MiR-26b inhibits hepatocellular carcinoma cell proliferation,migration, and invasion by targeting EphA2

Deregulated microRNAs (miRNAs) have been shown to play important roles in cancer progression as a result of changes in expression of their target genes. In this study, we investigated the expression of miR-16b in eight hepatocellular carcinoma (HCC) cell lines, revealed the roles of miR-26b on hepatocellular carcinoma (HCC) cell proliferation, migration, and invasion, and confirmed that EphA2 is a direct target of miR-26b. The miR-26b expression was decreased and EphA2 expression was evaluated in HCC cell lines. Luciferase assays revealed that miR-26b inhibited EphA2 expression by targeting the 3’-untranslated region of EphA2 mRNA. Overexpression of miR-26b dramatically inhibited the proliferation, invasion, and migration of HCC cells by targeting EphA2. Moreover, miR-26b down-regulated c-Myc and CyclinD1 expression, which was reversed by overexpressed EphA2. Taken together, our data demonstrated the mechanism of miR-26b contributed to HCC progression and implicated that miR-26b’s potential in HCC therapy.     

The expression and clinical significance of TPM4 in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is known as the fifth most common cancer in the world for its poor prognosis. New diagnostic markers and treatments are urgent to discover. To evaluate the protein expression of Tropomyosin4 (TPM4) and investigate its prognostic value in HCC, we collected 110 patients with different degrees of HCC and 10 patients with normal hepatic tissues and performed immunohistochemistry. Western bot was used to evaluate the expression of TPM4 in three HCC cell lines (HepG2, Huh7, SMMC-7721) and normal liver cell line LO2, as well as 7 HCC tissues and 7 normal hepatic tissues. The results of TPM4 staining revealed that TPM4 expression in HCC was higher than that in normal hepatic tissues, which was positive in 51.8% (n=57) and negative in 48.2% (n=53) while in normal hepatic tissues positive staining was in 10% (n=1) and negative staining was in 90% (n=9) (P=0.011). And the expression of TPM4 was related to pT status, grade and stage (P<0.001, P=0.015 and P<0.001, respectively). Western blot results indicated that TPM4 was high expressed in HCC cell line and HCC tissues. In conclusion, we believe that TPM4 can be applied as a diagnostic and prognostic marker to assist the management of HCC.     

CISD2 associated with proliferation indicates negative prognosis in patients with hepatocellular carcinoma

Background: An evolutionarily conserved gene, the CDGSH iron sulfur domain 2 (CISD2), functions to control mammalian life span and regulates human cells proliferation. However, the role of CISD2 in HCC remains unclear. This study was aimed at investigating the expression pattern and clinicopathological significance of CISD2 in patients with HCC. Methods: The mRNA and protein expression levels of CISD2 were analyzed in six HCC lines and eight paired hepatic cancer tumors by real-time PCR, Western blotting and immunohistochemical staining. Statistical analysis was used to evaluate the clinicopathological significance of CISD2 expression. Short hairpin RNA interfering approach was employed to suppress endogenous CISD2 expression in hepatic cancer cells to determine its role in proliferation. Results: CISD2 expression in liver cancer cell lines and tissues was significantly up-regulated at both the RNA and protein levels compared with that in normal cells and adjacent non-tumorous liver tissues (ANT). CISD2 was an independent prognostic factor for poor prognosis. It was correlated with tumor size (P=0.001), number of tumors (P=0.003), surgical margin (P=0.006), hepatitis B surface antigen (HBsAg) infection (P=0.002) and recurrence (P<0.001) of liver cancer. Multivariate analysis suggested that CISD2 expression was an independent prognostic indicator for the survival of patients with HCC. HCC patients with high CISD2 expression displayed a shorter overall survival and a higher recurrence rate than those with low CISD2 expression (P<0.05, respectively). Additionally, stable down-expression of CISD2 in hepatoma cells suppressed cell proliferation in vitro. Similarly, an in vivo assay showed that CISD2 down-regulation in hepatoma cells inhibited remarkably tumorigenic potential in tumor size and weight. Conclusions: CISD2 protein may serve as a candidate prognostic marker and a novel therapeutic target for HCC and play an important role in promoting proliferation and enhanced progression of HCC.     

Increased expression of microRNA-196a predicts poor prognosis in human ovarian carcinoma

Objective: Overexpression of MicroRNA-196a (miR-196a) has recently been reported in different types of human cancers. However, the prognostic value of miR-196a in ovarian carcinoma remains unknown. In this study, we investigated the expression of miR-196a in ovarian carcinoma and its relationship with tumor progression and clinical prognosis. Methods: The expression level of miR-196a was examined by quantitative Real-time PCR (qRT-PCR) in surgically removed ovarian cancer tissues and ovarian cancer cell lines. The correlation between miR-196a expression and clinical features and prognosis were statistically analyzed. Results: The results showed that the miR-196a expression was significantly upregulated in tumor tissues and ovarian cancer cell lines compared with that in normal ovarian surface tissues and normal ovarian epithelial cells. Moreover, miR-196a expression was positively correlated with FIGO stage (P <0.001), tumor size (P =0.020), and lymph nodes metastasis (P =0.019). Kaplan-Meier analysis demonstrated that high levels of miR-196a expression was associated with poorer overall survival (P <0.001) and recurrent-free survival (P =0.003), especially in patients with advanced disease (P =0.002). Multivariate analysis suggested that miR-196a expression was an independent prognostic factor for overall survival of patients with ovarian carcinoma. Conclusions: In conclusion, miR-196a may play an important role in the progression of ovarian carcinoma, and could be used as an independent prognostic biomarker for patients with ovarian carcinoma.     

LOXL4 is downregulated in hepatocellular carcinoma with a favorable prognosis

Lysyl oxidase like 4 (LOXL4), a member of the secreted copper-dependent amine oxidases that contribute to the assemble and maintenance of the extracellular matrix (ECM), was found to be up-regulated or down-regulated in different cancer types, suggesting its paradoxical roles in cancer. The specific role of LOXL4 in hepatocellular carcinoma (HCC), however, is still yet to be defined. Twenty-eight pairs of HCC specimens were used for LOXL4 mRNA expression analysis. The mRNA expression in HCC cell lines was examined, and HepG2 was selected for LOXL4 small interfering RNA (siRNA) interference to investigate the biological function of LOXL4, LOXL4 immunohistochemical staining was performed using a tissue microarray containing 298 HCC patients. The prognostic and diagnostic value of LOXL4 was evaluated using Cox regression and Kaplan-Meier analysis. LOXL4 mRNA or protein expression was significantly lower in HCC tissues than peritumoral tissues (LOXL4 mRNA expression, P = 0.018; LOXL4 protein expression, P < 0.001). Low LOXL4 expression was associated with lower overall survival (OS) rates and higher cumulative recurrence rates. Multivariate analysis indicated that LOXL4 was an independent prognostic indicator for OS and time to recurrence (TTR). Our results revealed that LOXL4 was down-regulated in HCC and correlated with aggressive tumors and a worse clinical outcome. LOXL4 may be a potential biomarker to identify the HCC patients with a higher risk of recurrence.     

Prognostic significance and anti-proliferation effect of microRNA-365 in hepatocellular carcinoma

MircroRNA functions as tumor suppressor or promoter in hepatocellular carcinoma (HCC). Researchers have found that miR-365 expression was lower in HCC tissues compared with that in adjacent normal tissues. However, its prognostic significance and anti-proliferation effect in HCC remain to be clarified. In this study, we firstly found that miR-365 expression was lower in HCC tissues compared with that in adjacent normal tissues. Then, we analyzed miR-365 expression level and its clinicopathological and prognostic significance. Finally, overexpression of miR-365 inhibits HCC cell proliferation and migration in vitro. Our findings suggest that miR-365 expression was an independent poor prognostic factor for HCC patient overall survival and suppressed tumor cell growth. Therefore, miR-365 may serve as a valuable prognostic marker and promising target for HCC.     

MicroRNA-338-3p inhibits cell proliferation in hepatocellular carcinoma by target forkhead box P4 (FOXP4)

MicroRNAs (miRNAs) are a class of small, non-coding RNAs, which have demonstrated to important gene regulators, and have critical roles in diverse biological processes including cancer cell proliferation. Previous studies suggested microRNA-338-3p (miR-338-3p) was down-regulated and play tumor suppressor roles in gastric cancer, colorectal carcinoma and lung cancer. However, the role of miR-338-3p in hepatocellular carcinoma (HCC) is still unclear. In this study, we analyzed the expression of miR-338-3p in HCC tissues and HCC cell lines. We find that miR-338-3p was downregulated in HCC tissues and cell lines. Then functional studies demonstrate ectopic miR-338-3p expression significantly suppressed the in vitro proliferation and colony formation of HCC cells and cause to cell cycle arrest. Using bio-informatic method and report assay we identified a novel miR-338-3p target, FOXP4 in HCC cells. Furthermore, knockdown of FOXP4 have the similar effects in HCC corrected with miR-338-3p. These findings suggest that miR-338-3p regulates survival of HCC cells partially through the downregulation of FOXP4. Therefore, targeting with the miR-338-3p/FOXP4 axis might serve as a novel therapeutic application to treat HCC patients.     

PRMT4 promotes hepatocellular carcinoma progression by activating AKT/mTOR signaling and indicates poor prognosis

Background: Protein arginine methyltransferase 4 (PRMT4) has been reported to play a role in several common cancers; however, the function and mechanism of PRMT4 in hepatocellular carcinoma (HCC) are not fully understood. This study aimed to investigate the role and mechanism of PRMT4 in the progression of HCC.Methods: PRMT4 expression and clinicopathological characteristics were investigated using an HCC tissue microarray (TMA) consisting of 140 patient samples analyzed by immunohistochemistry. CCK-8, crystal violet and Transwell assays were used to determine cell proliferation, colony formation, migration, and invasion of HCC cell lines in which PRMT4 was overexpressed or downregulated. The underlying mechanism of PRMT4 function was explored by Western blot assays.Results: PRMT4 was highly expressed in HCC tumor tissues compared to adjacent nontumor tissues. PRMT4 expression was significantly associated with alpha-fetoprotein levels, tumor size, satellite nodules, and microvascular invasion. Patients with higher PRMT4 expression had a shorter survival time and higher recurrence rate. Functional studies demonstrated that PRMT4 overexpression promoted HCC cell proliferation, migration, and invasion in vitro, while knocking down PRMT4 inhibited these malignant behaviors. Additional results revealed that PRMT4 promoted the progression of HCC cells via activation of the AKT/mTOR signaling pathway. Furthermore, inhibition of the AKT/mTOR signaling by MK2206 or rapamycin significantly attenuated PRMT4-mediated malignant phenotypes.Conclusions: This study suggests that PRMT4 may promote the progression of HCC cells by activating the AKT/mTOR signaling pathway, which may be a valuable biomarker and potential target for HCC.     

Augmented miR-10b expression associated with depressed expression of its target gene KLF4 involved in gastric carcinoma

Previous studies have revealed several targets of miR-10b, such as syndecan-1, HOXD10, TBX5, and E-cadherin. In this study, we aimed to assess whether Krüppel-like factor 4 (KLF4) is a target gene of miR-10b in gastric cancer (GC). Targeting of KLF4 by miR-10b was confirmed by dual-luciferase reporter assays. The expression levels of miR-10b and KLF4 mRNA in 5 different gastric cancer cell lines and 65 pairs of gastric cancer tissues were detected by Real-time PCR. In addition, KLF4 protein in gastric cancer cell lines and 30 GC tissues was measured by western blotting and immunochemistry, respectively. KLF4 is a direct target gene of miR-10b in GC, and its expression is reduced by miR-10b at both mRNA and protein levels. In addition, the expression level of miR-10b was tendentiously upregulated in GC tissues while the expression levels of KLF4 mRNA and protein were decreased in gastric cancer tissues compared with normal adjacent tissue. There was a dramatically inverse correlation between the expression levels of miR-10b and KLF4 mRNA in GC (r = -0.339, P = 0.006). These findings indicate that miR-10b was upregulated in GC and may have a key role in GC pathogenesis and development through the downregulation of its target gene KLF4.     

Decreased expression of serum miR-424 correlates with poor prognosis of patients with hepatocellular carcinoma

Background: MicroRNAs (miRNAs) play important roles in many important cellular processes and deregulation of miRNAs is linked to many human diseases including cancer. Although miR-424 has been demonstrated to inhibit progression of hepatocellular carcinoma (HCC), its expression level in serum samples and the potential clinical values remain unknown. Materials and methods: The expression level of miR-424 in the serum clinical samples from HCC patients and healthy volunteers were determined by qRT-PCR. Then the association of serum miR-424 expression level with various important clinicopathological parameters and survival rates was evaluated. Multivariate Cox regression analysis was used to identify the independent risk factors for HCC. Results: The expression level of serum miR-424 was significantly decreased in patients with HCC compared with the healthy volunteers (P<0.01). Reduced expression of serum miR-424 was associated with serum AFP (P=0.048), vein invasion (P=0.006) and TNM stage (P=0.003). In addition, survival analysis showed that HCC patients with lower serum miR-424 expression suffered poorer overall survival (P=0.018) and disease free survival (P=0.008). Moreover, serum miR-424 was demonstrated to be an independent risk factor for HCC. Conclusions: Our findings provide the compelling evidence that the decreased expression of serum miR-424 may serve as a novel biomarker to predict the unfavorable prognosis of HCC patients.     

miR-200b调控PDCD4的表达对乳腺癌细胞增殖和侵袭的影响

目的探讨miR-200b对乳腺癌细胞增殖及侵袭的影响及其可能的分子机制。方法利用荧光实时定量PCR检测人乳腺癌组织及乳腺癌细胞株中miR-200b的表达差异;利用生物信息学方法预测miR-200b的靶基因,并使用免疫蛋白印迹实验对靶基因的表达进行验证;分别将miR-200b siRNA、PDCD4 mimics以及相应对照miRNA转染MCF-7细胞,通过CCK-8实验检测细胞的增殖能力;采用Transwell侵袭实验检测细胞的侵袭能力。结果miR-200b在乳腺癌组织中呈低表达水平,进一步抑制miR-200b的表达,乳腺癌细胞的增殖及侵袭能力显著增强(P<0.05);将miR-200b siRNA、PDCD43′-UTR共转染到乳腺癌MCF-7细胞中,双荧光素酶报告基因结果显示抑制miR-200b的表达后,PDCD4的表达及活性相应上调(P<0.05);同时通过蛋白免疫印迹实验可以发现,抑制miR-200b表达后,PDCD4蛋白表达含量降低,乳腺癌细胞的增殖及侵袭能力明显增强(P<0.05);而过表达PDCD4后PDCD4蛋白表达含量增加(P<0.05),乳腺癌细胞的增殖及侵袭能力明显降低(P<0.05)。结论miR-200b可靶向上调PDCD4基因的表达,从而抑制乳腺癌细胞的增殖及侵袭能力。     

miR-107 promotes hepatocellular carcinoma cell proliferation by targeting Axin2

Background: A large number of studies demonstrated that microRNAs play important roles in the progression and development of human cancers. However, the expression level of miR-107 and its biological function in hepatocellular carcinoma (HCC) remains unclear. Method: Quantitative real-time PCR (qRT-PCR) was used to evaluate the expression level of miR-107 in HCC tissues and cell lines. Then, we explored the function of miR-107 to determine its potential roles on HCC cell proliferation in vitro. Luciferase reporter assay was used to confirm the target gene of miR-107, and the results were validated in cell lines. Results: miR-107 was significantly up-regulated in HCC tissues and cell lines. The enforced expression of miR-107 was able to promote cell proliferation in HepG2 cells. At the molecular level, our results suggested that expression of Axin2 was negatively regulated by miR-107. Conclusion: Our observations suggested that miR-107 could promote HCC cells proliferation via targeting Axin2 and might represent a potential therapeutic target for HCC.     

Roles of the miR-139-5p/CCT5 axis in hepatocellular carcinoma: a bioinformatic analysis

Background: MiRNAs are pivotal regulators involved in proliferation, apoptosis, invasion, metastasis, epithelial-mesenchymal transition (EMT), angiogenesis, drug resistance and autophagy in hepatocellular carcinoma (HCC). The aim of this study was to investigate the influence of miR-139-5p and its target genes on the outcomes of HCC.Methods: Survival analysis of miR-139-5p in HCC was conducted in Kaplan-Meier plotter. Target genes of miR-139-5p were identified in TargetScan, miRTarBase and starBase. Gene Expression Omnibus (GEO) series were used for the validation of miR-139-5p target genes. Cox proportional regression model was also established.Results: In Kaplan-Meier plotter, 163 HCC patients were included. MiR-139-5p downregulation was significantly associated with unfavorable overall survival (OS) and disease-free survival (DFS) in HCC patients (all P < 0.001). MiR-139-5p was significantly downregulated in HCC tumors and human hepatoma cell lines (all P < 0.05). As a target gene of miR-139-5p, CCT5 was overexpressed in HCC tumor tissues and peripheral blood mononuclear cells (all P < 0.05). A negative correlation between CCT5 and miR-139-5p was found in TCGA dataset. CCT5 overexpression was significantly associated with worse OS in HCC patients (P < 0.001), which was validated in the {"type":"entrez-geo","attrs":{"text":"GSE14520","term_id":"14520"}}GSE14520 dataset (P = 0.017). CCT5 mRNA was significantly overexpressed in HCC patients with alpha-fetoprotein (AFP) > 300 ng/ml, BCLC staging B-C, TNM staging III and main tumor size > 5 cm (all P < 0.05). According to the Cox regression model of CCT5-interacting genes, HCC patients with high risk had poor OS compared to those with low risk in the TCGA dataset (P < 0.001), with the 1-year, 3-year, and 5-year ROC curves of an area under the curve (AUC) equal to 0.704, 0.662, and 0.631, respectively.Conclusions: MiR-139-5p suppresses HCC tumor aggression and conversely correlated with CCT5. The miR-139-5p/CCT5 axis might perform crucial functions in the development of HCC.     

Long non-coding RNA LINC01296 promotes progression of oral squamous cell carcinoma through activating the MAPK/ERK signaling pathway via the miR-485-5p/PAK4 axis

IntroductionLong intergenic non-protein-coding RNA 1296 (LINC01296), a newly identified lncRNA, can function as an oncogenic driver to promote the development of multiple carcinomas. However, the effect of LINC01296 on oral squamous cell carcinoma (OSCC) is still unclear.Material and methodsWe determined the expression and role of LINC01296 in OSCC tissues and cell lines. The cell viability, migration and invasion were determined by MTT, wound healing assay and transwell assay, respectively. Flow cytometry was used for detecting cell cycle and apoptosis. The interaction and association between LINC01296, microRNA-485-5p (miR-485-5p) and p21 (RAC1) activated kinase 4 (PAK4) were analyzed by RNA immunoprecipitation (RIP) and luciferase reporter assays. The xenograft mouse model was established to detect the effect of LINC01296 on OSCC tumor growth.ResultsOur study showed that LINC01296 was over-expressed in OSCC tissues and cell lines. The level of LINC01296 was positively correlated with the patient’s tumor node metastasis (TNM) stage and nodal invasion. Knockdown of LINC01296 effectively inhibits cell viability, migration and invasion but promotes cell apoptosis in vitro. The in vivo experiment showed that LINC01296 knockdown inhibited OSCC tumor growth. The following analysis indicated that LINC01296 acted as a ceRNA for miR-485-5p, and PAK4 was identified as a direct target of miR-485-5p. Furthermore, we found that the effects of LINC01296 on OSCC progression were through regulating the expression of PAK4/p-MEK/p-ERK via sponging miR-485-5p.ConclusionsLINC01296 promote the cell cycle, proliferation, migration and invasion, and inhibit apoptosis of OSCC cells through activating the MAPK/ERK signaling pathway via sponging miR-485-5p to regulate PAK4 expression. These results suggested that the LINC01296/miR-485-5p/PAK4 axis was closely associated with OSCC progression. Our study provides a new insight into the molecular pathogenesis of OSCC, and may supply novel biomarkers for diagnosis and therapy of OSCC.