Over-Expression of miR-106b Promotes Cell Migration and Metastasis in Hepatocellular Carcinoma by Activating Epithelial-Mesenchymal Transition Process

Hepatocellular carcinoma (HCC) is one the the most fatal cancers worldwide. The poor prognosis of HCC is mainly due to the developement of distance metastasis. To investigate the mechanism of metastasis in HCC, an orthotopic HCC metastasis animal model was established. Two sets of primary liver tumor cell lines and corresponding lung metastasis cell lines were generated. In vitro functional analysis demonstrated that the metastatic cell line had higher invasion and migration ability when compared with the primary liver tumor cell line. These cell lines were subjected to microRNA (miRNAs) microarray analysis to identify differentially expressed miRNAs which were associated with the developement of metastasis in vivo. Fifteen human miRNAs, including miR-106b, were differentially expressed in 2 metastatic cell lines compared with the primary tumor cell lines. The clinical significance of miR-106b in 99 HCC clinical samples was studied. The results demonstrated that miR-106b was over-expressed in HCC tumor tissue compared with adjacent non-tumor tissue (p = 0.0005), and overexpression of miR-106b was signficantly correlated with higher tumor grade (p = 0.018). Further functional studies demonstrated that miR-106b could promote cell migration and stress fiber formation by over-expressing RhoGTPases, RhoA and RhoC. In vivo functional studies also showed that over-expression of miR-106b promoted HCC metastasis. These effects were related to the activation of the epithelial-mesenchymal transition (EMT) process. Our results suggested that miR-106b expression contributed to HCC metastasis by activating the EMT process promoting cell migration in vitro and metastasis in vivo.     

The miR-92a-2-5p in exosomes from macrophages increases liver cancer cells invasion via altering the AR/PHLPP/p-AKT/β-catenin signaling

Early studies indicated that the androgen receptor (AR) might play important roles in the regulating of the initiation and progression of hepatocellular carcinoma (HCC), but its linkage to the surrounding macrophages and their impacts on the HCC progression remain unclear. Here we found that macrophages in liver cancer might function via altering the microRNA, miR-92a-2-5p, in exosomes to decrease liver cancer cells AR expression, which might then lead to increase the liver cancer cells invasion. Mechanism dissection revealed that miR-92a-2-5p from the exosomes could target the 3′UTR of AR mRNA to suppress AR translation, altering the PHLPP/p-AKT/β-catenin signaling to increase liver cancer cells invasion. Preclinical studies demonstrated that targeting this newly identified signaling with miR-92a-2-5p inhibitors led to suppress liver cancer progression. Together, these findings suggest that macrophages in the liver cancer tumor microenvironment may function via exosomes to regulate liver cancer progression, and targeting this newly identified macrophages/exosomes-miR-92a-2-5p/AR/PHLPP/p-AKT/β-catenin signaling may help in the development of novel treatment strategies to better suppress liver cancer progression.Subject terms: Cancer microenvironment, Oncogenes     

Cisplatin-induced downregulation of miR-199a-5p increases drug resistance by activating autophagy in HCC cell

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Systemic chemotherapy plays an important role in the treatment of patients with advanced liver cancer. However, chemoresistance to cisplatin is a major limitation of cisplatin-based chemotherapy in the clinic, and the underlying mechanism of such resistance is not fully understood. In the study, we found that miR-199a-5p levels were significantly reduced in HCC patients treated with cisplatin-based chemotherapy. Cisplatin treatment also resulted in decreased miR-199a-5p levels in human HCC cell lines. Forced expression of miR-199a-5p promoted cisplatin-induced inhibition of cell proliferation. Cisplatin treatment activated autophagy in Huh7 and HepG2 cells, which increased cell proliferation. We further demonstrated that downregulated miR-199a-5p enhanced autophagy activation by targeting autophagy-associated gene 7 (ATG7). More important, autophagy inhibition abrogated miR-199a-5p downregulation-induced cell proliferation. These data demonstrated that miR-199a-5p/autophagy signaling represents a novel pathway regulating chemoresistance, thus offering a new target for chemotherapy of HCC.     

Long non-coding RNA 657 suppresses hepatocellular carcinoma cell growth by acting as a molecular sponge of miR-106a-5p to regulate PTEN expression

Previous study has identified the aberrant expression of LINC00657, a long non-coding RNA (lncRNA), in human breast cancer. However, the expression pattern, biological function and underlying mechanism of LINC00657 in human hepatocellular carcinoma (HCC) remain obscure. The expression levels of LINC00657 in HCC tissues and cell lines were determined by quantitative real-time PCR. CCK-8 assay, cell colony formation assay, cell cycle analysis, Transwell assay were performed to determine whether LINC00657 could affect HCC progression. Luciferase reporter assay was used to assess the target of LINC00657. Expressions of the relevant proteins were analyzed by Western blot. Herein, we found that LINC00657 was downregulated in HCC tissue specimens as well as in malignant HCC cell lines. LINC00657 overexpression inhibited the proliferation, migration and invasion of HCC cells, while LINC00657 depletion promoted both cell viability and cell invasion in vitro. We also found that LINC00657 could inhibit tumor growth in vivo. Further experiments demonstrated that down-regulated LINC00657 increased the expression of miR-106a-5p. miR-106a-5p decreased the abundances of PTEN protein, while had no impact on PTEN mRNA. Moreover, we identified that both LINC00657 and PTEN mRNA were targets of miR-106a-5p by using dual-luciferase reporter assay. Our results provide the new evidence supporting the tumor-suppressive role of LINC00657 in HCC, suggesting that LINC00657 might play a role in HCC and can be a novel therapeutic target for treating HCC.     

Long noncoding RNA TCL6 binds to miR-106a-5p to regulate hepatocellular carcinoma cells through PI3K/AKT signaling pathway

Long noncoding RNAs (lncRNAs) have been reported to dysregulate and involve in the pathology of hepatocellular carcinoma (HCC). Nonetheless, the functional role of lncRNA T cell leukemia/lymphoma 6 (TCL6) and its underlying mechanism in HCC remain unclear. Herein, we analyzed the expression of TCL6 and elucidated its mechanistic involvement in HCC. Bioinformatics analyses indicated TCL6 was evidently downregulated in HCC tissues compared with normal controls. TCL6 was downregulated while microRNA-106a-5p (miR-106a-5p) was upregulated in HCC cell lines. Moreover, knockdown or overexpression of TCL6 significantly raised or diminished the expression level of miR-106a-5p in HCC cells, similar to the effect of miR-106a-5p on TCL6 expression. Functionally, TCL6 inhibited the proliferative, migratory, and invasive potentials of HCC cells as analyzed by cell counting kit-8, scratch wound healing, and transwell assays, respectively. Conversely, miR-106a-5p exerted an opposite effect on the proliferative, migratory, and invasive potentials of HCC. RNA immune precipitation and luciferase reporter assays revealed TCL6 directly bound to miR-106a-5p and luciferase reporter assay verified phosphatase and tensin homolog (PTEN) was a target gene of miR-106a-5p. Mechanistically, TCL6 knockdown evidently reduced PTEN expression at both messenger RNA and protein levels, and miR-106a-5p inhibitor partially rescued this reduction effect in HCC cells. Additionally, western blot assays demonstrated miR-106a-5p downregulation or TCL6 overexpression promoted the protein level of PTEN, and suppressed the phosphorylation level of AKT, the protein level of phosphatidylinositol 3-kinase (PI3K). Collectively, these results revealed TCL6 as a tumor-suppressive lncRNA regulates PI3K/AKT signaling pathway via directly binding to miR-106a-5p in HCC. This mechanism provides a theoretical basis for HCC pathogenesis and a potential therapeutic strategy for HCC treatment.     

MicroRNA-500 as a potential diagnostic marker for hepatocellular carcinoma

We identified that microRNA expression changed dynamically during liver development and found that miR-500 is an oncofetal miRNA in liver cancer. miR-500 was abundantly expressed in several human liver cancer cell lines and 45% of human hepatocellular carcinoma (HCC) tissue. Most importantly, an increased amount of miR-500 was found in the sera of the HCC patients. In fact, miR-500 levels in sera of the HCC patients returned to normal after the surgical treatment in three HCC patients. Our findings reveal that diverse changes of miRNAs occur during liver development and, one of these, miR-500 is an oncofetal miRNA relevant to the diagnosis of human HCC.     

MicroRNA-29a-5p Is a Novel Predictor for Early Recurrence of Hepatitis B Virus-Related Hepatocellular Carcinoma after Surgical Resection

It is still difficult to predict the probability of tumor recurrence after resection of hepatocellular carcinoma (HCC). In this study, we set out to identify specific microRNA (miRNA) in microdissected hepatitis B virus (HBV)-related HCC tissue from formalin-fixed paraffin-embedded (FFPE) samples which might be used in predicting early recurrence after HCC resection. Taqman low density arrays were used to detect the 667 miRNA profiles in both the microdissected tumorous and adjacent non-tumorous liver tissues from 20 HCC patients (discovery set) including 10 patients with early tumor recurrence and 10 without early tumor recurrence and to identify the differentially expressed miRNAs related to HCC recurrence. Then quantitative real-time PCR (qRT-PCR) was used to verify the findings in 106 patients (training set), and to develop a predictive assay. The identified miRNAs were further validated in an independent cohort of 112 patients (validation set). Thirty seven miRNAs were identified from 20 HCC patients and validated in 106 HCC patients using qRT-PCR. A significant association was found between miR-29a-5p level in HCC tissues and early tumor recurrence (P = 0.0002). This association was further confirmed in the independent validation set of 112 patients (P = 0.0154). MiR-29a-5p level was significantly associated with both time to tumor recurrence (TTR) (P = 0.0015) and overall survival (OS) (P = 0.0079) in validation set. In the multivariate analyses, miR-29a-5p was identified as an independent factor for TTR, particularly for those patients with early stage of HCC. The sensitivity and specificity of miR-29a-5p for the prediction of early HCC recurrence of BCLC 0/A stage HCC were 74.2% and 68.2%, respectively. These suggest that miR-29a-5p might be a useful marker for the prediction of early tumor recurrence after HCC resection, especially in BCLC 0/A stage HCCs.     

Development and validation of serum exosomal microRNAs as diagnostic and prognostic biomarkers for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide. China accounts for over half of the new cases and deaths. Diagnostic imprecision and a lack of complimentary molecular biomarkers are partially responsible for this lack of progress. Herein, serum-derived exosomal microRNA (miRNA) profiling was performed on 80 patients which histologically confirmed HCC and 30 normal controls. A classification of 8 exosomal miRNAs had biologically and statistically significant differences between HCC and normal serum samples, including miR-122, miR-125b, miR-145, miR-192, miR-194, miR-29a, miR-17-5p, and miR-106a. Online algorithm showed strong independent classification accuracy (area under the curve) reached 0.535 to 0.850, separately. The significant correlation between serum exosomal miRNAs and tumor size was observed. In addition, the survival difference of HCC patients with high or low exosomal miR-106a was statistically significant using Kaplan-Meier analysis. Besides, we also measured the proliferation and invasion ability of HCC cells following exosomal miR-106a mimics or inhibitor treatment. After prediction with algorithms, mitogen-activated protein kinase and c-Jun N-terminal kinase pathways were identified associated with miR-106a’s function. In summary, differentially expressed serum exosomal miRNAs can be helpful for diagnostic and prognostic of HCC.     

Exosome-depleted MiR-148a-3p derived from Hepatic Stellate Cells Promotes Tumor Progression via ITGA5/PI3K/Akt Axis in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a major cause of cancer-related death worldwide. Although it has been known that hepatic stellate cells (HSCs) play critical roles in the development and progression of HCC, the molecular mechanism underlying crosstalk between HSCs and cancer cells still remains unclear. Here, we investigated the interactions between HSCs and cancer cells through an indirect co-culture system. The expressions of cellular and exosomal miR-148a-3p were evaluated by quantitative real-time PCR. Cell counting kit-8 was used for evaluating cell growth in vitro. Cell migration and invasion ability were evaluated by wound-healing and Transwell assays. Western blot, quantitative real-time PCR and Luciferase reporter assay were performed to determine the target gene of miR-148a-3p. A xenograft liver cancer model was established to study the function of exosomal miR-148a-3p in vivo.We found that miR-148a-3p was downregulated in co-cultured HSCs and overexpression of miR-148a-3p in HSCs impaired the proliferation and invasiveness of HCC both in vitro and in vivo. Moreover, further study showed that the miR-148a-3p was also downexpressed in HSCs-derived exosomes, and increased HSCs-derived exosomal miR-148a-3p suppressed HCC tumorigenesis through ITGA5/PI3K/Akt pathway. In conclusion, our study demonstrated that exosome-depleted miR-148a-3p derived from activated HSCs accelerates HCC progression through ITGA5/PI3K/Akt axis.     

MiR-27a-3p enhances the cisplatin sensitivity in hepatocellular carcinoma cells through inhibiting PI3K/Akt pathway

MicroRNAs (miRNAs) play an important role in drug resistance, and it is reported that miR-27a-3p regulated the sensitivity of cisplatin in breast cancer, lung cancer and ovarian cancer. However, the relationship between miR-27a-3p and chemosensitivity of cisplatin in hepatocellular carcinoma (HCC) was unclear, especially the underlying mechanism was unknown. In the present study, we analyzed miR-27a-3p expression levels in 372 tumor tissues and 49 adjacent tissues in HCC samples from TCGA database, and found that the miR-27a-3p was down-regulated in HCC tissues. The level of miR-27a-3p was associated with metastasis, Child–Pugh grade and race. MiR-27a-3p was regarded as a favorable prognosis indicator for HCC patients. Then, miR-27a-3p was overexpressed in HepG2 cell, and was knocked down in PLC cell. Next, we conducted a series of in vitro assays, including MTT, apoptosis and cell cycle assays to observe the biological changes. Further, inhibitor rate and apoptosis rate were detected with pre- and post-cisplatin treatment in HCC. The results showed that overexpression of miR-27a-3p repressed the cell viability, promoted apoptosis and increased the percentage of cells in G₀/G₁ phase. Importantly, overexpression of miR-27a-3p significantly increased the inhibitor rate and apoptosis rate with cisplatin intervention. Besides, we found that miR-27a-3p added cisplatin sensitivity potentially through regulating PI3K/Akt signaling pathway. Taken together, miR-27a-3p acted as a tumor suppressor gene in HCC cells, and it could be useful for modulating cisplatin sensitivity in chemotherapy.     

肝细胞癌患者血清外泌体microRNA表达鉴定

目的:探讨肝癌细胞外泌体中差异表达的microRNAs(miRNAs)在肝细胞癌(HCC)诊断中的应用价值。方法:通过高通量测序筛选肝癌细胞外泌体中差异表达的miRNAs。实时定量PCR验证差异表达分子;检测差异表达的miRNAs在健康人(Health)、慢性乙型肝炎患者(CHB)、肝硬化患者(LC)及乙型肝炎病毒阳性的肝细胞癌患者(HCC)血清外泌体中的表达。结果:高通量测序筛选到肝癌细胞外泌体中差异表达的miRNA共88种,其中58种表达上调,30种表达下调。选择其中8种差异表达的miRNAs进行q RT-PCR验证,结果显示,此8种miRNAs在细胞上清外泌体、细胞内、癌与癌旁组织中的表达趋势与测序结果一致。miR-221-3p和miR-224-5p在HCC组外泌体中的表达水平显著高于Health组、CHB组和LC组(P0.01),miR-124-3p和let-7a-5p在HCC组外泌体中的表达水平显著低于其他各组(P0.05)。四个组中,miR-21-5p、miR-191-5p、miR-34a-5p和miR-122-5p的表达水平不存在显著性差异(P0.05)。结论:血清外泌体中的miR-221-3p、miR-224-5p、miR-124-3p和let-7a-5p可能成为肝细胞癌的候选标志物。     

miR-130b Promotes CD133(+) liver tumor-initiating cell growth and self-renewal via tumor protein 53-induced nuclear protein 1

A novel paradigm in tumor biology suggests that cancer growth is driven by stem-like cells within a tumor, called tumor-initiating cells (TICs) or cancer stem cells (CSCs). Here we describe the identification and characterization of such cells from hepatocellular carcinoma (HCC) using the marker CD133. CD133 accounts for approximately 1.3%-13.6% of the cells in the bulk tumor of human primary HCC samples. When compared with their CD133? counterparts, CD133(+) cells not only possess the preferential ability to form undifferentiated tumor spheroids in vitro but also express an enhanced level of stem cell-associated genes, have a greater ability to form tumors when implanted orthotopically in immunodeficient mice, and can be serially passaged into secondary animal recipients. Xenografts resemble the original human tumor and maintain a similar percentage of tumorigenic CD133(+) cells. Quantitative PCR analysis of 41 separate HCC tissue specimens with follow-up data found that CD133(+) tumor cells were frequently detected at low quantities in HCC, and their presence was also associated with worse overall survival and higher recurrence rates. Subsequent differential microRNA expression profiling of CD133(+) and CD133? cells from human HCC clinical specimens and cell lines identified an overexpression of miR-130b in CD133(+) TICs. Functional studies on miR-130b lentiviral-transduced CD133? cells demonstrated superior resistance to chemotherapeutic agents, enhanced tumorigenicity in vivo, and a greater potential for self renewal. Conversely, antagonizing miR-130b in CD133(+) TICs yielded an opposing effect. The increased miR-130b paralleled the reduced TP53INP1, a known miR-130b target. Silencing TP53INP1 in CD133? cells enhanced both self renewal and tumorigenicity in vivo. Collectively, miR-130b regulates CD133(+) liver TICs, in part, via silencing TP53INP1.     

DNA methylation-regulated miR-193a-3p dictates resistance of hepatocellular carcinoma to 5-fluorouracil via repression of SRSF2 expression

Chemoresistance prevents effective cancer therapy and is rarely predictable prior to treatment, particularly for hepatocellular carcinoma (HCC). Following the chemoresistance profiling of eight HCC cell lines to each of nine chemotherapeutics, two cell lines (QGY-7703 as a sensitive and SMMC-7721 as a resistant cell line to 5-fluorouracil (5-FU) treatment) were systematically studied for mechanistic insights underpinning HCC 5-FU chemoresistance. Genomic profiling at both DNA methylation and microRNA (miR) levels and subsequent mechanistic studies illustrate a new mechanism for how DNA methylation-regulated miR-193a-3p dictates the 5-FU resistance of HCC cells via repression of serine/arginine-rich splicing factor 2 (SRSF2) expression. In turn, SRSF2 preferentially up-regulates the proapoptotic splicing form of caspase 2 (CASP2L) and sensitizes HCC cells to 5-FU. Forced changes of miR-193a-3p level reverse all of the phenotypic features examined, including cell proliferation, cell cycle progression, and 5-FU sensitivity, in cell culture and in nude mice. Importantly, the siRNA-mediated repression of SRSF2 phenocopies all of the miR-193a-3p mimic-triggered changes in QGY-7703. This newly identified miR-193a-3p-SRSF2 axis highlights a new set of companion diagnostics required for optimal 5-FU therapy of HCC, which involve assaying both the DNA methylation state of the miR-193a gene and the expression of miR-193a-3p and SRSF2 and the relative level of the proapoptotic versus antiapoptotic splicing forms of caspase 2 in clinical samples.     

MicroRNA-99a inhibits hepatocellular carcinoma growth and correlates with prognosis of patients with hepatocellular carcinoma

In our in-depth analysis carried out by the Illumina Solexa massive parallel signature sequencing, microRNA-99a (miR-99a) was found to be the sixth abundant microRNA in the miRNome of normal human liver but was markedly down-regulated in hepatocellular carcinoma (HCC). Compelling evidence has suggested the important roles of microRNAs in HCC development. However, the biological function of miR-99a deregulation in HCC remains unknown. In this study, we found that miR-99a was remarkably decreased in HCC tissues and cell lines. Importantly, lower miR-99a expression in HCC tissues significantly correlated with shorter survival of HCC patients, and miR-99a was identified to be an independent predictor for the prognosis of HCC patients. Furthermore, restoration of miR-99a dramatically suppressed HCC cell growth in vitro by inducing the G(1) phase cell cycle arrest. Intratumoral injection of cholesterol-conjugated miR-99a mimics significantly inhibited tumor growth and reduced the α-fetoprotein level in HCC-bearing nude mice. Insulin-like growth factor 1 receptor (IGF-1R) and mammalian target of rapamycin (mTOR) were further characterized as the direct targets of miR-99a. Furthermore, protein levels of IGF-1R and mTOR were found to be inversely correlated with miR-99a expression in HCC tissues. miR-99a mimics inhibited IGF-1R and mTOR pathways and subsequently suppressed expression of cell cycle-related proteins, including cyclin D1 in HCC cells. Conclusively, miR-99a expression was frequently down-regulated in HCC tissues and correlates with the prognosis of HCC patients, thus proposing miR-99a as a prospective prognosis predictor of HCC. miR-99a suppresses HCC growth by inducing cell cycle arrest, suggesting miR-99a as potential tumor suppressor for HCC therapeutics.     

Prognostic relevance of miR-137 and its liver microenvironment regulatory target gene AFM in hepatocellular carcinoma

MiR-137 has been identified as potential hepatocellular carcinoma (HCC) prognostic biomarkers. Highly relevant HCC prognostic biomarkers may be derived from combinations of miR-137 with its target genes involved in the regulation of liver microenvironment. This study aimed at the discovery of such a combination with improved HCC prognosis performance than miR-137 or its target gene alone in a significantly higher number of HCC patients than previous studies. Analysis of the differentially expressed micro RNAs (miRNAs) between cancer and noncancer tissues reconfirmed miR-137 to be among the most relevant prognostic miRNAs and the data of 375 HCC patients and 50 normal cases were from the Cancer Genome Atlas (TCGA) data sets. Target genes were identified by the established search methods and Kaplan–Meier survival analysis of HCC patients was used to evaluate the overall survival (OS) and recurrence-free survival (RFS). Cox proportional hazards regression indicated that the miR-137 and its target gene AFM combination is an independent prognostic factor for the OS and RFS in HCC. In vitro experiments validated that miR-137 could bind to 3′-untranslated region of the AFM and promote the invasion and metastasis of HCC cell lines. The expressions of miR-137 and its liver microenvironment regulatory target gene AFM in combination significantly correlated with HCC progression in a higher number of patients than in previous studies, which suggested their potential as prognostic biomarkers for HCC.     

MiR-23a-3p promoted G1/S cell cycle transition by targeting protocadherin17 in hepatocellular carcinoma

MiR-23a-3p has been shown to promote liver cancer cell growth and metastasis and regulate that of chemosensitivity. Protocadherin17 (PCDH17) is a tumor suppressor gene and plays an essential part in cell cycle of hepatocellular carcinoma (HCC). This study aimed at evaluating the effects of miR-23a-3p and PCDH17 on HCC cell cycle and underlining the mechanism. The level of miR-23a-3p was up-regulated, while PCDH17 level was down-regulated in HCC tissues compared to adjacent tissues. For the in vitro studies, high expression of miR-23a-3p down-regulated PCDH17 level; increased cell viability; promoted G1/S cell cycle transition; up-regulated cyclin D1, cyclin E, CDK2, CDK4, p-p27, and p-RB levels; and down-regulated the expression of p27. Dual luciferase reporter assay suggested PCDH17 was a target gene of miR-23a-3p. MiR-23a-3p inhibitor and PCDH17 siRNA led to an increase in cell viability and the number of cells in the S phase and up-regulated cyclin D1 and cyclin E levels, compared with miR-23a-3p inhibitor and NC siRNA group. For the in vivo experiments, high expression of miR-23a-3p promoted tumor growth and reduced PCDH17 level in the cytoplasm. These results indicated that high expression of miR-23a-3p might promote G1/S cell cycle transition by targeting PCDH17 in HCC cells. The miR-23a-3p could be considered as a molecular target for HCC detection.

     

LncRNA SNHG12 promotes proliferation and epithelial mesenchymal transition in hepatocellular carcinoma through targeting HEG1 via miR-516a-5p

Hepatocellular carcinoma (HCC) is the most common cancer and its prognosis is poor due to metastasis and recurrence. EMT is associated with metastasis. A deep understanding of regulatory mechanism of EMT is critical. LncRNA is involved in regulation of various biological processes including EMT. This study aimed to investigate the regulatory signal axis among lncRNA SNHG12, miR-516a-5p and the target gene HEG1 during EMT. Cell cycle and apoptosis were analyzed by flow cytometry. Tumorigenesis was analyzed by clone formation assay. Wound healing assay and transwell assay was performed to detect migration and invasion, respectively. Interaction among SNHG12, miR-516a-5p and HEG1 were analyzed by dual luciferase assay and RIP assay. We also detected expression of RNA and protein by QPCR and western blotting. Finally, tumor growth was analyzed by tumorigenesis assay in vivo. Ki-67 and HEG1 level in tumor tissues was analyzed by IHC. SNHG12 and HEG1 were upregulated, miR-516a-5p was downregulated in HCC cell lines. SNHG12 could interact with and inhibit miR-516a-5p. MiR-516a-5p could interact with HEG1 and inhibit HEG1 expression. Knock down SNHG12 inhibited proliferation, migration, invasion, EMT and promoted apoptosis of HCC cells. Such effects were antagonized by inhibiting miR-516a-5p. SNHG12 overexpression lead to opposite results. Similar results were observed in mice. SNHG12 could promote EMT in HCC through targeting and inhibiting miR-516a-5p, which eventually upregulated HEG1 expression, in both cell and mice.