidence that c-jun may be involved in the radioresistance of nasopharyngeal carcinoma (NPC) and knockdown of the c-jun gene may be a potential strategy to enhance the radiation sensitivity of NPC.

Evaluation of miR-29c, miR-124, miR-135a and miR-148a in predicting lymph node metastasis and tumor stage of gastric cancer.

MicroRNAs (miRNAs) are small noncoding RNA that have diverse functions in different biological process. The aim of this study was to evaluate the predictive ability of miR-29c, miR-124, miR-135a and miR-148a for lymph node metastasis (LNM) and tumor stage in gastric cancer. The expression of these miRNAs was detected and quantitated in gastric cancer tissues and in adjacent normal tissues from 60 patients by quantitative real-time reverse transcription-polymerase chain reaction. CT imaging and clinicopathologic characteristics of these patients were performed. The result of this study was that these miRNAs were down-regulated in gastric cancer tissues; The low expression of miR-124 and miR-135a in LNM group and tumor III-IV stages (P < 0.01) presented the potential correlation with LNM and tumor stage; The two miRNAs were highly correlated with r = 0.730. Receiver operating characteristic curve analysis showed that miR-124 had better predictive ability to identify LNM and tumor stage. It could discriminate non-LNM from LNM with 80.0% sensitivity and 80.0% specificity and discriminate tumor Ι-II stages from tumor III-IV stages with 71.9% sensitivity and 75.0% specificity at the best cut-off value of 0.0125. Compared with CT imaging, miR-124 had similar specificity (0.800 versus 0.900, P = 0.508) but higher sensitivity (0.800 versus 0.500, P = 0.022) for lymph node assessment; Combined of miR-124 and CT imaging, The sensitivity and specificity of assessing LNM were raised to 83.3% and 90.0% respectively. Taken together, miR-124 may be a predictor for LNM and tumor stage in gastric cancer.

Silencing of the hTERT gene by shRNA inhibits colon cancer SW480 cell growth in vitro and in vivo.

Human telomerase reverse transcriptase (hTERT) is the key enzyme responsible for synthesizing and maintaining the telomeres on the ends of chromosomes, and it is essential for cell proliferation. This has made hTERT a focus of oncology research and an attractive target for anticancer drug development. In this study, we designed a small interfering RNA (siRNA) targeting the catalytic subunit of hTERT and tested its effects on the growth of telomerase-positive human colon carcinoma SW480 cells in vitro, as well as on the tumorigenicity of these cells in nude mice. Transient and stable transfection of hTERT siRNA into colon cancer SW480 cells suppressed hTERT expression, reduced telomerase activity and inhibited cell growth and proliferation. Knocking down hTERT expression in SW480 tumors xenografted into nude mice significantly slowed tumor growth and promoted tumor cell apoptosis. Our results suggest that hTERT is involved in carcinogenesis of human colon carcinoma, and they highlight the therapeutic potential of a hTERT knock-down approach.

Effectiveness and safety profile of S-1-based chemotherapy compared with capecitabine-based chemotherapy for advanced gastric and colorectal cancer: A meta-analysis.

The aim of the present analysis was to compare the efficacy and safety profile of S-1-based chemotherapy (SBCT) versus capecitabine-based chemotherapy (CBCT) for advanced gastric cancer (AGC) and advanced colorectal cancer (ACRC). A meta-analysis was performed, which included eligible randomized controlled trials (RCTs) that were identified using RevMan 5.1.0 software. A total of 1,064 patients from 11 RCTs, comprising of 527 patients in the SBCT group and 537 patients in the CBCT group, were included in the analysis. For AGC, the meta-analysis of overall survival (OS) [hazard ratio (HR), 0.98; 95% confidence interval (CI), 0.85-1.12], time to progression (HR, 0.95; 95% CI, 0.80-1.12) and overall response rate (ORR) [odds ratio (OR), 1.06; 95% CI, 0.72-1.55] of patients in the SBCT group indicated no statistical significance when compared with those in the CBCT group. Furthermore, for ACRC, a pooled analysis demonstrated no significant difference between the SBCT and CBCT groups (OS: HR, 0.82; 95% CI, 0.61-1.10; progression-free survival: HR, 0.79; 95% CI=0.60-1.04; ORR: OR, 1.27; 95% CI, 0.91-1.78). The statistically significant differences identified in the overall meta-analysis indicated a low incidence of grade 3-4 hand-foot-syndrome (OR, 0.15; 95% CI, 0.06-0.36) in the SBCT group; however no statistically significant difference was observed in the incidence of grade 3-4 anemia, thrombocytopenia, leucopenia, neutropenia, diarrhea, stomatitis or nausea/vomiting. The SBCT treatment exhibited similar efficacy and an approximately equivalent safety profile compared with the CBCT treatment and was an alternative to CBCT for patients with AGC or ACRC; however, further investigation is required to provide confirmation.

Reduced FAF1 Expression and Helicobacter Infection: Correlations with Clinicopathological Features in Gastric Cancer.

Background. This study aimed to investigate possible associations between FAF1 expression and aspects of gastric cancer, in particular its clinical characteristics and Helicobacter infection. Materials and Methods. RT-PCR and immunohistochemistry were used to analyze expression of FAF1 mRNA and protein in 40 gastric cancer patients. H. pylori infection was detected by three staining protocols. Results. The expression level of FAF1 mRNA was significantly lower in gastric cancer tissue than in normal gastric mucosa from the same patient (P < 0.05). FAF1 mRNA expression was significantly lower in stage IV gastric cancer than in stage I+II or IIIA+IIIB (P = 0.004) and also significantly lower in gastric cancer with distant metastasis. FAF1 mRNA expression was higher in well-differentiated cancer than in poorly-differentiated cancer (0.39 ± 0.06 versus 0.19 ± 0.06, t = 9.966, P < 0.01). FAF1 protein was detected in 15 of 40 (37.5%) cancerous tissue samples and in 29 of 40 (72.5%) corresponding normal tissue samples (P < 0.01). FAF1 mRNA expression was lower in H. pylori-positive cancerous tissue samples than in H. pylori-negative ones (P < 0.05). Conclusions. Downregulation of FAF1 expression may be related to the carcinogenesis and progression of gastric cancer, and H. pylori infection during gastric carcinogenesis may downregulate FAF1 expression.

HBV A1762T, G1764A mutations are a valuable biomarker for identifying a subset of male HBsAg carriers at extremely high risk of hepatocellular carcinoma: a prospective study.

OBJECTIVES: Surveillance of hepatocellular carcinoma (HCC) can detect small tumors for resection but at a huge cost of health resources. The challenge is to reduce the surveillance population. We reported that 96% of HCC patients but only 24% of controls were infected with the hepatitis B virus (HBV) with A(1762)T, G(1764)A mutations in Guangxi, China. It is likely to be extremely beneficial in terms of cost and resources if a significant number of tumors can be detected early by screening this selected population. Our aim is to test this hypothesis. METHODS: A cohort of 2,258 hepatitis B surface antigen-positive subjects aged 30-55 yr was recruited in Guangxi. Following evaluation of virological parameters at baseline, HCC is diagnosed by 6-monthly measurements of serum alpha-fetoprotein levels and ultrasonographic examinations. RESULTS: Sixty-one cases of HCC were diagnosed after 36 months of follow-up. The HCC rate was higher in the mutant than wild-type group (P < 0.001, rate ratio [RR] 6.23, 95% confidence interval [CI] 2.83-13.68). The HCC rate in the male mutant group was higher than that in the male wild-type group (P < 0.001, RR 11.54, 95% CI 3.58-37.24). Specifically, 93.3% of male cases are infected with the mutant. Multivariate analyses showed that in men, increasing age and A(1762)T, G(1764)A double mutations are independently associated with developing HCC. CONCLUSIONS: HBV A(1762)T, G(1764)A mutations constitute a valuable biomarker to identify a subset of male HBsAg carriers aged >30 yr at extremely high risk of HCC in Guangxi, and likely elsewhere.

Telomerase activity and human telomerase reverse transcriptase expression in colorectal carcinoma.

AIM: To study the activity of telomerase and the expression of human telomerase reverse transcriptase (hTERT) in colorectal carcinoma and its adjacent tissues, normal mucosa and adenomatoid polyp, and to evaluate their relation with carcinogenesis and progression of colorectal carcinoma. METHODS: Telomerase activity and hTERT expression were determined in 30 samples of colorectal carcinoma and its adjacent tissues, normal mucosa and 20 samples of adenomatoid polyp by modified telomeric repeat amplification protocol (TRAP), enzyme-linked immunosorbent assay (ELISA) and immunohistochemical method. RESULTS: Telomerase activity and hTERT expression were 83.33% (25/30) and 76.67% (23/30) respectively in colorectal carcinoma, which were obviously higher than those in paracancerous tissues (13.33%, 16.67%), normal mucosa (3.33%, 3.33%) and adenomatoid polyp (10%, 10%). There was a significant difference between colorectal carcinoma and other tissues (P=0.027). The telomerase activity and hTERT expression were higher in colorectal carcinoma with lymphatic metastasis than in that without lymphatic metastasis (P=0.034). When the histological classification and clinical stage were greater, the telomerase activity and hTERT expression increased, but there was no significant difference between them. In colorectal carcinoma, the telomerase activity was correlated with hTERT expression (positive vs negative expression of telomerase activity and hTERT, P=0.021). CONCLUSION: Telomerase activity is closely correlated with the occurrence, development and metastasis of colorectal carcinoma. Overexpression of hTERT may play a critical role in the regulation of telomerase activity.