LncRNA miR663AHG represses the development of colon cancer in a miR663a-dependent manner.

The MIR663AHG gene encodes both miR663AHG and miR663a. While miR663a contributes to the defense of host cells against inflammation and inhibits colon cancer development, the biological function of lncRNA miR663AHG has not been previously reported. In this study, the subcellular localization of lncRNA miR663AHG was determined by RNA-FISH. miR663AHG and miR663a were measured by qRT-PCR. The effects of miR663AHG on the growth and metastasis of colon cancer cells were investigated in vitro and in vivo. CRISPR/Cas9, RNA pulldown, and other biological assays were used to explore the underlying mechanism of miR663AHG. We found that miR663AHG was mainly distributed in the nucleus of Caco2 and HCT116 cells and the cytoplasm of SW480 cells. The expression level of miR663AHG was positively correlated with the level of miR663a (r = 0.179, P = 0.015) and significantly downregulated in colon cancer tissues relative to paired normal tissues from 119 patients (P < 0.008). Colon cancers with low miR663AHG expression were associated with advanced pTNM stage (P = 0.021), lymph metastasis (P = 0.041), and shorter overall survival (hazard ratio = 2.026; P = 0.021). Experimentally, miR663AHG inhibited colon cancer cell proliferation, migration, and invasion. The growth of xenografts from RKO cells overexpressing miR663AHG was slower than that of xenografts from vector control cells in BALB/c nude mice (P = 0.007). Interestingly, either RNA-interfering or resveratrol-inducing expression changes of miR663AHG or miR663a can trigger negative feedback regulation of transcription of the MIR663AHG gene. Mechanistically, miR663AHG could bind to miR663a and its precursor pre-miR663a, and prevent the degradation of miR663a target mRNAs. Disruption of the negative feedback by knockout of the MIR663AHG promoter, exon-1, and pri-miR663A-coding sequence entirely blocked these effects of miR663AHG, which was restored in cells transfected with miR663a expression vector in rescue experiment. In conclusion, miR663AHG functions as a tumor suppressor that inhibits the development of colon cancer through its cis-binding to miR663a/pre-miR663a. The cross talk between miR663AHG and miR663a expression may play dominant roles in maintaining the functions of miR663AHG in colon cancer development.

Transient response of bridge piers under eccentric impact of near-fault earthquake.

To study the influence of near-fault earthquake on pier failure, establish a double-span continuous girder bridge. The seismic response of the bridge is calculated using the transient wave function expansion method and the indirect mode function superposition method. Solve the dynamic and displacement responses, and the effect of the vertical separation of the bridge on the eccentric compression of the pier is analyzed. The results show that under the near-fault vertical seismic action, the separation can significantly change the horizontal deformation at the top of the pier, and neglecting the separation may underestimate the eccentric compression of the pier. Calculations for different pier heights and girder spans show that separation has a greater effect on the longitudinal deformation of the pier top. Therefore, the reasonable design of longitudinal limit device on pier top in the near-fault area is helpful to reduce the damage of eccentric bridge impact.

Heart failure and cancer: From active exposure to passive adaption.

The human body seems like a "balance integrator." On the one hand, the body constantly actively receives various outside stimuli and signals to induce changes. On the other hand, several internal regulations would be initiated to adapt to these changes. In most cases, the body could keep the balance in vitro and in vivo to reach a healthy body. However, in some cases, the body can only get to a pathological balance. Actively exposed to unhealthy lifestyles and passively adapting to individual primary diseases lead to a similarly inner environment for both heart failure and cancer. To cope with these stimuli, the body must activate the system regulation mechanism and face the mutual interference. This review summarized the association between heart failure and cancer from active exposure to passive adaption. Moreover, we hope to inspire researchers to contemplate these two diseases from the angle of overall body consideration.

miR-2682-3p antagonizes its host lncRNA-MIR137HG by interacting with the same target FUS to regulate the progression of gastric cancer.

BACKGROUND: The mechanism of long non-coding RNA MIR137HG in human gastric cancer (GC) is currently unknown. In the present study, we aimed to explore the function and mechanism of MIR137HG in gastric cancer. METHODS: The expression of lncRNA-MIR137HG in 69 gastric cancer samples and their paired surgical margin (SM) tissue samples were tested by QRT-PCR. UCSC was used to find the gene location relationship among MIR137HG and its embedded miRNAs. TargetScan was used to predict the targets of miR-2682-3p. Starbase was used to predict the candidate proteins that interacted with MIR137HG. Western blot, co-focus, and RIP assay were used to verify the direct interaction between MIR137HG and FUS (fused in sarcoma/translocated in liposarcoma, FUS/TLS), while dual-luciferase reporter assay was used to confirm the interaction between miR-2682-3p and FUS. Cell migration assays, colony formation, and xenografts assay were used to investigate the function of MIR137HG and miR-2682-3p to tumor growth and metastasis. Western blot assay was used to explore the downstream candidate protein of FUS. RESULTS: Data showed that MIR137HG expressed significantly higher in GC than in SM. MIR137HG promoted colony formation and migration in vitro and promoted tumor formation and metastasis in vivo. MIR137HG is distributed in both the nucleus and cytoplasm. It was co-located with FUS and could directly interact with FUS, which might interact with other proteins, such as MET(MET-proto-oncogene, receptor tyrosine kinase), RHOC(ras homolog family member), and CTNNB1(catenin beta1). These proteins may involve different signaling pathways to regulate gastric cancer progression. By contrast, the embedded miR-2682-3p could antagonize the series functions of its host lncRNA-MIR137HG by targeting FUS. CONCLUSIONS: lncRNA-MIR137HG promoted growth and metastasis in gastric cancer by interacting with FUS, while miR-2682-3p could inhibit the function of MIR137HG via the same target FUS.

Author Correction: PBX3 is targeted by multiple miRNAs and is essential for liver tumour-initiating cells.

This Article contains an error in Figure 3. In panel g, images representing miR-222-TuD and miR-424-TuD were both taken from the miR-222-TuD image. The correct version of Figure 3 is shown in the accompanying Author Correction.

miR663a­TTC22V1 axis inhibits colon cancer metastasis.

An increasing number of studies have demonstrated that microRNAs (miRs) may act as oncogenes or anti­oncogenes in various types of cancer, including colon cancer (CC). However, the clinical and biological significance of miR663a in the prognosis of CC and its underlying molecular mechanisms remain unknown. Using the reverse transcription­quantitative polymerase chain reaction on CC and surgical margin tissue samples from 172 patients with CC, it was identified that miR663a was significantly downregulated in CC (P<0.001), particularly in metastatic CC (P=0.044). miR663a overexpression inhibited the proliferation and migration/invasion of CC cells in vitro, and also tumor growth and metastasis of CC cells in vivo. Additionally, miR663a target genes were analyzed. Inverse changes in tetratricopeptide repeat domain 22 variant 1 (TTC22V1) in response to alterations in miR663a expression were observed. miR663a decreased the reporter activity of the wild­type TTC22V1­3' untranslated region (UTR), but did not decrease that of a 3'UTR mutant. miR663a completely abolished cell migration/invasion induced by TTC22V1 containing the wild­type 3'UTR sequence, but not that induced by TTC22V1 containing the 3'UTR mutant. An inverse correlation between miR663a and TTC22 mRNA levels was observed in CC tissues. These results suggest that TTC22V1 mRNA is a crucial miR663a target that directly promotes cell migration/invasion. TTC22, which, to the best of our knowledge, has rarely been investigated, is located in the nuclei of epithelial cells in colon stem cell niches at crypt bases, and is significantly downregulated in CC, particularly in non­metastatic CC. High TTC22V1 expression is a significant poor survival factor for patients with CC. Collectively, the results of the present study suggested that TTC22V1 may be a metastasis­associated gene and that the miR663a­TTC22V1 axis inhibited CC metastasis.

MALAT1-miR663a negative feedback loop in colon cancer cell functions through direct miRNA-lncRNA binding.

The lncRNA MALAT1 has multiple biological functions, including influencing RNA processing, miRNA sponging, and cancer development. It is acknowledged that miR663a and its targets are inflammation-related genes frequently deregulated in many cancers. The associations between MALAT1 and miR663a and their target genes remain unknown. In this study, it was found that in colon cancer (CC) cells, MALAT1 and miR663a were reciprocally repressed in cDNA array screening and qRT-PCR analysis. However, MALAT1 was significantly upregulated in CC tissues, and miR663a was significantly downregulated relative to the corresponding surgical margin (SM) tissues. An inverse relationship between MALAT1 and miR663a expression was detected among CC tissue samples (n = 172, r = -0.333, p < 0.0001). The RNA-pulldown results showed MALAT1 lncRNA-miR663a binding. The results of luciferase-reporter analysis further revealed that the MALAT1 7038-7059 nt fragment was the miR663a seed sequence. Both miR663a knockdown and MALAT1 activation alone significantly upregulated the expression levels of miR663a targets, including TGFB1, PIK3CD, P53, P21, and JUND, in the CC cell lines HCT116 and SW480. A positive relationship was also observed between the expression levels of MALAT1 and these miR663a targets in the above 172 CC samples and 160 CC samples in publicly available databases. In addition, reciprocal abolishment of the effects of miR663a overexpression and MALAT1 activation on the proliferation, migration, and invasion of cancer cells was also observed, while miR663a upregulation and MALAT1 activation alone inhibited and promoted the behaviors of these CC cell lines, respectively. All these suggested that, as a competing endogenous lncRNA, MALAT1 maybe a dominant protector for the degradation of miR663a targets. miR663a and MALAT1 may consist of a negative feedback loop to determine their roles in CC development.

Serum Exosomal MiR-92b-5p as a Potential Biomarker for Acute Heart Failure Caused by Dilated Cardiomyopathy.

BACKGROUND/AIMS: MicroRNAs (miRNAs) can be used as biomarkers for cardiovascular diseases, especially for heart failure. However, there are few reports on serum exosomal miRNA biomarkers in patients with acute heart failure (AHF) due to Dilated cardiomyopathy (DCM). METHODS: We analyzed 3 different serum exosomal miRNAs (exo-miR-92b-5p, exo-miR-192-5p, and exo-miR-320a) in 43 patients with DCM-AHF and 34 healthy volunteers as a control group (CG) by using exosome separation followed by a quantitative reverse-transcript PCR assay. Exosomes were identified by electron microscopy, NaNOZS-90, and western blot analyses (CD63 and Hsp70). RESULTS: Serum exo-miR-92b-5p expression was increased in DCM-AHF patients compared to the CG (Mann-Whitney U-test: P < 0.001). Exo-miR-92b-5p was positively related to age and some ultrasound data (Spearman's correlation: exo-miR-92b-5p vs. age, r = 0.297, P = 0.014; exo-miR-92b-5p vs. left atrial diameter, r = 0.431, P < 0.001; exo-miR-92b-5p vs. left ventricular diastolic diameter, r = 0.419, P < 0.001; exo-miR-92b-5p vs. left ventricular systolic diameter, r = 0.446, P < 0.001). Exo-miR-92b-5p was also negatively related to other ultrasound data (Spearman's correlation: exo-miR-92b-5p vs. left ventricular fraction shortening, r = -0.497, P < 0.001; exo-miR-92b-5p vs. left ventricular ejection fraction, r = -0.482, P < 0.001). The discrimination of DCM-AHF patients from the CG by exo-miR-92b-5p was demonstrated by a receiver operating characteristic curve (exo-miR-92b-5p: cutoff value = 0.0023, area under the curve = 0.808, P < 0.001, sensitivity = 62.8%, specificity = 85.3%). CONCLUSION: Serum exo-miR-92b-5p is a potential biomarker for the diagnosis of DCM-AHF.

Circulating exosomal miR-92b-5p is a promising diagnostic biomarker of heart failure with reduced ejection fraction patients hospitalized for acute heart failure.

BACKGROUND: Circulating microRNA (miRNA) biomarkers have been extensively reported in cardiovascular diseases (CVDs). However, serum exosomal miRNA (exo-miRNA) as biomarker in patients with heart failure (HF) with reduced ejection fraction (HFrEF) remain largely unexplored. We sought to investigate the potential of three types of serum exo-miRNAs as biomarkers for diagnosis in HFrEF patients who were admitted in hospital because of acute heart failure (AHF). METHODS: A total of 28 HFrEF patients hospitalized for AHF, including de novo AHF and acute decompensated HF, and 30 volunteers as control group (CG) from 2015 to 2017 were enrolled in this study. Serum exo-miRNAs were extracted and analyzed by NaNOZS-90, electron microscopy, and western blotting. Three types of serum exo-miRNAs (exo-miR-92b-5p, -192-5p, and -320a) were assessed by quantitative real time polymerase chain reaction (qRT-PCR). RESULTS: The particle size was confirmed as 40-150 nm using NaNOZS-90 and transmission electron microscopy. Exosomal biomarkers CD63 and Hsp70 were readily detected. The expression level of serum exo-miRNAs were transformed into log2-delta CT in the qPCR assay. The data showed that exo-miR-92b-5p was elevated in HFrEF patients compared with controls. Moreover, exo-miR-92b-5p was inversely correlated with the left ventricular fraction shortening (LVFS) and left ventricular ejection fraction (LVEF), whereas it was positively correlated with left atrial diameter (LAD), left ventricular diastolic diameters (LVDD) and systolic diameters (LVSD). A receiver operating characteristic (ROC) curve was generated for discrimination between HFrEF patients and controls based on exo-miR-92b-5p (P<0.001, sensitivity =71.4%, specificity =83.3%). CONCLUSIONS: Exo-miR-92b-5p levels in the serum may serve as a marker for HFrEF diagnosis.

LncRNAs: From Basic Research to Medical Application.

This review aimed to summarize the current research contents about long noncoding RNAs (lncRNAs) and some related lncRNAs as molecular biomarkers or therapy strategies in human cancer and cardiovascular diseases. Following the development of various kinds of sequencing technologies, lncRNAs have become one of the most unknown areas that need to be explored. First, the definition and classification of lncRNAs were constantly amended and supplemented because of their complexity and diversity. Second, several methods and strategies have been developed to study the characteristic of lncRNAs, including new species identifications, subcellular localization, gain or loss of function, molecular interaction, and bioinformatics analysis. Third, based on the present results from basic researches, the working mechanisms of lncRNAs were proved to be different forms of interactions involving DNAs, RNAs, and proteins. Fourth, lncRNA can play different important roles during the embryogenesis and organ differentiations. Finally, because of the tissue-specific expression of lncRNAs, they could be used as biomarkers or therapy targets and effectively applied in different kinds of diseases, such as human cancer and cardiovascular diseases.

miR-137 plays tumor suppressor roles in gastric cancer cell lines by targeting KLF12 and MYO1C.

Aberrant expression of miR-137 has been reported in many kinds of cancers, but its mechanisms seem to be diversely. In the present study, we compared the expression level of miR-137 in 18 paired gastric cancer (GC) samples and surgical margin (SM) samples by RNA extraction and quantitative real-time PCR (QRT-PCR). Then, we investigated the effects of miR-137 on cell proliferation, cell cycle, and cell migration separately by cell growth counting assay, cell cycle analysis, and transwell assay. Candidate targets of miR-137 were selected by biological information analysis from the intersection of miRDB, Pictar, and TarScan. Finally, mRNA and protein expression level of Kruppel-like factor 12 (KLF12) and Myosin 1C (MYO1C) were tested by QRT-PCR and western blotting assay, followed by the Luciferase reporter assay to investigate the direct interaction between them and miR-137. The results showed that miR-137 was down-regulated in GC samples than in SM samples. The expression level of miR-137 was significantly higher in patients without the vascular embolus than those with vascular embolus. And the overall survival time of patients with high miR-137 expression was longer than those with low miR-137 expression. Over expression of miR-137 could inhibit the cell migration, proliferation, and promote cell cycle arrest in G0/G1 stage in BGC-823 and SGC-7901 cell lines. KLF12 and MYO1C might be the candidate target genes of miR-137 with direct interactions between them and miR-137. In conclusion, miR-137 plays tumor suppressor roles in gastric cancer cell lines by targeting KLF12 and MYO1C.

PBX3 is targeted by multiple miRNAs and is essential for liver tumour-initiating cells.

Tumour-initiating cells (TICs) are advocated to constitute the sustaining force to maintain and renew fully established malignancy; however, the molecular mechanisms responsible for these properties are elusive. We previously demonstrated that voltage-gated calcium channel α2δ1 subunit marks hepatocellular carcinoma (HCC) TICs. Here we confirm directly that α2δ1 is a HCC TIC surface marker, and identify let-7c, miR-200b, miR-222 and miR-424 as suppressors of α2δ1(+) HCC TICs. Interestingly, all the four miRNAs synergistically target PBX3, which is sufficient and necessary for the acquisition and maintenance of TIC properties. Moreover, PBX3 drives an essential transcriptional programme, activating the expression of genes critical for HCC TIC stemness including CACNA2D1, EpCAM, SOX2 and NOTCH3. In addition, the expression of CACNA2D1 and PBX3 mRNA is predictive of poor prognosis for HCC patients. Collectively, our study identifies an essential signalling pathway that controls the switch of HCC TIC phenotypes.

miR-129-1-3p promote BGC-823 cell proliferation by targeting PDCD2.

MicroRNAs (miRNAs) are the class of small noncoding RNAs, and play an important role in the regulation of gene expression at the posttranscriptional level. In this study, we explored the effect of miR-129-1-3p on the growth and cell cycle of human gastric cancer cell line BGC-823. The miR-129-1-3p mimics or inhibitors were transfected into the BGC-823 cell line, and the cell cycle and cell growth was measured by flow cytometry and real-time cell analyzer, respectively. The possible targets of miR-129-1-3p were analyzed by quantitative real time-PCR (QRT-PCR), Western blotting and Luciferase reporter assay. The results showed that miR-129-1-3p could promote the growth and cell cycle of BGC-823 cells. Although protein expression of programmed cell death 2 (PDCD2) was not changed with miR-129-1-3p, QRT-PCR showed that expression of PDCD2 mRNA was negatively related to the miR-129-1-3p. Luciferase reporter assay revealed that PDCD2 is one of the targets of miR-129-1-3p. Our results indicated that miR-129-1-3p might promote proliferation of BGC-823 cells by targeting PDCD2.

Characterization of human gastric carcinoma-related methylation of 9 miR CpG islands and repression of their expressions in vitro and in vivo.

BACKGROUND: Many miR genes are located within or around CpG islands. It is unclear whether methylation of these CpG islands represses miR transcription regularly. The aims of this study are to characterize gastric carcinoma (GC)-related methylation of miR CpG islands and its relationship with miRNA expression. METHODS: Methylation status of 9 representative miR CpG islands in a panel of cell lines and human gastric samples (including 13 normal biopsies, 38 gastritis biopsies, 112 pairs of GCs and their surgical margin samples) was analyzed by bisulfite-DHPLC and sequencing. Mature miRNA levels were determined with quantitative RT-PCR. Relationships between miR methylation, transcription, GC development, and clinicopathological characteristics were statistically analyzed. RESULTS: Methylation frequency of 5 miR CpG islands (miR-9-1, miR-9-3, miR-137, miR-34b, and miR-210) gradually increased while the proportion of methylated miR-200b gradually decreased during gastric carcinogenesis (Ps < 0.01). More miR-9-1 methylation was detected in 62%-64% of the GC samples and 4% of the normal or gastritis samples (18/28 versus 2/48; Odds ratio, 41.4; P < 0.01). miR-210 methylation showed high correlation with H. pylori infection. miR-375, miR-203, and miR-193b methylation might be host adaptation to the development of GCs. Methylation of these miR CpG islands was consistently shown to significantly decrease the corresponding miRNA levels presented in human cell lines. The inverse relationship was also observed for miR-9-1, miR-9-3, miR-137, and miR-200b in gastric samples. Among 112 GC patients, miR-9-1 methylation was an independent favourable predictor of overall survival of GC patients in both univariate and multivariate analysis (P < 0.02). CONCLUSIONS: In conclusion, alteration of methylation status of 6 of 9 tested miR CpG islands was characterized in gastric carcinogenesis. miR-210 methylation correlated with H. pylori infection. miR-9-1 methylation may be a GC-specific event. Methylation of miR CpG islands may significantly down-regulate their transcription regularly.

Epigenetic alterations as cancer diagnostic, prognostic, and predictive biomarkers.

Alterations of DNA methylation and transcription of microRNAs (miRNAs) are very stable phenomena in tissues and body fluids and suitable for sensitive detection. These advantages enable us to translate some important discoveries on epigenetic oncology into biomarkers for control of cancer. A few promising epigenetic biomarkers are emerging. Clinical trials using methylated CpG islands of p16, Septin9, and MGMT as biomarkers are carried out for predication of cancer development, diagnosis, and chemosensitivity. Circulating miRNAs are promising biomarkers, too. Breakthroughs in the past decade imply that epigenetic biomarkers may be useful in reducing the burden of cancer.

Upregulation of myosin Va by Snail is involved in cancer cell migration and metastasis.

Cell migration, which involves acto-myosin dynamics, cell adhesion, membrane trafficking and signal transduction, is a prerequisite for cancer cell metastasis. Here, we report that an actin-dependent molecular motor, unconventional myosin Va, is involved in this process and implicated in cancer metastasis. The mRNA expression of myosin Va is increased in a number of highly metastatic cancer cell lines and metastatic colorectal cancer tissues. Suppressing the expression of myosin Va by lentivirus-based RNA interference in highly metastatic cancer cells impeded their migration and metastasis capabilities both in vitro and in vivo. In addition, the levels of myosin Va in cancer cell lines are positively correlated with the expression of Snail, a transcriptional repressor that triggers epithelial-mesenchymal transition. Repression or overexpression of Snail in cancer cells caused reduced or elevated levels of myosin Va, respectively. Furthermore, Snail can bind to an E-box of the myosin Va promoter and induce its activity, which indicates that Snail might act as a transcriptional activator. These data demonstrate an essential role of myosin Va in cancer cell migration and metastasis, and suggest a novel target for Snail in its regulation of cancer progression.