Background
Hepatocellular carcinoma is a common digestive system tumor with biological characteristics of high invasiveness and high mortality [
1]. At present, the comprehensive treatment plan based on surgical resection and chemotherapy embolization is a great improvement in the prognosis of patients with HCC. However, most of the patients are diagnosed in the middle and late stage due to lack of effective early diagnostic biomarkers, and have poor prognosis despite treatment [
2,
3]. Therefore, it is urgent and important to investigate novel biomarkers to improve the rate of early diagnosis of HCC.
The human KIF26B, a member of the kinesin (KIF) family, is known for its function on adhesion and polarization of mesenchymal cells [
4]. Recent studies showed that overexpression of KIF26B was associated with poor prognosis of solid tumors including breast cancer [
5,
6], gastric cancer [
7] and colorectal cancer [
8]. In addition, Pu et al. found that KIF26B was essential for multi-drug resistance in osteosarcoma [
9]. However, no studies reported the biological role of KIF26B in HCC. In this study, we found KIF26B was overexpressed in HCC tissues and cell lines. Overexpression of KIF26B was associated with poor overall survival (OS), late TNM stage and poor differentiation. Suppression of KIF26B could inhibit cell viability, proliferation rate, invasion ability and tumor formation ability of HCC cells, and such phenomenon might be correlated with inhibition of PI3K/AKT signaling pathway. Furthermore, we proved that KIF26b was a target gene of miR-450b-5p in HCC cells. Taken together, our study found that miR-450b-5p/KIF26B/AKT axis is critical for tumorigenesis and progression of HCC, such discovery may provide a potential therapeutic target for HCC treatment.
Materials and methods
Patients and specimens
Tissue microarrays (TMA) containing 93 paired paraffin embedding HCC specimen and corresponding non-tumor tissues was obtained from Shanghai Core Super Biotechnology Co., Ltd. All these patients were available follow-up data. A total of 369 HCC patients and 50 normal patients, with follow-up data, from The Cancer Genome Atlas (TCGA,
https://tcga-data.nci.nih.gov/tcga/) database, were used for gene expression analysis and patients’ survival analyses according to our previous protocol [
10].
Cell culture
Normal (L02) and HCC cell lines (HepG2, SMMC-7721, Hep-3B and HCC-LM3) were purchased from ATCC (Manassas, USA) or Sibcb (Shanghai, China). Cells were cultured in DMEM supplied with 10% fatal bovine serum, and incubated in a humidified atmosphere of 95% air and 5% CO2 at 37 °C.
Western blot
Protein isolation and western blotting have been described before [
11]. Cells were collected at 48 h after transfection. Samples were probed with KIF26B, m-TOR, p-PI3K, p-AKT or GAPDH monoclonal antibody. Goat anti-mouse HRP antibodies were obtained from Zhongshan Jinqiao Company, Beijing. ECL detection system (Millipore, Bedford, MA, USA) is used to assess proteins expression.
Total RNA isolation and quantitative real-time PCR (qPCR)
Total RNA from HCC cells was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the standard RNA isolation protocol. Then reverse-transcribed with TransScript First-Strand cDNA Synthesis SuperMix (TransGen, Beijing, China). Quantitative real-time PCR (qRT-PCR) was performed using TransScript Top Green qPCR SuperMix (TransGen, Beijing, China) on 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA). GAPDH was used as the endogenous controls. Data were analyzed using the comparative Ct method (2−ΔΔCt). Each group included three repeated wells.
Immunohistochemistry
The Tissue microarrays (TMA) was rehydrated in a series of graded alcohol dilutions. Then heat epitope retrieval for 20 min in condition of Citrate salt solution. After closed by 5% BSA for 30 min, all tissues were incubated with a rabbit antibody to human anti-KIF26B, anti-Ki67, anti-m-TOR, anti-p-AKT and anti-p-PI3K overnight at 4 °C. Slides were then incubated with HRP at room temperature for 30 min and were visualized using DAB as chromogen for 5–10 min. The method for IHC score was according to the staining intensity score of 1–5: High KIF26B expression was defined as a staining score with 4–5, while low expression was defined as a staining score with 1–3.
Transfection of Hep-3B and HCC-LM3 cell lines
Human-specific shKIF26B (1–3) and negative control were obtained from Shanghai Genechem Co., Ltd. and used to generate stable KIF26B knockdown cell lines according to the instructions of manufacturer. The coding sequences of human KIF26B and negative control were amplified and cloned into pcDNA3.1 (+) to generate KIF26B overexpression and according negative control (GenePharma, Shanghai, China). miR-450b-5p mimics, inhibitors as well as respective negative controls (NC) were commercially purchased from Gene Pharma (Shanghai, China). The cell transfection was conducted according to the instruction manual.
Cell viability
Hep-3B and HCC-LM3 cell lines were transfected with KIF26B shRNA, Negative control and blank (or miR-450b-5p mimics, miR-450b-5p mimics and KIF26B and negative control). After 24 h, these cells were seeded into 96-well microplates with 5000 cells per well. Cell counting kit-8 (CCK-8) assay (Dojindo, Kumamoto, Japan) was then performed to detect cell viability in 24 h, 48 h and 72 h. The OD450 value was determined by using a MRX II microplate reader (Dynex, Chantilly, VA, USA).
Transmembrane invasion assay
Human Hep-3B and HCC-LM3 cells were transfected with KIF26B shRNA and negative control (or miR-450b-5p mimics, miR-450b-5p mimics and KIF26B and negative control). After 48 h, 5 × 106/mL cells were transferred into the upper chamber of the Millicell inserts pre-coated with 20 μg Matrigel and pre-incubated 1 h to reconstitute a basement membrane (Millipore, USA) in a serum-free DMEM. DMEM containing 10% fetal bovine serum was added to the lower chamber. After 24 h incubation, the cells that invaded through the membrane were fixed with methanol and stained with 0.5% crystal violet.
EDU assay
Proliferating HCC cell lines (Hep3B and HCC-LM3) were determined by using the Click-iTEdU Imaging Kit (Invitrogen; Carlsbad, CA, USA) according to the manufacturer’s protocol. Briefly, cells were transfected with KIF26B shRNA and negative control (or miR-450b-5p mimics, miR-450b-5p mimics and KIF26B and negative control). After 48 h, 10 μM EdU for 2 h before fixation, permeabilization, and EdU staining. Cell nuclei were stained with Hoechst 33342 (Invitrogen) at a concentration of 5 μg/mL for 30 min.
Hep-3B and HCC-LM3 were transfected with KIF26B shRNA and negative control (or miR-450b-5p mimics, miR-450b-5p mimics and KIF26B and negative control), then they were dissociated with trypsin, resuspended in DMEM complete medium with 10% FBS, and inoculated into a 6-well plate at a density of 2000 cells/well. After 12 days, colonies were washed twice with PBS, dyed with crystal violet and photographed.
Luciferase activity assay
The 3′UTR (3′ untranslated region) sequence of KIF26B containing the predicted miR-450b-5p-binding site and according the mutant sequence were subcloned into psiCHECK-2 luciferase reporter vector (Promega, Madison, WI, USA). The luciferase activity experiment was conducted according to the introduction manual.
In vivo tumorigenesis assay
All experimental procedures involving animals were in accordance with the Guide for the Care and Use of Laboratory Animals and were performed as described previously [
12]. The study protocol was also approved by the Committee on the Use of Live Animals in Teaching and Research, the First Affiliated Hospital of Zhengzhou University. For mice xenograft tumor model, 6- to 8-weeks-old male mice were used. HCC-LM3 cells transfected with shKIF26B or negative control were subcutaneously (s.c.) injected into the lower flank of the mice with 3.0 × 10
6. The tumor volume was calculated by the formula: Volume = (width)2 × length/2. Weeks 1, 2, and 3 after injection, mice were photographed with an IVIS@ Lumina II system (Caliper Life Sciences, Hopkinton, MA) 10 min after an intraperitoneal injection of 4.0 mg of luciferin (Gold Biotechnology, Inc., St. Louis, MO) in 50 μL of saline. After 3 weeks, tumors were surgically removed and weighed.
The gene set enrichment analysis (GSEA)
GSEA was used to determine which pathway was associated with KIF26B expression in TCGA data set. The expression profiles of 377 samples from TCGA data set was grouped two groups (KIF26B high and KIF26B low). GSEA v2.0 was used to determine whether the pathways from the MSigDB database v4.0 are randomly distributed at the top or bottom of the ranking. The significance threshold was set at p < 0.05.
Statistical analysis
All analyses were performed with SPSS 18.0 software (SPSS Inc., Chicago, IL). All experiments were repeated at least three times to calculate the mean and standard deviation (SD). For comparisons, the Student’s t test, paired-samples t-test, and ANOVA analysis were performed as appropriate. Survival probabilities were evaluated using the Kaplan–Meier method, and differences were assessed using the log-rank test. p (two-sided) values less than 0.05 were statistically significant. Data were presented as the mean ± standard deviation (SD).
Discussion
Initiation and progression of hepatocellular carcinoma is complex processes involving changes in gene expression, signaling pathways, and epigenetics. Abnormal expression of the core genes often results in dysregulated cell growth, differentiation, apoptosis, and migration [
13]. Therefore, investigating for the abnormally expressed core genes is important for developing new target for HCC treatment. Low early diagnosis rate and postoperative recurrence of liver cancer have been the two major factors affecting the prognosis of patients with liver cancer [
2,
3]. Therefore, it is important to investigate new therapeutic targets which could act as a potential biomarker for the early diagnosis and prognosis of patients with HCC.
The kinesin superfamily proteins (KIFs) are molecular motor proteins which correlated with microtubule binding and ATPase activities [
14]. Recent studies have proved that KIF26B acted as critical role in the regulation of many physiological events, including brain function [
15], developmental patterning [
4], and proliferation and migration of solid tumors [
5‐
8,
16,
17]. Other study demonstrated that KIF26B played important role in multi-drug resistance in osteosarcoma [
9]. However, there is limited evidence of the function of KIF26B during progression of HCC. In this study, we found KIF26B was overexpressed in HCC tissues, high expression of KIF26B was correlated with later TNM stage, poor tumor differentiation and prognosis. Suppression of KIF26B could inhibit proliferation rate and invasion ability of HCC cells in vitro, and affect tumor formation ability both in vitro and in vivo.
To further explore the molecular mechanism of knockdown of KIF26B on the proliferation and invasion of HCC cells, we used the KEGG and GO analysis through TCGA database to find the signaling pathway which was the most relevant to KIF26B expression levels. We found PI3K/AKT signaling pathway may regulated by KIF26B, and we proved that suppression of KIF26B could decrease expression of m-TOR, p-AKT and p-PI3K both in vitro and in vivo. Constitutive activation of the PI3K/AKT/mTOR signaling pathway acted as critical role in the progression of HCC, including cell proliferation, migration and invasion, angiogenesis and distant metastasis [
18]. Thus, we proved that suppression of KIF26B could inhibit proliferation, invasion and tumor formation ability of HCC cells through regulating activation of PI3K/AKT signaling pathway.
MiRNAs are noncoding RNAs with ~ 22 nucleotides which play vital role in regulating gene expression via inhibiting posttranscriptional translation of target mRNAs [
19]. To investigate upstream regulatory factor of KIF26B, we found miR-450b-5p could be specific complementary pairing with 3′UTR sequence of KIF26B mRNA. MiR-450b-5p has been reported associated with progression of several solid tumors, including colorectal cancer [
20], lung adenocarcinoma [
21], rectal cancer [
22], prostate cancer [
23], rhabdomyosarcoma [
24]. However, the function of miR-450b-5p in progression of HCC was unclear. In this study, we found miR-450b-5p was negative correlated with KIF26B expression in HCC tissues, and miR-450b-5p mimics and inhibitor could significantly affect KIF26B expression in HCC cells. Furthermore, we observed that miR-450b-5p could obviously inhibit cell viability, proliferation rate, invasion ability and tumor formation ability of HCC cells, while overexpression KIF26B could partially reverse tumor suppression function of miR-450b-5p. In addition, we found HCC patients who with miR-450b-5p
lowKIF26B
high always associated poor prognosis than those with miR-450b-5p
highKIF26B
low. Thus, our study demonstrated that KIF26B was one of the target genes of miR-450b-5p, low expression of miR-450b-5p could induce overexpression of KIF26B, and then promote activation of PI3K/AKT pathway.
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