Sage Journals: Discover world-class research

Abstract

MicroRNAs (miRNAs) have been demonstrated that play a critical role in tumorigenesis. The aim of this study is to identify the functional role of miR-378 in cholangiocarcinoma (CCA). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure the expression levels of miR-378 in human CCA tissue samples and CCA cell lines. The receiver operating characteristic (ROC) curve was established, and the area under the ROC curve (AUC) was calculated to estimate the capacity of miR-378 in distinguishing CCA patients with different TNM stages. Kaplan-Meier survival analysis and Cox regression assay were performed to explore the prognostic value of miR-378. Cell proliferation capacity was assessed by MTT assay. Cell migration and invasion were identified by Transwell assays. miR-378 was significantly elevated in CCA tissues when compared with adjacent normal tissues, and in CCA cell lines compared to HIBEC cells. And we found that the expression of miR-378 was significantly associated with TNM stage ( $P=$ 0.030) and lymph node metastasis ( $P=$ 0.018). ROC curve analysis result showed miR-378 could distinguish CCA patients with TNM stages III and IV from those with stages I and II, with the AUC was 0.816. Patients with high expression of miR-378 had a shorter overall survival rate (Log-rank $P=$ 0.030). The miR-378 was proven to be an independent prognostic predictor for the CCA patients (HR $=$ 1.735, 95% CI $=$ 1.007–2.988, $P=$ 0.041). Downregulation of miR-378 could inhibit cell proliferation, migration, and invasion. These results indicated that miR-378 function as an oncogene and promote CCA cells proliferation, migration, and invasion. The miR-378 could be a novel prognostic marker for CCA.

Keywords

microRNA-378 cholangiocarcinoma prognosis proliferation migration invasion

1. Introduction

Cholangiocarcinoma (CCA) is a rare malignant tumor originating in the epithelium of bile ducts prone to the lymph node metastasis [1]. It is the most frequent primary hepatic malignancy, which is classified as intrahepatic, extrahepatic or perihilar according to anatomical location [2, 3]. The incidence and mortality rate of CCA is increasing in recent years [4, 5]. Due to early metastasis and advanced stages diagnosis, the survival rate and prognosis of CCA patients are dismal. Surgical resection is perhaps the only potentially curative treatment, because of almost all types of CCA are resistant to chemotherapies [6]. However, the disease is difficult to be controlled given that CCA patients are usually diagnosed at an advanced stage. Despite advances in treatment, the 5-year survival rate of CCA patients is still unsatisfactory. Therefore, there is a crucial need to explore novel cancer-related biomarkers for early diagnostic and novel therapeutic applications to improve the poor prognosis and therapeutic efficacy.

MircroRNAs (miRNAs) are a class of small, endogenous, non-coding RNAs that regulate target genes expression at the post-transcriptional level by binding to the 3’ untranslated region (3’ UTR) [7]. Previous studies have shown that miRNAs were closely involved in the regulation of diverse physiological and pathological functions, including cell proliferation, cell differentiation, cell migration and invasion, cell apoptosis, and tumor metastasis [8, 9, 10, 11]. Dysregulated miRNA expression has been reported in several human diseases, including cancers [12, 13, 14, 15]. The expression of miR-378 was also found to be dysregulated in many diseases and tumors, such as ovarian cancer, liver cancer, breast cancer and so on [16, 17, 18]. miR-378-5p, a member of the miR-378 family, was found upregulated and influenced SULT2A1 expression in primary sclerosing cholangitis [19]. However, the underlying clinical significance of miR-378 in CCA is still unclear.

In the present study, we investigated the expression pattern of miR-378 in CCA tissues and cell lines. The prognostic impact of miR-378 expression was also tested. Then the effects of miR-378 on biological behavior were investigated. The aim of this study is to investigate the clinical role of miR-378 in CCA.

2. Materials and methods

2.1 Patients and tissue samples

The corresponding resected CCA tissues and adjacent normal tissues (all were confirmed by at least two pathologists) were collected from 120 CCA patients diagnosed in Yidu Central Hospital of Weifang from May 2008 to April 2012. All the patients did not receive adjuvant treatment prior to surgical resection. All the tissues and adjacent noncancerous tissues were snap frozen in liquid nitrogen subsequent stored at $-$ 80 ${}^{\circ}$ C until further use. A 5-year follow-up was enrolled after curative resection to collect survival information by telephone. The patients’ clinicopathological information were collected and summarized in Table 1. All samples collection and protocols of this study were approved by the Ethics Committee of Yidu Central Hospital of Weifang. Written informed consent was signed by each of the participants.

Table 1
Association between miR-378 expression and clinicopathological variables of CCA patients

Characteristics	Cases No.	miR-378 expression		$P$ values
	( $N=$ 120)	Low	High
		( $n=$ 54)	( $n=$ 66)
Age (Years)				0.441
$<$ 60	62	30	32
$\geqslant$ 60	58	24	34
Gender				0.447
Male	69	29	40
Female	51	25	26
Differentiation				0.508
Well $+$ moderate	56	27	29
Poor	64	27	37
TNM stage				0.030
I–II	58	32	26
III–IV	62	22	40
Lymph node metastasis				0.018
Negative	59	33	26
Positive	61	21	40

2.2 Cell lines and transfection

The human CCA cell lines (QBC939 and RBE), and normal human intrahepatic biliary epithelial cell line (HIBEC) were purchased from Cell Bank of Type Culture Collection (CTCC), Chinese Academy of Sciences (Shanghai, China). All cells were cultured in RPMI-1640 medium with 10% fetal bovine serum (FBS) and were incubated at 37 ${}^{\circ}$ C in 5% CO ${}_{2}$ incubator. CCA cells were seeded in 6-well plates 24 h (1 $\times$ 10 ${}^{5}$ cells per well). Then the cells were transfected with miR-378 mimics or mimic negative control (NC), miR-378 inhibitor or inhibitor NC using Lipofectamine 2000 Reagent (Invitrogen, USA) according to the manufacturer’s instructions, and cell transfected with only transfection reagent were used as the mock. Analyses were performed 48 h later and the transfection efficiency was detected by using quantitative real-time PCR (qRT-PCR).

2.3 RNA isolation and qRT-PCR analysis

Total RNA was isolated from the tissues and cell using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. And complementary DNA (cDNA) synthesis was performed using Transcriptor First Strand cDNA Synthesis Kit (Roche, Vilvoord, Brussel, Belgium). The relative miR-378 expression was analyzed with qRT-PCR on the 7300 Real-Time PCR System (Applied Biosystems, Foster City, USA) using SYBR Green PCR Master Mix (Applied Biosystems). The U6 gene was used as the endogenous control. The primers in this reaction referred to the study by Zeng et al. [20]. The relative expression levels were calculated with the 2 ${}^{-\Delta\Delta\text{Ct}}$ methods. All reactions were repeated three times.

Figure 1.

Expression of miR-378 was determined by qRT-PCR. A. The expression of miR-378 in CCA tissues was significantly higher than that in normal tissues ( $P<$ 0.001). B. miR-378 was overexpressed in CCA cell lines (QBC939 and RBE) compared to normal cell line HIBEC (* $P<$ 0.05, ** $P<$ 0.01, *** $P<$ 0.001).

2.4 Cell proliferation assays

The viability of CCA cells was measured with the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetra-zolium bromide (MTT) assay. After the transfection was stabilized, QBC939 and RBE cells were plated in 96-well plates (5 $\times$ 10 ${}^{4}$ cells per well). The plates were incubated in 10% FBS medium at 37 ${}^{\circ}$ C for 48 h, and then the medium was added with 20 $\mu$ L MTT (Sigma-Aldrich, 5 mg/mL) and incubated at 37 ${}^{\circ}$ C for 4 h. Subsequently, the medium was removed and 100 $\mu$ L dimethyl sulfoxide (DMSO, Sigma-Aldrich) was added to each well to dissolve formazan crystals. The colorimetric analysis was performed (wavelength, 490 nm) using a spectrophotometer (Multiskan MK3, Thermo, Waltham, MA, USA).

2.5 Migration and invasion assays

Cell migration and invasion studies were performed using the Transwell assays (Corning, Tewksbury, MA, USA, 8.0 $\mu$ m pores). For the migration assay, no Matrigel was added to the upper chamber. For invasion assay, the top chambers were coated with Matrigel (Bedford, MA, USA). The under chambers were filled with 600 $\mu$ L RMPI 1640 containing 20% FBS. 100 $\mu$ L cell suspension with 2 $\times$ 10 ${}^{5}$ cells were plated in the top chamber with serum-free RMPI 1640 medium at 37 ${}^{\circ}$ C for 24 h. Then the non-migrated/non-invading cells were removed from the upper sides of the membrane using cotton-tipped swabs. The migrated/invaded cells on the lower sides of the membrane were fixed in 3.7% formaldehyde for 5 min and stained with 0.1% crystal violet for 15 min. The cells were counted using a light microscope (Olympus, Tokyo, Japan).

2.6 Statistical analysis

All statistical analyses were performed using the SPSS 21.0 software (SPSS, Inc., Chicago, IL, USA) and presented by GraphPad Prism software (GraphPad Software, Inc. La Jolla, CA, USA). Data were presented as the mean $\pm$ standard deviation (SD). The differences between the two groups were compared with the paired Student’s t-test. The association between miR-378 expression and clinical parameters of CCA patients was applied using $\chi^{2}$ test. The receiver operating characteristic (ROC) curve was established to estimate the capacity of miR-378 in distinguishing CCA patients with different TNM stages. Survival curve was estimated using the Kaplan-Meier method to assess the association of miR-378 expression with the survival rate. Cox regression analysis was applied to explore the prognostic value of the miR-378 expression in CCA. A $P$ value less than 0.05 was considered significant.

3. Results

3.1 Expression of miR-378 in the CCA tissues and cell lines

To explore the expression pattern of miR-378 in CCA, we first determined the expression levels of miR-378 in CCA tissues and adjacent normal tissues using qRT-PCR. As shown in Fig. 1A, the expression level of miR-378 in CCA tissues was significantly higher than that in normal tissues ( $P<$ 0.001). Next, we detected the expression level of miR-378 in cell lines. Compared to the expression level in the normal human intrahepatic biliary epithelial cell line HIBEC, miR-378 has a low level of expression in CCA cell lines QBC939 and RBE (all $P<$ 0.01).

Figure 2.

The capacity of miR-378 in distinguishing CCA patients with different stages. A. Relative expression levels of miR-378 in CCA patients with different TNM stages (*** $P<$ 0.001). B. ROC curve of miR-378 in distinguishing CCA patients with TNM stages III and IV from those with stages I and II. The AUC is 0.816 (95% CI $=$ 0.741–0.891).

3.2 Correlation between miR-378 expression and clinicopathological characteristics of CCA patients

To measure the association between miR-378 expression and clinicopathological characteristics, the CCA patients were divided into two groups according to the mean value of miR-378 expression levels (3.896), including low-expression group ( $n=$ 54) and high-expression group ( $n=$ 66). The correlation between miR-378 expression and pathological characteristics of CCA patients was analyzed (Table 1). The results showed that the high expression of miR-378 was significantly associated with TNM stage ( $P=$ 0.030) and lymph node metastasis ( $P=$ 0.018). No significant differences were found between miR-378 expression and other features, such as age, gender, and differentiation (all $P<$ 0.05).

Figure 3.

Kaplan-Meier survival curves of patients with CCA based on miR-378 expression status. Patients with the high expression group have a significantly shorter overall survival rate than those in low expression group.

Table 2

Univariate and Multivariate Cox analysis of clinical parameters in relation to overall survival

Characteristics	Univariate analysis			Multivariate analysis
	HR	95% CI	$P$	HR	95% CI	$P$
miR-378	2.005	1.096–3.668	0.024	1.735	1.007–2.988	0.041
Age	0.866	0.515–1.458	0.589	–	–	–
Gender	0.753	0.445–1.273	0.289	–	–	–
Differentiation	1.142	0.676–1.928	0.620	–	–	–
TNM stage	1.308	0.764–2.239	0.327	–	–	–
Lymph node metastasis	1.287	0.742–2.230	0.369	–	–	–

–, indicated no related data.

Figure 4.

Effects of miR-378 on the proliferation of QBC939 and RBE cell line. A. qRT-PCR analysis of miR-378 in QBC939 and RBE cells. The expression of miR-378 was increased in cells transfected with miR-378 mimic, and down-regulated in cells transfected with miR-378 inhibitor. B. Cell proliferation was enhanced in the QBC939 and RBE cells transfected with miR-378 mimic but was inhibited by downregulation of miR-378 (* $P<$ 0.05, ** $P<$ 0.01, *** $P<$ 0.001).

Figure 5.

The effects of miR-378 on the migration and invasion abilities of QBC939 and RBE cells. A. The results of migration analysis.B. Overexpression of miR-378 by miR-378 mimic enhanced the cell migration, whereas downregulated miR-378 expression by miR-378 inhibitor suppressed the cell migration. C. The results of invasion analysis. D. Cell invasion was promoted by overexpression of miR-378 but was inhibited by downregulation of miR-378 in both cell lines (* $P<$ 0.05, ** $P<$ 0.01, *** $P<$ 0.001).

3.3 Expression levels of miR-378 among CCA patients with different TNM stages

Since the expression of miR-378 was significantly associated TNM stage, it may have capacity in distinguishing patients with different stages. The TNM stages of CCA patients were confirmed according to the American Joint Committee on Cancer (AJCC) edition staging system [21]. To investigate the capacity of miR-378 in distinguishing patients with different stages, CCA patients were divided into two groups, including low TNM stage (I and II) group and high (III and IV) group. The expression levels of miR-378 were 3.21 $\pm$ 0.99 in low stage group, 4.53 $\pm$ 1.06 in high stage group, which indicated the expression levels of miR-378 were increased in high stage group compared with that in low stage group (Fig. 2A). As shown in Fig. 2B, the AUC value was 0.816 (95% CI $=$ 0.741–0.891). Using the optimum cutoff value of 4.10, the sensitivity and specificity were 82.8% and 69.4%, respectively. The results showed that miR-378 had a relative accuracy in distinguishing high TNM stages CCA patients from low stages patients, indicating that high miR-378 is significantly correlated with the advanced TNM stage.

3.4 Correlations of miR-378 expression and survival of CCA patients

To determine the prognostic value of the miR-378 expression in CCA, we used the Kaplan-Meier survival analysis. As shown in Fig. 3, the overall survival of patients with high miR-378 expression was significantly worse than those with low miR-378 expression (log-rank test: $P=$ 0.030). Also, univariate and multivariate Cox regression analyses were performed to determine the prognostic parameters. The results confirmed that miR-378 was an independent prognostic factor for the patients with CCA (HR $=$ 1.735, 95% CI $=$ 1.007–2.988, $P=$ 0.041, Table 2).

3.5 In vitro effect of miR-378 on CCA cell proliferation, migration, and invasion

To investigate the functional significance of miR-378 in CCA, the QBC939 and RBE cells were transfected with miR-378 mimic or miR-378 inhibitor, and the viability of the cells was detected with an MTT assay. The qRT-PCR results indicated that the expression level of miR-378 in cells transfected with miR-378 mimic was significantly higher, and in those transfected with miR-378 inhibitor was decreased, compared to those transfected with negative control or mock ( $P<$ 0.05, Fig. 4A). The MTT result showed that transfection with a miR-378 mimic promoted the rate of proliferation in the QBC939 and RBE cells (all $P<$ 0.05), whereas transfection with a miR-378 inhibitor decreased the rate of proliferation in QBC939 and RBE cells (all $P<$ 0.05, Fig. 4B). This data suggested that miR-378 expression significantly promote cell proliferation in the two cell lines.

In addition to cell proliferation, the effects of miR-378 on CCA cell migration and invasion were also examined with a Transwell assay. As presented in Fig. 5, compared with the mock group, transfection with a miR-378 mimic increased the migration and invasion ability in the QBC939 and RBE cells, whereas transfection with a miR-378 inhibitor reduced the migration and invasion ability in QBC939 and RBE cells (all $P<$ 0.05). Taken together, miR-378 promotes the proliferation, migration, and invasion of human CCA cells.

4. Discussion

CCA has few distinguishable symptoms, although MRI and computed tomography (CT) are common methods used in the diagnosis of CCA, the patients are diagnosed at the advanced tumor stage, resulting to poor outcomes and a relatively low survival rate [22, 23]. Currently, numerous studies have devoted to exploring specific markers with diagnosis or/and prognosis in CCA [24, 25, 26]. For instance, Lee et al. indicated that HMGA2 expression and lymph node metastasis were associated with shorter patient survival and were an independent marker of poor prognosis in intrahepatic CCA [26]. Watanabe et al. indicated the EMT inducer FOXC2 expression contributed to a shorter cancer-specific survival in extrahepatic cholangiocarcinoma (EHCC) patients and might be a promising molecular target for regulating EHCC metastasis [27]. As novel biomarkers, the potential rules of miRNAs in cancers that have broad prospects has also been identified [28, 29, 30, 31]. These studies suggest that the progress of cancer-related biomarkers may be useful for improving the accuracy of diagnosis and prognosis, as well as help predict treatment strategies for cancer patients.

Numerous studies indicated miR-378 function as an oncogene in various cancers [16, 17, 18]. However, interestingly, in human colon cancer, miR-378 was downregulated in the cancer tissues and cell lines, indicating that miR-378 may act as a tumor suppressor to inhibit the proliferation, migration, and invasion of colon cancer cells by targeting SDAD1 [20]. Similarly, miR-378 was also found that serves as a tumor suppressor and played an important role in inhibiting tumor migration and invasion, such as in glioma, colorectal cancer [32, 33]. In the current study, which aimed to explore the functional significance of miR-378 in CCA, we demonstrated that miR-378 was upregulated in CCA tissues when compared with adjacent normal tissues, and in cancer cell lines compared with HIBEC cells. The data showed that miR-378 may act as an oncogene in CCA. Considering the multiple functions of miR-378 during the development of different cancers, we indicated that miR-378 may act as either an oncogene or a tumor suppressor gene depending on the cancer type. Moreover, the expression levels of miR-378 were significantly correlated with TNM stage and lymph node metastasis, which suggest miR-378 is involved in the development of CCA. Since the expression of miR-378 was significantly associated with TNM stage of patients, ROC curve analysis results showed miR-378 has the ability to distinguish CCA patients with high TNM stages from those with low TNM stages, indicating high miR-378 is significantly correlated with the advanced TNM stages.

To further investigate the prognostic role of miR-378 in CCA patients, Kaplan-Meier and Cox regression analysis were used. Firstly, we found that the levels of miR-378 were positively correlated with the TNM stage and lymph node metastasis, which showed miR-378 expression was associated with the progression of CCA. Then in the Kaplan-Meier survival analysis, the patients with higher miR-378 expression level had a significantly poorer prognosis than those with low miR-378 expression level ( $P=$ 0.030). In addition, we further performed a Cox proportional hazards regression analysis to determine the prognostic value of miR-378 in CCA patients. The result of multivariate analysis indicated that miR-378 was an independent prognostic biomarker for CCA patients.

Moreover, we performed cells experiments to explore the biological function of miR-378 in CCA cells. The results showed that miR-378 mimic promoted cell proliferation, migration, and invasion, whereas the miR-378 inhibitor could inhibit proliferation, migration, and invasion of CCA cells. Various studies have indicated the effects of miR-378 on biological behaviors during cancer progression, and the molecular pathways regulated by miR-378 [34, 35]. For instance, a study by Sun et al. demonstrated that miR-378 functioned as an onco-miRNA by targeting the ST7L/Wnt/ $\beta$ -catenin pathway and promoted cell proliferation in cervical cancer [34]. However, our present work didn’t perform the potential molecular mechanism of miR-378 in CCA. What’s more, the result of this study might be limited by the sample size. Further studies are needed to explore the specific role of miR-378 in CCA progression with more large-scale samples.

In conclusion, our present work showed that miR-378 expression was increased in CCA and was tightly associated with cancer cell proliferation, migration, and invasion. Our study provides miR-378 may be a prognostic biomarker for therapeutic strategies in the treatment of CCA.

Footnotes

Conflict of interest

The authors declare that they have no competing interests.

References

Gibiino

Fabbri

Fagiuoli

Ianiro

Fornelli

and Cennamo

, Defining the biology of intrahepatic cholangiocarcinoma: Molecular pathways and early detection of precursor lesions, Eur Rev Med Pharmacol Sci 21 (2017), 730–41.

Zhang

G.W.

Lin

J.H.

Qian

J.P.

and Zhou

, Identification of risk and prognostic factors for patients with clonorchiasis-associated intrahepatic cholangiocarcinoma, Ann Surg Oncol 21 (2014), 3628–37.

Ghouri

Y.A.

Mian

and Blechacz

, Cancer review: Cholangiocarcinoma, J Carcinog 14 (2015), 1.

Blechacz

, Cholangiocarcinoma: Current knowledge and new developments, Gut Liver 11 (2017), 13–26.

Marcano-Bonilla

Mohamed

E.A.

Mounajjed

and Roberts

L.R.

, Biliary tract cancers: Epidemiology, molecular pathogenesis and genetic risk associations, Chin Clin Oncol 5 (2016), 61.

Valle

J.W.

Wasan

Johnson

Jones

Dixon

Swindell

Baka

Maraveyas

Corrie

Falk

Gollins

Lofts

Evans

Meyer

Anthoney

Iveson

Highley

Osborne

and Bridgewater

, Gemcitabine alone or in combination with cisplatin in patients with advanced or metastatic cholangiocarcinomas or other biliary tract tumours: A multicentre randomised phase II study – The UK ABC-01 Study, Br J Cancer 101 (2009), 621–7.

Ambros

, The functions of animal microRNAs, Nature 431 (2004), 350–5.

Singh

R.P.

Massachi

Manickavel

Singh

Rao

N.P.

Hasan

Mc Curdy

D.K.

Sharma

Wong

Hahn

B.H.

and Rehimi

, The role of miRNA in inflammation and autoimmunity, Autoimmun Rev 12 (2013), 1160–5.

Smyth

L.A.

Boardman

D.A.

Tung

S.L.

Lechler

and Lombardi

, MicroRNAs affect dendritic cell function and phenotype, Immunology 144 (2015), 197–205.

10.

Chen

Song

Cui

Hou

Zheng

and Liu

, microRNAs regulate adipocyte differentiation, Cell Biol Int 37 (2013), 533–46.

11.

Xiang

D.S.

Wang

Z.Y.

Shen

Sun

T.Q.

Guan

and Wang

Y.J.

, miR-584 suppresses invasion and cell migration of thyroid carcinoma by regulating the target oncogene ROCK1, Oncol Res Treat 38 (2015), 436–40.

12.

Hayes

Peruzzi

P.P.

and Lawler

, MicroRNAs in cancer: Biomarkers, functions and therapy, Trends Mol Med 20 (2014), 460–9.

13.

Di Leva

Garofalo

and Croce

C.M.

, MicroRNAs in cancer, Annu Rev Pathol 9 (2014), 287–314.

14.

Fricke

Cimniak

A.F.V.

Ullrich

P.V.

Becherer

Bickert

Pfeifer

Heinz

Stark

G.B.

Bannasch

Braig

and Eisenhardt

S.U.

, Whole blood miRNA expression analysis reveals miR-3613-3p as a potential biomarker for dedifferentiated liposarcoma, Cancer Biomark 22 (2018), 199–207.

15.

Switlik

Karbownik

M.S.

Suwalski

Kozak

and Szemraj

, miR-30a-5p together with miR-210-3p as a promising biomarker for non-small cell lung cancer: A preliminary study, Cancer Biomark 21 (2018), 479–88.

16.

Chan

J.K.

Kiet

T.K.

Blansit

Ramasubbaiah

Hilton

J.F.

Kapp

D.S.

and Matei

, MiR-378 as a biomarker for response to anti-angiogenic treatment in ovarian cancer, Gynecol Oncol 133 (2014), 568–74.

17.

Lin

Qian

Yin

Yang

Tang

Chen

Wen

Guo

and Deng

, MiR-378 promotes the migration of liver cancer cells by down-regulating Fus expression, Cell Physiol Biochem 34 (2014), 2266–74.

18.

Browne

Dragon

J.A.

Hong

Messier

T.L.

Gordon

J.A.

Farina

N.H.

Boyd

J.R.

VanOudenhove

J.J.

Perez

A.W.

Zaidi

S.K.

Stein

J.L.

Stein

G.S.

and Lian

J.B.

, MicroRNA-378-mediated suppression of Runx1 alleviates the aggressive phenotype of triple-negative MDA-MB-231 human breast cancer cells, Tumour Biol 37 (2016), 8825–39.

19.

Wunsch

Klak

Wasik

Milkiewicz

Blatkiewicz

Urasinska

Barbier

Bielicki

Bogdanos

D.P.

Elias

and Milkiewicz

, Liver expression of sulphotransferase 2A1 enzyme is impaired in patients with primary sclerosing cholangitis: lack of the response to enhanced expression of PXR, J Immunol Res 2015 (2015), 571353.

20.

Zeng

Zhu

and Kang

, miR-378 suppresses the proliferation, migration and invasion of colon cancer cells by inhibiting SDAD1, Cell Mol Biol Lett 22 (2017), 12.

21.

Meng

Z.W.

Pan

Hong

H.J.

Chen

J.Z.

and Chen

Y.L.

, Macroscopic types of intrahepatic cholangiocarcinoma and the eighth edition of AJCC/UICC TNM staging system, Oncotarget 8 (2017), 101165–74.

22.

Nitta

Sato

Ren

X.S.

Harada

Sasaki

Hirano

and Nakanuma

, Autophagy may promote carcinoma cell invasion and correlate with poor prognosis in cholangiocarcinoma, Int J Clin Exp Pathol 7 (2014), 4913–21.

23.

Goyal

Govindan

Sheth

R.A.

Nardi

Blaszkowsky

L.S.

Faris

J.E.

Clark

J.W.

Ryan

D.P.

Kwak

E.L.

Allen

J.N.

Murphy

J.E.

Saha

S.K.

Hong

T.S.

J.Y.

Ferrone

C.R.

Tanabe

K.K.

Chong

D.Q.

Deshpande

Borger

D.R.

Iafrate

A.J.

Bardeesy

Zheng

and Zhu

A.X.

, Prognosis and clinicopathologic features of patients with advanced stage isocitrate dehydrogenase (IDH) mutant and IDH wild-type intrahepatic cholangiocarcinoma, Oncologist 20 (2015), 1019–27.

24.

Dong

Z.R.

Zhang

Cai

J.B.

Zhang

P.F.

Shi

G.M.

Gao

D.M.

Sun

H.C.

Qiu

S.J.

Zhou

A.W.

and Fan

, Role of 5-hydroxymethylcytosine level in diagnosis and prognosis prediction of intrahepatic cholangiocarcinoma, Tumour Biol 36 (2015), 2763–71.

25.

Song

Liu

Wang

Qiao

Cigliano

Utpatel

Ribback

Pilo

M.G.

Serra

Gordan

J.D.

Che

Zhang

Cossu

Porcu

Pascale

R.M.

Dombrowski

Calvisi

D.F.

Evert

and Chen

, Combined CDK4/6 and pan-mTOR inhibition is synergistic against intrahepatic cholangiocarcinoma, Clin Cancer Res (2018).

26.

Lee

C.T.

T.T.

Lohse

C.M.

and Zhang

, High-mobility group AT-hook 2: An independent marker of poor prognosis in intrahepatic cholangiocarcinoma, Hum Pathol 45 (2014), 2334–40.

27.

Watanabe

Suzuki

Yokobori

Altan

Kubo

Araki

Wada

Mochida

Sasaki

Kashiwabara

Hosouchi

and Kuwano

, Forkhead box protein C2 contributes to invasion and metastasis of extrahepatic cholangiocarcinoma, resulting in a poor prognosis, Cancer Sci 104 (2013), 1427–32.

28.

Sun

Zhu

Chen

Zhu

Cai

Guo

Wang

and Huang

, Diagnostic and prognostic value of microRNAs in cholangiocarcinoma: a systematic review and meta-analysis, Cancer Manag Res 10 (2018), 2125–39.

29.

Zhang

and Yu

, miR-19b serves as a prognostic biomarker of breast cancer and promotes tumor progression through PI3K/AKT signaling pathway, Onco Targets Ther 11 (2018), 4087–95.

30.

Wang

Zhang

Wang

Tan

and Zhang

, MicroRNA-384 inhibits proliferation migration and invasion of glioma by targeting at CDC42, Onco Targets Ther 11 (2018), 4075–85.

31.

Deng

and Chen

, Increased expression of mir-29a and its prognostic significance in patients with cholangiocarcinoma, Oncol Res Treat 40 (2017), 128–32.

32.

Wang

Sun

Wang

and Tao

, Decreased expression of miR-378 correlates with tumor invasiveness and poor prognosis of patients with glioma, Int J Clin Exp Pathol 8 (2015), 7016–21.

33.

Wang

Deng

Zhang

Gao

Sun

Chen

and Zhao

, MicroRNA-378-5p suppresses cell proliferation and induces apoptosis in colorectal cancer cells by targeting BRAF, Cancer Cell Int 15 (2015), 40.

34.

Sun

Wang

and Xiao

, MicroRNA-378 regulates epithelial-mesenchymal transition and metastasis of melanoma by inhibiting FOXN3 expression through the Wnt/beta-catenin pathway, Cell Biol Int (2018).

35.

Yang

Wang

Cao

and Yang

, miR-378 functions as an onco-miRNA by targeting the ST7L/Wnt/beta-catenin pathway in cervical cancer, Int J Mol Med 40 (2017), 1047–56.

miR-378 serves as a prognostic biomarker in cholangiocarcinoma and promotes tumor proliferation,migration,and invasion

Abstract

Keywords

1. Introduction

2. Materials and methods

2.1 Patients and tissue samples

Table 1 Association between miR-378 expression and clinicopathological variables of CCA patients

2.3 RNA isolation and qRT-PCR analysis

2.5 Migration and invasion assays

2.6 Statistical analysis

3. Results

3.1 Expression of miR-378 in the CCA tissues and cell lines

3.4 Correlations of miR-378 expression and survival of CCA patients

3.5 In vitro effect of miR-378 on CCA cell proliferation, migration, and invasion

4. Discussion

Footnotes

Conflict of interest

References

Table 1
Association between miR-378 expression and clinicopathological variables of CCA patients