MicroRNAs (miRNAs or miRs) play important roles in numerous cellular processes, including development, proliferation, tumorigenesis and apoptosis. It has been reported that miRNA expression is induced by ionizing radiation (IR) in cancer cells. However, the underlying molecular mechanisms are not yet fully understood. In this study, endogenous miR‑320a and its primary precursor (pri‑miR‑320a) were assayed by reverse transcription‑quantitative PCR (RT‑qPCR). Luciferase activities were measured using a dual‑luciferase reporter assay system. Western blot analysis was used to determine the protein expressions of upstream and downstream genes of miR‑320a. Cell apoptosis was evaluated by Annexin V apoptosis assay and cell proliferation was measured using the trypan blue exclusion method. The results revealed that miR‑320a expression increased linearly with the IR dose and treatment duration. Three transcription factors, activating transcription factor 2 (ATF2), ETS transcription factor (ELK1) and YY1 transcription factor (YY1), were activated by p38 mitogen‑activated protein kinase (MAPK) and mitogen‑activated protein kinase 8 (JNK) and by upregulated miR‑320a expression under IR conditions. In addition, it was identified that X‑linked inhibitor of apoptosis (XIAP) was an miR‑320a target gene during the IR response. By targeting XIAP, miR‑320a induced apoptosis and inhibited the proliferation of the cancer cells. On the whole, the results of this study demonstrated that miRNA‑320a, regulated by the p38 MAPK/JNK pathway, enhanced the radiosensitivity of cancer cells by inhibiting XIAP and this may thus prove to be a potential therapeutic approach with which to overcome radioresistance in cancer treatment.

Long noncoding RNAs (lncRNAs) are nonprotein coding transcripts longer than 200 nucleotides. Recently in mammals, thousands of long noncoding RNAs have been identified and studied as key molecular players in different biological processes with protein complexes. As a long noncoding RNA, maternally expressed gene 3 (MEG3) plays an important role in many cellular processes. However, the mechanism underlying MEG3 regulatory effects remains enigmatic. By using the specific interaction between MS2 coat protein and MS2 RNA hairpin, we developed a method (MS2-tagged RNA affinity purification and mass spectrometry (MTRAP-MS)) to identify proteins that interact with MEG3. Mass spectrometry and gene ontology (GO) analysis showed that MEG3 binding proteins possess nucleotide binding properties and take part in transport, translation, and other biological processes. In addition, interleukin enhancer binding factor 3 (ILF3) and poly(A) binding protein, cytoplasmic 3 (PABPC3) were validated for their interaction with MEG3. These findings indicate that the newly developed method can effectively enrich lncRNA binding proteins and provides a strong basis for studying MEG3 functions.

Maternally Expressed Gene 3 (MEG3) encodes a lncRNA which is suggested to function as a tumor suppressor. Previous studies suggested that MEG3 functioned through activation of p53, however, the functional properties of MEG3 remain obscure and their relevance to human diseases is under continuous investigation. Here, we try to illuminate the relationship of MEG3 and p53, and the consequence in hepatoma cells. We find that transfection of expression construct of MEG3 enhances stability and transcriptional activity of p53. Deletion analysis of MEG3 confirms that full length and intact structure of MEG3 are critical for it to activate p53-mediated transactivation. Interestingly, our results demonstrate for the first time that MEG3 can interact with p53 DNA binding domain and various p53 target genes are deregulated after overexpression of MEG3 in hepatoma cells. Furthermore, results of qRT-PCR have shown that MEG3 RNA is lost or reduced in the majority of HCC samples compared with adjacent non-tumorous samples. Ectopic expression of MEG3 in hepatoma cells significantly inhibits proliferation and induces apoptosis. In conclusion, our data demonstrates that MEG3 functions as a tumor suppressor in hepatoma cells through interacting with p53 protein to activate p53-mediated transcriptional activity and influence the expression of partial p53 target genes.

BACKGROUND:Thousands of long noncoding RNAs (lncRNAs) have been reported in mammalian genomes. These RNAs represent an important subset of pervasive genes involved in a broad range of biological functions. Aberrant expression of lncRNAs is associated with many types of cancers. Here, in order to explore the potential lncRNAs involved in hepatocellular carcinoma (HCC) oncogenesis, we performed lncRNA gene expression profile analysis in 3 pairs of human HCC and adjacent non-tumor (NT) tissues by microarray.METHODOLOGY:Differentially expressed lncRNAs and mRNAs were detected by human lncRNA microarray containing 33,045 lncRNAs and 30,215 coding transcripts. Bioinformatic analyses (gene ontology, pathway and network analysis) were applied for further study of these differentially expressed mRNAs. By qRT-PCR analysis in nineteen pairs of HCC and adjacent normal tissues, we found that eight lncRNAs were aberrantly expressed in HCC compared with adjacent NT tissues, which is consistent with microarray data.CONCLUSIONS:We identified 214 lncRNAs and 338 mRNAs abnormally expressed in all three HCC tissues (Fold Change ≥2.0, P<0.05 and FDR <0.05) with the genome-wide lncRNAs and mRNAs expression profile analysis. The lncRNA-mRNA co-expression network was constructed, which may be used for predicting target genes of lncRNAs. Furthermore, we demonstrated for the first time that BC017743, ENST00000395084, NR_026591, NR_015378 and NR_024284 were up-regulated, whereas NR_027151, AK056988 and uc003yqb.1 were down-regulated in nineteen pairs of HCC samples compared with adjacent NT samples. Expression of seven lncRNAs was significantly correlated to their nearby coding genes. In conclusion, our results indicated that the lncRNA expression profile in HCC was significantly changed, and we identified a series of new hepatocarcinoma associated lncRNAs. These results provide important insights about the lncRNAs in HCC pathogenesis.

Objective To develop and optimize a new method to extract miRNAs from plasma.Methods miRNAs were extracted from plasma by mixing it with the extraction solution that contained surfactant and by heating .Then the ribonuclease inhibitor was added into the extraction to prevent RNAs from degradation .The expression level of each miRNA was detected by real-time quantitative PCR in oder to evaluate the feasibility of this method .Results A method which extracted miRNAs from plamsa in just one step was established .The specificity , reproducibility and stability of this method have been demonstrated by real-time quantitative PCR .Conclusion The one-step method is simple , inexpensive , and plasma-saving.It seems like a new method for clinical examination of miRNAs from plasma .

Accumulating evidence suggests that microRNAs (miRNAs) can function as oncogenes or as tumor suppressor genes depending on the tissue type or target. Therefore, clarification of the specific roles of miRNAs is vital for the diagnosis and treatment of cancer. In the present study, miR-451 was found to be downregulated in hepatocellular carcinoma (HCC) tissues when compared to that in adjacent tissues. Functional analysis showed that, in vitro, miR-451 inhibited the migration of hepatoma cell lines HepG2 and SK-Hep-1. Further investigation of the molecular mechanisms identified activating transcription factor 2 (ATF2) as a target of miR-451. miR-451 inhibited ATF2 expression by binding to the 3'UTR. An in vivo assay revealed a significant negative correlation between miR-451 and ATF2 in liver cancer tissues. According to previous findings reported in the literature, the opposing functions of ATF2 are related to its subcellular localization. In the nucleus, ATF2 displays oncogenic activities in melanoma. In the present study, ATF2 exhibited a higher expression level in the nucleus in tumoral tissues of HCC as detected by immunohistochemistry. In conclusion, in this study, we identified a potential target of miR-451, ATF2, and revealed a novel role of miR-451 in the inhibition of the migratory ability of hepatoma cell lines.

目的 建立并优化检测真核细胞mRNA氧化损伤的方法——反转录阻断-双引物扩增法,检测p53 mRNA氧化损伤.方法 建立非细胞体系和细胞体系mRNA氧化损伤模型,提取RNA并反转录成cDNA,根据所检测mRNA的5’端和3’端各设计一对引物,以cDNA为模板进行实时定量PCR.结果 非细胞体系和细胞体系氧化处理组GAPDH和p53 mRNA的氧化损伤程度均明显高于对照组,且随着过氧化氢浓度增加RNA氧化损伤增强；同时一定限度内降低反转录酶用量能提高检测灵敏度.结论 本研究建立并优化的RNA氧化损伤检测方法能检测特定mRNA对氧化的敏感性及损伤程度,为分析相关疾病造成的RNA氧化损伤以及探索其发病机制和治疗方法提供了技术支持.

An increasing data indicates that altered microRNAs(miRNAs)participate in the radiation-induced DNA damage response.However,a correlation of mRNA and miRNA profiles across the entire genome and in response to irradiation has not been thoroughly assessed.We analyzed miRNA microarray data collected from HeLa cells after ionizing radiation(IR),quantified the expression profiles of mRNAs and performed comparative analysis of the data sets using target prediction algorithms,Gene Ontology(GO)analysis,pathway analysis,and gene network construction.The results showed that the altered miRNAs were involved in regulation of various cellular functions.miRNA-gene network analyses revealed that miR-186,miR-106b,miR-15a/b,CCND1and CDK6 played vital role in the cellular radiation response.Using qRT-PCR,we confirmed that twenty-two miRNAs showed differential expression in HeLa cells treated with IR and some of these miRNAs affected cell cycle progression.This study demonstrated that miRNAs influence gene expression in the entire genome during the cellular radiation response and suggested vital pathways for further research.

Objective To establish a new method of poly(U)-tailed RT-PCR to detect microRNA(miRNA).Methods The total RNA of cells was extracted and polyuridylylated by poly(U) polymerase,then the cDNA was obtained by reverse transcriptase with the universal primer,and the miRNA was detected by real-time quantitative PCR.Results This method of poly(U)-tailed RT-PCR could better detect miRNA with specificity and efficiency.Conclusion This method of poly(U)-tailed RT-PCR for detection of miRNAs is simple,economical and efficient.

MicroRNAs may function to promote or suppress tumor development, depending on the cellular context. The important role of microRNAs in regulating molecular pathways underlying tumorigenesis has been emphasized in hepatocellular carcinoma (HCC). MicroRNAs regulate gene expression via post-transcriptional mechanisms by inhibiting translation or by degrading mRNA. In this study, we show that microRNA-1 (miR-1) and microRNA-499 (miR-499) are capable of repressing the expression of the ets1 protooncogene, which plays a fundamental role in the extracellular matrix (ECM) degradation, a process required for tumor cell invasion and migration. We used luciferase reporter assays to demonstrate that miR-1 and miR-499 target the 3' untranslated region (UTR) of ets1. Overexpression of miR-1 and miR-499 in HepG2 cells led to downregulation of ets1 mRNA and protein as assessed by quantitative reverse transcription PCR and western blot analysis. Furthermore, overexpression of miR-1 and miR-499 inhibited the invasion and migration of HepG2 cells in matrigel invasion and transwell migration assays, respectively. These results suggest that miR-1 and miR-499 may play an important role in the pathogenesis of HCC by regulating ets1.

microRNAs (miRNAs) are a versatile class of non-coding RNAs involved in regulation of various biological processes. miRNA-122 (miR-122) is specifically and abundantly expressed in human liver. In this study, we employed 3′-end biotinylated synthetic miR-122 to identify its targets based on affinity purification. Quantitative RT-PCR analysis of the affinity purified RNAs demonstrated a specific enrichment of several known miR-122 targets such as CAT-1 (also called SLC7A1), ADAM17 and BCL-w. Using microarray analysis of affinity purified RNAs, we also discovered many candidate target genes of miR-122. Among these candidates, we confirmed that protein kinase, interferon-inducible double-stranded RNA-dependent activator (PRKRA), a Dicer-interacting protein, is a direct target gene of miR-122. miRNA quantitative-RT–PCR results indicated that miR-122 and small interfering RNA against PRKRA may facilitate the accumulation of newly synthesized miRNAs but did not detectably affect endogenous miRNAs levels. Our findings will lead to further understanding of multiple functions of this hepato-specific miRNA. We conclude that miR-122 could repress PRKRA expression and facilitate accumulation of newly synthesized miRNAs.

Cisplatin is a classic chemotherapy agent used for treating human non-small cell lung cancer (NSCLC). However, cisplatin resistance is a challenge against successful clinical use. Glutathione S-transferase P1 (GSTP1) has been reported to contribute to cisplatin resistance in many studies. MicroRNAs (miRNAs) are short non-coding RNAs that are 21–25 nucleotides in length. They play a role in post-transcriptional gene regulation by inducing repression and/or mRNA degradation. Recent studies have shown that miRNAs are responsible for cisplatin resistance. This study aims to determine whether deregulated miRNAs can sensitize human lung adenocarcinoma cells to cisplatin by targeting GSTP1. Real-time RT-PCR revealed that GSTP1 mRNA expression was 2.7±0.38 folds (p=0.039) upregulated in A549/CDDP cells, compared with the parental A549 cells, while miR-513a-3p expression was 0.34±0.03 folds (p=0.023) downregulated. Luciferase activity assay proved that GSTP1 was a target gene of miR-513a-3p, which was confirmed by Western blot analysis. Furthermore, CCK-8 assay showed that overexpression of miR-513a-3p could enhance cisplatin-induced apoptosis in human lung adenocarcinoma cell lines, A549/CDDP and SPC-A-1. In conclusion, our data demonstrated that miR-513a-3p can sensitize human lung adenocarcinoma cells to cisplatin by targeting GSTP1.

Ionizing radiation (IR) causes severe cellular damage both directly and indirectly and disrupts RNA integrity. RNA strand breaks are the most frequent type of damage caused by IR. RNA damage is involved in the development of degenerative diseases, including Alzheimer’s disease and Parkinson’s disease. However, the mechanism of mRNA damage and any resulting pathophysiological outcomes are poorly understood. This is partly because there is a lack of sensitive tools to monitor damage randomly occurring in RNA, especially RNA strand break damage in a given RNA. In this work, a method using the reverse transcription polymerase chain reaction (RT-PCR) after poly(A) addition to 3′-end of RNA to determine RNA strand break damage in a specific RNA by poly(A) polymerase has been developed. The levels of damage in specific mRNAs, including ABL1, TP53, GADD45A and ATR from IR-treated HeLa cells were examined. Strand breaks were detected in all mRNAs examined. The study provides a novel and sensitive method based on 3′-end poly(A)-tailing RT-PCR to monitor RNA strand break damage.

ObjectiveTo investigate miR-210 expression in HepG2 cells under three hypoxia models.MethodsHypoxia induced HepG2 cells under three different hypoxia models,including physical hypoxia and two chemical hypoxia-mimicking agents,such as CoCl2 and Na2SO3.qRT-PCR was used to assay the expression of miR-210.Results miR-210 could be up-regulated in these three hypoxia models.The expression of miR-210 changed with the concentration of CoCl2 and Na2SO3 in a dose-dependent manner.ConclusionHypoxia induced by physical hypoxia and chemical hypoxia-mimicking agents could upregulate the expression of miR-210 in HepG2 cells.All these three models could be applied to investigate the relationship among hypoxia,miRNA and cancer.These results further prove that the miR-210 expression is really caused by hypoxia.

Hepatocellular carcinoma(HCC) is one of the most malignant types of cancer.It has a rapid course and carries a poor prognosis.Hepatocarcinogenesis is a complex multi-step and multifactorial process.Recent studies have discovered the association between the dysregulation of insulin-like growth factor(IGF)-related signaling pathways and pathogenesis of liver cancer IGFs are multifunctional cell proliferation regulatory factors and play an important role in feta development,central nervous system development and cancer cell proliferation.The biological activity of IGFs is regulated by a complex regulatory network which consists of different types of receptors,IGF-binding proteins and IGF binding-related proteins.This review focuses on the changes in the IGF axis and IGF-related signaling pathways in liver tumorigenesis and their application in targeted therapy for liver cancer.

Objective To investigate the expression level and clinical significance of mRNA of alpha-fetoprotein(AFP),pyruvate kinase type M2(PKM2) and albumin(ALB) in the plasma of cirrhosis patients with hepatitis B virus(HBV).Methods Real-time quantitative PCR was used to detect the expression level of mRNA of AFP,PKM2 and ALB in the plasma of cirrhosis patients with HBV,and the correlation between the expression level and the clinical biochemical indexes was analyzed.Results The expression level of mRNA of AFP,PKM2 and ALB plasma was significantly different between the patients with HBV and the healthy people.The results were of statistical significance(P0.01).No significant correlation was found in the expression of mRNA of AFP and PKM2 with clinical biochemical indexes,but there were statistically significant relative risks between ALB mRNA expression and γ-glutamyl transpeptidase(GGT) or serum albumin.A higher expression level of mRNA was detected in the plasma of patients with HBV compared with alpha-fetoprotein expression.Conclusion The expression of mRNA of AFP,PKM2 and ALB in peripheral blood plasma can contribute to clinical diagnosis and treatment of liver disease.

MicroRNAs(miRNAs)are small single-stranded noncoding RNAs consisting of 21-23 nucleotides that play important gene-regulatory roles in eukaryotes by pairing to the mRNAs of protein-coding genes to direct their posttranscriptional repression.A growing body of evidence indicates that alterations in miRNA expression may occur following exposure to several oxidative stress including ionizing radiation.So miRNAs may serve as potential new targets for co-therapies aiming to improve the effects of radiation disease therapy in cancer patients.The progress in research on ionizing radiation-induced miRNAs is reviewed in this paper.

目的探讨microRNA-193b（miR-193b）在HepG2细胞中对K-Ras蛋白表达的调控作用。方法 Tar-getScan软件预测获得miR-193b调控候选靶基因K-ras;构建包含预测miR-193b作用靶位点的K-rasmRNA3′UTR野生型和突变型荧光素酶报告基因载体pGL-KRAS和pGL-Mu-KRAS,检测转染HepG2细胞的荧光素酶活性;合成miR-193b的dsRNA,转染HepG2细胞,实时定量PCR和Western印迹检测对K-rasmRNA和蛋白表达的影响。结果与结论 miR-193b可以与K-rasmRNA3′UTR结合。在HepG2中,miR-193b在mRNA和蛋白水平下调K-ras基因表达。

The pleckstrin homology (PH) domain-containing protein casein kinase 2 interacting protein-1 (CKIP-1) plays an important role in regulation of bone formation and muscle differentiation. How CKIP-1 localization is determined remains largely unclear. We observed that isolated CKIP-1-PH domain was predominantly localized in the nucleus and the C-terminus of CKIP-1 counteracted its nuclear localization. The net charge of basic residues and a serine-rich motif within the PH domain plays a pivotal role in the localization switch of both full-length CKIP-1 and the isolated PH domain. We propose that the N-terminal PH domain and C-terminal auto-inhibitory region of CKIP-1 coordinate to determine its subcellular localization and the nucleus-plasma membrane shuttling.

BACKGROUND AND AIMS:Recently, some miRNAs have been reported to be connected closely with the development of human hepatocellular carcinoma. However, the functions of these miRNAs in HCC remain largely undefined.METHODS:The expression profiles of miR-193b were compared between HCC tissues and adjacent normal liver tissues using qRT-PCR method. This method was also be used to screen the potential target genes of miR-193b. A luciferase reporter assay was conducted to confirm target association. Finally, the functional effect of miR-193b in hepatoma cells was examined further.RESULTS:miR-193b was significantly down-regulated in most of the HCC tissues compared to the matching non-tumoural liver tissues. Furthermore, ectopic expression of miR-193b dramatically suppressed the ability of hepatoma cells to form colonies in vitro and to develop tumours in nude mice. CCND1 and ETS1 were revealed to be regulated by miR-193b directly. By regulating the expressions of these oncogenes, miR-193b induced cell cycle arrest and inhibited the invasion and migration of hepatoma cells.CONCLUSIONS:miR-193b may function as a tumour suppressor in the development of HCC by acting on multiple tumourigenic pathways.