The expression profiling of circulating miR-204, miR-182, and lncRNA H19 as novel potential biomarkers for the progression of peptic ulcer to gastric cancer

Deregulation of noncoding RNAs, microRNAs (miRNAs) and long noncoding RNA (lncRNA), are implicated in the initiation and progression of gastric cancer (GC). This study is a pilot case-control study carried out on 75 subjects, 40 of them were Helicobacter pylori-gastric ulcer patients and 35 were GC patients recruited from the Gastrointestinal Endoscopy Unit in Al-Kasr Al-Aini Hospital, Cairo University in Egypt. Real-time PCR was performed to evaluate the expression level of serum miR-204, miR-182, and lncRNA H19 in patients with peptic ulcer-progressed GC vs nonprogressed peptic ulcer patients. Fibroblast growth factor 18 (FGF-18)/FGF receptor 2 (FGFR2) expression and their downstream immunological and inflammatory signaling markers were assessed and their association with the addressed noncoding RNAs investigated. As regards miR-204 and miR-182, they were significantly increased (12.5 and 2.6 folds, respectively) in GU samples, compared with those of healthy control levels. The elevated levels of these miRNAs were significantly de-escalated in GC samples compared with GU and the fold decrease valued 2.2 fold for miR-204 and 1.8 folds for miR-182. On the other hand, the significant escalation in the level of lnRNA H19 in GU recorded a 16.6 fold increase and further elevation in its levels was evident in GC samples. The herein assessed miRNAs are correlated with disease duration and FGFR2 with miR-182 being significantly correlated with all inflammatory markers, TAC, INF-γ, matrix metallopeptidase 9, and FGF-18. In terms of diagnostic accuracy of assessed miRNAs (stages III to IV), the receiver operating characteristic analysis indicated that serum lncRNA H19 showed the highest diagnostic accuracy (95.5%), specificity (100%), and sensitivity (90.9%), compared with miR-204 and miR-182, which showed the same specificity (60%), sensitivity (72.7%), and diagnostic accuracy (68.8%). Our findings conclude that lnRNA H19, miR-204, and miR-182 may function as novel prospective plasma biomarkers to detect GC and its progression from H. pylori-peptic ulcer, which would be helpful to improve the theranostics of GC.     

lncRNA ZFPM2-AS1 promotes proliferation via miR-18b-5p/VMA21 axis in lung adenocarcinoma

Lung cancer has been proved to be one of the most common kinds of cancers around the globe. Meanwhile, as the predominant type of lung cancer, lung adenocarcinoma (LUAD) has received increasing attention in cancer research. Long noncoding RNAs (lncRNAs) are known to be associated with oncogenesis and progression of various cancers. However, many lncRNAs have not been thoroughly detected in LUAD. In this study, through bioinformatics analysis we found that zinc finger protein multitype 2 antisense RNA 1 (ZFPM2-AS1) was associated with poor prognosis of LUAD patients. Also, ZFPM2-AS1 was detected to be overexpressed in LUAD tissues and cells. Furthermore, ZFPM2-AS1 could promote the proliferation of LUAD cells. Next, miR-18b-5p was found to bind with and negatively regulated by ZFPM2-AS1. VMA21, target gene of miR-18b-5p, could bind with and be negatively regulated by miR-18b-5p. More importantly, both ZFPM2-AS1 and VMA21 were found to be attached to the RNA-induced silencing complex constructed from miR-18b-5p and Ago2. Also, ZFPM2-AS1 could regulate the expression of VMA21. Therefore, ZFPM2-AS1 were confirmed to regulate VMA21 by competitively binding with miR-18b-5p. Finally, rescue assays confirmed that ZFPM2-AS1 could regulate LUAD cell proliferation via miR-18b-5p/VMA21 axis.     

LINC01433 promotes hepatocellular carcinoma progression via modulating the miR-1301/STAT3 axis

Long noncoding RNAs (lncRNAs) have been demonstrated to play significant roles in hepatocellular carcinoma (HCC) tumor progression. LINC01433 has been implicated in the progression of lung cancer. However, its biological role in HCC remains poorly understood. In our current study, we focused on the detailed mechanism of LINC01433 in HCC development. First, it was exhibited that LINC01433 was remarkably elevated in HCC cells, which indicated that LINC01433 was involved in HCC. Then, knockdown of LINC01433 was able to restrain HCC cell proliferation and cell colony formation and greatly induced cell apoptosis. On the contrary, overexpression of LINC01433 promoted HCC cell proliferation, increased cell colony formation, and enhanced cell invasion capacity. Subsequently, we found that miR-1301 was remarkably decreased in HCC cells, and it can serve as a target of LINC01433 according to bioinformatics analysis. In addition, the binding correlation between them was validated by performing RNA pull-down experiments and RIP assay. Moreover, STAT3 was predicted and validated as a target of miR-1301, and it was shown that miR-1301 mimics significantly suppressed STAT3 in HCC cells. Finally, in vivo models were established, and the results demonstrated that silencing of LINC01433 could repress HCC development through modulating miR-1301 and STAT3. Taken together, these results indicated in our study that LINC01433 participated in HCC progression through modulating the miR-1301/STAT3 axis and it might act as a novel biomarker in HCC diagnosis and treatment.     

lncRNA RBM5-AS1 promotes cell proliferation and invasion by epigenetically silencing miR-132/212 in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is regarded as one of the most common malignancies worldwide leading to cancer-related death. Long noncoding RNAs (lncRNAs) are a critical modulator affecting HCC progression. Whereas, the pathogenesis of lncRNA RBM5-AS1 in the development of HCC remains unclear. Quantitative RT-PCR or western blot assays were applied to detect the expression of genes and proteins, respectively. The proliferation and metastasis abilities were assessed using Cell counting kit-8 (CCK-8), EdU and transwell assays. RNA immunoprecipitation (RIP) experiment was employed to validate the molecular interactions. RBM5-AS1 is highly expressed in HCC tissues and cell lines, especially in Hep3B and HepG2 cells. RBM5-AS1 knockdown dramatically restrains cell proliferation, invasion and migration of HCC cells. Importantly, RBM5-AS1 acts as an epigenetic regulator to elevate the H3K27me3 level of miR-132/212 promoter regions via recruiting PRC2 (EZH2, SUZ12, EED), and eventually reducing miR-132/212 expressions. The recovery experiments demonstrated that downregulation of miR-132/212 markedly eliminate the antitumor effects mediated by RBM5-AS1 silencing in HCC cells. The data of this work illustrate that RBM5-AS1 acts as an epigenetic regulator to promote the HCC progression by repressing miR-132/212 expressions, which would provide a new insight for understanding the action mechanism of RBM5-AS1 in HCC development.     

DGCR5 promotes cancer stem cell-like properties of radioresistant laryngeal carcinoma cells by sponging miR-506 via Wnt pathway

Cancer stem cells (CSCs) have been recognized as the significant cause of tumor recurrence. Long noncoding RNAs (lncRNAs) are involved in various cancers, including human laryngeal cancer. So far the correlation between lncRNA DiGeorge syndrome critical region gene 5 (DGCR5) and CSC-like properties in human laryngeal cancer remains barely known. In our current study, two human larynx squamous carcinoma cell lines (Hep-2 and Hep-2R) with different radio sensitivities were cultured. Interestingly, CSC-like phenotypes were much more enriched in Hep-2R cells. We found that DGCR5 was upregulated and microRNA-506 (miR-506) was downregulated in Hep-2R cells. In addition, silence of DGCR5 could inhibit the stemness and enhance the radiosensitivity of Hep-2R cells. Meanwhile, overexpression of miR-506 also suppressed the CSC-like traits and the radiosensitivity was increased significantly. In addition, miR-506 was predicted as target of DGCR5 and the correlation between them was validated in our study. Finally, we observed that Wnt pathway exerted a significant role in human laryngeal CSCs and DGCR5 inhibition could repress Wnt signaling activity by sponging miR-506. In vivo assays were performed and we found that DCGR5 depressed stemness of human laryngeal cancer cells through modulating miR-506 and Wnt signaling pathway. Taken these together, we reported that DGCR5 induced CSC-like properties by sponging miR-506 through activating Wnt in human laryngeal carcinoma cells.     

LncRNA H19/miR-194/PFTK1 axis modulates the cell proliferation and migration of pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) remains a huge challenge due to its high mortality and morbidity; gene therapy might be a promising treatment for PDAC. The critical role of Wnt-signaling pathway in cancer pathogenesis has been widely recognized; cyclin-dependent kinase 14 (CDK14, PFTK1)-induced low-density lipoprotein receptor-related proteins 5/6 (LRP5/6) phosphorylation is an important issue in Wnt-signaling activation. Long noncoding RNA (LncRNA)-microRNA (miRNA)-messenger RNA (mRNA) modulating the pathogenesis of cancers has been regarded as a major mechanism. In the current study, upregulated lncRNAs positively correlated with PFTK1 were analyzed and selected using The Cancer Genome Atlas (TCGA) database. Of them, lncRNA H19 can activate Wnt signaling in cancers. In PDAC tissues, the expression of H19 and PFTK1 were upregulated; H19 knockdown suppressed the cell proliferation and migration of PDAC, while PFTK1 overexpression partially attenuated the suppressive effect of H19 knockdown. As analyzed by TCGA and predicted by online tools, miR-194 was negatively correlated with PFTK1 and might bind to both H19 and PFTK1, which was further confirmed by luciferase reporter and RNA immunoprecipitation assays. Moreover, the effect of H19 knockdown on PFTK1 protein and the cell proliferation and migration could be partially reversed by miR-194 inhibition; H19/miR-194 axis modulated PDAC cell proliferation and migration through PFTK1 downstream Wnt signaling. Results suggested that rescuing miR-194 expression in PDAC can inhibit lncRNA H19 and PFTK1 expression, subsequently suppressing PDAC cell proliferation and migration. Due to the complexity of the lncRNA-miRNA-mRNA network, further in vivo experiments examining potential side effects are needed in future study to explore the clinical application of these findings.     

Knockdown of DGCR5 enhances the radiosensitivity of human laryngeal carcinoma cells via inducing miR-195

Long noncoding RNAs (lncRNAs) exert critical roles in the development of various cancers, including human laryngeal cancer. Radioresistance contributes to the predominant causes of laryngeal cancer recurrence after radiotherapy. The aim of our study was to investigate the association of dysregulated lncRNA and radiation resistance in human larynx squamous carcinoma. Here, we investigated the biological roles of lncRNA DiGeorge syndrome critical region gene 5 (DGCR5) in radioresistance of human laryngeal cancer. Two human larynx squamous carcinoma cell lines (Hep-2 and Hep-2R), with different radiosensitivities in vitro were used in the present study. We observed that DGCR5 was significantly upregulated in Hep-2R cells. Inhibition of DGCR5 by LV-shDGCR5 transfection restrained Hep-2R cell proliferation and sensitized cells to radiation. Reversely, overexpression of DGCR5 exhibited an opposite phenomenon in vitro. In addition, microRNA (miR)-195 was predicted as a direct downstream target of DGCR5. Dual-luciferase reporter and RNA immunoprecipitation assays verified the direct interaction between them. Meanwhile, miR-195 was observed to be reduced in Hep-2R cells and miR-195 mimics repressed Hep-2 cell growth. Moreover, radiosensitivity of Hep-2R cells was greatly enhanced by overexpression of miR-195, which could be reversed by upregulation of DGCR5. Finally, in vivo experiments were used to validate that knockdown of DGCR5 suppressed laryngeal carcinoma via targeting miR-195. In conclusion, we indicated that DGCR5 could contribute to the radioresistance of human laryngeal carcinoma cells via sponging miR-195.     

Fluid shear stress regulates osteoblast proliferation and apoptosis via the lncRNA TUG1/miR-34a/FGFR1 axis

LncRNAs and microRNAs play critical roles in osteoblast differentiation and bone formation. However, their exact roles in osteoblasts under fluid shear stress (FSS) and the possible mechanisms remain unclear. The aim of this study was to explore whether and how miR-34a regulates osteoblast proliferation and apoptosis under FSS. In this study, FSS down-regulated miR-34a levels of MC3T3-E1 cells. MiR-34a up-regulation attenuated FSS-induced promotion of proliferation and suppression of apoptosis. Luciferase reporter assay revealed that miR-34a directly targeted FGFR1. Moreover, miR-34a regulated osteoblast proliferation and apoptosis via FGFR1. Further, we validated that lncRNA TUG1 acted as a competing endogenous RNA (ceRNA) to interact with miR-34a and up-regulate FGFR1 protein expression. Furthermore, lncRNA TUG1 could promote proliferation and inhibit apoptosis. Taken together, our study revealed the key role of the lncRNA TUG1/miR-34a/FGFR1 axis in FSS-regulated osteoblast proliferation and apoptosis and may provide potential therapeutic targets for osteoporosis.     

CircTIMELESS regulates the proliferation and invasion of lung squamous cell carcinoma cells via the miR-136-5p/ROCK1 axis

Numerous studies demonstrate that circular RNAs (circRNAs) are critical regulators of the occurrence and progression of tumors. However, research on the involvement of circRNAs in lung squamous cell carcinoma (LUSC) is limited. In our study, circTIMELESS (also named hsa_circ_0000408 in the Human circRNA Database) was upregulated in both LUSC tissues and LUSC cells, and circTIMELESS expression was positively associated with the TNM stage. Moreover, circTIMELESS silencing markedly suppressed invasion in vitro and disrupted proliferation in vitro as well as in vivo. Additional investigations have shown that circTIMELESS functions as a miR-136-5p “sponge” and regulates miR-136-5p expression. Furthermore, the impact of miR-136-5p upregulation was consistent with the results of circTIMELESS silencing, both of which inhibited the proliferation and invasion of LUSC cells. Additional results showed that Rho-associated coiled-coil containing protein kinase 1 (ROCK1) is targeted by miR-136-5p. The results of recovery experiments showed that ROCK1 overexpression partly rescued the impact of circTIMELESS silencing and miR-136-5p upregulation on proliferation and invasion. Consequently, our findings confirmed that circTIMELESS exists in LUSC and acts as a tumor promoter through the miR-136-5p/ROCK1 axis. Based on these findings, circTIMELESS may be potentially utilized as a therapeutic target for LUSC.     

lncRNA A1BG-AS1 suppresses proliferation and invasion of hepatocellular carcinoma cells by targeting miR-216a-5p

Extensive evidence indicate that long noncoding RNAs (lncRNAs) regulates the tumorigenesis and progression of hepatocellular carcinoma (HCC). However, the expression and biological function of lncRNA A1BG antisense RNA 1 (A1BG-AS1) were poorly known in HCC. Here, we found the underexpression of A1BG-AS1 in HCC via analysis of The Cancer Genome Atlas database. Further analyses confirmed that A1BG-AS1 expression in HCC was markedly lower than that in noncancerous tissues based on our HCC cohort. Clinical association analysis revealed that low A1BG-AS1 expression correlated with poor prognostic features, such as microvascular invasion, high tumor grade, and advanced tumor stage. Follow-up data indicated that low A1BG-AS1 level evidently correlated with poor clinical outcomes of HCC patients. Moreover, forced expression of A1BG-AS1 repressed proliferation, migration, and invasion of HCC cells in vitro. Conversely, A1BG-AS1 knockdown promoted these malignant behaviors in HepG2 cells. Mechanistically, A1BG-AS1 functioned as a competing endogenous RNA by directly sponging miR-216a-5p in HCC cells. Notably, miR-216a-5p restoration rescued A1BG-AS1 attenuated proliferation, migration and invasion of HCCLM3 cells. A1BG-AS1 positively regulated the levels of phosphatase and tensin homolog and SMAD family member 7, which were reduced by miR-216a-5p in HCC cells. Altogether, we conclude that A1BG-AS1 exerts a tumor suppressive role in HCC progression.     

miR-146a interacting with lncRNA EPB41L4A-AS1 and lncRNA SNHG7 inhibits proliferation of bone marrow-derived mesenchymal stem cells

Emerging evidence suggests that microRNA plays a pivotal role in cell proliferation. Our previous research has certified that miR-146a attenuates osteoarthritis through the regulation of cartilage homeostasis. However, little information about the function of miR-146a in bone marrow-derived mesenchymal stem cells (BMSCs) proliferation and the underlying mechanism was available. Therefore, this study aims at investigating the role of miR-146a on the proliferation of BMSCs and the possible mechanisms involved. The function of miR-146a on BMSCs proliferation was studied through overexpression and knockdown of miR-146a or the indicated long noncoding RNAs (lncRNAs) in BMSCs and then the proliferation rate of the BMSCs were detected by Cell Counting Kit-8 assay, colony formation assay. Besides, flow cytometry was used to test the cell cycle state of BMSCs modified by overexpression or knockdown of miR-146a or lncRNA EPB41L4A-AS1 (EPB41L4A Antisense RNA 1) and small nucleolar RNA host gene 7 (SNHG7). The expression level of marker genes involved in modulating cell proliferation was evaluated by quantitative polymerase chain reaction and western blot analysis. We discovered that the knockdown of miR-146a significantly promoted BMSCs proliferation. Moreover, miR-146a could bind to and inhibit endogenous expression of EPB41L4A-AS1 and SNHG7. Further study demonstrated that overexpression of EPB41L4A-AS1 and SNHG7 significantly enhanced proliferation of BMSCs. For the first time, we certified that miR-146a suppressed BMSCs proliferation, but EPB41L4A-AS1 and SNHG7 promoted BMSCs proliferation in the present study. Mechanistically, miR-146a significantly inhibited BMSCs proliferation partly through miR-146a/EPB41L4A-AS1 SNHG7/cell proliferation signaling pathway axis.     

LncRNA AC130710通过miR-129-5P/WNT4轴促进子宫内膜癌细胞增殖和上皮间质转化

目的探讨LncRNA AC130710通过miR-129-5P/WNT4轴对子宫内膜癌细胞（HEC-1A细胞）增殖、凋亡及上皮间质转化（EMT）的影响及机制研究。 方法通过实时荧光定量PCR检测LncRNA AC130710、miR-129-5P和WNT4在子宫内膜癌细胞（HEC-1A细胞）和人子宫内膜上皮细胞（HEEC）中的表达。细胞分别转染（1）siRNA NC、AC130710 siRNA、WNT4 siRNA;（2）inhibitor NC、miR-129-5P inhibitor;（3）pcDNA-3.1 （+）+mimics NC、pcDNA-AC130710+mimics NC、pcDNA-3.1 （+）+miR-129-5P mimics、pcDNA-AC130710+miR-129-5P mimics。MTT实验检测LncRNA AC130710、miR-129-5P和WNT4的表达对HEC-1A细胞增殖能力的影响;Western blot检测LncRNA AC130710、miR-129-5P和WNT4的表达对HEC-1A细胞凋亡相关蛋白B淋巴细胞瘤-2基因相关蛋白X （Bax）、剪切的半胱氨酰天冬氨酸特异性蛋白酶-3 （cleaved caspase-3）、cleaved caspase-9和B淋巴细胞瘤-2基因（Bcl-2）表达的影响;Western blot检测LncRNA AC130710、miR-129-5P和WNT4的表达对HEC-1A细胞EMT的影响。miRanda和双荧光素酶报告基因实验分析LncRNA AC130710和miR-129-5P之间的关系,TargetScan数据库分析miR-129-5P与WNT4的相关性,双荧光素酶报告基因检测miR-129-5P与WNT4的相互作用;RT-qPCR法检测LncRNA AC130710通过miR-129-5P对WNT4表达的影响。两组间比较采用独立样本t检验,多组间比较采用单因素方差分析,两两比较采用LSD-t检验。 结果与HEEC细胞比较,HEC-1A细胞中AC130710表达水平（1.86±0.21比0.85±0.06）、WNT4表达水平（1.88±0.26比1.08±0.12）升高;HEC-1A细胞中miR-129-5P表达水平（0.89±0.16比1.76±0.08）降低。与转染siRNA NC比较,转染AC130710 siRNA细胞内Bax、cleaved caspase-3、cleaved caspase-9、E-cadherin蛋白相对表达水平[（1.37±0.14比0.84±0.21）,（1.08±0.16比0.37±0.07）,（1.26±0.24比0.39±0.06）,（1.87±0.17比1.32±0.26）]上升,Bcl-2、N-cadherin、Snail和Vimentin蛋白相对表达水平[（0.38±0.08比1.18±0.14）,（0.36±0.04比0.85±0.24）,（0.35±0.09比1.12±0.18）,（0.42±0.10比1.26±0.27）]下降;与转染inhibitor NC比较,转染miR-129-5P inhibitor细胞的Bcl-2、N-cadherin、Snail和Vimentin蛋白相对表达水平[（0.98±0.07比0.65±0.08）,（1.39±0.15比0.68±0.09）,（0.95±0.08比0.42±0.06）,（1.16±0.16比0.54±0.02）]上升,Bax、cleaved caspase-3、cleaved caspase-9、E-cadherin蛋白相对表达水平[（0.27±0.09比0.85±0.13）,（0.48±0.05比1.16±0.28）,（0.52±0.14比1.19±0.15）,（0.43±0.09比1.08±0.26）]下降;与转染siRNA NC比较,转染WNT4 siRNA细胞的Bcl-2、N-cadherin、Snail和Vimentin蛋白相对表达水平[（0.23±0.08比0.84±0.12）,（0.28±0.09比1.14±0.17）,（0.42±0.23比1.06±0.15）,（0.35±0.08比1.13±0.08）]降低,Bax、cleaved caspase-3、cleaved caspase-9、E-cadherin蛋白相对表达水平[（0.96±0.12比0.42±0.08）,（1.13±0.25比0.45±0.06）,（1.54±0.23比0.72±0.12）,（1.87±0.24比1.26±0.18）]上升。 结论LncRNA AC130710可通过miR-129-5P/WNT4轴调控子宫内膜癌HEC-1A细胞增殖、凋亡及EMT。     

Involvement of NEAT1/miR-133a axis in promoting cervical cancer progression via targeting SOX4

NEAT1 is an important tumor oncogenic gene in various tumors. Nevertheless, its involvement remains poorly studied in cervical cancer. Our study explored the functional mechanism of NEAT1 in cervical cancer. NEAT1 level in several cervical cancer cells was quantified and we found NEAT1 was greatly upregulated in vitro. NEAT1 knockdown inhibited cervical cancer development through repressing cell proliferation, colony formation, capacity of migration, and invasion and also inducing the apoptosis. For another, microRNA (miR)-133a was downregulated in cervical cancer cells and NEAT1 negatively modulated miR-133a expression. Subsequently, we validated that miR-133a functioned as a potential target of NEAT1. Meanwhile, SOX4 is abnormally expressed in various cancers. SOX4 was able to act as a downstream target of miR-133a and silencing of SOX4 can restrain cervical cancer progression. In addition, in vivo assays were conducted to prove the role of NEAT1/miR-133a/SOX4 axis in cervical cancer. These findings implied that NEAT1 served as a competing endogenous RNA to sponge miR-133a and regulate SOX4 in cervical cancer pathogenesis. To sum up, it was implied that NEAT1/miR-133a/SOX4 axis was involved in cervical cancer development.     

lncRNA GAS5/miR-223/NAMPT axis modulates the cell proliferation and senescence of endothelial progenitor cells through PI3K/AKT signaling

Endothelial progenitor cells (EPCs) have been reported to replace the damaged endothelial cells to repair the injured or dead endothelium. However, EPC senescence might lead to the failure in EPC function. Thus, developing an in-depth understanding of the mechanism of EPC senescence might provide novel strategies for related vascular disorders’ treatments. Herein, nicotinamide phosphoribosyltransferase (NAMPT) overexpression could increase cell proliferation and suppress cell senescence in EPCs. miR-223 directly bound to the 3′-untranslated region of NAMPT to inhibit its expression, therefore modulating EPC proliferation and senescence through NAMPT and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling. Long noncoding RNA (lncRNA) GAS5 sponges miR-223, consequently downregulating miR-223 expression. GAS5 knockdown inhibited EPC proliferation and promoted senescence. GAS5 might serve as a competing endogenous RNA for miR-223 to counteract miR-223-mediated suppression on NAMPT, thus regulating EPC proliferation and senescence via the PI3K/AKT signaling pathway. In summary, our findings provide a solid experimental basis for understanding the role and mechanism of lncRNA GAS5/miR-223/NAMPT axis in EPC proliferation and senescence.