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miR-99b-3p promotes hepatocellular carcinoma metastasis and proliferation by targeting protocadherin 19
Gene 698 (2019) 141–149
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Gene journal homepage: www.elsevier.com/locate/gene
Research paper
miR-99b-3p promotes hepatocellular carcinoma metastasis and proliferation by targeting protocadherin 19 Xiaobin Yaoa,1, Hongguo Zhangb, Yujin Chengd
⁎,1
T
, Yujuan Liuc, Xiaoming Liua, Xinhui Wanga, Xiaofeng Suna,
a
Departments of General Surgery, General Hospital of Lanzhou Petrochemical Company, Lanzhou 730060, Gansu, China Department of Pharmacy, General Hospital of Lanzhou Petrochemical Company, Lanzhou 730060, Gansu, China c Teaching and research section of physiology and pathology in the basic department, Medical Sciences, Hexi University, Zhangye 734000, Gansu, China d Department of Science and Education, General Hospital of Lanzhou Petrochemical Company, Lanzhou 730060, Gansu, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Hepatocellular carcinoma miR-99b-3p PCDH19 Proliferation Metastasis
Hepatocellular carcinoma (HCC) is one of the most common cancers in the world, with characteristics of high morbidity and mortality. Identifying clinically practical targets and uncovering the potential mechanism for HCC were urgent for us. Though aberrantly expressed miR-99b-3p has been reported in several cancers, the expression and roles of miR-99b-3p in HCC remain uncovered. In the present study, we demonstrated for the first time that miR-99b-3p was overexpressed in HCC by our findings and data from GEO datasets. Statistical analysis revealed that highly expressed miR-99b-3p was closely related to malignant clinicopathological characteristics and poorer prognosis of HCC patients. Furthermore, results from Wound healing assay, Transwell assays, 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)assay and 5-ethynyl-2′-deoxyuridine (EdU) assay revealed that miR-99b-3p played a promoting role in migration, invasion and proliferation of HCC cells. Then, by bioinformatics tools, luciferase reporter gene assay, integrative database analysis, and Pearson correlation analysis and so on, protocadherin 19 (PCDH19) was identified as the target of miR-99b-3p in HCC cells. Furthermore, rescue experiments were conducted to confirm the mediator role of PCDH19 for miR-99b-3p. Collectively, we demonstrate that miR-99b-3p promotes HCC metastasis and proliferation by directly targeting PCDH19. MiR-99b-3p may become a potential therapy target for HCC.
1. Introduction Hepatocellular carcinoma (HCC), one of the most common cancers in the world, is the main type of primary liver cancer (Karaman et al., 2014; Erstad and Tanabe, 2017). Though last decades the diagnosis and treatment level of HCC has been improved largely, and lots of targets have been identified for HCC, the prognosis of HCC patients remain unsatisfactory (Karaman et al., 2014; Gupta et al., 2017; Rich et al., 2017). Thus, it's urgent for us to clarify the intrinsic mechanisms and explore the potential new targets for HCC therapy. As a class of non-coding RNAs, microRNAs (miRNAs), about 22 nucleotides in length, play an important role in tumor progression by regulating gene expression via binding to the 3′-untranslated regions
(3′-UTRs) of target mRNAs (Mao and Wang, 2015; Xu et al., 2018; Yang et al., 2018). At present, numerous miRNAs have been reported to be associated with HCC progression and development, such as miR-218 (Wu et al., 2018), miR-500a (Jiang et al., 2017) and miR-3662 (Chen et al., 2018). Recently, miR-99b-3p is found to be deregulated in papillary thyroid carcinoma (Dettmer et al., 2013), gastric cancer (Chang et al., 2015) and oral squamous cell carcinoma(He et al., 2015). And in these studies, miR-99b-3p expression is closely related to tumor progression and development. However, the expression and roles of miR99b-3p in HCC remain uncovered. Protocadherin 19 (PCDH19) is a member of the delta-2 protocadherin subclass of the cadherin superfamily (Pham et al., 2017). Previous studies reported that PCDH19 is under-expressed in HCC and
Abbreviations: HCC, Hepatocellular carcinoma; PCDH19, protocadherin 19; miRNA, microRNAs; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; EdU, 5-ethynyl-2′-deoxyuridine; DMEM, Dulbecco's Modified Eagle Medium; FBS, fetal bovine serum; RIPA, Radioimmunoprecipitation assay; 3′-UTR, 3′untranslated region; Real-time PCR, Real-time polymerase chain reaction; AFP, alpha-fetoprotein; TNM, tumor-node-metastasis ⁎ Corresponding author. E-mail address: [email protected] (H. Zhang). 1 Contributed equally. https://doi.org/10.1016/j.gene.2019.02.071 Received 24 August 2018; Received in revised form 11 February 2019; Accepted 17 February 2019 Available online 05 March 2019 0378-1119/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. miR-99b-3p is increased in HCC tissues and cell lines. (A) Real time-PCR indicated that the expression of miR-99b-3p in HCC tissues (n = 92) was much higher than that in adjacent non-tumor tissues (n = 92). (B) GEO dataset (GSE10694) showed that miR-99b-3p was significantly up-regulated in primary liver cancer tissues (n = 78), compared to that in normal liver tissues (n = 10) and adjacent noncancerous liver tissues (n = 78). (C) GEO dataset (GSE62007, n = 100) revealed that miR99b-3p was increased in 73% of the HCC tissues. (D) Real time-PCR revealed that the expression of miR-99b-3p was generally increased in HCC cell lines. *P < 0.05, **P < 0.01, ***P < 0.001.
2. Materials and methods
Table 1 Association of miR-99b-3p expression with different clinicopathological features of HCC patients (n = 92). Clinicopathological features
Cases
Expression of miR-99b-3p Low (n = 46)
Age (year) Gender AFP level (ng/ml) Tumor size (cm) Venous infiltration Edmondson-Steiner grading TNM stage
< 50 ≥50 Male Female < 400 ≥400 <5 ≥5 Absent Present I + II III + IV I + II III + IV
29 63 78 14 22 70 42 50 66 26 60 32 68 24
17 29 38 8 14 32 27 19 38 8 34 12 39 7
2.1. Patients and tissue samples
P
All of the 92 paired HCC tissue samples and adjacent non-tumor tissue samples, which were histopathologically confirmed, were collected from the HCC patients underwent surgery at the Department of General Surgery of General Hospital of Lanzhou Petrochemical Company from January 2005 to October 2013. The fresh tissue samples were stored at −80 °C. All of these patients didn't receive chemotherapy or radiotherapy before surgery. This study was approved by the Ethics Committees of General Hospital of Lanzhou Petrochemical Company. Informed consent was obtained from all individual participants included in the study.
High (n = 46) 12 34 40 6 8 38 15 31 28 18 26 20 29 17
0.262 0.562 0.143 0.012 0.021 0.080
2.2. Cell culture and transfection
0.018
Normal human liver cell (LO2) and HCC cell lines (HCCLM3, SMMC7721, HepG2, Hep3B, Huh7 and MHCC-97 L) were obtained from American Type Culture Collection (ATCC). All cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM, Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) and 1% penicillin/streptomycin. Cells were incubated in a humidified incubator (37 °C, 5% CO2). Transfections were conducted with Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA) according to the product specification. MiR-99b-3p mimics (#HmiR0307MR04), miR-99b-3p inhibitors (#HmiR-AN0856-AM01), clone for PTPRT (#E2229) and shRNA for PTPRT (#HSH058614-LVRH1GP) were purchased form GeneCopoeia Inc.(Guangzhou, China).
HCC, hepatocellular carcinoma; AFP, alpha-fetoprotein; TNM, tumor-nodemetastasis.
it is closely related to the prognosis of HCC patients, which means that PCDH19 may act as a tumor suppressor in HCC (Zhang et al., 2018). However, the exact function of PCDH19 in HCC remains unknown. In this study, we demonstrate that miR-99b-3p is overexpressed in both HCC tissues and cell lines. Highly expressed miR-99b-3p is closely associated with malignant clinicopathological characteristics and poorer prognosis of HCC patients. Furthermore, we found that miR-99b3p promotes HCC progression by directly targeting PCDH19.
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Fig. 2. HCC patients with higher miR-99b-3p have worse prognosis. (A) HCC patients in high miR-99b-3p group had worse 3-year overall survival. (B) HCC patients in high miR-99b-3p group had worse 3-year disease-free survival. (C) TCGA dataset (n = 362) from Oncolnc platform (http://www.oncolnc.org/) showed that HCC patients in high miR-99b-3p group had worse survival rate.
2.3. Real-time polymerase chain reaction (real-time PCR)
2.7. 5-ethynyl-2′-deoxyuridine (Edu) assay
The total RNA were extracted from tissue samples and cells with Trizol (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacture's instruction. For detection of miRNA expression, cDNA was synthesized using the qScript microRNA cDNA Synthesis Kit (Quantabio, Beverly, MA) according to manufacturer's instruction. Quantitative PCR was performed with miScript SYBR Green PCR Kit (Qiagen, Hilden, Germany) under the following thermocycler conditions: 95 °C for 2 min, and 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Primers for miR-99b-3p (#HmiRQP0856) and U6 snRNA (#HmiRQP9001) were purchased from GeneCopoeia (GeneCopoeia Inc., Guangzhou, China). U6 snRNA was used as housekeeping control for normalization. For detection of mRNA expression, 1 μg of total RNA was used to synthesize cDNA using cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA). The PCR conditions are as follows: 95 °C for 5 min, and 40 cycles of 95 °C 20 s and 62 °C 30 s, followed by 72 °C 3 min. The primers for PTPRT(#HQP058614) and GAPDH (#HQP006940) were purchased from GeneCopoeia (GeneCopoeia Inc., Guangzhou, China). The results was quantified with the 2−ΔΔCt method.
Cell-Light™ EdU Apollo®567 In Vitro Imaging Kit (RiboBio Co., Ltd. Guangzhou, China) was used for EdU assay. Briefly, transfected HCC cells (1 × 105) were cultured in 96-well plates. Cells were incubated with EdU labelling medium at moderate concentration for 2 h. Then, the cells were fixed with 4% paraformaldehyde, glycine and 0.5% TritonX-100 in PBS. Next, cells were stained with 100 μL Apollo dye solution for 30 min at room temperature. The cells were subsequently stained using Hoechst and incubated for 30 min. The photos were taken on a microscope. The percentage of EdU positive cells was calculated using ImageJ software. 2.8. Luciferase reporter assay pGL3-PTPRT-3′-UTR report/pGL3-PTPRT-3′-UTR Mutant report + TK100 Renilla reports were conducted. Then, the plasmids were transfected into 75% confluent HCC cells, along with miR-532-3p mimics or miR-532-3p inhibitors. The relative luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) and normalized to Renilla activity.
2.4. Wound healing assay 2.9. Western blot
When cells grew around 70–80% confluence in culture wells, 200 μL pipette tip was used to gently scratch the monolayer across the center of the well. Wells were briefly washed twice with medium to remove the detached cells. Fresh medium was added and cells grew for additional 24 h before photos were taken on a microscope.
Total protein was isolated by Radioimmunoprecipitation assay (RIPA) buffer, and quantified by Bradford assay (Sigma, St. Louis, MO). 20 μg of total protein was loaded into each well and transferred to PVDF membrane. Primary antibody used against human PTPRT (1:1000, ab115848, Abcam, Cambridge, UK) and anti-β-actin (1:1000; Cell Signaling Technology) were incubated with membrane at 4 °C overnight. Secondary anti-rabbit antibody (GE Healthcare Life Sciences, Beijing, China) was incubated with membrane at room temperature for 1 h. The protein bands were visualized using enhanced chemiluminescence kit (Amersham, Little Chalfont, UK) and Odyssey software (Infrared Imaging System LI-COR Biosciences).
2.5. Transwell assays The 24-well Transwell chambers (8 μm pore size, BD Biosciences) were used for Transwell migration and invasion assay. And, for Transwell invasion assay, the top chambers were coated with Matrigel matrix (Corning, Corning, NY). Approximate 2 × 104 cells were added into the top chambers. Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) was filled into the bottom chambers. After 24 h of incubation at 37 °C, the cells that did not pass through the membrane were removed by a cotton-tipped applicator, and cells on the lower surface of the membrane were subjected to crystal violet staining.
2.10. Statistical analyses Graphpad Prism 6 was used to perform statistical analyses. Unpaired t-test, one-way ANOVA, two-way ANOVA, and Kaplan-Meier method, Chi-square test, Spearman Pearson's correlation analysis and Log-Rank test were used to analyze the data. Differences were defined as statistically significant if P < 0.05.
2.6. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
3. Results Cells in 24 wells plate with different treatments were subjected to medium replacement with 10 μl sterile MTT dye (5 mg/mL). After incubation at 37 °C for 4 h, the MTT solution was removed, and 150 μl dimethyl sulfoxide was added to each well. Absorbance was read at 490 nm on an enzyme immunoassay analyzer (Biotek, Winooski, VT).
3.1. miR-99b-3p is up-regulated in HCC To investigate the expression levels of miR-99b-3p in HCC, we first detected the expression of miR-99b-3p in 92 paired HCC tissues and 143
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Fig. 3. The effects of miR-99b-3p on migration and invasion of HCC cells. (A) miR-99b-3p inhibitors significantly decreased the expression of miR-99b-3p in HCCLM3 cells. (B) Wound healing assay revealed that miR-99b-3p inhibitors obviously suppressed mobility of HCCLM3 cells. (C) Wound healing assay revealed that miR99b-3p mimics markedly enhanced mobility of Hep3B cells. (D) Transwell assays revealed that miR99b-3p inhibitors notably suppressed migration and invasion of HCCLM3 cells. (E) Transwell assays revealed that miR-99b-3p mimics notably promoted migration and invasion of Hep3B cells.Magnification:100 × .*P < 0.05, **P < 0.01, ***P < 0.001.
cell lines compared to the normal human liver cell LO2 (P < 0.001, respectively, Fig. 1D). Taken together, we demonstrate that miR-99b-3p is overexpressed in HCC and it's a potential oncogene in HCC.
adjacent non-tumor tissues by real-time PCR. The results indicated that miR-99b-3p expression was significantly upregulated in HCC tissues compared to the non-tumor tissues (P < 0.001, Fig. 1A). Consistently, GEO data from datasets GSE10694 (Fig. 1B) and GSE62007 (Fig. 1C) showed that miR-99b-3p was frequently upregulated in HCC tissues. In addition, we found that miR-99b-3p was generally upregulated in HCC 144
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Fig. 4. miR-99b-3p promotes proliferation of HCC cells. (A) MTT assay indicated that miR-99b-3p inhibitors notably suppressed viability of HCCLM3 cells. (B) MTT assay indicated that miR-99b-3p mimics markedly enhanced viability of Hep3B cells. Edu assay revealed that (C) miR-99b-3p inhibitors notably inhibited proliferation of HCCLM3 cells, (D) while miR-99b-3p mimics had the contrary effect on Hep3B cells. *P < 0.05, **P < 0.01, ***P < 0.001.
inhibited the healing capacity of HCCLM3 cells (P < 0.001, Fig. 3B). In contrast, the healing capacity of Hep3B cells was enhanced by miR-99b3p mimics (P < 0.01, Fig. 3C). Results from Transwell migration and invasion assays showed that miR-99b-3p inhibitors decreased the number of HCCLM3 cells passing through the membrance (P < 0.01, P < 0.5, Fig. 3D), while miR-99b-3p mimics had the contrary effect on Hep3B cells (P < 0.01, P < 0.5, Fig. 3E). Thus, these data demonstrate that miR-99b-3p promotes migration and invasion of HCC cells.
3.2. The clinical significance of miR-99b-3p in HCC Next, we attempted to explore whether miR-99b-3p was correlated with HCC progression. Based on the cutoff value, the median expression of miR-99b-3p in HCC tissues, the 92 HCC patients were sorted into two subgroups (low/high miR-99b-3p group). The data revealed that miR99b-3p was closely related to tumor size (P = 0.012, Table 1), venous infiltration (P = 0.021, Table 1) and TNM stage (P = 0.018, Table 1). In addition, HCC patients with higher miR-99b-3p expression had worse 3year overall survival rate (P = 0.0104, Fig. 2A) and disease-free survival rate (P = 0.0078, Fig. 2B), which was consistent with the result from TCGA dataset (http://www.oncolnc.org) (P = 0.0034, Fig. 2C). In conclusion, these findings suggest that miR-99b-3p is closely correlated with HCC progression.
3.4. miR-99b-3p promotes proliferation of HCC cells Next, we attempted to investigate whether miR-99b-3p had any effects on proliferation of HCC cells. MTT assay indicated that miR-99b3p inhibitors suppressed HCCLM3 cells viability (Fig. 4A), while miR99b-3p mimics had the contrary effect on Hep3B (Fig. 4B). Consistently, data from Edu assay showed that miR-99b-3p inhibitors decreased the percentage of Edu positive cells (Fig. 4C), while miR-99b-3p mimics increased the percentage (Fig. 4D). Taken together, the above results indicate that miR-99b-3p promotes migration, invasion and proliferation of HCC cells.
3.3. miR-99b-3p promotes migration and invasion of HCC cells Then, in order to explore the effects of miR-99b-3p on HCC cells, we first altered miR-99b-3p expressions of HCCLM3 and Hep3B cells by miR-99b-3p inhibitors or mimics. And the transfection efficiency in each cell line was measured with real-time PCR (Fig. 3A). Then, we attempted to explore the effects of miR-99b-3p on HCC cells migration and invasion. Wound healing assay showed that miR-99b-3p inhibitors 145
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Fig. 5. PCCDH19 acts as a downstream target of miR-99b-3p in HCC cells. (A) Results from bioinformatics tools (TargetScan, miRanda and PicTar) revealed that 3′UTR of PCDH19 had the bind site for miR-99b-3p. (B) Luciferase reporter assay revealed that miR-99b-3p inhibitors enhanced the fluorescence activity of PCDH19wt-3’UTR, while miR-99b-3p mimics repressed the fluorescence activity of PCDH19-wt-3’UTR. The alteration of miR-99b-3p expression had no effect on PCDH19mut-3’UTR. (C) The expression of PCDH19 mRNA was significantly lower in high miR-99b-3p HCC tissues. (D) Data from GEO dataset (GSE54236) and UALCAN platform (http://ualcan.path.uab.edu/index.html) revealed that PCDH19 expression was significantly lower in HCC tissues. (F) Result from UALCAN platform showed that PCDH19 was decreased with HCC progression. (G) Data analysis in HCC tissues of our cohort indicated that there existed a negative relationship between PCDH19 and miR-99b-3p. miR-99b-3p negatively regulated PCDH19 mRNA expression (H, I) and protein expression (J, K) in HCC cells. **P < 0.01, ***P < 0.001.
confirmed that the transfections were successful (Fig. 6A,B). Wound healing assay (Fig. 6C) and Transwell assays (Fig. 6E) showed that PCDH19 shRNAs enhanced the mobility of HHCLM3 cells, and reversed the functions of miR-99b-3p inhibitors. In contrast, PCDH19 clones inhibited the mobility of Hep3B cells, and receded the effects of miR99b-3p mimics (Fig. 6D,F). Similarly, MTT assay (Fig. 7A,B) and Edu assay (Fig. 7C,D) showed that PCDH19 suppressed the proliferation of HCC cells, and reversed the effects of miR-99b-3p. Collectively, these data indicate that PCDH19 suppresses HCC progression and mediates the effects of miR-99b-3p on HCC cells.
3.5. PCDH19 acts as the target of miR-99b-3p in HCC cells To elucidate the mechanism by which miR-99b-3p affects HCC cells metastasis and proliferation, we made an attempt to identify potential target genes of miR-99b-3p. Bioinformatics tools (TargetScan, miRanda and PicTar) were employed to search for the most potential gene under the control of miR-99b-3p. Among these predicted targets, protocadherin 19 (PCDH19) was focused. PCDH19 is a member of the delta-2 protocadherin subclass of the cadherin superfamily (Zhang et al., 2018). It has been reported that PCDH19 is downregulated in HCC and it is closely related to the prognosis of HCC patients (Zhang et al., 2018). As showed in Fig. 5A, there existed binding sites on 3′UTR of PCDH19 for miR-99b-3p. Luciferase reporter gene assay showed that miR-99b-3p negatively regulated the luciferase activity of WT-3′UTR of PCDH19, rather than MUT-3′UTR of PCDH19 (Fig. 5B). Then, we detected the mRNA expression of PCDH19 in HCC tissues and adjacent non-tumor tissues. The result from our cohort indicated that PCDH19 was decreased in HCC tissues (Fig. 5C), which was consistent with the data from GSE54236 data (Fig. 5D) and UALCAN platform (Fig. 5E). Besides, the data from UALCAN platform also showed that PCDH19 expression decreased as the HCC progressed (Fig. 5F). Furthermore, data from our cohort indicated that PCDH19 expression was negatively associated with miR-99b-3p (r = −0.8908, P < 0.001, Fig. 5G). Realtime PCR (Fig. 5H, I) and Western blot (Fig. 5J, K) revealed that both mRNA and protein expression levels of PCDH19 were negatively regulated by miR-99b-3p. Taken together, PCDH19 acts as the target of miR-99b-3p and may mediate its effects on HCC cells.
4. Discussion An increasing number of aberrantly expressed miRNAs have been reported to play a critical role in HCC progression by participating in various tumor cellular behaviors, such as migration, invasion, apoptosis, proliferation and cell cycle (Morishita and Masaki, 2015; Mizuguchi et al., 2016; Wang and Tian, 2016). For example, miR-367 acts as an oncogene to promote HCC cells migration and invasion by targeting FBXW7 (Wang et al., 2017). MiR-491-3p regulates drugs sensitivity of HCC cells by targeting Sp3 (Zhao et al., 2017). And, miR1301 suppresses HCC cell angiogenesis, migration and invasion by targeting BCL9 to inhibit Wnt pathway (Yang et al., 2017). Recently, some studies reported that miR-99b-3p is deregulated in several cancers, such as papillary thyroid carcinoma (Dettmer et al., 2013) and oral squamous cell carcinoma (He et al., 2015). These findings suggest that aberrantly expressed miR-99b-3p maybe related to tumor progression, as an oncogene or tumor suppressor. However, the exact expression and roles of miR-99b-3p in HCC remain to be explored. In this study, we found that miR-99b-3p was highly expressed in both HCC tissues and cell lines, which was validated by data from different datasets (GSE10694 and GSE62007). Then, we attempted to explore the clinical significance of miR-99b-3p in HCC by analyzing the
3.6. PCDH19 suppresses HCC progression and mediates the effects of miR99b-3p on HCC cells Next, to explore whether PCDH19 mediated the effects of miR-99b3p on HCC cells, co-transfections were performed. Western blot 146
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Fig. 6. PCDH19 suppresses HCC cells mobility and mediates the effects of miR-99b-3p. (A) The expression of PCDH19 in HCCLM3 cells co-transfected with PCDH19 shRNA, miR-99b-3p inhibitors and corresponding controls, was detected by Western blot. (B) PCDH19 expression in Hep3B cells co-transfected with PCDH19 clone, miR-99b-3p mimics, and corresponding controls, was detected by Western blot. Wound healing assay (C,D) and Transwell assays (E,F) revealed that PCDH19 negatively regulated cell mobility and mediated the effect of miR-99b-3p on mobility of HCC cells. *P < 0.05, **P < 0.01, ***P < 0.001.
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Fig. 7. PCDH19 suppresses HCC cells proliferation and mediates the effects of miR-99b-3p. (A,B) MTT assays revealed that PCDH19 reversed the effect of miR-99b-3p on viability of HCC cells. (C,D) Edu assay revealed that PCDH19 reversed the effect of miR-99b-3p on proliferation of HCC cells. *P < 0.05, **P < 0.01, ***P < 0.001.
99b-3p on HCC cells by gain- and loss-of-function analyses. Results indicated that downregulated miR-99b-3p suppressed HCC cells migration, invasion and proliferation, while overexpressed miR-99b-3p exerted the promoting effects. The above findings suggest that highly expressed miR-99b-3p is associated with HCC progression, and it acts
relation between miR-99b-3p expression and clinicopathological characteristics and HCC patients' prognosis. Data revealed that miR-99b-3p expression was closely related to tumor size, venous infiltration and TNM stage. And, patients in high miR-99b-3p group had much worse outcomes. Then, we attempted to assess the functional effects of miR148
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an oncogene in HCC by promoting cells migration, invasion and proliferation. It has been well documented that miRNAs exert their oncogene or anti-oncogene effects by repressing the expression of targets at a posttranscriptional level (Vasuri et al., 2018). Then, we attempted to explore the most potential target of miR-99b-3p in HCC cells by three bioinformatics tools (TargetScan, miRanda and PicTar). Then, numerous different targets were screened, and we selected the mutual genes predicted by all three databases. Give that PCDH19, a member of the delta-2 protocadherin subclass of the cadherin superfamily, has been identified as a tumor suppressor in HCC (Zhang et al., 2018), we focused on PCDH19 among these predicted targets. Here, consistently, data from both our cohort and Ualcan platform revealed that PCDH19 was downregulated in HCC tissues, and it decreased with the tumor progressing. We also found that there existed a negative relation between miR-99b-3p and PCDH19. And, PCDH19 was negatively regulated by miR-99b-3p at both mRNA and protein levels. Furthermore, miR-99b-3p negatively regulated the fluorescence activity of wild type of PCDH19–3′UTR, rather than the mutant type. And, rescue experiments revealed that PCDH19 mediated the effects of miR-99b-3p on HCC cells. Taken together, the above findings demonstrate that miR99b-3p promotes HCC progression by targeting PCDH19. In summary, our findings demonstrate the highly expressed miR99b-3p in HCC tissues and cell lines, which is closely related to malignant clinicopathological characteristics and poorer prognosis of HCC patients. Furthermore, overexpressed miR-99b-3p play the promoting role in migration, invasion and proliferation of HCC cells by directly targeting PCDH19. Thus, we conclude that miR-99b-3p maybe a promising therapeutic target for the treatment of HCC.
suppresses hepatocellular carcinoma growth through inhibition of HIF-1alphamediated Warburg effect. Cell Death Dis. 9, 549. Dettmer, M., Perren, A., Moch, H., Komminoth, P., Nikiforov, Y.E., Nikiforova, M.N., 2013. Comprehensive MicroRNA expression profiling identifies novel markers in follicular variant of papillary thyroid carcinoma. Thyroid 23, 1383–1389. Erstad, D.J., Tanabe, K.K., 2017. Hepatocellular carcinoma: early-stage management challenges. J Hepatocell Carcinoma 4, 81–92. Gupta, M.K., Behara, S.K., Vadde, R., 2017. In silico analysis of differential gene expressions in biliary stricture and hepatic carcinoma. Gene 597, 49–58. He, K., Tong, D., Zhang, S., Cai, D., Wang, L., Yang, Y., Gao, L., Chang, S., Guo, B., Song, T., Li, A., Huang, C., 2015. miRNA-99b-3p functions as a potential tumor suppressor by targeting glycogen synthase kinase-3beta in oral squamous cell carcinoma Tca8113 cells. Int. J. Oncol. 47, 1528–1536. Jiang, C., Long, J., Liu, B., Xu, M., Wang, W., Xie, X., Wang, X., Kuang, M., 2017. miR500a-3p promotes cancer stem cells properties via STAT3 pathway in human hepatocellular carcinoma. J. Exp. Clin. Cancer Res. 36, 99. Karaman, B., Battal, B., Sari, S., Verim, S., 2014. Hepatocellular carcinoma review: current treatment, and evidence-based medicine. World J. Gastroenterol. 20, 18059–18060. Mao, B., Wang, G., 2015. MicroRNAs involved with hepatocellular carcinoma (review). Oncol. Rep. 34, 2811–2820. Mizuguchi, Y., Takizawa, T., Yoshida, H., Uchida, E., 2016. Dysregulated miRNA in progression of hepatocellular carcinoma: a systematic review. Hepatol. Res. 46, 391–406. Morishita, A., Masaki, T., 2015. miRNA in hepatocellular carcinoma. Hepatol. Res. 45, 128–141. Pham, D.H., Tan, C.C., Homan, C.C., Kolc, K.L., Corbett, M.A., McAninch, D., Fox, A.H., Thomas, P.Q., Kumar, R., Gecz, J., 2017. Protocadherin 19 (PCDH19) interacts with paraspeckle protein NONO to co-regulate gene expression with estrogen receptor alpha (ERalpha). Hum. Mol. Genet. 26, 2042–2052. Rich, N.E., Yopp, A.C., Singal, A.G., 2017. Medical Management of Hepatocellular Carcinoma. J Oncol Pract 13, 356–364. Vasuri, F., Visani, M., Acquaviva, G., Brand, T., Fiorentino, M., Pession, A., Tallini, G., D'Errico, A., de Biase, D., 2018. Role of microRNAs in the main molecular pathways of hepatocellular carcinoma. World J. Gastroenterol. 24, 2647–2660. Wang, Y., Tian, Y., 2016. miRNA for diagnosis and clinical implications of human hepatocellular carcinoma. Hepatol. Res. 46, 89–99. Wang, Y., Liu, Z., Yao, B., Li, Q., Wang, L., Wang, C., Dou, C., Xu, M., Liu, Q., Tu, K., 2017. Long non-coding RNA CASC2 suppresses epithelial-mesenchymal transition of hepatocellular carcinoma cells through CASC2/miR-367/FBXW7 axis. Mol. Cancer 16, 123. Wu, J., Jiang, Z.M., Xie, Y., He, X.Q., Lin, X.H., Hu, J.L., Peng, Y.Z., 2018. miR-218 suppresses the growth of hepatocellular carcinoma by inhibiting the expression of proto-oncogene Bmi-1. J BUON 23, 604–610. Xu, T., Li, L., Hu, H.Q., Meng, X.M., Huang, C., Zhang, L., Qin, J., Li, J., 2018. MicroRNAs in alcoholic liver disease: recent advances and future applications. J. Cell. Physiol. 234 (1), 382–394. Yang, C., Xu, Y., Cheng, F., Hu, Y., Yang, S., Rao, J., Wang, X., 2017. miR-1301 inhibits hepatocellular carcinoma cell migration, invasion, and angiogenesis by decreasing Wnt/beta-catenin signaling through targeting BCL9. Cell Death Dis. 8, e2999. Yang, S., Sun, Z., Zhou, Q., Wang, W., Wang, G., Song, J., Li, Z., Zhang, Z., Chang, Y., Xia, K., Liu, J., Yuan, W., 2018. MicroRNAs, long noncoding RNAs, and circular RNAs: potential tumor biomarkers and targets for colorectal cancer. Cancer Manag. Res. 10, 2249–2257. Zhang, T., Guan, G., Chen, T., Jin, J., Zhang, L., Yao, M., Qi, X., Zou, J., Chen, J., Lu, F., Chen, X., 2018. Methylation of PCDH19 predicts poor prognosis of hepatocellular carcinoma. Asia Pac J Clin Oncol. Zhao, Y., Qi, X., Chen, J., Wei, W., Yu, C., Yan, H., Pu, M., Li, Y., Miao, L., Li, C., Ren, J., 2017. The miR-491-3p/Sp3/ABCB1 axis attenuates multidrug resistance of hepatocellular carcinoma. Cancer Lett. 408, 102–111.
Declaration of interest statement None. Conflict of interest The authors declare no conflicts of interest. Acknowledgements None. References Chang, H., Kim, N., Park, J.H., Nam, R.H., Choi, Y.J., Lee, H.S., Yoon, H., Shin, C.M., Park, Y.S., Kim, J.M., Lee, D.H., 2015. Different microRNA expression levels in gastric cancer depending on helicobacter pylori infection. Gut Liver 9, 188–196. Chen, Z., Zuo, X., Zhang, Y., Han, G., Zhang, L., Wu, J., Wang, X., 2018. MiR-3662
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Research paper
miR-99b-3p promotes hepatocellular carcinoma metastasis and proliferation by targeting protocadherin 19 Xiaobin Yaoa,1, Hongguo Zhangb, Yujin Chengd
⁎,1
T
, Yujuan Liuc, Xiaoming Liua, Xinhui Wanga, Xiaofeng Suna,
a
Departments of General Surgery, General Hospital of Lanzhou Petrochemical Company, Lanzhou 730060, Gansu, China Department of Pharmacy, General Hospital of Lanzhou Petrochemical Company, Lanzhou 730060, Gansu, China c Teaching and research section of physiology and pathology in the basic department, Medical Sciences, Hexi University, Zhangye 734000, Gansu, China d Department of Science and Education, General Hospital of Lanzhou Petrochemical Company, Lanzhou 730060, Gansu, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Hepatocellular carcinoma miR-99b-3p PCDH19 Proliferation Metastasis
Hepatocellular carcinoma (HCC) is one of the most common cancers in the world, with characteristics of high morbidity and mortality. Identifying clinically practical targets and uncovering the potential mechanism for HCC were urgent for us. Though aberrantly expressed miR-99b-3p has been reported in several cancers, the expression and roles of miR-99b-3p in HCC remain uncovered. In the present study, we demonstrated for the first time that miR-99b-3p was overexpressed in HCC by our findings and data from GEO datasets. Statistical analysis revealed that highly expressed miR-99b-3p was closely related to malignant clinicopathological characteristics and poorer prognosis of HCC patients. Furthermore, results from Wound healing assay, Transwell assays, 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)assay and 5-ethynyl-2′-deoxyuridine (EdU) assay revealed that miR-99b-3p played a promoting role in migration, invasion and proliferation of HCC cells. Then, by bioinformatics tools, luciferase reporter gene assay, integrative database analysis, and Pearson correlation analysis and so on, protocadherin 19 (PCDH19) was identified as the target of miR-99b-3p in HCC cells. Furthermore, rescue experiments were conducted to confirm the mediator role of PCDH19 for miR-99b-3p. Collectively, we demonstrate that miR-99b-3p promotes HCC metastasis and proliferation by directly targeting PCDH19. MiR-99b-3p may become a potential therapy target for HCC.
1. Introduction Hepatocellular carcinoma (HCC), one of the most common cancers in the world, is the main type of primary liver cancer (Karaman et al., 2014; Erstad and Tanabe, 2017). Though last decades the diagnosis and treatment level of HCC has been improved largely, and lots of targets have been identified for HCC, the prognosis of HCC patients remain unsatisfactory (Karaman et al., 2014; Gupta et al., 2017; Rich et al., 2017). Thus, it's urgent for us to clarify the intrinsic mechanisms and explore the potential new targets for HCC therapy. As a class of non-coding RNAs, microRNAs (miRNAs), about 22 nucleotides in length, play an important role in tumor progression by regulating gene expression via binding to the 3′-untranslated regions
(3′-UTRs) of target mRNAs (Mao and Wang, 2015; Xu et al., 2018; Yang et al., 2018). At present, numerous miRNAs have been reported to be associated with HCC progression and development, such as miR-218 (Wu et al., 2018), miR-500a (Jiang et al., 2017) and miR-3662 (Chen et al., 2018). Recently, miR-99b-3p is found to be deregulated in papillary thyroid carcinoma (Dettmer et al., 2013), gastric cancer (Chang et al., 2015) and oral squamous cell carcinoma(He et al., 2015). And in these studies, miR-99b-3p expression is closely related to tumor progression and development. However, the expression and roles of miR99b-3p in HCC remain uncovered. Protocadherin 19 (PCDH19) is a member of the delta-2 protocadherin subclass of the cadherin superfamily (Pham et al., 2017). Previous studies reported that PCDH19 is under-expressed in HCC and
Abbreviations: HCC, Hepatocellular carcinoma; PCDH19, protocadherin 19; miRNA, microRNAs; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; EdU, 5-ethynyl-2′-deoxyuridine; DMEM, Dulbecco's Modified Eagle Medium; FBS, fetal bovine serum; RIPA, Radioimmunoprecipitation assay; 3′-UTR, 3′untranslated region; Real-time PCR, Real-time polymerase chain reaction; AFP, alpha-fetoprotein; TNM, tumor-node-metastasis ⁎ Corresponding author. E-mail address: [email protected] (H. Zhang). 1 Contributed equally. https://doi.org/10.1016/j.gene.2019.02.071 Received 24 August 2018; Received in revised form 11 February 2019; Accepted 17 February 2019 Available online 05 March 2019 0378-1119/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. miR-99b-3p is increased in HCC tissues and cell lines. (A) Real time-PCR indicated that the expression of miR-99b-3p in HCC tissues (n = 92) was much higher than that in adjacent non-tumor tissues (n = 92). (B) GEO dataset (GSE10694) showed that miR-99b-3p was significantly up-regulated in primary liver cancer tissues (n = 78), compared to that in normal liver tissues (n = 10) and adjacent noncancerous liver tissues (n = 78). (C) GEO dataset (GSE62007, n = 100) revealed that miR99b-3p was increased in 73% of the HCC tissues. (D) Real time-PCR revealed that the expression of miR-99b-3p was generally increased in HCC cell lines. *P < 0.05, **P < 0.01, ***P < 0.001.
2. Materials and methods
Table 1 Association of miR-99b-3p expression with different clinicopathological features of HCC patients (n = 92). Clinicopathological features
Cases
Expression of miR-99b-3p Low (n = 46)
Age (year) Gender AFP level (ng/ml) Tumor size (cm) Venous infiltration Edmondson-Steiner grading TNM stage
< 50 ≥50 Male Female < 400 ≥400 <5 ≥5 Absent Present I + II III + IV I + II III + IV
29 63 78 14 22 70 42 50 66 26 60 32 68 24
17 29 38 8 14 32 27 19 38 8 34 12 39 7
2.1. Patients and tissue samples
P
All of the 92 paired HCC tissue samples and adjacent non-tumor tissue samples, which were histopathologically confirmed, were collected from the HCC patients underwent surgery at the Department of General Surgery of General Hospital of Lanzhou Petrochemical Company from January 2005 to October 2013. The fresh tissue samples were stored at −80 °C. All of these patients didn't receive chemotherapy or radiotherapy before surgery. This study was approved by the Ethics Committees of General Hospital of Lanzhou Petrochemical Company. Informed consent was obtained from all individual participants included in the study.
High (n = 46) 12 34 40 6 8 38 15 31 28 18 26 20 29 17
0.262 0.562 0.143 0.012 0.021 0.080
2.2. Cell culture and transfection
0.018
Normal human liver cell (LO2) and HCC cell lines (HCCLM3, SMMC7721, HepG2, Hep3B, Huh7 and MHCC-97 L) were obtained from American Type Culture Collection (ATCC). All cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM, Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) and 1% penicillin/streptomycin. Cells were incubated in a humidified incubator (37 °C, 5% CO2). Transfections were conducted with Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA) according to the product specification. MiR-99b-3p mimics (#HmiR0307MR04), miR-99b-3p inhibitors (#HmiR-AN0856-AM01), clone for PTPRT (#E2229) and shRNA for PTPRT (#HSH058614-LVRH1GP) were purchased form GeneCopoeia Inc.(Guangzhou, China).
HCC, hepatocellular carcinoma; AFP, alpha-fetoprotein; TNM, tumor-nodemetastasis.
it is closely related to the prognosis of HCC patients, which means that PCDH19 may act as a tumor suppressor in HCC (Zhang et al., 2018). However, the exact function of PCDH19 in HCC remains unknown. In this study, we demonstrate that miR-99b-3p is overexpressed in both HCC tissues and cell lines. Highly expressed miR-99b-3p is closely associated with malignant clinicopathological characteristics and poorer prognosis of HCC patients. Furthermore, we found that miR-99b3p promotes HCC progression by directly targeting PCDH19.
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Fig. 2. HCC patients with higher miR-99b-3p have worse prognosis. (A) HCC patients in high miR-99b-3p group had worse 3-year overall survival. (B) HCC patients in high miR-99b-3p group had worse 3-year disease-free survival. (C) TCGA dataset (n = 362) from Oncolnc platform (http://www.oncolnc.org/) showed that HCC patients in high miR-99b-3p group had worse survival rate.
2.3. Real-time polymerase chain reaction (real-time PCR)
2.7. 5-ethynyl-2′-deoxyuridine (Edu) assay
The total RNA were extracted from tissue samples and cells with Trizol (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacture's instruction. For detection of miRNA expression, cDNA was synthesized using the qScript microRNA cDNA Synthesis Kit (Quantabio, Beverly, MA) according to manufacturer's instruction. Quantitative PCR was performed with miScript SYBR Green PCR Kit (Qiagen, Hilden, Germany) under the following thermocycler conditions: 95 °C for 2 min, and 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Primers for miR-99b-3p (#HmiRQP0856) and U6 snRNA (#HmiRQP9001) were purchased from GeneCopoeia (GeneCopoeia Inc., Guangzhou, China). U6 snRNA was used as housekeeping control for normalization. For detection of mRNA expression, 1 μg of total RNA was used to synthesize cDNA using cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA). The PCR conditions are as follows: 95 °C for 5 min, and 40 cycles of 95 °C 20 s and 62 °C 30 s, followed by 72 °C 3 min. The primers for PTPRT(#HQP058614) and GAPDH (#HQP006940) were purchased from GeneCopoeia (GeneCopoeia Inc., Guangzhou, China). The results was quantified with the 2−ΔΔCt method.
Cell-Light™ EdU Apollo®567 In Vitro Imaging Kit (RiboBio Co., Ltd. Guangzhou, China) was used for EdU assay. Briefly, transfected HCC cells (1 × 105) were cultured in 96-well plates. Cells were incubated with EdU labelling medium at moderate concentration for 2 h. Then, the cells were fixed with 4% paraformaldehyde, glycine and 0.5% TritonX-100 in PBS. Next, cells were stained with 100 μL Apollo dye solution for 30 min at room temperature. The cells were subsequently stained using Hoechst and incubated for 30 min. The photos were taken on a microscope. The percentage of EdU positive cells was calculated using ImageJ software. 2.8. Luciferase reporter assay pGL3-PTPRT-3′-UTR report/pGL3-PTPRT-3′-UTR Mutant report + TK100 Renilla reports were conducted. Then, the plasmids were transfected into 75% confluent HCC cells, along with miR-532-3p mimics or miR-532-3p inhibitors. The relative luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) and normalized to Renilla activity.
2.4. Wound healing assay 2.9. Western blot
When cells grew around 70–80% confluence in culture wells, 200 μL pipette tip was used to gently scratch the monolayer across the center of the well. Wells were briefly washed twice with medium to remove the detached cells. Fresh medium was added and cells grew for additional 24 h before photos were taken on a microscope.
Total protein was isolated by Radioimmunoprecipitation assay (RIPA) buffer, and quantified by Bradford assay (Sigma, St. Louis, MO). 20 μg of total protein was loaded into each well and transferred to PVDF membrane. Primary antibody used against human PTPRT (1:1000, ab115848, Abcam, Cambridge, UK) and anti-β-actin (1:1000; Cell Signaling Technology) were incubated with membrane at 4 °C overnight. Secondary anti-rabbit antibody (GE Healthcare Life Sciences, Beijing, China) was incubated with membrane at room temperature for 1 h. The protein bands were visualized using enhanced chemiluminescence kit (Amersham, Little Chalfont, UK) and Odyssey software (Infrared Imaging System LI-COR Biosciences).
2.5. Transwell assays The 24-well Transwell chambers (8 μm pore size, BD Biosciences) were used for Transwell migration and invasion assay. And, for Transwell invasion assay, the top chambers were coated with Matrigel matrix (Corning, Corning, NY). Approximate 2 × 104 cells were added into the top chambers. Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) was filled into the bottom chambers. After 24 h of incubation at 37 °C, the cells that did not pass through the membrane were removed by a cotton-tipped applicator, and cells on the lower surface of the membrane were subjected to crystal violet staining.
2.10. Statistical analyses Graphpad Prism 6 was used to perform statistical analyses. Unpaired t-test, one-way ANOVA, two-way ANOVA, and Kaplan-Meier method, Chi-square test, Spearman Pearson's correlation analysis and Log-Rank test were used to analyze the data. Differences were defined as statistically significant if P < 0.05.
2.6. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
3. Results Cells in 24 wells plate with different treatments were subjected to medium replacement with 10 μl sterile MTT dye (5 mg/mL). After incubation at 37 °C for 4 h, the MTT solution was removed, and 150 μl dimethyl sulfoxide was added to each well. Absorbance was read at 490 nm on an enzyme immunoassay analyzer (Biotek, Winooski, VT).
3.1. miR-99b-3p is up-regulated in HCC To investigate the expression levels of miR-99b-3p in HCC, we first detected the expression of miR-99b-3p in 92 paired HCC tissues and 143
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Fig. 3. The effects of miR-99b-3p on migration and invasion of HCC cells. (A) miR-99b-3p inhibitors significantly decreased the expression of miR-99b-3p in HCCLM3 cells. (B) Wound healing assay revealed that miR-99b-3p inhibitors obviously suppressed mobility of HCCLM3 cells. (C) Wound healing assay revealed that miR99b-3p mimics markedly enhanced mobility of Hep3B cells. (D) Transwell assays revealed that miR99b-3p inhibitors notably suppressed migration and invasion of HCCLM3 cells. (E) Transwell assays revealed that miR-99b-3p mimics notably promoted migration and invasion of Hep3B cells.Magnification:100 × .*P < 0.05, **P < 0.01, ***P < 0.001.
cell lines compared to the normal human liver cell LO2 (P < 0.001, respectively, Fig. 1D). Taken together, we demonstrate that miR-99b-3p is overexpressed in HCC and it's a potential oncogene in HCC.
adjacent non-tumor tissues by real-time PCR. The results indicated that miR-99b-3p expression was significantly upregulated in HCC tissues compared to the non-tumor tissues (P < 0.001, Fig. 1A). Consistently, GEO data from datasets GSE10694 (Fig. 1B) and GSE62007 (Fig. 1C) showed that miR-99b-3p was frequently upregulated in HCC tissues. In addition, we found that miR-99b-3p was generally upregulated in HCC 144
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Fig. 4. miR-99b-3p promotes proliferation of HCC cells. (A) MTT assay indicated that miR-99b-3p inhibitors notably suppressed viability of HCCLM3 cells. (B) MTT assay indicated that miR-99b-3p mimics markedly enhanced viability of Hep3B cells. Edu assay revealed that (C) miR-99b-3p inhibitors notably inhibited proliferation of HCCLM3 cells, (D) while miR-99b-3p mimics had the contrary effect on Hep3B cells. *P < 0.05, **P < 0.01, ***P < 0.001.
inhibited the healing capacity of HCCLM3 cells (P < 0.001, Fig. 3B). In contrast, the healing capacity of Hep3B cells was enhanced by miR-99b3p mimics (P < 0.01, Fig. 3C). Results from Transwell migration and invasion assays showed that miR-99b-3p inhibitors decreased the number of HCCLM3 cells passing through the membrance (P < 0.01, P < 0.5, Fig. 3D), while miR-99b-3p mimics had the contrary effect on Hep3B cells (P < 0.01, P < 0.5, Fig. 3E). Thus, these data demonstrate that miR-99b-3p promotes migration and invasion of HCC cells.
3.2. The clinical significance of miR-99b-3p in HCC Next, we attempted to explore whether miR-99b-3p was correlated with HCC progression. Based on the cutoff value, the median expression of miR-99b-3p in HCC tissues, the 92 HCC patients were sorted into two subgroups (low/high miR-99b-3p group). The data revealed that miR99b-3p was closely related to tumor size (P = 0.012, Table 1), venous infiltration (P = 0.021, Table 1) and TNM stage (P = 0.018, Table 1). In addition, HCC patients with higher miR-99b-3p expression had worse 3year overall survival rate (P = 0.0104, Fig. 2A) and disease-free survival rate (P = 0.0078, Fig. 2B), which was consistent with the result from TCGA dataset (http://www.oncolnc.org) (P = 0.0034, Fig. 2C). In conclusion, these findings suggest that miR-99b-3p is closely correlated with HCC progression.
3.4. miR-99b-3p promotes proliferation of HCC cells Next, we attempted to investigate whether miR-99b-3p had any effects on proliferation of HCC cells. MTT assay indicated that miR-99b3p inhibitors suppressed HCCLM3 cells viability (Fig. 4A), while miR99b-3p mimics had the contrary effect on Hep3B (Fig. 4B). Consistently, data from Edu assay showed that miR-99b-3p inhibitors decreased the percentage of Edu positive cells (Fig. 4C), while miR-99b-3p mimics increased the percentage (Fig. 4D). Taken together, the above results indicate that miR-99b-3p promotes migration, invasion and proliferation of HCC cells.
3.3. miR-99b-3p promotes migration and invasion of HCC cells Then, in order to explore the effects of miR-99b-3p on HCC cells, we first altered miR-99b-3p expressions of HCCLM3 and Hep3B cells by miR-99b-3p inhibitors or mimics. And the transfection efficiency in each cell line was measured with real-time PCR (Fig. 3A). Then, we attempted to explore the effects of miR-99b-3p on HCC cells migration and invasion. Wound healing assay showed that miR-99b-3p inhibitors 145
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Fig. 5. PCCDH19 acts as a downstream target of miR-99b-3p in HCC cells. (A) Results from bioinformatics tools (TargetScan, miRanda and PicTar) revealed that 3′UTR of PCDH19 had the bind site for miR-99b-3p. (B) Luciferase reporter assay revealed that miR-99b-3p inhibitors enhanced the fluorescence activity of PCDH19wt-3’UTR, while miR-99b-3p mimics repressed the fluorescence activity of PCDH19-wt-3’UTR. The alteration of miR-99b-3p expression had no effect on PCDH19mut-3’UTR. (C) The expression of PCDH19 mRNA was significantly lower in high miR-99b-3p HCC tissues. (D) Data from GEO dataset (GSE54236) and UALCAN platform (http://ualcan.path.uab.edu/index.html) revealed that PCDH19 expression was significantly lower in HCC tissues. (F) Result from UALCAN platform showed that PCDH19 was decreased with HCC progression. (G) Data analysis in HCC tissues of our cohort indicated that there existed a negative relationship between PCDH19 and miR-99b-3p. miR-99b-3p negatively regulated PCDH19 mRNA expression (H, I) and protein expression (J, K) in HCC cells. **P < 0.01, ***P < 0.001.
confirmed that the transfections were successful (Fig. 6A,B). Wound healing assay (Fig. 6C) and Transwell assays (Fig. 6E) showed that PCDH19 shRNAs enhanced the mobility of HHCLM3 cells, and reversed the functions of miR-99b-3p inhibitors. In contrast, PCDH19 clones inhibited the mobility of Hep3B cells, and receded the effects of miR99b-3p mimics (Fig. 6D,F). Similarly, MTT assay (Fig. 7A,B) and Edu assay (Fig. 7C,D) showed that PCDH19 suppressed the proliferation of HCC cells, and reversed the effects of miR-99b-3p. Collectively, these data indicate that PCDH19 suppresses HCC progression and mediates the effects of miR-99b-3p on HCC cells.
3.5. PCDH19 acts as the target of miR-99b-3p in HCC cells To elucidate the mechanism by which miR-99b-3p affects HCC cells metastasis and proliferation, we made an attempt to identify potential target genes of miR-99b-3p. Bioinformatics tools (TargetScan, miRanda and PicTar) were employed to search for the most potential gene under the control of miR-99b-3p. Among these predicted targets, protocadherin 19 (PCDH19) was focused. PCDH19 is a member of the delta-2 protocadherin subclass of the cadherin superfamily (Zhang et al., 2018). It has been reported that PCDH19 is downregulated in HCC and it is closely related to the prognosis of HCC patients (Zhang et al., 2018). As showed in Fig. 5A, there existed binding sites on 3′UTR of PCDH19 for miR-99b-3p. Luciferase reporter gene assay showed that miR-99b-3p negatively regulated the luciferase activity of WT-3′UTR of PCDH19, rather than MUT-3′UTR of PCDH19 (Fig. 5B). Then, we detected the mRNA expression of PCDH19 in HCC tissues and adjacent non-tumor tissues. The result from our cohort indicated that PCDH19 was decreased in HCC tissues (Fig. 5C), which was consistent with the data from GSE54236 data (Fig. 5D) and UALCAN platform (Fig. 5E). Besides, the data from UALCAN platform also showed that PCDH19 expression decreased as the HCC progressed (Fig. 5F). Furthermore, data from our cohort indicated that PCDH19 expression was negatively associated with miR-99b-3p (r = −0.8908, P < 0.001, Fig. 5G). Realtime PCR (Fig. 5H, I) and Western blot (Fig. 5J, K) revealed that both mRNA and protein expression levels of PCDH19 were negatively regulated by miR-99b-3p. Taken together, PCDH19 acts as the target of miR-99b-3p and may mediate its effects on HCC cells.
4. Discussion An increasing number of aberrantly expressed miRNAs have been reported to play a critical role in HCC progression by participating in various tumor cellular behaviors, such as migration, invasion, apoptosis, proliferation and cell cycle (Morishita and Masaki, 2015; Mizuguchi et al., 2016; Wang and Tian, 2016). For example, miR-367 acts as an oncogene to promote HCC cells migration and invasion by targeting FBXW7 (Wang et al., 2017). MiR-491-3p regulates drugs sensitivity of HCC cells by targeting Sp3 (Zhao et al., 2017). And, miR1301 suppresses HCC cell angiogenesis, migration and invasion by targeting BCL9 to inhibit Wnt pathway (Yang et al., 2017). Recently, some studies reported that miR-99b-3p is deregulated in several cancers, such as papillary thyroid carcinoma (Dettmer et al., 2013) and oral squamous cell carcinoma (He et al., 2015). These findings suggest that aberrantly expressed miR-99b-3p maybe related to tumor progression, as an oncogene or tumor suppressor. However, the exact expression and roles of miR-99b-3p in HCC remain to be explored. In this study, we found that miR-99b-3p was highly expressed in both HCC tissues and cell lines, which was validated by data from different datasets (GSE10694 and GSE62007). Then, we attempted to explore the clinical significance of miR-99b-3p in HCC by analyzing the
3.6. PCDH19 suppresses HCC progression and mediates the effects of miR99b-3p on HCC cells Next, to explore whether PCDH19 mediated the effects of miR-99b3p on HCC cells, co-transfections were performed. Western blot 146
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Fig. 6. PCDH19 suppresses HCC cells mobility and mediates the effects of miR-99b-3p. (A) The expression of PCDH19 in HCCLM3 cells co-transfected with PCDH19 shRNA, miR-99b-3p inhibitors and corresponding controls, was detected by Western blot. (B) PCDH19 expression in Hep3B cells co-transfected with PCDH19 clone, miR-99b-3p mimics, and corresponding controls, was detected by Western blot. Wound healing assay (C,D) and Transwell assays (E,F) revealed that PCDH19 negatively regulated cell mobility and mediated the effect of miR-99b-3p on mobility of HCC cells. *P < 0.05, **P < 0.01, ***P < 0.001.
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Fig. 7. PCDH19 suppresses HCC cells proliferation and mediates the effects of miR-99b-3p. (A,B) MTT assays revealed that PCDH19 reversed the effect of miR-99b-3p on viability of HCC cells. (C,D) Edu assay revealed that PCDH19 reversed the effect of miR-99b-3p on proliferation of HCC cells. *P < 0.05, **P < 0.01, ***P < 0.001.
99b-3p on HCC cells by gain- and loss-of-function analyses. Results indicated that downregulated miR-99b-3p suppressed HCC cells migration, invasion and proliferation, while overexpressed miR-99b-3p exerted the promoting effects. The above findings suggest that highly expressed miR-99b-3p is associated with HCC progression, and it acts
relation between miR-99b-3p expression and clinicopathological characteristics and HCC patients' prognosis. Data revealed that miR-99b-3p expression was closely related to tumor size, venous infiltration and TNM stage. And, patients in high miR-99b-3p group had much worse outcomes. Then, we attempted to assess the functional effects of miR148
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an oncogene in HCC by promoting cells migration, invasion and proliferation. It has been well documented that miRNAs exert their oncogene or anti-oncogene effects by repressing the expression of targets at a posttranscriptional level (Vasuri et al., 2018). Then, we attempted to explore the most potential target of miR-99b-3p in HCC cells by three bioinformatics tools (TargetScan, miRanda and PicTar). Then, numerous different targets were screened, and we selected the mutual genes predicted by all three databases. Give that PCDH19, a member of the delta-2 protocadherin subclass of the cadherin superfamily, has been identified as a tumor suppressor in HCC (Zhang et al., 2018), we focused on PCDH19 among these predicted targets. Here, consistently, data from both our cohort and Ualcan platform revealed that PCDH19 was downregulated in HCC tissues, and it decreased with the tumor progressing. We also found that there existed a negative relation between miR-99b-3p and PCDH19. And, PCDH19 was negatively regulated by miR-99b-3p at both mRNA and protein levels. Furthermore, miR-99b-3p negatively regulated the fluorescence activity of wild type of PCDH19–3′UTR, rather than the mutant type. And, rescue experiments revealed that PCDH19 mediated the effects of miR-99b-3p on HCC cells. Taken together, the above findings demonstrate that miR99b-3p promotes HCC progression by targeting PCDH19. In summary, our findings demonstrate the highly expressed miR99b-3p in HCC tissues and cell lines, which is closely related to malignant clinicopathological characteristics and poorer prognosis of HCC patients. Furthermore, overexpressed miR-99b-3p play the promoting role in migration, invasion and proliferation of HCC cells by directly targeting PCDH19. Thus, we conclude that miR-99b-3p maybe a promising therapeutic target for the treatment of HCC.
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