MicroRNA-3163 targets ADAM-17 and enhances the sensitivity of hepatocellular carcinoma cells to molecular targeted agents

Molecular targeted agents, such as sorafenib, remain the only choice of an antitumor drug for the treatment of advanced hepatocellular carcinoma (HCC). The Notch signaling pathway plays central roles in regulating the cellular injury/stress response, anti-apoptosis, or epithelial–mesenchymal transition process in HCC cells, and is a promising target for enhancing the sensitivity of HCC cells to antitumor agents. The ADAM metalloprotease domain-17 (ADAM-17) mediates the cleavage and activation of Notch protein. In the present study, microRNA-3163 (miR-3163), which binds to the 3′-untranslated region of ADAM-17, was screened using online methods. miRDB and pre-miR-3163 sequences were prepared into lentivirus particles to infect HCC cells. miR-3163 targeted ADAM-17 and inhibited the activation of the Notch signaling pathway. Infection of HCC cells with miR-3163 enhanced their sensitivity to molecular targeted agents, such as sorafenib. Therefore, miR-3163 may contribute to the development of more effective strategies for the treatment of advanced HCC.


Introduction
Hepatocellular carcinoma (HCC) is one of the foremost threats to public health in China due to the high rate of hepatitis B virus infection in the Chinese population [1][2][3] . Regardless of the administration of anti-viral treatment, a large number of patients suffering from hepatitis B virusrelated chronic liver disease eventually progress to HCC, a fatal end-stage liver disease [4][5][6] . Unfortunately, a large proportion of patients with HCC often suffer from advanced-stage disease (e.g., advanced HCC, Barcelona Clinic Liver Cancer stage B or C) at initial diagnosis. This subset of patients is unsuitable for surgical resection and is associated with poor clinical outcome or prognosis 7,8 .
Moreover, advanced HCC is resistant to radiotherapy or cytotoxic chemotherapy, and the rapid or regressive recurrence after treatment may limit the application or efficiency of local therapies, such as transarterial chemoembolization or radiofrequency ablation [9][10][11] . Therefore, molecular targeted therapy plays important roles in the treatment of advanced HCC 12 . As the only first-line choice of an antitumor drug, the use of molecular targeted agents (i.e., oral administration of small molecular protein kinase inhibitors, such as sorafenib) has improved the overall survival or time to progression in patients with advanced HCC [13][14][15] . However, only a small proportion (20-40%) of patients with advanced HCC were initially sensitive to sorafenib. Of note, treatment with sorafenib is linked to a gradual increase in resistance 16 . Therefore, it is urgent to investigate and develop novel approaches to enhance the antitumor effects of molecular targeted therapies for the treatment of advanced HCC.
The Notch signaling pathway is a key regulator of cellular fate, survival, and cellular stress/cellular injury responses in HCC cells 17,18 . The aberrant expression of Notch protein or activation of the Notch pathway has been reported in various malignancies, such as prostate cancer, colorectal cancer, breast cancer, and especially in HCC [19][20][21][22][23] . During clinical treatment, radiotherapy (ionizing radiation) or chemotherapeutic agents (cellular toxicity) may function as cellular injuries to HCC cells, activating Notch. This leads to the development of stronger resistance to these antitumor strategies in HCC cells 24,25 . It has been confirmed that Notch protein is cleaved and activated by the ADAM metalloprotease domain-17 . This results in the release of the Notch intracellular domain (NICD) for translocation into the nucleus to mediate the transcription of pro-survival or anti-apoptosis genes, such as Survivin, B-cell lymphoma-2, or inhibitors of apoptosis proteins (IAPs) [26][27][28] . Increasing evidence demonstrated that inhibition of the activation of the Notch pathway may enhance the efficiency of antitumor agents in HCC cells 29,30 . Therefore, targeting ADAM-17 may be a novel strategy for inhibiting Notch activation and enhancing the sensitivity of HCC cells to antitumor treatment. In the present study, miR-3163, a microRNA targeting the 3′iuntranslated region (3′-UTR) of ADAM-17, was identified using an online tool (miRDB database). The in-vitro or in-vivo models showed that overexpression of miR-3163 enhanced the antitumor activation of molecular targeted agents.

Patients and agents
The collection of HCC clinical specimens and methods were approved by the Ethic Committee of the Fifth Medical Center of General Hospital, Chinese People's Liberation Army (formerly named the 302nd Hospital, Chinese People's Liberation Army). The HCC patients provided written informed consent for the collection and usage of specimens, which were previously described (Supplementary Table 1 33 . The cell lines were maintained in our lab under conditions, which were previously described 34,35 . Molecular targeted agents (i.e., sorafenib: catalog number S7397; regorafenib: catalog number S1178; lenvatinib: catalog number S1164; anlotinib: catalog number S8726; or apatinib: catalog number S5248) were purchased from Selleck Corporation (Houston, TX, USA). These agents (4 mg each) were dissolved in a mixture of dimethyl sulfoxide (15 μl), polyethylene glycol 400 (60 μl), and Tween80 (40 μl). Physiological saline was carefully added to the solution (agents dissolved in organic solvent) to a total volume of 20 ml 36,37 . Therefore, the concentration of agents was 0.2 mg/ml.

Subcellular fractionation and western blotting
Subcellular fractionation methods were used to examine the subcellular distribution of NICD in HCC cells 38,39 . HCC cells that were stably infected with control miRNA or miR-3163 by using lentivirus particles were collected and homogenized using a Dounce homogenizer. For subcutaneous tumor tissue formed by HCC cells, a 200mesh steel sieve was used to grind the tumor tissue and obtain a cell suspension. Subsequently, the cell suspension was washed with physiological saline to obtain single cells. The homogenate was centrifuged at 366 × g for 10 min at 4°C to collect the nuclear sub-fraction. Subsequently, the supernatant was centrifuged again at 13,201 × g for 15 min at 4°C and the final supernatant was the cytoplasmic subfraction. Western blotting experiments were performed following a standard protocol. The antibodies against Lamin A (catalog number ab8980), β-actin (catalog number ab205), or antibodies conjugated with horseradish peroxidase were purchased from Abcam PLC (Cambridge, UK). Moreover, the antibody of NICD (catalog number sc-373891) was obtained from Santa Cruz Corporation (Dallas, TX, USA). β-Actin was used as a cytoplasmic indicator and Lamin A was selected as the indicator of the nuclear fraction.

Extraction of RNA samples and qPCR experiments
Extraction of RNA samples and qPCR experiments were performed according to the methods described by Liang et al. 40 and Ji et al. 41 . Briefly, the total RNA sample of cultured HCC cells or tumor tissues was extracted and reverse-transcribed into cDNA using an RNeasy Mini kit (Qiagen, Valencia, CA, USA) according to the protocol provided by the manufacturer. The TaqMan miRNA qRT-PCR (Applied Biosystems, Foster City, CA, USA) was used to detect and quantify the miRNA expression of miR-3163 as previously described by Ji et al. 41 and Liang et al. 40 . The relative expression level of the miRNA was calculated using the comparative cycle threshold method. Universal small nuclear RNA U6 was used as the endogenous control for the miRNAs. The sequences of the primers used for the qPCR analysis are shown in Supplementary Table 2.

Examination of cell survival using the MTT method
Cells were cultured and collected to prepare a cell suspension. Subsequently, cells were seeded into 96-well plates (8000 cells per well). Following the full attachment of cells to the bottom of the plates, the cells were treated with the indicated concentrations of molecular targeted agents (i.e., 10, 3, 1, 0.3, 0.1, 0.03, and 0.01 μmol/l) for 48 h. Subsequently, the cells were analyzed through Thiazolyl Blue Tetrazolium Bromide [3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide] (MTT) analysis following previously described methods 42 . The inhibition rate was calculated as follows: (optical density [OD] 490 nm control group − OD 490 nm administration group)/(OD 490 nm control group) 43,44 .

In-vivo tumor model
The protocols of the animal experiments were approved by the Institutional Animal Care and Use Committee of the 302nd Hospital, Chinese People's Liberation Army, and were performed in accordance with the UK Animals (Scientific Procedures) Act, 1986, and its associated guidelines 45 . For the subcutaneous tumor model, MHCC97-H cells infected with lentivirus particles were injected into a subcutaneous location. Following the injection (4-5 days), the mice received oral administration of molecular targeted agents every 2 days. After 3 weeks of treatment (~10 administrations), the mice were collected and the tumor volumes/tumor weights were examined. The tumor volumes were calculated as follows: tumor width × tumor width × tumor length/2 46  For the intrahepatic migration model, MHCC97-H cells infected with lentivirus particles were injected into nude mice to form a subcutaneous tumor or into the liver via hepatic portal vein injection 47 . Following the injection (4-5 days), the mice received oral administration of molecular targeted agents every 2 days. After 3 weeks of treatment (~10 administrations), the mice were analyzed using micro positron emission tomography ( micro PET) according to the methods described by Li et al. 48 . Subsequently, the mice were collected and the livers with nodules formed by MHCC97-H of nude mice were collected. Photographs were captured and quantitatively analyzed to determine the total amount of nodules using the Image J software (version number: 1.51j8; the National Institutes of Health, Bethesda, MD, USA), according to the methods described by Shao et al. 47 . The radioactivity in the organs and blood (i.e., radio-activation of the liver to blood) was measured using a NaI (Tl) well counter (China Atom Corporation, Beijing, China). The inhibition rate was calculated as follows: [control group relative nodule area (percentages of nodules to the total area of the liver, %) − treatment group relative nodule area]/ (control group relative nodule area) × 100%; [control group relative radio-activation (the radio-activation of the liver to blood, folds) − treatment group relative radioactivation]/(control group relative radio-activation) × 100%

Statistical analysis
Statistical analysis was performed using Bonferroni's correction without two-way analysis of variance (SPSS software [Version Number 9.0]; IBM Corporation, Armonk, NY, USA). The half maximal inhibitory concentration (IC 50 ) values of agents were calculated using the Origin software (Version Number 6.1, OriginLab Corporation, Northampton, MA, USA). A P-value < 0.05 denoted statistical significance.

Results
High endogenous expression of ADAM-17 is associated with poor prognosis in patients with advanced HCC, who received sorafenib First, miR-3163 was identified as a microRNA targeting ADAM-17 using the online tool miRDB. As shown in Fig. 1, the bold and italicized fonts indicated the binding site of miR-3163 located in the 3′-UTR of ADAM-17 (Fig. 1a). Figure 1a also shows that mutations were introduced into the miR-3163-binding sites located in the 3′-UTR of ADAM-17. The expression of miR-3163 and ADAM-17 in HCC clinical specimens was examined to identify potential interactions. As shown in Fig. 1b, the expression of miR-3163 was negatively associated with ADAM-17 expression in the HCC specimens (Y = − 0.02488 × X + 0.0002473; P < 0.0001).
Subsequently, the involvement of ADAM-17 and miR-3163 in treatment with sorafenib was investigated. The endogenous level of ADAM-17 or miR-3163 was measured in clinical specimens obtained from patients with advanced HCC, who received sorafenib. By determining the median values of this expression level, the patients were divided into two groups for each factor: ADAM-17high group or ADAM-17-low group; miR-3163-high group or miR-3163-low group. The statistical data indicated that patients in the ADAM-17-high group were linked to a poor prognosis vs. those in the ADAM-17-low group (Table 1 and Fig. 1c-e). In contrast, patients in the miR-3163-high group were associated with a better prognosis vs. those in the miR-3163-low group (Table 2 and Fig. 1f-h). The results are shown as survival curves (Fig. 1c-h), mean + 95% confidence level of overall survival, or time to progression (Tables 1 and 2), or percentage of complete response, partial response, or stable disease (Tables 1 and 2).
Moreover, a high level of ADAM-17 was detected in HCC cell lines compared with L-02, a non-tumor haptic The bold and italicized fonts indicate the wild-type or mutant forms of putative miR-3163 targeting sequences. b The relationship between the expression level of miR-3163 and ADAM-17 in advanced HCC specimens was assessed through the Spearman's rank correlation analysis. c A total of 52 patients were divided into two groups (ADAM-17-high group or ADAM-17-low group) according to the median value of ADAM-17 expression. d, e Kaplan-Meier survival curves and log-rank tests were used to analyze the OS (d) or TTP (e) in advanced HCC patients with low or high levels of ADAM-17, who received treatment with sorafenib. f A total of 52 patients were divided into two groups (miR-3163-high group or miR-3163-low group) according to the median value of miR-3163 expression. g, h Kaplan-Meier survival curves and log-rank tests were used to analyze the OS (g) or TTP (h) in advanced HCC patients with low or high levels of miR-3163, who received treatment with sorafenib. *P < 0.05 Table 1  The expression vectors of ADAM-17 with mutated miR-3163-targeted sequences were also constructed to confirm whether miR-3163 targets ADAM-17. As shown in Fig. 2, compared with the control miRNA, miR-3163 significantly repressed the expression of ADAM-17 in MHCC97-H (Fig. 2a, b) or LM-3 (Fig. 2c, d). This effect was not observed for ADAM-17 Mut , which contains a mutation in the miR-3163-binding sites. Transfection of the miR-3163 inhibitor almost blocked the decreasing effect of miR-3163 on the expression of ADAM-17 (Fig. 2). Moreover, the interaction between the 3ʹ-UTR of ADAM-17 and miR-3163 was confirmed through luciferase experiments ( Supplementary Figs. 4 and 5). Therefore, ADAM-17 may be a target of miR-3163. It is suggested that miR-3163 may repress the expression of ADAM-17 in HCC cells by targeting the 3′-UTR of ADAM-17.

Overexpression of miR-3163 inhibits the activation of the Notch signaling pathway
The accumulation of NICD in the nucleus was examined to further identify the effect of miR-3163 on the activation of the Notch signaling pathway. As shown in Fig. 3, overexpression of miR-3163 significantly inhibited the expression of ADAM-17 in the cytoplasm of MHCC97-H (Fig. 3a) or LM-3 cells (Fig. 3b), and decreased the accumulation of the NICD of Notch protein in the nucleus of MHCC97-H (Fig. 3a) or LM-3 (Fig. 3b) cells. Transfection of ADAM-17 Mut or the inhibitor of miR-3163 almost blocked the inhibitory effect of miR-3163 on the cleavage of Notch protein and the accumulation of NICD in the nucleus (Fig. 3a, b). Subsequently, HCC cells infected with lentivirus particles were injected into nude mice to form subcutaneous tumors and the accumulation of NICD in the nucleus of single cells. As shown in Fig. 3, overexpression of miR-3163 significantly inhibited the expression of ADAM-17 in the cytoplasm of MHCC97-H (Fig. 3c) or LM-3 cells (Fig. 3d) separated from subcutaneous tumors. Moreover, it decreased the accumulation of the NICD of Notch protein in the nucleus of MHCC97-H (Fig. 3c) or LM-3 (Fig. 3d) cells. Transfection of ADAM-17 Mut almost blocked the inhibitory effect of miR-3163 on the cleavage of the Notch protein and the accumulation of NICD in the nucleus of cells separated from subcutaneous tumors (Fig. 3c, d).

Overexpression of miR-3163 enhances the sensitivity of HCC cells to molecular targeted agents
Subsequently, the effect of miR-3163 on the antitumor activity of molecular targeted agents was examined. As shown in Table 3, overexpression of miR-3163 enhanced the sensitivity of MHCC97-H cells to sorafenib. Of note, the IC 50 values of sorafenib decreased from 1.04 ± 0.05 μmol/l to 0.10 ± 0.01 μmol/l. Transfection of ADAM-   (Table 3). Similar results were obtained in LM-3 cells (Table 3). Subsequently, the effect of miR-3163 on the sensitivity of PDC cells to molecular targeted agents was examined in patient-derived cell lines. As shown in Table 4, overexpression of miR-3163 enhanced the sensitivity of five PDCs to the molecular targeted agents (i.e., sorafenib, regorafenib, lenvatinib, anlotinib, or apatinib).
To further examine the effect of miR-3163 on the antitumor activity of sorafenib, MHCC97-H cells were seeded into nude mice to form subcutaneous HCC tumors. As shown in Fig. 5, oral administration of sorafenib inhibited the subcutaneous growth of MHCC97-H cells. Overexpression of miR-3163 enhanced the sensitivity of HCC cells to sorafenib. Subsequently, the intrahepatic migration model was applied. As shown in Fig. 6, injection of MHCC97-H cells into the liver of nude mice via portal vein injection resulted in the formation of multiple disseminated lesions. Notably, the intrahepatic growth could be identified through micro PET. Oral administration of sorafenib inhibited the images of micro PET in the liver of nude mice and the area of lesions in the liver (Fig. 6). Overexpression of miR-3163 enhanced the antitumor effect of sorafenib on the intrahepatic growth of MHCC97-H cells (Fig. 6). Moreover, the specificity of miR-3163 on sorafenib was examined. As shown in Fig. 7 and Fig. 8, the expression of ADAM-17 Mut or NICD decreased the effect of miR-3163 on sorafenib. Similar results were obtained from PDCs: miR-3163 enhanced the sensitivity of PDCs to molecular targeted agents by targeting ADAM-17 (Table 5). To examine the specificity of miR-3163′ function, the expression level of downstream factors Notch pathways, pro-survival factors or EMT-related factors in the subcutaneous tumors of Fig. 8 were examined by western blotting experiments.

Discussion
In the present study, miR-3163 was identified as a microRNA potentially targeting ADAM-17. Overexpression of miR-3162 through infection lentivirus particles inhibited the cleavage of Notch protein and enhanced the sensitivity of HCC cells to molecular targeted agents such as sorafenib. The effect of miR-3163 on the Notch signaling pathway or sensitivity of HCC cells to sorafenib was almost blocked by transfection of mutated ADAM-17, the inhibitor of miR-3163, or NICD. This confirmed the effect of miR-3163 on ADAM-17 and the sensitivity of HCC cells to molecular targeted drugs by inhibiting the expression of ADAM-17. In addition, it confirmed that the miR-3163/ADAM-17 axis acts through the Notch signaling pathway. Therefore, our results indicated that miR-3163 may enhance the Moreover, our results showed that miR-3163 inhibited the EMT process in HCC cells. It is established that the EMT process in cancer cells is associated with poor patient survival. Mechanism data indicated that the EMT is a key step in the progression of cancer and participates in metastasis 55 . During the EMT process, the adhesion feature of cancer cells is decreased (e.g., E-cadherin loss). Furthermore, mesenchymal markers (i.e., Vimentin or N-Cadherin) decrease the polarity of cancer cells and accelerate migration and invasion 56,57 . Recently, the EMT process has been proposed as an important regulator of drug resistance [58][59][60] . Accumulating data have confirmed that mechanisms of resistance to sorafenib may involve the EMT and the Notch signaling pathway is a key regulator of the EMT process [61][62][63] . In this study, miR-3163 significantly inhibited the EMT process in HCC cells. This means that a decrease in the expression of ADAM-17 may inhibit the activation of the Notch signaling pathway, and enhance the sensitivity of HCC cells to antitumor agents by inhibiting the EMT process. In addition to EMT, we also investigated the expression of other cell-promoting and anti-apoptotic Notch downstream proteins, including Survivin, cIAP-1, and cIAP-2 25 . Downregulation of the activity of the Notch signaling pathway by various pathways can reduce the resistance of cells to various damaging factors. This increases the sensitivity of cells to molecular targeted drugs and offers safer and more effective treatments (i.e., cytotoxic chemotherapy drugs and radiation therapy) [64][65][66][67] .
Furthermore, patient-derived tumor cells are an important model of pharmacologically relevant research that reflects the actual conditions of patients 68,69 . Constructing appropriate research models, especially animal models, contributes to the development of relevant research and provides a basis for predicting patient sensitivity and prognosis in patients who received treatment. This study used a variety of tumor animal models, including subcutaneous tumor models and intrahepatic tumor models in nude mice. The former is a common model used in oncology research. Hepatic portal vein injection was used to inoculate HCC cells into the liver of nude mice, simulating the recurrence or metastasis of    (Leverkusen, Nordrhein-Westfalen, Germany), whereas lenvatinib is a first-line therapy for HCC developed by Eisai Official Corporate (Tokyo, Japan) 74,75 . Anlotinib and apatinib are molecular targeted drugs developed by Chinese manufacturers (HENGRUI Medicine, Lian-yunggang City, Jiangsu Province, China, or CHIATAI Tianqing Corporation, Nanjing City, Jiangsu Province, China) 76,77 . The mechanism of action of these drugs is similar. In the future, clinical studies investigating the use of anlotinib and apatinib for the treatment of advanced HCC may also be performed. This study found that the antitumor effect of lenvatinib may be superior to that of several other molecular targeted drugs. This provides a reference for future research.