Identication of the ASPM-miR-26b-5p network associated with the aggressive traits of HCC cells

Background: Even though earlier reports have revealed that abnormal spindle-like microcephaly associated (ASPM) exert essential roles in diverse malignancies, its relationship between specic microRNAs (miRNAs) in regulation of hepatocellular carcinoma (HCC) progression has never been elaborated. Methods: Bioinformatics analysis detected differentially expressed genes in HCC and normal. qRT-PCR was performed to detect expression of miR-26b-5p in HCC tissues and cells. HCC cells were transfected with plasmids and their proliferative ability and colony formation were detected with loss-of-function assay. The invasion of HCC cells was determined using Transwell assay. The expression of ASPM was detected by western blotting. Luciferase reporter gene assay was performed to detect the interaction between miR-26b-5p and ASPM. ASMP silencing cells were injected into mice to establish xenograft tumor model. Results: Herein, we proved that ASPM was upregulated in HCC and higher level of ASPM was signicantly associated with worse survival in HCC patients. ASPM silencing restrained HCC cell proliferation, migration and invasion capacities in vitro. In vivo, downregulation of ASPM also suppressed HCC cells growth. Mechanistic analyses illustrated that ASPM was a directly target of miR-26b-5p. The expression of ASPM was negatively modulated by miR-26b-5p. Rescues assays displayed that miR-26b-5p inhibited HCC cells growth and invasion via modulating the expression of ASPM. Conclusions: Our work validated that miR-26b-5p restrained the aggressiveness of HCC cells through targeting ASPM.


Background
Hepatocellular carcinoma (HCC), the common type of liver malignancies, is the leading cause of cancerassociated mortality [1]. A high postsurgical recurrence rate and metastasis lead to the poor prognosis of HCC patients. Unfortunately, limited effective treatments are available and patients often miss the optimal opportunities for clinical interventions, because HCC is frequently diagnosed at the advanced stages [2,3]. As the molecular mechanisms underlying HCC pathogenesis have not yet been completely understood, elucidating the pivotal cancer-promoting events would contribute to the comprehension of hepatocarcinogenesis and the development of novel effective targeted treatments [4].
Assembly factor for spindle microtubules (ASPM), also referred to as abnormal spindle microtubule assembly, is located on chromosome 1q31 and encodes the ASPM protein [5,6]. It has been reported that the ASPM gene is overexpressed in glioblastoma and malignant glioma compared with normal brain tissue [7]. Abnormalities in ASPM expression are associated with numerous cancer types [8]. ASPM mRNA is overexpressed in HCC and non-small cell lung cancer (NSCLC) tissues. It has also been proposed that upregulation of ASPM expression increases the invasive capacity of melanoma cells.
However, to date, the potential role of ASPM in the growth and metastatic-related phenotypes of HCC cells remains unknown.
MiRNAs are endogenous non-coding RNAs, with ∼22 nucleotides, which can bind to the 3′-untranslated region (3′-UTR) of target mRNA to regulate gene expression, leading to the degradation of the mRNA or translational inhibition of functional proteins [9,10]. An increasing number of evidences had revealed that miRNAs were extensively involved in numerous pathological processes, including diabetes mellitus, atherosclerosis, and tumors [11]. Meanwhile, there are many reports of miRNAs related to HCC, such as miR-17, miR-21, and miR-492. Currently, miR-26b-5p has been reported to act as tumor suppressor in many cancers. However, little is known about the regulatory mechanism of miR-26b-5p/ASPM axis in the progression of HCC.
In this study, we revealed an interaction between miR-26b-5p and ASPM which modulated HCC cells growth and invasion. Based on the results, a novel regulatory pathway composed of miR-26b-5p/ASPM was provided for HCC therapy.

Materials And Methods
HCC tissues and cell lines HCC samples and adjacent tissues were obtained from Shandong Provincial Hospital A liated to Shandong University. The clinicopathological features of patients with HCC were summarized in Table 1.
Written consent was obtained from all participants who were involved in the study. All tissues were frozen in liquid nitrogen after surgery and stored at -80 °C. This study was approved by the Ethics Committee of Shandong Provincial Hospital A liated to Shandong University. The HCC cell lines (HepG2, SNU449, SMMC-7721, Huh7 and PLC/PRF/5), HEK-293T and normal liver cell lines (LO2) were purchased from the Chinese Academy of Sciences (Shanghai, China). Cells were maintained in DMEM containing with 10% FBS (Thermo Fisher Scienti c, Waltham, MA, USA) and 100 U/ml penicillin/streptomycin in a humid incubator at 37 °C. Cell Transfection pcDNA3.1 vector, pcDNA3.1 vector carrying ASPM (ASPM), miR-26b-5p mimic, negative control oligonucleotides (miR-NC), miR-26b-5p inhibitor (anti-miR-26b-5p), negative control oligonucleotide (anti-miR-NC), small interfering RNA of ASPM (si-ASPM) and scramble siRNA (si-Con) were bought from RiboBio (Guangzhou, China). Cells were transfected utilizing lipofectamine 3000 (Thermo Fisher Scienti c) following to manufacturer's protocol. sh-NC and shRNA targeting ASPM (sh-ASPM) were purchased from RiboBio (Guangzhou, China) and transfected into SMMC-7721 cell. To stably overexpress miR-26b-5p in SMMC-7721 cell, the lentiviral packaging kit was used. Lentivirus containing miR-26b-5p was packaged following the manufacturer's manual. Lentivirus was packaged in HEK-293T cell and secreted into culture medium. SMMC-7721 cell was infected by lentivirus carrying miR-26b-5p with the presence of polybrene (Sigma) and selected by puromycin (Sigma) for two weeks. To overexpress ASPM in SMMC-7721 cells, lentivrius carrying ASPM cDNA (GeneCopoeia) were packaged and used to infect cell following to manufacturer's protocol.

Cell Proliferation And Colony Formation Assays
Cell growth was detected using CCK-8 Kit (Beyotime, Nanjing, China) according to the protocol. In colony formation assay, cells were cultured into six-well plates and incubated for two weeks. After that, cells were stained and photographed.

Migration And Invasion Assays
Cell migration assay was performed using transwell chamber (BD, Franklin Lakes, NJ, United States). 5 × 10 4 SMMC-7721 and PLC/PRF/5 cells were seeded into the upper chambers and the lower chambers were lled with medium with 20% FBS. Cell migration was assessed by counting migrative cells under an inverted microscope. Cell invasion assay was conducted using transwell chamber coated with Matrigel (BD). 5 × 10 4 SMMC-7721 and PLC/PRF/5 cells were seeded into the upper chambers and the lower chamber was lled with DMEM with 20% FBS. The number of invasion cell was counted under microscope.
Tumor Xenografts miR-NC or miR-26b-5p transfected SMMC-7721 cells were subcutaneously implanted into nude mice (Beijing Vital River Laboratory, Beijing, China). Tumor volume = 0.5 x length x width 2 . Tumor volume was examined every week. Mice were euthanized at 5 weeks post implantation. The tumor tissues were extracted for further immunohistochemical staining (IHC). All procedures involving experimental animals were performed in accordance with protocols that were approved by the Committee for Animal Research of Shandong Provincial Hospital A liated to Shandong University and complied with the Guide for the Care and Use of Laboratory Animals (NIH publication No. 86 − 23, revised 1985).

Immunoblotting Assay
Proteins were extracted from cells using RIPA buffer. 20 µg protein lysis were loaded onto 10% SDS-PAGE. After separated, the proteins were transferred to PVDF membrane (Millipore, Braunschweig, Germany).

Statistical analysis
GraphPad Prism software was used for statistical analyses. Data were presented as Mean ± SD.
Differences were calculated using Student's t-test or Mann-Whitney U test. The correlation analysis was performed by Spearman test. P < 0.05 was statistically signi cant.

ASPM is upregulated in HCC
Firstly, we assessed the GEO database GSE54236 and GSE14520. The volcano plot and heatmaps showed the dysregulated genes, including upregulated genes and downregulated genes (Fig. 1A-1B). ASPM was selected for investigation owing to its level was signi cantly upregulated in both GEO database and its biological roles in HCC needs to be explored (Fig. 1C). To con rm this observation, we measured the expression levels of ASPM in 46 pairs of HCC and adjacent samples using qRT-PCR.
Consistent with the results of bioinformatics analysis, ASPM was signi cantly upregulated in HCC tissues (Fig. 1D). The IHC data illustrated that ASPM expression was distinctly increased in HCC tissues as compared to that in adjacent tissues (Fig. 1E) Furthermore, overexpression of ASPM was associated with the aggressive clinic phenotypes (Fig. 1F-G). ASPM was also more highly expressed in HCC with advanced tumor stages (II-III) than that in HCC with early tumor stages (I). Using the online bioinformatics tool GEPIA (http://gepia.cancer-pku.cn/index.html), we found that patients with increased ASPM expression exhibited poor overall survival (OS) (Fig. 1H). All these results revealed that ASPM might exert a critical role in HCC.

Knockdown of ASPM reduces HCC cells growth and invasion ability
To reveal the function of ASPM in HCC, we explored its expressions in HCC cells (HepG2, SNU449, SMMC-7721, Huh7 and PLC/PRF/5) and normal liver cell line (LO2). The result of qRT-PCR implied that the levels of ASPM were higher in HCC cell lines than that in LO2 cell ( Fig. 2A). Next, we knocked-down the ASPM in SMMC-7721 and PLC/PRF/5 cells using siRNA (Fig. 2B). CCK-8 assay (Fig. 2C) and colony-formation assay (Fig. 2D) displayed that HCC cells growth was signi cantly inhibited after ASPM knockdown. Meanwhile, the migration and invasive capacity were decreased in si-ASPM transfected SMMC-7721 and PLC/PRF/5 cells as demonstrated by transwell migration (Fig. 2E) and invasion assay (Fig. 2F). Then, SMMC-7721 cells stable transfected with sh-ASPM were implanted into BALB/c nude mice. sh-ASPM markedly restrained tumor growth after cells implantation (Fig. 2G-2H). Nevertheless, we proved that upregulation of ASPM enhanced HCC cell proliferation and invasion of HCC cells in vitro (Fig. 2I-2K). All ndings suggest the oncogenic role of ASPM in HCC. miR-26b-5p binds to ASPM and negatively regulates its expression We predicted the miRNAs that have complementary base pairing with ASPM using miRTarBase (Fig. 3A) and we found that miR-26b-5p formed complementary base pairing with ASPM. Interestingly, after searching The Cancer Genome Atlas Program (TCGA), miR-26b-5p was signi cantly lower in HCC when compared with in normal (Fig. 3B). To analyze the relationship between ASPM and miR-26b-5p, luciferase reporter gene assay was conducted. Transfection of miR-26b-5p restrained the luciferase activities in HCC cells transfected with wt-ASPM. Nevertheless, miR-26b-5p had no inhibitive effect on the luciferase activities in HCC cells transfected with mut-ASPM (Fig. 3C). In addition, miR-26b-5p was downregulated in 46 cases of HCC samples (Fig. 3D) and lower level of miR-26b-5p was positively associated with advanced stage (Fig. 3E) and metastasis (Fig. 3F). Meanwhile, miR-26b-5p level was negatively connected to the mRNA level of ASPM in HCC (Fig. 3G). To verify the expression of ASPM was negatively modulated by miR-26b-5p, SMMC-7721 and PLC/PRF/5 cells were transfected with miR-26b-5p mimics (Fig. 3H) and we found that the expressions of ASPM were reduced by miR-26b-5p (Fig. 3I). In addition, upregulation of miR-26b-5p inhibited the growth and aggressive traits of HCC cells (Fig. 3J and K). All these observations indicate that miR-29b-5p negatively regulates the growth and invasion abilities of HCC cells.' miR-26b-5p suppresses HCC cells growth and invasion by targeting ASPM In xenograft tumor model, the tumor growth in nude mice inoculated with miR-26b-5p expressed SMMC-7721 cell was signi cantly inhibited (Fig. 4A). The result of IHC staining illustrated that ASPM expression was signi cantly declined in tumor tissue from mice injected with miR-26b-5p stable expressed SMMC-7721 cell (Fig. 4B). To study whether miR-26b-5p regulate HCC cells progression by modulating ASPM, SMMC-7721 cell was cotransfected with miR-26b-5p and pcDNA3.1-ASPM plasmid. The western blotting analysis disclosed that the level of ASPM was rescued in SW480 cells cotransfected with miR-26b-5p and pcDNA3.1-ASPM when compared to cells transfected with miR-26b-5p-5p alone (Fig. 4C). Upregulation of miR-26b-5p suppressed the colony formation and invasion of SMMC-7721 cell, which could be rescued by pcDNA3.1-ASPM (Fig. 4D-4E). PLC/PRF/5 cell was cotransfected with anti-miR-26b-5p and si-ASPM plasmid. The level of ASPM was decreased in PLC/PRF/5 cell cotransfected with anti-miR-26b-5p and si-ASPM when compared with cell transfected with anti-miR-26b-5p-5p alone (Fig. 4F). Downregulation of miR-26b-5p enhanced the colony formation and invasion of PLC/PRF/5 cell, which could be neutralized by si-ASPM (Fig. 4G-4H). These data suggest that miR-26b-5p regulates HCC cells growth and invasion by modulating ASPM.

Discussion
Increasing evidences have demonstrated that miR-26b-5p is associated with the progression of malignant tumors. For instance, miR-26b-5p suppresses the proliferation and induces apoptosis in multiple myeloma cells by targeting Jagged Canonical Notch Ligand 1 (JAG1) [12]. miR-26b-5p suppresses the growth and metastatic-associated traits of human papillary thyroid cancer (PTC) in a β-Catenin-dependent manner [13]. MiRNA-26b-5p enhances T cell responses through targeting Pim-2 Proto-Oncogene (PIM-2) in HCC [14]. All these ndings manifest that miRNA-26b-5p acts as an oncogenic miRNA in a variety of tumor types. Recently, ASPM has been identi ed to be upregulated in cancers and is involved into cancer cells process [15][16][17]. ASPM promotes prostate cancer stemness and progression by augmenting Wnt-Dvl-3-β-catenin signaling [18]. ASPM is a novel marker for vascular invasion, early recurrence, and poor prognosis of HCC [19]. However, the biological actions of miR-26b-5p-ASPM axis in HCC had not been reported so far.
Herein, we con rmed that ASPM was signi cantly upregulated in HCC tissues when compared to that in non-cancerous samples. Furthermore, we seek the association between ASPM level and the clinicalpathological parameters in HCC and veri ed that higher level of ASPM was related with advance malignant stage and more metastasis. Meanwhile, higher expression of ASPM resulted into worse OS of patients with HCC. All data displayed that ASPM served as a cancer-promoting gene in HCC. In vitro functional experimental assays, ASPM silencing markedly impaired HCC cells growth and the migration, invasion abilities of HCC cells. Consistently, knockdown of ASPM inhibited the tumor growth of HCC cells in mice. However, upregulation of endogenous ASPM caused completely opposite results.
By using bioinformatics prediction tools and luciferase reporter experiment, we certi ed that ASPM was a target of miR-26b-5p, which has been con rmed as a suppressive miRNA in a variety of cancers. The immunoblotting assay con rmed that ASPM was negatively modulated by miR-26b-5p in HCC cell. In human HCC tissues, we also revealed that miR-26b-5p was downregulated in cancer samples and its level was inversely associated with the expression of ASPM. Consistent with early reports, we demonstrated that miR-26b-5p impaired the aggressive phenotypes of HCC cells [12,13]. The correlation analysis further highlights the negative correlation between miR-26b-5p level and ASPM level in HCC tissues. Finally, the rescue experiments indicated that miR-26b-5p restrained the growth and invasion in HCC cells via intervening ASPM. However, the promoting impacts of miR-26b-5p inhibitor was neutralized by ASPM silencing.
Early studies disclosed that miRNAs have one or more downstream target genes and modulate mRNAs degradation or translational inhibition [20]. Our current study has several limitations. Although we revealed that miR-26b-5p suppressed the aggressive phenotypes of HCC cell through blocking ASPM, it might be better to examine other targets of miR-26b-5p. Additional, the biological function of miR-26b-5p-ASPM in other biological processes, including HCC cells apoptosis need to be further explored.

Conclusion
Altogether, our observations contribute to the understanding of the role of miR-26b-5p-ASPM axis involved into the aggressive-related traits of HCC cells.