Effects and mechanisms of shRNA interfered with expression of leucine-rich repeat containing G-protein coupled receptor 5 on the malignant behaviors of colorectal cancer stem cells / 中华消化外科杂志

Objective To investigate the effects and mechanisms of shRNA interfered with expression of leucine-rich repeat containing G-protein coupled receptor 5 (Lgr5) on the malignant behaviors of colorectal cancer stem cells (CSCs).Methods The experimental study was conducted.The CSCs expressing Lgr5+ were sorted by fluorescence activated cell sorting.Lgr5+ cells that were transfected with Lgr5-shRNA lentiviral vector and nontarget shRNA lentiviral vector were respectively allocated into the experimental group and control group.The percentage of Lgr5+ cells was analyzed by flow cytometery.The relative expression of Lgr5 mRNA was detected by fluorescence quantitative real-time polymerase chain reaction (qRT-PCR).The capacity of self-renewal was detected by sphere forming assay.The tumorigenesis in vitro and in vivo were respectively measured by colony formation assay and xenografting experiment.The mRNA expressions of stem cells related genes (Oct4,Sox2,Nanog,KLF4),CSCs genes (CD133,CD44,ALDH) and Wnt/β-catenin pathway key genes (Axin2,Wnt5a,Wnt3a,Fzd3,c-myc,VEGF,Ascl2,claudin-1) were detected by qRT-PCR.Measurement data with normal distribution were represented as-x±s.Comparison between groups was analyzed using the t test.Results (1)Transfection efficiency of shRNA lentiviral vector induced Lgr5 by flow cytometery was respectively 6.8％± 1.0％ in the experimental group and 92.7％±3.3％ in the control group,with a statistically significant difference (t =43.148,P＜0.05).The relative expression of Lgr5 mRNA measured by qPT-PCR was respectively 0.168±0.057 in the experimental group and 1.148±0.004 in the control group,with a statistically significant difference (t=28.778,P＜0.05).(2) The capacity of self-renewal was detected by sphere forming assay.The results of sphere forming assay:the number of spheres was 29±6 in the experimental group and 410± 10 in the control group,with a statistically significant difference (t =41.070,P＜0.05).The results of colony formation assay:the numbers of colonies in the experimental group and control group were respectively 72±4 and 412± 19,showing a statistically significant difference (t =31.433,P＜ 0.05).The results of tumorigenesis:the volumes of tumors in the experimental group and control group were respectively (81± 15)mm3 and (328±24)mm3,with a statistically significant difference (t=11.304,P＜0.05).(3) The effects of Lgr5 down-regulation on related genes,results of qRT-PCR detection:① The mRNA relative expressions of Oct4,Sox2,Nanog and KLF4 (stem cells related genes) were 0.377±0.093,0.662±0.104,3.591±0.300,0.425±0.091 in the experimental group and 1.957± 0.026,2.137±0.015,5.831±0.165,1.536±0.014 in the control group,with statistically significant differences (t=23.079,22.261,8.446,19.186,P＜0.05).② The mRNA relative expressions of CD133,CD44 and ALDH (CSCs genes) were 1.490±0.155,5.535±0.487,1.640±0.039 in the experimental group and 2.488± 0.061,9.908±0.332,5.718±0.292 in the control group,with statistically significant differences (t =8.170,9.667,27.849,P＜0.05).③The mRNA relative expressions of Axin2,Wnt5a,Wnt3a,Fzd3,c-myc,VEGF,Ascl2 and claudin-1 genes in the Wnt/β-catenin pathway were respectively 1.592±0.267,0.528±0.138,2.153±0.078,1.480±0.064,0.248±0.128,1.492±0.025,0.658±0.095,1.647±0.087 in the experimental group and 3.651±0.224,2.570±0.093,2.301±0.157,1.636±0.058,1.415±0.080,2.610±0.159,2.480±0.123,3.432±0.273 in the control group.There were statistically significant differences in the mRNA relative expressions of Axin2,Wnt5a,c-myc,VEGF,Ascl2 and claudin-1 genes between the 2 groups (t =7.316,15.332,12.649,12.320,14.831,9.063,P＜0.05),and no statistically significant difference in the mRNA relative expressions of Wnt3a and Fzd3 between the 2 groups (t =2.887,2.242,P＞0.05).Conclusion The malignant behaviors of colorectal CSCs are suppressed after shRNA lentivirus interfered with expression of Lrg5,and its mechanism is related to inhibiting activity of Wnt/β-catenin pathway.

Effect of restrictive intravenous fluid on the complications and quality of life in the advanced hepatocellular carcinoma patients / 中国医师杂志

Objective To investigate the effect of restrictive intravenous fluid on the complications and quality of life in the advanced hepatocellular carcinoma patients.Methods Clinical data of Three hundred and fifty-seven cases with advanced hepatocellular carcinoma from Mar 2010 to Mar 2016 was retrospectively analyzed.One hundred and sixty-eight cases were recruited in the restrictive intravenous fluid (RIF) group,and One hundred and eighty-nine cases were involved in the control group.The average volume of intravenous fluid of each day,plasma albumin concentration,splanchnocoel hydrops rate,phlebitis,incidence of vomiting,cancer related pain degree,anxiety degree were compared in the two groups.Results The average volume of intravenous fluid of each day in the RIF group [(720.29 ± 106.84) ml] were much lower than that in the control group [(1 820.36±342.12)ml] (P ＜0.05).The plasma albumin concentration in the RIF group [(35.65 ± 2.21)g/L] were higher than that in the control group [(32.25 ±2.32)g/L] (P ＜0.05).The rate of splanchnocoel hydrops,phlebitis,vomiting,bedsores,and hypstatic pneumonia in RIF group were 6.25％,4.69％,8.59％,3.9％,11.72％ and those in the control group were 13.97％,10.92％,17.47％,10.04％,and 24.45％,respectively (P ＜ 0.05).Moreover,the scores of cancer related pain and anxiety were much lower in the RIF group (5.21 ± 1.09,39.12 ± 5.54) than those in the control group(5.68 ± 1.18,41.56 ± 6.78) (P ＜ 0.01).Conclusions Restrictive intravenous fluid therapy can decrease the cancer associated complications and improved the quality of life in the advanced hepatocellular carcinoma patients.

Effect of restrictive intravenous fluid on the complications and quality of life in the advanced hepatocellular carcinoma patients / 中国医师杂志

Objective To investigate the effect of restrictive intravenous fluid on the complications and quality of life in the advanced hepatocellular carcinoma patients.Methods Clinical data of Three hundred and fifty-seven cases with advanced hepatocellular carcinoma from Mar 2010 to Mar 2016 was retrospectively analyzed.One hundred and sixty-eight cases were recruited in the restrictive intravenous fluid (RIF) group,and One hundred and eighty-nine cases were involved in the control group.The average volume of intravenous fluid of each day,plasma albumin concentration,splanchnocoel hydrops rate,phlebitis,incidence of vomiting,cancer related pain degree,anxiety degree were compared in the two groups.Results The average volume of intravenous fluid of each day in the RIF group [(720.29 ± 106.84) ml] were much lower than that in the control group [(1 820.36±342.12)ml] (P ＜0.05).The plasma albumin concentration in the RIF group [(35.65 ± 2.21)g/L] were higher than that in the control group [(32.25 ±2.32)g/L] (P ＜0.05).The rate of splanchnocoel hydrops,phlebitis,vomiting,bedsores,and hypstatic pneumonia in RIF group were 6.25％,4.69％,8.59％,3.9％,11.72％ and those in the control group were 13.97％,10.92％,17.47％,10.04％,and 24.45％,respectively (P ＜ 0.05).Moreover,the scores of cancer related pain and anxiety were much lower in the RIF group (5.21 ± 1.09,39.12 ± 5.54) than those in the control group(5.68 ± 1.18,41.56 ± 6.78) (P ＜ 0.01).Conclusions Restrictive intravenous fluid therapy can decrease the cancer associated complications and improved the quality of life in the advanced hepatocellular carcinoma patients.

Postoperativer TACE in hepatocellular carcinoma: a Meta-analysis / 中华肝胆外科杂志

Objective To evaluate the therapeutic effects of postoperative TACE (transarterial chemoembolization) in patients with hepatocellular carcinoma after liver resection.Methods The search was conducted on China biomedical literature database,Chinese CNKI,VIP,Wanfang and PubMed,OVID,Embase and Cochrane library.All the literatures were searched till the end of January 2016.The quality of the included studies were evaluated using the modified Jadad score for randomized controlled trials and the Newcastle-Ottawa Scale for case controlled studies.The trials were analyzed by Stata12.0.Results A total of 2184 patients from 1 randomized controlled trial (RCT) and 14 case-control studies were included.These patients were divided into two groups based on the treatment method.469 patients were included into the hepatic resection only group and 1087 patients were into the postoperative TACE group.The results showed that postoperative TACE improved the 1-year survival for hepatocellular carcinoma.Subgroup analyses were conducted on the risk factor group (tumor diameter greater than 5cm;multiple nodules and blood vessel invasion) and the no risk factor group.Postoperative TACE played an important role in the risk factor group (subgroup analysis for the no risk factor group:RR =1.10,95％ CI:0.97,1.25,P ＞0.05;subgroup analysis for the risk factor group:RR =1.16,95％ CI:1.02,1.32,P ＜ 0.05).Postoperative TACE significantly improved the 3 year survival rate in the postoperative TACE group (RR =1.33,95％ CI:1.15,1.53,P ＜0.05),but the five-year survival rate showed no significant difference between the two groups (RR=1.21,95％CI:0.95,1.15,P＞0.05).Conclusions Postoperative TACE prolonged the survival rate of patients with hepatocellular carcinoma,especially those who had the following risk factors:tumor diameter greater than 5 cm;multiple nodules;blood vessel invasion.Postoperative TACE can be recommended as a routine treatment.

Role of portal venous pressure changes in the liver dysfunction caused by hepatic congestion after extended liver resection / 中华消化外科杂志

Objective To explore the role of portal venous pressure changes in the liver dysfunction caused by hepatic congestion after extended liver resection.Methods The experimental study was adopted.According to the random number table,90 Sprague-Dawley rats were divided into 3 groups,30 in each group:30 rats in the non-congestion group received 70％ of liver resection (median lobe + left lobe),30 rats in the congestion group received 70％ of liver resection (median lobe + left lobe) with whole caudal lobe congestion by ligation of veins and 30 rats in the congestion + splenectomy group received 70％ of liver resection (median lobe + left lobe) with whole caudal lobe congestion by ligation of veins and splenectomy.(1) Twenty rats in each group were used to make postoperative survival analysis.Ten rats in each group were used for related experiments.The portal venous pressures (PVPs) of 5 rats in each group were detected at postoperative 12 hours and 24 hours,and then blood and liver specimens were collected.(2) PVP changes were detected at postoperative 12 hours and 24 hours.(3) Clinical and biochemical test:level of total bilirubin (TBil) was tested at postoperative 12 hours and 24 hours.(4) Pathological examination:liver pathological damage was detected by HE staining.(5) The expression of CD68 macrophagocyte was detected by immunohistochemical staining.(6) The relative expressions of Cleaved Casepase-3 and hypoxia inducible factor-1α (HIF-1α) proteins at postoperative 24 hours were detected by Westein blot.(7) The relative expressions of mRNA of vascular regulation related genes (ET-1/eNOS) and inflammatory factors (TNF-α and IL-6) were detected by real-time polymerase chain reaction (RT-PCR).(8)The hyaluronic acid (HA) was measured by enzyme-linked immuno-sorbent assay (ELISA).Measurement data with normal distribution were represented as （x） ± s.Comparison among 3 groups was done using the ANOVA,and pairwise comparison was done by the LSD test.The postoperative 5-day survival curve was drawn by the KaplanMeier method,and the survival was compared using the Log-rank test.Results (1) Survival analysis:5-day survival rate in the non-congestion group,congestion group and congestion + splenectomy group were respectively 75％,10％ and 55％,with a statistically significant difference among the 3 groups (x2=18.21,P ＜0.05).(2)Changes of PVPs and TBil:levels of PVP and TBil in the non-congestion group,congestion group and congestion + splenectomy group were respectively (15.77 ±0.67)cmH2O,(18.33 ±0.28) cmH2O,(14.87 ± 0.58) cmH2O,(1.48 ±0.10)μmol/L,(1.76±0.15) μ mol/L,(1.62 ±0.11) μmol/L at postoperative 12 hours and (13.49 ± 0.45) cmH2 O,(16.96 ± 0.82) cmH2 O,(15.69 ± 0.85) cmH2 O,(1.47 ± 0.11) μmol/L,(1.94 ± 0.07) μmol/L,(1.67 ± 0.11) μmol/L at postoperative 24 hours,showing statistically significant differences among 3 groups (F =56.53,29.01,6.81,27.85,P ＜ 0.05).(3) Results of pathological examination:compared with noncongestion group,there were a lot of vacuolar cells with degeneration appearing in non-congestion liver tissues,severe liver cell swelling and hepatic sinus congestion in the congestion group at postoperative 24 hours.Compared with congestion group,vacuolar degeneration appearing in non-congestion liver tissues have some improvement in the congestion + splenectomy group.(4) Immunohistochemical staining:compared with non-congestion group and congestion + splenectomy group,the positive CD68 marked macrophages in the congestion group were increased at postoperative 24 hours.(5) Western blot assay:the relative expressions of Cleaved Casepase-3 and HIF-1α proteins in the non-congestion group,congestion group and congestion + splenectomy group were 0.63 ± 0.05,1.17 ± O.18,0.95 ± 0.17 and 0.63 ± 0.14,1.48 ± 0.08,1.13 ± 0.17,respectively,showing statistically significant differences among 3 groups (F =17.42,50.58,P ＜ 0.05).(6) Results of RT-PCR:the relative expression of mRNA of ET-1/eNOS in the non-congestion group,congestion group and congestion + splenectomy group was respectively 1.01 ± 0.63,2.09 ± 0.27,0.82 ± 0.12 at postoperative 12 hours and 0.73 ± 0.17,2.16 ± 0.94,0.80 ± 0.24 at postoperative 24 hours,showing statistically significant differences among 3 groups (F =62.91,10.65,P ＜0.05).The relative expression of mRNA of TNF-α in the non-congestion group,congestion group and congestion + splenectomy group was respectively 0.99 ± 0.08,127.80 ± 13.15,7.34 ± 1.56 at postoperative 12 hours and 0.99 ± 0.06,116.62 ± 13.32,58.62 ± 12.12 at postoperative 24 hours,showing statistically significant differences among 3 groups (F =436.77,154.54,P ＜ 0.05).The relative expression of mRNA of IL-6 in the non-congestion group,congestion group and congestion + splenectomy group was respectively 0.98 ±0.06,1.87 ±0.34,1.54 ±0.15 at postoperative 12 hours and 0.99 ±0.05,2.02 ±0.27,1.51 ±0.11at postoperative 24 hours,with statistically significant differences among 3 groups (F =22.08,46.71,P ＜ 0.05).(7) Results of ELISA:the level of HA in the non-congestion group,congestion group and congestion + splenectomy group was respectively (149 ± 9) ng/L,(200 ± 19) ng/L,(174 ± 9) ng/L at postoperative 12 hours and (136 ± 16) ng/L,(202 ± 13) ng/L,(91 ± 11) ng/L at postoperative 24 hours,with statistically significant differences among 3 groups (F =19.23,34.68,P＜0.05).Conclusions On the basis of extended liver resection,a wide range of liver congestion through increasing PVP causes hepatic microcirculation disorders,hypoxia,inflammation,vacuoles degeneration cells,increased cells apoptosis,aggravated damage of liver function and increased mortality of rats.Splenectomy could reduce PVP and then improve the liver tissues damage caused by liver congestion,meanwhile,increase the survival rate of rats.

LY294002 decreases the proliferation of cancer stem cell-enriched spheroid cells from human hepatocel-lular carcinoma via inhibiting of PI3K-Akt signaling pathway / 国际肿瘤学杂志

Objective To investigate the impact of LY294002 on the proliferation of cancer stem cell-enriched spheroid cells from human hepatocellular carcinoma via regulating phosphatidylinositol-3-kinaseprotein kinase B(PI3K-Akt)signaling pathway. Methods The cancer stem cell-enriched spheroid cells were genera-ted by culturing HepG2 cells in serum-free medium. LY294002(10,20,30 μmol/ L),an inhibitor of PI3K-Akt signaling pathway,was used in the experimental groups,without used in the control group. The impact of LY294002 on the spheroid cells proliferation was confirmed by cell counting kit(CCK-8 kit). The expression of Akt was tested by Western blotting. The expression of PI3K-Akt signaling pathway downstream genes such as decoy receptor 3(DcR3),mammalian target of rapamycin(mTOR),B-cell lymphoma(Bcl)-2 and Cyclin D1 were tested by real-time PCR. Results 30 μmol/ L LY294002 could inhibit the proliferation of spheroid cells, and significant difference in the absorbance(A value)was observed between the experimental group and control group[(0. 14 ± 0. 03)vs(0. 56 ± 0. 01),t = - 8. 915,P = 0. 000]. The expression level of phosphorylated Akt protein increased[(0. 57 ± 0. 08)vs(0. 16 ± 0. 42),t = 6. 027,P = 0. 026]. The mRNA of DcR3 [(0. 38 ± 0. 08)vs 1,t = 13. 060,P = 0. 006],mTOR[(0. 37 ± 0. 04)vs 1,t = 30. 363,P = 0. 001],Bcl-2 [(0. 26 ± 0. 04)vs 1,t = 33. 554,P = 0. 001]and Cyclin D1[(0. 10 ± 0. 02)vs 1,t = 63. 528,P = 0. 000] decreased. Conclusion LY294002 could inhibit the proliferation of cancer stem cell-enriched spheroid cells from human hepatocellular carcinoma via inhibiting PI3K-Akt signaling pathway.