LncRNA MT1JP Plays a Protective Role in Cholangiocarcinoma by Regulating miR-18a-5p/FBP1 Axie

Background: Cholangiocarcinoma is a common malignant tumor of digestive system. LncRNA MT1JP has been reported to play tumor-suppressing roles in multiple cancers. However, the effect of MT1JP on cholangiocarcinoma has not been evaluated. Methods: The expression of MT1JP in cholangiocarcinoma specimens and paired para-carcinoma tissues were detected by real-time PCR. The overexpression plasmid and siRNA of MT1JP were transfected into cholangiocarcinoma cells to change the expression levels of MT1JP. CCK-8, ow cytometry and transwell assays were performed to measure proliferation, cell cycle transition, apoptosis, migration and invasion in cholangiocarcinoma cells. Dual-luciferase reporter assay, real-time PCR and western blot were carried out to screen the miRNA bound by MT1JP. In addition, xneograft experiment was used to determine the tumorigenesis of cholangiocarcinoma cells in nude. Results: MT1JP was downregulated in cholangiocarcinoma specimens, compared with para-carcinoma tissues, and its expression was related with tumor size, TNM stage and lymph node metastasis. Overexpression of MT1JP inhibited proliferation, cell cycle transition, migration and invasion, and induced apoptosis in cholangiocarcinoma cells. The knockdown of MT1JP led to opposite results. MT1JP bound to miR-18a-5p to facilitate the expression of fructose-1,6-bisphosphatase 1 (FBP1). MiR-18a-5p was increased in cholangiocarcinoma samples, and its expression was negatively correlated with that of MT1JP. In addition, MT1JP also suppressed tumorigenesis in nude mice. Conclusions: MT1JP alleviated proliferation, migration and invasion, and induced apoptosis in cholangiocarcinoma cells by regulating miR-18a-5p/FBP1 axie. These ndings may provide novel insights for diagnosis and treatment of cholangiocarcinoma in terminal deoxynucleoitidyl transferase mediated nick end labeling; TNM stage, tumor-node-metastasis stage.

Long noncoding RNAs (lncRNAs) are a group of noncoding RNAs with length of more than 200 nucleotides. LncRNAs have been known abundant in lives, and to widely regulate gene expression via various ways [8,9]. LncRNA metallothionein 1J pseudogene (MT1JP) was rst reported as a tumor suppressor in liver cancer cells by regulating the translation of p53 [10]. Subsequently, MT1JP was found as a ceRNA to bind to miR-214-3p to facilitate runt related transcription factor 3 (RUNX3) expression, and suppressed cell proliferation, invasion and migration in gastric cancer cells [11]. The tumor-suppressing role of MT1JP has been veri ed in multiple cancer types, but its effect on cholangiocarcinoma has not been evaluated.
In our previous study, MT1JP was found downregulated in cholangiocarcinoma specimens, compared with paired para-carcinoma tissues. In addition, MT1JP was predicted to bind to miR-18a-5p seed sequence. In the present study, the effect of MT1JP on cholangiocarcinoma cells was investigated via gain-and loss-of-function experiments, and the relation between MT1JP and miR-18a-5p was also studied.

Methods
Clinical specimens Thirty paired of cholangiocarcinoma and para-carcinoma tissues were collected from January 2016 to December 2019 in hepatological surgery department, a liated hospital of Qiangdao Unversity via surgery resection. The patients had not received chemotherapy or radiotherapy treatment. The specimens were identi ed as cholangiocarcionma by pathological examination. Informed consent was obtained from each patient. The specimen collection and experimental procedure were line with Declaration of Helsinki, and approved by Ethics Committee of A liated Hospital of Qingdao University.
The expression levels of MT1JP and miR-18a-5p in the cholangiocarcinoma specimens were detected with real-time PCR.
The expression level of MT1JP in another 58 cholangiocarcinoma samples was analyzed with the patients' clinicalpathological characteristics by Chi-square test.

Real-time PCR
Total RNA was extracted with TRIpure reagent (BioTeke, Beijing, China), and reversely transcribed into cDNA with M-MLV reverse transcriptase (BioTeke), in presence of Olig(dT) and random, or speci c miRNA primers (GenScript, Nanjing, China). The cDNA was used for real-time PCR to detect the expression levels of MT1JP and miR-18a-5p, and mRNA level of FBP1 with 2×Power Taq PCR MasterMix (BioTeke) and SYBR Green (Sigma,St. Louis, USA). The data were analyzed using 2 -ΔΔCt method. β-actin served as the internal control of MT1JP, and 5S served as the internal control of miR-18a-5p. The sequence information of primers was shown in Table 1.
Cell transfection was performed with Lipofectamine 2000 reagent (Invitrogen, Carlsbad, USA) in serumfree medium according to the manufacturer's protocol.
To obtain the stably transfected cell line, HCCC-9810 cells were transfected with MT1JP overexpression plasmid, and treated with 400 μg/ml G418 for 2 weeks. The single cells were selected out, and cultured without G418. After veri cation of MT1JP in RNA levels, the MT1JP-stably expressed cell lines were obtained.
Cell counting kit-8 (CCK-8) assay CCK-8 assay was performed to measure the cell viability. The cells were cultured in 96-well plates at 37 ℃ with 5% CO 2 . After culture for 0 h, 24 h, 48 h or 72 h, the cells were incubated with CCK-8 reagent (10 μl per well) (KeyGEN, Nanjing, China) for 2 h. The optical density of medium was detected with a microplate reader (BioTek, Winooski, VT, USA) at 450 nm.

Flow cytometry
Flow cytometry was used for detection of apoptosis and cell cycle.
The cells were collected, and incubated with Annextin V-FITC reagent and propidium iodide at room temperature for 20 min in the dark. Then the cells were detected by ow cytometer (BD, Franklin Lakers, NJ, USA).
For cell cycle detection, the cells were collected and immobilized with 70% ethanol at 4 ℃ overnight. Then the cells were incubated with PI/RNaseA buffer at room temperature for 60 min in the dark, and used for detection by ow cytometer.

Transwell assay
Transwell assay was used for detection of migration and invasion.
The cells were collected and counted. About 3×10 3 cells were seeded into the upper chamber with serumfree medium, and the lower chamber was added with medium with 30% FBS. After culture for 24 h, the cells on the reverse surface of transwell membrane was xed with 4% paraformaldehyde (Aladdin, Shanghai, China) and stained with 0.4% crystal violet (Amresco, Solon, OH, USA). The cells were photographed and counted under a microscope at 100× magni cation.
For invasion analysis, the polycarbonate membrane of transwell chambers (Corning, NY, USA) was precoated with Matrigel at 37 ℃. Approximately 1.5×10 4 cells were seeded into upper chamber with serumfree medium, and medium with 30% FBS were added into the lower chamber. After culture for 24 h, the cells on the reverse surface was xed and stained, and the cell number was counted under a microscope.

Immuno uorescent staining
The cells were pre-seeded on glass slides. After culture for certain times, the cells were xed with 4% paraformaldehyde for 15 min, permeated with 0.1% TritonX-100 (Beyotime) for 30 min, and blocked with goat serum for 10 min at room temperature. Subsequently, the cells were incubated with antibody against FBP1 (1:100; cat. no. 12842-1-AP, Proteintech) at 4 ℃ overnight, incubated with secondary antibody labeled with Cy3 (1:200; cat. no. A0516, Beyotime) at room temperature in the dark for 60 min, and counterstained with DAPI (Aladdin). Finally, the glass slide were mounted with anti-fading reagent (Solarbio, Beijing, China), and the cells were observed under a uorescent microscope (Olympus, Tokyo, Japan) at 400× magni cation.

Xneograft model
Healthy BALB/c mice were purchased from HFK Biotechnology Co. Ltd. (Beijing, China), and kept in a controlled environment (12 h/12 h light/dark, 22±1 ℃) with free access to food and water. The animals were taken care of according to Guide for the Care and Use of Laboratory animal (8th edition, NIH), and the experimental procedures were approved by Ethics Committee of A liated Hospital of Qingdao University.
After accommodation for one week, the mice were subcutaneously injected with HCCC-9810 cells (5×10 5 each mouse) stably transfected with MT1JP overexpression plasmid or pcDNA3.1 vector. One week later, the tumor size was measured every 3 days. Three weeks after injection, the mice were euthanized via overdose of pentobarbital sodium (200 mg/kg, intraperitoneal injection), and the tumors were isolated for detection.

HE staining
The tumor isolated from mice were xed in 4% paraformaldehyde overnight, and washed with ow water for 4 h. Then the tissue was dehydrated with ethanol of grading concentrations and xylene, embedded with para n, and cut into sections of 5 μm. The sections were depara nized with xylene and ethanol, and stained with hematoxylin (Solarbio). After soaking in 1% hydrochloric acid/ethanol for several seconds, the sections were stained with eosin (Sangon, Shanghai, China). Finally, the sections were dehydrated again, mounted with gum, and observed under a microscope at 200× magni cation.

TUNEL staining
The tumor tissue was made into para n sections as described previously. After depara nization, the sections were permeated with 0.1% Triton X-100, and blocked with 3% H 2 O 2 at room temperature. Then the sections were incubated with TUNEL buffer (Roche, Basel, Switzerland) for 60 min at 37 ℃ in the dark, and incubated with Converter-POD reagent for 30 min at 37 ℃. Subsequently, the sections were reacted with DAB substrate (Solarbio), and counterstained with hematoxylin. Finally, the sections were dehydrated, mounted and photographed with a microscope at 400× magni cation.

Immunohistochemical staining
The tumor tissues were used for immunohistochemical staining for detection of Ki-67 and FBP1. The tissues were made into sections as previous description. The sections were reacted with antigen repair buffer in boiling for 10 min, and blocked with 3% H 2 O 2 and goat serum. Then the section were incubated with antibody against Ki-67 (1:100; cat. no. AF0198, A nity) or FBP1 (1:100; cat. no. 12842-1-AP, Proteintech) at 4 ℃ overnight. After washing with PBS, the sections were incubated with secondary antibody labeled with HRP (1:500; Beyotime) at 37 ℃ for 60 min, and reacted with DAB substrate. After counterstaining with hematoxylin, the sections were dehydrated, mounted and observed with a microscope at 400× magni cation.

Statistical analysis
The data in this study were presented as meas±SD, and analyzed with GraphPad Prism 8.0. The data from two independent groups were analyzed with student's t test. Comparisons among multiple groups were performed with one-way or two-way analysis of variance followed with Bonferroni post-hoc test. The correlation between MT1JP and clinical features were analyzed by Pearson χ 2 test. The correlation between MT1JP and miR-18a-5p was analyzed with Pearson test. A p value less than 0.05 was considered as statistically signi cantly.

MT1JP was downregulated in cholangiocarcinoma specimens
The expression of MT1JP in cholangiocarcinoma tissues were detected by real-time PCR. As shown in Fig. 1A, the MT1JP was downregulated in cholangiocarnoma tissues, compared with para-carcinoma tissues. On the contrary, the expression of miR-18a-5p was increased in cholangiocarcinoma samples (Fig. 1B). In addition, the correlation between MT1JP and miR-18a-5p was analyzed with pearson test, and Fig. 1C revealed that the expression of MT1JP was negatively related with that of miR-18a-5p.
Next, the correlation between MT1JP expression and clinicopathological characteristics were analyzed. As shown in Table 2, the expression level of MT1JP was signi cantly related with tumor size, TNM stage and lymph node metastasis.

MT1JP inhibited proliferation and promoted apoptosis in cholangiocarcinoma cells
As MT1JP was decreased in cholangiocarcinoma tissues, its expression was examined in several cholangiocarcinoma cell lines. As shown in Fig. 2A, MT1JP was lowest expressed in HCCC-9810 cells, and highest expressed in HUCCT1 cells, which was opposite with that of miR-18a-5p (Fig. 2B). In order to investigate the roles of MT1JP in cholangiocarcinoma cells, MT1JP overexpresion plasmid was transfected into MT1JP low-expressed HCCC-9810 cells, and its siRNA was transfected into highexpressed HUCCT1 cells. The effectiveness of overexpression and silencing was con rmed by real-time PCR (Fig. 2C).
Next, the proliferation and apoptosis was detected. CCK-8 assay showed that MT1JP inhibited cell ability in cholangiocarcinoma cells (Fig. 2D and 2E). The expression level of PCNA was decreased after MT1JP overexpression and increased after MT1JP knockdown (Fig. 2F). The ow cytometry results revealed that MT1JP delayed G1/S and S/G2 transition in HCCC-9810 cells, and the silencing of MT1JP accelerated cell cycle transition in HUCCT1 cells (Fig. 2G), which was supported by expression of cyclin B1and cyclin E (Fig. 2H). In addition, the ectopic expression of MT1JP enhanced apoptosis in HCCC-9810 cells, evidenced by expression changes of cleaved caspase-3 and cleaved PARP ( Fig. 2I and 2J). The knockdown slightly inhibited the apoptosis in HUCCT1 cells (Fig. 2I).

MT1JP suppressed migration and invasion in cholangiocarcinoma cells
Next, the migration and invasion ability of cholangiocarcinoma cells were determined by transwell assay with or without Matrigel. As shown in Fig 3, the overexpression of MT1JP suppressed migration and invasion in HCCC-9810 cells, and the silencing of MT1JP enhanced migration and invasion in HUCCT1 cells.
The effect of MT1JP was attenuated by overexpression of miR-18a-5p or knockdown of FBP1 In order to further investigate the mechanism of MT1JP function, the MT1JP overexpression plasmid was cotransfected with miR-18a-5p or FBP1 siRNA. As shown in Fig. 5, increase of cell viability and invade ability and the decrease of apoptosis of induced by MT1JP was abolished by transfection of miR-18a-5p mimics in HCCC-9810 cells (Fig. 5A-C). Similarly, the effect of MT1JP on cell viability, apoptosis and invasion in HCCC-9810 cells were attenuated by FBP1 siRNA (Fig. 5D-F). These results demonstrated that MT1JP played its role by binding to miR-18a-5p and facilitated the expression of FBP1.

MT1JP restrained tumorigenesis in nude mice
To investigate the effect of MT1JP on tumorigenesis of cholangiocarcinoma cells, the HCCC-9810 cells with stable expression of MT1JP were subcutaneously injected into nude mice (n=6). The tumors were isolated 3 weeks after injection. Fig. 6A and 6B showed that MT1JP signi cantly inhibited tumorigenesis in nude mice. HE staining, TUNEL and immunohistochemical staining of Ki-67 revealed that MT1JP led to cell necrosis and apoptosis, and suppressed cell proliferation in tumors (Fig. 6C-E). The real-time PCR and immunohistochemical staining results demonstrated that in MT1JP-overexpressed tumors, the expression of miR-18a-5p was declined, and that of FBP1 was elevated (Fig. 6F-G), which supported the hypothesis that MT1JP bound to miR-18a-5p and facilitated the expression of FBP1.

Discussion
In this study, we found that MT1JP was downregulated in clinical cholangiocarcinoma specimens, and its expression was correlated with tumor size, TNM stage and lymph node metastasis. Gain-and loss-offunction experiments demonstrated that MT1JP inhibited cell proliferation, cell cycle transition, migration and invasion, and promoted apoptosis in cholangiocarcinoma cells. Xenograft experiments showed that MT1JP suppressed tumorigenesis in nude mice. In addition, miR-18a-5p was increased in cholangiocarcinoma tissues, which was negatively correlated with that of MT1JP. MT1JP bound to miR-18a-5p as a sponge, and enhanced the expression of a target of miR-18a-5p, FBP1.
The tumor-suppressing role of MT1JP has been reported in multiple cancer cells, including breast cancer, glioblastoma, bladder cancer and gastric cancer [11][12][13][14]. The expression of lncRNA is often tissuespeci c. We rst demonstrated its effect on cholangiocarcinoma cells. The MT1JP gene locates in chromosome 16 in a cluster consisting of several homologous protein-coding genes of the metallothionein family [10]. MT1JP has been known to bind to miRNAs to play its roles. In this study, we demonstrated that MT1JP bound to miR-18a-5p to facilitate the expression of FBP1. MiR-18a-5p belongs to miR-17-92 cluster, plays tumor-promoting roles in colorectal cancer, lung cancer and renal cell carcinoma cells [15][16][17]. However, one study reported that miR-18a-5p was downregulated in breast cancer tissues, and inhibited migration and invasion in breast cancer cells [18]. These reporters suggested that the role of miR-18a-5p may be different in various cancers. In our study, miR-18a-5p was increased in cholangiocarcinoma specimens, and its overexpression enhanced proliferation and invasion, and reduced apoptosis in cholangiocarcinoma cells. FBP1 was con rmed as a target of miR-18a-5p. FBP1 is gluconeogenesis regulatory enzyme, and catalyzes the hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate and inorganic phosphate [19]. In our previous study, FBP1 was demonstrated to inhibit proliferation and metastasis in cholangiocarcinoma cells by regulating Wnt/β-catenin signaling pathway [20]. MT1JP may function via miR-18a-5p/FBP1/Wnt/β-catenin axis.
In this study, we analyzed the correlation between MT1JP expression and clinical characteristics of 58 cholangiocarcinoma patients, and found that the expression level of MT1JP was related with tumor size, TNM stage and lymph node metastasis. The MT1JP low expression is more common in patients with larger tumor, advanced stage or/and existence of lymph node metastasis. Moreover, the expression of MT1JP was signi cantly decreased in cholangiocarcinoma samples. These nding suggested that We came up with a hypothesis that MT1JP acted as a miRNA sponge, and bound to miR-18a-5p. In our results, the miR-18a-5p level was inhibited by MT1JP, suggested that MT1JP may be a degradable sponge. The expression of FBP1 was inhibited by miR-18a-5p, but enhanced by MT1JP, suggested that MT1JP competed with FBP1 for binding to miR-18a-5p. This hypothesis needs to be supported by more experiment results.

Conclusions
In this study, we demonstrated that lncRNA MT1JP was downregulated in cholangiocarcinoma tissues. MT1JP inhibited proliferation, migration, invasion and tumorigenesis and enhanced apoptosis in cholangiocarcinoma cells as a miRNA sponge to bind to miR-18a-5p to facilitate the expression of FBP1.

Declarations
Ethics approval and consent to participate All patients that participated to this study provided informed writing consent. This study was approved by Ethic Committee of a liated hospital of Qingdao University.

Consent for publication
Not applicable.

Availability of data and materials
The data in this study will not be shared due to the policy of the Ethic Committee.