Triple-negative breast cancer (TNBC) is a nasty disease with extremely high malignancy and poor prognosis. Annexin A3 (ANXA3) is a potential prognosis biomarker, displaying an excellent correlation of ANXA3 overexpression with patients' poor prognosis. Silencing the expression of ANXA3 effectively inhibits the proliferation and metastasis of TNBC, suggesting that ANXA3 can be a promising therapeutic target to treat TNBC. Herein, we report a first-in-class ANXA3-targeted small molecule (R)-SL18, which demonstrated excellent anti-proliferative and anti-invasive activities to TNBC cells. (R)-SL18 directly bound to ANXA3 and increased its ubiquitination, thereby inducing ANXA3 degradation with moderate family selectivity. Importantly, (R)-SL18 showed a safe and effective therapeutic potency in a high ANXA3-expressing TNBC patient-derived xenograft model. Furthermore, (R)-SL18 could reduce the β-catenin level, and accordingly inhibit the Wnt/β-catenin signaling pathway in TNBC cells. Collectively, our data suggested that targeting degradation of ANXA3 by (R)-SL18 possesses the potential to treat TNBC.

ObjectiveTo determine whether HBV DNA polymerase is associated with T-cell failure and thus mediates the immune escape of HBV-related hepatocellular carcinoma （HCC） tumor cells， and to investigate the specific molecular mechanisms. MethodsLiver cancer cell lines Huh7 and HepG2 stably transfected with HBV DNA polymerase expression plasmid with Flag （Flag-HBV-P） and intercellular adhesion molecule-1 （ICAM1） were co-cultured with Jurkat cells， and MTT assay， qRT-PCR， and ELISA were used to measure Jurkat cell proliferation， activation （CD69 expression）， and secretion of the cytokine IFN-γ. RNA-seq was used to screen for differentially expressed immune-associated molecules between stably transfected cell lines and control cells， and mRNA half-life and protein half-life assays were used to determine the specific levels of the immune-associated molecules that were affected by HBV DNA polymerase. Related websites were used to predict the transcription factors that may bind to the promoter region of this immune-associated molecule， Western blot was used to verify the effect of transcription factors on the immune-associated molecule， and rescue experiment was used to determine whether HBV DNA polymerase affects the expression level of the immune-associated molecule through this transcription factor. The independent-samples t test was used for comparison between two groups. ResultsThe experimental group had significant reductions in Jurkat cell proliferation， activation， and cytokine secretion compared with the control group （all P<0.01）. Compared with the control group， the experimental group （Huh7 and HepG2 cell lines） had significant reductions in the mRNA and protein expression levels of ICAM1 （all P<0.01）. Website prediction identified the ICAM1 promoter and preliminarily highlighted NFKB1， RELA， and STAT3. Compared with the control group， the experimental group （Huh7 and HepG2 cell lines） had a significant reduction in the protein expression level of p65 （all P<0.01）. After p65 overexpression， there was a significant increase in the protein expression level of ICAM1， and after the expression of p65 was reduced， there was a significant reduction in the protein expression level of ICAM1 （all P<0.01）. In the rescue experiment， there was no significant difference in the protein expression level of ICAM1 between the control group and the experimental group after p65 overexpression （all P>0.05）. After the overexpression of ICAM1， there were no significant differences in the proliferation， activation， and cytokine secretion of Jurkat cells between the control group and the experimental group （Huh7 and HepG2 cell lines） （all P>0.05）. ConclusionHBV DNA polymerase downregulates the level of ICAM1 to mediate HCC immune escape by inhibiting the expression of p65 in NF-κB.

Objective:To compare the safety and short-term efficacy of percutaneous thermal ablation for liver cancer at hepatic dome guided by ultrasound with assisted technologies or computed tomography.Methods:Patients who underwent thermal ablation of liver cancer at hepatic dome from January 2016 to October 2019 in the Xixi Hospital affiliated to Zhejiang Chinese Medical University were studied. Using the different guidance methods, the patients were divided into the ultrasound guidance combined with assisted technologies group (the ultrasound guided group) and the CT guided group. Assisted technologies included contrast-enhanced ultrasound, fusion imaging and artificial ascites. The complications during and within one week of ablation were recorded to compare the safety between the two guidance methods. One month after ablation, contrast-enhanced CT or contrast-enhanced MRI was performed to evaluate the short-term efficacy.Results:Of 52 patients who were included in the study, 39 had no previous treatment while 13 had recurrent liver cancer (7 underwent previous surgical resection and 6 thermal ablation). There were 41 males and 11 females, age ranged from 38.0 to 76.0 (57.3±10.0) years. The tumor size was 12.0 to 46.0 (30.7±8.8) mm. Of the 34 patients in the ultrasound guided group, the age ranged from 38.0 to 73.0 (56.6±10.2) years, and the tumor size ranged from 17.0 to 46.0 (30.6±8.7) mm. Of the 18 patients in the CT guided group, the age ranged from 39.0 to 76.0 (58.1±9.8) years, and the tumor size ranged from 12.0 to 45.0 (30.9±9.2) mm. There were no significant differences in age and tumor size between the two groups ( P>0.05). During and within one week of ablation, 3 patients developed hydrothorax in the ultrasound guided group and 8 patients developed hydrothorax, and 7 patients pneumothorax in the CT guided group. The difference between the two groups was significant ( P<0.05). All complications were successfully treated with conservative treatment. One month after ablation, the complete ablation rate was 91.2% (31/34) in the ultrasound guided group and 88.9% (16/18) in the CT guided group. There was no significant difference between the two groups ( P>0.05). Conclusion:The two methods of guidance resulted in similar short-term efficacy after thermal ablation for liver cancer at hepatic dome. Compared with computed tomography guidance, ultrasound guidance with assisted technologies significantly reduced the incidence of thoracic complication rates.

Objective: To investigate the regulation of hypoxia-inducible factor-1α (HIF-1α) on permeability of rat vascular endothelial cells and the mechanism. Methods: Twelve male Sprague-Dawley rats aged 35 to 38 days were collected and vascular endothelial cells were separated and cultured. The morphology of cells was observed after 4 days of culture, and the following experiments were performed on the 2nd or 3rd passage of cells. (1) Rat vascular endothelial cells were collected and divided into blank control group, negative control group, HIF-1α interference sequence 1 group, HIF-1α interference sequence 2 group, and HIF-1α interference sequence 3 group according to the random number table (the same grouping method below), with 3 wells in each group. Cells in negative control group, HIF-1α interference sequence 1 group, HIF-1α interference sequence 2 group, and HIF-1α interference sequence 3 group were transfected with GV248 empty plasmid, recombinant plasmid respectively containing HIF-1α interference sequence 1, interference sequence 2, and interference sequence 3 with liposome 2000. Cells in blank control group were only transfected with liposome 2000. After transfection of 24 h, expression levels of HIF-1α mRNA and protein of cells in each group were respectively detected by reverse transcription real-time fluorescent quantitative polymerase chain reaction and Western blotting (the same detecting methods below) . The sequence with the highest interference efficiency was selected. (2) Another batch of rat vascular endothelial cells were collected and divided into blank control group, negative control group, and HIF-1α low expression group, with 3 wells in each group. Cells in blank control group were only transfected with liposome 2000, and cells in negative control group and HIF-1α low expression group were respectively transfected with GV248 empty plasmid and low expression HIF-1α recombinant plasmid selected in experiment (1) with liposome 2000. After 14 days of culture, the mRNA and protein expressions of HIF-1α in each group were detected. (3) Another batch of rat vascular endothelial cells were collected and divided into blank control group, negative control group, and HIF-1α high expression group, with 3 wells in each group. Cells in blank control group were transfected with liposome 2000, and cells in negative control group and HIF-1α high expression group were respectively transfected with GV230 empty plasmid and HIF-1α high expression recombinant plasmid with liposome 2000. After 14 days of culture, the mRNA and protein expressions of HIF-1α of cells in each group were detected. (4) After transfection of 24 h, cells of three groups in experiment (1) and three groups in experiment (2) were collected, and mRNA and protein expressions of myosin light chain kinase (MLCK), phosphorylated myosin light chain (p-MLC), and zonula occludens 1 (ZO-1) of cells were detected. Data were processed with one-way analysis of variance and t test. Results: After 4 days of culture, the cells were spindle-shaped, and rat vascular endothelial cells were successfully cultured. (1) The interference efficiencies of HIF-1α of cells in HIF-1α interference sequence 1 group, HIF-1α interference sequence 2 group, and HIF-1α interference sequence 3 group were 47.66%, 45.79%, and 62.62%, respectively, and the interference sequence 3 group had the highest interference efficiency. After transfection of 24 h, the mRNA and protein expression levels of HIF-1α of cells in interference sequence 3 group were significantly lower than those in blank control group (t=18.404, 9.140, P<0.01) and negative control group (t=15.099, 7.096, P<0.01). (2) After cultured for 14 days, the mRNA and protein expression levels of HIF-1α of cells in HIF-1α low expression group were significantly lower than those in blank control group (t=21.140, 5.440, P<0.01) and negative control group (t= 14.310, 5.210, P<0.01). (3) After cultured for 14 days, the mRNA and protein expression levels of HIF-1α of cells in HIF-1α high expression group were significantly higher than those in blank control group (t=19.160, 7.710, P<0.01) and negative control group (t= 19.890, 7.500, P<0.01). (4) After transfection of 24 h, the mRNA expression levels of MLCK and p-MLC of cells in HIF-1α low expression group were significantly lower than those in blank control group (t=2.709, 4.011, P<0.05 or P<0.01) and negative control group (t=2.373, 3.744, P<0.05 or P<0.01). The mRNA expression level of ZO-1 of cells in HIF-1α low expression group was significantly higher than that in blank control group and negative control group (t=4.285, 5.050, P<0.01). The mRNA expression levels of MLCK and p-MLC of cells in HIF-1α high expression group were significantly higher than those in blank control group (t=9.118, 11.313, P<0.01) and negative control group (t=9.073, 11.280, P<0.01). The mRNA expression level of ZO-1 of cells in HIF-1α high expression group was significantly lower than that in blank control group and negative control group (t=2.889, 2.640, P<0.05). (5) After transfection of 24 h, the protein expression levels of MLCK and p-MLC of cells in HIF-1α low expression group were significantly lower than those in blank control group (t=2.652, 3.983, P<0.05 or P<0.01) and negative control group (t=2.792, 4.065, P<0.05 or P<0.01). The protein expression of ZO-1 of cells in HIF-1α low expression group was significantly higher than that in blank control group and negative control group (t=3.881, 3.570, P<0.01). The protein expression levels of MLCK and p-MLC of cells in HIF-1α high expression group were 1.18±0.24 and 0.68±0.22, which were significantly higher than 0.41±0.21 and 0.35±0.14 in blank control group (t=5.011, 3.982, P<0.05 or P<0.01) and 0.43±0.20 and 0.36±0.12 in negative control group (t= 4.880, 3.862, P<0.05 or P<0.01). The protein expression level of ZO-1 of cells in HIF-1α high expression group was 0.08±0.06, which was significantly lower than 0.20±0.09 in blank control group and 0.19±0.09 in negative control group (t=4.178, 3.830, P<0.05 or P<0.01). Conclusions HIF-1α up-regulates expressions of MLCK and p-MLC and down-regulates expression of ZO-1, thereby increasing the permeability of rat vascular endothelial cells.

Objective To explore the mechanism of hypertonic salt solution (HS) alleviates lung injury of rats at the early stage of severe scald.Methods Thirty-two female Sprague-Dawley (SD) rats were randomly assigned to sham group, lactated Ringer solution (LR) group, HS200 group (200 mmol/L HS group, 1 L 200 mmol/L HS contained 955 mL LR and 45 mL 10％ NaCl) and HS400 group (400 mmol/L HS group, 1 L 400 mmol/L HS contained 846 mL LR and 154 mL 10％ NaCl), with 8 rats in each group. A 30％ total body surface area (TBSA)Ⅲ degree scalded model was reproduced by scalded on the back with 98℃ boiling water for 12 seconds, whereas those in the sham group were exposed to 37 ℃ water without liquid resuscitation. Rats in the three drug intervention groups were resuscitated with LR, 200 mmol/L HS and 400 mmol/L HS by caudal vein according to the Parkland formula, respectively. All rats were sacrificed at 8 hours after scald injury to harvest abdominal aorta blood and lung tissues. Interleukins (IL-6, IL-10 and IL-17) in serum were determined by enzyme-linked immunosorbent assay (ELISA). Samples from the lung tissue were used to measure malondialdehyde (MDA) and superoxide dismutase (SOD) levels by ultraviolet spectrophotometer. Expressions of p38 mitogen-activated protein kinase (p38MAPK) and extracellular regulated protein kinase 1/2 (ERK1/2) in the lung were determined by Western Blot. The lung tissue was stained with hematoxylin and eosin (HE), and the pathological changes were observed with a light microscope.Results Compared with the sham group, the lung tissues in the LR group were damage obviously, which accompanied with more inflammatory cell infiltration, cell edema and pulmonary septum thickening, and the levels of IL-6, IL-10, IL-17 in serum and MDA content, the phosphorylation of p38MAPK and ERK1/2 in lung tissues were increased whereas the activity of SOD was decreased. Compared with the LR group, the lung injury was significantly alleviated, the levels of IL-6, IL-17 in serum and MDA content and the phosphorylation of p38MAPK and ERK1/2 were decreased, and the levels of IL-10 and SOD were increased in both HS groups with a dose-dependent manner. There were significant difference in above parameters between HS400 group and LR group [serum IL-6 (ng/L): 3.76±0.12 vs. 6.72±0.90, serum IL-10 (ng/L): 33.76±3.71 vs. 16.77±3.19, serum IL-17 (ng/L): 103.52±2.78 vs. 124.96±4.96, lung MDA (nmol/mg): 5.59±0.24 vs. 7.09±0.39, lung SOD (U/mg):226.7±3.9 vs. 172.7±3.4, lung phosphorylation of p38MAPK (p-p38MAPK)/p38MAPK: 0.15±0.09 vs. 0.35±0.19, lung phosphorylation of ERK1/2 (p-ERK1/2)/ERK1/2: 0.27±0.01 vs. 0.70±0.01, allP < 0.01].Conclusion HS protected against lung injury induced by severe burns in rats with a dose-dependent manner, and it was better than LR, and its possible mechanism was related with reducing the expression of p38MAPK and ERK1/2 pathway in lung tissue, increasing the level of anti-inflammatory cytokines and decreasing the release of pro-inflammatory cytokines, thus inhibiting excessive inflammation and oxidative stress injury in lung.

Objective To investigate the methods and results of hand reconstruction by transplanting the fostered residual finger and the second toe,which share the same blood supply system.Methods From January,2014 to January,2016,3 cases with destructive hand injuries in our hospital;and debridement was performed in one session during the early stage of trauma,with skin grafting to cover the wrist stump and foster the residual finger to a branch of the dorsalis pedis artery.The first dorsal metatarsal artery around the toe web was identified and then gradually isolated proximally with dissection of the branches of the dorsalis pedis artery supplying the fostered finger until the convergence of the two blood vessels.Then,reconstructed hand by residual finger and combined second toe transplantation.Results After a follow-up period of 3 months to 2 years,the reconstructed hand had a good appearance and was able to complete basic mundane actions.The function of the limb was restored to a large extent andthe patient was satisfied with the reconstructed hand.Conclusion Transfer of a fostered residual finger and combined second toe which have the same blood supply system for hand reconstruction as a new method need to anastomose only one set of arteries,and it is feasible with the good appearance and function of the reconstructed hand.

Autophagy plays a dual role in tumor therapy.A variety of chemotherapy drugs can induce tumor autophagy by activating endoplasmic reticulum stress,and hyperactive autophagy can cause tumor cell apoptosis and plays an anti-tumor effect.Autophagy has a protective effect on tumor cells,which can maintain the survival of tumor cells and reduce the sensitivity of chemotherapy drugs by reusing the degradation of the damaged organelles.Therefore,the promotion or inhibition of autophagy modulators combined with chemotherapy drugs will have the potential for development in tumor therapy.

Objective To evaluate the role of two-dimensional ultrasound combined with contrast-enhanced ultrasonography (CEUS) in the classification of liver nodules in cirrhotic patients.Methods Consecutively cirrhotic patients with intrahepatic nodules at Xixi Hospital of Hangzhou were included from November 2015 to December 2016.All (142 nodules in 109 patiens) presented as non-cancerous focal lesions on conventional magnetic resonance imaging and CT examination and had available information of liver biopsy.Each lesion was percutaneous biopsied under the guidance of two-dimensional ultrasound.Ultrasonographic parameters evaluated were as following:(1) sizes of nodules under US;(2) ultrasonographic characteristics of the nodular;(3) CEUS enhancement features of the nodules.Four types of hepatic nodule suggesting different histology were defined according to the ultrasonographicparameters.x2 test was used to compare the difference of hepatocellular carcinoma (HCC) incidence among liver nodules with varying sizes and nodules with different enhancement features under CEUS.As for the statistical differences of HCC and high-grade dysplastic nodule (HGDN) incidence between type Ⅲ & Ⅳ nodules and type Ⅰ & Ⅱ nodules,x2 test was also used for analysis.Results A total of 142 eligible nodules were detected in 109 patients with cirrhosis,including 16 HCCs,2 intrahepatic cholangiocellular carcinomas (ICC),41 HGDNs,40 low-grade dysplastic nodules (LGDN) and 43 regenerative nodules (RN).In terms of diameter,all (6/6) the nodules larger than 2.0 cm,20.0％ (8/40) of middle size nodules (1.5-2.0 cm),were HCCs.The remained 2 lesions of HCC came from two subgroups with even small size nodules [1.0-1.4 cm (n=93),and ＜ 1.0 cm (n=3),in diameter],respectively.Two lesions of ICC were attributed to nodules with a 1.0-1.4 cm diameter.About 28 nodules with a diameter of 1.5-2.0 cm,13 nodules with a diameter of 1.0-1.4 cm were HGDN.HCC incidences between these 4 groups were different significantly (x2=61.425,P ＜ 0.001).Asfor the CEUS,14 nodules exhibited a rapid enhancement feature in arterial phase,12 of which were HCC.In56 nodules with a slow enhancement feature,4 nodules were HCC.HCC incidences between these 3 groups were different significantly (x2=75.752,P ＜ 0.001).Under the combined ultrasonography,HCC incidences of type Ⅲ and type Ⅳ nodules were significantly higher than that of type Ⅰ and type Ⅱ lesions [21.9％ (16/73)vs 0 (0/65),x2=15.222,P ＜ 0.001],similar result was observed in the comparison of HGDN incidences between type Ⅲ & Ⅳ and type Ⅰ & Ⅱ nodules[53.4％ (39/73) vs 3.1％ (2/65),x2=38.842,P ＜ 0.001].Conclusion The classification presented by this study,combining the three ultrasonographic parameters,which is nodule size,nodular echo characteristics and enhancement features of the nodules under CEUS,could be helpful for the diagnosis of HCC in cirrhotic patients with ill-defined nodule on routine image examination.

Objective To investigate the methods of repair the lateral skin and soft tissue defect of the middle and rear foot with the plantar medial flap transferred before the achilles tendon.Methods Fifteen cases with the lateral skin and soft tissue defect of the middle and rear foot were repaired with the plantar medial flap pedicled with posterior tibia artery transferred before the achilles tendon from January,2012 to October,2015.All the 15 patients were followed up in the way by telephone or back to hospital postoperatively 3 months to 3 years,including 6 cases by telephone follow-up and 9 cases of back-to-hospital follow-up,to observe the appearance,functions of the skin flap and whether the skin flap survived well.Results The flaps of 15 cases survived well.Postoperative followup of 3 months to 3 years,the sensation of the flap was good,without any ulcer or necrosis.The patients walked well and were satisfied with the flap.Conclusion To repair the lateral skin and soft tissue defect with the plantar medial flap transferred before the achilles tendon as a new method is feasible with the good ability of faster sensory recovery,the good appearance and function.

Objective To investigate the clinical effect of individual prefabricated reconstructive titanic plate combined with vascularized iliac bone-muscular flap for reconstruction of mandibular defects.Methods From Aug.2010 to Dec.2014,12 cases with mandibular tumor received preoperative maxillofacial CT scans and 3-dimensional reconstruction.Based on the CT results,mirror imaging technology was used to simulate the reconstruction of the defect at affected side.The individual reconstructive titanium plate was prefabricated on the model.The iliac bone-muscular flap was designed according to the defect shape and size,combined with reconstructive plate.All patients underwent CT scan and oral curved surface tomography postoperatively.The maxillofacial function and appearance were also evaluated.Results According to the CT scan and curved surface tomography,the 12 iliac bone-muscular flaps survived completely with good bone union and good condyle position.Both the functional and cosmetic results were satisfactory.There was no complication on the donor sites.Conclusions Individual prefabricated reconstructive titanic plate combined with vascularized iliac bone-muscular flap provides an precise method for reconstruction of mandibular defects.The cosmetic and functional results could be expected.