Biobanking of fresh-frozen human colon tissues: impact of tissue ex-vivo ischemia times and storage periods on RNA quality.

BACKGROUND: Biobanking plays an important role in translational cancer research. The impact of tissue ex-vivo ischemia time and storage period on RNA integrity is not well documented. METHODS: Fresh-frozen colon tissues were collected in Taizhou Hospital of Zhejiang Province in China since 2004. Fifty-one colon cancer tissues with tumor cell content higher than 70 % and matched normal tissues during four storage periods (less than 15 months, 16-20 months, 21-25 months, and 26-40 months) were chosen to detect RNA quality. Fresh colon cancer tissues from 5 patients were cut into pieces and kept at room temperature or on ice for 0.5, 1, 2, and 4 h before snap freezing. RNA integrity was determined by microcapillary electrophoresis by the RNA integrity number (RIN) algorithm. RESULTS: Sixty-seven percent of normal colon tissues and 94 % of colon cancer specimens yielded RNA with a RIN of ≥7. Matched colon cancer and normal tissues showed significant difference in RNA quality. RNA remained stable in colon cancer tissues kept at room temperature and on ice for up to 4 h, and long-term storage of banked colon specimens did not negatively influence RNA quality (RNA with RIN of ≥7 banked less than 15 months, 83 %; 16-20 months, 78 %; 21-25 months, 77 %; 26-40 months, 90 %). CONCLUSIONS: Frozen colon tissues yield high-quality RNA in approximately 80 % of specimens. Ex-vivo ischemia times and storage periods did not adversely affect RNA quality. This study showed that standard operation protocols and the maintenance of high-quality tissue repositories were the keys to translational medicine research.

Adhesion-regulating molecule 1 (ADRM1) can be a potential biomarker and target for bladder cancer.

Adhesion-regulating molecule 1 (ADRM1) has been implicated in tumor development, yet its specific role in bladder cancer (BC) remains undefined. This study aimed to elucidate the function of ADRM1 in BC through a combination of bioinformatics analysis and immunohistochemical analysis (IHC). Utilizing R version 3.6.3 and relevant packages, we analyzed online database data. Validation was conducted through IHC data, approved by the Institutional Ethics Committee (Approval No. K20220830). In both paired and unpaired comparisons, ADRM1 expression was significantly elevated in BC tissues compared to adjacent tissues, as evidenced by the results of TCGA dataset and IHC data. Patients with high ADRM1 expression had statistically worse overall survival than those with low ADRM1 expression in TCGA dataset, GSE32548 dataset, GSE32894 dataset, and IHC data. Functional analysis unveiled enrichment in immune-related pathways, and a robust positive correlation emerged between ADRM1 expression and pivotal immune checkpoints, including CD274, PDCD1, and PDCD1LG2. In tumor microenvironment, samples with the high ADRM1 expression contained statistical higher proportion of CD8 + T cells and Macrophage infiltration. Meanwhile, these high ADRM1-expressing samples displayed elevated tumor mutation burden scores and stemness indices, implying potential benefits from immunotherapy. Patients with low ADRM1 expression were sensitive to cisplatin, docetaxel, vinblastine, mitomycin C, and methotrexate. According to the findings from bioinformatics and IHC analyses, ADRM1 demonstrates prognostic significance for BC patients and holds predictive potential for both immunotherapy and chemotherapy responses. This underscores its role as a biomarker and therapeutic target in BC.

[Expression of extracellular matrix metalloproteinase inducer in the unstable plaque of patients with acute coronary syndrome].

OBJECTIVE: To observe the expression of extracellular matrix metalloproteinase inducer (EMMPRIN) in the unstable plaque of patients with acute coronary syndrome (ACS), and the impact of leukotriene B4 (LTB4) on the EMMPRIN expression in macrophages. METHODS: The EMMPRIN expression was detected by immunohistochemistry in 11 unstable plaques from patients with ACS. Protein expression of EMMPRIN was evaluated by Western blot on macrophages differentiated from THP-1 which were stimulated with LTB4 in the absence or presence of LTB4 antagonist U75302. There are 8 study groups: 1-THP-1, 2-8-the macrophages derived from THP-1, 2-6-macrophages were stimulated by LTB4 (0, 10(-10), 10(-9), 10(-8) and 10(-7) mol/L) for 24 h, 7-8-the macrophages were pretreated by 10(-6) mol/L or 10(-7) mol/L U75302 2 h before the LTB4 (10(-7) mol/L) stimulation. RESULTS: Abundant EMMPRIN expression was detected in macrophages and smooth muscle cells of unstable plaques from ACS patients. As to the THP-1 derived macrophages, EMMPRIN expression was significantly upregulated in a concentration-dependent manner in LTB4 stimulated groups, which was significantly higher in group 3-6 than in the THP-1 group (group 1) and macrophages group (group 2) (all P < 0.05) and pretreatment with U75302 significantly reduced the LTB4 induced upregulation of EMMPRIN in a dose-dependent manner (P < 0.05). CONCLUSION: EMMPRIN expression is enhanced in macrophages and smooth muscle cells on unstable coronary artery plaques from ACS patients. LTB4 could stimulate EMMPRIN expression on THP-1 derived macrophages suggesting that LTB4 and EMMPRIN might be both involved in the formation and progression of unstable plaques, future studies are warranted to explore if LTB4 and EMMPRIN antagonists are effective or not for treating patients with ACS.

[Nucleolar localization of human acetyltransferase-like protein and its expression in tumors].

OBJECTIVE: To confirm the nucleolar localization of telomerase-regulation associated protein-human N-acetyltransferase-like protein (hALP) and its associated functions. METHODS: Immunofluoresent staining and immunoelectron microscopy were used to detect the distribution of hALP in HeLa and Saos2 cells, and the co-localization of hALP and rDNA was analyzed by fluorescence in situ hybridization and immunofluoresence. RNAi was performed to further verify the nucleolar localization of hALP. A series of eukaryotic expression plasmids carrying various portions of hALP sequence were constructed and transiently transfected to HeLa and Saos2 cells. The expression of hALP in tumor tissues was stained by immunohistochemistry. RESULTS: hALP distributed predominantly in the nucleoli of HeLa and Saos2 cells, and colocalized with rDNA. Granular component was the precise distribution of hALP in the nucleolus under electron microscope. Nucleolar signals for hALP reduced significantly in cells transfected with hALP siRNA. The carboxy terminus of hALP including residues 549-834 was necessary for its nucleolar localization. hALP could be detected in the nucleoli of many kinds of tumor cells, including leiomyosarcoma, primitive neuroectodermal tumor, neuroblastoma, melanoma, prostatic cancer, and clear cell renal carcinoma. CONCLUSION: hALP is a nucleolar protein, and the nucleolar localization is mediated by its carboxy terminal domain, and hALP could be detected in the nucleoli of many tumor tissues, which is worthy of further investigation.

[Correlation of telomere length and the expression of its regulating proteins in mesenchymal sarcomas].

OBJECTIVE: To explore the significance in the change of telomere length in mesenchymal sarcomas, through analyzing telomere length and expression of its associated proteins, including TRF1, POT1, hTERT, P53 and c-myc. METHODS: The telomere length in 20 cases of osteosarcomas, 25 of chondrosarcomas, 19 of rhabdomyosarcomas, 26 of liposarcomas was measured by telomere fluorescence in situ hybridization (Telo-FISH), and the expression of TRF1, POT1, hTERT, p53 or c-myc was analyzed by immunohistochemistry, respectively. RESULTS: The telomere length in osteosarcomas was significantly shorter than that of either chondrosarcomas or liposarcomas (P<0.05). Similarly, the telomere length of rhabdomyosarcoma was shorter than that of chondrosarcoma (P<0.05). Meanwhile, telomere shortening was positively correlated with down expression of telomere binding proteins TRF1 and POT1 (P<0.05), but trends were detected more frequently in positive expression of hTERT (P<0.05) and in nuclear accumulation of P53 or expression of c-myc. With advancing in histological grading, telomere length was shortened markedly in chondrosarcomas, especially in liposarcomas (P<0.05). CONCLUSION: The shortening of telomere could prevail in mesenchymal sarcoma and reflect the malignant potential. Telomere attrition usually correlated with down expression of POT1, TRF1 and with increased levels of hTERT, P53 and c-myc.

[Expression and significance of metallothionein in the placenta of women with low level lead exposure during pregnancy].

OBJECTIVE: To investigate the significance of metallothionein (MT) expression in the placenta of women exposed to low level lead during pregnancy. METHODS: Sixty-seven pregnant women with blood lead level ranging from 1.5 micromol/L to 4.8 micromol/L were randomly selected from the Department of Obstetrics of Qingdao Municipal Hospital between Mar 2005 and Mar 2006. Among them, 35 were with blood lead level less than 2.9 micromol/L (group A) and 32 more than 2.9 micromol/L (group B). Immunohistochemical streptavidin-peroxidase-biotin methods were used to observe the expression of MT in the placental tissue. RESULTS: (1) Among the 67 pregnant women, the highest level of blood lead was 4.7 micromol/L, and the lowest level was 1.6 micromol/L. The blood lead level of groups A and B was (1.7 +/- 0.3) micromol/L, and (3.1 +/- 0.4) micromol/L, with a significant difference between them (P < 0.05). (2) The positive expression of MT was mainly cytoplastic in the cytotrophoblast, decidual cell and small vascular endothelial cells. The positive cell staining was diffuse or scattered. (3) The positive staining of MT was 91% (29/32) and 74% (26/35) in the placental tissue of groups A and B, respectively, with a significant difference between them (P < 0.05). (4) The blood lead level of pregnant women was correlated with the expression of MT of placenta. CONCLUSIONS: Lead can induce the expression of MT in the placental tissue in a dose-dependent manner. MT is mainly located in the cytotrophoblast, decidual cell and small vascular endothelial cells of the placenta. MT expression in the placenta is important to the structural integrity and function of the placenta.

[Effects of lead exposure to rat placenta and pups during different gestation periods].

OBJECTIVE: To investigate the effects of lead exposure to rat placenta and pups during different gestation periods. METHODS: All 108 Wistar rats (72 females, 36 males) were randomly divided into four groups. All rats were orally fed with 0.025% lead acetate during different gestation periods. Blood was obtained from the abdominal vena cava and the lead level in maternal blood was measured by means of atomic absorption spectrometry at the end of the pregnancy. The number of pups, their body weight, body length and tail length were measured. The effects of lead to rat placenta were observed by level of microscopy, optical microscopy and electronic microscopy. RESULTS: Experimental groups the blood lead level at the end of gestation were above 0.483 micromol/L. There were significant differences among, of pups, during different groups (P < 0.01). Among them the drinking lead group of whole distant was the lowest in placenta weight [(0.31 +/- 0.13) g] body weight of pups [(2.08 +/- 0.88) g] length and tail length of pups [(2.37 +/- 0.32) cm, (0.98 +/- 0.09) cm]. There were significantly differences between the experimental groups and controls. Maternal blood lead level was negatively related to placenta weight (r = 0.652, P < 0.01), and had no relation with the body weight of pups (r = -0.107, P = 0.46). In the experimental groups of lead poisoned rats, the placenta showed focus necrosis in the deciduas, and increased the trophoblastic giant cells and light staining cells in the trophospongium. Trophoblast in the labyrinth and trophospongium showed degeneration; fibrin deposition around the villi was increased. Microvilli around the trophoblast were shorter and less, mitochondrion was swollen and decreased in number, rough endoplasmic reticulum was distended and ribosomal number on membrane decreased. CONCLUSION: Lead exposure during different gestation periods should have a traumatic effect on the trophoblast, leading to interference of nutrition and oxygen exchange. Furthermore, the blood supply to the placenta and nutrition and oxygen exchange between mother and pups were also interfered, leading to reduction of placenta weight and retardation of development of pups.

Expression of hypoxia-inducible factor prolyl hydroxylase 3 HIFPH3 in human non-small cell lung cancer (NSCLC) and its correlation with prognosis.

PURPOSE: To investigate the expression of hypoxia-inducible factor prolyl hydroxylase 3 (HIFPH3) in non-small cell lung cancer (NSCLC) and explore the correlation of HIFPH3 expression with lymph node metastasis and microvessel density (MVD). MATERIALS AND METHODS: A total of 73 cases of NSCLC specimens, 24 cases of para- cancerous tissues, and 20 normal pulmonary tissues were collected for HIFPH3 and CD31 immunohistochmical (IHC) study. Microvessel density (MVD) of the NSCLC tissues was also determined based on the expression of CD31. RESULTS: The expression of HIFPH3 in carcinoma tissue was statistically higher than para-cancerous and normal pulmonary tissues (χ2=48.806, p<0.05). Compared withthe negative lymph node metastasis group, the lymph node metastasis group showed significantly higher HIFPH3 expression (χ2=6.300, p<0.05). The strong HIFPH3+group displayed a significantly higher MVD than weak HIFPH3+ and HIFPH3- groups (p<0.05). No differences in positive HIFPH3 expression were noted regarding the tumor diameter, age, smoking status, gender of NSCLC patients, tumor size, histopathology, or differentiation. CONCLUSIONS: HIFPH3 expression in human NSCLC lesions is significantly higher than that in para-cancerous and normal lung tissues and is positively associated with lymph node metastasis and MVD.