Triple-negative breast cancer is therapeutically challenging due to the low expression of tumor markers and 'cold' tumor immunosuppressive microenvironment. Here, we present a dual-targeting peptide-drug conjugate (PDC) for tumor inhibition. Our PDC efficiently and selectively delivers cytotoxic Monomethyl Auristatin E (MMAE) into tumor cells via C-X-C chemokine receptor type 4 (CXCR4) and folate receptor 1 (FOLR1) for synergistic inhibition of growth and metastasis. Our results show that the dual-targeting PDC has potent antitumor activity in cultured human cells and several murine transplanted tumor models without apparent toxicity. The combination of dual-targeting PDC and radiotherapy modulates the tumor immunosuppressive microenvironment by increasing CD8⁺ T cell infiltration and attenuating the proportion of myeloid-derived suppressor and regulatory T cells. Therefore, our dual-targeting PDC represents a promising new strategy for cancer therapy that rebalances the immune system and promotes tumor regression.

Objective:To analyze the expression of tuftelin protein (TUFT1) and its clinical value in hepatocellular carcinoma (HCC)-related liver cancer tissues.Methods:The biological information data of TUFT1 mRNA expression in liver cancer and non-cancer tissues were analyzed from the TCGA and Oncomine database. After the approval of the ethics committee, the self-pairing method was used to collect the postoperative cancer and para-carcinoma tissues of 132 HCC cases hospitalized between January 2009 and December 2014. Tissue microarray and immunohistochemistry (IHC) were used to analyze the expression of TUFT1 in liver tissues. According to IHC staining, liver cancer was divided into high TUFT1 and low/no expression group. Combined with clinical data, the clinicopathological characteristics were statistically analyzed between and within the groups. The 5-year overall survival (OS) and disease-free survival (DFS) was analyzed by correlation analysis.Results:IHC staining showed that TUFT1 in cancer tissue was localized in the cytoplasm and cell membrane, and its positive expression rate was significantly higher in the liver cancer group (87.1%) than the para-carcinoma group (64.4%) ( χ2 = 18.563, P < 0.001). TUFT1 expression intensity in patients with liver cancer was significantly correlated with HBeAg positive ( χ2 = 4.080, P = 0.043), tumor size ( χ2 = 9.388, P = 0.002), vascular invasion ( χ2 = 14.885, P < 0.001), TNM stage ( χ2 = 13.516, P < 0.001) and ascites ( χ2 = 5.940, P = 0.015). TUFT1 high expression was negatively correlated with OS and DFS ( P < 0.001). Conclusion:The overexpression of TUFT1 is closely related to HBV replication, vascular invasion and poor prognosis, and it is expected to become a useful marker for liver cancer diagnosis and prognosis.

Objective:To analyze the expression of CD44 in non-alcoholic fatty liver disease (NAFLD) accompanied with hepatitis B virus (HBV) infection and its clinical significance.Methods:Blood sample of hospitalized patients with NAFLD, chronic hepatitis B, cirrhosis, and healthy population (control) was collected. The study was approved by the hospital ethics committee. Serum CD44 level and clinopathological characteristics were analyzed quantitatively by enzyme-linked immunosorbent-assay. Flow cytometry was used to analyze the proportion of CD44 +T lymphocytes in patients with NAFLD and chronic hepatitis B. NAFLD model was prepared with high-fat diet to verify the abnormal expression of CD44. Results:Compared with the healthy control group, the expression of serum CD44 in the cirrhosis group, chronic hepatitis B group and NAFLD group was increased, and the difference between the groups were statistically significant ( P < 0.01). NAFLD patients graded as mild or severe group were equally accompanied by hepatocyte injury, abnormal blood glucose, lipid or CD44. In NAFLD patients accompanied with HBV infection, serum CD44 concentrations were significantly higher in HBsAg, HBeAg and HBV DNA positive group than HBsAg, HBeAg and HBV DNA negative group ( P < 0.01). The proportion of CD44 +T lymphocytes in peripheral blood of NAFLD and chronic hepatitis B group were 78.2% ± 16.3% and 68.5% ± 20.9%, respectively, and both groups (NAFLD and chronic hepatitis B) were significantly higher than the healthy control group (46.5% ± 20.5%) ( P < 0.05). The high-fat diet model confirmed that in rat liver tissues the CD44 was overexpressed with fat deposition accompanied with liver cell damage, especially remarkable in liver tissues containing carcinogens. Conclusion:The abnormal expression of CD44 in patients with NAFLD may be related to the malignant transformation of HBV-related liver disease.

The early diagnosis and effective treatment of hepatocellular carcinoma (HCC) still remains a difficult problem that plagues the medical community. Exosomes are microvesicles with a diameter of 40~100 nm, and contains proteins, lipids and nucleic acids (mRNAs, lncRNAs, circRNAs, and microRNAs). They serve as an information exchange carrier, and play an important role in regulating and controlling the biomolecular function to maintain the stability of the intracellular environment. The function of exosomes in HCC includes intercellular communication, neoangiogenesis, cancer cell metastasis and multidrug resistance, which mediates the transformation of microRNAs (miRNA) and regulate the microenvironment of tumor progression, and then affect the pathophysiological behavior of cancer cells. Exosome-derived miRNA can be used for HCC monitoring or potential specific markers of early diagnosis. In addition, with the development and application prospects it could be a therapeutic goal for HCC. This paper summarizes the recent progress in the study of HCC-derived exosomal miRNA.

Objective: To quantitatively detect CD44 expression in patients with nonalcoholic fatty liver disease (NAFLD) for comparative analysis. Methods: Patients with chronic liver diseases accompanied with or without NAFLD, including chronic hepatitis B, cirrhosis and hepatocellular carcinoma after chronic hepatitis B, and healthy blood donors as normal controls who admitted to the Affiliated Hospital of Nantong University from May to October 2018 were selected. The proportion of CD44 positive cells was analyzed by flow cytometry. CD44 level was quantified by an enzyme-linked immunosorbent assay, and the biochemical indicators such as serum aspartate aminotransferase, alanine aminotransferase activity, total cholesterol and triglyceride were routinely analyzed. The cancerous and adjacent cancerous tissues of patients accompanied with or without NAFLD were collected by self-matching method and analyzed by immunoblotting and histochemistry and compared by CD44 integrated optical density. Image-Pro Plus version 6.0, Image J, GraphPad Prism 5.0, Photoshop, Microsoft Excel and IBM SPSS statistics 23 were used to analyze and draw pictures. An independent sample t-test was used to compare the differences between groups. Results: Patients accompanied with NAFLD had hepatocyte injury and dyslipidemia. NAFLD and chronic liver disease patients had significantly elevated serum CD44 levels than normal control group (P < 0.01). CD44 positive lymphocyte ratio was 78.19 % ± 16.33 % in NAFLD patients and 68.47% ± 20.91% in chronic hepatitis B group, which was higher than the control group (46.51% ± 20.52%). Chronic hepatitis B group with steatosis had significantly higher CD44 concentration (181.42 ± 49.36) ng/ml than chronic hepatitis B group (142.52 ± 53.87) ng/ml and normal control group (99.47 ± 15.23) ng/ml. CD44/GAPDH ratio in the liver cancer group (1.306 ± 0.614) was significantly higher than paracancerous group (0.477 ± 0.291) and the difference between the two groups was statistically significant (t = 3.451, P = 0.004). The integrated optical density of CD44 in the NAFLD-related liver cancer and paracancerous group were 25.721 ± 5.881 and 14.155 ± 4.001 and the difference between the groups was statistically significant (t = 14.544, P < 0.001). The pathological features of high expression of CD44 in patients with hepatocellular carcinoma were significantly correlated with HBV infection, tumor size, single/multi-center, and lymph node metastasis, degree of differentiation, TNM grade, Child-Pugh score, portal vein tumor thrombus and extrahepatic metastasis. HCC patients with NAFLD had significantly higher serum CD44 (234.62 ± 69.40) ng/ml than patients without NAFLD (186.49 ± 58.89) ng/ml (t = -3.191, P = 0.002), but there was no statistically significant difference in the clinicopathological characteristics between the high/low CD44 groups of HCC patients with NAFLD. Conclusion The results suggest that CD44 is abnormally activated and its mechanism may play an important role in the progression of NAFLD.

Objective: To investigate the Wnt3a expression in tissues of HCC and its gene knockout on effects of HepG2 cell proliferation or xenograft tumor growth. Methods: Hepatic Wnt3a expressions in 87 HCC and their matched surrounding tissues were observed by tissue microarray and immunohistochemistry for analyzing its clinicopathological characteristics; Wnt3a-knockout HepG2 cell lines were established by Crispr/cas9-sgRNA system and genomic cleavage efficiency was verified at gene level by surveyor assay. The relative proteins were confirmed by Western blotting; Cell Counting Kit-8 assay was used to examine cell proliferation after knocking-out Wnt3a successfully, and the nude mice HepG2 cell xenograft tumors delete that the relationship between Wnt3a and HCC growth. Results: The positive Wnt3a with brown staining particles was mainly distributed in cytosol and membrane of hepatocytes. The incidence of hepatic Wnt3a expression in cancerous tissues (95.4%) was significantly higher (χ ² = 47.754, P < 0.001) than that in their surrounding tissues (49.4%). The high Wnt3a expression was 70.1% in the HCC and only 14.9% in the surrounding tissues. High Wnt3a expression was associated with poorly-differentiated grade, liver cirrhosis, HBV infection, portal vein invasion, TNM stage and 5-year survival rate. After knocked-out by Crispr/cas9-sgRNA system successfully, Wnt3a expression was down-regulated significantly at gene or protein level. Key molecule β-catenin in cytoplasma was obviously inhibited. HepG2 cell lines proliferation was suppressed in time-dependent manner. The nude mice HepG2 cell xenograft tumors confirmed that the knock-out of Wnt3a could significantly supressed HCC growth with slower speed (t = 6.418, P < 0.001), smaller volume(869.4 ± 222.5 mm³ vs 355.0 ± 99.9 mm³, t = 5.168, P < 0.001), and lighter weight (0.88 ± 0.20 g vs 0.35 ± 0.11 g, t = 5.628, P < 0.001)compared with the control group. Conclusion Abnormal expression of Wnt3a could be expected as a promising target for HCC gene therapy.

BACKGROUND: Cardiospecific proteins of the troponin-tropomyosin complex in the contractile system of the cardiomyocytes have challenged creatine kinase isoenzyme MB (CK-MB) as the "gold standard" for the early biochemical detection of acute myocardial injury.OBJECTIVE: To investigate cardiac troponin Ⅰ messager RNA (cTnI-mRNA) in peripheral blood and its clinical values in diagnosis of patients with myocardial injury.DESIGN: A basic and observational study for set up a method to analyze cTnI-mRNA.SETTING: Department of Cardiology, Affiliated Hospital, Nantong University.MATERIALS: The project was accomplished from May 2003 to May 2005 in Research Center of Clinical Molecular Biology, and Department of Pathology, Affiliated Hospital, Nantong University. The cTnI-mRNA was detected from blood by a nested PCR assay, and its clinical values as a sensitive myocardial diagnostic marker were confirmed in patients with myocardial injury.METHODS: Pathologic features and microstructure of cardiac myocytes were examined by H&E staining or electron microscopy. The cTnI-mRNA was extracted from blood and synthesized to cDNA through random primers and reverse transcriptase, and amplified by a nested PCR assay, and its clinical values as a myocardial diagnostic marker were investigated in patients with myocardial injury.MAIN OUTCOME MEASURES: Microstructure of cardiomyocytes, sensitivity of analysis method and diagnostic values.RESULTS: Microstructure of cardiomyocytes with mitochondria swell,rupture, vacancy-like denaturation, nucleus abnormality, and chromatin condensed were observed by electron microscopy. The cTnI-mRNA fragments from heart and blood were successfully amplified and the sensitivity was 2 pg/μL. The product sequences from tissues or blood were confirmed by sequencing. The cTnI-mRNA from cardiac myocytes was found that it present in blood plasma and not in circulating nucleus cell. The incidence of blood cTnI-mRNA of chronic cardiomyopathy was significantly higher (P ＜ 0.05) than that of serum enzymatic patterns or cTnI quality,respectively.CONCLUSION: The analysis of blood cTnI-mRNA is a sensitive marker for diagnosis and monitoring of myocardial injury.