胃癌中显著下调的miR-433的作用靶点研究

目的为了筛选胃癌中miRNAs的表达标记,验证胃癌相关miRNAs的作用靶点,建立一种新的诊断和治疗胃癌的方法。方法运用基因芯片技术检测3个正常胃组织标本,24个胃癌组织标本,胃癌细胞SGC7901和正常胃黏膜细胞GES-1中328个miRNAs的表达情况。用以上方法检测出在胃癌组织和SGC7901中,miR-433的表达水平显著下调。为了确保结果的准确性,采用实时荧光定量PCR对其进行验证。并用基因克隆和Western印迹方法分析miR-433的作用靶点。结果共有26个miRNAs在胃癌标本（包括24个胃癌组织和SGC7901）中异常表达。其中19个miRNAs下调,7个miRNAs上调。实时荧光定量PCR检测出miR-433在胃癌标本中的表达水平显著下调,该结果和基因芯片检测结果一致。另外,在本实验中发现miR-433与Grb2（growth factor receptor—bound protein 2）的表达呈负相关。结论胃癌相关miRNAs已进行了初步筛选。其中,miR-433可能是胃癌中的标记性miRNAs之一,Grb2是其作用靶点。这为建立新的以miRNAs为基础的诊断和治疗胃癌的方法提供了相关信息。     

MicroRNA let-7f inhibits tumor invasion and metastasis by targeting MYH9 in human gastric cancer

Background

MicroRNAs (miRNAs) are important regulators that play key roles in tumorigenesis and tumor progression. A previous report has shown that let-7 family members can act as tumor suppressors in many cancers. Through miRNA array, we found that let-7f was downregulated in the highly metastatic potential gastric cancer cell lines GC9811-P and SGC7901-M, when compared with their parental cell lines, GC9811 and SGC7901-NM; however, the mechanism was not clear. In this study, we investigate whether let-7f acts as a tumor suppressor to inhibit invasion and metastasis in gastric cancers.

Methodology/Principal

Real-time PCR showed decreased levels of let-7f expression in metastatic gastric cancer tissues and cell lines that are potentially highly metastatic. Cell invasion and migration were significantly impaired in GC9811-P and SGC7901-M cell lines after transfection with let-7f-mimics. Nude mice with xenograft models of gastric cancer confirmed that let-7f could inhibit gastric cancer metastasis in vivo after transfection by the lentivirus pGCsil-GFP- let-7f. Luciferase reporter assays demonstrated that let-7f directly binds to the 3′UTR of MYH9, which codes for myosin IIA, and real-time PCR and Western blotting further indicated that let-7f downregulated the expression of myosin IIA at the mRNA and protein levels.

Conclusions/Significance

Our study demonstrated that overexpression of let-7f in gastric cancer could inhibit invasion and migration of gastric cancer cells through directly targeting the tumor metastasis-associated gene MYH9. These data suggest that let-7f may be a novel therapeutic candidate for gastric cancer, given its ability to reduce cell invasion and metastasis.     

Mad2beta, an alternative variant of Mad2 reducing mitotic arrest and apoptosis induced by adriamycin in gastric cancer cells

Mad2beta is an alternative splicing variant of spindle checkpoint gene mad2, which was previously found by us and was related to the drug resistance in gastric cancer cells. In this paper, we explored the molecular mechanisms that Mad2beta variant promoted the formation of multidrug resistance in gastric cancer cells. We found that Mad2beta variant was detected only in the two human drug resistant gastric cancer cell sublines SGC7901/VCR and SGC7901/ADR, and it did not appear in its parental cell line SGC7901 and other detected gastric cancer cell lines. Expressions of Mad2 mRNA and protein in SGC7901 cells transfected with Mad2beta, SGC7901/VCR and SGC7901/ADR were significantly lower than that in SGC7901 cells. Moreover, SGC7901 cells overexpressing Mad2beta variant became more resistant to adriamycin, vincristine and mitomycin by abrogating mitotic arrest and apoptosis. This suggests that expression of Mad2beta variant decreases the relative expression of efficient MAD2, which may help gastric cancer cells to develop the phenotype of multidrug resistance.     

Depression of MAD2 inhibits apoptosis of gastric cancer cells by upregulating Bcl-2 and interfering mitochondrion pathway

Mitotic arrest deficient 2 (MAD2) is an essential component of the mitotic spindle checkpoint pathway. It was previously shown to be associated with drug resistance of tumor cells. To further explore the roles of MAD2 in responses of gastric cancer cells to chemotherapy drugs, we constructed the siRNA vectors of MAD2 and transfected them into gastric cancer SGC7901 cells to inhibit expression of MAD2. MTT assay showed that the downregulation of MAD2 increased the resistance of SGC7901 cells to spindle inhibitors and DNA damaging agents. The apoptosis rates of gastric cancer cells transfected with MAD2-siRNA were 10.7% and 10%, respectively, after treated by 1.0microg/ml VCR and cisplatin. In contrast, the apoptosis rates of SGC7901 and SGC7901/psilencer3.1 induced by VCR were 43.2%, 38.7%; and that induced by cispaltin were 34.1%, 31.4%. The ratio of Bcl-2 to Bax was much higher in the MAD2-siRNA transfectants compared with the SGC7901/psilencer. In SGC7901/psilencer, cytochrome c and cleaved caspase 3 protein levels increased along with the exposure time increased. However, these protein levels of SGC7901/MAD2-siRNA had no changes during the drug treatment. These results indicate that down regulation of MAD2 could promote the drug resistance of gastric cancer cells and inhibit anticancer drugs induced-apoptosis by upregulating Bcl-2 and interfering the mitochondrion apoptosis pathway.     

长链非编码RNA UCA1在胃癌多药耐药中的作用研究

目的:研究胃癌耐药细胞及其亲本细胞中长链非编码RNA UCA1的表达差异,探讨UCA1在胃癌多药耐药中的作用。方法:通过实时荧光定量PCR(q RT-PCR)检测胃癌耐药细胞SGC7901/ADR、SGC7901/VCR及其亲本细胞SGC7901中UCA1的表达差异;通过si RNA转染降低SGC7901/ADR中UCA1表达,MTT法检测细胞半数抑制浓度(IC50)的变化,流式细胞仪检测细胞凋亡变化。结果:QRT-PCR结果显示,UCA1在SGC7901/ADR和SGC7901/VCR胃癌耐药细胞表达显著高于SGC7901胃癌亲本细胞;MTT实验表明,干扰UCA1的SGC7901/ADR相对于阴性对照(NC)组的IC50显著降低;凋亡检测结果显示,在相同剂量化疗药物作用下,干扰UCA1后SGC7901/ADR凋亡率显著高于NC组;Western blot证实,干扰UCA1表达可显著降低BCL-2蛋白表达。结论:长链非编码RNA UCA1在胃癌耐药细胞表达显著升高,干扰UCA1表达可明显逆转胃癌耐药,UCA1可作为治疗胃癌耐药的重要分子靶标。     

MiR-935通过其靶基因Notch1调控胃癌SGC7901细胞的发生发展

目的:探究Mi R-935调控胃癌SGC7901细胞的增殖和浸润与Notch1基因表达的关系。方法:分别检测40例正常人胃粘膜组织与40例胃印戒细胞癌的Notch1表达情况,并分析胃印戒细胞癌组织中Notch1表达与患者年龄、性别、组织进展程度、TNM分期、有无淋巴结转移的关系;采用Mi R-935转染体外培养的SGC7901细胞系,检测Notch1的表达情况,其后采用Mi R-935抑制剂处理,通过Transwell实验检测胃癌细胞的侵袭能力,细胞划痕实验检测胃癌细胞迁移能力。结果:正常人胃粘膜组织中Notch1表达呈阴性,而胃印戒细胞癌组织中Notch1表达呈阳性;Notch1的表达与胃印戒细胞癌的TNM分期、有无淋巴结转移存在着显著的相关性;转染Mi R-935的SGC7901细胞Notch1表达明显上调,采用Mi R-935抑制剂处理后,Notch1的表达显著下降。结论:Mi R-935可能通过调控Notch1的表达调控胃癌的扩增和浸润。     

胃癌多药耐药相关microRNA的筛选和鉴定

目的:研究胃癌多药耐药相关microRNA并对其进行鉴定、靶基因预测和预测靶基因的生物信息学分析。方法:运用microRNA芯片对胃癌多药耐药细胞SGC7901/ADR和其亲本细胞SGC7901进行microRNA表达谱分析;采用实时定量PCR的方法对差异表达的miRNA进行验证;再运用生物信息学方法对差异表达的miRNA进行靶基因预测;再对预测的靶基因进行GO和KEGG通路分析。结果:与SGC7901相比SGC7901/ADR表达上调超过2倍的miRNA有6个,表达下调超过2倍的有11个。实时定量PCR对共同差异表达的microRNA进行验证显示与芯片结果的一致性。对这17个差异表达的miRNA进行靶基因预测,再对预测得到的靶基因进行GO和KEGG通路分析显示预测的靶基因参与了肿瘤相关通路、MAPK通路、Focal Adhesion通路等。结论:我们初步筛选得到了胃癌多药耐药相关miRNA并对其进行了生物信息学分析,为进一步地探索miRNA在胃癌多药耐药中的作用及其分子机制奠定了基础。     

CXXC finger protein 4 inhibits the CDK18‐ERK1/2 axis to suppress the immune escape of gastric cancer cells with involvement of ELK1/MIR100HG pathway

Gastric cancer, is the fourth most common tumour type yet, ranks second in terms of the prevalence of cancer‐related deaths worldwide. CXXC finger protein 4 (CXXC4) has been considered as a novel cancer suppressive factor, including gastric cancer. This study attempted to investigate the possible function of CXXC4 in gastric cancer and the underlying mechanism. The binding of the ETS domain‐containing protein‐1 (ELK1) to the long non‐coding RNA MIR100HG promoter region was identified. Then, their expression patterns in gastric cancer tissues and cells (SGC7901) were detected. A CCK‐8 assay was used to detect SGC7901 cell proliferation. Subsequently, SGC7901 cells were co‐cultured with CD3+ T cells, followed by measurement of CD3+ T cell proliferation, magnitude of IFN‐γ+ T cell population and IFN‐γ secretion. A nude mouse model was subsequently developed for in vivo validation of the in vitro results. Low CXXC4 expression was found in SGC7901 cells. Nuclear entry of ELK1 can be inhibited by suppression of the extent of ELK1 phosphorylation. Furthermore, ELK1 is able to bind the MIR100HG promoter. Overexpression of CXXC4 resulted in weakened binding of ELK1 to the MIR100HG promoter, leading to a reduced proliferative potential of SGC7901 cells, and an increase in IFN‐γ secretion from CD3+ T cells. Moreover, in vivo experiments revealed that CXXC4 inhibited immune escape of gastric cancer cells through the ERK1/2 axis. Inhibition of the CXXC4/ELK1/MIR100HG pathway suppressed the immune escape of gastric cancer cells, highlighting a possible therapeutic target for the treatment of gastric cancer.     

Cycloxygenase-2 is Essential for the Survival and Proliferation of Gastric Cancer Cells

Cycloxygenase-2 catalyzes the synthesis of prostaglandins from arachidonic acid and this enzyme has been implicated in the metastasis of gastric cancer. In order to examine the significance of cycloxygenase-2 (Cox-2) in the survival and proliferation of gastric cancer cells, we have stably overexpressed an antisense Cox-2 in two gastric cancer cell lines, SGC7901 and AGS, in order to reduce the expression of this protein. The sense and antisense Cox-2 expression vectors were created by cloning COX-2 cDNA, in pIRES2-EGFP plasmid. Cox-2 gene expression was monitored by RT-PCR and Western blotting and the results indicated that cells with antisense Cox-2 construct had significantly reduced Cox-2 expression in comparison to the cells that received sense-Cox-2 plasmid. Reduction of Cox-2 expression in SGC7901 and AGS gastric cancer cells led to markedly decreased proliferation. The metastatic capability of the two cell lines, as assessed by in vitro colony formation assay, is also significantly compromised by lowered Cox-2 expression. Thus, this study demonstrates that Cox-2 activity is necessary for the proliferation and metastasis of gastric cancer cells.     

Class I phosphatidylinositol 3-kinase inhibitor LY294002 activates autophagy and induces apoptosis through p53 pathway in gastric cancer cell line SGC7901

We aimed to study the effects of LY294002, an inhibitor of class I phosphatidylinositol 3-kinase (PI3K), on proliferation, apoptosis, and autophagy in gastric cancer cell line SGC7901. In this study, we showed that LY294002 inhibited the viability of gastric cancer SGC7901 cells. We also showed that LY294002 increased the expression of microtubule-associated protein 1 light chain 3 (LC3), and increased monodansylcadaverine (MDC)-labeled vesicles. LY294002 activated autophagy by activating p53 and caspase-3, and induced apoptosis by up-regulatingp53 and p53-up-regulated modulator of apoptosis ( PUMA ). Therefore, LY294002 might induce cytotoxicity in SGC7901 cells through activation of p53 and the downstream point PUMA . These findings suggest that inhibition of the class I PI3K signaling pathway is a potential strategy for managing gastric cancers.