Background:To determine the congenital heart defect (CHD) prevalence and identify the associated risk factors in children within the multi-ethnic Yunnan Region of China.Methods:This is a prospective matched case-control screening study. Screening for CHD in children residing within 28 county districts of Yunnan Province during the period of January 2001 to December 2016 was conducted. A total of 2,421 and CHD cohort and 24,210 control cohort were derived from a total population of 400,855 children (under 18 years of age).Results:A total of 2,421 children were diagnosed with CHD, yielding a CHD prevalence of 6.04 cases per 1,000 children. The prevalence of CHD by sex was 6.54 per 1,000 females versus 5.59 per 1,000 males. The ethnic groups displaying the highest CHD prevalence were the Lisu (15.51 per 1,000), Achang (13.18 per 1,000), Jingpo (12.32 per 1,000), Naxi (9.68 per 1,000), and Tibetan (8.57 per 1,000), respectively. The most common CHD was atrial septal defect, amounting to 1.94 instances per 1,000 children. We identified a number of child-associated parameters that significantly correlated with greater CHD risk, such as lower mass at birth, shorter duration of gestation, and younger age at the time of screening. We also identified a number of maternal and familial risk factors.Conclusions:This ultrasonic color Doppler imaging study revealed a relatively commonplace prevalence of CHD. Moreover, the prevalence of CHD in Yunnan Region significantly varied with sex and ethnic status. Certain child-associated, maternal, and familial risk factors may contribute to CHD risk.

Background: Peripheral atherosclerotic disease (PAD) is the narrowing or blockage of arteries that supply blood to the lower limbs. Given its complex nature, bioinformatics can help identify crucial genes involved in the progression of peripheral atherosclerosis. Materials and Methods: Raw human gene expression data for 462 PAD arterial plaque and 23 normal arterial samples were obtained from the GEO database. The data was analyzed using an integrated, multi-layer approach involving differentially-expressed gene analysis, KEGG pathway analysis, GO term enrichment analysis, weighted gene correlation network analysis, and protein-protein interaction analysis. The monocyte/macrophage-expressed leukocyte immunoglobulin-like receptor B2 (LILRB2) was strongly associated with the human PAD phenotype. To explore the role of the murine LILRB2 homologue PirB in vivo, we created a myeloid-specific PirB-knockout Apoe −/− murine model of PAD (PirB MΦKO) to analyze femoral atherosclerotic burden, plaque features of vulnerability, and monocyte recruitment to femoral atherosclerotic lesions. The phenotypes of PirB MΦKO macrophages under various stimuli were also investigated in vitro. Results: PirB MΦKO mice displayed increased femoral atherogenesis, a more vulnerable plaque phenotype, and enhanced monocyte recruitment into lesions. PirB MΦKO macrophages showed enhanced pro-inflammatory responses and a shift toward M1 over M2 polarization under interferon-γ and oxidized LDL exposure. PirB MΦKO macrophages also displayed enhanced efferocytosis and reduced lipid efflux under lipid exposure. Conclusion: Macrophage PirB reduces peripheral atherosclerotic burden, stabilizes peripheral plaque composition, and suppresses macrophage accumulation in peripheral lesions. Macrophage PirB inhibits pro-inflammatory activation, inhibits efferocytosis, and promotes lipid efflux, characteristics critical to suppressing peripheral atherogenesis.

目的 探讨目前国际上基因改造程度最大的基因编辑猪在临床前异种器官移植中的应用前景.方法 将1只猪内源性逆转录病毒(PERV)敲除联合3种主要异种抗原基因敲除以及抑制补体活化、调节凝血紊乱、抗炎抗吞噬的9种人源化基因转入猪(PERV-KO/3-KO/9-TG)作为供体,获取其心脏、肝脏和肾脏,分别移植给3只恒河猴受体,建立猪-猴异种器官移植临床前研究模型.观察血流重建后各移植物的功能状态并总结受体存活情况;监测移植物的血流动力学情况;比较各器官移植受体的血液学指标变化;观察移植物组织病理学表现.结果 血流重建后各移植器官颜色红润、质地柔软、血流灌注状态良好.术后1 d,移植心脏、肝脏和肾脏均表现为动、静脉血流状态充盈,灌注情况良好.心脏、肝脏和肾脏移植受体的术后存活时间分别为7 d、26 d和1 d.心脏移植受体术后1 d肌酸激酶、肌酸激酶同工酶以及乳酸脱氢酶水平均升高,至术后6 d逐渐恢复至接近正常水平.术后7 d各项指标均急剧升高.肝脏移植受体术后2 d天冬氨酸转氨酶水平升高,术后10 d转氨酶基本恢复正常,但总胆红素持续升高.术后12 d天冬氨酸转氨酶和丙氨酸转氨酶水平均出现升高,至术后15 d达到高峰.肾脏移植受体术后1 d出现轻微蛋白尿,后因突发严重心律失常死亡.组织病理学显示心脏和肾脏移植物组织结构接近正常,移植肝脏表现为片状坏死,肝组织结构出现紊乱,并伴有炎症损伤、间质出血和血栓性微血管病形成.结论 PERV-KO/3-KO/9-TG猪在克服超急性排斥反应、缓解体液性排斥反应及凝血紊乱方面具有一定优势,但其能否作为临床异种器官移植潜在供体需进一步评估.

Cardiac hypertrophy, characterized by the enlargement of cardiomyocytes, is initially an adaptive response to physiological and pathological stimuli. Decompensated cardiac hypertrophy is related to fibrosis, inflammatory cytokine, maladaptive remodeling, and heart failure. Although pathological myocardial hypertrophy is the main cause of hypertrophy-related morbidity and mortality, our understanding of its mechanism is still poor. Long noncoding RNAs (lncRNAs) are noncoding RNAs that regulate various physiological and pathological processes through multiple molecular mechanisms. Recently, accumulating evidence has indicated that lncRNA-H19 is a potent regulator of the progression of cardiac hypertrophy. For the first time, this review summarizes the current studies about the role of lncRNA-H19 in cardiac hypertrophy, including its pathophysiological processes and underlying pathological mechanism, including calcium regulation, fibrosis, apoptosis, angiogenesis, inflammation, and methylation. The context within which lncRNA-H19 might be developed as a target for cardiac hypertrophy treatment is then discussed to gain better insight into the possible biological functions of lncRNA-H19 in cardiac hypertrophy.

Expression of indoleamine 2,3-dioxygenase (IDO) in mesenchymal stem cells (MSC) is thought to contribute to MSC-mediated immunosuppression. A lentiviral-based transgenic system was used to generate bone marrow stem cells (BMSC) which stably expressed IDO (IDO-BMSCs). Coculture of IDO-BMSCs with dendritic cells (DC) or T cells was used to evaluate the immunomodulatory effect of IDO-BMSCs. A heterotopic heart transplant model in rats was used to evaluate allograft rejection after IDO-BMSC treatment. Mechanisms of IDO-BMSC-mediated immunosuppression were investigated by evaluating levels of proinflammatory and anti-inflammatory cytokines, and production of Tregs. A significant decrease in DC marker-positive cells and a significant increase in Tregs were observed in IDO-BMSC cocultured. Treatment of transplanted rats with IDO-BMSCs was associated with significantly prolonged graft survival. Compared with the control groups, transplanted animals treated with IDO-BMSCs had a (1) significantly higher ejection fraction and fractional shortening, (2) significantly lower expression of CD86, CD80, and MHCII, and significantly higher expression in CD274, and Tregs, and (3) significantly higher levels of interleukin-10 (IL-10), transforming growth factor beta-1 (TGF-β1), TGF-β2, and TGF-β3, and significantly lower levels of IL-2 and interferon gamma. Our results expand our understanding of the molecular mechanisms underlying suppression of heart allograft rejection via IDO-expressing BMSCs.

BACKGROUND:Aberrant endothelial function is a major contributing factor in cardiovascular disease. Dyslipidemia leads to decreased nitric oxide (NO) bioavailability, an early sign of endothelial failure. Low insulin gene enhancer protein (ISL1) levels decrease healthy NO bioavailability. We hypothesized that the microRNA miR-652-3p negatively regulates endothelial ISL1 expression and that dyslipidemia-induced miR-652-3p upregulation induces aberrant endothelial functioning via ISL1 downregulation. METHODS:Various in vitro experiments were conducted in human umbilical vein endothelial cells (HUVECs). Luciferase assays were performed in HEK293 cells. We constructed a high-fat diet (HFD) Apoe-/- murine model of dyslipidemia and a rat model of low-density lipoprotein (LDL)-induced dyslipidemia to conduct in vivo and ex vivo experiments. RESULTS:Luciferase assays confirmed miR-652-3p's targeting of the ISL1 3'-untranslated region (3'-UTR). Simvastatin blocked oxidized LDL (ox-LDL)-induced increases in miR-652-3p and ox-LDL-induced decreases in ISL1 protein expression, endothelial NO synthase (eNOS) activation, and NO production. Simvastatin's effects were abrogated by miR-652-3p overexpression and phenocopied by miR-652-3p inhibition. The dyslipidemic mouse model exhibited increased miR-652-3p and decreased ISL1 protein levels in the endothelium, effects opposed by simvastatin or miR-652-3p inhibition. The impact of simvastatin in vivo was abolished by overexpressing miR-652-3p or knocking-down ISL1. The rat model of dyslipidemia exhibited a similar pattern of miR-652-3p upregulation, attenuated ISL1 protein levels, decreased eNOS activation, and decreased NO production, effects mitigated by simvastatin. CONCLUSIONS:Dyslipidemia upregulates endothelial miR-652-3p, which decreases ISL1 protein levels, eNOS activation, and NO production. Simvastatin therapy lowers endothelial miR-652-3p expression to protect endothelial function under dyslipidemic conditions.

Objective To investigate the changes of differentially expressed genes before and after in the liver xenotransplantation, and they were screened and verified. Methods Four cases of xenograft splenic fossa heterotopic auxiliary liver transplantation were performed with a -1,3- galactoside transferase gene knockout (GTKO) mini-pig as donor and Tibetan macaque as recipient. Appropriate preoperative liver specimens were harvested from the donor liver at the back table procedure,and postoperative liver specimens were harvested by biopsy at 48 h after opening the blood flow of the graft. High throughput chip analysis of miRNAs and mRNAs was performed on the transplanted liver specimens. The key differential expression of miRNAs and mRNAs were screened, and real-time quantitative PCR and Western Blot tests were performed. Results There were 165 miRNAs differentially expressed after surgery,and 112 of them were up-regulated and 53 were down-regulated. Twelve differentially expressed miRNAs,such as mi-23b,were screened and verified. In addition to miR-150 and miR-126-3p,the expression changes of other miRNAs were basically consistent with the results of miRNAs chip. Compared with preoperative data,2 035 mRNAs showed significant differential expression after operation, 922 of them were up-regulated and 1 113 were down-regulated,5 differentially expressed genes which were closely related to liver transplantation, such as IGFBP6, CDK2AP1, TNFAIP6, FⅦ and NKG2D,were screened at mRNA and protein levels. Except for NKG2D, the changes in the protein level of the other 4 genes were basically consistent with the changes in the mRNA level (P＜0.05), but the changes in the CDK2AP1 protein were small, and there was no statistical difference (P＝0.073). Conclusion The expression of miRNAs and mRNAs in transplanted liver after xenotransplantation has greatly changed. These differentially expressed genes may be related to immune injury and coagulation dysfunction.

Background: The immunosuppressive activity of mesenchymal stem cells (MSCs) has been exploited to induce tolerance after organ transplantation. The indoleamine 2,3-dioxygenase (IDO) may have beneficial effects in the immunoregulatory properties of MSCs. It was recently revealed that exosomes derived from MSCs play important roles in mediating the biological functions of MSCs. This study aimed to explore the roles of exosomes derived from MSCs in the induction of immune tolerance. Methods: Dendritic cells (DCs) and T-cells were cultured with exosomes derived from rat bone marrow MSCs (BMSCs) overexpressing IDO1 or controls. For the in-vivo study, rats received heart transplants and were treated with exosomes from IDO-BMSCs and heart function was evaluated. Flow cytometry was used to detect expression of cell surface markers. Cytokine levels were detected in culture supernatants or serum samples. Protein and microRNA expressions in exosomes were investigated by chips. Results: Exosomes from IDO-BMSCs cultured with DCs and T-cells (1) downregulated CD40, CD86, CD80, MHC-II, CD45RA, CD45RA+CD45RB, OX62, and upregulated CD274 expression, (2) increased the number of regulatory T-cells (Tregs) and decreased the number of CD8+ T-cells, and (3) decreased the levels of pro-inflammatory cytokines, but increased the levels of anti-inflammatory cytokines compared with the other groups. Transplanted rats, which were injected with exosomes from IDO-BMSCs, had reduced allograft-targeting immune responses and improved cardiac allograft function. Exosomes secreted by IDO-BMSCs exhibited significant upregulations of the immunoregulatory protein FHL-1, miR-540-3p, and a downregulation of miR-338-5p. Conclusion: Exosomes derived from IDO-BMSCs can be used to promote immunotolerance and prolong the survival of cardiac allografts.

Background: Pig-to-nonhuman primate orthotopic liver xenotransplantation is often accompanied by thrombocytopenia and coagulation disorders. Furthermore, the release of cytokines can trigger cascade reactions of coagulation and immune attacks within transplant recipients. To better elucidate the process of inflammation in liver xenograft recipients, we utilized a modified heterotopic auxiliary liver xenotransplantation model for xeno-immunological research. We studied the cytokine profiles and the relationship between cytokine levels and xenograft function after liver xenotransplantation.Methods: Appropriate donor and recipient matches were screened using complement-dependent cytotoxicity assays. Donor liver grafts from alpha 1,3-galactosyltransferase gene-knockout (GTKO) pigs or GTKO pigs additionally transgenic for human CD47 (GTKO/CD47) were transplanted into Tibetan macaques via two different heterotrophic auxiliary liver xenotransplantation procedures. The cytokine profiles, hepatic function, and coagulation parameters were monitored during the clinical course of xenotransplantation.Results: Xenograft blood flow was stable in recipients after heterotopic auxiliary transplantation. A Doppler examination indicated that the blood flow speed was faster in the hepatic artery (HA) and hepatic vein (HV) of xenografts subjected to the modified Sur II (HA-abdominal aorta+HV-inferior vena cava) procedure than in those subjected to our previously reported Sur I (HA-splenic artery+HV-left renal vein) procedure. Tibetan macaques receiving liver xenografts did not exhibit severe coagulation disorders or immune rejection. Although the recipients did suffer from a rapid loss of platelets, this loss was mild. In blood samples dynamically collected after xenotransplantation (post-Tx), dramatic increases in the levels of monocyte chemoattractant protein 1, interleukin (IL)-8, granulocyte-macrophage colony-stimulating factor, IL-6, and interferon gamma-induced protein 10 were observed at 1 hour post-Tx, even under immunosuppression. We further confirmed that the elevation in individual cytokine levels was correlated with the onset of graft damage. Finally, the release of cytokines might contribute to leukocyte infiltration in the xenografts.Conclusion: Here, we established a modified auxiliary liver xenotransplantation model resulting in near-normal hepatic function. Inflammatory cytokines might contribute to early damage in liver xenografts. Controlling the systemic inflammatory response of recipients might prevent early post-Tx graft dysfunction.

To investigate the changes in the expression levels of scavenger receptor class B member 1 (SCARB 1) in the liver tissues before and after liver xenotransplantation and analyze the relationship between the variations in the SCARB1 expression and coagulation regulating dysfunction in the recipients.Methods The Wuzhishan miniature pig with α-1,3-galactosyltransferase gene-knockout(GTKO) was utilized as the donor and Macaca thibetana was chosen as the recipient.Heterotopic auxiliary liver xenotransplantation models were established.The liver tissue specimen was collected before and after liver xenotransplantation.Primary hepatocytes were extracted from the pig using collagenase digestion method.Human peripheral blood mononuclear cells were obtained by immunomagnetic bead sorting.These two types of cells were co-cultured and supplemented with human plasma to establish cell models with coagulation regulating dysfunction following liver xenotransplantation.Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were performed to quantitatively measure and statistically compared the expression levels of messenger ribonucleic acid (mRNA) and protein of SCARB1 in the tissue and cell samples.At the cellular level,the expression of SCARB 1 was interfered by lentiviral vector.The coagulation time was detected to validate the effect upon coagulation function.Results The expression levels of SCARB1 mRNA and protein were significantly down-regulated after liver xenotransplantation (both P＜0.05).In the cell models,the expression levels of SCARB1 mRNA and protein in the porcine hepatocytes co-cultured with human monocytes were significantly down-regulated compared with those in porcine hepatocytes without intervention (both P＜0.05).Compared with the non-intervention group,the coagulation time was significantly prolonged after the expression of SCARB1 was interfered by lentiviral vector (P＜0.05).Conclusions The down-regulated expression of SCARB1 in the liver graft is one of the main causes of mediating coagulation regulating dysfunction.Intervention of SCARB1 expression contributes to resolve the coagulation regulating dysfunction in the recipients after liver xenotransplantation.

MicroRNA-150 (miR-150) is frequently dysregulated in cancer and is involved in carcinogenesis and cancer progression. In this study, we found that miR-150 was significantly downregulated in hepatocellular carcinoma (HCC) tissues compared to adjacent noncancerous tissues. Low levels of miR-150 were significantly associated with worse clinicopathological characteristics and a poor prognosis for patients with HCC. miR-150 overexpression inhibited cell proliferation, migration and invasion in vitro and tumor growth and metastasis in vivo. Further experiments indicated that Grb2-associated binding protein 1 (GAB1) was a direct target of miR-150 in HCC cells. In addition, GAB1 expression was increased in HCC tissues and inversely correlated with miR-150 levels. Knockdown of GAB1 mimicked the tumor-suppressive effects of miR-150 overexpression on HCC cells, whereas restoration of GAB1 expression partially abolished the inhibitory effects. Moreover, miR-150 overexpression decreased GAB1 expression, subsequently downregulated phospho-ERK1/2 and suppressed epithelial-mesenchymal-transition (EMT). These effects caused by miR-150 overexpression were alleviated by exogenous GAB1 expression. Taken together, this study demonstrates that miR-150 may be useful as a prognostic marker and that the identified miR-150-GAB1-ERK axis is a potential therapeutic target for HCC.

Hepatocellular carcinoma (HCC) is one of the most common human malignancies and also the leading cause of cancer-related death in the world. The mechanisms underlying the progression and metastasis of HCC remain unclear. The E3 ubiquitin ligase F-box and WD repeat domain-containing 7 (Fbxw7) is broadly considered as a tumor suppressor gene. However, the role of Fbxw7 in HCC is not clear. To investigate the expression and biological functions of Fbxw7 in HCC, we examined Fbxw7 expression level using HCC tissue microarray and immunohistochemistry. Our data showed that Fbxw7 expression is significantly reduced in HCC compared with non-cancerous tissues (P<0.05). Fbxw7 levels were significantly associated with tumor differentiation (P=0.013), the incidence of portal or hepatic venous invasion (P=0.031), metastasis (P=0.027) and AJCC cancer stage (P=0.047). Then, we observed a strong correlation between low Fbxw7 expression and a worse 5-year survival of HCC patients (P<0.001). Furthermore, multivariate Cox regression analyses demonstrated that the Fbxw7 expression (P<0.001) was an independent factor for the prediction of the overall survival of HCC patients. We also found that both Fbxw7 mRNA and protein levels were significantly reduced in HCC cell lines compared with human liver non-tumor cell line. Moreover, our in vitro experiments showed a remarkable increase of cell migration and invasion in Fbxw7-knockdown cells and a decrease in Fbxw7-overexpress cells. In addition, the present study demonstrated that Fbxw7 is involved in the migration and invasion of HCC cells via regulating Notch1 and the downstream molecules of Notch1. Taken together, our findings indicate that Fbxw7 can be used as a prognostic marker; it has an important role in HCC progression and inhibits HCC cell migration and invasion through the Notch1 signaling pathway.

MicroRNAs (miRNAs) are associated with human carcinogenesis and tumor development. Moreover, serum miRNAs can reflect the level of tissue miRNAs and be potential tumor markers. Serum microRNA-21 (miR-21) is overexpressed in many human cancers including hepatocellular carcinoma (HCC). However, how serum miR-21 changes during the HCC formation and whether miR-21 plays a regulatory role in this whole process are unknown. The current study evaluated the prognostic and diagnostic potential of serum miR-21 in HCC patients. Next, we established a HCC rat model and collected the blood and liver tissues at regular time points. AFP from the serum, RNA from the serum and liver tissues were collected and quantified separately. The results revealed that tissue and serum miR-21 was upregulated significantly in the groups of cirrhosis, early and advanced HCC compared with normal and fibrosis groups. The AFP levels were increased in early and advanced HCC compared with other groups. Then, the changes of miR-21 downstream proteins (i.e., programmed cell death 4 [PDCD4] and phosphatase and tensin homolog [PTEN]) in the liver tissues were measured. PDCD4 and PTEN expression was decreased gradually after tumor induction and negatively correlated with miR-21 expression. All these results suggested that serum miR-21 was associated with the prognosis of HCC; the changes in serum miR-21 were earlier and more accurately reflected the pathogenesis of HCC than AFP; therefore, it could be used as an early diagnostic marker for HCC. Our in vivo experiments further confirmed that miR-21 plays an important role in promoting the occurrence and development of HCC by regulating PDCD4 and PTEN.

Neural precursor cell expressed, developmentally downregulated 9 (NEDD9) plays an integral role in natural and pathological cell biology. Overexpression of NEDD9 protein has been correlated with poor prognosis in various types of cancer. However, few available data address the precise function of the NEDD9 gene in hepatocellular carcinoma (HCC). In the present study, we investigated NEDD9 expression in 40 primary human HCC tissues compared with matched adjacent non-tumor hepatic tissues using RT-qPCR and western blot analysis. Immunohistochemistry was performed to analyze the correlations between NEDD9 expression and clinicopathological factors. Statistical analyses were applied to derive prognostic values of NEDD9 in HCC. The results showed that the NEDD9 mRNA and protein expression levels in HCC tissues were significantly higher than those in matched adjacent non-tumor hepatic tissues. High NEDD9 expression was correlated with larger tumor size, advanced tumor grade, metastasis, intrahepatic venous invasion and high UICC TNM stages in HCC patients. Patients with high NEDD9 expression levels exhibited poorer recurrence-free and overall survival than those with a low NEDD9 expression. Additionally, NEDD9 expression status was an independent prognostic factor for survival. This correlation remained significant in patients with early-stage HCC or with normal serum AFP levels. The results of this study suggest that NEDD9 may be a valuable prognostic biomarker for HCC, including early-stage and AFP-normal patients.

As a highly aggressive malignant disease, the prognosis of patients with pancreatic ductal adenocarcinoma (PDAC) is poor. Yet, the mechanisms underlying the progression of PDAC remain unclear. MicroRNAs (miRNAs) may be involved in various human cancers as cancer suppressors or oncogenes. MicroRNA-183 (miR-183) was recently reported to be dysregulated in various types of cancer and to play an important role in the processes of cancer. However, the effects and potential mechanisms of action of miR-183 in PDAC have not been explored. In the present study, low expression of miR-183 was observed in PDAC tissues and cell lines. Low expression of miR-183 in PDAC was significantly associated with tumor grade, metastasis and TNM stage. Kaplan-Meier survival analysis demonstrated that patients harboring low expression of miR-183 had a significantly reduced overall survival than patients with a high level of miR-183 expression. The present study revealed that B-cell-specific Moloney murine leukemia virus insertion site 1 (Bmi-1) expression was inversely correlated with miR-183. Our findings also demonstrated that a low level of miR-183 expression effectively suppressed the growth of PDAC cells via regulation of Bmi-1. Following Bmi-1 silencing or upregulation of miR-183, the expression levels of cyclin D1, cyclin-dependent kinase (CDK)2 and CDK4 were decreased. It is reasonable to conclude that alteration of miR-183 expression may regulate the function of PDAC cells by the downregulation of Bmi-1 expression.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal solid tumor due to the lack of reliable early detection markers and effective therapies.MicroRNAs (miRNAs), noncoding RNAs that regulate gene expression, are involved in tumorigenesis and have a remarkable potential for the diagnosis and treatment of malignancy.In this study, we investigated aberrantly expressed miRNAs involved in PDAC by comparing miRNA expression profiles in PDAC cell lines with a normal pancreas cell line and found that miR-135a was significantly down-regulated in the PDAC cell lines.The microarray results were validated by qRT-PCR in PDAC tissues, paired adjacent normal pancreatic tissues, PDAC cell lines, and a normal pancreas cell line.We then defined the tumor-suppressing significance and function of miR-135a by constructing a lentiviral vector to express miR-135a.The overexpression of miR-135a in PDAC cells decreased cell proliferation and clonogenicity and also induced G1 arrest and apoptosis.We predicted Bmi1 may be a target of miR-135a using bioinformatics tools and found that Bmi1 expression was markedly up-regulated in PDAC.Its expression was inversely correlated with miR-135a expression in PDAC.Furthermore, a luciferase activity assay revealed that miR-135a could directly target the 3'-untranslated region (3'-UTR) of Bmi1.Taken together, these results demonstrate that miR-135a targets Bmi1 in PDAC and functions as a tumor suppressor.miR-135a may offer a new perspective for the development of effective miRNA-based therapy for PDAC.

Objective To investigate the mechanisms of erythropoietin-producing hepatocellular A3 (EphA3) in the invasion of hepatocellular carcinoma (HCC) cells.Methods Hepatic cell HL-7702 and HCC cell and HCC cell lines HepG2 and MHCC97H were cultured.The expression of EphA3 in the HepG2 and MHCC97H cells was suppressed by siRNA interference,and then were divided into the untreated group,the control group and the siRNA intervention group.The expression of EphA3 was detected by RT-PCR and Western blot.The invasion ability of HepG2 and MHCC97H was detected by Transwell chamber.The protein expression of VEGF and activity of vascular endothelial growth factor (VEGF) were detected by western blot and ELISA.All data were analyzed using the analysis of variance or LSD-t test.Results The relative mRNA expressions of EphA3 in HL-7702,HepG2,and MHCC97H cells were 0.94 ±0.13,1.76 ±0.16 and 3.62 ±0.14,respectively,and the protein expressions of EphA3 in the 3 cells were 0.96 ±0.12,1.59 ±0.11 and 3.82 ±0.11.There was significant difference in the EphA3 expression between HL-7702 cells and HepG2,MHCC97H cells (t =2.511,6.437 ; 2.321,6.895,P ＜ 0.05).The relative mRNA expressions of EphA3 in the HepG2 cells in the untreated group,the control group and the siRNA intervention group were 0.95 ±0.11,0.96 ±0.12 and 0.31 ±0.15,respectively.There was significant difference in the mRNA expression of EphA3 in the HepG2 cells between the siRNA intervention group and the control group (t =4.051,P ＜ 0.05).The relative mRNA expressions of EphA3 in the MHCC97H cells in the untreated group,the control group and the siRNA intervention group were 0.97 ± 0.16,0.95 ± 0.14 and 0.40 ± 0.11,respectively.There was significant difference in the mRNA expression of EphA3 in the MHCC97H cells between the siRNA interference group and the control group (t =5.237,P ＜0.05).The relative protein expressions of EphA3 in the HepG2 cells in the untreated group,the control group and the siRNA intervention group were 0.97 ± 0.16,0.95 ± 0.15 and 0.32 ± 0.17,respectively.There was significant difference in the protein expression of EphA3 in the HepG2 cells between the siRNA interference group and the control group (t =4.145,P ＜ 0.05).The relative protein expressions of EphA3 in the MHCC97H cells in the untreated group,the control group and the siRNA intervention group were 0.95 ± 0.11,0.96 ± 0.12 and 0.38 ±0.17,respectively.There was significant difference in the protein expressions of EphA3 in the MHCC97H cells between the siRNA interference group and the control group (t =4.327,P ＜ 0.05).The numbers of HepG2 cells penetrated the Watrigel in the untreated group,the control group and the siRNA intervention group were (111 ±4)/10HPF,(109 ±5)/10HPF and (51 ±3)/10HPF,respectively.There was significant difference in the number of HepG2 cells between the siRNA interference group and the control group (t =7.582,P ＜ 0.05).The numbers of MHCC97H cells penetrated the Watrigel in the untreated group,the control group and the siRNA intervention group were (402 ± 6)/10HPF,(397 ± 7)/10HPF and (152 ± 7)/10HPF,respectively.There was significant difference in the number of MHCC97H cells between the siRNA interference group and the control group (t =9.479,P ＜ 0.05).The relative protein expressions of VEGF in the HepG2 cells in the untreated group,the control group and the siRNA intervention group were 0.98 ± 0.11,0.96 ± 0.13 and 0.57 ± 0.11,respectively.There was significant difference in the protein expression of VEGF of the HepG2 cells between the siRNA interference group and the control group (t =3.167,P ＜ 0.05).The relative protein expression of VEGF in the MHCC97H cells in the untreated group,the control group and the siRNA intervention group were 0.97 ±0.14,0.98 ±0.12 and 0.34 ± 0.15,respectively.There was significant difference in the protein expression of VEGF of the MHCC97H cells between the siRNA interference group and the control group (t =4.278,P ＜ 0.05).The relative activities of VEGF proteins of HepG2 cells in the untreated group,the control group and the siRNA intervention group were 0.96 ±0.15,0.94 ±0.11 and 0.47 ±0.13,respectively.There was significant difference in the activity of VEGF protein in the HepG2 cells between the siRNA interference group and the control group (t =3.981,P ＜ 0.05).The relative activities of VEGF proteins in MHCC97H cells in the untreated group,the control group and the siRNA intervention group were 0.98 ±0.12,0.97 ±0.12 and 0.38 ±0.14,respectively.There was significant difference in the activity of VEGF protein in the MHCC97H cells between the siRNA interference group and the control group (t =4.059,P ＜ 0.05).Conclusions EphA3 plays an important role in the invasion of HCC cells via regulating the protein expression and activity of VEGF.EphA3 might be a new target for the treatment of HCC.