BACKGROUND Previous research has highlighted correlations between blood cell counts and chronic liver disease. Nonetheless, the causal relationships remain unknown. AIM To evaluate the causal effect of blood cell traits on liver enzymes and nonalcoholic fatty liver disease (NAFLD) risk. METHODS Independent genetic variants strongly associated with blood cell traits were extracted from a genome-wide association study (GWAS) conducted by the Blood Cell Consortium. Summary-level data for liver enzymes were obtained from the United Kingdom Biobank. NAFLD data were obtained from a GWAS meta-analysis (8434 cases and 770180 controls, discovery dataset) and the Fingen GWAS (2275 cases and 372727 controls, replication dataset). This analysis was conducted using the inverse-variance weighted method, followed by various sensitivity analyses. RESULTS One SD increase in the genetically predicted haemoglobin concentration (HGB) was associated with a β of 0.0078 (95%CI: 0.0059-0.0096), 0.0108 (95%CI: 0.0080-0.0136), 0.0361 (95%CI: 0.0156-0.0567), and 0.0083 (95%CI: 00046-0.0121) for alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase, and gamma-glutamyl transferase, respectively. Genetically predicted haematocrit was associated with ALP (β = 0.0078, 95%CI: 0.0052-0.0104) and ALT (β = 0.0057, 95%CI: 0.0039-0.0075). Genetically determined HGB and the reticulocyte fraction of red blood cells increased the risk of NAFLD [odds ratio (OR) = 1.199, 95%CI: 1.087-1.322] and (OR = 1.157, 95%CI: 1.071-1.250). The results of the sensitivity analyses remained significant. CONCLUSION Novel causal blood cell traits related to liver enzymes and NAFLD development were revealed through Mendelian randomization analysis, which may facilitate the diagnosis and prevention of NAFLD.

Nasopharyngeal carcinoma (NPC) is a malignant tumor commonly associated with Epstein-Barr virus (EBV) infection, and its early diagnosis as well as its differentiation from nasopharyngitis (NPG) remains challenging due to the insufficient sensitivity of routine screening methods in clinical practice. To date, circulating extracellular vesicles (EVs, 40-1000 nm) have shown appealing potential in liquid biopsy for cancer diagnosis and prognosis. Herein, nanoflow cytometry (nFCM) capable of single EV analysis was applied to examine the expression of surface proteins with very low copy numbers on individual EVs as small as 40 nm. The particle concentrations of five EV subsets exposing EBV-encoded latent membrane proteins (LMP1 and LMP2A) and tumor markers (PD-L1, EGFR, and EpCAM) in plasma were determined rapidly via single-particle enumeration. We identified a five-marker panel named EVSUM5 (an unweighted sum of the concentration of the five individual EV subsets) that significantly surpassed the traditional VCA-IgA assay in discriminating NPC patients from both healthy donors and NPG patients with accuracies of 96.3 and 83.1%, respectively. Moreover, EVSUM2 (an unweighted sum of virus-specific LMP1- and LMP2A-positive EVs) could achieve the diagnosis of NPG with an accuracy of 82.6%. Collectively, the work presented a rapid, reliable, and noninvasive method as well as two diagnostic markers to help more accurately differentiate NPC from NPG patients and healthy donors in clinical practice.

BACKGROUNDEpstein-Barr virus (EBV) infection has a role in the development and progression of nasopharyngeal carcinoma (NPC); however, it is unclear whether EBV load correlates with tumor prognosis or the need for immunotherapy. This study evaluated whether the EBV DNA concentration in peripheral blood mononuclear cells (PBMC) or programmed cell death-ligand1 (PD-L1) expression in tumor-infiltrating lymphocytes (TIL) could predict the clinical outcomes of patients with NPC.METHODSClinicopathological parameters of 198 patients with NPC were analyzed retrospectively from June 2012 to May 2018. Patients' EBV loads were determined by droplet digital PCR. TIL PD-L1 was analyzed by immunohistochemistry.RESULTSA log value of 1.98 log IU/mL for PBMC EBV DNA and a percentage of PD-L1 expression of 15% in TILs marked distinguishing cutoffs in NPC prognosis. The 5-year progression-free survival (PFS) rates in patients with high vs low log (PBMC EBV DNA) were 68.2% and 93.1%, respectively (P = 0.002). The 5-year PFS rates in patients with high vs low TIL PD-L1 expression were 66.3% and 33.7%, respectively (P = 0.03). The 5-year PFS rates of the high-risk group (high log [PBMC EBV DNA] and low TIL PD-L1), low-risk group (low log [PBMC EBV DNA] and high TIL PD-L1), and those in between (intermediate group) were 0%, 91.9%, and 71.4%, respectively (P < 0.001).CONCLUSIONConcentrations of PBMC EBV DNA and TIL PD-L1 expression can be used as prognostic markers in NPC. The combination of both an increased EBV DNA concentration and suppressed TIL PD-L1 expression is associated with metastasis or relapse.

Abstract Tumor specific alterations in miRNA provide ideal biomarkers for early diagnosis of colorectal cancer(CRC). Methylation changes play an important role in the miRNA regulation mechanism involved in tumorigenesis. Therefore, it is important to find miRNA methylation markers related to tumor proliferation and metastasis, and to explore its key regulatory mechanisms. Previous integrated bioinformatics analysis suggested that reduced expression and hypermethation of miR-146a have detected in majority of CRC. However, its biological behavior in CRC remains unclear. This article aims to explore its epigenetic regulation and mechanism in the development of CRC. We collected peripheral blood and tissues from 60 patients with advanced primary CRC, analyzed their clinicopathological characteristics. The mRNA and methylation difference was detected by qPCR and MSP respectively, and the significance in the diagnosis of CRC was analyzed. The research was further confirmed in SW480, SW620, HCT116 and HT29 CRC cell lines, as well as CRC animal models treated with demethylated drugs–5-aza-miR2-oxyeytidineine(5-AZA) and various gene transfection, in order to explore the key downstream regulatory network of miR-146a methylation changes and the effect of methylation inhibitors in the development of CRC. We found that the percentage of down-regulated and methylation of miR-146a in both tissues and plasmas of CRC was significantly higher than that of normal samples, and the expression of miR-146a could be recovered after demethylation treatment in cancer, which was associated with clinical stage. miR-146a methylation was significantly correlated with grading, but not gender, tumor location, histological type or TNM, it may be used as a predictor of poor prognosis. Diagnostic evaluation of miR-146a methylation in tissue and plasma was evaluated using ROC curve, area under regional curve, which indicated that miR-146a methylation could be used as a biomarker for the diagnosis of CRC. Fluorescein report system showed that miR-146a targeted VASN protein, and while miR-146a was overexpressed after transfection into CRC cells, the expression levels of VASN, TGF-β, VEGF-C, MMP9, and Vimentin were significantly reduced, while the expression of TIMP1 and Ecadherin were up-regulated. Additionally, the miR-146a mRNA in CRC tissues was also negatively correlated with the expression of VASN. VASN Silencing down-regulated TGF-β expression, accompanied by a decrease of MMP9. At the same time, after 5-aza demethylation or siVASN, proliferation activity of CRC cells decreased, accompanied by varying degrees of decline in membrane penetrating and migration ability, suggesting that miR-146a may regulate the proliferation and metastasis by regulating VASN/TGF-β relatived pathway. In vivo animal models, it was further confirmed that overexpressed miR-146a was consistent with the effect of 5-aza intervention or siVASN, which could significantly inhibit the occurrence of liver metastasis of CRC. Demethylated drugs treatment or VASN knockdown also significantly inhibited tumor growth in nude mice with CRC. miR-146a was a potential epigenetic silencing tumor suppressor in CRC, and the methylation of miR-146a may play an important role in the diagnosis and prediction of tumor progression in CRC as a new tumor marker. Citation Format: Weiwei Tang, Dan Zhou, Hanxiang An, Jiapeng Kang, Mingquan Cai, Ru Zeng, Jing Song, Bin Hu, Jiabian Lian, Qin Lin, Lilin Chen, Feng Ye. miR-146a methylation regulates proliferation and metastasis by targeted activation of VASN/TGF-beta signaling pathway in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2443.

As a malignant disease that seriously threatens human health, hepatocellular carcinoma (HCC) lacks effective early screening and prognostic assessment methods. Herein, we developed a method for efficient capture and multiphenotype analysis of circulating tumor cells (CTCs) of hepatocellular carcinoma. The anti-ASGPR antibody and the anti-EpCAM antibody were modified in parallel on a deterministic lateral displacement (DLD)-patterned microfluidic Synergetic-Chip to enhance capture efficiency by a complementary effect. CTCs were detected in 45 out of 45 (100%) HCC patients, with a sensitivity and specificity of 97.8 and 100%, respectively. Patients with more total CTCs and nonepithelial CTCs were in later stages of HCC and had more malignant progression. This strategy proposes a feasible approach for early diagnosis and prognosis of hepatocellular carcinoma.

161 Background: A real-world study to observe the efficacy and safety of apatinib treatment in clinical practice, and explored patients (pts) who could benefit from apatinib. Methods: This is an open-label, prospective, observational study, pts (age ≥ 18 yrs) with pathologically or histologically diagnosed GC who were given apatinib treatment met the inclusion criteria. Results: From September 1, 2017 to September 1, 2018. 954 pts were enrolled. 679 pts received palliative treatment were included in the analysis. 499 (73.6%) were males. The age of the pts was (63.1 ± 10.8) yrs. Pts with ECOG PS 0/1 accounted for 78.1%. Pts received first-line treatment, second-line treatment, third-line treatment or above , respectively were 42.2%, 30.5%, 27.3%. 509 (75.1%) pts were treated with apatinib at dose of 500 mg. There were 375 pts available for efficiency evaluation. Among them, 1 achieved CR, 35 achieved PR, 198 had SD, and 57 had PD, resulting in an ORR of 11.9% and a DCR of 77.4%. The mPFS is 4.2 m (95% CI: 3.5-4.8), OS data was still in follow-up. This study showed its efficacy for pts with monotherapy compared with combined chemotherapy (3.3 m vs. 5.0 m, P = 0.003). The mPFS for patients with hypertension were longer than without hypertension (7.1m vs. 3.7m, P = 0.038). And pts also had longer mPFS with good ECOG PS (0-1) compared to those with poor ECOG PS (2-4) (4.7 m vs. 2.9 m, P = 0.003). The incidence of AEs was 402 (88.2%) and the grade 3/4 treatment-related AEs was 14.7%. The most common treatment-related AEs were debilitation (31.2%), hand-foot syndrome (27.5%), hypertension (25.3%), anorexia (11.5%), diarrhea (10.3%), albumin decreased (38.5%), uric acid elevated (31.6%), and total bilirubin increased (29.1%). Conclusions: In the real world, apatinib is confirmed to be effective and safe for GC pts. Factors associated with better prognosis were apatinib combined chemotherapy, ECOG PS 0/1 and occurrence of hypertension. Further analysis is needed to identify pts who benefit from apatinib treatment. Preliminary results of the study were released in ESMO 2018 Congress (683P). Clinical trial information: NCT 03333967.

BackgroundThe incidence of intrahepatic cholangiocarcinoma (ICC) has increased worldwide in recent years, but the risk factors of ICC have not yet been clearly defined. The aim of this study was to identify risk factors for ICC.MethodsA literature search was conducted in PubMed database for eligible studies published from January 2000 to November 2018. Summary odds ratio (OR) with their corresponding 95% confidence intervals (CI) were calculated using a random-effects model.ResultsThirty-two studies (5 cohort, 27 case control) were included in this meta-analysis. Pooled estimates indicated that cirrhosis (OR=11.96, 95% CI 7.53-19.00), hepatitis B virus (HBV) infection (OR=4.09, 95% CI 3.13-5.34), hepatitis C virus (HCV) infection (OR=3.94, 95% CI 2.85-5.45), alcohol consumption (OR=2.68, 95% CI 1.97-3.66), hepatolithiasis (OR=14.94, 95% CI 6.41-34.83), choledochal cyst (OR=21.67, 95%CI 10.83-43.36), primary sclerosing cholangitis (PSC) (OR=93.91, 95%CI 43.99-200.47), diabetes mellitus (OR=1.82, 95% CI 1.57-2.11), non-alcoholic fatty liver disease (OR=2.23, 95% CI 1.65-3.02), liver fluke infection (OR=2.28, 95% CI 1.30-4.01) and smoking (OR=1.26, 95% CI 1.07-1.49) were significantly associated with ICC. Non-significant association was found between ICC and history of cholecystectomy, hypertension, coffee and aspirin.ConclusionsCirrhosis, HBV infection, HCV infection, alcohol consumption, hepatolithiasis, choledochal cyst, PSC, diabetes mellitus, non-alcoholic fatty liver disease, liver fluke infection and smoking are risk factors for ICC.

The tumor microenvironment affects the processes involved in the development of gastric cancer and contributes to multidrug resistance (MDR). Although the metabolism of gastric cancer cells is known to be associated with the development of the tumor microenvironment, the exact role of metabolism in microenvironment‑induced MDR formation remains unclear. In the present study, conditioned medium (CM) formed through the metabolism of SGC‑7901 gastric carcinoma cells was used to mimic the tumor microenvironment. The effects of CM on drug resistance were evaluated in gastric carcinoma cells. The results revealed that CM was not only able to upregulate the expression levels of ATP‑binding cassette subfamily G member 2 (ABCG2) and MDR‑associated protein 2 (MRP2), but also upregulated the expression of certain anti‑apoptotic proteins in SGC‑7901 cells. In addition, CM activated the ataxia‑telangiectasia mutated (ATM) and NF‑κB pathways, while CM‑induced ABCG2, MRP2 and anti‑apoptotic protein upregulation was impaired by ATM and NF‑κB inhibitors. The results of the present study indicated that CM augmented chemotherapeutic resistance by activating the ATM and NF‑κB pathways in gastric cancer cells, and that these pathways may be potential therapeutic targets for cases of chemotherapeutic resistance in gastric cancer.

Background and objective HSF1 is reported to be overexpressed in various solid tumors and play a pivotal role in cancer progression. A meta-analysis was conducted to assess the potential prognostic role of HSF1 in patients with solid tumors. Methods An extensive electronic search of three databases was performed for relevant articles. The pooled hazard ratios (HRs) or odds ratios with their corresponding 95% CI were calculated with a random-effects model. Heterogeneity and publication bias analyses were also conducted. Results A total of 3,159 patients from 10 eligible studies were included into the analysis. The results showed that positive HSF1 expression was significantly correlated with poor overall survival in all tumors (HR=2.09; 95% CI: 1.62–2.70; P<0.001). Subgroup analysis revealed that there was a significant association between HSF1 overexpression and poor prognosis in esophageal squamous cell carcinoma (ESCC) (HR=1.83; 95% CI: 1.21–2.77; P=0.004), breast cancer (BC) (HR=1.52; 95% CI: 1.24–2.86; P<0.001), hepatocellular carcinoma (HR=3.02; 95% CI: 1.77–5.18; P<0.001), non-small-cell lung cancer (HR=2.19; 95% CI: 1.20–3.99; P=0.01), and pancreatic cancer (HR=2.58; 95% CI: 1.11–6.03; P=0.03) but not in osteosarcoma (HR=1.58; 95% CI: 0.47–5.35; P=0.46). In addition, HSF1 overexpression was significantly associated with some phenotypes of tumor aggressiveness including TNM stage, histological grade, lymph node metastasis, and vascular invasion. Conclusion HSF1 overexpression may prove to be an unfavorable prognostic biomarker for solid tumor patients.

Virus-mimicking particles have made great contribution to the development of nanomedicine. Herein, several modularized peptides (lipophilic NS5A peptide, 6xHis tag, and tumor-targeting peptide RGD) were genetically inserted into the C-terminus and the major immunodominant loop region (MIR) of hepatitis B core protein (HBc), respectively. This study demonstrated that the recombinant HBc-based VLPs could participate in self-assembly of monodisperse nanoparticles (33.6±3.5nm) with well-defined morphology, and DOX can be packaged into VLNPs without any chemical modification. Moreover, the HBc-based VLPs could specifically target to cancer cells via the interaction with overexpressed integrin αvβ3. The treatment with DOX-loaded HBc-based VLPs showed a significant inhibition of tumor growth (90.7% TGI) and less cardiotoxicity in B16F10 tumor-bearing mice models than that with the free DOX. Importantly, the results may offer an easy way to give a variety of ideal functional modulations for VLPs, thereby extending its potential biomedicine applications.

PURPOSE:To investigate the association of pretreatment Ki67 expression and the derived neutrophil-lymphocyte ratio (dNLR) with clinical outcomes in nasopharyngeal carcinoma (NPC).PATIENTS AND METHODS:For the study, 46 patients diagnosed with NPC at our hospital were recruited between April 2013 and December 2015. All patients were histologically confirmed to have non-keratinizing undifferentiated NPC. The expression of Ki67 proteins in NPC tissue was analyzed immunohistochemically, and the dNLR was assessed in the peripheral blood, both before treatment. Stage I and II disease was treated with radiotherapy with or without concurrent chemotherapy, and stage III and IV disease was treated with cisplatin-based radiochemotherapy and neoadjuvant chemotherapy regimens.RESULTS:Forty-five of the 46 patients met the criteria, and the median follow-up period was 41 months (15-56 months). The cutoff values for Ki67 and dNLR were 77.5% and 2.01%, respectively. The 3-year overall survival (OS) and progression-free survival rates in the high versus low Ki67 expression groups were 62.5% vs. 93.1% (P = 0.009) and 56.3% vs. 93.1% (P = 0.003), respectively. The 3-year OS rate of patients with high dNLR vs. low dNLR was 64.3% vs. 90.3% (P = 0.023). In the Cox risk ratio model, Ki67 expression and dNLR were independent prognostic factors for OS. Patients were then divided into three groups based on Ki67 expression and dNLR (high risk, both factors were high; intermediate risk, one factor was high; and low risk, neither factor was high). The 3-year OS rates were 20%, 85%, and 95% for the high, intermediate, and low risk groups, respectively (P < 0.001).CONCLUSION:Pretreatment Ki67 and dNLR levels can be used as independent prognostic markers in NPC, and elevated values are associated with poor prognosis. Concurrently, high Ki67 expression and dNLR predict a significantly adverse outcome.

BackgroundDelayed gastric emptying (DGE) is one of the most frequent complications following pancreaticoduodenectomy. This meta-analysis aimed to evaluate the impact of Braun enteroenterostomy on DGE following pancreaticoduodenectomy.MethodsA systematic review of the literature was performed to identify relevant studies. Statistical analysis was carried out using Review Manager software 5.3.ResultsEleven studies involving 1672 patients (1005 in Braun group and 667 in non-Braun group) were included in the meta-analysis. Braun enteroenterostomy was associated with a statistically significant reduction in overall DGE (odds ratios [OR] 0.32, 95% confidence intervals [CI] 0.24 to 0.43; P <0.001), clinically significant DGE (OR 0.27, 95% CI 0.15 to 0.51; P <0.001), bile leak (OR 0.50, 95% CI 0.29 to 0.86; P=0.01), and length of hospital stay (weighted mean difference -1.66, 95% CI -2.95 to 00.37; P=0.01).ConclusionsBraun enteroenterostomy minimizes the rate and severity of DGE following pancreaticoduodenectomy.

The impact of tumor size on prognosis for surgically treated patients with pancreatic ductal adenocarcinoma (PDAC) remains controversial. A systematic review and meta-analysis was performed to evaluate this issue.

Fetal aneuploidy and other chromosomal aberrations affect 9 in 1000 live births. Unlike the invasive diagnosis with high risk of miscarriage, non-invasive prenatal diagnosis (NIPD) sampling from maternal blood becomes a promising way for fetal genetic screening. However, fetal cell-based NIPD has a major challenge due to the small number of fetal cells present in maternal blood. We designed a frequency-enhanced transferrin receptor antibody-labelled microfluidic chip (FETAL-Chip) for efficient enrichment and identification of circulating fetal cells, i.e., circulating nucleated red blood cells (cNRBCs) from maternal blood. The FETAL-Chip can dramatically enhance the interaction of fetal cells with antibody-coated microposts to increase the capture efficiency while minimizing nonspecific adsorption. With the help of immunostaining, we can identify cNRBCs from as little as 2 milliliter maternal blood. Various numbers of cNRBCs were detected from volunteers as early as 7 weeks after conception and throughout the entire pregnancy. Gene analysis was also carried out to confirm the fetal origin of captured cells. With easy, non-invasive and highly efficient enrichment of cNRBCs, the method presented here offers great potential for non-invasive prenatal diagnosis.

A majority of studies have indicated that microRNA-125b (miR-125b) is aberrantly expressed in various types of cancer. However, there are no studies on the expression and function of miR-125b in human laryngeal squamous cell carcinoma (LSCC). In the present study, miR-125b expression in LSCC sample tissues, corresponding adjacent non-neoplastic tissues, LSCC cell lines and a normal human keratinocyte cell line was measured using the reverse transcription-quantitative polymerase chain reaction. Following transfection with miR-125b mimics, the Cell Counting Kit-8, cell migration, cell invasion, western blotting and dual-luciferase reporter assays were performed on LSCC cell lines. According to the results, miR-125b was observed to be significantly downregulated in LSCC, and its expression was significantly associated with clinical stage and alcohol history. miR-125b was also observed to decrease cell growth, migration and invasion in LSCC cells by directly targeting signal transducer and activator of transcription 3. The results of the present study suggested that miR-125b may be a potential treatment target of LSCC in the future.

Abnormal expression levels of microRNA-204 (miR-204) have been identified in various types of human cancer. However, the expression and functions of miR-204, and the underlying molecular mechanism involved in the initiation and progression of hepatocellular carcinoma (HCC), require further investigation. The results of the present study demonstrated that miR-204 is downregulated in HCC tissues and cell lines. Notably, zinc finger E-box binding homeobox 2 (ZEB2) was identified as a direct target of miR-204 in HCC. In addition, miR-204 negatively regulates ZEB2 expression level in HCC cells at the post-transcriptional level. In functional studies, the overexpression of miR-204 inhibited the proliferation, migration and invasion of HCC cells. Furthermore, the knockdown of ZEB2 may mimic the functions of miR-204 in HCC cells, suggesting that ZEB2 is a direct functional target of miR-204. In conclusion, the results of the present study indicated that miR-204 suppresses the tumor growth, migration and invasion of HCC cells by directly targeting ZEB2, and may serve as a novel therapeutic target for HCC.

Primary liver cancer is globally the sixth most frequent cancer, and the second leading cause of cancer death and its incidence is increasing in many countries, thus, becoming serious threat to human health. Substantial research has focused on the treatment and prevention of liver cancer. However, the underlying molecular mechanism of liver cancer are still not fully understood, and therefore development of treatments are delayed. Akt has been suggested to play an essential role in the progression of inflammation response and apoptosis. Hence, in the present study, Akt knockout mice and cell lines were used as a model to investigate the molecular mechanism of Akt-associated inflammatory and apoptotic signaling pathway with NF-κB and Bad in the progression of liver cancer. Western blotting, quantitative RT-PCR (qRT-PCR), immunohistochemistry, ELISA and flow cytometric analysis were used to determine the key signaling pathway in the development of liver cancer. The results indicated that, compared to the normal liver cells, the expression of Akt was significantly higher in liver cancer cell lines. In addition, Akt-knockout liver cancer cells showed lower Akt expression. we also, found that Akt-knockout cancer cell lines modulated inflammation response and apoptosis via inhibiting NF-κB expression and suppressing apoptotic activation. Our results indicated that the downstream signals, including cytokines regulated by NF-κB signaling pathway and caspase-3-activated apoptosis affected by Bad were downregulated for knockout of Akt. These findings demonstrated that Akt is related to NF-κB and Bad signaling pathway possibly playing a direct role in the progression of liver cancer. Thus, Akt might be an important and potential treatment choice for the clinical diagnosis and treatment in the future.

Up to date, there is no adeno-associated virus (AAV) vector-mediated gene therapy targeting Hepatitis B virus (HBV). In this study, adeno-associated virus 2 vector, AAV2, targeting Hepatitis B surface antigen (HBsAg) was constructed, and its effect of mediating the short hairpin RNA (shRNA) inhibition of HBV gene expression was investigated. Phage display technology was used to screen polypeptides that binded to HBsAg. Using PCR and cloning, a specific polypeptide was inserted at aa587 of nucleocapsid protein of AAV2, and AAV2 containing shRNA of HBsAg was also constructed. Using these two plasmids and a helper plasmid, a recombinant virus, rAAVssyU6-shRNA- hrGFP, with both specific polypeptide and shRNA was constructed. Flow cytometry was employed to evaluate the specificity of HepG2.215 cells infection by rAAVssyU6-shRNA-hrGFP and ELISA to detect its inhibitory effect to HBsAg and Hepatitis B e antigen (HBeAg) expression. The specific HBsAg polypeptide sequence is SSYAPYVWQPIA, and rAAVssyU6-shRNA-hrGFP containing both specific polypeptide and shRNA was successfully constructed and the produced virus titer was more than 109 v.g/ml. The infection rate of rAAVssyU6-shRNA-hrGFP virus was higher than that of AAV2 control virus in both HepG2 and HepG2.215. Compared to HepG2, HepG2.215 obtained higher infection rate. Both HBsAg and HBeAg secretions were inhibited in HepG2.215 infected by rAAVssyU6-shRNA-hrGFP virus. Furthermore, blocking with Hepatitis B surface antibody (HBsAb) significantly inhibited the infection rate of rAAVssyU6-shRNA-hrGFP virus in HepG2.215 cells. It is promising to develop AAV-mediated gene therapy targeting HBV.