SIRT1 promotes the progression and chemoresistance of colorectal cancer through the p53/miR-101/KPNA3 axis.

INTRODUCTION: Sirtuin 1 (SIRT1) is a key modulator in several types of cancer, including colorectal cancer (CRC). Here, we probed into the molecular mechanism of SIRT1 regulating the development and chemoresistance of CRC. METHODS: Differentially expressed genes related to the growth, metastasis and chemoresistance of CRC were identified by bioinformatics analysis. The expression of SIRT1 in clinical tissues from CRC patients and CRC cell lines was detected by RT-qPCR. Interactions among SIRT1, p53, miR-101 and KPNA3 were analyzed. The effect of SIRT1 on the cell viability, migration, invasion, epithelial-mesenchymal transformation and chemoresistance to 5-FU was evaluated using loss-function investigations in CRC cells. Finally, a xenograft model of CRC and a metastasis model were constructed for further exploration of the roles of SIRT1 in vivo. RESULTS: SIRT1 was elevated in CRC tissues and cell lines. SIRT1 decreased p53 via deacetylation, and consequently downregulated the expression of miR-101 while increasing that of the miR-101 target gene KPNA3. By this mechanism, SIRT1 enhanced the proliferation, migration, invasion, epithelial-mesenchymal transformation, and resistance to 5-FU of CRC cells. In addition, in vivo data also showed that SIRT1 promoted the growth, metastasis and chemoresistance to 5-FU of CRC cells via regulation of the p53/miR-101/KPNA3 axis. CONCLUSIONS: In conclusion, SIRT1 can function as an oncogene in CRC by accelerating the growth, metastasis and chemoresistance to 5-FU of CRC cells through the p53/miR-101/KPNA3 axis.

Distributed optical fiber sensing in coherent Φ-OTDR with a broadband chirped-pulse conversion algorithm.

We propose a broadband chirped-pulse conversion algorithm (BCPCA) to convert a finite-step scanning probe pulse into an equivalent broadband chirped probe pulse by convolving a chirp factor on the received signal in coherent phase-sensitive optical time domain reflectometry (Φ-OTDR). Combined with Rayleigh interference pattern (RIP) demodulation in chirped-pulse Φ-OTDR (CP-ΦOTDR), environmental perturbations, such as strain and temperature along the sensing fiber, can be quantitatively measured. The equivalent broadband chirped pulse is generated by digital processing, and its bandwidth can be increased by changing the composition of the scanning pulse. Thus, the measurable perturbation range of the system can be expanded. As a proof-of-concept experiment, a high-performance distributed strain measurement was realized on a 10 km fiber, the frequency response was 5 kHz, which is only limited by the fiber length, and the strain resolution was 8.04 p ε/H z. The proposed method of generating equivalent broadband chirped pulse through the digital domain can be used as a supplement to CP-ΦOTDR.

Signal Activity Detection for Fiber Optic Distributed Acoustic Sensing with Adaptive-Calculated Threshold.

The key point on analyzing the data stream measured by fiber optic distributed acoustic sensing (DAS) is signal activity detection separating measured signals from environmental noise. The inability to calculate the threshold for signal activity detection accurately and efficiently without affecting the measured signals is a bottleneck problem for current methods. In this article, a novel signal activity detection method with the adaptive-calculated threshold is proposed to solve the problem. With the analysis of the time-varying random noise's statistical commonality and the short-term energy (STE) of real-time data stream, the top range of the total STE distribution of the noise is found accurately for real-time data stream's ascending STE, thus the adaptive dividing level of signals and noise is obtained as the threshold. Experiments are implemented with simulated database and urban field database with complex noise. The average detection accuracies of the two databases are 97.34% and 90.94% only consuming 0.0057 s for a data stream of 10 s, which demonstrates the proposed method is accurate and high efficiency for signal activity detection.

Improvement in the condition of patients with primary liver cancer with transcatheter arterial chemoembolization before and after microwave ablation interventional therapy.

BACKGROUND: We compared the clinical efficacy and safety of transcatheter arterial chemoembolization (TACE) combined with microwave ablation (MWA) and TACE alone for the treatment of patients with primary liver cancer (PLC). MATERIALS AND METHODS: A total of 160 patients with PLC were enrolled and randomized into a study group (n=80) and a control group (n=80). Patients in the study group were treated with TACE combined with MWA, whereas those in the control group were treated with TACE alone. Treatment efficacy, changes in hepatic function indices after the treatment, incidence of adverse reactions, quality of life after treatment, and 3-year survival rates of the two groups were compared. Cox proportional hazards model was used for analyzing the patients' prognostic factors. RESULTS: The total effective rate in the study group was higher than that in the control group (P<0.05). Patients in the study group had lower alanine aminotransferase and total bilirubin levels (P<0.05) and higher albumin levels (P<0.05) than those in the control group. The 1-, 2-, and 3-year overall survival rates in the study group were higher than those in the control group (P<0.05). Cox proportional hazards model showed that tumor size, extrahepatic metastasis, portal vein tumor thrombosis, severity of liver cirrhosis, and therapeutic methods were independent risk factors for patients with PLC. CONCLUSIONS: TACE combined with MWA is more effective than TACE alone in treating PLC, reducing the damage to the patients' cardiac function and prolonging survival.

ZEB2-AS1 Accelerates Epithelial/Mesenchymal Transition Through miR-1205/CRKL Pathway in Colorectal Cancer.

Background: Accumulating reports have demonstrated that long-noncoding RNAs (lncRNAs) play critical roles in the pathological progression of colorectal cancer (CRC). However, the role of lncRNA zinc finger E-box binding homeobox 2 antisense RNA 1 (ZEB2-AS1) in CRC remains largely unknown. Methods: The authors detected the ZEB2-AS1 expression in CRC tissue sample and CRC cell lines. The effects of ZEB2-AS1 on CRC were identified through in vitro assays (i.e., transwell assay, wound-healing assay, immunofluorescence assay, and Western blot) in a ZEB2-AS1 knockdown system. The molecular mechanism of ZEB2-AS1 was explored via bioinformatic tools, quantitative real-time polymerase chain reaction (qRT-PCR), dual-luciferase reporter assay, RNA immunoprecipitation assay, and so on. Moreover, a series of gain-of-function experiments were performed to identify the effect of ZEB2-AS1 and miR-1205 on epithelial-to-mesenchymal transition (EMT) in CRC cells. Results: This analysis clarified that ZEB2-AS1 was upregulated in both CRC tissue sample and cells lines; meanwhile, the high expression of ZEB2-AS1 was correlated with poor overall survival rate. ZEB2-AS1 knockdown significantly suppresses the EMT in CRC cells. Furthermore, the authors identified that the expression of ZEB2-AS1 was negatively correlated with expression of miR-1205, and CRKL could be a direct target of miR-1205. Through the gain-of-function experiments, they found that ZEB2-AS1 accelerates EMT in CRC cells via modulating the expression of miR-1205 and CRKL. Conclusion: Taken together, this study revealed that ZEB2-AS1 accelerates EMT in CRC through the miR-1205/CRKL pathway, suggesting that ZEB2-AS1 may potentially serve as a target of CRC.

Effects of RNA binding protein QKI on pancreatic cancer ductal epithelial cells and surrounding activation fibroblasts.

To determine the correlation between QKI and pancreatic cancer tissues, the QKI expression of pancreatic cancer cells and fibroblasts in the tumor-surrounding microenvironment were detected. Then, QKI overexpression and interference with QKI short hairpin RNA in LX-2 (a fibroblast cell line) were established in vitro. Meanwhile, to observe the cell proliferation, invasion, migration, and other changes, QKI, and related epithelial-mesenchymal transition (EMT) molecules were detected by a polymerase chain reaction and Western blot analysis. In addition, an in vivo tumorigenicity test in node mice was performed to confirm whether QKI expression can promote the proliferation, invasion, and metastasis of pancreatic cancer ductal epithelial cells. Finally, the autophagy levels of fibroblasts with QKI overexpression were observed by electron microscopy to further explore the QKI pathogenic mechanism. It was found that cell proliferation, invasion, migration, and EMT-related markers were increased in QKI-overexpressed fibroblasts LX-2. Furthermore, in vivo, liver and peritoneal metastasis decreased overall survival rate and increasing autophagy levels in QKI-overexpressing nude mice were observed. Meanwhile, knock down QKI with small interfering RNA can reverse all the above effects. QKI can promote the proliferation, metastasis, and invasion of pancreatic cancer through activating fibroblasts surrounding pancreatic cancer and accelerating EMT and increasing the autophagy in pancreatic cancer. QKI may become a potential target for the treatment of pancreatic cancer.

Transcutaneous electroacupuncture alleviates postoperative ileus after gastrectomy: A randomized clinical trial.

AIM: To investigate the efficacy and safety of transcutaneous electroacupuncture (TEA) to alleviate postoperative ileus (POI) after gastrectomy. METHODS: From April 2014 to February 2017, 63 gastric cancer patients were recruited from the Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China. After gastrectomy, the patients were randomly allocated to the TEA (n = 33) or control (n = 30) group. The patients in the TEA group received 1 h TEA on Neiguan (ST36) and Zusanli (PC6) twice daily in the morning and afternoon until they passed flatus. The main outcomes were hours to the first flatus or bowel movement, time to nasogastric tube removal, time to liquid and semi-liquid diet, and hospital stay. The secondary outcomes included postoperative symptom assessment and complications. RESULTS: Time to first flatus in the TEA group was significantly shorter than in the control group (73.19 ± 15.61 vs 82.82 ± 20.25 h, P = 0.038), especially for open gastrectomy (76.53 ± 14.29 vs 87.23 ± 20.75 h, P = 0.048). Bowel sounds on day 2 in the TEA group were significantly greater than in the control group (2.30 ± 2.61/min vs 1.05 ± 1.26/min, P = 0.017). Time to nasogastric tube removal in the TEA group was earlier than in the control group (4.22 ± 1.01 vs 4.97 ± 1.67 d, P = 0.049), as well as the time to liquid diet (5.0 ± 1.34 vs 5.83 ± 2.10 d, P = 0.039). Hospital stay in the TEA group was significantly shorter than in the control group (8.06 ± 1.75 vs 9.40 ± 3.09 d, P = 0.041). No significant differences in postoperative symptom assessment and complications were found between the groups. There was no severe adverse event related to TEA. CONCLUSION: TEA accelerated bowel movements and alleviated POI after open gastrectomy and shortened hospital stay.

Azoramide, a novel regulator, favors adipogenesis against osteogenesis through inhibiting the GLP-1 receptor-PKA-ß-catenin pathway.

BACKGROUND: The reciprocal fate decision of mesenchymal stem cells (MSCs) to either bone or adipocytes is determined by Wnt-related signaling and the glucagon-like peptide-1 receptor (GLP-1R). Azoramide, an ER stress alleviator, was reported to have an antidiabetic effect. In this study, we investigated the function of azoramide in regulating the lineage determination of MSCs for either adipogenic or osteogenic differentiation. METHODS: In this study, microcomputed tomography and histological analysis on bone morphogenetic protein (BMP)2-induced parietal periosteum bone formation assays, C3H10T1/2 and mouse bone marrow MSC-derived bone formation and adipogenesis assays, and specific staining for bone tissue and lipid droplets were used to evaluate the role of azoramide on the lineage determination of MSC differentiation. Cells were harvested for Western blot and quantitative real-time polymerase chain reaction (PCR), and immunofluorescence staining was used to explore the potential mechanism of azoramide for regulating MSC differentiation. RESULTS: Based on MSC-derived bone formation assays both in vivo and in vitro, azoramide treatment displayed a cell fate determining ability in favor of adipogenesis over osteogenesis. Further mechanistic characterizations disclosed that both the GLP-1R agonist peptide exendin-4 (Ex-4) and GLP-1R small interfering (si)RNA abrogated azoramide dual effects. Moreover, cAMP-protein kinase A (PKA)-mediated nuclear ß-catenin activity was responsible for the negative function of azoramide on bone formation in favor of adipogenesis. CONCLUSIONS: These data provide the first evidence to show that azoramide may serve as an antagonist against GLP-1R in MSC lineage determination.

Quaking Deficiency Amplifies Inflammation in Experimental Endotoxemia via the Aryl Hydrocarbon Receptor/Signal Transducer and Activator of Transcription 1-NF-κB Pathway.

Macrophages, characterized by considerable diversity and plasticity, play a crucial role in a broad spectrum of biological processes, including inflammation. However, the molecular mechanisms underlying the diverse phenotypes of macrophages are not well defined. Here, we show that the RNA-binding protein, quaking (QKI), dynamically modulates macrophage polarization states. After lipopolysaccharide (LPS) stimulation, QKI-silenced RAW 264.7 cells displayed a pro-inflammatory M1 phenotype characterized by increased expression of iNOS, TNF-α, and IL-6 and decreased expression of anti-inflammatory factors, such as IL-10, found in inflammatory zone (Fizz1), and chitinase-like 3 (Chil3 or Ym1). By contrast, QKI5 overexpression led to a suppressive phenotype resembling M2 macrophages, even under M1 differentiation conditions. Moreover, myeloid-specific QKI-deficient mice tended to be more susceptible to LPS-induced endotoxic shock, while the exogenous transfer of macrophages overexpressing QKI5 exerted a significant improving effect. This improvement corresponded to a higher proportion of M2 macrophages, in line with elevated levels of IL-10, and a decrease in levels of pro-inflammatory mediators, such as IL-6, TNF-α, and IL-1ß. Further mechanistic studies disclosed that QKI was a potent inhibitor of the nuclear factor-kappa B (NF-κB) pathway, suppressing p65 expression and phosphorylation. Strikingly, reduced expression of the aryl hydrocarbon receptor (Ahr) and reduced phosphorylation of signal transducer and activator of transcription 1 in QKI-deficient cells failed to restrain the transcriptional activity of NF-κB and NRL pyrin domain containing 3 (NLRP3) activation, while restoring QKI expression skewed the above M1-like response toward an anti-inflammatory M2 state. Taken together, these findings suggest a role for QKI in restraining overt innate immune responses by regulating the Ahr/STAT1-NF-κB pathway.

MOTS-c peptide increases survival and decreases bacterial load in mice infected with MRSA.

Sepsis is a life-threatening disease characterized by uncontrolled inflammatory responses upon pathogen infections, especially for the antibiotic-resistant strains, such as Methicillin-resistant S. aureus (MRSA). Here we demonstrated that a Mitochondria-derived peptide (MOTS-c) could significantly improve the survival rate and decrease bacteria loads in MRSA-challenged mice, accompanied with declined levels of pro-inflammatory cytokines, such as TNF-α, IL-6 and IL-1ß, but with increased level of anti-inflammatory cytokine IL-10. Moreover this peptide enhanced bactericidal capacity of macrophages. Meanwhile, MOTS-c inhibited the phosphorylation mitogen-activated protein kinases (MAPK), and enhanced the expression of aryl hydrocarbon receptor (AhR) and signal transducer and activator of transcriptional 3 (STAT3) in macrophages. Overall, MOTS-c plays a beneficial role in curbing the overwhelming inflammatory bursts in the fight against MRSA infection. It may serve as a potential therapeutic agent in sepsis treatment. Highlight.

Downregulation of Rab27A contributes to metformin-induced suppression of breast cancer stem cells.

Cancer stem cells (CSCs) are associated with tumor initiation, therapeutic resistance, relapse and metastasis. However, the underlying mechanisms CSCs use to preserve stemness are not yet fully understood. The present study demonstrated that the expression of RAB27A, member RAS oncogene family (Rab27a), which was reported to promote tumor progression by upregulating exocytosis of extracellular vesicles, was higher in mammosphere cells than in adherent MDA-MB-231 breast cancer cells. Downregulation of Rab27A inhibited mammosphere formation by decreasing the proportion of CD44+CD24-/low cells of the MDA-MB-231 cell line. Furthermore, Rab27A overexpression redistributed the cell cycle of breast (b) CSCs. The present study revealed that downregulation of Rab27A enhanced the capacity of metformin, the most widely used oral hypoglycemic drug for the treatment of type II diabetes, to inhibit mammosphere growth. Metformin reduced the expression of Rab27A dose-dependently. These data suggested that Rab27A acts as a mediator of human bCSCs by promoting the growth of mammospheres and that synergistic suppression of Rab27A, alone or in combination with metformin, holds promise for therapeutically targeting bCSCs.

Antibiotics-induced gut microbiota dysbiosis promotes tumor initiation via affecting APC-Th1 development in mice.

Gut microbiota is critical for maintaining body immune homeostasis and thus affects tumor growth and therapeutic efficiency. Here, we investigated the link between microbiota and tumorgenesis in a mice model of subcutaneous melanoma cell transplantation, and explored the underlying mechanism. We found disruption of gut microbiota by pretreating mice with antibiotics promote tumor growth and remodeling the immune compartment within the primary tumor. Indeed, gut microbial dysbiosis reduced the infiltrated mature antigen-presenting cells of tumor, together with lower levels of co-stimulators, such as CD80, CD86 and MHCII, as well as defective Th1 cytokines, including IFNγ, TNFα, IL12p40, and IL12p35. Meantime, splenic APCs displayed blunted ability in triggering T cell proliferation and IFNγ secretion. However, oral administration of LPS restored the immune surveillance effects and thus inhibited tumor growth in the antibiotics induced gut microbiota dysbiosis group. Taken together, these data highly supported that antibiotics induced gut microbiota dysbiosis promotes tumor initiation, while LPS supplementation would restore the effective immune surveillance and repress tumor initiation.

Rab27A mediated by NF-κB promotes the stemness of colon cancer cells via up-regulation of cytokine secretion.

Recent evidences have unveiled critical roles of cancer stem cells (CSCs) in tumorigenicity, but how interactions between CSC and tumor environments help maintain CSC initiation remains obscure. The small GTPases Rab27A regulates autocrine and paracrine cytokines by monitoring exocytosis of extracellular vesicles, and is reported to promote certain tumor progression. We observe that overexpression of Rab27A increased sphere formation efficiency (SFE) by increasing the proportion of CD44+ and PKH26high cells in HT29 cell lines, and accelerating the growth of colosphere with higher percentage of cells at S phase. Mechanism study revealed that the supernatant derived from HT29 sphere after Rab27A overexpression was able to expand sphere numbers with elevated secretion of VEGF and TGF-ß. In tumor implanting nude mice model, tumor initiation rates and tumor sizes were enhanced by Rab27A with obvious angiogenesis. As a contrast, knocking down Rab27A impaired the above effects. More importantly, the correlation between higher p65 level and Rab27A in colon sphere was detected, p65 was sufficient to induce up-regulation of Rab27A and a functional NF-κB binding site in the Rab27A promoter was demonstrated. Altogether, our findings reveal a unique mechanism that tumor environment related NF-κB signaling promotes various colon cancer stem cells (cCSCs) properties via an amplified paracrine mechanism regulated by higher Rab27A level.

Curcumin promotes browning of white adipose tissue in a norepinephrine-dependent way.

Brown adipose tissue converts energy from food into heat via the mitochondrial uncoupling protein UCP1, defending against cold. In some conditions, inducible 'brown-like' adipocytes, also known as beige adipocytes, can develop within white adipose tissue (WAT). These beige adipocytes have characteristics similar to classical brown adipocytes and thus can burn lipids to produce heat. In the current study, we demonstrated that curcumin (50 or 100 mg/kg/day) decreased bodyweight and fat mass without affecting food intake in mice. We further demonstrated that curcumin improves cold tolerance in mice. This effect was possibly mediated by the emergence of beige adipocytes and the increase of thermogenic gene expression and mitochondrial biogenesis in inguinal WAT. In addition, curcumin promotes ß3AR gene expression in inguinal WAT and elevates the levels of plasma norepinephrine, a hormone that can induce WAT browning. Taken together, our data suggest that curcumin can potentially prevent obesity by inducing browning of inguinal WAT via the norepinephrine-ß3AR pathway.

An improved fluorescence polarization assay in 5'-nuclease reaction for gene promoter methylation detection.

The detection of gene promoter methylation plays increasing roles in personalized medicine. In this study, an improved gene promoter methylation assay based on fluorescence polarization in 5'-nuclease reaction was developed. The novel assay offered a homogeneous annealing and cleavage reaction fully integrated with PCR which used a probe labeled with fluorescence without quencher to obtain the decreased fluorescence polarization values. In this platform, gene promoter methylated and unmethylated alleles were detected simultaneously in a tube. O(6)-methylguanine-DNA methyltransferase gene promoter methylation in 103 glioma tissue samples and epidermal growth factor receptor gene promoter methylation in 116 primary non-small-cell lung carcinoma tissue samples were detected by the novel assay and sequencing, absolute quantitative analysis of methylated allele in parallel. The accuracy of the results measured by the improved fluorescence polarization assay was evaluated using the paired-samples t test. No significant difference was found ( P>0.05). Therefore, the improved fluorescence polarization assay in 5'-nuclease reaction demonstrated a homogeneous, reliable and cost-effective method for gene promoter methylation analysis in clinic. That would provide a scientific basis for applying a reasonable therapeutic regimen in future treatment.

EGFR Promoter Methylation, EGFR Mutation, and HPV Infection in Chinese Cervical Squamous Cell Carcinoma.

Therapy strategy toward epidermal growth factor receptor (EGFR) inhibition in cervical cancer has been ongoing. EGFR promoter methylation status and EGFR tyrosine kinase inhibitor-sensitive mutations in cervical cancer may be significant for clinical outcome prediction using anti-EGFR treatment. In this study, EGFR tyrosine kinase inhibitor-sensitive mutations, EGFR exons 18, 19, and 21 mutations, were detected by sequencing in a total of 293 Chinese cervical squamous cell carcinoma tissue samples. EGFR promoter methylation status was detected by an EGFR asymmetric PCR and hybridization-fluorescence polarization assay and sequencing in 293 Chinese cervical squamous cell carcinoma tissue samples. High-risk human papillomavirus (HPV) genotypes in 293 Chinese cervical squamous cell carcinoma tissue samples were detected by an asymmetric GP5+/6+ PCR and hybridization-fluorescence polarization assay. No EGFR exons 18, 19, and 21 mutations were detected, EGFR promoter methylation status was identified in 98 samples, and HPV 16 infection was the first frequent HPV genotype. The methylated EGFR promoter was identified most frequently in cervical squamous cell carcinoma samples with HPV 16 infection (53.4%). Statistical significant difference of EGFR promoter methylation prevalence was found between HPV 16 and other HPV genotypes (P<0.01). This study suggested that there was no EGFR tyrosine kinase inhibitor-sensitive mutation in EGFR exons 18, 19, and 21 in Chinese cervical squamous cell carcinoma tissue samples. EGFR promoter methylation was common and it might be associated with HPV 16 infection in Chinese cervical squamous cell carcinoma. The results provided a novel understanding and an applicable pharmacogenomic tool for individualized management of cervical cancer patients.

EGFR promoter methylation detection in cervical cancer by a hybridization-fluorescence polarization assay.

The methylation status of the epidermal growth factor receptor (EGFR) promoter is of potential predictive value for benefitting from EGFR inhibition therapy. Stratified therapy assignment for cervical cancer patients based on the EGFR promoter methylation status requires a simple detection method in the daily practice of diagnosis. A novel assay detecting the EGFR promoter methylation status in cervical cancer tissue samples using a hybridization-fluorescence polarization (FP) technique was developed. A pair of primers was used to amplify a 156 bp fragment in the promoter region of EGFR. Two probes specific for either methylated or unmethylated EGFR promoter DNA labeled with different fluorophores hybridized, respectively, with their target amplicons. The EGFR promoter methylation status was determined by the FP values. A total of 273 cervical cancer tissue samples were simultaneously analyzed using the new assay technique and combined bisulfite restriction analysis. The new assay was more sensitive compared with the combined bisulfite restriction analysis, and it allowed the discrimination of the EGFR promoter methylation status directly in solution without the restriction enzyme digestion. Sensitivity, specificity, and stability of the hybridization-FP assay had been recorded. The minimum detection level established with the new assay was 50 copies/µL, and it was able to detect the minor population of the EGFR promoter methylation status even when its contents were as low as 10%. No cross-reaction was observed in the assay when the amount of plasmids used accounted for no more than 10(9) copies/µL. The coefficient of variation of the reproducibility for the assay was <10%.

Detection of primary YMDD mutations in HBV-related hepatocellular carcinoma using hybridization-fluorescence polarization.

Lamivudine is used for the treatment of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). However, HBV-related HCC patients with mutations in the tyrosine-methionine-aspartate-aspartate (YMDD) motif have no response to lamivudine therapy. The detection of YMDD mutations in HBV-related HCC patients may help guide the treatment of HCC. In this study, a simple, sensitive, reliable and cost-effective hybridization-fluorescence polarization assay for the detection of YMDD mutations in HCC was developed. A pair of general primers within the highly conserved region of the HBV polymerase gene was used in an asymmetric PCR. Three probes specific for the corresponding YMDD mutations labeled with different fluorescent reporters hybridized to their target amplicons, and hybridization was indicated by higher fluorescence polarization. The hybridization-fluorescence polarization assay was capable of detecting YMDD mutations at a limit of detection of 10 copies per reaction, and the assay was able to detect minor populations of viruses with primary YMDD mutations as low as 10%. The rates of primary YMDD mutations and the correlation between YMDD mutations and HBV genotypes in 251 HBV-related HCC patients were investigated using the hybridization-fluorescence polarization assay.