LncRNA HOXC-AS3通过miR-15b-5p/E2F3生物学轴促进胃癌细胞的增殖和迁移

背景与目的：胃癌是一种发病率和死亡率均较高的恶性肿瘤。长链非编码RNA（long non-coding RNA,lncRNA）是一类具有调控功能的大分子非编码RNA,在胃癌的转移中发挥着重要的作用。探讨lncRNA HOXC-AS3（lnc-HOXC-AS3）在胃癌中的表达模式,以及通过miR-15b-5p/E2F3生物学轴促进胃癌细胞增殖和转移的分子机制。方法：根据生物信息学分析的方法寻找收集2017年4月—2018年12月安徽医科大学第一附属医院收治并经病理学检查诊断为胃癌的90例患者的胃癌组织中异常表达的lncRNA,并且利用实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR）检测lnc-HOXC-AS3的表达水平;在对lnc-HOXC-AS3功能的研究中,采用细胞计数试剂盒（cell counting kit-8,CCK-8）和transwell等方法确定了lnc-HOXC-AS3对胃癌细胞（MGC803）增殖和迁移的影响;在机制上,采用双荧光素酶报告基因检测lnc-HOXC-AS3、miR-15b-5p和E2F3的靶向调控关系。结果：Lnc-HOXC-AS3在胃癌组织和胃癌细胞系中异常高表达。功能上,lnc-HOXC-AS3促进胃癌细胞增殖和迁移,相反,敲低lnc-HOXC-AS3则明显抑制胃癌细胞增殖和迁移。在机制的探讨中,通过双荧光素酶报告基因和一系列体外实验证实lnc-HOXC-AS3通过靶向下调miR-15b-5p的表达,进而解除miR-15b-5p对E2F3的抑制作用,促进了胃癌细胞增殖和迁移。结论：Lnc-HOXC-AS3在胃癌中异常高表达,并通过调控miR-15b-5p/E2F3生物学轴促进胃癌细胞增殖和迁移,渴望成为研究胃癌早期诊断和治疗的靶点。     

Frequent Amplification of the Cyclin E Gene in Human Gastric Carcinomas

We searched for genetic alterations of the cyclin D1 and cyclin E genes in 45 human gastric carcinoma tissues. Expression of cyclin E mRNA and protein was also analyzed in eight of them by Northern and Western blots and immunohistochemical staining. The cyclin E gene was amplified 3–10 fold in seven gastric cancer tissues (15.6%), of which six were advanced gastric cancers. All of the cases with the cyclin E gene amplification displayed lymph node metastasis. Moreover, the case with the gene amplification overexpressed the cyclin E mRNA and protein. One of eight gastric cancer cell lines, MKN-7, shared the cyclin E gene amplification, and all of the gastric cancer cell lines expressed high levels of the cyclin E mRNA and protein even without gene amplification. Amplification of the cyclin D1 gene was not observed in any of the gastric carcinoma tissues or gastric carcinoma cell lines. These results suggest that the gene amplification and overexpression of cyclin E play an important role in the abnormal growth and progression of gastric carcinoma.     

Genetic Status and Expression of the Cyclin-dependent Kinase Inhibitors in Human Gastric Carcinoma Cell Lines

Deregulation of cyclin, cyclin-dependent kinases (CDKs) and their inhibitors could have a pivotal role in the development of diverse human cancers. We examined the genetic status and the expression of CDK inhibitors ( p21, p27, pl6 and p15 ), CDK2 and cyclins (A, D1 and E) in eight gastric carcinoma cell lines, in comparison with the status of p53 gene alterations. All the cell lines (except MKN-28) that contained a p53 gene abnormality expressed very low or undetectable levels of p21 mRNA, while the cell lines (MKN-45 and -74) with wild-type p53 gene expressed high levels of p21 mRNA. An inverse correlation was found between the level of p21 mRNA and the expression of mRNAs for CDK2 and G1 cyclins. MKN-28 was an exception; it contained mutated p53 , and expressed mRNAs for p21 , CDK2 and G1 cyclins at high levels. Only MKN-45 and -74, with wild-type p53 , expressed considerable levels of p21 protein. Homozygous deletion of the p16 and p15 genes was detected in two (MKN-45 and HSC-39) of the eight gastric carcinoma cell lines. p16 protein was not expressed in three cell lines (MKN-28, MKN-74 and KATO-III), as well as MKN-45 and HSC-39. Rearrangement of the p15 gene was found in TMK-1. Rearrangement of the p27 gene was detected in MKN-45, although the expression of p27 protein was well preserved in all the gastric carcinoma cell lines. The expression of pRb was also preserved in all the cell lines except KATO-III. No obvious correlation was observed between the p53 gene status and the expression of p27 and p16 . These findings suggest that abnormal regulation of CDK2/cyclins and CDK inhibitors might be involved in deregulated growth of gastric carcinomas.     

Germ-line p53-targeted disruption inhibits helicobacter-induced premalignant lesions and invasive gastric carcinoma through down-regulation of Th1 proinflammatory responses

p53 is a tumor suppressor gene that is mutated in many human malignancies, including gastric cancer. It remains unclear why patients with germ-line p53 mutations (i.e., Li-Fraumeni syndrome) are not at increased risk for gastric adenocarcinoma, despite the fact that they show a high rate of many other tumors. Furthermore, the precise relationship between germ-line p53 mutations and the response to chronic bacterial infections (such as Helicobacter spp.) has not been investigated. To assess the role of germ-line p53 deletions in modulating the progression to gastric cancer, p53(+/-) and wild-type (WT) C57BL/6 mice were infected with H. felis. The gastric pathology and immune response in these two groups of mice were analyzed for up to 15 months postinfection. The gastric fundus and antrum were evaluated independently using a 0-4 scale to score inflammation, parietal and chief cell loss, mucus metaplasia, and helicobacter colonization. Nonparametric statistical analysis was performed to determine the effects of p53(+/-), infection status, and postinoculation (p.i.) time on inflammation, preneoplastic changes, invasive lesions, and helicobacter colonization. mRNA expression for gammaIFN, interleukin (IL)-1, IL-10, and IL-4 was quantified by PCR. Sera were also evaluated for H. felis antibody by ELISA. Antral inflammation increased significantly with time in infected mice. There was a significant, protective effect on the development of preneoplastic fundic lesions and invasive carcinoma attributable to the deletion of one p53 allele (P < 0.05). Submucosal invasive foci were observed in 9 of 11 WT-infected mice ranging from 13 to 15 months p.i.; invasion of adjacent submucosal blood vessels by glandular epithelia also was present in 5 of these mice. None of these lesions were observed in 33 p53(+/-) mice, infected or not, at any time p.i. p53(+/-) mice had significantly higher helicobacter colonization consistent with a Th2 host response. In sera from WT mice, IgG2a, considered a proinflammatory Th1 response, continued to rise throughout the 15-month study (P < 0.004). In contrast, IgG2a levels of the p53(+/-) mice were 50-60% lower than those of the WT mice at each time point (P range, <0.012 to 0.002) and did not progress in magnitude between 12 and 15 months of chronic H. felis infection (P = 0.167). mRNA levels for gammaIFN and IL-1 were significantly up-regulated in WT mice infected with H. felis (P < 0.05) but were slightly elevated or were at background levels in p53(+/-) mice. IL-10 and IL-4 mRNA expression was not significantly different from control samples. Our results support the hypothesis that germ-line deletion of one p53 allele results in a down-regulated Th1 response to gastric helicobacter infection, possibly because of T-cell senescence, which may indirectly protect against the development of gastric cancer and other epithelial-derived neoplasms associated with chronic inflammation.     

Overexpression of MAPK15 in gastric cancer is associated with copy number gain and contributes to the stability of c-Jun

This study was aimed at understanding the functional and clinicopathological significance of MAPK15 alteration in gastric cancer. Genome-wide copy number alterations (CNAs) were first investigated in 40 gastric cancers using Agilent aCGH-244K or aCGH-400K, and copy number gains of MAPK15 found in aCGH were validated in another set of 48 gastric cancer tissues. The expression of MAPK15 was analyzed using immunohistochemistry in concurrent lesions of normal, adenoma, and carcinoma from additional 45 gastric cancer patients. The effects of MAPK15 on cell cycle, c-Jun phosphorylation, and mRNA stability were analyzed in gastric cancer cells. Copy number gains of MAPK15 were found in 15 (17%) of 88 tumor tissues. The mRNA levels of MAPK15 were relatively high in the gastric cancer tissues and gastric cancer cells with higher copy number gains than those without. Knockdown of MAPK15 using siRNA in gastric cancer cells significantly suppressed cell proliferation and resulted in cell cycle arrest at G₁-S phase. Reduced c-Jun phosphorylation and c-Jun half-life were observed in MAPK15-knockdowned cells. In addition, transient transfection of MAPK15 into AGS gastric cancer cells with low copy number resulted in an increase of c-Jun phosphorylation and stability. The overexpression of MAPK15 occurred at a high frequency in carcinomas (37%) compared to concurrent normal tissues (2%) and adenomas (21%). In conclusion, the present study suggests that MAPK15 overexpression may contribute to the malignant transformation of gastric mucosa by prolonging the stability of c-Jun. And, patients with copy number gain of MAPK15 in normal or premalignant tissues of stomach may have a chance to progress to invasive cancer.     

Expression of cyclooxygenase-2 and peroxisome proliferator-activated receptor-gamma and levels of prostaglandin E2 and 15-deoxy-delta12,14-prostaglandin J2 in human breast cancer and metastasis

Cyclooxygenase-2 (COX-2) expression and peroxisome proliferator-activated receptor-gamma (PPARgamma) inactivation are linked to increased risk of human breast cancer. The purpose of our study was to examine the relationship between COX-2 (with the resulting prostaglandins E(2), PGE(2)) and PPARgamma (and its natural endogenous ligand 15-Deoxy-Delta(12,14)-prostaglandin J(2), 15d-PGJ(2)) at various stages during the development of human breast cancer and its progression to metastasis. Human breast tissue specimens were collected from normal breasts or from individuals with fibrocystic disease and served as controls (n = 22). Tissues were also collected from uninvolved (n = 25), tumor (n = 25) and lymph node metastasis (n = 15) regions from breast cancer patients. COX-2 and PPARgamma mRNA expression were increased and downregulated, respectively, in tissues from cancer patients compared to controls. Metastatic tissues tended to have higher alterations compared to non-metastatic tissues (p < 0.05). These altered expressions in COX-2 and PPARgamma were paralleled by increases in the tissue levels of PGE(2) and decreases in 15d-PGJ(2). A significant inverse correlation was found between PGE(2) and 15-d-PGJ(2) (r = -0.51, p < 0.05). Significant correlations (p < 0.05) were also obtained between COX-2 and PPARgamma mRNA (inverse, r = -0.72) and between COX-2 and PGE(2) (direct, r = 0.68). Increases in COX-2 mRNA expression and levels of PGE(2) and down-regulation of PPARgamma mRNA expression and 15d-PGJ(2) levels were characterized as predictors of breast cancer risk (p < 0.05). Our results suggest that the altered expression of COX-2 and PPARgamma and the subsequent modulation in the tissue levels of PGE(2) and 15-d-PGJ(2) may influence the development of human breast cancer and its progression to metastasis.     

Combined evaluation of expressions of cyclin E and p53 proteins as prognostic factors for patients with gastric cancer.

BACKGROUND: There is a lack of consistency regarding the prognostic value of cyclin E overexpression in gastric cancer (gastric cancer). Our aim was to report on this overexpression and to analyze its correlations with the clinicopathologic variables. Another aim was to examine if aberrant expression of both cyclin E and p53 might increase the malignant potential of gastric cancer. METHODS: Specimens from 89 patients with gastric cancer treated with "curative" intent were evaluated for cyclin E and p53 expressions using immunohistochemical method. The correlations between cyclin E overexpression alone or in combination with p53 expression and the patient's clinicopathologic variables were analyzed. RESULTS: Cyclin E overexpression and p53 expression were shown in 35 (39.3%) and 46 (51.7%) tumors, respectively. The incidence of cyclin E overexpression was significantly higher in deeply invasive cancers (P < 0.0001), in cancers with lymph node metastasis (P = 0.003), and in cancers with advanced stages (P < 0.0001). There were no significant correlations with other clinicopathologic variables. Patients in whom their tumors showed cyclin E overexpression alone or in combination with p53 survived less than patients with negative cyclin E tumors. Multivariate analysis revealed that combined cyclin E overexpression and p53 expression was significantly associated with poor survival after adjusting for other variables (hazard ratio, 3.12; P = 0.009). CONCLUSIONS: Cyclin E overexpression is a common event in gastric cancer. Gastric cancer with cyclin E overexpression exhibit increased aggressiveness in the presence of aberrant p53. The combination of cyclin E overexpression with the p53 expression in gastric cancer further distinguished a subgroup of patients with poor prognosis.     

Oncolysis of human gastric cancers by an E1B 55 kDa-deleted YKL-1 adenovirus

Advanced gastric cancer cannot be treated with surgery or conventional cancer therapy, which has prompted a search for new therapeutic modalities. Previously, we and other groups showed that E1B 55 kDa-deleted recombinant adenoviruses, such as YKL-1, effectively replicate and induce cytotoxicity in p53-deficient cancer cells while sparing normal cells. Here, we investigated selective YKL-1 replication and resultant cytolysis in human gastric cancer cells. The cytopathic effects were obvious in all five gastric cancer cell lines we examined. Evaluation of p53 expression indicated that only the AGS cell line retained functionally normal p53. Nevertheless, AGS was 10-fold more sensitive to YKL-1 than the other cell lines. Transmission electron microscopy showed typical morphological alterations along with efficient replication of YKL-1 in AGS cells. Therefore, YKL-1 induces preferential cytotoxic effects in human gastric cancer cells in a p53-independent manner, making YKL-1 a promising therapeutic agent for human gastric cancers.     

Tim-3 Up-regulation in Patients with Gastric Cancer and Peptic Ulcer Disease

Background: T-cell immunoglobulin and mucin domain protein-3 (Tim-3), an inhibitory immunoregulatory receptor, has been recently implicated in tumor biology and tumor-associated immune suppression. In the present study, expression of Tim-3 was evaluated in gastric cancer (GC) and peptic ulcer disease (PUD) at both mRNA and protein levels. Methods: A total of 133 gastric tissue biopsies, comprising 43 from GC cases, 48 from PUD and 42 from non-ulcer dyspepsia (NUD) serving as controls were collected. Additionally, non-neoplastic adjacent tissue biopsies were also obtained from 6 patients with GC. Infection with Helicobacter pylori was determined by the rapid urease test for all participants and H&E staining was conducted for GC and PUD patients. Tim-3 relative mRNA expression was determined by SYBR Green based Real-Time PCR using β-actin as a reference gene. Tim-3 protein expression was also studied by immunohistochemistry in 7 GC, 7 PUD and 10 NUD tissue samples. Results: Tim-3 was expressed at higher levels in GC (p=0.030) and PUD (p=0.022) cases compared to he NUD group. Among paired samples obtained from gastric cancer patients, tumor tissues showed elevated Tim-3 expression (p=0.019) in comparison with adjacent non-neoplastic biopsies. Tim-3 mRNA findings were supported by detection of more Tim-3 protein in cancerous (p=0.002) and ulcerative (p=0.01) tissues than in controls. Tim-3 was similarly expressed in H. pylori positive and negative cases.Conclusion: Higher Tim-3 expression in patients with gastric cancer and peptic ulcer implies that it might be involved in immune regulation and establishment of these gastrointestinal diseases. Targeted immunotherapy by blocking of inhibitory receptors like Tim-3 could be a promising approach for gastric cancer treatment.     

mdm2、p14基因的扩增与胃癌的关系

背景与目的:肿瘤的发生与基因的变化密切相关,p53基因的变化被认为是肿瘤发生的一个重要原因,mdm2和p14被认为是调控p53功能的两个重要基因。目前,关于mdm2和p14在胃癌发病中作用的报道较少。本研究通过观察胃癌细胞中癌基因mdm2和抑癌基因p14的扩增水平,分析mdm2和p14与胃癌发生、发展的关系。方法:取10例正常胃粘膜(对照组)和45例胃癌组织(经病理检查证实为胃腺癌,其中高分化、中分化、低分化各15例),采用RT-PCR方法检测全部标本中p14和mdm2mRNA水平的变化。结果:(1)mdm2基因扩增阳性率在胃癌组织中为73.3%,与正常组织的40.0%比较,差异有显著性(P<0.05)。胃癌组织中高分化的93.3%、中分化的80.0%、低分化的46.6%,三组之间两两比较差异有显著性(P<0.05)。(2)p14基因扩增阳性率在胃癌组织中60.0%,与正常组织的50.0%比较差异无显著性(P>0.05),其中高分化的60.0%、中分化的66.6%、低分化的53.3%,三组之间两两比较差异无显著性(P>0.05)。结论:(1)mdm2基因扩增与胃癌的发生发展有密切的关系;(2)本研究未能证明p14与胃癌有关。     

Decreased expression of the carboxyl terminus of heat shock cognate 70 interacting protein in human gastric cancer and its clinical significance

The carboxyl terminus of heat shock cognate 70 interacting protein (CHIP) is an E3 ubiquitin ligase, which can promote ubiquitylation and degradation of many tumor-related proteins. However, the expression of CHIP in human gastric cancer has not been investigated. In this study, the mRNA and protein levels of CHIP expression in 53 cases of gastric cancer and matched normal tissues were determined by quantitative real-time PCR, western blotting and immunohistochemistry. We showed that CHIP was registered from basal to middle portions of normal gastric mucosa. CHIP expression was notably decreased or lost in human gastric cancer samples compared with the matched normal non-cancer samples. The correlations between CHIP downregulation and the clinicopathological characteristics were also evaluated. The expression of CHIP was significantly lower in the gastric cancer samples compared to the matched normal samples at both mRNA and protein levels (P<0.05 and P<0.05, respectively). More importantly, the downregulation of CHIP was correlated with TNM stage (P=0.048) and lymph node metastasis (P=0.010) at the mRNA levels. In addition, the downregulation of CHIP was correlated with lymph node metastasis (P=0.021) and tumor differentiation (P=0.009) at the protein levels. Taken together, at both mRNA and protein levels, the decreased expression of CHIP was correlated with lymph node metastasis. Furthermore, our study suggests that a negative correlation exists between CHIP expression and tumor malignancy in human gastric cancer.     

Activation of p53 in cervical cancer cells by human papillomavirus E6 RNA interference is transient, but can be sustained by inhibiting endogenous nuclear export-dependent p53 antagonists

p53 is degraded in cervical cancer cells by the human papillomavirus E6 and can be stabilized with short interfering RNA (siRNA) molecules targeting E6 mRNA. In this in vitro study, we show that E6 siRNA-induced p53 activation is transient in HeLa cervical cancer cells despite continuous suppression of E6 mRNA; activation can be sustained if the endogenous p53 antagonists COP1, MDM2, Pirh2, and c-Jun-NH(2)-kinase are also targeted by siRNAs or by inhibiting the nuclear export of p53 with leptomycin B. The direct targeting of any one of these four cellular p53 antagonists had no effect on p53 activity when E6 was intact, but inhibited the fading off of E6 siRNA-induced p53 activation in nonstress conditions. The effect was additive when multiple cellular antagonists were concomitantly inhibited, indicating that all these proteins degrade p53 when E6 is inactivated. The antiproliferative effect induced by E6 silencing was enhanced when the endogenous p53 antagonists were additionally targeted. In conclusion, if human papillomavirus E6 is inhibited under nonstress conditions, the subsequent p53 activation is quickly reversed by the endogenous p53 degenerative machinery. The present results indicate that several cellular p53 antagonists must be inhibited for sustained p53 activity if E6 siRNA therapy is attempted and if no combined genotoxic therapy is applied.     

Expression of eukaryotic initiation factor 4E in gastric adenocarcinoma and its association with clinical outcome

BACKGROUND AND OBJECTIVES: Overexpression of eIF4E can result in oncogenic transformation and uncontrolled growth of mammalian cells, presumably by facilitating the expression of growth-control gene products, which are normally translationally repressed. Overexpression of eIF4E was present in human breast carcinoma and human head and neck squamous cell carcinoma, and may be of prognostic value in breast carcinomas. In order to elucidate the clinical significance of eIF4E expression, this study was conducted to quantify expression of eIF4E in human gastric cancer tissue and correlate them with clinicopathological factors and patient survival. METHODS: Specimens from sixty-nine patients with gastric adenocarcinoma were analyzed and eIF4E overexpression was quantified by Western blot analysis. Quantification of eIF4E levels in cancer was expressed relative to controls from non-tumorous mucosa of the same patients. Confirmation of eIF4E overexpression at the cellular level was performed using immunohistochemical staining. The association of clinicopathologic factors and survival with eIF4E expression was analyzed. RESULTS: In non-tumorous parts of specimens, overexpression of eIF4E was always present in gastric glands but not in gastric pits lining mucosa, and it was also expressed in the areas of intestinal metaplasia and dysplasia. In the 69 specimens, the mean eIF4E expression was 5.77 +/- 8.55-fold (mean +/- standard deviation), ranged from from 0.1-fold to 38-fold. The degree of eIF4E expression appeared to be independent of invasion depth of tumor, lymph node metastasis, Lauren classification, Borrmann types, and Helicobacter pylori infection. Marked overexpression of eIF4E (more than seven-fold) was correlated with tumor vascular invasion (P = 0.046, Fisher exact-test). The survival rate of the patients with underexpression or mild overexpression of eIF4E (less than sevenfold) was significantly higher than that with marked eIF4E overexpression (more than sevenfold) (P = 0.01734, log rank test). CONCLUSIONS: Marked eIF4E overexpression in gastric cancer was found to be associated with vascular invasion. The prognosis for gastric cancer patients with marked overexpression of eIF4E was worse than those with underexpression. It may serve as an additional prognostic and therapeutic factor in gastric cancer, and deserves further investigation.     

雌二醇对胃癌细胞周期的影响

目的:初步探讨雌二醇（E2）对胃癌细胞SGC-7901增殖的影响。方法:SGC-7901细胞分为E2干预组和对照组,E2干预组加入不同浓度E2（0.1μmol/L、1.0μmol/L）干预24小时,对照组加入等体积含无水乙醇的培养基。CCK8检测E2对胃癌细胞增殖能力的影响,流式细胞术检测细胞周期的分布,PCR检测E2对胃癌细胞雌激素受体ERα和ERβ mRNA表达水平的影响,Oncomine公共数据库查询ERα的表达情况。结果:E2对胃癌细胞的增殖抑制率具有浓度依赖性。E2处理组SGC-7901细胞G0/G1期细胞比例［(68.866±3.336)%］较对照［(59.333±0.294)%］显著增加,G2期和S期细胞总比例下降,1.0μmol/L E2作用可使胃癌细胞SGC-7901发生G1期阻滞（P<0.01）。两种雌激素受体（ERα和ERβ）均表达于胃癌细胞SGC-7901,且其相关基因ERα和ERβ mRNA表达水平不随E2浓度的增加而改变。利用Oncomine公共数据库查询发现ERα在胃癌中的表达高于癌旁。结论:E2直接作用可抑制胃癌细胞SGC-7901的增殖,引起G1期阻滞。