Cell-free DNA testing for early hepatocellular carcinoma surveillance.

BACKGROUND: Liver cirrhosis (LC) is the highest risk factor for hepatocellular carcinoma (HCC) development worldwide. The efficacy of the guideline-recommended surveillance methods for patients with LC remains unpromising. METHODS: A total of 4367 LCs not previously known to have HCC and 510 HCCs from 16 hospitals across 11 provinces of China were recruited in this multi-center, large-scale, cross-sectional study. Participants were divided into Stage Ⅰ cohort (510 HCCs and 2074 LCs) and Stage Ⅱ cohort (2293 LCs) according to their enrollment time and underwent Tri-phasic CT/enhanced MRI, US, AFP, and cell-free DNA (cfDNA). A screening model called PreCar Score was established based on five features of cfDNA using Stage Ⅰ cohort. Surveillance performance of PreCar Score alone or in combination with US/AFP was evaluated in Stage Ⅱ cohort. FINDINGS: PreCar Score showed a significantly higher sensitivity for the detection of early/very early HCC (Barcelona stage A/0) in contrast to US (sensitivity of 51.32% [95% CI: 39.66%-62.84%] at 95.53% [95% CI: 94.62%-96.38%] specificity for PreCar Score; sensitivity of 23.68% [95% CI: 14.99%-35.07%] at 99.37% [95% CI: 98.91%-99.64%] specificity for US) (P < 0.01, Fisher's exact test). PreCar Score plus US further achieved a higher sensitivity of 60.53% at 95.08% specificity for early/very early HCC screening. INTERPRETATION: Our study developed and validated a cfDNA-based screening tool (PreCar Score) for HCC in cohorts at high risk. The combination of PreCar Score and US can serve as a promising and practical strategy for routine HCC care. FUNDING: A full list of funding bodies that contributed to this study can be found in Acknowledgments section.

The development of a cSMART-based integrated model for hepatocellular carcinoma diagnosis.

BACKGROUND: Hepatocellular carcinoma (HCC) generally arises from a background of liver cirrhosis (LC). Patients with cirrhosis and suspected HCC are recommended to undergo serum biomarker tests and imaging diagnostic evaluation. However, the performance of routine diagnostic methods in detecting early HCC remains unpromising. METHODS: Here, we conducted a large-scale, multicenter study of 1675 participants including 490 healthy controls, 577 LC patients, and 608 HCC patients from nine clinical centers across nine provinces of China, profiled gene mutation signatures of cell-free DNA (cfDNA) using Circulating Single-Molecule Amplification and Resequencing Technology (cSMART) through detecting 931 mutation sites across 21 genes. RESULTS: An integrated diagnostic model called "Combined method" was developed by combining three mutation sites and three serum biomarkers. Combined method outperformed AFP in the diagnosis of HCC, especially early HCC, with sensitivities of 81.25% for all stages and 66.67% for early HCC, respectively. Importantly, the integrated model exhibited high accuracy in differentiating AFP-negative, AFP-L3-negative, and PIVKA-II-negative HCCs from LCs.

The Landscape of Cell-Free HBV Integrations and Mutations in Cirrhosis and Hepatocellular Carcinoma Patients.

PURPOSE: Intratumoral hepatitis B virus (HBV) integrations and mutations are related to hepatocellular carcinoma (HCC) progression. Circulating cell-free DNA (cfDNA) has shown itself as a powerful noninvasive biomarker for cancer. However, the HBV integration and mutation landscape on cfDNA remains unclear. EXPERIMENTAL DESIGN: A cSMART (Circulating Single-Molecule Amplification and Resequencing Technology)-based method (SIM) was developed to simultaneously investigate HBV integration and mutation landscapes on cfDNA with HBV-specific primers covering the whole HBV genome. Patients with HCC (n = 481) and liver cirrhosis (LC; n = 517) were recruited in the study. RESULTS: A total of 6,861 integration breakpoints including TERT and KMT2B were discovered in HCC cfDNA, more than in LC. The concentration of circulating tumor DNA (ctDNA) was positively correlated with the detection rate of these integration hotspots and total HBV integration events in cfDNA. To track the origin of HBV integrations in cfDNA, whole-genome sequencing (WGS) was performed on their paired tumor tissues. The paired comparison of WGS data from tumor tissues and SIM data from cfDNA confirmed most recurrent integration events in cfDNA originated from tumor tissue. The mutational landscape across the whole HBV genome was first generated for both HBV genotype C and B. A region from nt1100 to nt1500 containing multiple HCC risk mutation sites (OR > 1) was identified as a potential HCC-related mutational hot zone. CONCLUSIONS: Our study provides an in-depth delineation of HBV integration/mutation landscapes at cfDNA level and did a comparative analysis with their paired tissues. These findings shed light on the possibilities of noninvasive detection of virus insertion/mutation.

Development of a Novel Serum Exosomal MicroRNA Nomogram for the Preoperative Prediction of Lymph Node Metastasis in Esophageal Squamous Cell Carcinoma.

Preoperative prediction of lymph node (LN) metastasis is accepted as a crucial independent risk factor for treatment decision-making for esophageal squamous cell carcinoma (ESCC) patients. Our study aimed to establish a non-invasive nomogram to identify LN metastasis preoperatively in ESCC patients. Construction of the nomogram involved three sequential phases with independent patient cohorts. In the discovery phase (N = 20), LN metastasis-associated microRNAs (miRNAs) were selected from next-generation sequencing (NGS) assay of human ESCC serum exosome samples. In the training phase (N = 178), a nomogram that incorporated exosomal miRNA model and clinicopathologic was developed by multivariate logistic regression analysis to preoperatively predict LN status. In the validation phase (n = 188), we validated the predicted nomogram's calibration, discrimination, and clinical usefulness. Four differently expressed miRNAs (chr 8-23234-3p, chr 1-17695-5p, chr 8-2743-5p, and miR-432-5p) were tested and selected in the serum exosome samples from ESCC patients who have or do not have LN metastasis. Subsequently, an optimized four-exosomal miRNA model was constructed and validated in the clinical samples, which could effectively identify ESCC patients with LN metastasis, and was significantly superior to preoperative computed tomography (CT) report. In addition, a clinical nomogram consisting of the four-exosomal miRNA model and CT report was established in training cohort, which showed high predictive value in both training and validation cohorts [area under the receiver operating characteristic curve (AUC): 0.880 and 0.869, respectively]. The Hosmer-Lemeshow test and decision curve analysis implied the nomogram's clinical applicability. Our novel non-invasive nomogram is a robust prediction tool with promising clinical potential for preoperative LN metastasis prediction of ESCC patients, especially in T1 stage.

Biodiversity and richness shifts of mucosa-associated gut microbiota with progression of colorectal cancer.

The host-associated gut microbiota is considered critical for the occurrence and progression of colorectal cancer (CRC); however, systematic evaluations of the changes in the biodiversity and richness of mucosa-associated gut microbiota with the development of CRC have been limited. Twenty-three paired samples from colorectal tumor sites and the surrounding non-tumor tissues were collected from stage I to IV CRC patients. The microbial compositions of the samples were analyzed by Illumina MiSeq sequencing of the V4 region of the 16S rRNA gene. Gut bacterial alterations at the tumor sites and surrounding healthy tissue sites collected from the different stages of CRC patients were analyzed. No significant differences were observed in the overall microbial richness and biodiversity between the CRC tissue and surrounding non-CRC tissue samples, however, composition and community segregation of the gut microbiota with the progression of CRC were observed. A general increasing trend of Bacteroidetes, Firmicutes, and Fusobacteria and decreasing trend of Proteobacteria were observed at the phylum level with the development of CRC. Further analysis revealed that thirty-four taxa differed significantly with the progression of CRC. Conclusively, our findings provide a comprehensive view of the human mucosa-associated gut microbiota, in association with the different stages of CRC.

Obesity and pancreatic cancer: An update of epidemiological evidence and molecular mechanisms.

Despite advances in therapy and achievements in translational research, pancreatic cancer (PC) remains an invariably fatal malignancy. Risk factors that affect the incidence of PC include diabetes, smoking, obesity, chronic pancreatitis, and diet. The growing worldwide obesity epidemic is associated with an increased risk of the most common cancers, including PC. Chronic inflammation, hormonal effects, circulating adipokines, and adipocyte-mediated inflammatory and immunosuppressive microenvironment are involved in the association of obesity with PC. Herein, we systematically review the epidemiology of PC and the biological mechanisms that may account for this association. Included in this review is a discussion of adipokine-mediated inflammation, lipid metabolism, and the interactions of adipocytes with cancer cells. We consider the influence of bariatric surgery on the risk of PC risk as well as potential molecular targets of therapy. Our review leads us to conclude that targeting adipose tissue to achieve weight loss may represent a new therapeutic strategy for preventing and treating PC.

Increased expression of the long noncoding RNA CRNDE-h indicates a poor prognosis in colorectal cancer, and is positively correlated with IRX5 mRNA expression.

BACKGROUND: The long noncoding RNA (lncRNA) colorectal neoplasia differentially expressed - h (CRNDE-h) plays important roles in the early stages of human development and cancer progression. We investigated the expression and clinical significance of lncRNA CRNDE-h in colorectal cancer (CRC). METHODS: The expression level of lncRNA CRNDE-h was analyzed in 142 CRC tissues and 142 paired adjacent nontumorous tissues, along with 21 inflammatory bowel diseases, 69 hyperplastic polyp, and 73 colorectal adenoma samples, using quantitative real-time polymerase chain reaction. The association between lncRNA CRNDE-h, and Iroquois homeobox protein 5 (IRX5) mRNA was examined in the same 142 CRC tissues. RESULTS: We found that lncRNA CRNDE-h level was elevated in the CRC and adenoma groups compared with the other groups (all at P<0.001). In CRC, upregulation of lncRNA CRNDE-h was significantly correlated with large tumor size, positive regional lymph node metastasis, and distant metastasis (all at P<0.05). Area under the curve for lncRNA CRNDE-h showed diagnostic capability for distinguishing CRC from other groups. Patients with CRC with high lncRNA CRNDE-h expression level had poorer overall survival than those with low lncRNA CRNDE-h expression (log-rank test, P<0.001). Further, multivariable Cox regression analysis suggested that increased expression of lncRNA CRNDE-h was an independent prognostic indicator for CRC (hazard ratio [HR]=2.173; 95% confidence interval [CI], 1.282-3.684, P=0.004). Furthermore, lncRNA CRNDE-h expression was positively correlated with IRX5 mRNA in CRC tissues. CONCLUSIONS: Our data offers convincing evidence for the first time that lncRNA CRNDE-h is associated with adverse clinical characteristics and poor prognosis, which suggests that it might play an important role in CRC development and progression and might have clinical potential as a useful prognostic predictor.

MicroRNA-218 is a prognostic indicator in colorectal cancer and enhances 5-fluorouracil-induced apoptosis by targeting BIRC5.

One major reason for the failure of advanced colorectal cancer (CRC) treatment is the occurrence of chemoresistance to fluoropyrimidine (FU)-based chemotherapy. Various reports showed that ectopic expression and function of microRNAs (miRNAs) played key roles to mediate apoptosis at the post-transcriptional level. To further explore the possible mechanisms, we evaluated the prognostic effect of miR-218 in patients with CRC receiving 5-FU-based treatment and investigated the proapoptotic role of miR-218 in vitro. Primary tumour specimens and adjacent non-tumour sites were used to determine miR-218 expression distribution and explore its potential prognostic value in response to 5-FU-based treatment in patients with CRC. HCT116 and HT29 cells were transfected with precursor miR-218 or negative control, followed by assays to investigate its influence on apoptosis, cell proliferation and pathways involved in molecular mechanisms of chemoresistance to 5-FU. Results showed that high miR-218 expression was associated with positive response to firstline 5-FU treatment in CRC patients. MiR-218 promoted apoptosis, inhibited cell proliferation and caused cell cycle arrest in CRC cells by suppressing BIRC5 expression. Furthermore, miR-218 enhanced 5-FU cytotoxicity in CRC cells by suppressing the 5-FU targeted enzyme, thymidylate synthase (TS). In conclusion, we demonstrated that high miR-218 expression had a positive prognostic value in 5-FU-based treatments for CRC patients and discovered a novel mechanism mediated by miR-218 to promote apoptosis and to function synergistically with 5-FU to promote chemosensitivity by suppressing BIRC5 and TS in CRC. These suggest the unique potential of miR-218 as a novel candidate for developing miR-218-based therapeutic strategies in CRC.

Combined detection of sialic acid and hydroxyproline in diagnosis of ovarian cancer and its comparison with human epididymis protein 4 and carbohydrate antigen 125.

BACKGROUND: Elevated serum sialic acid (SA) and hydroxyproline (Hyp) concentrations have been found in a variety of malignant cancers. We simultaneously detect serum concentrations of SA and Hyp (SA&Hyp) in ovarian cancer, and compare its diagnostic value with classic tumor markers-human epididymis protein 4 (HE4) and carbohydrate antigen 125 (CA125). METHODS: Serum concentrations of SA&Hyp, HE4 and CA125A were detected in a total of 767 serum samples collected from 484 patients with gynecologic diseases, 180 healthy individuals, 45 pregnant women and 58 patients with renal failure using chemical colorimetry and electrochemiluminescence immunoassay (ECLIA), respectively. Risk of ovarian malignancy algorithm (ROMA) was calculated based on HE4 and CA125 values. RESULTS: Serum SA&Hyp concentrations were influenced significantly by renal failure and pregnancy but not age and menopausal status. The median concentrations of SA&Hyp, HE4 and CA125 in patients with ovarian cancer were 119.0 U/ml, 190.2 pmol/l and 366.0 pmol/l, which were significantly higher than concentrations in patients with benign gynecologic diseases (P<0.001). SA&Hyp showed a significantly higher AUC than HE4 and CA125 in the diagnosis of gynecologic malignancies (P<0.001), while no significance was found when compared with ROMA. Specially, SA&Hyp in 48.3% subjects (29/60) diagnosed as positive before primary surgery showed negative after surgery. CONCLUSIONS: Renal failure and pregnancy are the main source for increased false positive of SA and Hyp. Compared with HE4 and CA125, SA&Hyp shows a better diagnosis value and can be used in the diagnosis and dynamic monitoring of gynecologic pelvic malignancies, while no statistical significance was found compared with ROMA.

Overexpression of miR-223 correlates with tumor metastasis and poor prognosis in patients with colorectal cancer.

The purpose of the study was to investigate microRNA-223 (miR-223) expression in colorectal cancer (CRC) and its relationship with tumorigenesis and disease prognosis. Quantitative real-time PCR was used to measure levels of miR-223 in tumor samples and adjacent non-cancerous tissues from 62 patients undergoing radical resection for the treatment of CRC. The associations between miR-223 expression and patient age, sex, as well as clinicopathologic parameters, such as tumor size, differentiation, location, invasion depth, metastasis, tumor-node-metastasis (TNM) stage, and overall patient survival, were analyzed by Mann-Whitney U and Kruskal-Wallis tests. Kaplan-Meier method and Cox proportional hazards regression analyses were performed to estimate the prognostic factors for patient survival prediction. The expression of miR-223 was significantly upregulated in CRC tissues compared with adjacent non-cancerous tissues (P < 0.05). This overexpression was associated with TNM stage and lymph node and distant metastases, (P < 0.05). Moreover, Kaplan-Meier analysis demonstrated that patients with high miR-223 expression had a poorer overall survival (OS) than those with low miR-223 expression (P = 0.002). Univariate analysis revealed a statistically significant correlation between OS and miR-223 level, histology grade, metastasis and TNM stage (P < 0.001). Furthermore, miR-223 levels and histology grade were independently associated with OS (HR 0.204, 95 % CI 0.101-0.415, P < 0.05 and HR 2.252, 95 % CI 1.429-3.546, P < 0.05, respectively). The overexpression of miR-223 may play an important role in the progression of CRC and can be used as an independent factor to determine CRC prognosis.

Decreased expression of miR-133a correlates with poor prognosis in colorectal cancer patients.

AIM: To investigate microRNA-133a (miR-133a) expression in colorectal cancer (CRC) and its relationship with tumorigenesis and disease prognosis. METHODS: Quantitative real-time polymerase chain reaction was used to measure levels of miR-133a in tumor samples and adjacent non-cancerous tissues from 169 patients undergoing radical resection for CRC. The associations between miR-133a expression and patient age, sex, as well as clinicopathologic parameters, such as tumor size, differentiation, location, invasion depth, metastasis, tumor-node-metastasis (TNM) stage and overall patient survival, were analyzed by Mann-Whitney U and Kruskal-Wallis tests. The Kaplan-Meier method and Cox proportional hazards regression analyses were performed to estimate the prognostic factors for patient survival prediction. RESULTS: The expression of miR-133a was significantly downregulated in CRC tissues compared with adjacent non-cancerous tissues (P < 0.05). This reduction was associated with the depth of the local invasion, poor differentiation, lymph node metastasis and advanced disease (P < 0.05). Moreover, Kaplan-Meier analysis demonstrated that patients with low miR-133a expression had poorer overall survival (OS) than those with high miR-133a expression (P < 0.001). Univariate analysis revealed statistically significant correlations between OS and miR-133a level, tumor local invasion, lymph node metastasis and TNM stage (P < 0.001). Furthermore, miR-133a levels and TNM stage were independently associated with OS (HR = 0.590, 95%CI: 0.350-0.995, P < 0.05; and HR = 6.111, 95%CI: 1.029-36.278, P < 0.05, respectively). CONCLUSION: The downregulation of miR-133a may play an important role in the progression of CRC and can be used as an independent factor to determine CRC prognosis.

Limited diagnostic value of microRNAs for detecting colorectal cancer: a meta-analysis.

BACKGROUND: MicroRNAs have been demonstrated to play important roles in the development and progression of colorectal cancer. Several studies utilizing microRNAs as diagnostic biomarkers for colorectal cancer (CRC) have been reported. The aim of this meta-analysis was to comprehensively and quantitatively summarize the diagnostic value of microRNAs for detecting colorectal cancer. METHODS: We searched PubMed, Embase and Cochrane Library for published studies that used microRNAs as biomarkers for the diagnosis of colorectal cancer. Summary estimates for sensitivity, specificity and other measures of accuracy of microRNAs in the diagnosis of colorectal cancer were calculated using the bivariate random effects model. A summary receiver operating characteristic (SROC) curve was also generated to summarize the overall effectiveness of the test. RESULT: Thirteen studies from twelve published articles met the inclusion criteria and were included. The overall sensitivity, specificity, positive likelihood ratio, negative likelihood ratio and diagnostic odd ratio of microRNAs for the diagnosis of colorectal cancer were 0.81 (95%CI: 0.79-0.84), 0.78 (95%CI: 0.75-0.82), 4.14 (95%CI: 2.90- 5.92), 0.24 (95%CI: 0.19-0.30), and 19.2 (95%CI: 11.7-31.5), respectively. The area under the SROC curve was 0.89. CONCLUSIONS: The current evidence suggests that the microRNAs test might not be used alone as a screening tool for CRC. Combining microRNAs testing with other conventional tests such as FOBT may improve the diagnostic accuracy for detecting CRC.

Krüppel-like factor 8 overexpression is correlated with angiogenesis and poor prognosis in gastric cancer.

AIM: To investigate Krüppel-like factor 8 (KLF8) expression in gastric cancer and its relationship with angiogenesis and prognosis of gastric cancer. METHODS: One hundred and fifty-four patients with gastric cancer who underwent successful curative resection were retrospectively enrolled in the study. Fifty tumor-adjacent healthy gastric tissues (≥ 5 cm from the tumor margin) obtained during the original resection were randomly selected for comparative analysis. In situ expression of KLF8 and CD34 proteins were examined by immunohistochemistry. The intratumoral microvessel density (MVD) was determined by manually counting the immunostained CD34-positive endothelial cells in three consecutive high-magnification fields (× 200). The relationship between differential KLF8 expression and MVD was assessed using Spearman's correlation coefficient test. χ² test was performed to evaluate the effects of differential KLF8 expression on clinicopathologic factors. Kaplan-Meier and multivariate Cox survival analyses were used to assess the prognostic value of differential KLF8 expression in gastric cancer. RESULTS: Significantly higher levels of KLF8 protein were detected in gastric cancer tissues than in the adjacent non-cancerous tissues (54.5% vs 34.0%, P < 0.05). KLF8 expression was associated with tumor size (P < 0.001), local invasion (P = 0.005), regional lymph node metastasis (P = 0.029), distant metastasis (P = 0.023), and tumor node metastasis (TNM) stage (P = 0.002), as well as the MVD (r = 0.392, P < 0.001). Patients with KLF8 positive expression had poorer overall survival (P < 0.001) and cancer-specific survival (P < 0.001) than those with negative expression. Multivariate analysis demonstrated that KLF8 expression independently affected both overall and cancer-specific survival of gastric cancer patients (P = 0.035 and 0.042, respectively). CONCLUSION: KLF8 is closely associated with gastric tumor progression, angiogenesis and poor prognosis, suggesting it may represent a novel prognostic biomarker and therapeutic target for gastric cancer.

Aberrant expression of chemokine receptor CCR4 in human gastric cancer contributes to tumor-induced immunosuppression.

The chemokine receptor CCR4 is preferentially expressed on certain immune cells and some hematological tumor cells, which play pivotal roles in suppression of host immune response. However, the reasons for the upmodulation of CCR4 and its immune functions in solid tumors remain unclear. Herein, we aimed to determine the expression profiles of CCR4 in gastric cancer cells and its role in regulating antitumor immunity. CCR4 expression was assessed in 63 cases of gastric carcinomas by immunohistochemistry. We found cancer cells in lymphocyte-rich carcinomas more frequently showed moderate to strong positive staining for CCR4 than those in conventional carcinomas (P = 0.041), and also found a positive relationship between expression of CCR4 and tumor necrosis factor-α (P = 0.012). Stimulation of gastric cell lines with various cytokines showed that tumor necrosis factor-α uniquely upmodulated CCR4 expression through activation of nuclear factor-κB. Additional coculture experiments showed the forced expression of CCR4 in SGC-7901 cells caused a significant reduction of γ-interferon and elevation of interleukin-10 secretion in the supernatants from cocultured SGC-7901 cells and PBMCs. In addition, granzyme A production in cancer cell-cocultured CD56(+) natural killer cells was significantly downregulated. Inhibition of the overexpressed CCR4 in cancer cells by an inhibitor of CCR4, compound 39, proved to partly restore the antitumor immunity in respect of the inverse changes in those factors. Our studies suggest that the aberrant expression of CCR4 in human gastric cancer could contribute to tumor-induced immunosuppression. Conceivably, downmodulation of CCR4 expression could be a promising immunotherapy for human gastric cancer.

RNA interference of human papillomavirus type 16 E7 increases HLA class I antigen expression in HaCaT-E7 cells.

BACKGROUND: High-risk human papillomaviruses (HPVs) are the major causative agents of cervical cancer. The E7 protein of high-risk HPV disturbs cell cycle control and down-regulates components of the antigen presentation pathway, suggesting an ideal target for development of the immunotherapy in HPV-positive cervical cancers. We previously reported that HPV16 E7 could down-regulate cell-surface HLA class I antigen accompanying decreased expression of transporter associated with antigen processing 1 (TAP-1). The purpose of this study was to determine whether knockdown of HPV16 E7 could up-regulate surface HLA class I antigen expression in HPV16 E7 expressing HaCaT cells (HaCaT-E7). METHODS: An E7-specific small interfering RNA (siRNA) was transfected into the HaCaT-E7 cells, and the expression of HPV16 E7 was measured by real-time reverse transcriptase polymerase chain reaction and Western blot. With the use of flow cytometry analysis, the levels of cell surface HLA class I antigen and intracellular TAP-1 expression were detected. RESULTS: It was found that transfection of HPV16 E7-siRNA reduced HPV16 E7 expression as measured on messenger RNA and protein levels. The flow cytometry analysis showed that, compared with mock transfection, a statistically significant increase of approximately 75% in surface HLA class I levels was observed in HaCaT-E7 cells at 72 hours after transfection of E7 siRNA. Moreover, he knockdown of E7 in HaCaT-E7 cells could result in an increase of intracellular TAP-1 expression, which is essential for the expression of HLA class I at cell surface. CONCLUSIONS: Our study showed that the knockdown of HPV16 E7 could increase cell surface HLA class I antigen expression in HaCaT-E7 cells. In addition, for HPV-positive human cervical cancer, our observations indicate that the HPV E7 gene is a target of choice.

Overexpression of Bmi-1 in uterine cervical cancer: correlation with clinicopathology and prognosis.

INTRODUCTION: B-cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1) is a member of polycomb group, which participates in axial patterning, hematopoiesis, cell cycle regulation, and senescence. Recently, overexpression of Bmi-1 has been reported in various human cancers and proved to be associated with poor survival. The aim of this study was to investigate the expression of Bmi-1 protein in human uterine cervical cancer (UCC) and explore its associations with clinicopathological factors and prognosis. METHODS: Western blot was used to detect the expression of Bmi-1 in 4 human cervical cancer cell lines (Hela, SiHa, CasKi, and C33A) and a normal cervical epithelial cell line. In addition, 152 UCC and 30 adjacent normal cervical paraffin-embedded samples were collected to detect Bmi-1 expression by immunohistochemistry. RESULTS: Western blot analysis showed Bmi-1 was overexpressed in 4 human UCC cell lines but not in the normal cervical epithelial cell line. Moreover, immunohistochemical staining revealed Bmi-1 was overexpressed in 63.2% UCC tissues (Bmi-1 ++ or +++), and the overexpression of Bmi-1 protein was significantly correlated with tumor size (P = 0.046), clinical stage (P = 0.021), and regional lymph nodes metastasis (P = 0.010). Survival analysis showed a significant difference between Bmi-1 protein overexpression and poor survival (P = 0.021). Cox proportional hazards risk analysis indicated that Bmi-1 protein overexpression was an independent prognostic factor for overall survival. CONCLUSIONS: B-cell-specific Moloney murine leukemia virus integration site 1 is overexpressed in UCC and correlated with adverse clinical characteristics and poor prognosis, which suggests that the Bmi-1 might participate in the development and progression of UCC and have clinical potential not only as a useful predictor of aggressive phenotype but also a promising prognostic predictor.

[Construction of double-strand break repair protein hKu70 deficient cell strain and its biologic characters].

OBJECTIVE: To construct DNA double-strand break (DSB) repair protein hKu70 deficient cell strain and to observe its biological characters for studying the functions of hKu70 gene and the effects of occupational harmfulness factors on DSB repair. METHODS: Human lung fibroblasts (HLF) were transfected with the eukaryotic expression plasmids of hKu70 gene antisense RNA (pEGFP-C1-K) to construct hKu70 protein deficient cells (named as "HLFK"). The protein expression levels of hKu70 gene in HLFC and HLFK were detected by the Western blotting to estimate the effects of antisense inhibition. Morphology, growth character and growth status in soft agar of transfected HLFK were observed. RESULTS: pEGFP-C1-K vector was successfully expressed in HLF. The protein expression level of hKu70 gene in HLFK was decreased by 42% as compared with that in HLFC. No obvious changes of the biologic characters were observed in HLFK. CONCLUSION: The hKu70 protein deficient cell strain was successfully constructed. The hKu70 protein deficiency alone didn't induce obvious changes of the biological characters in HLFK.