Extracellular vesicles-encapsulated let-7i shed from bone mesenchymal stem cells suppress lung cancer via KDM3A/DCLK1/FXYD3 axis

Accumulating evidence has suggested that extracellular vesicles (EVs) play a crucial role in lung cancer treatment. Thus, we aimed to investigate the modulatory role of bone marrow mesenchymal stem cell (BMSC)-EV-derived let-7i and their molecular mechanism in lung cancer progression. Microarray-based analysis was applied to predict lung cancer-related miRNAs and their downstream genes. RT-qPCR and Western blot analyses were conducted to determine Let-7i, lysine demethylase 3A (KDM3A), doublecortin-like kinase 1 (DCLK1) and FXYD domain-containing ion transport regulator 3 (FXYD3) expressions, after which dual-luciferase reporter gene assay and ChIP assay were used to identify the relationship among them. After loss- and gain-of-function assays, the effects of let-7i, KDM3A, DCLK1 and FXYD3 on the biological characteristics of lung cancer cells were assessed. Finally, tumour growth in nude mice was assessed by xenograft tumours in nude mice. Bioinformatics analysis screened out the let-7i and its downstream gene, that is KDM3A. The findings showed the presence of a high expression of KDM3A and DCLK1 and reduced expression of let-7i and FXYD3 in lung cancer. KDM3A elevated DCLK1 by removing the methylation of H3K9me2. Moreover, DCLK1 suppressed the FXYD3 expression. BMSC-EV-derived let-7i resulted in the down-regulation of KDM3A expression and reversed its promoting role in lung cancer development. Consistently, in vivo experiments in nude mice also confirmed that tumour growth was suppressed by the BMSC-EV-derived let-7i. In conclusion, our findings demonstrated that the BMSC-EV-derived let-7i possesses an inhibitory role in lung cancer progression through the KDM3A/DCLK1/FXYD3 axis, suggesting a new molecular target for lung cancer treatment.     

KDM5B promotes self-renewal of hepatocellular carcinoma cells through the microRNA-448–mediated YTHDF3/ITGA6 axis

Histone methylation plays important roles in mediating the onset and progression of various cancers, and lysine-specific demethylase 5B (KDM5B), as a histone demethylase, is reported to be an oncogene in hepatocellular carcinoma (HCC). However, the mechanism underlying its tumorigenesis remains undefined. Hence, we explored the regulatory role of KDM5B in HCC cells, aiming to identify novel therapeutic targets for HCC. Gene Expression Omnibus database and StarBase were used to predict important regulatory pathways related to HCC. Then, the expression of KDM5B and microRNA-448 (miR-448) in HCC tissues was detected by RT-qPCR and Western blot analysis. The correlation between KDM5B and miR-448 expression was analysed by Pearson's correlation coefficient and ChIP experiments, and the targeting of YTH N6-methyladenosine RNA binding protein 3 (YTHDF3) by miR-448 was examined by luciferase assay. Additionally, the effect of KDM5B on the proliferation, migration, invasion and apoptosis as well as tumorigenicity of transfected cells was assessed using ectopic expression and depletion experiments. KDM5B was highly expressed in HCC cells and was inversely related to miR-448 expression. KDM5B demethylated H3K4me3 on the miR-448 promoter and thereby inhibited the expression of miR-448, which in turn targeted YTHDF3 and integrin subunit alpha 6 (ITGA6) to promote the malignant phenotype of HCC. Moreover, KDM5B accelerated HCC progression in nude mice via the miR-448/YTHDF3/ITGA6 axis. Our study uncovered that KDM5B regulates the YTHDF3/ITGA6 axis by inhibiting the expression of miR-448 to promote the occurrence of HCC.     

miR-126-3p is essential for CXCL12-induced angiogenesis

Atherosclerosis, in the ultimate stage of cardiovascular diseases, causes an obstruction of vessels leading to ischemia and finally to necrosis. To restore vascularization and tissue regeneration, stimulation of angiogenesis is necessary. Chemokines and microRNAs (miR) were studied as pro-angiogenic agents. We analysed the miR-126/CXCL12 axis and compared impacts of both miR-126-3p and miR-126-5p strands effects in CXCL12-induced angiogenesis. Indeed, the two strands of miR-126 were previously shown to be active but were never compared together in the same experimental conditions regarding their differential functions in angiogenesis. In this study, we analysed the 2D-angiogenesis and the migration assays in HUVEC in vitro and in rat's aortic rings ex vivo, both transfected with premiR-126-3p/-5p or antimiR-126-3p/-5p strands and stimulated with CXCL12. First, we showed that CXCL12 had pro-angiogenic effects in vitro and ex vivo associated with overexpression of miR-126-3p in HUVEC and rat's aortas. Second, we showed that 2D-angiogenesis and migration induced by CXCL12 was abolished in vitro and ex vivo after miR-126-3p inhibition. Finally, we observed that SPRED-1 (one of miR-126-3p targets) was inhibited after CXCL12 treatment in HUVEC leading to improvement of CXCL12 pro-angiogenic potential in vitro. Our results proved for the first time: 1-the role of CXCL12 in modulation of miR-126 expression; 2-the involvement of miR-126 in CXCL12 pro-angiogenic effects; 3-the involvement of SPRED-1 in angiogenesis induced by miR-126/CXCL12 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.     

Silencing of long noncoding RNA LEF1-AS1 prevents the progression of hepatocellular carcinoma via the crosstalk with microRNA-136-5p/WNK1

Long noncoding RNAs (lncRNAs) have been recognized as cancer-associated biological molecules, favoring hepatocellular carcinoma (HCC) progression. This study was conducted to elucidate the effects lncRNA lymphoid enhancer-binding Factor 1 antisense RNA (LEF1-AS1) on the pathological development of HCC, along with the crosstalk involving microRNA-136-5p (miR-136-5p) and with-no-K (lysine) kinase 1 (WNK1). The study recruited primary HCC tissues and their corresponding nonneoplastic liver tissues. The gain- and loss-of-function studies were performed in HCC cells HuH-7 and tumor xenografts in nude mice. The dual luciferase reporter gene assay system, RNA pull-down, and radioimmunoprecipitation assays were applied to detect their interactions among lncRNA LEF1-AS1, miR-136-5p, and WNK1. 5-Ethynyl-2′-deoxyuridine staining, scratch test, Transwell assays, and in vitro tube formation assays were conducted to examine HCC cell proliferation, migration, and invasion and HUVEC angiogenesis. HCC tissues and cells contained high lncRNA LEF1-AS1 expression. LncRNA LEF1-AS1 upregulation triggered markedly increased HCC cell proliferation, migration, and invasion and human umbilical vein endothelial cell angiogenesis. In vivo silencing lncRNA LEF1-AS1 resulted in reduced tumor cell vitality and matrix metalloproteinase-9 and the vascular endothelial growth factor expression. Additionally, the role of lncRNA LEF1-AS1 was found to be largely dependent on WNK1. Association of lncRNA LEF1-AS1 with WNK1 blocked the inhibitory effect of miR-136-5p on WNK1, which was confirmed by in vivo experiments. Altogether, our results revealed an important role of lncRNA LEF1-AS1 in regulating the HCC progression by regulating WNK1, providing a potential biomarker for the therapeutic modalities regarding HCC.     

Overexpression of let-7d explains down-regulated KDM3A and ENO2 in the pathogenesis of preeclampsia

Pre-eclampsia (PE) is the leading cause of maternal death; however, the causative molecular basis remains largely unknown. Recent studies have revealed the important role microRNAs (miRNAs) play in PE. We aimed to explore the effects of let-7d on trophoblast proliferation, migration, invasion and apoptosis in PE and its underlying mechanism. Placental tissues were collected from PE patients and healthy pregnant women, and it was found that let-7d expression was increased, while KDM3A and ENO2 expression was decreased in PE tissues and cells. Bioinformatics analysis indicated the interaction among let-7d, KDM3A and ENO2, confirmed by dual luciferase reporter gene assay; ChIP experiment identified methylated modification to ENO2 by KDM3A. With gain- and loss-function method, silencing of let-7d increased KDM3A expression and enhanced the binding between KDM3A and ENO2. Furthermore, overexpression of let-7d suppressed cell proliferation, migration and invasion of trophoblasts, and induced apoptosis of trophoblasts, while these capacities were restored upon additional treatment of overexpressed ENO2. PE rat models were established to explore the effects of let-7d and ENO2 on PE in vivo. The results established that the silencing of let-7d alleviated the tissue injury and PE-related symptoms when reducing urine protein, TUNEL-positive cells and increasing ENO2, and KDM3A expression in rats. Cumulatively, let-7d suppressed cell progression of trophoblasts, and induced apoptosis through the down-regulation of KDM3A to promote ENO2 methylation, thereby promoting progression of PE. Such an epigenetic network of let-7d, KDM3A and ENO2 in the pathogenesis of PE might provide novel insight into targeted therapy against this disorder.     

miR-224-5p inhibits proliferation,migration, and invasion by targeting PIK3R3/AKT3 in uveal melanoma

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. Accumulating investigations have identified the aberrant expression of miRNAs (microRNAs) in UM, such as miR-181, miR-20a, miR-144, miR-146a. The purpose of this study is to investigate the biological function of miR-224-5p in UM. The expression of miR-224-5p, PIK3R3, and AKT3 in 30 tumor tissues and paired adjacent noncancerous tissues were analyzed using Western blot analysis and quantitative real-time polymerase chain reaction (qRT-PCR) assays. Cell proliferation assay, transwell assay, and wound healing assay were used to measure the effects of miR-224-5p on the motility of UM in vitro. Western blot analysis and luciferase assays were used to detect the expression of PIK3R3 and AKT3 as miR-224-5p downstream targets. The results of Western blot analysis and qRT-PCR assays indicated that the expression of miR-224-5p was lower in UM tissues compared to normal tissue, while the expression of PIK3R3 and AKT3 were simultaneously increased. Upregulation of miR-224-5p significantly inhibited capacities of proliferation, invasion, and migration of OCM-1A cells and decreased expression of PIK3R3 and AKT3. Luciferase assay demonstrated PIK3R3 and AKT3 as downstream targets of miR-224-5p. Moreover, upregulating PIK3R3 and AKT3 restrained miR-224-5p-induced inhibition of the motility of OCM-1A cells. Thus, our study proved that miR-224-5p was involved in proliferation, invasion, and migration of UM cells via regulation the expression of PIK3R3 and AKT3. And the results also established a miR-224-5p/PIK3R3/PI3K/AKT axis in the regulation of UM progression, providing an experimental basis for further exploring the miR-224-5p as a therapeutic and diagnosis target for patients with UM.     

DNA Methylation-mediated Repression of miR-941 Enhances Lysine (K)-specific Demethylase 6B Expression in Hepatoma Cells

MicroRNAs (miRNAs) have been shown to play important roles in carcinogenesis. However, their underlying mechanisms of action in hepatocellular carcinoma (HCC) are poorly understood. Recent evidence suggests that epigenetic silencing of miRNAs through tumor suppression by CpG island hypermethylation may be a common hallmark of human tumors. Here, we demonstrated that miR-941 was significantly down-regulated in HCC tissues and cell lines and was generally hypermethylated in HCC. The overexpression of miR-941 suppressed in vitro cell proliferation, migration, and invasion and inhibited the metastasis of HCC cells in vivo. Furthermore, the histone demethylase KDM6B (lysine (K)-specific demethylase 6B) was identified as a direct target of miR-941 and was negatively regulated by miR-941. The ectopic expression of KDM6B abrogated the phenotypic changes induced by miR-941 in HCC cells. We demonstrated that miR-941 and KDM6B regulated the epithelial-mesenchymal transition process and affected cell migratory/invasive properties.     

Retracted: Long noncoding LINC01551 promotes hepatocellular carcinoma cell proliferation,migration, and invasion by acting as a competing endogenous RNA of microRNA-122-5p to regulate ADAM10 expression

Hepatocellular carcinoma (HCC) is a severe disease with high mortality in the world. It has been shown that long noncoding RNA (lncRNA) might play a role in HCC. The aim of the present study was to identify the role of long intergenic noncoding RNA 01551 (LINC01551) in the HCC development and explore the underlying mechanism of LINC01551/miR-122-5p/ADAM10 axis. The differentially expressed lncRNAs associated with HCC were screened out by a microarray analysis. The expression of LINC01551, miR-122-5p, and ADAM10 was determined in HCC tissues and cells. The potential miRNA (miR-122-5p) regulated by LINC01551 was explored, and the target relationship between miR-122-5p and ADAM10 was confirmed. To evaluate the effect of LINC01551 and miR-122-5p on proliferation, migration, invasion, and apoptosis of HCC, different plasmids were delivered into MHCC97-H cells. High expression of LINC01551 and ADAM10 yet low-expression of miR-122-5p were revealed in HCC tissues and cells. Overexpression of miR-122-5p could downregulate ADAM10. Biological prediction websites and fluorescence in situ hybridization assay verified that LINC01551 was mainly expressed in the cytoplasm. Silencing LINC01551 reduced HCC cell viability, proliferation, migration, invasion, and cell cycle entry yet induce cell apoptosis. Upregulation of LINC01551 increased its ability of competitively binding to miR-122-5p, thus reducing miR-122-5p and upregulating ADAM10 expression, as well as promoting the proliferative, migrative, and invasive ability. Taken together the results, it is highly possible that LINC01551 functions as an competing endogenous RNA (ceRNA) to regulate the miRNA target ADAM10 by sponging miR-122-5p and therefore promotes the development of HCC, highlighting a promising competitive new target for the HCC treatment.     

Circ_0005615 promotes cervical cancer cell growth and metastasis by modulating the miR-138-5p/KDM2A axis

Cervical cancer (CC) is a highly fatal gynecological malignancy due to its high metastasis and recurrence rate. Circular RNA (circRNA) has been regarded as a regulator of CC. However, the underlying molecular mechanism of circ_0005615 in CC remains unclear. The levels of circ_0005615, miR-138-5p, and lysine demethylase 2A (KDM2A) were measured using qRT-PCR or western blot. Cell proliferation was assessed by Cell Counting Kit-8, 5-ethynyl-2′-deoxyuridine, and colony formation experiments. Cell invasion and migration were tested by transwell assay and wound healing assay. Flow cytometry and Caspase-Glo 3/7 Assay kit were used to analyze cell apoptosis. The expression of proliferation-related and apoptosis-related markers was detected by western blot. The binding relationships among circ_0005615, miR-138-5p, and KDM2A were verified by dual-luciferase reporter assay or RNA immunoprecipitation assay. Xenograft assay was applied to detect the effect of circ_0005615 in vivo. Circ_0005615 and KDM2A were upregulated, while miR-138-5p was downregulated in CC tissues and cells. Circ_0005615 knockdown retarded cell proliferation, migration, and invasion, while promoting apoptosis. Besides, circ_0005615 sponged miR-138-5p, and miR-138-5p could target KDM2A. miR-138-5p inhibitor reversed the regulation of circ_0005615 knockdown on CC cell growth and metastasis, and KDM2A overexpression also abolished the inhibitory effect of miR-138-5p on CC cell growth and metastasis. In addition, we also discovered that circ_0005615 silencing inhibited CC tumor growth in vivo. Circ_0005615 acted as a tumor promoter in CC by regulating the miR-138-5p/KDM2A pathway.     

FXYD6: a novel therapeutic target toward hepatocellular carcinoma

FXYD6, FXYD domain containing ion transport regulator 6, has been reported to affect the activity of Na⁺/K⁺-ATPase and be associated with mental diseases. Here, we demonstrate that FXYD6 is up-regulated in hepatocellular carcinoma (HCC) and enhances the migration and proliferation of HCC cells. Up-regulation of FXYD6 not only positively correlates with the increase of Na⁺/K⁺-ATPase but also coordinates with the activation of its downstream Src-ERK signaling pathway. More importantly, blocking FXYD6 by its functional antibody significantly inhibits the growth potential of the xenografted HCC tumors in mice, indicating that FXYD6 represents a potential therapeutic target toward HCC. Altogether, our results establish a critical role of FXYD6 in HCC progression and suggest that the therapy targeting FXYD6 can benefit the clinical treatment toward HCC patients.     

FtMt promotes glioma tumorigenesis and angiogenesis via lncRNA SNHG1/miR-9-5p axis

ObjectiveThis study is to investigate the effects and the mechanisms of mitochondrial ferritin (FtMt) on the glioma tumorigenesis and angiogenesis.MethodsFtMt expression was detected in glioma tissues and cells as well as in nude mouse tissues. Cell proliferation and apoptosis rate were observed following transfection of LV-FtMt or sh-FtMt in glioma cell line. Moreover, glioma cells with FtMt over-expression/knockdown were co-cultured with human umbilical vein endothelial cells (HUVECs) to observe its function on HUVEC proliferation, angiogenic ability and the vascular endothelial growth factor (VEGF) content. Gain and loss of function of small nucleolar RNA host gene 1 (SNHG1) and miR-9-5p were performed in glioma cells and GBM nude mice to observe its effect on glioma cell proliferation and HUVEC angiogenic ability. Luciferase reporter gene and RIP assay were employed to inspect the interactions among SNHG1, FtMt and miR-9-5p. Additionally, a xenograft mouse model was applied to determine the role of FtMt in glioma.ResultsIn this work, FtMt was strongly expressed in glioma tissues and cells as well as in nude mouse tumor tissues. The employment of the loss-of and gain-of functions assays illustrated that FtMt enhanced glioma tumorigenesis and angiogenesis. Mechanistically, our findings showed that FtMt positively related to SNHG1 while negatively correlated with miR-9-5p, and both SNHG1 and FtMt can competitively bind with miR-9-5p. Besides, the inhibition effects of sh-FtMt on glioma were surveyed in vivo experiments.ConclusionEvidence in this study suggested that FtMt promotes glioma tumorigenesis and angiogenesis via SNHG1 mediated miR-9-5p expression, which may provide a theoretical basis for glioma treatment.     

MicroRNA-26a Inhibits Angiogenesis by Down-Regulating VEGFA through the PIK3C2α/Akt/HIF-1α Pathway in Hepatocellular Carcinoma

Background & Aims

microRNAs (miRNAs) have been reported to regulate angiogenesis by down-regulating the expression of pro-angiogenic or anti-angiogenic factors. The aims of this study were to investigate whether miR-26a inhibited angiogenesis by down-regulating vascular endothelial growth factor A (VEGFA) and its clinical relevance in hepatocellular carcinoma (HCC).

Methods

The expression of miR-26a was modified in HepG2 and HCCLM3 cell lines respectively, and a panel of angiogenic factors was measured by real-time PCR in the cells. A luciferase reporter assay was used to validate the target gene of miR-26a. Specific inhibitors of signal transduction pathway and siRNA approaches were used to explore the regulatory mechanism of miR-26a. Migration and tube forming assays were conducted to show the changes of angiogenesis induced by miR-26a and its target genes. Finally animal studies were used to further validate those findings.

Results

Ectopic expression of miR-26a exhibited decreased levels of VEGFA in HepG2 cells. Migration and tube forming of human umbilical vein endothelial cells (HUVECs) were decreased in the conditioned medium from ectopic expression of miR-26a in HepG2 cells compared to control HepG2 cells. The pro-angiogenic effects of the conditioned medium of HepG2 cells on HUVECs were specifically decreased by LY294002, YC-1, and bevacizumab. Integrated analysis disclosed PIK3C2α as a downstream target gene of miR-26a. Ectopic expression of miR-26a suppressed ectopic and orthotopic tumor growth and vascularity in nude mice. The results in HCCLM3 were consistent with those in HepG2. miR-26a expression was inversely correlated with VEGFA expression in HCC patients.

Conclusions

miR-26a modulated angiogenesis of HCC through the PIK3C2α/Akt/HIF-1α/VEGFA pathway. The expression of VEGFA was inversely correlated with miR-26a expression in HCC tumors.     

CircMTO1 suppresses hepatocellular carcinoma progression via the miR-541-5p/ZIC1 axis by regulating Wnt/β-catenin signaling pathway and epithelial-to-mesenchymal transition

CircRNA mitochondrial tRNA translation optimization 1 (circMTO1) functions as a tumor suppressor usually and is related to the progression of many tumors, including hepatocellular carcinoma (HCC). CircMTO1 is downregulated in HCC as compared to adjacent nontumor tissue, which may suppress the HCC progression by certain signal pathways. However, the underlying signal pathway remains largely unknown. The interactions between circMTO1 and miR-541-5p were predicted through bioinformatics analysis and verified using pull-down and dual-luciferase reporter assays. CCK-8, transwell, and apoptosis assays were performed to determine the effect of miR-541-5p on HCC progression. Using bioinformatic analysis, dual-luciferase reporter assay, RT-qPCR, and western blot, ZIC1 was found to be the downstream target gene of miR-541-5p. The regulatory mechanisms of circMTO1, miR-541-5p, and ZIC1 were investigated using in vitro and in vivo rescue experiments. The results depicted that silencing circMTO1 or upregulating miR-541-5p expression facilitated HCC cell proliferation, migration, and invasion and inhibited apoptosis. CircMTO1 silencing upregulated the expression of downstream ZIC1 regulators of the Wnt/β-catenin pathway markers, β-catenin, cyclin D1, c-myc, and the mesenchymal markers N-cadherin, Vimentin, and MMP2, while the epithelial marker E-cadherin was downregulated. MiR-541-5p knockdown had the opposite effect and reversed the effect of circMTO1 silencing on the regulation of downstream ZIC1 regulators. Intratumoral injection of miR-541-5p inhibitor suppressed tumor growth and reversed the effect of circMTO1 silencing on the promotion of tumor growth in HCC. These findings indicated that circMTO1 suppressed HCC progression via the circMTO1/ miR-541-5p/ZIC1 axis by regulating Wnt/β-catenin signaling and epithelial-to-mesenchymal transition, making it a novel therapeutic target. Subject terms: Cancer therapy, Liver cancer, Tumour-suppressor proteins     

Integrin alpha 7 correlates with poor clinical outcomes,and it regulates cell proliferation,apoptosis and stemness via PTK2-PI3K-Akt signaling pathway in hepatocellular carcinoma

This study aimed to evaluate the correlation of integrin alpha 7 (ITGA7) with clinical outcomes and its effect on cell activities as well as stemness in hepatocellular carcinoma (HCC). HCC tumor tissues and paired adjacent tissues from 90 HCC patients were obtained and ITGA7 expression was detected using immunohistochemistry assay. Cellular experiments were conducted to examine the effect of ITGA7 on cell activities, astemness via ITGA7 ShRNA transfection, and compensation experiments were further performed to test whether ITGA7 functioned via regulating PTK2-PI3K-AKT signaling pathway. ITGA7 was overexpressed in tumor tissues compared with paired adjacent tissues and its high expression was correlated with larger tumor size, vein invasion and advanced Barcelona Clinic Liver Cancer stage, and it also independently predicted worse overall survival in HCC patients. In cellular experiments, ITGA7 was upregulated in SMMC-7721, Hep G2, HuH-7 and BEL-7404 cell lines compared with normal human liver cells HL-7702. ITGA7 knockdown suppressed cell proliferation but promoted apoptosis, and it also downregulated CSCs markers (CD44, CD133 and OCT-4) as well as PTK2, PI3K and AKT expressions in SMMC-7721 and Hep G2 cell lines. ITGA7 overexpression promoted cell proliferation but inhibited apoptosis, and it also upregulated CSCs markers in HL-7702 cells. Further compensation experiments verified that ITGA7 regulated cell proliferation, apoptosis and CSCs markers via PTK2-PI3K-Akt signaling pathway. ITGA7 negatively associates with clinical outcomes in HCC patients, and it regulates cell proliferation, apoptosis and CSCs markers via PTK2-PI3K-Akt signaling pathway.     

Correlation of MicroRNA-16, MicroRNA-21 and MicroRNA-101 Expression with Cyclooxygenase-2 Expression and Angiogenic Factors in Cirrhotic and Noncirrhotic Human Hepatocellular Carcinoma

Background

Hepatocellular carcinoma (HCC) is a classical example of inflammation-linked cancer and is characterized by hypervascularity suggesting rich angiogenesis. Cycloxygenase-2 (COX-2) is a potent mediator of inflammation and is considered to upregulate angiogenesis. The aims of the study are (1) to analyze expression of Cox-2 mRNA, Cox-2 protein, miR-16, miR-21 and miR-101 in HCC and adjacent liver parenchyma in cirrhotic and noncirrhotic liver, (2) to investigate the relation between COX-2 expression, miR-21 expression and angiogenic factors in these tissues and (3) to investigate the association between miR-16 and miR-101 and COX-2 expression.

Methods

Tissue samples of HCC and adjacent liver parenchyma of 21 noncirrhotic livers and 20 cirrhotic livers were analyzed for COX-2 expression at the mRNA level (qRT-PCR) and at the protein level by Western blot and immunohistochemistry. Gene expression of VEGFA, VEGFR1, VEGFR2, Ang-1, Ang-2 and Tie-2 were correlated with COX-2 levels. miR-16, miR-21 and miR-101 gene expression levels were quantified in HCC tumor tissue.

Results

COX-2 mRNA and protein levels were lower in HCC as compared to adjacent liver parenchyma both in cirrhotic and noncirrhotic liver. COX-2 protein localized mainly in vascular and sinusoidal endothelial cells and in Kupffer cells. At the mRNA level but not at the protein level, COX-2 correlated with mRNA levels of angiogenic factors VEGFR1, Ang-1, and Tie2. miR-21 expression was higher in cirrhotic tissues versus noncirrhotic tissues. MiR-101 expression was lower in cirrhotic versus noncirrhotic adjacent liver parenchyma. None of the miRNAs correlelated with COX-2 expression. miR-21 correlated negatively with Tie-2 receptor in adjacent liver parenchyma.

Conclusions

In human HCC, COX-2 mRNA but not COX-2 protein levels are associated with expression levels of angiogenic factors. MiR-21 levels are not associated with angiogenic molecules. MiR-16 and miR-101 levels do not correlate with COX-2 mRNA and protein levels.