miR-25在人胃癌组织中的表达分析及其靶基因TOB1的鉴定

目的:分析miR-25在胃癌组织中的表达水平并鉴定其新的靶基因。方法:选取10例胃癌患者的胃癌及癌旁正常组织样本,用qRT-PCR法检测miR-25的表达水平。利用miRNA靶基因预测数据库预测miR-25的靶基因。构建荧光素酶载体(pMIR-TOB1)及绿色荧光蛋白报告载体(pMIR-GFP-TOB1)(含有miR-25结合位点的TOB1的3'UTR片段),利用荧光素酶活性实验和GFP荧光强度报告实验鉴定miR-25的预测靶基因(TOB1)。qRT-PCR法和Western blot法分别检测TOB1的mRNA和蛋白质的表达水平。结果:miR-25在80%(8/10)的胃癌组织样本中表达上调。miR-25模拟物显著抑制了荧光素酶的活性(P<0.01)和GFP荧光强度。miR-25模拟物显著下调了AGS细胞中TOB1的mRNA(P<0.05)和蛋白质的表达。结论:miR-25在人胃癌组织中表达上调,miR-25能结合TOB1的3'UTR并下调TOB1的mRNA和蛋白质的表达。

Down-regulation of miR-622 in gastric cancer promotes cellular invasion and tumor metastasis by targeting ING1 gene

AIM:To evaluate the biological and clinical characteristics of miR-622 in gastric cancer. METHODS:We analyzed the expression of miR-622 in 57 pair matched gastric neoplastic and adjacent non-neoplastic tissues by quantitative real-time polymerase chain reaction. Functional analysis of miR-622 expression was assessed in vitro in gastric cancer cell lines with miR-622 precursor and inhibitor. The roles of miR-622 in tumorigenesis and tumor metastasis were analyzed using a stable miR-622 expression plasmid in nude mice. A luciferase reporter assay was used to assess the effect of miR-622 on inhibitor of growth family,member 1 (ING1) expression. RESULTS:Expression of miR-622 was down-regulated in gastric cancer. MiR-622 was found involved in differentia-tion and lymphatic metastasis in human gastric cancer. Ectopic expression of miR-622 promoted invasion,tumorigenesis and metastasis of gastric cancer cells both in vitro and in vivo. ING1 is a direct target of miR-622. CONCLUSION:These findings help clarify the molecular mechanisms involved in gastric cancer metastasis and indicate that miR-622 modulation may be a bona fide treatment of gastric cancer.

LncRNA cancer susceptibility 20 regulates the metastasis of human gastric cancer cells via the miR-143-5p/MEMO1 molecular axis

BACKGROUND Gastric cancer(GC)is considered as one of the most widespread malignancies.Emerging evidence has shown that lncRNAs can function as important oncogenes or tumor suppressors during GC progression.AIM To investigate the effect and mechanism of lncRNA cancer susceptibility 20(CASC20)in the proliferation and metastasis of GC cells.METHODS Data mining and clinical samples were used to evaluate the expression of CASC20 in GC and adjacent tissues.CASC20 was down-regulated in GC cells by shortinterfering RNA.Cell proliferation was evaluated by CCK-8 assay,and cell migration and invasion were detected by wound healing and Transwell assays.The expressions of proteins related to epithelial-mesenchymal transition were detected by western blot assay.RESULTS The expression of CASC20 was increased in GC tumor tissues and various GC cell lines.High CASC20 expression was correlated with a high risk of lymphatic metastasis and poor prognosis in GC patients.In vitro assays showed that silencing CASC20 reduced cell proliferation,migration,and invasion in GC cells.Mechanistic studies revealed that CASC20 exhibits oncogenic functions by regulating MEMO1 expression through competitive endogenous binding to miR-143-5p,leading to induction of epithelial-mesenchymal transition.CONCLUSION Our findings indicate that CASC20 serves as a tumor promoter by regulating metastasis in GC via the miR-143-5p/MEMO1 axis.CASC20 may be a potential therapeutic target for GC.

Overexpression of MiR-633 Suppresses the Tumorigenicity of Gastric Cancer Cells and Induces Apoptosis by Targeting MAPK1

Objective MicroRNA(miRNA/miR)-633 is dysregulated in several types of cancers and is involved in tumorigenesis.However,the function and role of this miRNA in gastric cancer(GC)are not fully understood.The aim of the present study was to evaluate miR-633 expression in GC cell lines and in GC tissue vs.adjacent normal tissue,and to determine its association with clinicopathological data.This work was extended to investigate the effects of miR-633 overexpression on tumor cells in vitro.Methods Reverse transcription-quantitative PCR(RT-qPCR)was used to detect and compare the expression level of miR-633 in GC cells,as well as in GC and normal adjacent tissue samples.The clinical significance of miR-633 was also analyzed.MiR-633 lentivirus(LV-miR-633)and negative control lentivirus(LV-NC)were generated and used to transduce SGC-7901 and HGC-27 GC cells in order to analyze the effect of miR-633 on their phenotype.The effects of miR-633 overexpression on GC cell proliferation,apoptosis,migration and invasion were investigated.The target gene of miR-633 was predicted,then confirmed using a dual luciferase reporter gene assay,RT-qPCR and Western blotting.Results MiR-633 was significantly downregulated in GC cell lines,as well as in GC tissue compared with adjacent normal tissue.Moreover,miR-633 expression was associated with the tumor/node/metastasis(TNM)stage,invasion depth,Borrmann classification and lymph node metastasis(P<0.05).Compared with the LV-NC group,transduction with LV-miR-633 reduced the proliferation,the number of clones,the wound healing rate,the number of invading cells and the number of cells in the G1 phase of the cell cycle(P<0.01).LV-miR-633 also increased the apoptosis rate(P<0.01).The expression level of mitogen-activated protein kinase(MAPK)1,high-mobility group box 3(HMGB3),claudin 1(CLDN1)and MAPK13 were downregulated in LV-miR-633-transduced cells(P<0.01).The dual luciferase reporter assay confirmed that the 3′-untranslated region of MAPK1 was the target site of miR-633(P<0.01).Conclusion MiR-633 acts as a tumor suppressor in GC,and its expression level is associated with TNM stage,invasion depth,Borrmann type and lymph node metastasis.Overexpression of miR-633 inhibits the proliferation and migration of GC cells and induces apoptosis and cell cycle arrest at the in G1 phase.In addition,miR-633 negatively regulates the expression of MAPK1,HMGB3,CLDN1 and MAPK13 and directly targets MAPK1.

MiR-503 regulates cisplatin resistance of human gastric cancer cell lines by targeting IGF1R and BCL2

Background Studies have shown that the drug resistance of gastric cancer cells can be modulated by abnormal expression of microRNAs （miRNAs）.We investigated the role of miR-503 in the development of cisplatin resistance in human gastric cancer cell lines.Methods MiR-503 expression was measured by quantitative real-time PCR.MTT （3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide） and clonogenic assays were used to examine changes in cell viability and the drug resistance phenotype of cancer cells associated with upregulation or downregulation of the miRNA.A dual-luciferase activity assay was used to verify target genes of miR-503.Immunohistochemistry,Western blotting analysis,and a flow cytometric apoptosis assay were used to elucidate the mechanism by which miR-503 modulates drug resistance in cancer cells.Results MiR-503 was significantly downregulated in gastric cancer tissues and several gastric cancer cell lines.Additionally,downregulation of miR-503 in the cisplatin （DDP）-resistant gastric cancer cell line SGC7901/DDP was concurrent with the upregulation of insulin-like growth factor-1 receptor （IGF1R） and B-cell lymphoma 2 （BCL2） expression compared with the parental SGC7901 cell line.An in vitro drug sensitivity assay showed that overexpression of miR-503 sensitized SGC7901/DDP cells to cisplatin.The luciferase activity of reporters driven by IGF1R and BCL2 3＇-untranslated regions in SGC7901/DDP cells suggested that IGF1R and BCL2 were both direct target genes of miR-503.Enforced miR-503 expression in SGC7901/DDP cells reduced expression of the target proteins,inhibited proliferation,and sensitized the cells to DDP-induced apoptosis.Conclusion Our findings suggest that hsa-miR-503 modulates cisplatin resistance of human gastric cancer cells at least in part by targeting IGF1R and BCL2.

1α,25(OH)_2D_3通过阻滞细胞周期抑制胃癌细胞系增殖

目的研究1α,25(OH)2D3对胃癌细胞增殖和细胞周期的影响,并探讨其相关的作用机制。方法 BGC-823和SGC-7901胃癌细胞系分别给予终浓度为1×10-10～1×10-7mol/L的1α,25(OH)2D3处理72 h,用MTT法检测细胞抑制率;用流式细胞仪检测细胞周期;用RT-qPCR技术检测细胞周期相关基因P21、cyclin D1、cyclin E1和CDK6 mRNA表达。结果 1α,25(OH)2D3对胃癌细胞的增殖抑制率具有浓度依赖性,1α,25(OH)2D3干预导致BGC-823和SGC-7901细胞系G1期细胞比例升高,S期细胞比例降低(P<0.05);1α,25(OH)2D3干预后,BGC-823和SGC-7901胃癌细胞P21 mRNA表达升高(P<0.01),而cyclin D1、cyclin E1和CDK6 mRNA表达降低(P<0.01)。结论 1α,25(OH)2D3抑制胃癌细胞增殖,诱导细胞周期阻滞,可能与上调P21,下调cyclin D1、cyclin E1和CDK6的表达有关。

MicroRNA-638 inhibits cell proliferation by targeting phospholipase D1 in human gastric carcinoma

MicroRNAs （miRNAs） are a type of small non-coding RNAs that are often play important roles in carcinogene- sis, but the carcinogenic mechanism of miRNAs is still unclear. This study will investigate the function and the mechanism of miR-638 in carcinoma （GC）. The expres- sion of miR-638 in GC and the DNA copy number of miR- 638 were detected by real-time PCR. The effect of miR-638 on cell proliferation was measured by counting kit-8 assay. Different assays, including bioinformatics algo- rithms （TargetScan and miRanda）, luciferase report assay and Western blotting, were used to identify the target gene of miR-638 in GC. The expression of miR-638 target gene in clinical CRC tissues was also validated by immunohistochemical assay. From this research, we found that miR-638 was downregulated in GC tissues compared with corresponding noncancerous tissues （NCTs）, and the DNA copy number of miR-638 was lower in GC than NCTs, which may induce the corresponding downregulation of miR-638 in GC. Ectopic expression of miR-638 inhibited GC cell growth in vitro. Subsequently, we identified that PLD1 is the target gene of miR-638 in GC, and silencing PLD1 expression phenocopied the inhibitory effect of miR-638 on GC cell proliferation. Fur- thermore, we observed that PLD1 was overexpressed inGC tissues, and high expression of PLDt in GC predicted poor overall survival. In summary, we revealed that miR- 638 functions as a tumor suppressor in GC through inhibiting PLDI.