MiR-375 is epigenetically downregulated due to promoter methylation and modulates multi-drug resistance in breast cancer cells via targeting YBX1.

OBJECTIVE: In this study, we firstly verified how miR-375 is downregulated in breast cancer cells with multi-drug resistance (MDR) and further investigated the regulative effect of miR-375 on Ybx1 expression. MATERIALS AND METHODS: MiR-375 expression and promoter methylation status were studied by retrieving data in NCBI GEO Datasets, qRT-PCR and Methylation-Specific PCR (MSP) assay. Drug sensitivity of the cancer cells was assessed using MTT assay. The binding between miR-375 and YBX1 gene was predicted using Targetscan 7.1 and verified using western blot and dual luciferase assay. RESULTS: MiR-375 is significantly downregulated in both MCF-7/ADM and MCF-7/PTX cells than in MCF-7 cells. MCF-7/ADM and MCF-7/PTX cells had significantly higher level of promoter methylation than MCF-7 cells. 5-AZA-dC treatment significantly reduced the methylation in MCF-7/ADM and MCF-7/PTX cells and increased miR-375 expression. MiR-375 can directly target 3'UTR of YBX1 and thereby decrease its expression in MCF-7/ADM and MCF-7/PTX cells. Both miR-375 overexpression and YBX1 knockdown significantly decreased P-gp expression and increased chemosensitivity of the cancer cells. CONCLUSIONS: MiR-375 is downregulated in MCF-7/ADM and MCF-7/PTX cells, and its downregulation is a result of promoter methylation. MiR-375 can directly target 3'UTR of YBX1 and thereby decrease its expression, which might be an important mechanism of MDR in breast cancer cells.

Preparation and activity of conjugate of monoclonal antibody HAb18 against hepatoma F(ab')(2) fragment and staphylococcal enterotoxin A.

AIM: To prepare the conjugate of staphylococcal enterotoxin A (SEA) protein which is a bacterial SAg and the F(ab')(2) fragment of mAb HAb18 against human hepatocellular carcinoma (HCC), and identify its activity in order to use SAg in the targeting therapy of HCC. METHODS: MAb HAb18 was extracted from the abdominal dropsy of Balb/c mice, and was purified through chromatography column SP 40HR with Fast protein liquid chromatography (FPLC) system. The F(ab')(2) fragment of mAb HAb18 was prepared by papainic digestion method. The conjugate of mAb HAb18 F(ab')(2) fragment and SEA was prepared with chemical conjugating reagent N succinimidyl 3 (2-pyridyldithio) propionate (SPDP) and purified through chromatography column Superose 12 with FPLC system. The molecular mass and purity of each collected peak were identified with SDS-PAGE assay. The protein content was assayed by Lowry's method. The antibody activity of HAb18 F(ab')(2) against HCC in the conjugate was identified by indirect immunocytochemical ABC method, and the activity of SEA in the conjugate to activate peripheral blood mononuclear cells (PBMC) was identified with MTT assay. RESULTS: The IgG mAb HAb18 was extracted, and purified successfully. Immunocytochemical staining demonstrated that it reacted with most of HHCC cells of human HCC cell line. There were two peaks in the process of purification of the prepared HAb18 F(ab')(2) SEA conjugate. SDS-PAGE assay demonstrated that the molecular mass of the first peak was about 130 ku, and the second peak was the mixture of about 45 ku and a little 100 ku proteins. The immunocytochemical staining was similar in HAb18 F(ab') (2) SEA conjugate and HAb18 F(ab') (2), i.e.the cytoplasm and/or cell membranes of most HHCC cells were positively stained. The MTT assay showed that the optical absorbance (A) value at 490 nm of HAb18 F(ab') (2) SEA conjugate was 0.182 +/- 0.012, that of negative control was 0.033 +/- 0.009, and there was significant difference between them (P < 0.05). CONCLUSION: SPDP is a good protein conjugating reagent and can be used in preparing protein conjugate. The conjugate of mAb HAb18 F(ab') (2) fragment and SEA protein was prepared successfully in present study and can be used in the experimental study of HCC targeting therapy with the conjugate of SAg and anti HCC mAbs or their fragments.

Immunohistochemical demonstration of CCAAT/enhancer binding protein (C/EBP) in human liver tissues of various origin.

C/EBP is a sequence-specific DNA-binding protein. In order to identify its distribution and localization, immunohistochemical technique (ABC method) was done using anti-C/EBP polypeptide antibodies 1103#, 425# in liver specimens from 20 normal adults, 5 neonates, 6 patients with hepatitis, 25 with liver cirrhosis, 80 with hepatocellular carcinoma (40 cases were associated with surrounding nontumorous tissues) and 26 patients with cholangiocarcinoma (15 cases were associated with surrounding nontumorous tissues). The results showed that C/EBP was diffusely distributed in nuclei and cytoplasm of differentiated liver cells and very low or undetectable in liver cancer cells. The manifestation of C/EBP correlated with degree of differentiation of tumour cells, and was obviously weaker than that in surrounding nontumorous tissues. C/EBP positive staining has also been found in regenerating epithelial cells of bile ductules. The results suggested that C/EBP should play an important role in establishing and maintaining the differentiation of liver cells.

[Determination and significance of C/EBP in hepatoma and various liver tissues].

C/EBP is a sequence-specific DNA-binding protein. In order to identify its distribution, localization and function in liver specimens from 18 normal adult, 5 neonates and 79 hepatocellular carcinoma patients (40 cases associated with surrounding nontumorous tissues), immunocytochemical studies (ABC method) were performed by using anti-C/EBP polypeptide antibodies 1103#, 425#. Northern blot using synthesized C/EBP cDNA probe in liver tissues of 3 normal adult, one neonate and 10 hepatocellular carcinoma tissues (8 cases were associated with surrounding nontumorous tissues) were also studied. The results of immunocytochemical staining showed that C/EBP was diffusely distributed in nuclei and cytoplasm of differentiated liver cells and very low or undetectable in liver cancer cells. The expression of C/EBP was in proportion to the degree of tumor cell differentiation and was much less than in the nontumorous surrounding tissues. C/EBP positive staining has also been found in the regenerating epithelial cells of bile ductules. Northern blot examination matched closely with the immunocytochemical examination. The results suggest that C/EBP may play an important role in establishing and maintaining the differentiation of liver cells and may exert an inhibiting effect against transformation of liver cells and proliferation of neoplastic tissue.

[Immunofluorescent localization of associated antigen of anti-human nasopharyngeal carcinoma cell monoclonal antibody].

The immunofluorescent localization of associated antigen of anti-human nasopharyngeal carcinoma (NPC) cell monoclonal antibody (McAb) was performed in different tissues (NPC, non-NPC tumor, chronic inflammation of nasopharyngeal mucosa, adult normal tissue and embryo tissue) with 5 control experiments. McAb (CN-1, Cs-C1) was produced by Department of Microbiology of our college and all the specimens were confirmed by pathology. The results revealed that Cs-C1 antigen was mainly found on the surface of NPC cells with a positive rate of 80.3%. Majority of the positive cells showed yellow-greenish linear fluorescence surrounding the cell membrane while some cells manifested granular fluorescence. In addition, Cs-C1 antigen was also found in a few embryo tissues, epithelial cells of the normal gastric mucosa and cells of the gastric cancer. It is suggested that Cs-C1 antigen be a kind of molecular structure, being gradually produced or increased in quantity during carcinogenesis, and belong to the tumor associated antigen. Cs-C1 antigen could be important in the study of carcinogenic mechanism of NPC cells and valuable to clinical immunodiagnosis.

[Primary malignant lymphoma of the central nervous system--a case report and review of literature].

One case of primary malignant lymphoma of the central nervous system is reported. The patient, a nine year old boy having headache, vomiting, seizures in the right limbs and unconsciousness, was admitted into our hospital and died the next day. A tumor at the base of the left frontal lobe was found on autopsy. There was no evidence of tumor elsewhere. A pathological diagnosis of T-cell malignant lymphoma was established. In this paper, the view that the T-cell lymphoma can arise from the central nervous system is proposed for the first time. Its clinical features, pathomorphology and histogenesis are discussed. Yet, how follicles are formed in the tumor tissue and its significance await further study.