miR-216b-5p is Down-Regulated in Human Breast Cancer and Inhibits Proliferation and Progression by Targeting HDAC8 Oncogene

Background Previous studies showed the role of histone deacetylases (HDACs) on the progression of some malignancies. Recently, there is more attention to therapeutic applications of epigenetic factors such as microRNAs (miRs). To the best of our knowledge, there are no other results regarding the contribution of miR-216b-5p and its potential target, HDAC8, in progression of cancer. Aim In the present study, we investigated the role of miR-216b-5p on HDAC8 and there following impacts on breast cancer (BC) progression. Methods Human BC specimens and noncancerous tissues were acquired from Iran Tumor Bank (I.T.B). The MDA-MB-231, MCF-7 and MCF-10A BC cell lines were also prepared. The tissue and cell line expression levels of miR-216b-5p and HDAC8 were determined by quantitative real-time PCR (qPCR). Protein levels of HDAC8 were also measured by Western blotting assay. The cell cycle, cell proliferation and colony formation assay were determined and the role of HDAC8 was investigated using a knockout vector. Targeting the 3′ untranslated region (3′UTR) of HDAC8 by miR-216b-5p were confirmed using a luciferase reporter assay. Results Our results show the significant decline in miR-216b-5p and highly increase in HDAC8 levels in human breast cancer tissues and cell lines. Overexpression of miR-216b-5p in BC cell lines inhibited cellular proliferation and progression and inhibition of miR-216b-5p reverse these effects. We also confirmed that HDAC8 is directly down-regulated by miR-216b-5p. Knockout of HDAC8 had also the effects as miR-216b-5p overexpression. Furthermore, we found that lower levels of miR-216b-5p are negatively correlated with lymph node metastasis and advanced tumor size. Conclusion Taken together, our data shown that targeted overexpression of miR-216b-5p can suppress the growth of BC cells down-regulation of HDAC8. Therefore, inhibition of HDAC8 by miR-216b-5p will be helpful developing newer therapies for the effective treatment of BC.

healthy subjects. Besides, the present study attempts to assess the potential role of miR-216 (as a potential inhibitor of the HDAC8) on tumor growth in human breast adenocarcinoma.
Scientific) according to the manual. Quantitative real-time PCR was performed with a SYBR Premix ExTaqII kit (Takara, Japan) and the Rotor-Gene 6000 apparatus (Corbett Research, Mortlake, NSW, Australia). Quantification of miRs expression was carried out using an YBR R Premix ExTaq TM kit (Takara, Japan) according to the manufacturer's instruction. HPRT and SNORD47 (U47) were used for mRNA and miR data normalization respectively. Primer sequences are listed in the Table 3. CRISPR/Cas9 mediated knockout of the HDAC8 gene in MCF-7 and MDA-MB-231cell line: To knockout (KO) the expression of HDAC8 in the MCF-7 and MDA-MB-231cell lines, CRISPR/Cas9 based KO strategy was applied. CRISPR sgRNAs were designed using crispr.mit.edu and the oligos were made by Macrogen Inc. (Seoul, Korea). pCAG-eCas9-GFP-U6-gRNA vector (Addgene 79145) without ITR element (Hojland Knudsen et al., 2018) was digested with BbsI enzyme for gRNA cloning. The gRNAs sequences used for knocking out of the HDAC8 gene are illustrated in table 3. The prepared eCas9/gRNA expression vectors co-transfected to the cell lines using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). The eCas9 expression vector without any cloned gRNA was used for the control.

Transient transfection:
Mcf-7 and MDA-MB-231 cell lines were seeded in six-well plates at a density of 1.5 × 10 5 cells/well and grown to 90% confluency. Then, the cells transiently transfected with 100 nM miR-216b-5p mimics, negative control and inhibitor (Exiqon). Transfection was performed by Lipofectamine 2000 according to the manufacturer's instructions. After transfection, cells were kept in a culture medium containing 10% FBS for up to 72 h. Subsequently, total RNA and protein were extracted and used for further investigations.
Luciferase reporter assay: The PCR purified HDAC8-3′UTR (WT-UTR) containing the predicted miR-216b-5p binding sites were cloned into the psiCHECK™-2 Vector (Promega, Madison, USA) using specific primers for HDAC8-3′UTR (Table 3). The vector was digested by SgfI and NotI restriction enzymes. A mutant luciferase vector with miR-216b-5p changed pairing site (Mut-UTR) was also constructed. Wild and mutant types of HDAC8-UTR plasmid were confirmed by DNA sequencing. When HEK293T cells reached 80% confluence in 24-well plate, cells were co-transfected with luciferase vector and miR-216b-5p mimic, inhibitor and negative control using Lipofectamine 2000. After 48 h, luciferase activities were detected by a dual-luciferase reporter assay system (Promega, Madison, US) according to the manufacturer's instructions.
Cell cycle analysis: Flow cytometry was applied for cell cycle assessment in different groups. Briefly, 72h after transfection, cells were collected and fixed with 70% ethanol at 1 h at 4 °C. The fixed cells washed with PBS, then incubated with propidium iodide (PI) (Sigma-Aldrich) at room temperature for 1h and samples were analyzed using a BD FACScalibur flow cytometer (BD Biosciences, Mountain View, CA).
Cell proliferation assay: Mcf-7 and MDA-MB-231 cell lines were seeded in a 96-well plate at 5000 cells per well then transfected with miR-216b-5p mimic, negative control and inhibitor and incubated for 96 h. Cell viability was assessed after transfection using a commercial 3-2, 5-diphenyl tetrazolium bromide (MTT) assay kit (Sigma, St Louis, MO) according to the manufacturer's instructions at designated times (6 consecutive days). The absorbance of each well was measured with a microplate reader set at 450 nM.

Colony formation assay:
Firstly, the Mcf-7 and MDA-MB-231 cell lines were transfected with miR-216b-5p mimic, negative control, inhibitor and HDAC8 knockout vector in 6-well culture dishes. The plates were covered with a layer of 0.6% agar in a medium supplemented with 20% FBS. A total of 1,000 cells were prepared in 0.3% agar and incubated for 14 days at 37 °C and 5% CO2 conditions. The resulting colonies stained with 0.04% crystal violet for 40 minutes, then rinsed with PBS again. Finally, the numbers of colonies per well were counted.
Western Blotting: All treated and control groups were harvested using a RIPA lysis buffer supplemented with a protease inhibitor cocktail and phosphatase inhibitor cocktail (Sigma-Aldrich, USA) at 4 °for 10 minutes. Protein concentrations of the cell lysates in different groups was further determined with BCA protein assay kit (Bio basic, Canada). The aliquots of 40 µg of lysates were electrophoresed by 12% SDS-PAGE and subsequently transferred onto a PVDF membrane (Millipore, USA). Non-fat milk powder (5%) was used for blocking the membranes at room temperature for 1h, then the membrane were incubated overnight with primary antibodies (Minneapolis, MN 55413). They were then incubated with horseradish peroxidase-labeled secondary antibodies (Santa Cruz, 10513) at room temperature. Finally, the signals were detected using an ECL detection kit (Sigma-Aldrich), and the membranes were scanned and analyzed using a Flowjo imaging system with imaging software (version quantity 1). Protein expression was normalized to an endogenous reference (GAPDH, Santa Cruz, D1717) and relative to the control. A protein ladder (Fermentas, Republic of Lithuania) was used as a molecular marker.

Statistical analysis:
Statistical analyses were performed using SPSS 16 (SPSS Inc., Chicago, USA). Results were presented as Mean ± SD. Comparison of the possible differences between studied groups were done by the independent samples T test. One Way ANOVA followed by Post Hoc, Tukey, and Dunnett tests were used to analyze mean differences between more than two groups. The association between two variables was calculated using the Spearman correlation coefficient. In all performed hypothesis tests, a P value less than 0.05 was considered as statistically significant.
Diagnostic value of HDAC8 and miR-216b-5p as potential tumor marker in breast cancer: The means and standard deviations of HDAC8 for cancerous and normal tissues were 4.86±0.44 (r.u.) and 3.52±0.43 (r.u.), respectively. The corresponding values for miR-216b-5p were 0.0024±0.00025 and 0.004±0.00044 in BC and healthy specimens, respectively. Using a cut-off level of 3.47 (r.u.) for HDAC8, sensitivity and specificity were 69% and 60% respectively. The corresponding values for miR-216b-5p, using a cut-off level of 0.0020 (r.u.), were 81% and 60%. Areas under the curve (AUC) for HDAC8 and miR-216b-5p were 0.687 and 0.752 respectively. The Positive Predictive Value (PPV) and Negative Predictive Value (NPV), likelihood ratio (positive and negative) for both HDAC8 and miR-216b-5p are shown in Table 4. Besides, our results showed that decreased level of miR-216b-5p was significantly associated with tumor size and lymph node invasion (Spearman's ρ= -0.382, p value=0.041 and Spearman's ρ= -0.373, p value=0.039). There was not any significant association between HDAC8 tissue level and clinical outcomes ( Table 5).
Effects of ectopic expression of miR-216b-5p on cell proliferation and cell cycle: To study the role of miR-216b-5p in BC cells, MCF-7 and MDA-MB-231 cell lines were transiently transfected with miR-216b-5p mimics and inhibitor, then the following overexpression or inhibition of miR-216b-5p by mimics or inhibitor were detected by qRT-PCR. As shown in figures 2A  while miR-216b-5p inhibitor transfection suppressed this effect (p Value < 0.0001).
HDAC8 is involved in miR-216b-5p -regulated proliferation, cell cycle arrest and colony formation inhibition in breast cancer cells: To identify whether HDAC8 involves in miR-216b-5p-suppressed breast cancer progression, HDAC8 was knocked out by CRISPR/Cas9 method in MDA-MB-231 and MCF7 cell lines and BC cellular functions including proliferation, cell cycle and colony formation were checked on the knocked out selected population of the cell lines. Decline in HDAC8 expression after transfection with knockout vector were detected in RNA and protein levels using qRT-PCR and Western blot methods, respectively.
qRT-PCR analysis of HDAC8 expression showed significant decrease in HDAC8 expression levels after transfection of BC cell lines with HDAC8 knockout vector compared to empty vector and mock controls (0.096±0.005 and 0.14±0.004 (r.u.), respectively) ( Figures 3A and C). Besides, the corresponding protein levels also confirmed the highly decline in HDAC8 in knockout group as compared to controls (0.76±0.03 and 0.63±0.04, respectively) ( Figures 3B and D). As shown in figure 3C  respectively) compared with empty vector (73±3 and 51±2 colonies, respectively) and mock group (78±9 and 56±2.1 colonies,respectively) (p value= 0.0035 and 0.0003, respectively). (Figures 3G and H) miR-216b-5p directly targets 3'UTR of HDAC8 mRNA: In order to search for the effective functional target of miR-216b-5p and its mechanism we used online computational miRNA target prediction algorithm (TargetScan 6.0). This algorithm predicted the 3′-UTRs of HDAC8 mRNA contain putative miR-216b-5p binding sites. We evaluated three different predicted miR-216b-5P binding sites that were found in 3'UTR region of HDAC8 gene ( Figure 4A). We then investigated the mechanism by which miR-216b-5p suppresses the progression of breast cancer tumors. To confirm whether HDAC8 is a direct target of miR-216b-5p, we inserted the wild-type and the 550bp-mutant form of HDAC8 3′-UTR into the psiCHECK-2 vector at the downstream of the Renilla luciferase coding sequence separately. As illustrated in figure 4B miR-216b-5p rather than control significantly suppressed the Renilla luciferase activity in HEK293T cells, which was compromised when the binding site of miR-216b-5p was mutated. Furthermore, we found that overexpression of miR-216b-5p reduced the endogenous HDAC8 protein expression in both MCF-7 and MDA-MB-231 cells, whereas transfection with miR-216b-5p inhibitor increased the level of HDAC8 mRNA in MCF-7 and MDA-MB-

Discussion:
In the present study, we examined the functional significance of HDAC8 and miR-216b-5p in breast cancer. In this study we showed that miR-216b-5p is significantly down-regulated in human breast cancer tissue and breast cancer cell lines and has a negative correlation with the HDAC8 expression level.
Bioinformatics analyses identified various tumor suppressive miRs that can target the 3ʹ-UTR of HDAC8 and among them we showed that the expression of miR-216b-5p is highly correlated with the expression of HDAC8 in different BC cell lines. Hence, we report miR-216b-5p to be one of the most potent miR targeting HDAC8. Our results demonstrate that miR-216b-5p down regulates the expression of HDAC8 by directly binding to its 3ʹ-UTR sequence. In addition, a significant decrease in the HDAC8 expression level was observed upon miR-216b-5p overexpression. Also, a significant down-regulation of HDAC8 expression was seen while the miR-216b-5p mimics transfected to the cells.
Although the prognosis of breast cancer has improved due to advances in treatment concepts and treatment methods, the benefit of current approaches is limited given the emergence of resistance to treatments (Jiang et al., 2014). The beginning and development of cancer, conventionally seen as a genetic disease, has been determined to involve epigenetic abnormalities in addition to genetic alterations (Shen et al., 2016). The epigenetic mechanisms of cancer consist of DNA methylation, histone modification, nucleosome positioning, and noncoding RNA expression, specifically microRNA expression (Shen et al., 2016). Recent studies revealed the direct correlation between alterations in histone proteins and breast tumorigenesis (Connolly and Stearns, 2012;Kanwal et al., 2015;Koeneke et al., 2015). In breast cancer, HDAC8 is highly over expressed in triple-negative breast cancer (Hsieh et al., 2016) and responsible for late stage particularly in triple-negative breast cancer, poor prognosis and poor treatment response (Hsieh et al., 2016). Therefore regulation of HDAC8 at the protein level can play a critical role in tumor development. Recently, it has been more attention to targeted inhibition of HDAC8 as a potential cancer cells growth suppressor in vitro and in vivo (Rettig et al., 2015).
In line with previous studies, we showed that HDAC8 is overexpressed in clinical specimens and breast cancer cell lines. Furthermore, to confirm the oncogenic activity of HDAC8 in breast cancer we performed the knocking out of the HDAC8 using the CRISPR/Cas9 method and found out that by partial knocking out of the endogenous HDAC8 at the cells population level inhibit the oncogenic functions of HDAC8.
Altered expressions of different miRNAs have been also reported in breast cancer pathogenesis (Bischoff et al., 2014;Liang et al., 2014). For better therapeutic approach and better understanding of the molecular mechanism of cancer growth, the roles of miRs in cancer progression have been studied extensively.
In human cancer growth and metastasis miR-216b-5p takes a big role as a tumor suppressor in nasopharyngeal carcinoma (Deng et al., 2011), colorectal cancer (Kim et al., 2012, breast cancer (Zheng et al., 2014) and hepatocellular carcinoma (Liu et al., 2015). On the other hand it also regulates the apoptosis by mediating the activity of c-Jun (Xu et al., 2016) and Autophagy (Chen et al., 2016).
However the role of miR-216b-5p for the regulation of HDAC8 has not been studied yet. Our result, for the first time, explain that the miR-216b-5p function as a tumor suppressor and inhibit the proliferation, cell growth and colony formation by regulating the expression of HDAC8 in breast cancer.
Despite diagnostic and therapeutic advances during the last decades, unsuccessful chemotherapy because of fail to respond or resistance to chemotherapeutic agents are still remain in about 50% of the BC patients. This has caused to BC still remains as the second death-leading cause from cancer among women. Drug resistance is still a major clinical problem to successful treatment in breast cancer patients.
In addition to the chemoresistance, the metastasis of cancerous cells in patients with solid tumors likes those created from breast tissue, is responsible for about 90% of deaths. As a result there is more attention to development of a new therapeutic target for the treatment of BC patients. By these observations we found that miR-216b-5p may be a potent therapeutic molecule for the treatment of metastatic breast cancer by regulating the activity of HDAC8.
In conclusion, our study gives the idea of molecular mechanism involved in metastatic breast cancer growth via dysregulation of miR-216b-5p resulted HDAC8 overexpression. Down-regulation of HDAC8 expression in breast cancer suppresses cell proliferation, cell cycle arrest and colony formation. HDAC8 suppression via the miR-216b-5p possibly open a new therapeutic approach to treat the metastatic breast cancer.

Acknowledgments:
The author wish to thank all patients and health stuffs who participated in this study. Financial support from Kurdistan University of medical sciences is highly appreciated. Financial disclosure: The author has no financial relationships relevant to this article to disclose.
Author Contributions: All authors contributed equally in this work.