Anticancer bioactive peptide-3 inhibits human gastric cancer growth by targeting miR-338-5p

Background

Cancer incidence and mortality have been increasing in China, making cancer the leading cause of death since 2010 and a major public health concern in the country. Cancer stem cells have been studied in relation to the treatment of different malignancies, including gastric cancer. Anticancer bioactive peptide-3 (ACBP-3) can induce the apoptosis of gastric cancer stem cells (GCSCs) and reduce their tumorigenicity. In the present study, for the first time, we used a miRNA microarray and bioinformatics analysis to identify differentially expressed miRNAs in ACBP-3-treated GCSCs and GCSC-derived tumors in a xenograft model and functionally verified the identified miRNAs. miR-338-5p was selected based on its significant upregulation by ACBP-3 both in cultured GCSCs and in tumor tissues.

Results

miR-338-5p was downregulated in GCSCs compared with normal gastric epithelial cells, and the ectopic restoration of miR-338-5p expression in GCSCs inhibited cell proliferation and induced apoptosis, which correlated with the upregulation of the pro-apoptotic Bcl-2 proteins BAK and BIM. We also found that ACBP-3-treated GCSCs could respond to lower effective doses of cisplatin (DDP) or 5-fluorouracil (5-FU), possibly because ACBP-3 induced the expression of miR-338-5p and the BAK and BIM proteins and promoted GCSC apoptosis.

Conclusions

Our data indicate that miR-338-5p is part of an important pathway for the inhibition of human gastric cancer stem cell proliferation by ACBP-3 combined with chemotherapeutics. ACBP-3 could suppress GCSC proliferation and lower the required effective dose of cisplatin or 5-fluorouracil. Therefore, this study provides not only further evidence for the remarkable anti-tumor effect of ACBP-3 but also a possible new approach for the development of GCSC-targeting therapies.

     

MicroRNA-338 Inhibits Growth,Invasion and Metastasis of Gastric Cancer by Targeting NRP1 Expression

NRP1 as multifunctional non-tyrosine-kinase receptors play critical roles in tumor progression. MicroRNAs (miRNAs) are an important class of pervasive genes that are involved in a variety of biological functions, particularly cancer. It remains unclear whether miRNAs can regulate the expression of NRP1. The goal of this study was to identify miRNAs that could inhibit the growth, invasion and metastasis of gastric cancer by targeting NRP1 expression. We found that miR-338 expression was reduced in gastric cancer cell lines and in gastric cancer tissues. Moreover, we found that miR-338 inhibited gastric cancer cell migration, invasion, proliferation and promoted apoptosis by targeting NRP1 expression. As an upstream regulator of NRP1, miR-338 directly targets NRP1. The forced expression of miR-338 inhibited the phosphorylation of Erk1/2, P38 MAPK and Akt; however, the expression of phosphorylated Erk1/2, P38 MAPK and Akt was restored by the overexpression of NRP1. In AGS cells infected with miR-338 or transfected with SiNRP1, the protein levels of fibronectin, vimentin, N-cadherin and SNAIL were decreased, but the expression of E-cadherin was increased. The expression of mesenchymal markers in miR-338-expressing cells was restored to normal levels by the restoration of NRP1 expression. In vivo, miR-338 also decreased tumor growth and suppressed D-MVA by targeting NRP1. Therefore, we conclude that miR-338 acts as a novel tumor suppressor gene in gastric cancer. miR-338 can decrease migratory, invasive, proliferative and apoptotic behaviors, as well as gastric cancer EMT, by attenuating the expression of NRP1.     

The Effect of miR-338-3p on HBx Deletion-Mutant (HBx-d382) Mediated Liver-Cell Proliferation through CyclinD1 Regulation

Objective

Hepatitis B Virus (HBV) DNA integration and HBV X (HBx) deletion mutation occurs in HBV-positive liver cancer patients, and C-terminal deletion in HBx gene mutants are highly associated with hepatocarcinogenesis. Our previous study found that the HBx-d382 deletion mutant (deleted at nt 382–400) can down-regulate miR-338-3p expression in HBx-expressing cells. The aim of the present study is to examine the role of miR-338-3p in the HBx-d382-mediated liver-cell proliferation.

Methods

We established HBx-expressing LO2 cells by Lipofectamine 2000 transfection. A miR-338-3p mimics or inhibitor was transfected into LO2/HBx-d382 and LO2/HBx cells using miR-NC as a control miRNA. In silico analysis of potential miR-338-3p targets revealed that miR-338-3p could target the cell cycle regulatory protein CyclinD1. To confirm that CyclinD1 is negatively regulated by miR-338-3p, we constructed luciferase reporters with wild-type and mutated CyclinD1-3′UTR target sites for miR-338-3p binding. We examined the CyclinD1 expression by real-time PCR and western blot, and proliferation activity by flow cytometric cell cycle analysis, Edu incorporation, and soft agar colony.

Results

HBx-d382 exhibited enhanced proliferation and CyclinD1 expression in LO2 cells. miR-338-3p expression inhibited cell proliferation in LO2/HBx-d382 cells (and LO2/HBx cells), and also negatively regulated CyclinD1 protein expression. Of the two putative miR-338-3p binding sites in the CyclinD1-3′UTR region, the effect of miR-338-3p on the second binding site (nt 2397–2403) was required for the inhibition.

Conclusion

miR-338-3p can directly regulate CyclinD1 expression through binding to the CyclinD1-3′UTR region, mainly at nt 2397–2403. Down-regulation of miR-338-3p expression is required for liver cell proliferation in both LO2/HBx and LO2/HBx-d382 mutant cells, although the effect is more pronounced in LO2/HBx-d382 cells. Our study elucidated a novel mechanism, from a new miRNA-regulation perspective, underlying the propensity of HBx deletion mutants to induce hepatocarcinogenesis at a faster rate than HBx.     

上皮性卵巢癌组织miR-338-3p、miR-1294的表达及临床意义

摘要 目的：探讨上皮性卵巢癌组织中微小RNA（miR）-338-3p、miR-1294表达与患者临床病理参数的关系,并分析其表达对上皮性卵巢癌预后的影响。方法：收集2010年6月～2015年6月我院行手术治疗的上皮性卵巢癌患者的石蜡组织样本（癌组织和癌旁正常组织）,实时荧光定量PCR（RT-PCR）技术检测组织样本中miR-338-3p、miR-1294的表达情况;分析癌组织中miR-338-3p、miR-1294表达与患者临床病理参数的关系;Kaplan-Meier生存曲线分析miR-338-3p、miR-1294表达与患者预后的关系;Cox回归模型分析上皮性卵巢癌患者的预后影响因素。结果：与癌旁正常组织相比,上皮性卵巢癌组织中miR-338-3p、miR-1294表达均降低（均P<0.05）;miR-338-3p表达与组织学分级、淋巴结转移、FIGO分期相关（均P<0.05）;miR-1294表达与肿瘤直径、淋巴结转移、FIGO分期相关（均P<0.05）。Kaplan-Meier生存曲线结果显示,miR-338-3p低表达组患者、miR-1294低表达组患者的预后较差（均P<0.05）。Cox回归模型分析结果显示,淋巴结转移、较高FIGO分期、miR-338-3p低表达、miR-1294低表达是上皮性卵巢癌预后的危险因素（均P<0.05）。结论：上皮性卵巢癌组织中miR-338-3p、miR-1294均低表达,且均与较差临床病理参数、预后不良相关;miR-338-3p、miR-1294可能是上皮性卵巢癌患者预后预测的潜在指标。     

Growth inhibitory effects of three miR-129 family members on gastric cancer

Reduced expression of microRNA-129 (miR-129) has been reported in several types of tumor cell lines as well as in primary tumor tissues. However, little is known about how miR-129 affects cell proliferation in gastric cancer. Here, we show that all miR-129 family members, miR-129-1-3p, miR-129-2-3p, and miR-129-5p, are down-regulated in gastric cancer cell lines compared with normal gastric epithelial cells. Furthermore, using the real-time cell analyzer assay to observe the growth effects of miR-129 on gastric cancer cells, we found that all three mature products of miR-129 showed tumor suppressor activities. To elucidate the molecular mechanisms underlying down-regulation of miR-129 in gastric cancer, we analyzed the effects of miR-129 mimics on the cell cycle. We found that increased miR-129 levels in gastric cancer cells resulted in significant G₀/G₁ phase arrest. Interestingly, we showed that cyclin dependent kinase 6 (CDK6), a cell cycle-associated protein involved in G₁-S transition, was a target of miR-129. We also found that expression of the sex determining region Y-box 4 (SOX4) was inversely associated with that of miR-129-2-3p and miR-129-5p but not of miR-129-1-3p. Together, our data indicate that all miR-129 family members, not only miR-129-5p, as previously thought, play an important role in regulating cell proliferation in gastric cancer.     

miR-145-5p affects the differentiation of gastric cancer by targeting KLF5 directly

Krüppel-like factor 5 (KLF5) takes part in the pathologic processes of many types of cancer; however, its expression and roles in the biological behavior of gastric cancer remain unknown. TargetScan suggested that miR-145-5p is the predicted effective and conserved microRNA (miRNA) that binds to KLF5 through its 3′-untranslated region (UTR). We investigated the expression of KLF5 and miR-145-5p messenger RNA (mRNA) in gastric cancer and then analyzed its role in the biological behavior of gastric cancer cells. Our results indicated that KLF5 expression was detected by immunohistochemistry in 39.7% of the gastric cancer cases and was increased compared with that of the corresponding noncancerous normal mucosa (0.01 < p < 0.05). The poorly differentiated subtype showed positive KLF5 expression, whereas the differentiated subtype showed negative KLF5 expression (p < 0.05). Dual-luciferase reporter assay suggested KLF5 3′-UTR was the direct target of miR-145-5p. Compared with the differentiated gastric cancer, miR-145-5p was downregulated in undifferentiated gastric cancer (p < 0.05). The downregulation of KLF5 expression and differentiation of MGC-803 and BGC-823 caused by siKLF5 or miR-145-5p mimic transfection. Our results indicated that miR-145-5p/KLF5 3′-UTR affected the differentiation of gastric cancer. miR-145-5p was able to promote gastric cancer differentiation by targeting KLF5 3′-UTR directly. Our data suggest a novel mechanism for cancer differentiation and a new facet to the role of miR-145-5p/KLF5 in gastric cancer.     

MicroRNA-149 Inhibits Proliferation and Cell Cycle Progression through the Targeting of ZBTB2 in Human Gastric Cancer

An increasing body of evidence indicates that miR-149 can both suppress and promote tumor growth depending on the tumor type. However, the role of miR-149 in the progression of gastric cancer (GC) remains unknown. Here we report that miR-149 is a tumor suppressor in human gastric cancer. miR-149 expression is decreased in GC cell lines and clinical specimens in comparison to normal gastric epithelial cell and tissues, respectively. The expression levels of miR-149 also correlate with the differentiation degree of GC cells and tissues. Moreover, ectopic expression of miR-149 in gastric cancer cells inhibits proliferation and cell cycle progression by down-regulating ZBTB2, a potent repressor of the ARF-HDM2-p53-p21 pathway, with a potential binding site for miR-149 in its mRNA''s 3′UTR. It is also found that ZBTB2 expression increases in GC cells and tissues compared to normal gastric epithelial cell and tissues, respectively. Silencing of ZBTB2 leads to suppression of cell growth and cell cycle arrest in G0/G1 phase, indicating that ZBTB2 may act as an oncogene in GC. Furthermore, transfection of miR-149 mimics into gastric cancer cells induces down-regulation of ZBTB2 and HDM2, and up-regulation of ARF, p53, and p21 compared to the controls. In summary, our data suggest that miR-149 functions as a tumor suppressor in human gastric cancer by, at least partially through, targeting ZBTB2.     

外周血miR-17-92基因簇对于早期胃癌的诊断及预测价值

目的:探讨外周血mi R-17-92簇对早期胃癌的诊断价值,为胃癌的早期诊断及治疗提供参考依据。方法:收集胃癌125例(Ⅰ期35例,Ⅱ期28例,Ⅲ期39例,Ⅳ期23例)和癌前病变24例(包括肠化生及上皮内瘤变),同时选择65例慢性胃炎作为对照组。采用实时荧光定量PCR技术(Real-time quantitative PCR,RT-qPCR)检测患者血清中的mi R-17-92基因簇的表达水平。通过受试者工作曲线(Receiver Operating Curve, ROC)及曲线下的面积(Area Under the Curve,AUC)评估mi R-17-92基因簇表达水平诊断早期胃癌的敏感性和特异性。结果:(1)慢性胃炎与癌前病变mi R-17-92基因簇表达比较无显著差异(P0.05);(2)早期胃癌及进展期胃癌mi R-17-5p表达明显高于慢性胃炎(P0.05),mi R-19a-3p、mi R-19b-3p、mi R-20a-5p和mi R-92a-3p表达则显著低于慢性胃炎及进展期胃癌(P0.05);(3)miR-17-5p诊断早期胃癌的曲线下面积较mi R-19a-3p、mi R-19b-3p、mi R-20a-5p、mi R-92a-3p及CEA更高;(4)miR-19a-3p、mi R-19b-3p、mi R-20a-5p、mi R-92a-3p高低表达组与在胃癌的浸润深度间有显著性差异(P0.05),mi R-19b-3p高低表达组在胃癌的临床分期间有显著性差异(P0.05);(5)miR-17-5p、mi R-19a-3p、mi R-19b-p、mi R-20a-5p、mi R-92a-3p诊断早期胃癌的阳性率较CEA、CA199高。结论:外周血mi R-17-92基因簇对于早期胃癌的诊断价值明显优于CEA和CA199,这可能为胃癌的早诊早治提供新的策略。     

Oncogenic miRNA-182-5p Targets Smad4 and RECK in Human Bladder Cancer

Onco-miR-182-5p has been reported to be over-expressed in bladder cancer (BC) tissues however a detailed functional analysis of miR-182-5p has not been carried out in BC. Therefore the purpose of this study was to: 1. conduct a functional analysis of miR-182-5p in bladder cancer, 2. assess its usefulness as a tumor marker, 3. identify miR-182-5p target genes in BC. Initially we found that miR-182-5p expression was significantly higher in bladder cancer compared to normal tissues and high miR-182-5p expression was associated with shorter overall survival in BC patients. To study the functional significance of miR-182-5p, we over-expressed miR-182-5p with miR-182-5p precursor and observed that cell proliferation, migration and invasion abilities were increased in BC cells. However cell apoptosis was inhibited by miR-182-5p. We also identified Smad4 and RECK as potential target genes of miR-182-5p using several algorithms. 3′UTR luciferase activity of these target genes was significantly decreased and protein expression of these target genes was significantly up-regulated in miR-182-5p inhibitor transfected bladder cancer cells. MiR-182-5p also increased nuclear beta-catenin expression and while Smad4 repressed nuclear beta-catenin expression. In conclusion, our data suggests that miR-182-5p plays an important role as an oncogene by knocking down RECK and Smad4, resulting in activation of the Wnt-beta-catenin signaling pathway in bladder cancer.     

miR-338-3p suppresses neuroblastoma proliferation,invasion and migration through targeting PREX2a

MicroRNAs (miRNA) can regulate cancer cell proliferation and metastasis. Here, we show that miR-338-3p is down-regulated in metastatic tumor tissues compared to primary tumors, and that that miR-338-3p can inhibit cell proliferation by inducing cell cycle arrest, as well as restrain cell migration and invasion. PREX2a is confirmed as a direct target of miR-338-3p. Knockdown of PREX2a inhibits cell proliferation, migration and invasion through the PTEN/Akt pathway. miR-338-3p-dependent inhibition of proliferation and invasion can be rescued by PREXa. Overall, this study demonstrates that miR-338-3p affects the PTEN/Akt pathway by down-regulating PREX2a. This newly identified function of miR-338-3p provides novel insights into neuroblastoma and may foster therapeutic applications.     

肝癌组织中miR-338-3p的表达及与临床病理参数的关系

目的:探讨肝癌组织中微小RNA-338-3p(miR-338-3p)的表达及与临床病理参数的关系。方法:选取2015年1月至2016年6月我院手术获得的67例肝癌组织标本,同时每例标本均取癌旁正常组织标本作为配对对照,采用实时定量逆转录聚合酶链反应(RT-qPCR)对两组组织标本中的miR-338-3p进行检测,并分析其与肝癌临床病理特征的关系。结果:45例(67.16%)miR-338-3p表达下调,22例(32.85%)表达上调;RT-qPCR结果显示,肝癌组织中miR-338-3p的相对含量为(0.76±0.38),低于癌旁正常组织中的(1.23±0.45),差异有统计学意义(t=-6.259,P=0.000)。miR-338-3p在低分化、TNM分期Ⅲ+Ⅳ期、肿瘤浸润深度T3+T4期、有淋巴结转移肝癌患者肝癌组织中的表达下调率高于中高分化、Ⅰ+Ⅱ期、T1+T2期、无淋巴结转移肝癌患者,差异有统计学意义(P0.05)。不同性别、年龄、病理类型、肿瘤大小肝癌患者肝癌组织中miR-338-3p表达下调率差异无统计学意义(P0.05)。结论:miR-338-3P在肝癌组织中呈低表达水平,与分化程度、TNM分期、肿瘤浸润深度、淋巴结转移有关,可能参与了肝癌的发生发展过程,早期检测可作为评估肝癌病情的指标。     

MiR-17-5p and MKL-1 modulate stem cell characteristics of gastric cancer cells

Effectively targeting cancer stem cells to treat cancer has great therapeutic prospects. However, the effect of microRNA miR-17/MKL-1 on gastric cancer stem cells has not been studied yet. This study preliminarily explored the mechanism of miR-17/MKL-1 in gastric cancer stem cells. Many previous reports have indicated that microRNA and EMT regulated cancer stem cell characteristics, and miR-17 and MKL-1 were involved as a critical gene in migration and invasion in the EMT pathway. Through RT-PCR, Western Blot, flow cytometry, immunofluorescence, sphere formation xenograft tumor assays and drug resistance, the role of miR-17-5p and MKL-1 on promoting stem cell-like properties of gastric cancer were verified in vivo and vitro. Next, MKL-1 targets CD44, EpCAM, and miR -17-5p promoter verified by luciferase assay and ChIP. Besides, the TCGA database analysis found that both miR-17-5p and MKL-1 increased in gastric cancer, and the prognostic survival of the MKL-1 high expression group was reduced. It is found that MKL-1 promotes expression by targeting miR-17, CD44 and EpCAM promoters. Besides, the TCGA database analysis found that both miR-17-5p and MKL-1 increased in gastric cancer, and the prognostic survival of the MKL-1 high expression group was reduced. These findings reveal new regulatory signaling pathways for gastric cancer stem cells, thus it give new insights on potential early diagnosis and/or molecular therapy for gastric cancer.     

miR-1207-5p and miR-1266 suppress gastric cancer growth and invasion by targeting telomerase reverse transcriptase

hTERT is the catalytic subunit of the telomerase complex. Elevated expression of hTERT is associated with the expansion and metastasis of gastric tumor. In this study, we aimed to identify novel tumor suppressor miRNAs that restrain hTERT expression. We began our screen for hTERT-targeting miRNAs with a miRNA microarray. miRNA candidates were further filtered by bioinformatic analysis, general expression pattern in different cell lines, gain-of-function effects on hTERT protein and the potential of these effects to suppress hTERT 3′ untranslated region (3′UTR) luciferase activity. The clinical relevance of two miRNAs (miR-1207-5p and miR-1266) was evaluated by real-time RT-PCR. The effects of these miRNAs on cell growth, cell cycle and invasion of gastric cancer cells were measured with CCK-8, flow cytometry and transwell assays. Finally, the ability of these miRNAs to suppress the transplanted tumors was also investigated. Fourteen miRNAs were identified using a combination of bioinformatics and miRNA microarray analysis. Of these fourteen miRNAs, nine were expressed at significantly lower levels in hTERT-positive cell lines compared with hTERT-negative cell lines and five could downregulate hTERT protein expression. Only miR-1207-5p and miR-1266 interacted with the 3′ UTR of hTERT and the expression levels of these two miRNAs were significantly decreased in gastric cancer tissues. These two miRNAs also inhibited gastric tumor growth in vitro and in vivo. Altogether, miR-1207-5p and miR-1266 were determined to be hTERT suppressors in gastric cancer, and the delivery of these two miRNAs represents a novel therapeutic strategy for gastric cancer treatment.