MicroRNA-196a/-196b regulate the progression of hepatocellular carcinoma through modulating the JAK/STAT pathway via targeting SOCS2

microRNAs (miRNAs) play essential roles in progression of hepatocellular carcinoma (HCC). However, the roles of miR-196a and miR-196b as well as mechanism in HCC progression remain poorly understood. The expressions of miR-196a, miR-196b and suppressor of cytokine signaling 2 (SOCS2) were measured in HCC tissues and cells by quantitative real-time polymerase chain reaction or immunohistochemistry. HCC progression was investigated by cell proliferation, glycolysis, cycle, clones, apoptosis, and necrosis. The interaction between SOCS2 and miR-196a or miR-196b was explored by luciferase activity and RNA immunoprecipitation analyses. The expressions of proteins in Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway were measured by western blot. A xenograft model was established to investigate the roles of miR-196a or miR-196b in vivo. We found that miR-196a and miR-196b were highly expressed in HCC tissues and cells. High expression of miR-196a or miR-196b was correlated with tumor size, tumor-node-metastasis stage, lymph node metastasis, albumin–bilirubin grade and poor 5-year survival. Knockdown of miR-196a or miR-196b suppressed cell proliferation, glycolysis, cell cycle process, colony formation but induced apoptosis or necrosis in HCC cells. SOCS2 was targeted by miR-196a and miR-196b and its interference ablated abrogation of miR-196a or miR-196b-mediated inhibitory effect on HCC progression. SOCS2 was negatively associated with activation of the JAK/STAT pathway. Besides, knockdown of miR-196a or miR-196b limited xenograft tumor growth by blocking the JAK/STAT pathway. We concluded that downregulation of miR-196a or miR-196b inhibited HCC progression through regulating the JAK/STAT pathway via targeting SOCS2, providing novel targets for prognosis and therapeutics of HCC.


Introduction
Hepatocellular carcinoma (HCC) is one of the most common malignant tumors and the third leading cause of cancer death with rising incidence worldwide 1 . Despite great advances in diagnosis and treatment of HCC, the recurrence rate remains more than 80% and the survival rate of patients is still unsatisfactory 2 . Hence, it is urgent to explore novel strategies for diagnosis and therapeutics of HCC. microRNAs (miRNAs) are a class of small noncoding RNAs with~23 nucleotides in length, which play essential roles in development and progression of HCC 3 . miR-196 has been suggested to have an impact on the outcome of development and therapeutics of cancers 4 . Moreover, accruing works have indicated miR-196 as an oncogene to promote progression of various cancers, including glioblastoma, breast cancer, oral cancer, and digestive tract cancers [5][6][7][8] . The miR-196 family consists of miR-196a and miR-196b sharing regulatory efficacy 9 . Previous study has reported that miR-196a is notably upregulated in HCC tissues compared with that in matched nontumor samples 10 . Furthermore, miR-196b has been suggested to be highly expressed in HCC tissues and associated with HCC risk factors 11 . However, little is known about whether and how miR-196a and miR-196b could regulate HCC progression.
The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway has been regarded as one of the main molecular pathways in HCC progression 12 . STAT proteins, including STAT1-6, exhibit promoting or suppressive effect on antiviral response, inflammation, and tumorigenesis in liver, one of which, STAT5, could promote liver damage 13 . Suppressor of cytokine signaling (SOCS) family, such as SOCS1, SOCS2, and SOCS3, has been indicated as key negative regulator of the JAK/STAT signaling pathway in HCC [14][15][16] . The available evidence indicated the importance of SOCS2 in various diseases and cancers by regulating biological processes through addressing the JAK/STAT pathway or other signaling 17 . Hence, we hypothesized that miR-196a and miR-196b might regulate progression of HCC by targeting SOCS2. In the present study, we measured the expression of miR-196a and miR-196b and explored their roles as well as underlying mechanism in HCC progression.

Patients and tissue samples
A total of 72 patients with HCC who had not received any chemotherapy, radiotherapy, or related treatment before surgery were recruited from Luoyang Central Hospital Affiliated to Zhengzhou University. Written informed consent was obtained from all participants and the work was accepted by the Research Ethics Committee of Luoyang Central Hospital Affiliated to Zhengzhou University. Paired tumor and peri-tumor samples collected from patients were collected for hematoxylin and eosin (H&E) staining or immediately stored at −80°C until used. The clinicopathological characteristics of patients are shown in Table 1. The expression data of miR-196a and miR-196b were provided from database of YM500v3 (http://driverdb.tms.cmu.edu.tw/ym500v3/). The 5-year survival rate was investigated in every group. The correlation between clinicopathological characteristics of patients and miR-196a or miR-196b expression is presented in Tables 2 and 3. The liver damage was investigated according to the albumin-bilirubin (ALBI) score 18 .
Quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was isolated from tissues or cells by using TRIzol reagent (Invitrogen) following the manufacturer's instructions. The TaqMan microRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) or M-MLV Reverse Transcription Kit (Invitrogen) was used for synthesis of complementary DNA (cDNA). The diluted cDNA was amplified using SYBR green (Applied Biosystems) with the following amplification protocol: 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 1 min. The expressions of miR-196a, miR-196b, and SOCS2 were normalized by U6 small RNA or β-actin with 2 −ΔΔCt method 19 . All primers were obtained from Sangon   (Forward, 5′-CAGC CTTCCTTCTTGGGTAT-3′ Rev erse, 5′-TGGCATAGAGGTCTTTACGG-3′).

Measurement of glucose consumption and lactate production
SMMC-7721 and HepG2 cells were seeded in 96-well plates overnight. After 72 h, cells were washed three times in phosphate buffer saline (Gibco), and then collected for analyses of glucose consumption and lactate production via using the Glucose Uptake Colorimetric Assay Kit or Lactate Assay Kit (Sigma, St. Louis, MO, USA) according to the manufacturer's instructions, respectively. The concentrations of glucose consumption and lactate production were measured by using a microplate reader and normalized by the total protein concentration determined via the bicinchoninic acid (BCA) assay kit (Thermo Fisher).

Flow cytometry
Flow cytometry was conducted to measure cell apoptosis or necrosis. After 72 h, transfected cells were washed with PBS. Cells were resuspended in binding buffer and double stained with the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (Sigma) according to the manufacturer's instructions. The apoptotic or necrotic cells were analyzed through a flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) with CellQuest software.

RNA immunoprecipitation (RIP)
The RIP assay was conducted in HepG2 cells by using the RNA-binding protein immunoprecipitation kit (Millipore) according to the manufacturer's protocols. HepG2 cells transfected with miR-196a, miR-196b, or miR-NC were lysed in RIP buffer with anti-Ago2-or IgG-bound magnetic beads. The mRNA level of SOCS2 enriched on beads was measured by qRT-PCR after treatment of TRIzol reagent.

Immunohistochemistry
Tumor and peri-tumor tissues were fixed with 4% polyoxymethylene (Sigma), embedded in paraffin and sectioned to 4 μm thick. Following blocking endogenous peroxidase using 3% H 2 O 2 (Sigma), sections were incubated with primary antibodies against SOCS2 (ab74533, 1:100 dilution, Abcam, Cambridge, UK) for 2 h and incubated with antirabbit IgG for 30 min, followed by stained with diaminobenzidine and hematoxylin (Sigma). The positive cells were detected under a light microscopy (Olympus).

Murine xenograft model
BALB/c nude mice (male, four-week-old) were obtained from Vital River Laboratory Animal Technology (Beijing, China) and housed in specific pathogen-free conditions with a 12 h light/dark cycle and free access to water and food. Every experiment was made to minimize animals (n = 7 per group) under the approval of the Animal Research Committee of Luoyang Central Hospital Affiliated to Zhengzhou University. HepG2 cells were transfected with the lentiviral vectors with anti-miR-196a (Lenti-anti-miR-196a), anti-miR-196b (Lenti-anti-miR-196b), anti-miR-NC (Lenti-anti-miR-NC), or negative control (Lenti-NC) constructed by GeneCopoeia (Rockville, MD, USA). Stably transfected cells (5 × 10 6 ) were subcutaneously injected into the nude mice. Tumor volumes were measured every week for 5 weeks with the formula: volume (mm 3 ) = width 2 × length/2. After 5 weeks following the inoculation, the mice were killed and tumor samples were collected and weighted. The collected tumor tissues were used for further molecular studies. Cell apoptosis and proliferation in tumor tissues were measured by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining with in situ cell death detection kit (Roche, Mannheim, Germany) or Ki67 immunohistochemistry with anti-Ki67 antibody (ab16667, 1:1000 dilution, Abcam) following the manufacturer's instructions as previous study 20 .

Statistical analysis
The data were presented as the mean ± standard deviation (S.D.) from three independent experiments. The survival curve was generated via Kaplan-Meier method. The statistical differences between groups were investigated by Student's t test or one-way analysis of variance (ANOVA) using SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA). P < 0.05 was regarded as statistically significant.
Results miR-196a and miR-196b expressions are enhanced and SOCS2 level is decreased in HCC tissues and cells The morphology investigation of HCC and peri-tumor tissues using H&E staining displayed that stained HCC tissues showed typical HCC morphology with many formed balloon-like lesions and the peri-tumor tissues exhibited normal liver cell morphology (Fig. S1). Bioinformatics analysis suggested that miR-196a and miR-196b were highly expressed in HCC tissues compared with that in normal tissues by YM500v3 ( Fig. S2A and S2B). To explore the roles of miR-196a and miR-196b in HCC progression, their expression levels were measured in HCC tissues. Compared with peri-tumor group, HCC tissues showed higher expression levels of miR-196a and miR-196b (Fig. 1a, b). Similarly, their levels were also increased in HCC cells compared with those in control group (Fig. 1c, d). Furthermore, patients were classified as high expression and low expression group according to the mean value of miR-196a or miR-196b expression level, named as high miR-196a expression (n = 40) and low miR-196a expression (n = 32) or high miR-196b expression (n = 39) and low miR-196b expression (n = 33) group, respectively. Results showed that high expression of miR-196a or miR-196b was associated with tumor size, Fig. 1 The expressions of miR-196a, miR-196b and SOCS2 in HCC tissues and cells. a, b The expressions of miR-196a and miR-196b were measured in tumor and peri-tumor tissues of HCC patients by qRT-PCR. c, d The expressions of miR-196a and miR-196b were detected in HCC cells by qRT-PCR. e, f The overall survival rate of patients was analyzed in low miR-196a, high miR-196a, low miR-196b, or high miR-196b group by Kaplan-Meier method. g-i The expression of SOCS2 was measured in tissues or cells by qRT-PCR or immunohistochemistry. ***P < 0.001 Fig. 2 The effect of miR-196a or miR-196b knockdown on cell proliferation and glycolysis in HCC cells. SMMC-7721 and HepG2 cells were transfected with anti-miR-196a, anti-miR-196b, or their corresponding anti-miR-NC. Their expression levels (a, b), cell proliferation (c, d), glucose consumption and lactate production (e, f) were measured in the two cells by qRT-PCR, the MTT assay, or the special commercial kit. **P < 0.01, ***P < 0.001 tumor-node-metastasis stage, lymph node metastasis, and ALBI grade (Tables 2, 3 and Fig. S2C-F). In addition, high expression of miR-196a or miR-196b displayed lower overall survival rate (P = 0.0025 or 0.0039, respectively) (Fig. 1e, f). Besides, the expression of SOCS2 mRNA and protein were significantly decreased in HCC compared with that in corresponding control (Fig. 1g-i).

miR-196a and miR-196b regulate HCC progression by targeting SOCS2
To explore the potential mechanism underlies miR-196a or miR-196b participating in progression of HCC, the promising targets were wanted in HCC cells. Bioinformatics analysis provided the potential targets by Tar-getScan Release 7.2 (Table 4). Therein, SOCS2 with the potential binding sites is an important target involved in pathogenesis of HCC (Fig. 4a, b). To validate this prediction, luciferase activity and RIP analyses were conducted into HepG2 cells. Results showed that overexpression of miR-196a or miR-196b significantly reduced luciferase activity in HepG2 cells transfected with SOCS2-WT, whereas the efficacy was lost in term of SOCS2-MUT (Fig. 4c, d). Moreover, the enrichment of SOCS2 abundance was notably elevated in miR-196a or miR-196b-transfected group compared with that in miR-NC group after Ago2 RIP, while IgG failed to show efficacy of enrichment (Fig. 4e, f). Meanwhile, SOCS2 displayed higher ability of miR-196a or miR-196b enrichment than SOCS1 (Fig. S4A and S4B). Moreover, western blot data showed that accumulation of miR-196a or miR-196b limited SOCS2 protein level, while deficiency of miR-196a or miR-196b played an opposite effect (Fig. 4g, h).
To investigate whether SOCS2 was required for miR-196a or miR-196b-mediated progression of HCC, SMMC-7721 and HepG2 cells were cotransfected with anti-miR-196a or anti-miR-196b and si-SOCS2, or si-NC. Fig. 3 The effect of miR-196a or miR-196b depletion on cell apoptosis or necrosis in HCC cells. SMMC-7721 and HepG2 cells were treated as described in Fig. 2. a, b Cell apoptosis or necrosis was analyzed in transfected cells by flow cytometry. c, d The expressions of apoptosis-related proteins were measured in SMMC-7721 and HepG2 cells by western blot. ***P < 0.001, ****P < 0.0001 Fig. 4 The association of SOCS2 and miR-196a or miR-196b. a, b The potential binding sites of SOCS2 and miR-196a or miR-196b were predicted by TargetScan Release 7.2. c, d Luciferase activity was analyzed in HepG2 cells cotransfected with SOCS2-WT or SOCS2-MUT and miR-196a, miR-196b or miR-NC. e, f The enrichment of SOCS2 was detected in HepG2 cells transfected with miR-196a, miR-196b or miR-NC after Ago2-RIP. g, h The expression of SOCS2 protein was detected in the two cells transfected with miR-196a, miR-196b, anti-miR-196a, anti-miR-196b, or their corresponding negative control by western blot. ***P < 0.001, ****P < 0.0001  Fig. S5A-D).

Depletion of miR-196a or miR-196b inhibits HCC progression by regulating the JAK/STAT pathway
To explore the potential signaling pathway participating in HCC progression in this study, the expressions of p-STAT5, STAT5, p-JAK2, and JAK2 proteins were measured in SMMC-7721 and HepG2 cells transfected with SOCS2, pcDNA, si-SOCS2, or si-NC. As a result, the abundance of SOCS2 protein was effectively elevated by transfection of SOCS2 expression vector and reduced by introduction of si-SOCS2 compared with their corresponding control, respectively (Fig. 7a, b). Moreover, overexpression of SOCS2 inhibited phosphorylation of JAK2 and STAT5, whereas its knockdown promoted the expressions of p-JAK2 and p-STAT5 in SMMC-7721 and HepG2 cells (Fig. 7a, b).
To further investigate the roles of miR-196a and miR-196b in HCC, the murine xenograft model was established by subcutaneously injecting with HepG2 cells infected with Lenti-NC, Lenti-anti-miR-NC, Lenti-anti-miR-196a, or Lenti-anti-miR-196b. Results displayed that knockdown of miR-196a or miR-196b decreased tumor volume and weight compared with treatment of Lenti-anti-miR-NC (Fig. 8a, b). Then the expression of miR-196a or miR-196b was measured in tumor tissues. Impaired expression of miR-196a or miR-196b was Fig. 5 The regulatory role of SOCS2 interference in miR-196a-mediated HCC progression. SMMC-7721 and HepG2 cells were transfected with anti-miR-NC, anti-miR-196a, or along with si-NC or si-SOCS2. The SOCS2 protein level a, cell proliferation b, glucose consumption c, lactate production d, and apoptosis e, f were detected in the transfected cells by western blot, the MTT assay, the commercial kit, or flow cytometry. *P < 0.05, **P < 0.01, ***P < 0.001 exhibited in Lenti-anti-miR-196a or Lenti-anti-miR-196b compared with that in Lenti-anti-miR-NC group, respectively (Fig. 8c). Subsequently, the abundances of p-STAT5, STAT5, p-JAK2, JAK2, and SOCS2 proteins were measured in tumor tissues. Results revealed that inhibition of miR-196a or miR-196b promoted SOCS2 protein level but blocked phosphorylation of JAK2 and STAT5 (Fig. 8d). In addition, tissues showed more apoptotic cells and less cell proliferation in Lenti-anti-miR-196a or Lenti-anti-miR-196b compared with that in Lenti-anti-miR-NC group, respectively (Fig. 8e). Besides, knockdown of miR-196a and miR-196b led to obvious decrease of Bcl-2 protein level and increase of Bax protein abundance in xenograft tumor tissues ( Fig.  S6A and S6B).

Discussion
miRNAs have been reported to play pivotal roles in regulating development, diagnosis, and antitumor treatments of HCC 21 . In this study, we found that miR-196a and miR-196b expressions were enhanced in HCC tissues and cells, which is also in agreement with previous efforts 10, 11 . This indicated that miR-196a and miR-196b might serve as oncogenes to promote HCC progression. However, the exact roles of miR-196a and miR-196b in HCC remain largely unknown. Here we explored the effect of miR-196a or miR-196b on HCC cell progression and first revealed the mechanism possibly associated with the SOCS2/JAK2/STAT5 signaling pathway.
The former finding has suggested that miR-196a knockdown inhibited cell proliferation, colony formation, Fig. 6 The restorative effect of SOCS2 silence in miR-196b-mediated HCC progression. SMMC-7721 and HepG2 cells were transfected with anti-miR-NC, anti-miR-196b, or along with si-NC or si-SOCS2. The SOCS2 protein abundance a, cell proliferation b, glucose consumption c, lactate production d, and apoptosis e, f were analyzed in the two transfected cells by western blot, the MTT assay, the commercial kit, or flow cytometry, respectively. **P < 0.01, ***P < 0.001 migration, and invasion but induced cell apoptosis by targeting forkhead transcription factor O1 (FOXO1) in HCC 22 . Moreover, miR-196a was reported to contribute to cell proliferation through regulating cell cycle via targeting FOXO1 in HCC 23 . As for miR-196b, it was shown to predict poor prognosis and increase cell migration as well as invasion in HCC by regulating FOXP2 24 . Consistently with these reports, we also found that miR-196a and miR-196b knockdown could induce proliferation and apoptosis inhibition and indicate poor outcome of patients. Cell cycle process and colony formation are necessary for normal proliferation, while in this study the two miRNAs knockdown arrested cell cycle process and colony formation. Moreover, elevated glycolysis is an important progress in HCC development, which could support metabolic needs for rapid proliferation of cancer cells 25 . The intrinsic apoptosis pathway is addressed by proapoptotic protein Bax and antiapoptotic protein Bcl-2. The disturbance of Bcl-2/Bax balance contributes to apoptosis production. Our results revealed that knockdown of miR-196a and miR-196b inhibited glycolysis and Bcl-2 expression but promoted Bax level. These all findings suggested the poor outcomes of miR-196a and miR-196b in prognosis and progression of HCC.
Functional miRNAs are known to regulate targets expressions by binding their 3′-UTR sequences 26 . This research explored multiple targets of miR-196a and miR-196b and previous studies have validated the interaction between them and FOXO1, FOXP2, or lysosomeassociated protein transmembrane-4β (LAPTM4B) in HCC [22][23][24]27 . SOCS proteins mediate inflammation and apoptosis via regulating immune homeostasis in human cancers. Although Zhao et al. have revealed SOCS2 as a target of miR-196b in human laryngeal squamous cell carcinoma 28 , here we focused on the interaction between miR-196a or miR-196b and SOCS2, which displayed higher miRNA-bound ability than SOCS1. Increasing evidences demonstrated that SOCS2 might function as a tumor suppressor in HCC by regulating tumor progression [29][30][31][32] . By luciferase activity and RIP assays we identified SOCS2 as a functional target of miR-196a and miR-196b in HCC cells. And recuse experiment by further SOCS2 silence uncovered that miR-196a and miR-196b regulated HCC progression by targeting SOCS2.
Active JAKs recruit STAT proteins to mediate gene transcription, participating in cell proliferation, apoptosis and glucose metabolism. SOCS2 along with other members of SOCS family are regarded as importantly negative regulator of the JAK/STAT pathway 33 . Although STAT3 signaling might be more important than STAT5 in HCC progression and miR-196b could activate STAT3 by targeting SOCS2 in macrophages 34,35 , previous work showed that importance of JAK2/STAT5 signaling in liver metabolism, liver diseases, and HCC 36 . Hence, we Fig. 7 The effect of SOCS2 on the JAK/STAT pathway in HCC cells. a, b The expressions of p-STAT5, STAT5, p-JAJ2, JAK2, and SOCS2 protein were measured in SMMC-7721 and HepG2 cells transfected with SOCS2, pcDNA, si-SOCS2, or si-NC by western blot. **P < 0.01, ***P < 0.001 assumed JAK/STAT signaling was implicated in miR-196a-or miR-196b-mediated progression of HCC. This study uncovered that SOCS2 negatively regulated activation of the JAK/STAT pathway in HCC cells and miR-196a or miR-196b inhibition decreased HCC progression by regulating the SOCS2/JAK/STAT pathway revealed by a xenograft model.
In conclusion, high expressions of miR-196a and miR-196b were shown in HCC and predicted poor outcome of patients. Knockdown of miR-196a or miR-196b inhibited HCC progression, possibly by the SOCS2/JAK2/STAT5 pathway. This study indicates that miR-196a and miR-196b might serve as promising therapeutic therapy for HCC treatment.