Prognostic significance of microRNA-203 in cholangiocarcinoma.

MircroRNA functions as a tumor suppressor or a promoter in cholangiocarcinoma (CCA). Researchers have found that miR-203 functioned as tumor suppressor in many types of cancer. However, the role of miR-203 that plays in CCA remains to be clarified. We aimed to detect the expression level and the prognostic significance of miR-203 in CCA tissues. qRT-RCR was performed to examine the miR-203 expression levels in CCA tissue specimens and corresponding normal tissues. Our findings suggest that miR-203 expression was an independent poor prognostic factor for CCA patient overall survival. Therefore, miR-203 may serve as a valuable prognostic marker and promising treatment target for CCA.

Prognostic significance and anti-proliferation effect of microRNA-365 in hepatocellular carcinoma.

MircroRNA functions as tumor suppressor or promoter in hepatocellular carcinoma (HCC). Researchers have found that miR-365 expression was lower in HCC tissues compared with that in adjacent normal tissues. However, its prognostic significance and anti-proliferation effect in HCC remain to be clarified. In this study, we firstly found that miR-365 expression was lower in HCC tissues compared with that in adjacent normal tissues. Then, we analyzed miR-365 expression level and its clinicopathological and prognostic significance. Finally, overexpression of miR-365 inhibits HCC cell proliferation and migration in vitro. Our findings suggest that miR-365 expression was an independent poor prognostic factor for HCC patient overall survival and suppressed tumor cell growth. Therefore, miR-365 may serve as a valuable prognostic marker and promising target for HCC.

Differential expression of microRNAs in dorsal root ganglia after sciatic nerve injury.

This study investigated the possible involvement of microRNAs in the regulation of genes that participate in peripheral neural regeneration. A microRNA microarray analysis was conducted and 23 microRNAs were identified whose expression was significantly changed in rat dorsal root ganglia after sciatic nerve transection. The expression of one of the downregulated microRNAs, microRNA-214, was validated using quantitative reverse transcriptase-PCR. MicroRNA-214 was predicted to target the 3'-untranslated region of Slit-Robo GTPase-activating protein 3. In situ hybridization verified that microRNA-214 was located in the cytoplasm of dorsal root ganglia primary neurons and was downregulated following sciatic nerve transection. Moreover, a combination of in situ hybridization and immunohistochemistry revealed that microRNA-214 and Slit-Robo GTPase-activating protein 3 were co-localized in dorsal root ganglion primary neurons. Western blot analysis suggested that Slit-Robo GTPase-activating protein 3 was upregulated in dorsal root ganglion neurons after sciatic nerve transection. These data demonstrate that microRNA-214 is located and differentially expressed in dorsal root ganglion primary neurons and may participate in regulating the gene expression of Slit-Robo GTPase-activating protein 3 after sciatic nerve transection.

miR-33a is up-regulated in chemoresistant osteosarcoma and promotes osteosarcoma cell resistance to cisplatin by down-regulating TWIST.

BACKGROUND: miRNAs are involved in osteosarcoma (OS) chemoresistance, and TWIST reportedly enhances cisplatin-induced OS cell apoptosis by inhibiting multiple signaling pathways. In this study, we profiled miRNAs differentially expressed in chemoresistant OS, with a focus to identify miRNAs that regulate TWIST expression and OS chemoresistance. METHODS: OS patients who showed <90% tumor necrosis after neochemotherapy were defined as poor responders (chemoresistant), and those who showed ≥90% tumor necrosis were defined as good responders (control). miRNA microarray analysis was carried out with a discovery cohort (n = 12) of age-, sex- and tumor stage-matched chemoresistant and control OS patients. RESULTS: Among the up-regulated miRNAs in chemoresistant OS samples, miR-33a was verified to down-regulate TWIST expression, which was supported by an inverse miRNA-33a/TWIST expression trend in the validation cohort (n = 70), target-sequence-specific inhibition of TWIST-3' untranslated region-luciferase reporter activity by miR-33a, and alteration of TWIST expression by overexpression or inhibition of miR-33a in human OS cell lines. In Saos-2 cells treated with cisplatin, inhibition of miR-33a by antagomir-33a markedly increased cell apoptosis, which was enhanced by overexpression of TWIST. The apoptosis-inducing effect of TWIST overexpression was reversed by overexpression of miR-33a. In MG-63 cells, overexpression of miR-33a significantly decreased cisplatin-induced cell apoptosis, which was enhanced by knockdown of TWIST. Antagomir-33a significantly increased cisplatin-induced cell apoptosis, which was reversed by knockdown of TWIST. CONCLUSIONS: We have demonstrated in this study that miR-33a is up-regulated in chemoresistant OS and that the miR-33a level is negatively correlated with the TWIST protein level in OS. Our in vitro data indicate that miR-33a promotes OS cell resistance to cisplatin by down-regulating TWIST; on the other hand, inhibition of miR-33a by antagomir-33a enhances cisplatin-induced apoptosis in OS cells by up-regulating TWIST expression. The findings suggest that inhibition of miR-33a/TWIST signaling could be a potential new strategy to enhance neoadjuvant chemotherapy for OS.

Endothelin-1 single nucleotide polymorphisms and risk of pulmonary metastatic osteosarcoma.

Pulmonary metastases are the major cause of death of osteosarcoma (OS) patients. Endothelin-1 (ET-1) reportedly plays an important role in OS metastasis. In the present study, we for the first time explored the association of ET-1 SNPs with the risk of pulmonary metastatic OS. We genotyped three SNPs (rs1800541, rs2070699 and rs5370) in the ET-1 gene in a case-control study, using 260 pairs of age-, sex-, residence area- and tumor location-matched subjects. Patients with pulmonary metastatic OS and patients with localized high-grade (stage IIB) OS were enrolled as cases and controls, respectively. The G allele at rs1800541 was found associated with reduced risk of pulmonary metastatic OS after adjustment for body mass index, systolic blood pressure, diastolic blood pressure and the plasma ET-1 level (P=10(-4); adjusted OR, 0.55; 95% CI, 0.42-0.70), while the G allele at rs2070699 was not significantly associated with the risk of pulmonary metastatic OS (P=0.15; adjusted OR, 1.15; 95% CI, 0.87-1.50). The mRNA and the secreted protein levels of ET-1 in primary OS cell cultures (POCCs) established from surgically resected primary OS in the rs1800541 TT homozygotes were higher than those from the TG heterozygotes (P<0.05), who in turn showed higher ET-1 mRNA and secreted ET-1 levels than the GG homozygotes (P<0.05). In the control subjects, the rs1800541 TT homozygotes showed an 18.4% relapse rate, significantly higher than that of the GG homozygotes (0%) (P<0.01). On the other hand, the GG homozygotes showed a 71.4% complete recovery rate, significantly higher than that of the TG heterozygotes (7.3%) and the TT homozygotes (0%) (P<0.01). This study provides the first evidence of an association between the ET-1 gene SNPs and the risk of pulmonary metastatic OS.

TWIST interacts with endothelin-1/endothelin A receptor signaling in osteosarcoma cell survival against cisplatin.

Both TWIST and the endothelin-1 (ET-1)/endothelin A receptor (ETAR) signaling are important in osteosarcoma (OS) progression. In the present study, the interaction between TWIST and ET-1/ETAR signaling in OS cells was investigated, and the impact of the functional interaction on OS cell survival against chemotherapy agent-induced apoptosis was assessed. TWIST was overexpressed and knocked down in Saos-2 and MG-63 OS cells, respectively. In Saos-2 cells, overexpression of TWIST significantly decreased ET-1 mRNA and protein expression levels, cell survival against cisplatin and phosphorylation of Akt at serine 473 (ser473), which was abolished by the selective phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, or the selective ETAR inhibitor, BQ123. In MG-63 cells, knockdown of TWIST significantly increased ET-1 expression, cell survival against cisplatin and phosphorylation of Akt at ser473. However, exogenous ET-1 only partially rescued cell survival against cisplatin-induced apoptosis in the cells in which TWIST had been knocked down in the presence of LY294002. In conclusion, we have demonstrated that TWIST significantly, although only partially, decreases OS cell survival against cisplatin by downregulating ET-1/ETAR signaling via inhibition of the PI3K/Akt pathway. To the best of our knowledge, the present study has provided the first evidence of a functional interaction between TWIST and ET-1/ETAR signaling in OS cells. This finding adds novel insights into the molecular mechanisms underlying OS progression, cell survival and chemoresistance.

[Effect of anti-ICOS monoclonal antibody combined with low-dose CsA on chronic rejection of heart grafts in rats].

OBJECTIVE: To evaluate the effect of anti-inducible costimulator monoclonal antibody (anti-ICOS-Ab) combined with low-dose cyclosporine (CsA) on the survival quality and chronic rejection of heart allografts in rats. METHODS: The rats' heterotopic cardiac transplantation model was established by Ono's method. The recipient rats were randomly divided into an isotransplantation control group and an allotransplantation experiment group. The experiment group was re-classified into a placebo group, a normal-dose CsA group, an anti-ICOS-Ab group, a low-dose CsA group, and an anti-ICOS-Ab combined with low-dose CsA group. The survival time of grafts was monitored. The cardiac grafts were harvested for histological analysis. Flow cytometric analysis was employed to detect the population of CD25+CD4+ in peripheral lymphocytes from recipients with a long-term surviving graft. RESULTS: The survival time of the cardiac allografts in CsA-treated groups was significantly longer than that in placebo group (P<0.05). The survival time of the cardiac allografts in anti-ICOS-Ab combined with low-dose CsA group was significantly longer than that in low dose CsA-treated group (P<0.05). There was no significant difference in the survival time of the cardiac grafts between the anti-ICOS-Ab group and the placebo group (P>0.05). Compared with the normal-dose CsA group, the chronic rejection lesions of the anti-ICOS-Ab combined with low-dose CsA treatment group significantly were alleviated in the long-term survival grafts, and the proportion of CD4+CD25+ regulatory T cell increased in peripheral blood. CONCLUSION: The anti-ICOS-Ab combined with low-dose CsA can prolong the survival of cardiac allografts and alleviate the chronic rejection significantly. The high expression level of CD4+CD25+ regulatory T cell is beneficial to the long-term survival of grafts.

[Experimental study on induction of skin allograft tolerance in mice by heat shock protein 60].

OBJECTIVE: To investigate the role and mechanism of heat shock protein 60 (HSP60) in induction of murine skin allograft tolerance. METHODS: At the age of 8-12 weeks, inbred female BALB/C (H-2d) mice (n=45) and CBA/N (H-2k)mice (n=15) were used as transplantation donors and C57BL/6 (H-2b) mice (n=60) as recipients. Recipients C57BL/6 (H-2b) mice were randomized into 4 groups (n=15). In group A, 1 cm x 1 cm Wolfe-Krause skin graft was excised from the back of BALB/C (H-2d) mice and hypoderma was scraped off aseptically, and then transplanted to the back of C57BL/6 (H-2b) mice. The method of skin transplantation in the other 3 groups was the same as to group A. In group B, C57BL/6 (H-2b) mice were treated with incomplete Freund's adjuvant (IFA) administration into the back 2 weeks before transplantation of BALB/C (H-2d) mice skin. In group C, C57BL/6 (H-2b) mice were administered HSP60 emulsified in IFA into the back 2 weeks before transplantation of BALB/C (H-2d) mice skin. In group D, C57BL/6 (H-2b) mice were treated with HSP60 emulsified in IFA into the back and followed by skin transplantation of CBA/N (H-2k) mice 2 weeks later. The delayed type hypersensitivity was determined 7 days after transplantation. One-way mixed lymphocyte reaction, the concentration of cytokines in the mixed lymphocyte reaction culture supernatant was determined 7 days and 25 days after transplantation. The survival time of skin allograft was observed. RESULTS: The survival time of skin allograft in groups A, B, C and D was 12.4 +/- 0.5, 11.6 +/- 0.8, 29.3 +/- 2.6 and 27.6 +/- 2.1 days, respectively. There was significant difference between groups A, B and groups C, D (P < 0.05), while there was no significant difference between group A and group B as well as between group C and group D (P > 0.05). The counts of per minute impulse (cpm) of mixed lymphocyte reaction 7 days after transplantation in groups A, B, C and D was 12 836 +/- 1357, 11876 +/-1265, 6581 +/- 573 and 6843 +/- 612, respectively. There was significant difference between groups A, B and group C and group D (P < 0.05), while there was no significant difference between group A and group B as well as between group C and group D (P > 0.05). The cpm of mixed lymphocyte reaction at 25 days after transplantation in group A, B, C and D was 13286 +/- 1498, 12960 +/- 1376, 11936 +/- 1265 and 12374 +/- 1269, respectively. There was no significant difference among 4 groups (P > 0.05).The concentration of IL-10 in the mixed lymphocyte reaction culture supernatant in groups C, D were higher than that in groups A, B, and IL-2 and IFN-gamma were lower than that in groups A, B 7 days after transplantation (P < 0.05), while there was no significant difference between group A and group B as well as between group C and group D (P > 0.05). There was no significant difference in cytokines among the 4 groups 25 days after transplantation (P > 0.05). The delayed type hypersensitivity in groups A, B, C and D 7 days after transplantation was 0.84 +/- 0.09, 0.81 +/- 0.07, 0.43 +/- 0.05 and 0.46 +/- 0.03 mm, respectively. There was significant differences between groups A, B and groups C, D (P < 0.05). While there was no significant difference between group A and group B as well as between group C and group D (P > 0.05). CONCLUSION: HSP60 may play a role in induction and maintenance of murine skin allograft tolerance.

[Effect of mono and combination therapy with FTY720 and ICAM-1 mAb for mouse-to-rat cardiac xenotransplantation].

OBJECTIVE: To observe the effect of FTY720 and ICAM-1 mAb mono and combination therapy in mouse-to-rat cardiac xenotransplantation. METHODS: Cardiac xenotransplantation was performed in abdominal site with micro-surgical technique. Recipients with xenografts were treated with different doses of FTY720 and/or ICAM-1 mAb. Graft survival, histopathology, infiltration of CD4+, and CD8+ T cells and levels of serum IL-2, IFN-gamma, IL-4, and IgM were investigated. RESULTS: Survival time of xenografts was (2.75+/- 0.43)d in the controls, survival of grafts treated with ICAM-1 mAb did not significantly improve. Treatment with large dose FTY720 led to a survival of (4.25+/- 0.71)d (P<0.01). Combination therapy with large dose FTY720 and ICAM-1 mAb achieved a significant prolongation of graft survival with (10.25+/- 2.12)d (P<0.01). Levels of serum IL-2, IFN-gamma and rat-anti-mouse IgM decreased in the combined therapy group. Pathologic lesion and infiltration of T cells in xenografts showed mitigated in the large dose combined therapy group. There was a significant negative correlation between the antibody level and the graft survival time (R=-0.754, P<0.01). CONCLUSION: The combined therapy of FTY720 and ICAM-1 mAb can achieve a significant effect in the prolongation of heart xenograft survival and inhibition of xenoantibodies.

[Immune tolerance induced by combined heart-thymus transplantation for heart allograft in rats].

OBJECTIVE: To explore the role of combined heart-thymus transplantation for heart allograft in rats. METHODS: Vascularized heart-thymus combined transplantation was performed with microsurgical technique. Graft survival, histopathology, infiltration of CD4+, CD8+ T cells, level and mRNA expressions of IL-2 and IL-4 in the serum and cardiac grafts were investigated. RESULTS: Heart allograft in the controls had a survival time of (6.0+/-0.76) d. heart-thymus combined transplantation in non-thymectomized rats had a survival time of (6.88+/-0.64)d (P<0.05). Heart-thymus combined transplantation in thymectomized rats led to an evident survival time of (14.13+/-5.82)d (P<0.01) for cardiac graft, which further obtained long term survival after short course of treatment with cyclosporine. Pathologic lesion and infiltration of CD4+ and CD8+ T cells in cardiac grafts showed mitigated in the long term survival group. IL-2 level in the serum and cardiac grafts maintained low level in the long term survival group, whereas IL-4 maintained high level. CONCLUSION: Whether thymectomized or not in recipient rats, heart-thymus combined transplantation has a positive effect to protect cardiac graft. Furthermore, in thymectomized rats heart-thymus combined transplantation may lead to evident survival prolongation of the heart grafts, which induces immune tolerance in short course of treatment with cyclosporine.

Preoperative risk factor analysis in orthotopic liver transplantation with pretransplant artificial liver support therapy.

AIM: To assess the value of pre-transplant artificial liver support in reducing the pre-operative risk factors relating to early mortality after orthotopic liver transplantation (OLT). METHODS: Fifty adult patients with various stages and various etiologies undergoing OLT procedures were treated with molecular adsorbent recycling system (MARS) as preoperative liver support therapy. The study included two parts, the first one is to evaluate the medical effectiveness of single MARS treatment with some clinical and laboratory parameters, which were supposed to be the therapeutical pre-transplant risk factors, the second part is to study the patients undergoing OLT using the regression analysis on preoperative risk factors relating to early mortality (30 d) after OLT. RESULTS: In the 50 patients, the statistically significant improvement in the biochemical parameters was observed (pre-treatment and post-treatment). Eight patients avoided the scheduled Ltx due to significant relief of clinical condition or recovery of failing liver function, 8 patients died, 34 patients were successfully bridged to Ltx, the immediate outcome of this 34 patients within 30 d observation was: 28 kept alive and 6 patients died. CONCLUSION: Pre-operative SOFA, level of creatinine, INR, TNF-alpha, IL-10 are the main preoperative risk factors that cause early death after operation, MARS treatment before transplantation can relieve these factors significantly.

[Immune tolerance induced by combined heart-thymus transplantation with intrathymic inoculation of thymocytes in rats].

OBJECTIVE: To explore the role of allo heart and thymus transplantation by intrathymic inoculation of thymocytes. METHODS: Wistar recipients were given intrathymic injection of allo thymocytes (2 x 10(7)) 14 days before the heart and/or thymus transplantation. Graft survival, histopathology, levels and mRNA expressions of IL-2, IL-4 in serum and cardiac-grafts were investigated. RESULTS: Heart transplantation and heart-thymus composite transplantation with the treatment of CysA for 7 or 14 days prolonged graft survival. Heart transplantation and heart-thymus composite transplantation with intrathymic thymocytes injection induced the long-term survival of allo-grafts transiently immunosuppressed with CysA; IL-4 maintained at high levels but IL-2 kept at low levels in grafts in long-term survivals. CONCLUSION: Intrathymic inoculation of allo thymoctyes can induce immune tolerance for both cardiac transplantation and heart-thymus combined transplantation in rats. Thymus graft may play a role in the induction and maintenance of central tolerance.