[Effect of silent information regulator 1 on the LPS induced lncRNA expression of macrophages in mice].

Objective: To investigate the effect of Silent information regulator 1 (Sirt1) on the expression profile of long non-coding RNA (lncRNA) in macrophages upon lipopolysaccharide (LPS) treatment. Methods: Peritoneal macrophages (PM) were isolated from nine wild-type C57BL/6 male mice (wild-type group) and nine myeloid-specific Sirt1 knock-out mice (knock-out group). RNA samples were extracted from macrophages stimulated with 1 µg/ml LPS. Sequencing and the differentially expressed lncRNA were screened after the RNA was quantified. The threshold set for up-and down-regulated genes was a fold change (wild-type group/knock-out group) ≥2 and P≤0.05. Afterwards, gene ontology (GO) and pathway enrichment analysis were conducted and co-expression network map was constructed. Results: Four hundred and forty five lncRNA genes were differentially expressed (185 lncRNA genes were up-regulated and 260 lncRNA genes were down-regulated). Two hundred mRNA genes were differentially expressed (113 mRNA genes were up-regulated and 87 mRNA genes were down-regulated). It was found that the differentially expressed lncRNA genes and the predicted corresponding target genes were mainly distributed in the regions of biological processes of macrophage inflammatory response, macrophage chemotaxis and cell metabolism by GO and pathway enrichment analysis. Conclusion: lncRNA expression profile changes significantly in LPS induced macrophages isolated from Sirt1 knock out mice, which is closely related to the function of macrophages.

Clinical significance of CA 19.9 and LINC01197 in pancreatic cancer.

OBJECTIVE: This study aimed to explore the expression and clinical significance of LINC01197 in serum of patients with pancreatic cancer (PC). PATIENTS AND METHODS: Fifty PC patients (patient group) treated in our hospital from March 2012 to April 2014 were collected, and another 50 normal people (normal group) were collected for physical examination. The LINC01197 expression in serum of the two groups was detected by qRT-PCR method, and the CA 19.9 expression in serum was detected by Roche automatic biochemistry. The expression and diagnostic value of CA 19.9 and LINC01197 in PC were analyzed, and the relationship between LINC01197 and prognosis of PC patients was observed. RESULTS: The CA 19.9 expression in the patient group was significantly higher than that in the normal group (p<0.001). Their area under the curve was 0.791 and 0.944 respectively. The incidence of phases III+IV, lymphatic invasion, and distant metastasis in patients with low expression of LINC01197 is significantly higher than that in those with high expression, and has higher diagnostic value. With the progress of clinical staging, the TNM expression decreased gradually and there were differences between groups (p<0.001). Spearman's test analysis found that the decreased TNM staging of LINC01197 increased gradually (r=-0.816, p<0.001), and the area under the curve of LINC01197 distinguishing phase I and phase II+phase+III+phase IV was 0.930. The 1-year survival rate and 5-year survival rate of patients in low expression group were lower than those in high expression group (p1 year =0.037, p5 year =0.014). Distant metastasis is an independent prognostic factor for PC patients to survive for 1 to 5 years. Differentiation, TNM staging, and LINC01197 are independent prognostic factors for PC patients to survive for 5 years. CONCLUSIONS: The low expression of LINC01197 in PC patients indicates poor prognosis of patients and is expected to be a potential diagnostic and prognostic indicator of PC.

[A retrospective clinical study of 3 382 cases of malignant oral maxillofacial tumors].

To investigate the incidence of malignant tumors in oral and maxillofacial region and the pathological features of various tumors, a total of 3 382 cases of malignant tumors in oral and maxillofacial region admitted to Jilin University from Januarary 2000 to December 2017. The characteristics of age, sex, location and pathological types of all kinds of tumors were analyzed. The median onset age is 57 years old, 51 to 70 years old is a high-risk age group, the ratio of male to female was 1.9∶1. The primary tumor location is tongue, gingiva and floor of mouth. Epithelial, lymphatic hematopoietic system, bone and soft tissue were the three major sources of tumor tissue, and squamous cell carcinoma was the most common pathological type (65.1%), followed by mucoepidermoid carcinoma and adenoid cystic carcinoma. In summary, oral and maxillofacial malignancies have a high incidence in elderly men, and tongue is the most common site of disease. Epithelial-origin and squamous cell carcinomas are the first of their origins and pathological types, respectively.

[Effects of microRNA-34a on regulating silent information regulator 1 and influence of the factor on myocardial damage of rats with severe burns at early stage].

Objective: To explore the effects of microRNA-34a on regulating silent information regulator 1 (SIRT1) and influence of SIRT1 on myocardial damage of rats with severe burns at early stage. Methods: (1) Twenty-four Sprague-Dawley (SD) rats were divided into sham injury (SI) group, simple burns (SB) group and SIRT1 agonist (SA) group according to the random number table (the same grouping method below), with 8 rats in each group. Rats in groups SB and SA were inflicted with 30% total body surface area full-thickness scald (hereinafter referred to as burns) on the back, and rats in group SI were sham injuried on the back. Immediately after injury, rats in groups SI and SB were intraperitoneally injected with normal saline of 50 mL/kg, and rats in group SA were intraperitoneally injected with normal saline of 50 mL/kg and 1 mg/mL resveratrol of 50 mg/kg. At 6 h post injury, abdominal aortic blood was collected to make serum and myocardial tissue of rats was collected. (2) Myocardial cells of twelve neonatal SD rats were collected and divided into microRNA-34a mimic control (MMC) group, microRNA-34a mimic (MM) group, microRNA-34a inhibitor control (MIC) group, and microRNA-34a inhibitor (MI) group, which were respectively transfected with gene sequences of mimic control, mimic, inhibitor control, and inhibitor of microRNA-34a. The microRNA-34a expression level and protein expression level of SIRT1 in myocardial cells were respectively detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. Another batch of myocardial cells were divided into microRNA-34a inhibitor control+ burn serum (MCB) group, microRNA-34a inhibitor+ burn serum (MB) group, and microRNA-34a inhibitor+ burn serum + EX527 (MBE) group. Myocardial cells in group MCB were transfected with gene sequence of inhibitor control, and myocardial cells in the later groups were transfected with gene sequence of inhibitor of microRNA-34a. After transfection of 48 h, myocardial cells in group MBE were cultured in Dulbecco's modified Eagle's medium (DMEM) solution for 6 hours, with serum in group SB of volume fraction of 10% and final amount-of-substance concentration of 1 mol/L, and myocardial cells in the other 2 groups were cultured in DMEM solution with serum from rats of group SB of volume fraction of 10%. The protein expression levels of myocardial cells of SIRT1, cleaved-caspase-3, and Bax were detected by Western blotting. (3) Myocardial tissue from (1) was collected to detect expression levels of microRNA-34a and mRNA of SIRT1 in groups SI and SB by real-time fluorescence quantitative RT-PCR. Morphology of myocardial tissue of rats in groups SI, SB, and SA was observed with biological image navigator. The mRNA expression levels of interleukin 1ß (IL-1ß) and tumor necrosis factor (TNF-α) of rats in groups SI, SB, and SA were detected by real-time fluorescence quantitative RT-PCR. The expression levels of cleaved-caspase-3, and Bax of myocardial tissue of rats in groups SI, SB, and SA were detected by Western blotting. Data were processed with one-way analysis of variance and least-significant difference test. Results: (1) After transfection of 48 h, the expression level of microRNA-34a of myocardial cells in group MM was 4.67±0.92, significantly higher than 1.03±0.04 in group MMC (P<0.01); the protein expression level of SIRT1 of myocardial cells in group MM was 0.35±0.06, significantly lower than 1.12±0.11 in group MMC (P<0.01). After transfection of 48 h, the expression level of microRNA-34a of myocardial cells in group MI was 0.26±0.07, significantly lower than 1.33±0.07 in group MIC (P<0.01); the protein expression level of SIRT1 of myocardial cells in group MIC was 1.12±0.16, significantly lower than 1.74±0.34 in group MI (P<0.01). At 6 h after culture, compared with those in group MCB, the SIRT1 protein expression level of myocardial cells in group MB was significantly increased (P<0.05), while cleaved-caspase-3 and Bax protein expression levels of myocardial cells in group MB were significantly decreased (P<0.05). Compared with those in group MB, the SIRT1 protein expression level of myocardial cells in group MBE was with no significantly statistical difference (P>0.05), and cleaved-caspase-3 and Bax protein expression levels were significantly increased (P<0.05). (2) At 6 h post injury, compared with that in group SI, the microRNA-34a expression level of myocardial tissue in group SB was significantly increased (P<0.01), and the mRNA expression level of SIRT1 of myocardial tissue in group SB was significantly decreased (P<0.01). At 6 h post injury, myocardial cells in group SI arranged neatly with normal nucleus and no inflammatory cells infiltration; myocardial cells in group SB arranged disorderly, with no abnormal nucleus, and obvious inflammatory cells infiltration; myocardial cells in group SA arranged neatly, with normal nucleus and little inflammatory cells infiltration. At 6 h post injury, compared with those in group SB, the mRNA expression levels of IL-1ß and TNF-α, and the protein expression levels of cleaved-caspase-3 and Bax of myocardial tissue in groups SI and SA were significantly decreased (P<0.01). Conclusions: The microRNA-34a expression level of myocardial tissue of rats with severe burns at early stage increases, which decreases the expression level of SIRT1, and increases the expression levels of IL-1ß, TNF-α, cleaved-caspase-3 and Bax, leading to obvious myocardial damage. Activation of SIRT1 can alleviate myocardial damage of rats with severe burns at early stage through decreasing expression levels of IL-1ß, TNF-α, cleaved-caspase-3, and Bax.

[Effects of activating silent information regulator 1 on early kidney damage in rats with severe burn].

Objective: To investigate the effects of activating silent information regulator 1 (SIRT1) on the early kidney damage in rats with severe burn. Methods: Thirty healthy male SD rats were divided into sham injury group (SI), pure burn group (PB), and SIRT1 activator group (SA) according to the random number table, with 10 rats in each group. Rats in groups PB and SA were inflicted with 30% total body surface area full-thickness scald (hereinafter referred to as burn) on the back. Immediately after injury, rats in group PB were intraperitoneally injected with normal saline in the dosage of 50 mL/kg, and those in group SA with 1 mg/mL (final mass concentration) resveratrol in the dosage of 50 mL/kg. Rats in group SI were sham injured and intraperitoneally injected with normal saline in the dosage of 50 mL/kg immediately after injury. Kidney tissue and abdominal aorta blood of rats in the three groups were collected at 24 hours after injury. The morphology of kidney tissue was observed after HE staining. The serum content of creatinine and urea nitrogen was determined with enzyme-linked immunosorbent assay. Protein expressions of SIRT1, Bax, and Bcl-2 in kidney tissue were determined with Western blotting. mRNA expressions of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), and IL-10 in kidney tissue were determined with real-time fluorescent quantitative reverse transcription polymerase chain reaction. Data were processed with one-way analysis of variance and LSD-t test. Results: (1) In rats of group SI, structures of kidney tubules and glomeruli were intact. In rats of group PB, structures of kidney tubules were not clear with casts in them, and glomeruli showed pyknosis. In rats of group SA, structures of kidney tubules were relatively intact, and the pyknosis of glomeruli were slighter as compared with that of group PB with fewer glomeruli showing pyknosis. (2) The serum content of creatinine and urea nitrogen in rats of group PB was (67±14) µmol/L and (22.0±4.4) mmol/L, respectively, which was significantly higher than that of group SI [(28±7) µmol/L and (5.5±1.2) mmol/L respectively, with t values respectively 6.07 and 11.53, P values below 0.01]. The serum content of creatinine and urea nitrogen in rats of group SA was (39±9) µmol/L and (14.1±1.7) mmol/L, respectively, significantly lower than that of group PB (with t values respectively 4.09 and 4.17, P values below 0.01). (3) Compared with those of group SI, protein expressions of SIRT1 and Bcl-2 in kidney tissue of rats in group PB were significantly decreased (with t values respectively 16.32 and 19.58, P values below 0.01), while the protein expression of Bax was significantly increased (t=5.98, P<0.01). Compared with those of group PB, protein expressions of SIRT1 and Bcl-2 in kidney tissue of rats in group SA were significantly increased (with t values respectively 6.94 and 5.37, P values below 0.01), while the protein expression of Bax was significantly decreased (t=3.44, P<0.01). (4) mRNA expressions of TNF-α, IL-1ß, and IL-10 in kidney tissue of rats in group PB were 17.0±4.0, 2.27±0.59, and 2.5±0.9, respectively, significantly higher than those of group SI (1.0, 1.00, and 1.0, respectively, with t values from 3.27 to 8.93, P<0.05 or P<0.01). mRNA expressions of TNF-α and IL-1ß in kidney tissue of rats in group SA were 6.8±1.2 and 1.18±0.26, respectively, significantly lower than those of group PB (with t values respectively 4.59 and 4.32, P values below 0.01). mRNA expression of IL-10 in kidney tissue of rats in group SA was 5.0±1.0, significantly higher than that of group PB (t=5.51, P<0.01). Conclusions: Activating SIRT1 on early stage of severe burn in rats can decrease levels of creatinine and urea nitrogen, thus improving the kidney function. It can down-regulate the protein expression of Bax and up-regulate the protein expression of Bcl-2, thus reducing the apoptosis in kidney tissue. Meanwhile, it can inhibit expressions of TNF-α and IL-1ß and promote the expression of IL-10, thus alleviating the inflammatory response in kidney.

Molecular cloning, characterization, and expression of vacuolar-type-H⁺-ATPase B1 (VHAB1) gene in the gill of Anguilla marmorata.

We explored the molecular mechanism of the regulation of vacuolar-type-H+-ATPase B1 (VHAB1) in elvers in the response to salinity. The full-length cDNA of VHAB1 in Anguilla marmorata (designated as AmVHAB1), which was 1741 base pairs (bp) in length, was found to encompass a 1512-bp open reading frame encoding a polypeptide with 503 amino acids (55.9 kDa), an 83-bp 5'-untranslated region, and a 146-bp 3'-untranslated region. The mRNA and protein expression levels of AmVHAB1 in the gill were evaluated at different time points (0, 1, 3, 6, 12, 24, 48, 72, and 96 h, and 15 days) during the exposure to various salinity levels (0, 10, and 25‰). The results indicated that the expression levels of AmVHAB1 mRNA in the gill significantly increased and reached the highest level at 1 h exposure in the brackish water (BW, 10‰) group and at 6 h exposure in the seawater (SW, 25‰) group. The salinity level affected the relative expression level of AmVHAB1 mRNA in the gill, which was increased by approximately 44-fold in the SW group when compared with that in fresh water. Immunoblotting analysis showed that VHA expression was significantly higher in the BW and SW groups, with the highest expression level was detected at 96 h exposure. We found that the AmVHAB1 gene in elvers from A. marmorata plays an important role in the adaptation to seawater.

An Agrobacterium gene involved in tumorigenesis encodes an outer membrane protein exposed on the bacterial cell surface.

A gene designated as aopB was identified which was involved in tumorigenesis of Agrobacterium tumefaciens. aopB is located on the circular chromosome as a single copy. This gene shares high homology with ropB, a Rhizobium leguminosarum gene encoding an outer membrane protein. A transposon mutant CGI1 containing a gfp-tagged transposon insertion at aopB caused attenuated tumors on plants when inoculated at a low cell concentration (5x10(7) cells/ml). The mutation did not affect the bacterial growth on different media. A broad host range plasmid containing the wild type aopB could restore the tumor formation ability of CGI1 to the wild type level. When both aopB-gfp and aopB-phoA fusions were used to study the aopB gene expression, we found that the aopB gene was inducible by acidic pH but not by plant phenolic compound acetosyringone. aopB encodes a putative protein of 218 amino acids with a predicted molecular weight of 22.8 kDa. TnphoA transposon mutagenesis of aopB, subcellular fractionation and whole cell ELISA experiments indicated that AopB is an outer membrane protein exposed on the bacterial cell surface. It appeared that AopB was exclusively present in the outer membrane and not in other fractions. The vir gene induction assays showed that the aopB gene was not required for the expression of the Ti plasmid encoded vir genes that are essential for tumorigenesis. The C-terminal half of AopB is slightly homologous to some of the bacterial porin proteins and some of plant dehydrins. The role of AopB in Agrobacterium-plant interaction is discussed.

[The application of transposition of the gastrocnemius muscle in the limb-salvage operations of the proximal tibial tumors].

OBJECTIVE: To evaluate the efficacy of transposition of the gastracnemius muscle in the limb-salvage operation of the proximal tibial tumor. METHODS: From 1995, transposition of the gastrocnemius muscle was applied to cover the devitalized bone, bone cement or autologous bone graft in 15 cases with tumors of the proximal tibia (transposition of medial heads of gastrocnemius muscle in 12 cases, and lateral heads of gastrocnemius muscle in 3 cases respectively). Among them, there were 7 osteosarcomas, 5 giant cell tumors, 1 malignant fibrous histocytoma, 1 chondrosarcoma and 1 osteoblastoma. The operations included segmental devitalization with 95% alcohol in 7 cases, knee reconstruction of the unilateral tibial plateau with iliac graft in 5 cases, segmental devitalization with microwave in 2 cases, local resection and bone graft in 1 cases. RESULTS: Apart from 2 cases whose wounds needed suturing again due to the liquefaction of the subcutaneous fat around the incision, no wound complications were occured in other 13 patients. No significant loss in the function of the leg and ankle was observed after transposition of the gastrocnemius muscle. There was no local recurrence, but 3 patients died due to lung metastases. CONCLUSION: Transposition of the gastrocnemius muscle after resection of promixal tibial tumors can improve the local blood supply, cover the deep structures and prevent from the failure of limb-salvage operation due to wound complications.

Molecular mechanism about lymphogenous metastasis of hepatocarcinoma cells in mice.

AIM: To investigate the correlation between lymphogenous metastasis and matrix metalloproteinases (MMPs) activity and the expression of Fas ligand of tumor cells in lymph nodes. METHODS: Fifty-six inbred 615-mice were equally divided into 2 groups and inoculated with Hca-F and Hca-P cells. Their lymph node metastatic rates were examined. Growth fraction of lymphocytes in host lymph nodes was detected by flow cytometry. The Hca-F and Hca-P cells were cultured with extract of lymph node, liver or spleen. The quantity of MMPs in these supernatants was examined by zymographic analysis. The expression of Fas ligand, PCNA, Bcl-2 protein of Hca-F and Hca-P cells in the mice were examined by immunohistochemistry. The apoptosis signals of macro-phages in lymph nodes were observed with in situ DNA fragmentation. RESULTS: On the 28th day post-inoculation, the lymph node metastatic rate of HcaF was 80%(16/20), whereas that of Hca-P was 25%(5/20). The growth fraction of lymphocytes was as follows: in the Hca-F cells, the proliferating peak of lymphocytes appeared on the 14th day post inoculation and then decreased rapidly, while in HcaP cells, the peak appeared on the 7th day post inoculation and then kept at a high level. With the extract of lymph node, the quantity of the MMP-9 activity increased (P<0.01) and active MMP-9 and MMP-2 were produced by both Hca-F and Hca-P tumor cells, which did not produce MMPs without the extract of lymph node or with the extracts of the liver and spleen. The expression of Fas Ligand of Hca-F cells was stronger than that of Hca-P cells (P <0.01). The expressions of PCNA and Bcl-2 protein of Hca-F cells in the tumors of inoculated area were the same as that of Hca-P cells. In situ DNA fragmentation showed that the positive signals of macrophages were around Hca-F cells. CONCLUSION: Secretion of MMPs which was associated with metastatic ability of Hca-F and Hca-P tumor cells depends on the environment of lymph nodes. The increased expression of Fas ligand protein of Hca-F tumor cells with high lymphogenous metastatic potential in lymph nodes may help tumor cells escape from being killed by host lymphocytes.