Background and purpose:Retinoic acid-induced gene G (RIG-G) is a tumor suppressor gene which is cloned by NB4 cell line from a acute promyelocytic leukemia cell. This study aimed to investigate the effect ofRIG-G in lung cancer cells A549 by constructing a lentiviral vector expressing RIG-G under doxycycline (DOX) regulation.Methods:RIG-G gene ampliifcation was performed by quantitative real-time PCR (qRT-PCR). pLenti6/TO/V5-GIM-RIG-G lentiviral vector withGFP was built by LR recombination system. The concentration of pLenti6/TO/V5-GIM-RIG-G lentiviral vector andTet-on lentiviral vector were measured by virus titer method. After infecting A549 cells, stably transfected lines were selected via limiting dilution analysis.RIG-G gene expression was examined by immunolfuorescence staining and Western blot assay. Cellular proliferation was determined by CCK-8 assay.Results:The concentrations of pLenti6/TO/V5-GIM-RIG-G lentiviral vector andTet-on lentiviral vector were 1.0×108TU/mL and 4×109 VP/mL, respectively. RIG-G was expressed in lentivirus infected A549 cells after adding DOX, and the amount of cells withGFP could be observed by lfuorescence microscopy.After the expression of RIG-G protein, the prolif-eration activity of A594 cell was signiifcantly inhibited compared to the control group (1.168±0.107vs 2.099±0.162, P<0.05).Conclusion:The regulated expression ofRIG-G gene was established in A549 lung cancer cell line. The RIG-G protein has potential abilities to inhibit the proliferation of lung cancer cell A549.

Objective:To investigate the mechanisms of IL-1β promoted lung cancer cells proliferation. Methods: The“Transwell? Inserts” system was used to coculture lung cancer cells A549,NCI-H520 with macrophages. BrdU ELISA used to measure the effect of macrophages promoted lung cancer cells proliferation. Expression of mRNA of IL-1β in A549 and NCI-H520 cells were analysed by Real-time PCR analysis. IL-1β was responsible for macrophage-promoted lung cancer cells growth, IL-1β neutralizing antibody was added. The autophagy marker Beclin1 protein was detected by Western blot. Results:The BrdU ELISA assay showed that after coincubation with macrophages in the proportion of 1:0. 5,the OD value of A549 increased from(0. 41±0. 06)to(1. 13±0. 10). There was statistical significance(P<0. 05). It also showed that the growth of the A549 cell was dependent on the macrophage number (P<0. 05). The OD value variability of NCI-H520 cells was as same as A549 cell upon cocultured with macrophages. Real-time PCR results showed that the expression of IL-1β mRNA in macrophages was remarkably enhanced in a time dependent manner upon coincubated with lung cancer cell,and the expression level was higher than lung cancer cells. Addition of IL-1β neutralizing antibody markedly inhibited macrophage-promoted lung cancer cells proliferation. The OD value of these two cells were decreased from ( 3. 63 ± 0. 33) to (1. 46±0. 18),from (2. 94±0. 38) to (1. 53±0. 20),respectively (P<0. 05). After treatment with IL-1β,the expression of Beclin1 was significantly inhibited in tumor cells. Conclusion:Over-expression of IL-1βfrom macrophages and lung cancer cells is re-sponsible for proliferation of tumor cells in coculture condition. Inhibition of autophagy in tumor cells may be the important mechanisms of IL-1β promotes lung cancer cells proliferation.

Objective To establish a TaqMan-MGB fluorescent probe characterized real-time polymerase chain reaction ( qPCR) method for detecting retinoic acid induced genes G ( RIG-G) in human acute promyelocytic leukemia ( M3 ) .Analyze RIG-G expression levels in peripheral blood of both normal persons and M3 patients and explore its diagnosis value for M 3.Methods Methodology establishment study.A detection method and standard curve of TaqMan-MGB real-time PCR were established after designing specific primers and TaqMan-MGB fluorescence probe of human RIG-G gene and using reverse transcription complementary DNA ( cDNA) as a template.The performance of this method was evaluated in specificity, accuracy, precision, analytical sensitivity and interference substances . Twenty clinical specimens with M3 were quantified RIG-G expression so as to evaluate the correlation between peripheral blood and bone marrow samples .Meanwhile , the results of RIG-G expression in peripheral blood of 40 normal specimens and 20 patients with M3 were analyzed by t-test.And receiver-operating characteristic curve ( ROC ) was used to analyze the detection efficiency of M 3.Results There was a good linear relationship between log value of RIG-G standard substance and threshold cycle number ( Ct ) ( standard curve equation:Y=-3.539X+42.952,R2 =0.999).New method was used to detect standard substance . The deviation between observed and expected values was <5% (r=0.999).Three concentration samples (107 ,104 ,101 copies/μl) were selected for precision test.Intra-assay coefficients of variation were 1.38%, 2.31% and 1.38%, respectively , and intre-assay coefficients of variation were 0.71%, 1.17% and 5.07%, separately.All were less than 10%.The sensitivity of this method was 101 copies/μl.There was a good correlation of RIG-G results between peripheral blood and bone marrow in M 3 patients(r=0.996, b=0.973).But there was no significant difference between this two group results (t=0.099, P>0.05). However , there was obvious difference of RIG-G value in peripheral blood between control group and M 3 patient group (U=18,P<0.001), 3.62 ×104(1.61 ×104 -4.90 ×104)copies/μl for controls and 7.10 ×102 (5.43 ×102 -2.21 ×103 ) copies/μl for M3 patients, respectively.Conclusions Successfully establishe a TaqMan-MGB real-time PCR method for detecting RIG-G gene in peripheral blood.The accuracy, precision, sensitivity and specificity are good .It could provide necessary help in early diagnosis and monitor treatment of clinical M3 patients.

Objective To evaluate the serum level of antimicrobial peptide human cationic antimicrobial protein 18 ( hCAP18 ) in non-small cell lung cancer ( NSCLC ) patients and its auxiliary diagnosis and prognosis value.Methods Case-control study was used.The serum level of hCAP18 was measured by enzyme linked immunosorbent assay ( ELISA) in 50 cases with NSCLC patients of department of thoracic surgery and 50 cases healthy people of department of physical examination from January 2011 to January 2012 in Tongji Hospital of Tongji University.The concentrations of hCAP18 in serum of NSCLC patients before and after surgery were analyzed.The sensitivity and specificity of serum hCAP18 for the diagnosis of NSCLC were evaluated using the receiver operating characteristic ( ROC ) curves.Data was analyzed by using the t-test and Log-rank test.Results Serum hCAP18 concentration in NSCLC patients (6 733 ±771.8) μg/L was significantly higher than in healthy controls (253 ±6.9) μg/L (t=8.396, P<0.05) .However, the concentration of hCAP18 showed no significant difference between squamous cell carcinoma and adenocarcinoma[(6 300.0 ±1 221.0) μg/L and (7 074.0 ±1 005.0) μg/L, respectively;t=0.494 2, P <0.05 ] .hCAP18 levels had significantly decreased in serum of NSCLC patients after 30 d surgery compared to preoperative results[from (6 733.0 ±771.8) μg/L to (433.6 ±38.2)μg/L;t=8.512, P<0.05].ROC analysis of serum hCAP18 yielded an AUC (Area under the ROC curve) of 0.931 ( 95% CI =0.884 -0.978 ) with 95% sensitivity and 96.3% specificity, which was higher than the CYFRA21-1[0.873 (95%CI=0.758-0.917)].The relapse rate of NSCLC patients with serum hCAP18≤390.0 μg/L was 12.5%(4/32), while 44.4%(8/18) in NSCLC patients with serum hCAP18>390.0μg/L (χ2 =22.64,P<0.05).Conclusions Detection of serum hCAP18 shows a good sensitivity and specificity for the auxiliary diagnosis of NSCLC. It is possible to be a potential detection index for noninvasive diagnosis and monitoring progression of lung cancer.

Objectve To evaluate the serum level of antimicrobial peptide human cationic antimicrobial protein 18 (hCAP18) in colorectal patients and it auxiliary diagnosis and prognosis value.Methods Case-control study was used.The serum level of hCAP18 was measured by enzyme linked immunosorbent assay(ELISA) in 68 cases with colorectal patients of department of gastrointestinal surgery and 40 cases healthy people of department of physical examination from January 2014 to Junc 2015 in Tongji Hosptial of Tongji University.The concentrations of hCAP18 in serum of colorectal patients before and surgery were analyzed.Immunohistochemistry was used to detect hCAP18 expression in colorectal carcinoma.The effect of hCAP18 on colon carcinoma cell proliferation was detected by BrdU-ELISA and soft agar colony formation assay.The sensitivity and specificity of serum hCAP18 for the diagnosis of eolorectal were evaluated using the receiver operating characteristic curves(ROC).Date was analyzed by using the ttest and one-way analysis of variance.Results hCAP18 serum levels in colon cancer of stage Ⅰ,Ⅱ,llⅢ and Ⅳ patients were (0.46 ± 0.18) mg/L,(0.65 ± 0.45) mg/L,(1.26 ± 0.68) mg/L and (2.35 ± 1.06)mg/L.Mean value was(1.16 ±0.88) mg/L,which was significantly higher than in normal people (0.19 ±0.07) mg/L (t =5.290,P ＜ 0.05).hCAP18 levels had significantly decreased in serum of colorectal patients after 30 d surgery compared to preoperative results [from (1.16 ± 0.88) mg/L to (0.26 ± 0.06) mg/L;t =3.971,P ＜ 0.05].Immunohistochemistry results showed hCAP18 was high expression in colon cancer tissue compared with adjacent tissues;BrdU-ELISA assay results showed HCTll6 and SW480 cell proliferation increased significantly after 0.05-1 mg/L of hCAP18 treatment;Soft agar clone formation experiment proved hCAP18 could significant enhance clone formation of HCT116 and SW480 colon cancer cell lines.The size of clonal cluster of HCT116 was increased from (145.40 ± 35.20) μm to (370.80 ± 32.65) μm (t =10.50,P ＜ 0.05) and SW480 was increased from (101.00 ± 27.10) μm to (369.00 ± 27.29) μm (t =15.58,P ＜0.05);The numbers of clonal cluster of HCT116 was increased from 8.50 ± 2.30 to 42.80 ± 6.60 (t =3.945,P ＜ 0.05) and SW480 was increased from 6.20 ± 1.70 to 46.00 ± 7.20 (t =4.775,P ＜ 0.05).ROC analysis of serum hCAP18 yielded an AUC (area under the ROC curve) of 0.93 (95％ CI =0.859-0.999)with 91.17％ sensitivity and 80.00％ specificity,which was higher than the CEA[0.78 (95％ CI =0.699-0.933)].Conclousions Detection of serum hCAP18 shows a good sensitivity and specificity for the auxiliary diagnosis of colon cancer.It is possible to be potential detection index for noninvasive diagnosis and monitoring progression of colon cancer.hCAP18 could promote the proliferation of colon cancer cells,it played an important role in the progression of colon cancer.

Objective To investigate the association between cumulative exposure blood to pressure (cum BP) and new-onset chronic kidney disease (CKD).Methods In this prospective cohort study,101 510 employees of Kailuan Group receiving annual health examination during 2006 to 2007 were observed.The participants received the second,third,and fourth annual health examinations during 2008 to 2009,2010 to 2011,and 2012 to 2013 year respectively.Their urinary and serum creatinine were tested,and participants with incomplete SBP,DBP data and CKD were excluded.Further excluding those who somehow failed to take annual health examination,with incomplete data,or new-onset CKD 27 809 participants were selected in the analysis.According to cum BP exposure quintile grouping:Q1 ＜ 3.70 scores;Q2:3.70-6.16 scores;Q3:6.17-8.45 scores;Q4:8.46-10.95scores;Q5 ≥ 10.96 scores.Multivariate Logistic regression was used to analyze the association between cum BP level and new-onset CKD by cum BP exposure quintile grouping.Results The rise of cum BP exposure level caused the increased incidence of CKD.The incidences of CKD in the five quintile groups were 2.59％,3.11％,4.19％,5.81％,and 7.73％ respectively (P＜ 0.01).Compared with Q1 group,multivariate logistic regression analysis showed that after the adjustment of age,gender,education,income,smoking,drinking,BMI,FBG,TC,TG,LDL,HDL,UA and CRP,the incidences of CKD gradually increased in the Q2,Q3,Q4,and Q5 cum BP quintile groups,and OR(95％CI) values were 1.08(0.86-1.35),1.26(1.01-1.58),1.57(1.27-1.95),1.78(1.43-2.21) respectively (P for trend ＜0.01).Similar results were obtained in different genders.For each single point increase of cum BP exposure level,the incidence of CKD increased 6％ in the general population (P for trend ＜ 0.01),increased 8％ in male (P for trend ＜ 0.01),and 3％ in female (P for trend=0.12).Conclusion As the cumulative exposure to blood pressure increases,the risk of CKD incidence rises,especially in men.

Objective: To isolate and identify exosomes from human serum, explore the feasibility of exosomal CEA for the diagnosis of colorectal cancer. Methods: Retrospective study.64 cases with colorectal cancer patients(41 cases with normal CEA results and 23cases with high CEA results), 20 cases with benign colorectal diseases patients and 40 cases with healthy people of department of physical examination from October 2015 to December 2016 in Tongji Hospital of Tongji University. Exosomes were isolated from these serum using ExoQuick, and then identified by using transmission electron microscopy, and Western Blot for morphology and molecular phenotype.The serum level of CEA and exosomal CEA was measureed by enzyme linked immunosorbent assay (ELISA). The diagnostic efficacy of serum Exosomal CEA concentration in the colorectal cancer by using U test and the subject work characteristic curve (ROC). Results: Exosomes in human serum was successfully extracted with ExoQuick kit.Exosomal CEA concentration in 64 cases with colorectal cancer patients (1 056.36-28 637.78)pg/ml was significantly higher than in cases with benign colorectal diseases patients (394.61-2 437.83)pg/ml and healthy controls(610.89-2 076.70)pg/ml(U=124.000, U=119.000, P<0.01). Exosomal CEA concentration in 41 colorectal cancer patients with normal serum CEA concentration(1 056.36-5 832.07)pg/ml was significantly higher than in benign colorectal diseases patients and healthy controls(U=113.000, U=119.000; P<0.01). ROC analysis of exosomal CEA yielded an AUC(area under the ROC curve)of 0.954(95% CI=0.919-0.988), which was higher than the serum CEA.The area under the ROC curve of serum Exosomal CEA concentration for colorectal cancer diagnosis is 0.954(95% CI=0.919-0.988), which is superior to the serum CEA concentration[0.636 (95% CI=0.531-0.742)]. Conclusions Isolation and detection of serum Exosomal CEA concentrations have a good diagnostic efficacy in colorectal cancer. It′s possible to be a marker for early diagnosis of colorectal cancer.(Chin J Lab Med, 2018, 41: 503-508)

Objective To explore the structure and component changes of colonization bacterium in colorectal cancer (CRC) and analyze the relationship between the colonization bacteria and the development of CRC.Methods Clinical data of this retrospective study were obtained from Tongji Hospital of Tongji University (1/2016-10/2017).Forty matching tissues,including normal intestinal mucosa tissues (n=12) and CRC tissues (n=28),were collected in the present study.The V3-V4 region of bacteria 16S rRNA gene was detected using Illumina Miseq sequencing technologies.The colonized bacterium structure and composition were analyzed and compared between the two groups.Results It was found that there is significant difference in structure of the colonized bacterium between the two groups.In the level of Phylum,the abundance of Firmicutes and Bacteroidetes were significant lower (Z=-1.964,P＜0.05),and Proteobacteria was significant higher (Z=-1.997,P＜0.001) in the tissues of CRC.In the level of genus,Bacteroides,Blautia,Prevotella,and Faecalibacterium were significant lower in the tissues of CRC (Z=-3.008,P＜0.05).Buchnera,Prevotella,and Prevotellaceae only existed in normal intestinal mucosa tissues and the abundance was top three.Catonella,Kurthia,and Brachymonas only existed in CRC tissues and the abundance was top three.Conclusions The overall structure and component were significant difference between the two groups.Some bacterium disappeared or reduced,and others new emerged or increased.The change of colonized bacterium structure and component in intestinal mucosa may play an important role in the development of CRC.Therefore,it may be an,accurate and low cost of early diagnosis marker by identifing the structure and component change of intestinal mucosa colonized bacterium for CRC,which may find a new way for prevention and treatment of CRC.

Objective: To investigate the effect and mechanism of the antibacterial peptide cathelicidin secreted by tumor associated macrophages on the growth of colorectal cancer in mice. Methods: Azoxymethane (AOM)/ dextran sodium sulfate (DSS) method was used to establish a mouse model of colitis associated colon cancer. To induce tumor formation, cathelicidin antibody, IgG antibody (positive control) or PBS (negative control) was respectively injected into mice once every 3 days and lasted one month. Then the pictures of mice colon were taken, and the numbers of tumor were counted and evaluated. Expressions of cathelicidin in tumor associated macrophages isolated from tumor and adjacent normal tissues of mice were examined by quantitative RT-PCR (qRT-PCR) and Western blot. Expressions of the tumor proliferating antigen Ki-67, macrophage marker CD68 and cathelicidin in tumor and non-tumor tissues were determined by immunohistochemistry analysis. Apoptosis of cells from tumor tissues was analyzed by using TdT-mediated dUTP nick-end labeling (TUNEL). Results: In colon tumor tissues, cathlicidin strongly expressed in inflammatory cells (macrophages), but weakly expressed in tumor cells. The tumor number and size in mice injected with cathelicidin neutralizing antibody were 4.50±1.18 and (1.74±0.18) mm, respectively, significantly lower than 13.88±1.98 and (3.74±0.38) mm of mice injected with PBS (t=4.07, t=4.72; P< 0.01) and 15.25±1.82 and (3.40±0.36) mm of mice injected with IgG antibody (t=4.96, t=4.08; P<0.01). The Ki-67 positive rate of cells in tumor tissues of mice injected with cathelicidin neutralizing antibody was (28.20±3.44) %, significantly lower than (68.20±3.51) % of mice injected with PBS (t=8.135, P<0.01) and (69.20±3.41) % of mice injected with IgG antibody (t=8.461, P<0.01). Immunohistochemistry analyses showed that the expression of CD68 in tumor tissues of mice injected with cathelicidin antibody was significantly lower than that of mice injected with IgG antibody or PBS. TUNEL result showed that treatment with cathelicidin neutralizing antibody had negligible effect on the apoptosis of tumor cells. Conclusions Cathelicidin secreted by tumor associated macrophages can promote the growth of colorectal cancer in mice, and neutralizing cathelicidin activity can inhibit the growth and proliferation of colorectal cancer. Cathelicidin mediated promotion of colon cancer proliferation may mainly be exerted by recruiting inflammatory cells such as macrophages into the tumor microenvironment.

Objective: To investigate the effect and molecular mechanism of antimicrobial peptide LL-37 secreted by stromal cells on the growth of colorectal cancer cells. Methods: Colorectal cancer cells SW480 or HCT116 were co-cultured with human macrophages using Transwell^® maxicell inserts to mimic the tumor microenvironment. The effect of macrophages on the proliferation of colorectal cancer cells was detected by Bromodeoxyuridine and enzyme-linked immunosorbent assay (BrdU-ELISA). The expression of LL-37 mRNA and protein in macrophages and colorectal cancer cells was evaluated by reverse transcription-real-time quantitative PCR (RT-qPCR) and Western blot. LL-37 neutralizing antibody was added to abrogate the LL-37 activation. Additionally, macrophages were transfected with LL-37 shRNA plasmids to inhibit LL-37 expression. And then, the proliferation of colorectal cancer cells was observed. Furthermore, the growth-related signaling pathways were detected by Western blot. Results: The BrdU-ELISA results showed that the absorbance of SW480 cells increased from 1.072±0.097 to 5.121±0.407 after co-culture (P<0.001), and that of HCT116 cells increased from 1.229±0.073 to 3.495±0.228 (P<0.001). RT-qPCR results showed that LL-37 mRNA expression in macrophages significantly increased from 2.682±0.191 to 6.117±0.768 after co-incubation (P<0.05), whereas that in SW480 had no significant difference. Consistently the protein expression of LL-37 in macrophages was significantly increased by Western blot, while it did not change in SW480. The proliferation rate of SW480 cells was repressed by adding LL-37 neutralizing antibody or LL-37 shRNA plasmid. Furthermore, Western blot analysis showed that the expression of non-phosphorylated (activated) β-catenin and its target genes cyclin D1 as well as c-myc were distinctly increased in co-cultured SW480 cells, which could be reversed by anti-LL-37 antibodies. Conclusion Macrophages promote the in vitro proliferation of colorectal cancer cells by enhancing the expression and secretion of antimicrobial peptides LL-37, and it seems that LL-37 activates colorectal cancer cells via Wnt/β-catenin pathway.