Department of Gastroenterology, Changzheng Hospital, Second Military Medical University, Shanghai, China
Address reprint requests to: Wei-Fen Xie, M.D., Ph.D., Department of Gastroenterology, Changzheng Hospital, Second Military Medical University, 415 Fengyang Road, Shanghai 200003, China. E-mail: email@example.com; fax: +86-21 8188-9624.
Potential conflict of interest: Nothing to report.
This work was supported by grant no. 81071842, Distinguished Young Scholars (30825020), Key Program (81230011), and Creative Research Groups (81221061) from the National Natural Science Foundation of China and grants from Shanghai Science and Technology Committee for the key projects (11JC1416200 and 10431903600) and the Rising-Star Program (11QA1408800).
MicroRNA 370 (miR-370) is located within the DLK1/DIO3 imprinting region on human chromosome 14, which has been identified as a cancer-associated genomic region. However, the role of miR-370 in malignances remains controversial. Here, we report that miR-370 was repressed in human hepatocellular carcinoma (HCC) tissues and hepatoma cell lines. Using gain-of-function and loss-of-function experiments, we demonstrated that miR-370 inhibited the malignant phenotype of HCC cells in vitro. Overexpression of miR-370 inhibited growth and metastasis of HCC cells in vivo. Moreover, the RNA-binding protein, LIN28A, was identified as a direct functional target of miR-370, which, in turn, blocked the biogenesis of miR-370 by binding to its precursor. LIN28A also mediated the suppressive effects of miR-370 on migration and invasion of HCC cells by post-transcriptionally regulating RelA/p65, which is an important effector of the canonical nuclear factor kappa B (NF-κB) pathway. Interleukin-6 (IL-6), a well-known NF-κB downstream inflammatory molecule, reduced miR-370 but increased LIN28A levels in HCC. Furthermore, miR-370 levels were inversely correlated with LIN28A and IL-6 messenger RNA (mRNA) levels, whereas LIN28A mRNA expression was positively correlated with IL-6 expression in human HCC samples. Interestingly, reduction of miR-370 expression was associated with the development of HCC in rats, as well as with aggressive tumor behavior and short survival in HCC patients. Conclusions: These data demonstrate the involvement of a novel regulatory circuit consisting of miR-370, LIN28A, RelA/p65 and IL-6 in HCC progression. Manipulating this feedback loop may have beneficial effect in HCC treatment. (Hepatology 2013; 58:1977–1991)
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nonobese diabetic severe compromised immunodeficient
plasmid DNA with a cytomegalovirus promoter
polymerase chain reaction
RNA-binding protein immunoprecipitation assay
short interfering RNA
tumor necrosis factor alpha
Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, especially in Asia. Most HCCs develop on a background of chronic inflammation caused by hepatitis virus, toxins, metabolic impairment, or autoimmune hepatopathy. Inflammatory molecules can provide signals that promote the proliferation and metastasis of HCC cells.[2, 3] The transcription factor, nuclear factor kappa B (NF-κB), is a key modulator of inflammatory response and plays a pivotal role in the regulation of inflammatory signal transduction pathways in the liver. Activation of NF-κB is also widely viewed as a link between inflammation and the pathogenesis of various cancers, including HCC.[4, 5]
MicroRNAs (miRNAs) are a class of small noncoding RNA molecules that regulate post-transcriptional events. Aberrant expression of many miRNAs is implicated in the onset and development of HCC.[7, 8] MicroRNA 370 (miR-370) is located within the DLK1/DIO3 imprinting region on human chromosome 14. It was first cloned from human embryonic stem cells, but had a very low expression level. Several studies have identified the DLK1/DIO3 domain as a cancer-associated genomic region, implicating the involvement of miR-370 in cancer pathogenesis. Nevertheless, the role of miR-370 in malignances remains controversial. Substantial evidence demonstrates that miR-370 serves as a tumor suppressor in malignant cholangiocytes,[12, 13] leukemia cells, and oral squamous carcinoma cells. In contrast, several studies have reported that overexpression of miR-370 contributes to the progression of gastric carcinoma, prostate cancer, and acute myeloid leukemia.[16-18] In addition, miR-370 has been shown to affect lipid metabolism in the liver by directly targeting carnitine palmitoyl transferase 1 alpha (Cpt1α) and up-regulating liver-enriched miRNA miR-122, indicating that miR-370 may be important for hepatic function.
Lin28, consisting of Lin28 homolog A (Lin28A) and its homolog, Lin28B, is a functionally conserved RNA-binding protein originally characterized in Caenorhabditis elegans as a major regulator of developmental timing.[20, 21] Emerging evidence suggests that Lin28 plays crucial roles not only in development, but also in pluripotency, metabolism, and carcinogenesis in mammals. Despite its wide expression in the early stage of developing tissues, Lin28 is undetectable in most adult organs. Interestingly, both LIN28A and LIN28B are up-regulated in diverse human malignancies, including ovarian, breast, colon, lung, and liver cancer, as well as in chronic myeloid leukemia and germ cell tumors.[23-26] Higher expression of LIN28A/LIN28B is associated with more-advanced tumor grade and poorer prognosis.[23, 27] Functional studies have also suggested that LIN28A and LIN28B facilitate the carcinogenesis and development of cancers, including HCC.[23, 24, 26, 28-32] Both LIN28A and LIN28B promote the proliferation of HCC cells, whereas LIN28B also enhances the transformation and invasion of HCC.[23, 24, 31, 32] However, the tumor-promoting mechanisms of LIN28 in HCC remain largely unknown.
In this study, we clarified the role of miR-370 in HCC and elucidated the contribution of the miR-370/LIN28A/NF-κB circuit to the progression of HCC. We speculate that manipulation of this feedback loop could be explored as a novel strategy for the treatment of HCC.
Materials and Methods
Human liver tissue samples (excluding the samples on the tissue microarray) were obtained from patients who underwent surgical resection and were diagnosed by professional pathologists at the Eastern Hepatobiliary Surgery Hospital (Shanghai, China) and Changzheng Hospital (Shanghai, China), with written informed consent. HCC tissues with typical macroscopic features were collected from the central part of tumor nodules, which were also examined with hematoxylin and eosin (H&E) staining to confirm the diagnosis. The paired adjacent nontumoral tissues without histopathologically identified tumor cells were collected from at least 5 cm away from the tumor border. All human experiments were approved by the ethics committee of the Second Military Medical University (Shanghai, China).
To detect the effect of miR-370 on tumorigenicity in vivo, HCC cells infected with adenovirus expressing miR-370 (Ad-miR-370) or control virus adenovirus containing green fluorescent protein (Ad-GFP) were transplanted subcutaneously (SC) into both flanks of Balb/c nude mice. To explore the effect of miR-370 on metastasis, MHCC-LM3 cells stably expressing luciferase and infected with Ad-GFP or Ad-miR-370 were injected by the tail vein into nonobese diabetic severe compromised immunodeficient (NOD/SCID) mice. Mice were monitored using the IVIS200 imaging system (Caliper Life Sciences, Hopkinton, MA) once a week and sacrificed 8 weeks after cell transplantation. Tumor nodules on the lungs were counted and histopathologically analyzed with H&E staining. To investigate the antitumor effect of miR-370 in vivo, Ad-miR-370 or Ad-GFP was injected into the xenografts of an SC-implanted tumor model in Balb/c nude mice. All animal experiments were performed according to protocols approved by the institutional animal care and use committee at the Second Military Medical University.
RNA-Binding Protein Immunoprecipitation Assay
RNA-binding protein immunoprecipitation (RIP) assays were performed as described previously, with minor modifications. Briefly, Lin28A primary antibody (ab46020; Abcam, Cambridge, MA) was used for endogenous LIN28A immunoprecipitation (IP) in PLC/PRF/5 cells; anti-FLAG Ab-conjugated agarose beads (A2220; Sigma-Aldrich, St. Louis, MO) were used for IP of ectopically expressing LIN28A in MHCC-97H cells transfected with pFlag-cytomegalovirus (CMV)-2 empty vector, pFlag-CMV-LIN28A vector, and pFlag-CMV-LIN28A vector with C161A mutation.
In Situ miRNA Hybridization Assay
Tissue microarray slides of 50 paired HCC and adjacent cancer-free samples were obtained from Xinchao Biotechnology (Shanghai, China). In situ miRNA hybridization (ISH) assays were performed according to the manufacturer's instructions. Stained slides were scanned using a ScanScopeXT (Aperio Technologies, Vista, CA) scanner and analyzed with Aperio Spectrum software (Aperio Technologies).
All statistical analyses were performed using SPSS software (version 17.0; SPSS, Inc., Chicago, IL). Statistical tests for data analysis included two-tailed Student t, log-rank, Wilcoxon's matched pairs, Mann-Whitney's U, and chi-square tests. A P value <0.05 was considered statistically significant.
Detailed materials and methods can be found in the Supporting Materials.
miR-370 Inhibits Malignant Phenotype of HCC Cells In Vitro
We examined miR-370 expression in 20 paired primary HCC and surrounding nontumorous liver specimens. miR-370 was down-regulated (HCC/nontumorous specimens <0.5) in 16 cases (80%; Fig. 1A). We also detected miR-370 expression in nontransformed immortalized human hepatocytes (IMH) and hepatoma cell lines. miR-370 levels were significantly decreased, relative to the IMH control, in all of the 11 tested hepatoma cell lines (Supporting Fig. 1A). Enforced expression of miR-370 by transfecting cells with plasmid DNA with a cytomegalovirus promoter (pcDNA)-miR-370 reduced colony formation of HCC cells (Fig. 1B and Supporting Fig. 1B). Overexpression of miR-370 significantly decreased proliferation of HCC cells (Fig. 1C and Supporting Fig. 1C,D). In contrast, inhibition of miR-370 enhanced cell proliferation (Fig. 1D and Supporting Fig. 1E). Flow cytometry assay showed that overexpression of miR-370 promoted apoptosis of HCC cells, whereas inhibition of miR-370 attenuated serum starvation-induced apoptosis of HCC cells (Supporting Fig. 2). We also examined the effects of miR-370 on migration and invasion of the highly invasive MHCC-LM3 and YY-8103 cells. miR-370 overexpression markedly reduced, whereas miR-370 inhibition increased, migration and invasion of these cells (Fig. 1E,F and Supporting Fig. 3A-D). Interestingly, miR-370 inhibition also markedly enhanced migration and invasion of IMH cells (Supporting Fig. 3E,F).
miR-370 Suppresses Growth and Metastasis of HCC Cells In Vivo
To further investigate the effect of miR-370 on tumorigenesis of HCC cells in vivo, MHCC-97H or YY-8103 cells infected with Ad-miR-370 or control adenovirus Ad-GFP were SC transplanted into the flanks of Balb/c nude mice. Xenografts were detected in 37.5% (3 of 8) of mice as early as day 14 and in all subjects by day 33 after inoculation in mice receiving MHCC-97H cells infected with Ad-GFP (Fig. 2A). No xenografts were observed until day 33 in mice receiving MHCC-97H cells infected with Ad-miR-370, and only small nodules were identified in 50% (4 of 8) of mice by day 38 (Fig. 2A). Xenografts were significantly smaller in the Ad-miR-370 group, compared to the control group, at every time point (Supporting Fig. 4A). Consistently, xenograft weight was significantly reduced in the Ad-miR-370 group (Fig. 2B). Real-time polymerase chain reaction (PCR) analysis showed a significant increase in miR-370 levels in the Ad-miR-370 group, relative to the Ad-GFP control (Supporting Fig. 4B). Similar results were obtained with YY-8103 cells (Supporting Fig. 4C,D). We further investigated the effect of miR-370 on HCC metastasis in vivo in NOD/SCID mice injected with luciferase-labeled MHCC-LM3 cells infected with Ad-miR-370 or Ad-GFP. Luciferase signals were detected in lungs in all mice in the Ad-GFP group by ex vivo imaging, but in only 2 of 5 mice in the Ad-miR-370 group 8 weeks after cell transplantation (Fig. 2C and Supporting Fig. 4E). Number of tumor foci on lungs was also significantly reduced in the Ad-miR-370 group (Fig. 2D). Histologic analysis confirmed reduced tumor foci, which were composed of paratypic HCC cells in the Ad-miR-370 group (Fig. 2D). We then explored the antitumor effect of miR-370 on an established HCC cell transplanted SC tumor model in Balb/c nude mice. Intratumoral injection of Ad-miR-370 significantly reduced the growth and weight of MHCC-97H xenografts (Fig. 2E and Supporting Fig. 4F). Real-time PCR confirmed the increased expression of miR-370 in Ad-miR-370-treated tumor nodules (Supporting Fig. 4G). Histological analysis revealed that the tumor nodules were composed of HCC cells arranged in a trabecular pattern, as proved by H&E staining (Fig. 2F). Additionally, Ad-miR-370-treated tumor nodules displayed decreased Ki-67 expression (Fig. 2F) and contained more apoptotic cells (Supporting Fig. 5).
LIN28A Is a Functional Mediator of miR-370
To investigate the underlying molecular mechanisms by which miR-370 exerts its antitumor effect, we screened for putative targets of miR-370 by performing an in silico complementarity search using TargetScan (www.targetscan.org/) and PicTar (http://picta.mdc-berlin.de/). This approach identified LIN28A, an evolutionarily conserved molecule across many species, as a potential downstream target of miR-370 (Fig. 3A and Supporting Fig. 6A). LIN28A messenger RNA (mRNA) and protein levels were decreased in HCC cells by ectopic expression of miR-370 (Fig. 3B) and increased by miR-370 inhibitor (Fig. 3C). Immunohistochemical (IHC) analysis also revealed decreased LIN28A in Ad-miR370-treated MHCC-97H xenografts (Supporting Fig. 6B). Reporter assay revealed that overexpression of miR-370 decreased the luciferase activity of the wild-type (WT) LIN28A 3′ untranslated region (UTR) by 59.4% (P < 0.0001; Fig. 3D). Deletion or point mutation of the target sequence on the LIN28A 3′ UTR diminished the effect of miR-370 on LIN28A, indicating that LIN28A is a direct downstream target of miR-370 (Fig. 3D and Supporting Fig. 6C,D).
Enforced expression of LIN28A promoted proliferation of MHCC-97H cells, whereas knockdown of LIN28A inhibited their proliferation (Supporting Fig. 7A,B). In addition, overexpression of LIN28A significantly augmented, whereas down-regulation of LIN28A suppressed, migration and invasion of HCC cells (Supporting Fig. 7C,D). Importantly, the suppressive effects of miR-370 on migration and invasion of HCC cells were substantially reduced by infection with a lentiviral expression vector of LIN28A without the 3′ UTR (Fig. 3E,F and Supporting Fig. 7E). Overall, these findings demonstrate that down-regulation of LIN28A contributes to the functional role of miR-370 in HCC cells.
LIN28A Blocks Maturation of miR-370
LIN28 has been shown to function as an oncoprotein by forming a double-negative feedback loop with let-7 in breast cancer. Identification of LIN28A as a target of miR-370 in HCC cells raises the possibility that LIN28A may block the biogenesis of miR-370. Indeed, our results showed that overexpression of LIN28A significantly decreased miR-370 level, whereas substitution of a single amino acid (C161A) required for the RNA-binding affinity of LIN28A efficiently reversed the effect of LIN28A on miR-370 (Fig. 4A). As a positive control, let-7 level was also reduced upon ectopic expression of LIN28A, but not by C161A mutation (Fig. 4A). However, as a negative control, miR-21 level was not influenced by LIN28A (Fig. 4A). In contrast, knockdown of LIN28A by small interfering RNA (siRNA) substantially raised levels of miR-370 and let-7, but not miR-21 (Fig. 4B). RIP assay revealed that both miR-370 and let-7 precursors, but not miR-21 precursor, were highly enriched in LIN28A immunoprecipitates from PLC/PRF/5 cells (Fig. 4C), suggesting direct binding between endogenous LIN28A and pre-miR-370 in HCC cells. To confirm the specificity of binding, MHCC-97H cells were transfected with Flag-LIN28A or empty vector. Subsequent RIP assay displayed significant enrichment of pre-miR-370 and pre-let-7 in Flag-LIN28A immunoprecipitates, which were abolished by C161A mutation (Fig. 4D).
LIN28A Activates NF-κB Pathway by Posttranscriptional Regulation of RelA/p65
NF-κB has been reported to transcriptionally activate the expression of LIN28B, but not LIN28A, in breast cancer.[28, 34] However, the effect of LIN28 on NF-κB has not been reported. Our experiments showed that LIN28A overexpression enhanced, whereas LIN28A knockdown suppressed, the activity of a NF-κB luciferase reporter in HCC cells (Fig. 5A and Supporting Fig. 8A). LIN28A inhibition resulted in down-regulation of NF-κB target genes, including interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and matrix metalloproteinase 9 (MMP-9; Fig. 5B), indicating that LIN28A is implicated in activation of the NF-κB pathway in HCC.
LIN28A is a post-transcriptional modulator of mRNAs, and we therefore sought to determine its effect on the translation of RelA/p65, which plays an important role in canonical NF-κB pathway transduction. Real-time PCR failed to detect any significant effect of LIN28A on RelA/p65 mRNA levels (data not shown). However, RelA/p65 protein levels were significantly increased by LIN28A overexpression and decreased by LIN28A knockdown (Fig. 5C). Direct binding of RelA/p65 mRNA and LIN28A, which was abolished by C161A mutation, was detected by RIP assay (Fig. 5D,E). Furthermore, LIN28A overexpression increased, whereas LIN28A repression decreased, the activity of the luciferase reporter gene carrying the RelA/p65 3′ UTR (Fig. 5F). Interestingly, the effects of LIN28A on HCC cell migration and invasion were reversed by inhibition of RelA/p65 NF-κB transcriptional activity with oridonin and 4-methyl-N1-(3-phenylpropyl)benzene-1,2-diamine (JSH-23; Supporting Fig. 8B-E). Overall, these findings suggest that direct binding of LIN28A to RelA/p65 mRNA promotes the translation of RelA/p65, which contributes, at least in part, to the functional role of LIN28A in HCC.
Positive Feedback Loop Involving miR-370, LIN28A, RelA/p65, and IL-6 Aggravates Malignant Phenotype of HCC Cells
In view of the effect of LIN28A on the NF-κB pathway, we speculated that miR-370 may exert its inhibitory effect on HCC by suppression of the NF-κB pathway. As expected, miR-370 overexpression decreased, whereas miR-370 inhibition increased, RelA/p65 protein expression and activity of the NF-κB luciferase reporter in HCC cells (Fig. 6A,B), but RelA/p65 mRNA was unaffected (data not shown). RelA/p65 protein levels were also repressed in MHCC-97H xenografts treated with Ad-miR-370 (Supporting Fig. 9A). Consistently, ectopic expression of miR-370 led to down-regulation of NF-κB target genes (i.e., IL-6, TNF-α, and MMP-9), whereas inhibition of miR-370 exerted the opposite effect (Fig. 6C). Reduced expression of these NF-κB target genes was also observed in MHCC-97H xenografts treated with Ad-miR-370 (Supporting Fig. 9B). Interestingly, the effect of miR-370 on RelA/p65 protein level, activity of the NF-κB luciferase reporter, and NF-κB downstream genes in HCC cells could be abrogated by nontargetable LIN28A (Fig. 6D and Supporting Fig. 9C-E). More important, effects of miR-370 on HCC cell migration and invasion were abrogated by oridonin or JSH-23 (Supporting Fig. 10), supporting the hypothesis that the NF-κB pathway is involved in the suppressive effects of miR-370 on HCC.
IL-6 is a downstream target gene of NF-κB that plays a crucial role in hepatocarcinogenesis.[4, 5] Previous studies reported that IL-6 inhibited miR-370 through modulation of DNA methylation in human cholangiocarcinoma (CCA).[12, 13] In this study, we confirmed that treatment of HCC cells with IL-6 significantly decreased miR-370 levels, followed by an increase in LIN28A protein (Fig. 6E and Supporting Fig. 11A,B). Furthermore, the methylation inhibitor, 5-aza-2′-deoxycytidine markedly increased expression of primary miR-370 (Supporting Fig. 11C), suggesting that miR-370 is down-regulated in HCC in an epigenetic manner. These results indicate that a positive feedback loop, consisting of miR-370, LIN28A, RelA/p65, and IL-6, is involved in the progression of HCC (Fig. 6F).
Down-Regulation of miR-370 Is Associated With Development of HCC in Rats and With Aggressive Tumor Behavior and Short Survival in HCC Patients
We further validated the roles of miR-370 and LIN28A in the development of HCC by using real-time PCR to examine miR-370 and LIN28A mRNA levels in liver tissues from diethylinitrosamine (DEN)-treated rats, 86 paired primary HCC and adjacent nontumorous liver tissues from primary HCC patients with complete clinical data (excluding the 20 pairs of samples referred to above), and 24 healthy human liver tissue samples. miR-370 expression was significantly down-regulated in cirrhotic liver tissues from rats at week 11 after DEN administration, compared to normal rat livers, and was further reduced in HCC tissues, relative to adjacent fibrotic tissues (Fig. 7A). In contrast, LIN28A mRNA was gradually up-regulated during the development of DEN-induced HCC (Fig. 7B). Consistently, miR-370 expression was substantially repressed in the surrounding nontumorous livers from HCC patients, compared to healthy human livers (median, 0.859 and 0.003, respectively; P < 0.001; Mann-Whitney's U test), and was further reduced in HCC tissues (Fig. 7C). In contrast, LIN28A mRNA levels were increased 19-fold in nontumorous livers, compared to healthy livers. LIN28A mRNA was only slightly augmented in HCC tissues, relative to surrounding nontumorous tissues (median, 3.59 × 10−4 and 6.08 × 10−4, respectively; Fig. 7D). Correlation studies displayed that miR-370 levels were inversely correlated with LIN28A and IL6 mRNA levels in human HCC specimens (Fig. 7E), and LIN28A expression was positively correlated with IL-6 expression (Fig. 7E). Specifically, low expression of miR-370 was more likely in human HCC specimens with high levels of LIN28A and IL-6 mRNAs, whereas high expression of LIN28A was more likely in human tissues with high IL-6 levels. More interesting, clinicopathologic analysis demonstrated that down-regulation of miR-370 in human HCCs was significantly correlated with aggressive pathologic characteristics, including larger tumor size (P = 0.028), advanced tumor stage (P = 0.004), presence of venous invasion (P = 0.003), tumor microsatellite formation (P = 0.009), and presence of capsular invasion (P = 0.003; Table 1). To further validate the relation between miR-370 levels and survival of HCC patients, a tissue microarray, which contained 50 paired HCC samples and adjacent cancer-free samples obtained from HCC patients with a median follow-up of 33.5 months (range, 2.0-72.0; standard deviation [SD]: 24.4), was used for in situ hybridization of miR-370. Kaplan-Meier's analysis revealed that lower miR-370 levels were correlated with shorter overall survival (OS) in HCC patients (Fig. 7F).
Table 1. miR-370 Expression Correlates With Aggressive Phenotype
Previous studies have demonstrated reduced miR-370 levels in gastrointestinal stromal tumors, bladder cancer, neuroblastoma cells, and oral squamous cell carcinoma. However, the role of miR-370 in hepatocarcinogenesis remains elusive. The current study revealed that miR-370 expression was gradually reduced during the development of HCC in DEN-treated rats. This decrease in miR-370 was observed in all tested hepatoma cells and in most HCC tumor samples. Moreover, we also demonstrated the suppressive effects of miR-370 on the malignant phenotype of HCC cells both in vitro and in vivo by gain-of-function and loss-of-function experiments. Low expression of miR-370 in HCCs was associated with an aggressive disease phenotype, including advanced tumor stage, larger tumor size, and the presence of venous invasion, microsatellite tumors, and capsular invasion. Importantly, HCC patients with lower levels of miR-370 had shorter OS. All these data suggest that miR-370 may play a crucial role in the carcinogenesis and progression of HCC and may represent a novel therapeutic target and prognostic marker for HCC.
However, our findings seem to be in conflict with other studies that have reported a tumor-promoting function for miR-370.[16-18] miRNAs primarily exert their effects by regulating multiple target mRNAs. Known targets of miR-370 include mitogen-activated protein kinase kinase kinase 8, Wingless-type MMTV integration site family, member 10B, forkhead box M1, insulin receptor substrate 1, transforming growth factor beta receptor II, forkhead box protein O1, neurofibromin 1, and Cpt1α. Most of these targets are implicated in cancer pathogenesis, some as oncogenes and others as tumor suppressors. The opposing effects of miR-370 on tumors may thus be attributed to the different functional natures of their target genes in a given cell type or under specific circumstances, making it a context-dependent effector. The RNA-binding protein, LIN28A, and its paralog, LIN28B, are oncoproteins that are involved in many aspects of malignancies.[23, 24, 26, 28-32] The results of the current study suggested that LIN28A was a bona-fide target of miR-370 and promoted the proliferation, migration, and invasion of HCC cells. More important, the nontargetable LIN28A reversed the miR-370-mediated suppression of HCC cell migration and invasion, suggesting that inhibition of the oncoprotein, LIN28A, contributes to the suppressive effects of miR-370 on HCC. The involvement of LIN28A may thus explain, at least in part, the inhibitory roles of miR-370 in HCC.
Lin28 promotes tumor development in at least two independent manners. First, it selectively blocks the biogenesis of a class of miRNAs, such as let-7. Second, it acts as a post-transcriptional regulator by directly binding specific mRNAs. The Lin28/let-7 double-negative feedback loop is one of the best-characterized examples of the modulation between an miRNA and its post-transcriptional regulator. To our knowledge, let-7 is the only miRNA that has been reported to interact reciprocally with Lin28. The current study demonstrated that LIN28A blocked the biogenesis of miR-370 by binding to its precursor. The mutual regulation of LIN28A and miR-370 thus represents another paradigm of the direct interaction between LIN28 and miRNA. The identification of this novel LIN28A/miRNA loop suggests that the double-negative feedback loop between tumor-suppressive miRNA and LIN28A may be a ubiquitous phenomenon in cancer pathogenesis. On the other hand, direct translational modulation of mRNAs is another crucial mechanism by which Lin28 regulates gene expression. Most documented mRNA targets of LIN28A, including insulin-like growth factor-2, Oct4, cyclin A, cyclin B, cyclin-dependent kinase 4, and human epidermal growth factor receptor 2, are important for cell growth, metabolism, and cancer development.[21, 26, 40] Interestingly, we demonstrated that direct binding of LIN28A to RelA/p65 mRNA promoted the translation of RelA/p65. RelA/p65 is the key subunit of the NF-κB family, which functions as an important promoter of liver carcinogenesis. Thus, post-transcriptional modulation of this crucial oncoprotein represents a novel and important mechanism whereby LIN28A may exert its tumor-promoting function, in addition to its effect on miRNAs.
Most cases of HCC arise in cirrhotic livers with persistent inflammation. Deeper understanding of the mechanistic link between inflammation and HCC would help to identify potential therapeutic targets for HCC. Proinflammatory transcription factors, such as NF-κB and signal transducer and activator of transcription 3, and nontranscriptional elements, such as miRNAs, often cooperate in the regulatory networks that link inflammation to cancers.[28, 41, 42] The results of our current study demonstrated that miR-370 suppressed the NF-κB pathway by inhibiting LIN28A, and the biological functions of both miR-370 and LIN28A were reversed by inactivation of the NF-κB pathway. IL-6 is a well-known target of NF-κB and plays a crucial role in inflammation, wound healing, and hepatocarcinogenesis.[4, 5] Consistent with previous studies in CCA that demonstrated reduced miR-370 expression by IL-6 through modulation of DNA methylation,[12, 13] we also observed a decrease in miR-370 levels, followed by an increase in Lin28A protein levels, induced by IL-6 in HCC cells. Therefore, we identified a novel regulatory circuit in HCC consisting of miR-370, LIN28A, RelA/p65, and IL-6. This regulatory loop is perturbed in human HCC tissues, suggesting that the self-reinforcing regulatory feedback circuit is involved in the progression of HCC.
In conclusion, the present study highlights the biological significance of miR-370 in HCC and elucidates a previously unrecognized molecular mechanism underlying the development of HCC. These findings suggest that early intervention to disrupt this loop may have therapeutic potential for HCC.