Expression and clinical significances of Beclin1, LC3 and mTOR in colorectal cancer

Autophagy is related to cancer and other diseases, and compromised autophagy could promote chromosome instability associated with carcinogenesis and tumor progression. The role of autophagy in the growth and metastasis of colorectal cancer (CRC) remains poorly understood. Beclin1 mediates autophagic initiation, and LC3 is a specific marker for autophagy. Inactivation of mTOR caused by cellular hypoxia or energy deficiency induces autophagic activity. This study aims to examine the expression and clinical significance of these proteins in CRC. Immunohistochemistry results showed that the positive expression rates of Beclin1, LC3, and mTOR in cancer tissues were 90.50%, 87.19%, and 46.28%, respectively, which were higher than those in adjacent tissues (P < 0.05). Differentiation degree and lymph node metastasis were associated with LC3 overexpression (P < 0.05) but not with Beclin1 (P > 0.05). Lymph node metastasis was also related to mTOR. Spearman analysis results showed that LC3 expression was positively correlated with Beclin1 but negatively correlated with mTOR (r = 0.593 and -0.165, respectively; P < 0.01). Beclin1 expression was also not associated with mTOR (P > 0.05). Survival analysis further indicated that LC3, mTOR, and lymph node metastasis were independent prognostic factors in CRC. Real-time PCR results and Western blot indicated that Beclin1, LC3, and mTOR expression in CRC was significantly higher than that in adjacent tissues (P < 0.01). The aberrant protein expression may be associated with the development and progression of CRC. The LC3 and mTOR genes must be simultaneously detected to evaluate progression and prognosis of CRC.     

Intestinal apical polarity mediates regulation of TORC1 by glucosylceramide in C. elegans

TORC1 (target of rapamycin complex 1) plays a central role in regulating growth, development, and behavior in response to nutrient cues. We previously showed that leucine-derived monomethyl branched-chain fatty acids (mmBCFAs) and derived glucosylceramide promote intestinal TORC1 activity for post-embryonic development and foraging behavior in Caenorhabditis elegans. Here we show that clathrin/adaptor protein 1 (AP-1)-dependent intestinal apical membrane polarity and polarity-dependent localization of the vacuolar-type H⁺-ATPase (V-ATPase) mediate the impact of the lipid pathway on intestinal TORC1 activation. Moreover, NPRL-3 represses mmBCFA-dependent intestinal TORC1 activity at least partly by regulating apical membrane polarity. Our results provide new insights into TORC1 regulation by lipids and membrane polarity in a specific tissue.     

TMP21 modulates cell growth in papillary thyroid cancer cells by inducing autophagy through activation of the AMPK/mTOR pathway

Objective: To investigate the role of transmembrane protein (TMP) 21 in human thyroid cancer. Methods: The recombinant expression vector pcDNA3.1 (+)-TMP21 and specific small interfering RNAs (siRNA) against TMP21 were transfected into a papillary thyroid cancer cell line (TPC1). After transfection, the expression of TMP21 was confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. Moreover, cell viability and apoptosis rate were respectively determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) colorimetric assay and flow cytometry (FCM). Additionally, Western blotting was performed to analyze the adenosine monophosphate (AMP)-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathways associated protein (P-AMPKα^Thr172, P-mTOR^Ser2448, light chain (LC)-II/LC3-I, and P-S6K^Thr389) after pre-treatment with AMPK inhibitor, compound C (Com C) and siTMP21. Results: The TMP21 protein level and cell viability were significantly higher, but apoptotic rate was significantly lower by transfection with pcDNA3.1-TMP21 than those in control group (P < 0.05), and reverse results were obtained by transfection with siTMP21. However, qRT-PCR showed different results due to the feedback inhibition of mRNA. Besides, silencing of TMP21 significantly reduced the levels of P-mTOR^Ser2448 and P-S6K^Thr389 (P < 0.05), but significantly increased the levels of P-AMPKα^Thr172 and LC3-II/LC3-I compared with the control group (P < 0.01). Whereas, the levels of P-AMPKα^Thr172 and LC3-II/LC3-I were significantly decreased by Com C compared with the control group (P < 0.05). Conclusion: TMP21 modulates cell growth in TPC1 cells by inducing autophagy, which may be associated with activation of AMPK/mTOR pathway.     

Low MiR-149 expression is associated with unfavorable prognosis and enhanced Akt/mTOR signaling in glioma

microRNAs (miRs) play critical roles in the progression of glioma. Previous in vitro studies have described the anti-tumor role of miR-149 in cancer cells including glioma. In this study, we aimed to investigate whether miR-149 is associated with the prognosis of glioma patients. A total of 163 glioma patients who underwent tumor resection were included in our follow-up study. We found that the miR-149 expression was significantly lower in tumor tissues compared with that in normal tissues (P<0.05). Kaplan-Meier and analysis showed that the miR-149 expression status was significantly associated with the survival duration (logrank test, P<0.001), and multivariate Cox regression revealed that patients with low miR-149 expression were exposed to a 1.825 fold higher death risk (HR=1.825, 95% CI=1.031-3.229, P=0.039) compared with those with high miR-149 expression. Further study showed that Akt/mTOR signaling was hyperactive in low miR-149 expressing tissues. Our study thus demonstrates that miR-149 expression in glioma tissues is critically associated with the prognosis of patients, suggesting its potential clinical significance.     

国产雷帕霉素对人淋巴瘤细胞Raji增殖的影响

目的探讨国产雷帕霉素（宜欣可）对人淋巴瘤细胞株Raji细胞体外生长及对mTOR/p 70S6K信号通路的影响。方法MTT法检测不同浓度（0、1、5、10、20、40、50、100 nmol/L）国产雷帕霉素作用不同时间（24、48、72 h）对Raji 细胞增殖的影响。光学显微镜观察Raji细胞形态学变化。流式细胞仪测定国产雷帕霉素对Raji细胞周期分布和凋亡的影响。Western blot 方法检测国产雷帕霉素处理前后对Raji 细胞mTOR、p70S6K、p-p70S6K蛋白的影响。结果国产雷帕霉素对Raji细胞增殖有明显的抑制作用（不同浓度P<0.01或P<0.05）,呈现明显的剂量-效应和时间-效应依赖关系。国产雷帕霉素明显抑制Raji细胞周期发展（P<0.05）,但没有发生明显的凋亡（P>0.05）。0、10、50、100 nmol/L国产雷帕霉作用于Raji细胞的mTOR、p-p70S6K,其蛋白量随药物浓度增大而降低（P<0.05）,p70S6K随药物浓度增大而升高（P<0.05）。结论人淋巴瘤细胞株Raji中存在mTOR/p70S6K信号通路激活状态,宜欣可可抑制该通路激活并通过阻滞细胞周期发展抑制Raji细胞增殖。     

Donor derived HLA-G polymorphisms have a significant impact on acute rejection in kidney transplantation

Human leucocyte antigen G (HLA-G) is a non-classical HLA-class I antigen that exerts immunoregulatory functions. The polymorphisms 14-base pair (bp) insertion/deletion (ins/del) (rs1704) and +3142C > G (rs1063320) could modify the expression level of HLA-G.We genotyped 175 kidney recipients (41 with acute rejection and 134 without rejection) and additionally the corresponding donors for both polymorphisms in order to assess their impact on acute rejections one year after transplantation. In addition, we analyzed soluble HLA-G (sHLA-G) levels in sera of 32 living kidney donors and compared the sHLA-G levels in terms of the present genotype.In kidney transplant recipients we did not observe an impact of the 14-bp ins/ins and the +3142GG genotypes on acute rejection. In contrast, we found a higher frequency of these genotypes in the donors of the no-rejection collective compared to the rejection collective (4.9% vs. 24.6%; p = 0.010; 9.8% vs. 31.3%; p = 0.006). Soluble HLA-G levels were highest in healthy kidney donors homozygous for the 14-bp insertion.We conclude that the HLA-G polymorphisms of the donor are of importance for susceptibility of acute rejection in kidney transplantation. We suggest that the 14-bp ins/ins and the +3142GG genotypes are protective against kidney transplant rejection.     

Multicenter trial of everolimus in pediatric renal transplant recipients: results at three year

Abstract:  There are few prospective clinical trials of mTOR inhibitors (or proliferation signal inhibitors) combined with CNI inhibitors in de novo pediatric renal transplantation. Results reported here are from a multicenter, open-label study in de novo pediatric renal transplant patients (≤16 yr), in which patients received everolimus with cyclosporine and corticosteroids for one yr, then entered an extension study for a further two yr. Nineteen patients completed the one-yr study, of whom three discontinued study medication. Fifteen of the remaining 16 patients entered the extension study, eight of whom were aged <10 yr (Group 1) and seven were aged 10–16 yr (Group 2). Mean daily dose of everolimus during the first 36 months was 1.53 mg/m² BSA. Biopsy-proven acute rejection occurred in three patients in Group 2 and in one patient in Group 1. Biopsy-proven chronic allograft rejection was reported in four patients (two in each age group). Graft survival at one yr was 100%; one patient in Group 2 lost their graft subsequently during the extension. For patients entering the extension, patient survival at three yr was 100%. There were three cases of viral infection, including one case of cytomegalovirus infection. At three yr, mean total cholesterol was 5.5 ± 0.8 m m /L (213 ± 31 mg/dL) and four patients received statin therapy. Mean serum creatinine at 36 months was 96 ± 36 μ m /L (1.1 ± 0.4 mg/dL). This is the first long-term prospective study to demonstrate that a regimen of everolimus, cyclosporine, and corticosteroids provides good efficacy, tolerability, and safety in de novo pediatric renal transplant patients.     

Pik3cb在小鼠下颌第一磨牙牙胚发育不同阶段的表达研究

目的:检测Pik3cb在小鼠下颌第一磨牙牙胚发育不同阶段的表达分布及变化规律,进一步揭示Pik3cb对牙形态发生的影响。方法:制备原位杂交探针,及小鼠牙胚发育标志时间点的牙胚冰冻切片,通过原位杂交方法进一步显示Pik3cb在小鼠下颌第一磨牙牙胚发育不同时期的表达情况。结果:蕾状期Pik3cb明显表达于牙板上皮,帽状期开始大量表达于釉上皮与牙乳头,进入钟状期表达部位趋于集中,尤以内釉上皮及后续新形成硬组织周围的高柱状成釉细胞处最为明显。结论:Pik3cb在牙源性上皮及成釉细胞表达,并可能通过PI3K/PTEN/AKT/mTOR信号通路与成釉细胞瘤行为相关。     

Kisspeptins and the metabolic control of reproduction: Physiologic roles and physiopathological implications

In this presentation, we have provided a succinct state-of-the-art of our knowledge on kisspeptins, the newly identified neuropeptide system with key roles in the control of the gonadotropic axis, in the metabolic regulation of puberty onset and fertility. The experimental evidence revised herein, gathered mostly in rodent models, supports the contention that hypothalamic Kiss1 neurons do operate as a central conduit for conveying metabolic information onto the centers governing reproductive function, through a putative leptin-kisspeptin-GnRH pathway, which is likely to involve Crtc1 and/or mTOR as molecular mediators.     

Mammalian life-span determinant p66shcA mediates obesity-induced insulin resistance

Obesity and metabolic syndrome result from excess calorie intake and genetic predisposition and are mechanistically linked to type II diabetes and accelerated body aging; abnormal nutrient and insulin signaling participate in this pathologic process, yet the underlying molecular mechanisms are incompletely understood. Mice lacking the p66 kDa isoform of the Shc adaptor molecule live longer and are leaner than wild-type animals, suggesting that this molecule may have a role in metabolic derangement and premature senescence by overnutrition. We found that p66 deficiency exerts a modest but significant protective effect on fat accumulation and premature death in lep^Ob/Ob mice, an established genetic model of obesity and insulin resistance; strikingly, however, p66 inactivation improved glucose tolerance in these animals, without affecting (hyper)insulinaemia and independent of body weight. Protection from insulin resistance was cell autonomous, because isolated p66KO preadipocytes were relatively resistant to insulin desensitization by free fatty acids in vitro. Biochemical studies revealed that p66shc promotes the signal-inhibitory phosphorylation of the major insulin transducer IRS-1, by bridging IRS-1 and the mTOR effector p70S6 kinase, a molecule previously linked to obesity-induced insulin resistance. Importantly, IRS-1 was strongly up-regulated in the adipose tissue of p66KO lep^Ob/Ob mice, confirming that effects of p66 on tissue responsiveness to insulin are largely mediated by this molecule. Taken together, these findings identify p66shc as a major mediator of insulin resistance by excess nutrients, and by extension, as a potential molecular target against the spreading epidemic of obesity and type II diabetes.