Ink4a and Arf differentially affect cell proliferation and neural stem cell self-renewal in Bmi1-deficient mice

The Polycomb group (PcG) gene Bmi1 promotes cell proliferation and stem cell self-renewal by repressing the Ink4a/Arf locus. We used a genetic approach to investigate whether Ink4a or Arf is more critical for relaying Bmi1 function in lymphoid cells, neural progenitors, and neural stem cells. We show that Arf is a general target of Bmi1, however particularly in neural stem cells, derepression of Ink4a contributes to Bmi1(-/-) phenotypes. Additionally, we demonstrate haploinsufficient effects for the Ink4a/Arf locus downstream of Bmi1 in vivo. This suggests differential, cell type-specific roles for Ink4a versus Arf in PcG-mediated (stem) cell cycle control.     

Aging and cancer resistance in lymphoid progenitors are linked processes conferred by p16Ink4a and Arf

Lymphoid progenitors exhibit severe growth defects during aging while myelopoiesis is relatively unperturbed. These effects are due in part to the preferential expression of p16^Ink4a and Arf in aged lymphoid progenitors. Their increased expression contributes to reduced growth and survival of lymphoid progenitors and makes them refractory to malignant transformation. Down-regulation of p16^Ink4a and Arf in aged lymphoid progenitors reverted the senescent phenotype and restored susceptibility to transformation. These data provide a molecular explanation for the preferential effects of aging on lymphopoiesis, suggest that inhibiting p16^Ink4a and Arf expression can rejuvenate B lymphopoiesis, and link aging and cancer resistance.     

Bmi-1 regulates self-renewal,proliferation and senescence of human fetal neural stem cells in vitro

Knockout and knockdown studies have shown that the polycomb gene Bmi-1 is important for mouse postnatal and prenatal neural stem cells (NSCs) self-renewal and proliferation. Different downstream targets of Bmi-1 gene have been identified in mouse, including Ink4a/Arf locus in adult NSCs and p21 gene in embryonic NSCs. However, little is known regarding the role of Bmi-1 in human NSCs. Here, using lentiviral-delivered shRNA knockdown and over-expression techniques, we examined whether Bmi-1 is required for the self-renewal and proliferation of human fetal NSCs (hfNSCs) in vitro. Our results showed that shRNA-mediated Bmi-1 reduction profoundly impaired hfNSCs self-renewal and proliferation, whereas Bmi-1 over-expression promoted hfNSCs self-renewal capacity. Interestingly, different from mouse embryonic NSCs, Bmi-1 repressed Ink4a/Arf locus instead of p21 gene in human fetal NSCs. Moreover, Bmi-1 knockdown induced obvious senescence phenotype in hfNSCs. Further studies on the Bmi-1 pathways would help to understand the molecular mechanisms underlying hfNSCs self-renewal and human brain development.     

Two mechanisms underlying the loss of p16(Ink4a) function are associated with distinct tumorigenic consequences for WS MEFs escaping from senescence

Werner syndrome (WS) mouse embryonic fibroblasts (MEFs) can spontaneously escape from senescence and become immortalized, either tumorigenic or non-tumorigenic. Our data revealed a single p53^N236S point mutation in the tumorigenic cell lines, which was correlated with the down-regulation of p21^Waf1/Cip1. p16^Ink4a expression was significantly decreased in all immortalized cell lines.Bisulfate sequencing indicated that the p16^Ink4a gene was methylated in the tumorigenic cells. Exogenous overexpression of p21^Waf1/Cip1 demethylated p16^Ink4a and restored its expression, which induced cell growth arrest and senescence. While in non-tumorigenic immortalized cells, the Ink4a loci and adjacent genomic DNA were found to be deleted.These data suggest that the loss of p16^Ink4a function by either genomic DNA deletion or methylation have been adopted by senescent WS MEFs escaping from senescence, with distinct tumorigenic consequences. The fact that cells that had escaped senescence via the spontaneous biallelic deletion of the Ink4a loci could not form tumors suggests that the functional loss of p16^Ink4aper se might not be sufficient for tumorigenesis; most likely, it is a byproduct and passenger mutation. The mutations in factors regulating p16^Ink4a methylation might be the driver mutation. These findings shed light on the strategy of anti-aging by regulating p16^Ink4a expression.     

Combined detection of p16INK4a and IMP3 increase the concordance rate between cervical cytologic and histologic diagnosis

Currently, there are discrepancies in the interpretation between cervical liquid-based cytology (LBC) and histologic diagnoses. The aim of our study was to evaluate the utility of p16^INK4a (p16) and IMP3 staining of LBC specimens to increase the concordance rate. A total of 98 cell blocks with biopsy results, including 37 low-grade squamous intraepithelial lesions (LSIL), 36 high-grade squamous intraepithelial lesions (HSIL), and 25 squamous cell carcinomas (SCC), were selected for the immunocytochemical analysis of p16 and IMP3. The LBC diagnoses corresponded with histological diagnoses for 59.5% (22/37), 63.9% (23/36), and 88.0% (22/25) of LSIL, HSIL, and SCC lesions, respectively. We found a high frequency of p16 positivity in HSIL (72.2%) and SCC (100%), but not LSIL (29.7%). IMP3 was frequently expressed in SCC (84.0%), but rarely in LSIL (8.1%) and HSIL (25.0%). Cervical intraepithelial neoplasia 1 (CIN1) was negative for both p16 and IMP3, CIN2/3 tended to be positive for p16 and negative for IMP3, and SCC was positive for both p16 and IMP3. The combination of p16 and IMP3 immunostaining had a higher sensitivity and specificity for detecting CIN1 and CIN2/3 than cytology. For detecting SCC, p16/IMP3 had a higher sensitivity than cytology, but a lower specificity. IMP3 is a useful diagnostic immunomarker that can be used to identify SCC and the combination of p16/IMP3 expression was found to improve the discrepant results between cytologic and histologic diagnoses.     

小鼠p16INK4a基因外显子1α胚胎干细胞条件打靶研究

目的 研究打靶载体的结构与打靶效率的关系，探讨小鼠p16^INK4a基因在活体水平上抑制种瘤发生和发展的功能。方法 利用筛选基因组文库得到的小鼠p16^INK4a基因组DNA片段，设计并构建了针对小鼠p16^INK4a基因外显子1α的条件打靶载体，其短臂为2.0kb EcoR Ⅰ/Xba Ⅰ片段，长臂为5.9kb Spe Ⅰ/Not Ⅰ片段，上游交叉位点（locus of crossing-over,loxP）位于外显子1α起始密码上游240bp处，下游loxP位于外显子1α起始密码下游1633bp处，经重组酶（Cre）介导后可将外显子1α和选择标记Neo基因同时删除。结果 将此条件打靶通过电穿孔转导小鼠胚胎干细胞，获得24个药物抗性克隆，其中1个经Southern杂交证实为正确同源重组克隆。结论 在同源臂两侧各用一个TK基因可以获得较高的正确同源重组率。     

Cyclin-dependent kinase inhibitor p16(INK4a) and telomerase may co-modulate endothelial progenitor cells senescence

Endothelial cells (ECs) damage is an initial and pivotal step in the formation of atherosclerosis. Endothelial progenitor cells (EPCs), which have been considered as the precursor of ECs, can migrate and home to the site of injured ECs to divide into mature ECs and keep the integrity of the endothelial monolayer. It has been shown that the number and function of EPCs are negatively correlated with various atherosclerotic risk factors. This finding may be explained partly by accelerated senescence of EPCs induced by telomere attrition or shortening owning to oxidative stress and accumulative ROS. However, elevated telomerase activity which extends the telomere cannot lead to cellular immortal in the presence of the cyclin-dependent kinase inhibitor p16(INK4a). Researchers have the opinion that senescence is the balance between the regeneration and cancer. High expression of phosphorylated p16(INK4a), which is caused by oxidative stress and accumulative ROS, can prevent tumor cells from unlimited division and becoming malignant ones by accelerating premalignant cells premature senescence. It has been demonstrated that the expression of p16(INK4a) increases remarkably with age due to oxidative stress and accumulative ROS in some stem and progenitor cells, and regulates these cells age-dependent senescence. It is observed that telomeres shortening exists in these cells. Therefore, it can be hypothesized that p16(INK4a), together with telomerase, may co-modulate EPCs senescence.     

P16INK4a overexpression and survival in osteosarcoma patients: a meta analysis

Osteosarcoma is one of the most common primary bone malignancies. Although there is a significant improvement of survival on osteosarcoma patients in the past decades, treatment of osteosarcoma is still unsatisfactory for the development of pulmonary metastasis. The potential prognostic value of p16^INK4a in osteosarcoma has been investigated, however, the results from different studies were somewhat controversial. To elucidate whether p16^INK4a is indeed a prognostic factor of osteosarcoma, we conducted a meta-analysis of the published literatures to provide a comprehensive evaluation of the significance of p16^INK4a expression in patients with osteosarcoma. Eight studies with a total of 354 patients with osteosarcoma were examined. The pooled odds ratio (OR) with corresponding 95% confidence interval (95% CI) was calculated to evaluate the effect of p16^INK4a expression on overall survival. Meta-analysis showed that patients with high p16^INK4a expression were significantly associated with favourable overall survival when compared to their counterparts with low or undetectable p16^INK4a expression (OR = 0.270, 95% CI 0.162-0.451, P < 0.001). Sensitivity analysis suggested the pooled OR was stable and not significantly changed when a single study was removed. In conclusion, the results from this meta-analysis highlight that p16^INK4a is an effective biomarker of survival in patients with osteosarcoma.     

p16INK4a expression in basal-like breast carcinoma

BLBC represents a distinctive group of invasive breast carcinomas with specific genotype and immunopro-file. BLBC is usually defined by gene expression profiling and is currently associated with poor outcome. BLBCs are estrogen receptor (ER) negative, progesterone receptor (PgR) negative, HER2 negative, and usually show a variable expression of basal cytokeratins (CKs), EGFR and CD117. p16 ^INK4a is a tumor suppressor protein, encoded by the CDKN2A gene, which regulates cell cycle. The reported association of abnormalities in the p16/Rb pathway with increased risk of malignancy prompted us to determine the expression of p16^INK4a in a group of BLBC; the results were compared with a group of high-grade invasive carcinoma (HG-IC) of breast. Tissue microarrays (TMA) were constructed in triplicate including 18 BLBC and 18 HG-IC. All BLBC cases were ER-/PgR-/HER2-. Seventeen (94%) BLBC were CK 5/6+/CK 14+; 14 (78%) BLCB showed EGFR expression and 13 (72%) were CD117 positive. BLBCs showed a strong positive reaction with p16 ^INK4a antibody in 16 of 18 (89%) cases. Although the significance of p16 ^INK4a expression in breast cancer is not fully understood, we have shown that p16^INK4a is strongly expressed in breast cancers with basal-like phenotype. Since it is known that p16^INK4a is associated with aggressive behavior in human carcinomas, these data suggest that p16^INK4a play a role in the poor prognosis of BLBC.     

Comments on Bu et al. “P16INK4a overexpression and survival in osteosarcoma patients: a meta analysis”

Recently, we read the article “P16^INK4a overexpression and survival in osteosarcoma patients: a meta analysis” by Jie Bu and his colleagues, published in the recent issue of International Journal Of Experimental Pathology. This research performed a meta-analysis to uncover the role of P16^INK4a expression in overall survival rate in patients with osteosarcoma. The investigators concluded as follows: (i) the pateints with overexpression of P16^INK4a had a longer overall survival rate than that with loss expression of P16^INK4a; (ii) P16^INK4a was an effective biomarker of prognosis in patients with osteosarcoma.The findings are valubale and encouraging. However, some flaws and imperfections rooted in this work.     

Evaluation of p16INK4a and pRb expression in cervical squamous and glandular neoplasia

p16(INK4a) is known to play a critical role as a negative regulator of cell cycle progression and differentiation by controlling the activity of the tumor-suppressor protein pRb. The present study evaluated the expression of p16(INK4a) and pRb in cervical squamous and glandular neoplasia. Immunohistochemical staining was performed for p16(INK4a) and pRb in formalin-fixed, paraffin-embedded tissue sections of the uterine cervix using an indirect immunoperoxidase method. p16(INK4a) staining was detected in 7 of 108 sections (6.5%) of normal squamous mucosa, in scattered ciliated columnar cells in 33 of 88 sections (37.5%) of normal endocervical glands, in 9 of 30 sections (30%) with Nabothian cysts, and in 4 of 4 areas (100%) of tubal metaplasia. In contrast, strong p16(INK4a) staining was found in 13 of 18 cases (72.2%) of cervical intraepithelial neoplasia (CIN) I and in all cases of CIN II/III (n = 46), squamous cell carcinoma (n = 18), endocervical glandular dysplasia (n = 10), adenocarcinoma in situ (n = 23), and invasive adenocarcinoma (n = 12). pRb expression was detected in each diagnostic category; however, the proportion of pRb-positive cells was relatively decreased in high-grade premalignant and malignant lesions of the squamous and endocervical mucosa and showed a generally inverse correlation with the expression of p16(INK4a) at the tissue level. These findings confirm a correlation between the expression of p16(INK4a) and pRb in cervical neoplasias and indicate that p16(INK4a) is a specific marker for premalignant and malignant lesions of the squamous and endocervical mucosa.     

Role of INK4a locus in normal eye development and cataract genesis

The murine INK4a locus encodes the critical tumor suppressor proteins, p16(INK4a) and p19(ARF). Mice lacking both p16(INK4a) and p19(ARF) (INK4a-/-) in their FVB/NJ genetic backgrounds developed cataracts and microophthalmia. Histopathologically, INK4a-/- mice showed defects in the developmental regression of the hyaloid vascular system (HVS), retinal dysplasia, and cataracts with numerous vacuolations, closely resembling human persistent hyperplastic primary vitreous (PHPV). Ocular defects, such as retinal fold and abnormal migration of lens fiber cells, were observed as early as embryonic day (E) 15.5, thereby resulting in the abnormal differentiation of the lens. We also found that ectopic expression of p16(INK4a) resulted in the induction of gammaF-crystallin, suggesting an important role of INK4a locus during mouse eye development, and also providing insights into the potential genetic basis of human cataract genesis.     

Methylation of CpG islands of p16(INK4a) and cyclinD1 overexpression associated with progression of intraductal proliferative lesions of the breast

P16(INK4a) is a tumor suppressor gene frequently inactivated by aberrant promoter hypermethylation. In this study, p16(INK4a) methylation was evaluated in intraductal proliferative lesions of the breast, using real-time quantitative polymerase chain reaction (MethyLight) and methylation-sensitive restriction endonuclease polymerase chain reaction. Immunohistochemistry was performed to compare and validate the methylation analysis. P16(INK4a) methylation associated with oncogene cyclinD1 expression, detected through the use of in situ hybridization and immunohistochemistry, was likewise characterized. P16(INK4a) methylation displayed varying significance among different types of intraductal proliferative lesions. Both the positive rate and the median quantitative methylation value increased with the evolution of intraductal proliferative lesions through the use of quantitative and qualitative assays. P16(INK4a) methylation was positively correlated to cyclinD1 overexpression. This study demonstrated that p16(INK4a) methylation served as the silencing mechanism of p16(INK4a) protein expression and played a crucial role in the intraductal proliferative lesions' progression. In the differential diagnosis of intraductal proliferative lesions, quantitative DNA methylation analysis of p16(INK4a) by MethyLight may be used as a surrogate, especially to distinguish atypical ductal hyperplasia from usual ductal hyperplasia and low-grade ductal carcinoma in situ. Furthermore, this study discovered that flat epithelial atypia do not share similar molecular profiles of p16(INK4a) epigenetic modification with atypical ductal hyperplasia and low-grade ductal carcinoma in situ.     

Expression of p21<Superscript>cip1</Superscript>, p27<Superscript>kip1</Superscript>, and p16<Superscript>INk4a</Superscript> Cyclin-Dependent Kinase Inhibitors in Papillary Thyroid Carcinoma: Correlation with Clinicopathological Factors

In a variety of human malignancies, aberrant expression of proteins involved in the control of cell-cycle progression has been reported. In this study, p21^cip1, p27^kip1, and p16^INk4a cyclin-dependent kinase inhibitors were analyzed to evaluate their usefulness in clinical management of papillary thyroid carcinoma (PTC). Archived material derived from 46 cases of PTC was analyzed immunohistochemically. Protein expression was ascertained on tissue microarrays, and results were correlated with clinicopathological features of the patients. Positive immunostaining was observed in 14 (30,4%) p21^cip1, 26 (56,5%) p27^kip1, and 14 (30,4%) p16^INk4a cases. No significant correlation between p21^cip1 or p27^kip1 and clinical factors was found. In contrast, p16^INk4a expression showed a significant correlation with initial extension of the disease. Therefore, 45.8% of patients with loco-regional extension were p16^INk4a positive, whereas overexpression was only seen in 15.7% of cases with intrathyroid disease (p < 0.05). Moreover, all patients with simultaneous p16^INk4a positivity and lack of p27^kip1 staining (four patients) presented lymph node metastases. In contrast, only 12 (28.5%) of the remaining patients showed lymph node tumor involvement. In conclusion, p16^INk4a expression suggests extrathyroid neck extension of PTC. This effect is enhanced when p27^kip1 is negative. We think that their analysis by immunohistochemistry could be useful in the management of patients with PTC.     

The influence of anthracosis and p16 ink4a gene aberrant methylation on small-sized pulmonary adenocarcinoma

Aims

Anthracosis is the deposition of black dusty material in the pulmonary parenchyma. Previous reports showed anthracosis and p16^ink4a gene aberrant methylation are closely related to the promotion and progression of small-sized pulmonary adenocarcinoma. In this study, we investigated the influence of anthracosis and p16^ink4a gene aberrant methylation on clinical samples from patients with small-sized adenocarcinoma.

Methods and results

DNA was bisulfite modified and methylation-specific PCR was performed to detect p16^ink4a gene aberrant methylation; black dusty material was extracted from lung tissues. Anthracotic index (AI) was defined as the absolute absorbance by densitometry. The histopathological diagnosis was concluded according to Noguchi's classification for small-sized pulmonary adenocarcinoma. The mean AI and the frequency of p16^ink4a gene aberrant methylation of heavy smokers were significantly higher than that of nonsmokers (Ｐ < 0.01 andＰ < 0.05, respectively). The frequency of p16^ink4a gene aberrant methylation of early stage small-sized adenocarcinoma was lower than that of advanced and poorly differentiated, while p16^ink4a protein expression level of early stage small-sized adenocarcinoma was significantly higher than that of poorly differentiated small-sized adenocarcinoma (P < 0.05).

Conclusions

AI and p16^ink4a gene aberrant methylation may provide a potential universal biomarker for small-sized adenocarcinoma.     

Immunostaining of p16INK4a/Ki-67 and L1 Capsid Protein on Liquid-based Cytology Specimens Obtained from ASC-H and LSIL-H Cases

Background: Atypical squamous cell cannot exclude high-grade squamous intraepithelial lesion (ASC-H) and low-grade intraepithelial lesion cannot exclude high-grade squamous intraepithelial lesion (LSIL-H) are ambiguous diagnostic entities for the prediction of high-grade cervical lesion. Objective and reproducible tests for predicting high-grade cervical lesions are needed to reduce unnecessary colposcopic referrals or follow-ups.Objective: We aimed to identify an adequate set of adjunctive markers to predict cervical intraepithelial neoplasia grade 2+ (CIN2+) in residual liquid-based cytology specimens (LBCS).Methods: We conducted p16 ^INK4a/Ki-67 and L1 capsid protein immunostaining and human papillomavirus (HPV) DNA typing on 56 LBCS diagnosed with ASC-H or LSIL-H, all of which were subjected to histologic confirmation or follow-up cytologic examination.Results: Positivity for p16 ^INK4a/Ki-67 was associated with a histology of CIN2+ (P=0.047) and CIN3+ (P=0.002). Negativity for L1 capsid protein was associated with CIN2+ confirmed at follow-up (P=0.02).Positivity for high-risk HPV (HR-HPV) was associated with CIN2+ confirmed at follow-up (P=0.036) and a histology of CIN2+ (P=0.037). The sensitivity, specificity, positive predictive value, and negative predictive value for predicting follow-up CIN2+ were 76.2%, 51.4%, 48.5%, and 78.3%, respectively, for p16 ^INK4a/Ki-67 immunostaining; 95.2%, 34.3%, 46.5%, and 92.3%, respectively, for L1 capsid protein; and 66.7%, 67.7%, 54.5%, and 77.8%, respectively, for HR-HPV. The classification and regression tree analysis showed that the combined results of p16 ^INK4a/Ki-67 andL1 capsid protein immunostaining and the HR-HPV test, conducted sequentially, correctly classified 81.8% of samples (27/33)in the prediction of a histology of CIN2 + in ASC-H or LSIL-H. For determination of the histology of cervical intraepithelial neoplasia grade 3+ (CIN3+)in ASC-H or LSIL-H, we found that the combined results of p16 ^INK4a/Ki-67 and L1 capsid protein immunostaining correctly classified 78.8% (26/33) of samples.Conclusions: p16^INK4a/Ki-67 and L1 capsid protein immunostaining and HR-HPV testing of residual LBCS diagnosed with ASC-H or LSIL-H are useful objective biomarkers for predicting CIN2+. Immunostaining for p16^INK4a/Ki-67 and L1 capsid protein are sufficient to predict CIN3+.     

p16(INK4a) promoter methylation and 9p21 allelic loss in colorectal carcinomas: relation with immunohistochemical p16(INK4a) expression and with tumor budding

In colorectal carcinomas, p16(INK4a) inactivation is known to occur by allelic loss and by promoter methylation, but mutations are rare. p16(INK4a) is up-regulated in tumor buds, and the consequent shutdown of proliferation may be a prerequisite for tumor budding. Fifty-seven colorectal carcinomas from a consecutive series were investigated. Using DNA from tissue homogenates, p16(INK4a) promoter methylation was seen in 17 of 57 tumors by methylation-specific polymerase chain reaction, and this could be confirmed using DNA from laser-capture microdissected material in 16 of these cases. A total loss of immunohistochemical p16(INK4a) expression was seen in 6 of 17 tumors with promoter methylation. Quantification of immunohistochemical p16(INK4a) expression for the remaining 11 cases revealed statistically lower frequencies of expression as compared with cases without p16(INK4a) promoter methylation. 9p21 allelic loss was observed in 9 cases, but p16(INK4a) expression in these carcinomas was not reduced. Attempted linear regression of p16(INK4a) expression in tumor buds on the degree of tumor budding, as counted on pan-cytokeratin immunostains, did not show a correlation. p16(INK4a) promoter methylation can completely abrogate p16(INK4a) expression in colorectal carcinomas. In many cases, however, it has an appreciable but only modulatory influence on p16(INK4a) expression. Possibly, methylations are heterozygous, and/or mosaic in colorectal carcinomas and/or methylations are not totally stable but can be lost between carcinoma cell replication cycles. Up-regulation of p16(INK4a) does not seem to be a strict requirement for tumor budding, hence, the absence of a correlation.     

Human papilloma virus (HPV) status, p16INK4a, and p53 overexpression in epithelial malignant and borderline ovarian neoplasms

This investigation is the first to evaluate simultaneously human papilloma virus (HPV) status, p16(INK4a), and p53 immunoreactivity in epithelial ovarian neoplasms. The results were analyzed and correlated with histological type, histological grade, and survival of patients. Subtypes considered are papillary serous and mucinous. Polymerase chain reaction (PCR) analysis, performed in our previous study, had already demonstrated a small number of HPV-positive epithelial ovarian neoplasms. No significant correlation was found between the presence of HPV DNA and subtypes of ovarian neoplasms; thus, HPV cannot be considered responsible for epithelial ovarian neoplasm. Since p16 immunoreactivity was present in many other HPV-negative cases of epithelial ovarian neoplasms, this study suggests that p16 overexpression in some neoplasms of the female genital tract is not related to HPV carcinogenesis. A higher p53 expression rate observed between borderline and malignant serous tumors and between serous and mucinous neoplasms can confirm a recent dualistic model of ovarian carcinogenesis. According to this theory, low-grade serous carcinomas (serous intraepithelial carcinomas, serous borderline neoplasm, and ovarian mucinous neoplasms) (type I tumors) develop from mutations of KAS and BRAF, while high-grade serous carcinomas (type II tumors) develop from mutation of p53. In malignant neoplasms, for univariate analysis, patient survival seems to be related to p53, strong and diffuse p16 overexpression, and the stage of development of neoplasms at the diagnosis. In multinomial logistic regression, used to evaluate the role of staging, grading, p16 and p53 immunopositivity as predictor variables of unfavorable outcome of the disease, only p16 positivity was significantly related to the poor prognosis of the cancer.     

MicroRNA-450a-3p通过抑制Bub1基因的表达调控小鼠细胞增殖和胚胎发育

目的:探讨microRNA-450a-3p(miR-450a-3p)对小鼠细胞增殖和胚胎发育的调控是否通过抑制Bub1基因的表达实现。方法:用萤光素酶报告基因实验检测miR-450a-3p能否特异性结合于Bub1基因的3’-非翻译区(untranslated region, UTR);用Western blotting和实时荧光定量RT-PCR检测miR-450a-3p对Bub1蛋白和mRNA表达;分别通过MTT、Hoechst染色、流式细胞术等分析miR-450a-3p对小鼠胚胎成纤维细胞(mouse embryonic fibroblasts,MEFs)增殖、凋亡和细胞周期等生物学功能的调控;利用染色体核型分析技术检测miR-450a-3p对MEFs染色体数目的影响。结果:miR-450a-3p能与靶基因Bub1的3’-UTR结合,在翻译水平抑制MEFs中Bub1的表达,然而其转录水平的表达却不受影响。miR-450a-3p通过下调靶基因Bub1的表达,抑制MEFs的增殖,促进细胞的凋亡;而且miR-450a-3p还能使大多数细胞停滞在G₁/G₀期,使细胞分裂受阻,导致细胞染色体数目异常。结论:miR-450a-3p能够调控靶基因Bub1的表达,抑制MEFs增殖,并最终影响小鼠胚胎的发育。     

Wnt signaling in neuroprotection and stem cell differentiation

In the past several years, we postulated that the loss of Wnt signaling was implicated in the pathology of Alzheimer's disease (AD). Since then, our lab and other groups have confirmed the involvement of the Wnt signaling in some aspects of AD. So far, we have demonstrated that activation of Wnt signaling protects neurons against neurotoxic injuries, including both amyloid-beta (Abeta) fibrils and Abeta oligomers by using either lithium, an inhibitor of the glycogen-synthase-kinase-3beta (GSK-3beta), or different Wnt ligands. Also, we have found that several molecules which activate well known neurotransmitter systems and other signaling system, are able by crosstalk to activate Wnt/beta-catenin signaling in order to protect neurons against both Abeta fibrils or Abeta oligomers. In particular, the activation of non-canonical Wnt signaling was able to protect postsynaptic regions and dendritic spines against Abeta oligomers. Furthermore Wnt signaling ligands also affect stem cells, and they are also involved in cell fate decision during neurogenesis and embryonic development as well as in adult stem cells differentiation in the nervous system. The Wnt signaling plays a key role modulating their cell differentiation or proliferation states. Altogether, these findings in both stem cell biology and neuroprotection, may introduce new approaches in the treatment of neurodegenerative diseases, including drug screening and therapies against neurodegenerative diseases which activates the Wnt signaling pathway.