MicroRNA signature in patients with hepatocellular carcinoma associated with type 2 diabetes

BACKGROUND Nonalcoholic steatohepatitis-related cirrhosis is one of the liver complications in type 2 diabetes mellitus(T2 DM) and reported to be a risk factor for developinghepatocellular carcinoma(HCC). A reliable screening biomarker of liver cirrhosis(LC) and HCC among T2 DM patients is important to reduce the morbidity and mortality of this disease. Micro RNA(mi RNA) is considered a key player in HCC and T2 DM, and it might be a hidden culprit in diabetes-associated HCC, making it a promising reliable prognostic tool.AIM To investigate the signature of serum mi RNAs as early biomarkers for the screening of HCC among diabetic patients.METHODS Expression profiles of mi RNAs in serum samples of diabetic LC and diabetic HCC patients were assessed using Illumina sequencing; then, RT-q PCR was used to validate significantly altered mi RNAs between the two groups. Candidate mi RNAs were tested in serum samples of 200 T2 DM patients, 270 LC patients,200 HCC patients, and 225 healthy control subjects. Additionally, receiver operating characteristic(ROC) analysis, with area under the curve(AUC), was performed to assess the diagnostic performance of the screened mi RNAs for discriminating HCC from LC and nonmalignant patients(LC + T2 DM).RESULTS Expression of the sequenced mi RNAs in serum was different in HCC vs LCpositive T2 DM patients. Two mi RNAs(mi R-34 a, mi R-221) were significantly upregulated and five mi RNAs(mi R-16, mi R-23-3 p, mi R-122-5 p, mi R-198, mi R-199 a-3 p) were significantly down-regulated in HCC compared to LC patients. Analysis of ROC curve demonstrated that the combination of these seven mi RNAs can be used as a reliable biomarker for detection of HCC in diabetic patients, as it could identify HCC with high diagnostic accuracy in diabetic LC patients(AUC =0.993) and in diabetic nonmalignant patients(AUC = 0.961).CONCLUSION This study validates a panel of serum mi RNAs that can be used as a reliable noninvasive screening biomarker of HCC among T2 DM cirrhotic and noncirrhotic patients. The study recommends further research to shed light on a possible role of c-Met in T2 DM-associated HCC via the mi RNA regulatory pathway.     

Disease-specific mi R-34a as diagnostic marker of nonalcoholic steatohepatitis in a Chinese population

AIM To assess disease-specific circulating micro RNAs(mi RNAs) in non-alcoholic steatohepatitis(NASH) patients.METHODS A total of 111 biopsy-proven non-alcoholic fatty liver disease(NAFLD) or chronic hepatitis B(CHB) patients and healthy controls from mainland China were enrolled to measure their serum levels of mi R-122,-125 b,-146 b,-16,-21,-192,-27 b and-34 a. The correlations between serum mi RNAs and histological features of NAFLD were determined. The diagnostic value of mi RNA in NASH and significant fibrosis was analyzed and compared with that of cytokeratin-18(CK-18), fibrosis-4(FIB-4), and aspartate aminotransferase to platelet ratio index(APRI), respectively.RESULTS Circulating mi R-122,-16,-192 and-34 a showed differential expression levels between NAFLD and CHB patients, and mi R-34 a had an approximately 2-fold increase in NAFLD samples compared with that of CHB samples(P 0.01). Serum mi R-122,-192 and-34a levels were correlated with steatosis(R = 0.302, 0.323 and 0.470, respectively, P 0.05) and inflammatory activity(R = 0.445, 0.447 and 0.517, respectively, P 0.01); only serum mi R-16 levels were associated with fibrosis(R = 0.350, P 0.05) in patients with NAFLD. The diagnostic value of mi R-34 a for NASH(area under the receiver operating characteristic, 0.811, 95%CI: 0.670-0.953) was superior to that of alanine aminotransferase, CK-18, FIB-4 and APRI in NAFLD, but mi R-16 showed a limited performance in the diagnosis of significant fibrosis in NASH.CONCLUSION Circulating mi R-34 a may serve as a disease-specific noninvasive biomarker for the diagnosis of NASH.     

Novel serum microRNAs panel on the diagnostic and prognostic implications of hepatocellular carcinoma

AIM To determine a panel of serum micro RNAs(mi RNAs) that could be used as novel biomarkers for diagnosis of hepatocellular carcinoma(HCC).METHODS We initially screened 9 out of 754 serum mi RNAs by Taq Man Low Density Array in two pooled samples respectively from 35 HCC and 35 normal controls, and then validated individually by RT-qP CR in another 114 patients and 114 controls arranged in two phases. The changes of the selected mi RNAs after operation and their prognostic value were examined.RESULTS miR-375, miR-10 a, miR-122 and miR-423 were found to be significantly higher in HCC than in controls(P 0.0001), and the area under the receiver-operating-characteristic curve for the 4-miR NA panel was 0.995(95%CI: 0.985-1). All the four mi RNAs were significantly reduced after surgical removal of the tumors(P 0.0001), while still higher than normal controls(at least P 0.05)CONCLUSION The four serum miR NAs(miR-375, miR-10 a, miR-122 and miR-423) could potentially serve as novel biomarkers for the diagnostic and prognostic of HCC.     

hsa-mir-183 is frequently methylated and related to poor survival in human hepatocellular carcinoma

AIM To screen clinically relevant micro RNAs (mi RNAs) silenced by DNA methylation in human hepatocellular carcinoma(HCC).METHODS Knockdown of DNA methyltransferases (DNMTs) using si RNAs and mi RNA profiling in HCC cell lines were performed to identify DNA hypermethylation-mediated mi RNA downregulation. Confirmation using individual quantitative real-time PCR (qR T-PCR) assays was thenperformed followed by DNA methylation quantification at the promoter of the mi RNA genes. Quantification of DNA methylation and mi RNA expression was then performed in primary HCC tumor samples and related with clinicopathological variables.RESULTS mi RNA profiling after DNMT knockdown in HCC cell lines revealed upregulation of mi R-23, mi R-25 and mi R-183. After q RT-PCR confirmation and Cp G island methylation quantification of these miR NAs in cell lines, further analysis in primary HCC specimens showed that hsa-mi R-183 is hypermethylated in 30% of HCC (n = 40). Expression of mature miR-183 showed an inverse correlation with DNA methylation levels. In HCC cells, DNMT knockdown and 5-aza-2'-deoxycytidine treatment reduced methylation and stimulated expression of mi R-183. In HCC patients, hypermethylation at hsami R-183 promoter significantly correlates with poor survival (log-rank test P = 0.03). DNA methylation analysis in healthy liver, benign liver tumors (hepatocellular adenoma and focal nodular hyperplasia) and their corresponding adjacent tissues showed absence of hypermethylation supporting the notion that aberrant methylation at hsa-miR-183 is specific for the malignant transformation of hepatocytes.CONCLUSION Our data indicate that hypermethylation of hsa-miR-183 is a frequent event in HCC and potentially useful as a novel surrogate diagnostic and prognostic marker.     

Micro RNA aberrations: An emerging field for gallbladder cancer management

Gallbladder cancer(GBC) is infrequent but most lethal biliary tract malignancy characterized by an advanced stage diagnosis and poor survival rates attributed to absence of specific symptoms and effective treatment options. These necessitate development of early prognostic/predictive markers and novel therapeutic interventions. Micro RNAs(mi RNAs) are small, noncoding RNA molecules that play a key role in tumor biology by functioning like tumor suppressor- or oncogenes and their aberrant expression are associated with the pathogenesis of several neoplasms with overwhelming clinical implications. Since mi RNA signature is tissue specific, here, we focused on current data concerning the mi RNAs abberations in GBC pathogenesis. In GBC, mi RNAs with tumor suppressor activity(mi R-135-5p, mi R-335, mi R-34 a, mi R-26 a, mi R-146b-5p, Mir-218-5p, mi R-1, mi R-145, mir-130a) were found downregulated, while those with oncogenic property(mi R-20 a, mi R-182, mir-155) were upregulated. The expression profile of mi RNAs was significantly associated with GBC prognosis and prediction, and forced over-expression/ inhibition of these mi RNAs was shown to affect tumor growth and development. Further, differential expression of mi RNAs in the blood samples of GBC patients suggest mi RNAs as promising noninvasive biomarker. Thus, mi RNAs represent potential candidate for GBC management, though many hurdles need to be overcome before mi RNAs therapy can be clinically applied to GBC prevention and treatment.     

mi R-122 negatively correlates with liver fibrosis as detected by histology and FibroScan

AIM: To investigate whether expression of selected mi RNAs obtained from fibrotic liver biopsies correlate with fibrosis stage.METHODS: Altogether, 52 patients were enrolled in the study representing various etiologic backgrounds of fibrosis: 24 cases with chronic hepatitis infections(types B, C), 19 with autoimmune liver diseases(autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, overlapping syndrome cases), and 9 of mixed etiology(alcoholic and nonalcoholic steatosis, cryptogenic cases). Severity of fibrosis was determined by both histologic staging using the METAVIR scoring system and noninvasive transient elastography. Following RNAisolation, expression levels of mi R-21, mi R-122, mi R-214, mi R-221, mi R-222, and mi R-224 were determined using Taq Man Micro RNA Assays applying mi R-140 as the reference. Selection of mi RNAs was based on their characteristic up- or downregulation observed in hepatocellular carcinoma. Relative expression of mi RNAs was correlated with fibrosis stage and liver stiffness(LS) value measured by transient elastography, as well as with serum alanine aminotransferase(ALT) level.RESULTS: The expression of individual mi RNAs showed deregulated patterns in stages F1-F4 as compared with stage F0, but only the reduced level of mi R-122 in stage F4 was statistically significant(P 0.04). When analyzing mi RNA expression in relation to fibrosis, levels of mi R-122 and mi R-221 showed negative correlations with fibrosis stage, and mi R-122 was found to correlate negatively and mi R-224 positively with LS values(all P 0.05). ALT levels displayed a positive correlation with mi R-21(P 0.04). Negative correlations were observed in the fibrosis samples of mixed etiology between mi R-122 and fibrosis stage and LS values(P 0.05), and in the samples of chronic viral hepatitis, between mi R-221 and fibrosis stage(P 0.01), whereas mi R-21 showed positive correlation with ALT values in the samples of autoimmune liver diseases(P 0.03). The results also revealed a strong correlation between fibrosis stage and LS values(P 0.01) when etiology of fibrosis was not taken into account.CONCLUSION: Reduced expression of mi R-122 in advanced fibrosis and its correlation with fibrosis stage and LS values seem to be characteristic of hepatic fibrosis of various etiologies.     

MiR-96-5p inhibition induces cell apoptosis in gastric adenocarcinoma

BACKGROUND Gastric adenocarcinoma(GAC) mortality rates have remained relatively changed over the past 30 years, and it continues to be one of the leading causes of cancerrelated death.AIM To search for novel mi RNAs related to GAC prognosis and further investigate the effect of mi R-96-5 p on MGC-803 cells.METHODS The mi RNA expression profile data of GAC based on The Cancer Genome Atlas were obtained and used to screen differently expressed mi RNAs(DEMs) and DEMs related to GAC prognosis. Then, the expression of DEMs related to GAC prognosis was identified in GAC tumor samples and adjacent normal samples by q RT-PCR. The target gene, ZDHHC5, of mi R-96-5 p was predicted using Target Scan, mi RTar Base, and mi RDB databases and confirmed by luciferase reporter assay. Furthermore, MGC-803 cells were transfected with inhibitor NC,mi R-96-5 p inhibitor, si-ZDHHC5, or mi R-96-5 p inhibitor + si-ZDHHC5, and then cell apoptosis was detected by flow cytometry. The expression of ZDHHC5, Bcl-2, and COX-2 was detected using western blotting.RESULTS A total of 299 DEMs and 35 DEMs related to GAC prognosis were screened based on The Cancer Genome Atlas. Then compared with adjacent normal samples, the levels of mi R-96-5 p, mi R-222-5 p, and mi R-652-5 p were remarkably increased,while mi R-125-5 p, mi R-145-3 p, and mi R-379-3 p levels were reduced in GAC tumor samples(P 0.01), which were consistent with bioinformatics analysis.Furthermore, ZDHHC5 was defined as a direct target gene of mi R-96-5 p. mi R-96-5 p inhibition increased the number of apoptotic cells as well as promoted the expression of ZDHHC5, Bcl-2, and COX-2 in MGC-803 cells(P 0.01). After ZDHHC5 inhibition, the number of apoptotic cells and the expression of ZDHHC5, Bcl-2, and COX-2 were reduced. The addition of an mi R-96-5 p inhibitor partly reversed these effects(P 0.01).CONCLUSION Our findings identified six mi RNAs related to GAC prognosis and suggested that downregulated mi R-96-5 p might induce cell apoptosis via upregulating ZDHHC5 expression in MGC-803 cells.     

miRNAs as Potential Biomarkers for Viral Hepatitis B and C

Around 257 million people are living with hepatitis B virus (HBV) chronic infection and 71 million with hepatitis C virus (HCV) chronic infection. Both HBV and HCV infections can lead to liver complications such as cirrhosis and hepatocellular carcinoma (HCC). To take care of these chronically infected patients, one strategy is to diagnose the early stage of fibrosis in order to treat them as soon as possible to decrease the risk of HCC development. microRNAs (or miRNAs) are small non-coding RNAs which regulate many cellular processes in metazoans. Their expressions were frequently modulated by up- or down-regulation during fibrosis progression. In the serum of patients with HBV chronic infection (CHB), miR-122 and miR-185 expressions are increased, while miR-29, -143, -21 and miR-223 expressions are decreased during fibrosis progression. In the serum of patients with HCV chronic infection (CHC), miR-143 and miR-223 expressions are increased, while miR-122 expression is decreased during fibrosis progression. This review aims to summarize current knowledge of principal miRNAs modulation involved in fibrosis progression during chronic hepatitis B/C infections. Furthermore, we also discuss the potential use of miRNAs as non-invasive biomarkers to diagnose fibrosis with the intention of prioritizing patients with advanced fibrosis for treatment and surveillance.     

MicroRNA-mediated interactions between host and hepatitis C virus

Micro RNAs(mi RNAs) are small noncoding RNAs. More than 2500 mature mi RNAs are detected in plants, animals and several types of viruses. Hepatitis C virus(HCV), which is a positive-sense, singlestranded RNA virus, does not encode viral mi RNA. However, HCV infection alters the expression of host mi RNAs, either in cell culture or in patients with liver disease progression, such as liver fibrosis, cirrhosis, and hepatocellular carcinoma. In turn, host mi RNAs regulate HCV life cycle through directly binding to HCV RNAs or indirectly targeting cellular m RNAs. Increasing evidence demonstrates that mi RNAs are one of the centered factors in the interaction network between virus and host. The competitive viral and host RNA hypothesis proposes a latent cross-regulation pattern between host m RNAs and HCV RNAs. High loads of HCV RNA sequester and de-repress host mi RNAs from their normal host targets and thus disturb host gene expression, indicating a means of adaptation for HCV to establish a persistent infection. Some special mi RNAs are closely correlated with liver-specific disease progression and the changed levels of mi RNAs are even higher sensitivity and specificity than those of traditional proteins. Therefore, some of them can serve as novel diagnostic/prognostic biomarkers in HCVinfected patients with liver diseases. They are also attractive therapeutic targets for development of new anti-HCV agents.     

Long non-coding RNA highly up-regulated in liver cancer promotes exosome secretion

BACKGROUND Highly upregulated in liver cancer(HULC) is a long non-coding RNA(lnc RNA)which has recently been identified as a key regulator in hepatocellular carcinoma(HCC) progression. However, its role in the secretion of exosomes from HCC cells remains unknown.AIM To explore the mechanism by which HULC promotes the secretion of exosomes from HCC cells.METHODS Serum and liver tissue samples were collected from 30 patients with HCC who had not received chemotherapy, radiotherapy, or immunotherapy before surgery.HULC expression in serum exosomes and liver cancer tissues of patients was measured, and compared with the data obtained from healthy controls and tumor adjacent tissues. The effect of HULC upregulation in HCC cell lines and the relationship between HULC and other RNAs were studied using q PCR and dualluciferase reporter assays. Nanoparticle tracking analysis was performed to detect the quantity of exosomes.RESULTS HULC expression in serum exosomes of patients with HCC was higher than that in serum exosomes of healthy controls, and HULC levels were higher in liver cancer tissues than in tumor adjacent tissues. The expression of HULC in serum exosomes and liver cancer tissues correlated with the tumor-node-metastasis(TNM) classification, and HULC expression in tissues correlated with that in serum exosomes. Upregulation of HULC promoted HCC cell growth and invasion and repressed apoptosis. Notably, it also facilitated the secretion of exosomes from HCC cells. Moreover, q PCR assays showed that HULC repressed micro RNA-372-3 p(mi R-372-3 p) expression. We also identified Rab11 a as a downstream target of mi R-372-3 p. Dual-luciferase reporter assays suggested that mi R-372-3 p could directly bind both HULC and Rab11 a.CONCLUSION Our findings illustrate the importance of the HULC/mi R-372-3 p/Rab11 a axis in HCC and provide new insights into the molecular mechanism regulating the secretion of exosomes from HCC cells.     

Oncogenic role of microRNA-423-5p in hepatocellular carcinoma

BACKGROUND: It has been found that micro RNA-423-5p(mi R423-5p) is an oncogenic factor and frequently upregulated in gastric carcinoma. However,the involvement of mi R423-5p in hepatocellular carcinoma(HCC) has been rarely reported. The aim of this study was to assess whether mi R423-5p is aberrantly expressed in HCC tissues,and to characterize its roles in the cancerous biology of HCC.METHODS: HCC and corresponding nonmalignant tissues were obtained from 115 patients during liver transplantation to detect the expression level of mi R423-5p. The mi R423-5p mimic and inhibitor were transfected into LM3 cell line. Cell viability assay,cell cycle analysis,transwell invasion and migration experiments were used to evaluate the oncogenic role of mi R423-5p.RESULTS: mi R423-5p was significantly upregulated in HCC compared with nonmalignant tissues,and this upregulation was negatively associated with recurrence-free survival. For patients beyond the Milan criteria,low expression of mi R423-5p was correlated with better prognosis. Functional analysis showed that mi R423-5p enhanced the proliferative,invasive and migratory capacity of HCC cells.CONCLUSIONS: mi R423-5p contributed to the tumorigenesis and progression of HCC. It could be a new predictor in HCC patients beyond the Milan criteria and would help to improve patient outcomes and enlarge recipient pools of liver transplantation.     

Knockdown of lncRNAXLOC＿001659 inhibits proliferation and invasion of esophageal squamous cell carcinoma cells

BACKGROUND Studies have shown that long non-coding RNAs(lnc RNAs) play a key role in almost all key physiological and pathological processes, including different types of malignant tumors. Our previous lnc RNA microarray results have shown that lnc RNA XLOC_001659 is upregulated in esophageal cancer(EC) tissues, with a fold change of 20.9 relative to normal esophageal tissues. But its effect and the molecular biological mechanisms on proliferation and invasion of EC cells remain unclear.AIM To investigate the effect of lnc RNA XLOC_001659 on esophageal squamous cell carcinoma(ESCC) cells and explore the molecular biological mechanisms involved.METHODS RT-q PCR assay was used to quantify the expression levels of lnc RNAXLOC-001659 and mi R-490-5 p. The proliferative capacity of the cells was determined using CCK8 and colony formation assays, and the effect of lnc RNAXLOC-001659 on the invasion of ESCC cells was determined by Transwell assay. Dualluciferase reporter assay was used to detect the target genes of lnc RNAXLOC-001659 and mi R-490-5 p.RESULTS The results of RT-q PCR showed that the expression of lnc RNAXLOC_001659 was upregulated in ESCC cells. CCK-8 assay showed that knockdown of lnc RNAXLOC_001659 significantly inhibited ESCC cell proliferation. Colony formation and Transwell invasion assays showed that knockdown of lnc RNAXLOC_001659 or overexpression of mi R-490-5 p significantly inhibited ESCC cell growth and invasion. Furthermore, lnc RNAXLOC_001659 acts as an endogenous sponge by competitively binding to mi R-490-5 p to downregulate mi R-490-5 p. Further results confirmed that mi R-490-5 p targeted PIK3 CA, and therecovery of PIK3 CA rescued lnc RNAXLOC_001659 knockdown or mi R-490-5 p overexpression-mediated inhibition of cell proliferation and invasion, which suggested the presence of an lnc RNAXLOC_001659/mi R-490-5 p/PIK3 CA regulatory axis.CONCLUSION Knockdown of lnc RNA XLOC_001659 inhibits proliferation and invasion of ESCC cells via regulation of mi R-490-5 p/PIK3 CA, suggesting that it may play a role in ESCC tumorigenesis and progression.     

Circulating micro RNAs as diagnostic and prognostic tools for hepatocellular carcinoma

Hepatocellular carcinoma(HCC) is an aggressive malignancy and the second leading cause of cancerrelated deaths worldwide. Conventional biomarkers exhibit poor performance in the surveillance,diagnosis,and prognosis of HCC. Micro RNAs(mi RNAs) are a class of evolutionarily conserved small non-coding RNAs that are involved in the regulation of gene expression and protein translation,and they play critical roles in cell growth,differentiation,and the development of various types of cancers,including HCC. Recent evidence revealed the role of mi RNAs as potential novel and ideal biomarkers for HCC. mi RNAs are released to extracellular spaces,and they are extremely stable in bodily fluids,including serum or plasma,where they are packaged into various microparticles or associated with RNA-binding proteins. Numerous studies have demonstrated that circulating mi RNAs have potential applications as minimally invasive biomarkers for HCC diagnosis and prognosis. The present review highlights current understanding of mi RNA biogenesis and the origins and types of circulating mi RNAs. We summarize recent progress in the use of circulating mi RNAs as diagnostic and prognostic biomarkers for HCC. We also discuss the challenges and perspectives of the clinical utility of circulating mi RNAs in HCC.