Clinical significance of exosomal miR-1231 in pancreatic cancer / 中华肿瘤杂志

Objective@#To investigate the expression and clinical significance of exosomal miR-1231 in plasma of pancreatic cancer (PC) patients and pancreatic cancer cells.@*Methods@#A total of 16 patients who were diagnosed with pancreatic cancer in Hunan Cancer Hospital were collected from April 2016 to August 2017. Meanwhile, 16 healthy volunteers were recruited as the healthy control group at the same period. The plasma exosomes were extracted, and the levels of miR-1231 were detected by qRT-PCR in PC and healthy control groups. Moreover, the clinicopathological significance of exosomal miR-1231 expression was analyzed. Furthermore, the expression of exosomal miR-1231 was detected in several pancreatic cancer cells (MIA PaCa-2, PANC-1, SW1990, AsPC-1 and BxPc-3) and two normal pancreatic epithelial cells (HPDE and human primary pancreatic epithelial cell).@*Results@#qRT-PCR results showed that the expression level of miR-1231 in plasma exosomes of pancreatic cancer patients (1.06±0.46) was significantly lower than that in healthy controls (2.30±0.99; P<0.05). The levels of exosomal miR-1231 in patients with stage Ⅰ-Ⅱ (1.515±0.531), no distant metastasis (1.236±0.461) and no lymph node metastasis (1.337±0.522) were significantly higher than those with stage Ⅲ-Ⅳ (0.848±0.224), distant metastasis (0.757±0.278) and lymph node metastasis (0.838±0.261), respectively (P<0.05 for all). In addition, there were no correlation between exosomal miR-1231 expression and age, sex, smoking history, CA19-9 levels and tumor sites (P>0.05). Furthermore, the expression level of exosomal miR-1231 in pancreatic cancer cell lines (0.142±0.135) was significantly lower than that in normal epithelial cells (1.127±0.179; P<0.05).@*Conclusions@#The downregulation of exosomal miR-1231 in plasma of pancreatic cancer patients and pancreatic cancer cells suggests that it is related to the initiation and development of PC. It may be a new diagnostic and prognostic marker for PC.

Effect of EMS1-siRNA on the proliferation and migrationof human gastric cancer cell line MGC803 / 临床与实验病理学杂志

Purpose To explore the effect of EMS1-siRNA on the growth,invasion and migration of human gastric cancer cell line MGC803.Methods It used the Colony formation assay to determine the abilities of proliferation,and flow cytometry analysis to asses cell cycle distribution and apoptosis,transwell invasion and migration experiment to determine the ability of cell invasion and migration after knockdown the expression of EMS1 in MGC803.Results These results suggested that EMS1 gene down-regulated have no affect on cell cycle and cell apoptosis,but the ability of colony formation depressed and migration lowerd obviously (P ＜ 0.05).Conclusion The results shows EMS1 gene is related to proliferation and migration of tumor.

Effects of RNAi-mediated Cofilin-1 Gene Silencing on proliferation and invasiveness in hepatocellular carci-noma Huh-7 cells / 医学研究生学报

[Abstract ] Objective Cofilin-1 is involved in the pathogenesis of various tumours .However, the expression and effect of Cofilin-1 in hepatocellular carcinoma is not clear .The aim of this study is to observe the Cofilin-1 gene expression in human hepatocel-lular carcinoma (HCC) tissues, and to explore the effect of Cofilin-1 gene expression on invasion and metastasis of HCC HuH-7 cells. Methods Real-time quantitative PCR was used to assess the Cofilin-1 gene expression in human HCC tissues and normal tumor-ad-jacent tissues.The specific small interfering RNA ( siRNA) of Cofilin-1 sequence was synthetized in vitro , and was transfected into HCC HuH-7 cells using liposome transfection.The experiment was divided into Cofilin-1-siRNA group, Ctrl-siRNA group and un-transfected group.Western blot assay was used to detect the protein expression of Cofilin-1.Migration and invasion experiments in vitro were used to investigate the invasive ability of transfected cells. Results Compared with the adjacent liver tissue , Cofilin-1 gene ex-pression in human liver cancer tissue was significantly increased (0.698 ±0.156 vs 3.523 ±0.412, P and in untransfected group (0.961 ±0.020) (P<0.05).The results of migration and invasion experiments in vitro showed that the amount of migration and invasion cells in Cofilin-1-siRNA group were significantly lower than Ctrl-siRNA group or untransfected group (58.50 ±1.78 vs 79.00 ±1.33, 74.50 ±1.35,P<0.05; 36.50 ±0.83 vs 60.20 ±1.60, 51.50 ±1.14, P<0.05). Conclusion Cofilin-1 is highly expressed in HCC, and the invasion and metastasis of HCC HuH-7 cells is suppressed by inhibiting the Cofilin-1 gene expression.

Effects of DADS on the xenograft growth of NCI-H446 human small cell lung cancer cells in nude mice / 医学研究生学报

Objective Diallyl disulfide ( DADS) has achieved remarkable effects in treatment and research of diverserfied cancers.The article was to explore the effects and the mechanism of DADS on the xenograft growth of human small cell lung cancer ( SCLC) cells in nude mice . Methods A total of 25 nude mice were selected to establish xenograft model of NCI-H446 human SCLC cells.The nude mice bearing with SCLC H446 were divided into 5 groups by random selection:positive control group(DDP 66 mg/kg), negative control group(physiological saline), 20 mg/kg DADS group, 60 mg/kg DADS group and 180 mg/kg DADS group, which is 40.6%, 53.1%and 66.4%, respectively.The growth of xenograft tumor in mice was observed after being treated with differ-ent concentrations of DADS .The morphological changes of the tumors were examined under light microscopy .Phase distribution and apoptosis of xenograft cells were analyzed by flow cytometry ( FCM) . Results The growth of xenograft tumor were inhibited signifi-cantly by DADS, resulting in decreased cell density and cellular atypia .Moreover, xenograft cell cycle was blocked in G 2/M and cell apoptosis rate was enhanced . Conclusion DADS can significantly inhibit the growth of NCI-H446 cells and lead to apoptosis .

Characterization of Key Regulatory Elements of LCRG1 Promoter / 生物化学与生物物理进展

LCRG1(laryngeal carcinoma related gene1,LCRG1),a new candidate tumor suppressor gene of laryngeal carcinoma.However,it is known little about the possible regulatory mechanisms of LCRG1 gene expression.Restriction endonuclease digestion was used to obtain a set of the 5',or 3'deletion mutants from the region(-169～+127) of the LCRG1 gene.It has been found that the minimal promoter of the LCRG1 gene is mapped at the region from-169～-57.Linker scanning mutational analysis in the region(-169～+127) of the LCRG1 gene was used to identify the crucial cis-elements within the promoter region,The key cis-elements are within the region from-137～-122.SP1,E2F1/DP1,EKLF and ZF9 transcription factor binding site sites were predicted in the region by bioinformatics analysis.Co-transfection with each of a panel of the expression plasmids of the known transcription factors with the relevant reporter construct indicates Sp1 is potent transcription factor for enhancement of the promoter activity,SP1 can also up-regulate the endogenous expression of LCRG1 gene.Electrophoretic mobility shift assay(EMSA) was applied to verify that the key cis-elements of LCRG1 gene exist sequence of Sp1 binding sites.The findings,which showed that the key cis-elements within the region from 137～-122 play an important role in expression of the LCRG1 gene,provide a novel evidence for further study of the function of LCRG1 gene.

Cloning and Identification of Promoter of Human LCRG1 Gene / 生物化学与生物物理进展

Laryngeal carcinoma related gene 1(LCRG1) is a candidate tumor suppressor gene of Laryngeal carcinoma. To further investigate its transcriptional regulation, the transcriptional start sites for LCRG1 gene have been identified by 5′ RACE (rapid amplification of cDNA ends) based on the bioinformation analysis of LCRG1. Then eleven luciferase expression vectors which contained potential human LCRG1 gene promoter were constructed. Luciferase reporter assay indicated that LCRG1 promoter region was mainly located in -169～+127 region nearby the major transcriptional start site. These results suggested that the region (-169～+127) includes an essential promoter for human LCRG1 gene transcription.

Study on the gene expression profile of poorly differentiated gastric carcinoma using cDNA microarray / 中国癌症杂志

Background and purpose:The molecular mechanism of the pathogenesis of poorly differentiated gastric carcinoma is unclear, because the key genes related to poorly differentiated gastric carcinoma have not been identified. The study was designed to establish the gene expression profile of poorly differentiated gastric carcinoma, isolate gastric carcinoma-related genes, and investigate the relationship between gastric carcinoma-related genes and gastric carcinoma. Methods:The changes of gene expression profile between poorly differentiated gastric carcinoma and adjacent normal tissue of gastric epithelia were analyzed by cDNA microarray which represented approximately 10 000 known genes that would be tested in the assay. Immunohistochemistry were employed to validate the relationship between gastric carcinoma-related genes and gastric carcinoma.Results:212 genes that were differentially expressed in cancer and non cancerous tissues were identified; 169 genes were highly expressed in cancer tissues by more than 2.0 fold, 43 genes were lower expressed in cancer tissues by more than 2.0 fold. EMS1 protein was located in cytoplasma. The positive rate of EMS1 protein expression was 20% (4/20) in normal gastric mucosa and 89.72% (131/146) in the gastric carcinoma, the difference was significant (P

Regulation of LCRG1 promoter transcription activity by Sp1 and Egr-1 / 医学研究生学报

Objective:The regulatory mechanisms of LCRG1 gene expression is undear,this study was to investigate the transcription regulation of the promoter of human LCRG1 gene by transcription factors Sp1 and Egr-1.Methods:Analysis of putative binding sites of transcription factors in the promoter of human LCRG1 gene by online program MatInspector,Co-transfection with the expression plasmids of the known transcription factors such as Sp1、wtEgr-1、mtEgr-1with the LCRG1 reporter construct analyze potent transcription factor for enhancement of the promoter activity.Results:SP1 and Egr-1 transcription factor binding site sites were predicted in the region by bioinformatics analysis,mtEgr-1 can enhance the promoter activity.Conclusion:mtEgr-1 may be involved in the regulatory mechanisms of LCRG1 gene expression

Effects of silencing cortactin expression by siRNA on the proliferation and invasion in Hep-2 cells / 中国癌症杂志

Background and purpose:The cortical actin-binding protein,cortactin,participates in several functions in the cytoskeleton system,cellular signal transduction and cell adhesion.There is also increasing evidence that it regulates tumor invasion and metastasis.However,the role played by cortactin in laryngeal carcinoma has not been clearly delineated.The purpose of this experiment was to investigate the effect of silencing cortactin expression on the proliferation and invasion in the human laryngeal carcinoma cell line Hep-2.Methods:A plasmid from a siRNA targeting cortactin was constructed and transfected into a Hep-2 cell line.The siRNA interference efficiency of cortactin was determined by Western blot.The proliferation was measured by MTT assay and plate colony formation. The Transwell test was used to detect the migration and invasion ability of the Hep-2 cells.Empty plasmid-transfected Hep-2 and normal Hep-2 were used as control groups.Results:Compared to Hep-2 cells,the cortactin expression of pSilencer3.1-cortactin-siRNA/Hep-2 was 11.22%(P

Cloning of 5'flank upstream regulation region of LCRG1 and the measurement of promoter activity / 中国癌症杂志

Background and purpose:The molecular regulation mechanism of the LCRG1 gnen is unclear;The study was designed to clarify the regulatory elements of LCRG1 gene in its 5'flanking region.Methods:Bioinformatics approaches were adopted and a putative promoter region was found in 5'flank upstream fragment of LCRG1 gene,5'flank upstream regulation fragment 1.776 kb(from-1 191 bp to +585 bp)of LCRG1 gene was amplified by PCR using genomic DNA as template,construct was obtained by cloning DNA fragments into pGL3 reporter vector.The construct was then introduced into COS7 cells by Lipofectemin method for transient expression of reporter gene,and luciferase activities was measured by luciferase assay.Results:The sequence of 5'flank upstream regulation fragment 1.776 kb(from-1 191 bp to +585 bp)of LCRG1 gene was successfully cloned and proved to be correct by DNA sequencing,the activity of pGL3-1776 was about 0.16-fold higher than that of pGL3-control cotransfection with PRL-TK and 35-fold higher than that of pGL3-basic cotransfection with PRL-TK.Conclusions:5'flank upstream regulation region 1.776 kb(from-1 191 bp to +585 bp)of LCRG1 was cloned successfully,the fragment presented promoter activity.