ObjectiveTo investigate the effects of overexpression of alpha/beta hydrolase domain-containing protein 5 (ABHD5) on the invasion and migration of human colon cancer cell line HCT116 and the pathway of adenosine monophosphate-activated protein kinase (AMPK)/mechanistic target of rapamycin (mTOR).MethodsThe expression of ABHD5 in colon cancer tissues and its relationship with clinicopathological features was analyzed by UALCAN database. HCT116 cells were cultured in vitro and transfected with ABHD5 recombinant plasmid, then they were divided into control group, negative transfection group and ABHD5 transfection group. Real time quantitative PCR (qRT-PCR) was used to detect the expression of ABHD5 mRNA in HCT116 cells. The proliferation of HCT116 cells was detected by CCK-8 method. Transwell assay was used to detect the invasion and migration of HCT116 cells. The expression of matrix metalloprotein 9 (MMP-9), E-cadherin, Snail, and AMPK/mTOR pathway proteins p-AMPK, AMPK, p-mTOR and mTOR were detected by Western blot.ResultsThe results of the UALCAN showed that compared with normal colon tissues, the expression of ABHD5 mRNA in colon cancer tissues was decreased (P<0.05), and which in the adenocarcinoma and the N1 stage was lower than that of the mucinous adenocarcinoma (P<0.05) and N0 stage (P<0.05), respectively. Compared with the control group and the negative transfection group, the expression of ABHD5 mRNA in the ABHD5 transfection group was increased (P<0.05), the proliferation inhibition rate of HCT116 cells in the ABHD5 transfection group was increased (P<0.05), the numbers of migration and invasion cells in the ABHD5 transfection group were decreased (P<0.05), the expressions of MMP-9, Snail, p-mTOR and mTOR were reduced, and the expressions of E-cadherin, p-AMPK and AMPK were increased (P<0.05).ConclusionsThe overexpression of ABHD5 can inhibit the invasion and migration of colon cancer HCT116 cells, activate AMPK, and inhibit the expression of mTOR. It suggests that ABHD5 may play a role in inhibiting colon cancer by affecting AMPK/mTOR pathway.
ObjectiveTo explore the protective effect of rapamycin on pancreatic damage in severe acute pancreatitis (SAP) and further to explain its protective mechanism.MethodsNinety selected SPF males SD rats were randomly divided into 3 groups: sham-operated group (SO group), SAP group, and rapamycin group (RAPA group), with 30 rats in each group. Then each group of rats were randomly divided into 3 subgroups of 24 h, 36 h, and 48 h, 10 rats in each subgroup. Rats in each group underwent laparotomy, the model was prepared by retrograde injection of solutions into biliopancreatic duct, rats of the SO group were injected with 0.9% normal saline, rats of the SAP group and RAPA group were injected with 5% sodium taurocholate solution, but rats of the RAPA group were injected with rapamycin at 30 min before the injection of 5% sodium taurocholate. All the survival rats in corresponding subgroup were killed at 24 h,36 h, and 48 h after operation respectively, then serum and pancreas tissues of rats were collected, serum inflammatory factors content of IL-1β, IL-6, and TNF-α were detected by ELISA method, expression levels of p-mTOR and p-S6K1 in pancreas were detected by Western blot, pancreas tissues were stained by Hematoxylin-Eosin Staining and pathological changes of pancreas were scored under light microscope.Results① At the timepoint of 24 h, 36 h, and 48 h, the order of the expression levels of p-mTOR and p-S6K1 in pancreatic tissues of 3 groups were all as follows: SO group<RAPA group<SAP group, there were significant difference among any 2 groups (P<0.05). ② IL-1β: at the timepoint of 48 h, the order of the content of IL-1β in 3 groups were as follows: SO group<RAPA group<SAP group, there were significant differences among any 2 groups (P<0.05); IL-6: at the timepoint of 36 h and 48 h, the order of the content of IL-6 in3 groups were as follows: SO group<RAPA group<SAP group, there were significant differences among any 2 groups (P<0.05); TNF-α: at the timepoint of 48 h, the order of the content of TNF-α in 3 groups was as follows: SO/RAPA group<SAP group (P<0.05), but there was no significant difference between the SO group and RAPA group (P>0.05). ③ Pancreatic histological score: at the timepoint of 24 h, 36 h, and 48 h, the order of the pancreatic histological score in3 groups was all as follows: SO group<RAPA group <SAP group, there were significant differences among any 2 groups (P<0.05). ④ The expression levels of p-mTOR and p-S6K1 in pancreatic tissue were positively correlated with the pathological scores of pancreatic tissue (r=0.97, P<0.01; r=0.89, P<0.01).ConclusionRapamycin can reduce the degree of pancreatic damage in SAP and has protective effect on pancreatic tissue.
Objective To study the p-mammalian target of rapamyein(p-roTOR)expression and its prognostic significance in non-small cell lung cancer(NSCLC)．Methods Immunohistochemical staining of EnVision was applied to investigate the expression of p-roTOR in lung specimens from 59 cases with NSCLC and 10 cases with benign pulmonary diseases(3 pulmonary tuberculosises and 7 inflammatory pseudotumors 1．Results The positive rate of p-mTOR was 40．7％ in NSCLC which was significantly higher than that in the benign pulmonary diseases(x =6．237，P=0．013)．The expression of p-mTOR was closely correlated with age，sex and pTNM stage．Kaplan-Meire survival analysis revealed that the expression of p-mTOR was not correlated significantly with survival days(Log rank test P =0．055)．Conclusion P-mTOR might be a biomark for differential diagnosis of malignant lung disease，but has poor prognostic value．
Objective To evaluate the short and long term effectiveness and safety of rapamycin-based immunosuppression regimes with CsA preserving versus CsA withdrawal. Methods We searched MEDLINE, EMBASE, The Cochrane Library and CNKI from Jan. 1995 to Dec. 2005. We identified randomized controlled trials of rapamycin-hased immunosuppression regimes with CsA preserving versus CsA withdrawal for renal transplantation patients. The quality of included trials was evaluated by two reviewers. Meta-analysis was conducted on homogeneous studies. Results Ten studies （1 121 patients） undergoing renal transplantation were included. All included studies were graded in term of randomization, allocation concealment and bhnding. Six studies were graded A and the other 4 were graded B. Meta-analysis results showed CsA withdrawal in sirolimus-based therapy in renal transplantation patients survival rate OR.（95% CI ） values were 0,77（0.17, 3.52）, 1.24（0.48, 3.16）, 1.32（0.57, 3.08）, 1.21（0.60, 2.41） at the end of 6, 12, 24, 36 months respectively; renal allografts survival rate OR. （95% CI） values were 1.79 （0.63, 5.06）, 1.15 （ 0.56, 2.36） , 1.39 （0.68, 2.85）, 1.80（0.99, 3.29）, 2. 13（1.16, 3.89）, 2.01（1.15, 3.51） at the end of 6, 12, 24, 36, 48, 54 months respectively; and acute rejection OP,（95% CI） values were 0.92（0.48, 1.78）, 1.90（1.25, 2.89）, 2. 01 （0.94,4.27）, 1.93（0.93, 4.00）, 1.52（0.77, 3.02） at the end of6, 12, 24, 36, 48 months respectively. Conclusions Available evidence shows that compared with CsA preserving, CsA withdrawal in rapamycin-based immunosuppression regimes can lead to higher incidence rates of acute rejection at the end of one year while there is no statistical difference to survival rate of patients/renal allograft in cases with stabilized renal function post-transplantation. And CsA withdrawal is of benefit to allografts for long term survival rate and is helpful to recovery of renal function. Owing to high possibility of selection bias and measurement bias in included studies, there must be a negative impact on evidence intensity of our results. We expect best evidence from with high quality double blind randomized control trials.
ObjectiveTo explore the involvement of miR-126 and the role of mammalian target of rapamycin (mTOR)/hypoxia-induced factor 1 α (HIF-1 α) pathway in regulating human umbilical cord mesenchymal stem cells (hUCMSCs) exosomes (Exo) on vascular endothelial growth factor (VEGF)-A levels in high glucose-induced human retinal vascular endothelial cells (HRECs). MethodsThe hREC was cultured in EGM-2-MV endothelial cell culture medium with 30 mmol/L glucose and placed in hypoxic cell incubator by 1% oxygen concentration. The cell model of high glucose and low oxygen was established. After modeling, divided HRECs into Exo group, phosphate buffer saline (PBS) group, PBS+anti-miR126 group, Exo+anti-miR126 group, PBS+anti-mTOR group, and PBS+anti-HIF-1 α group. High-glucose and hypoxia-induced hREC in the PBS and Exo groups were respectively co-cultured with PBS and 100 μg/ml hUCMSC Exo. PBS+anti-mTOR group, PBS+anti-HIF-1 α group: 500 nmol/L mTOR inhibitor ADZ2014, 25 μmol/L HIF-1 α inhibitor YC-1 pretreatment for hREC 12 h, and then co-culture with PBS after High-glucose and hypoxia-induced. PBS+anti-miR126 group, Exo+anti-miR126 group: miR-126 LNA power inhibitor probe was transfected with high glucose, and co-cultured with PBS and hUCMSC Exo 6 h after transfection. Real-time polymerase chain reaction (qPCR) measured miRNA-126 expression levels in PBS, and Exo groups for 0, 8, 16 and 24 h. After 24 hof co-culture, the levels of mTOR and HIF-1 α in the cells of PBS and Exo groups were detected by immunofluorescence, Western blot and qPCR, respectively. Western blot, qPCR detection of VEGF-A expression levels in cells of the PBS+anti-mTOR and PBS+anti-HIF-1 α groups. The expression of VE GF-A, mTOR, and HIF-1 α mRNA was measured in cells of PBS+anti-miR126 group and Exo+anti-miR126 group by qPCR. Comparison between two groups was performed by t-test; one-way ANOVA was used for comparison between multiple groups. ResultsAt 0, 8, 16 and 24 h, the relative mRNA expression of miR-126 gradually increased in the Exo group (F=95.900, P＜0.05). Compared with the PBS group, The mTOR, HIF-1 α protein (t=3.466, 6.804), mRNA in HRECs in the Exo group, VEGF-A mRNA expression (t=8.642, 7.897, 6.099) were all downregulated, the difference was statistically significant (P＜0.05). The relative expression level of VEGF-Aprotein (t=3.337, 7.380) and mRNA (t=8.515, 10.400) was decreased in HRECs of the anti-mTOR+PBS group and anti-HIF-1 α+PBS group, differences were statistically significant (P＜0.05). The relative expression of VEGF-A, mTOR, and HIF-1 α mRNA was significantly increased in the cells of the Exo+anti-miR126 group, the differences were all statistically significant (t=4.664, 6.136, 6.247; P＜0.05). ConclusionsmiR-126 plays a role in regulating the effect of hUCMSCs exosomes on VEGF-A levels in high glucose-induced HRECs via mTOR-HIF-1 α pathway.
Objective To observe efficacy of rapamycin combined with sorafenib in hepatocellular carcinoma (HCC) patients with tumor recurrence after liver transplantation beyond Milan criteria. Methods Forty-one beyond Milan criteria HCC patients who underwent the classic orthotopic liver transplantation without bypass and the tumor postoperatively recurred in the Tianjin First Center Hospital from February 1, 2012 to August 31, 2015 were collected retrospectively, then were divided into a local treatment group (n=21) and a comprehensive treatment group (n=20). The local treatment included the surgical resection, radiofrequency ablation, transcatheter arterial chemoembolization, radioactive seed implantation, etc.. The comprehensive treatment was on the basis of the local treatment plus rapamycin in combination with sorafenib. Results There were 12 patients with stable disease and 9 patients with progressive disease in the local treatment group. There were 12 patients with partial response, 10 patients with stable disease and 8 patients with progressive disease in the comprehensive group. In the local treatment group and the comprehensive treatment group, the median survival time were 9 months and 12 months, and the 1-year and 2-year survival rates after the recurrence were 14% versus 55%, 0 versus 15%, respectively. The survival of the comprehensive treatment group was significantly better than that of the local treatment group (P<0.01). Conclusion Combination of rapamycin and sorafenib in HCC patients with tumor recurrence after liver transplantation beyond Milan criteria can significantly improve survival time of patient with recurrence.