hsa-MicroRNA-28-5p Inhibits Diffuse Large B-Cell Lymphoma Cell Proliferation by Downregulating 14-3-3ζ Expression

MicroRNAs (miRNAs) participate in the comprehensive biological process of several cancer types. In our former study, we found that hsa-microRNA- (miR-)28-5p was downregulated, but tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activating protein zeta (14-3-3ζ or YWHAZ) was upregulated in diffuse large B-cell lymphoma (DLBCL) tissues. We predicted that YWHAZ was a target gene for hsa-miR- 28-5p using bioinformatics analysis. Our goal was to reveal the role of hsa-miR-28-5p in DLBCL. YWHAZ was tested by immunohistochemistry (IHC) in formalin-fixed paraffin-embedded (FFPE) tissues of 137 DLBCL tissues, and the expression of hsa-miR-28-5p and YWHAZ was examined by quantitative real-time polymerase chain reaction (qRT-PCR) in 15 fresh and frozen DLBCL tissues. To study the functional roles of the downregulated hsa-miR-28-5p in DLBCL, a Cell Counting Kit-8 assay was conducted to estimate cell proliferation. Transient transfection of miRNA mimics was performed to overexpress hsa-miR-28-5p, and flow cytometry was performed to examine cell apoptosis and cell cycle progression. A dual-luciferase reporter assay was employed to explore the relationship between hsa-miR-28-5p and YWHAZ. Western blotting and qRT-PCR were used to investigate the function of hsa-miR-28-5p in YWHAZ expression. hsa-miR-28-5p was found to be significantly downregulated in DLBCL tissues and cell lines. Functional studies showed that hsa-miR-28-5p overexpression inhibited cell viability and proliferation, and YWHAZ was predicted to be a target of hsa-miR-28-5p. Dual-luciferase reporter assay, Western blotting, and qRT-PCR verified that hsa-miR-28-5p negatively regulated YWHAZ expression by directly targeting its 3′ untranslated regions in DLBCL cells. hsa-miR-28-5p may suppress the growth of DLBCL cells by inhibiting YWHAZ expression. These findings could provide novel targets for DLBCL diagnosis and therapy.


Introduction
Diffuse large B-cell lymphoma (DLBCL) is the most common pathological subtype of non-Hodgkin's lymphoma (NHL), accounting for 30%-58% of NHLs [1]. It is also the most common type of lymphoma. In the domestic lymphoma cooperation group, the incidence of DLBCL is as high as 40-50%, which is higher than the international incidence of 30%. DLBCL is a highly heterogeneous and aggressive group of B-cell lymphomas with diverse clinical features, histological morphology, and gene and molecular phenotypes [2]. Although the R-CHOP regimen can significantly improve the prognosis of most patients, there are individual differences among patients in the drug resistance of tumor cells stimulated by chemotherapeutic drugs. One-third of patients relapse within a short period of time after the current treatment regimen and reach the advanced tumor stage [3]. For relapsed/refractory (R/R) DLBCL patients, although salvage autologous hematopoietic stem cell transplantation (ASCT) after chemotherapy can achieve longer-term remission, the significant toxicity of high-dose chemotherapy drugs limits the further treatment of patients with complications and elderly patients. e existing salvage chemotherapy combined with ASCT can cure only approximately 10% of patients [4], and the prognosis of patients diagnosed with R/R DLBCL remains poor. Prolonging the survival time of the remaining 30% of patients and improving their prognosis has become the focus of research at home and abroad. In the last few years, the Ki67 index, International Prognostic Index (IPI) score, serum lactate dehydrogenase (LDH), Ann Arbor stages, and the classification of Han's model have been used clinically and pathologically, but these evaluation systems are unable to analyze patients who do not respond to treatment. erefore, there is an urgent need for new biomarkers that have better stratification methods to analyze different levels of patients. e roles of microRNAs (miRNAs) in the development and function of T and B cells have been confirmed [5]. hsa-microRNA-(miR-) 28-5p is an intragenic miRNA that targets a variety of tumor-related genes and participates in cell proliferation, migration, invasion, and epithelial-mesenchymal transformation [6]. e expression of hsa-miR-28-5p in carcinomas is high, low, or lost [6][7][8][9], but its expression is downregulated, lost, or upregulated in B-cell lymphoma [7][8][9]. For example, hsa-miR-28-3p and hsa-miR-28-5p are upregulated in GCB-DLBCL [9]. Recent research showed that high expression of hsa-miR-28-5p hampers tumor growth in DLBCL [8]. Previous studies have characterized the genes regulated by hsa-miR-28 in other human B-cell lymphoma cells by transcriptomic and proteomic analysis [8]. However, the exact mechanism of hsa-miR-28-5p in DLBCL is not clearly known.
Our research group showed that hsa-miR-28-5p was downregulated in DLBCL tissues (GSE173080) using an Agilent Human miRNA Microarray [10]. e series record GSE173080 provides access to all of the current data (https:// www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc�GSE173080). Furthermore, we found that the protein tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activating protein zeta (also named 14-3-3ζ or YWHAZ) was differentially expressed based on isobaric tags for relative and absolute quantitation (iTRAQ). Parallel reaction monitoring (PRM) verified that YWHAZ was upregulated in DLBCL tissues. e high expression of YWHAZ was related to a poor prognosis of DLBCL [11]. According to the TargetScan database (http://www.targetscan.org) and starBase (https:// starbase.sysu.edu.cn), YWHAZ is a direct target of hsa-miR-28-5p. We predicted that the hsa-miR-28-5p/YWHAZ axis might be involved in the pathogenesis of DLBCL. However, no studies have evaluated how hsa-miR-28-5p relates to the occurrence and development of DLBCL.

Tissue Samples.
e study was approved by the Medical Ethics Committee of the Tumor Hospital Affiliated to Xinjiang Medical University, and 15 patients and/or their families signed informed consent before the histological biopsy was carried out by the patients' chief physician. Fresh and frozen DLBCL samples were stored at −80°C, other samples were from formalin-fixed paraffin-embedded (FFPE) tissues, and treatments were not given before this research. e features of 15 patients with fresh and frozen samples are described in Table 1.

Tissue Microarray and Immunohistochemistry (IHC).
ree tissue microarray (TMA) blocks were constructed using a tissue arrayer. Each individual case was represented by two tumor cores with a diameter of 0.6 mm that had been taken from the original paraffin blocks. Serial sections of 3 µm were prepared from the tissue array blocks and used in the immunohistochemical analysis, including YWHAZ (ab51129, Abcam, 1 : 100, cytoplasm). Pretreatment in 1 mm Tris/EDTA buffer (pH 9.0, no. ZLI-9069, ZSBIO, China) was conducted for 25 minutes at 98°C. Using 3% hydrogen peroxide to block nonspecific peroxidase reactions, tissues were placed in a humidified incubator at 37 C for 20 minutes. After washing three times with phosphate-buffered saline (PBS) for 5 minutes each time, the tissues were dried, and a small amount of YWHAZ monoclonal antibody (1 : 100, Abcam, ab51129) was added at 4°C overnight. e tissues were incubated with rabbit anti-mouse IgG antibody (PV-6000, ZSBIO, China) for 20 minutes at room temperature as a secondary antibody application. e staining intensity of tumor tissues was scored as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong) for antigens present in the cytoplasm using a light microscope (magnification, ×400), determined separately by two independent pathologists. DLBCL was classified according to Han's algorithm: the germinal center B-cell (GCB) subtype had a CD10+ or CD10−, Bcl-6+, and MUM1− phenotype, whereas the Bcl-6or Bcl-6+, CD10−, and MUM1+ phenotype represented the non-GCB subtype (including the activated B-cell-like (ABC) subtype and unclassified subtype in this study). IHC was performed following the manufacturer's protocols.

Cell Transfection.
e YWHAZ overexpression plasmid and siRNAs against YWHAZ were purchased from General Biol (Anhui, China), with scramble plasmid and siRNA employed as negative controls. hsa-miR-28-5p mimics and hsa-miR-28-5p inhibitors were synthesized by Gene Pharma (Suzhou, China). All of the above reagents were transfected into cells via Lipofectamine 3000 ® Transfection Reagent (L3000-008, Invitrogen, USA) according to the manufacturer's recommendations. e transfection conditions were as follows: hsa-miR-28-5p mimics or inhibitor negative control labeled with 0.1 μm red fluorescence was transfected into cells 48 hours after transfection. Under a fluorescence microscope, the transfection efficiency was determined by naked eye observation of the red fluorescence expression, and the transfection efficiency was more than 70%.

Western Blotting Assay.
Protein was extracted from cells and transferred to polyvinylidene fluoride (PVDF) membranes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). en, membranes were blocked with 5% nonfat milk for 1 hour and incubated overnight at 4°C with the following primary antibodies: YWHAZ (1 : 500, Abcam, ab51129) and β-actin (38 KD, Sino Biological Inc, 100166-MM1). After washing the membranes three times with Tris Buffered Solution Tween (TBST), a secondary antibody was incubated with the membranes for 1 hour at room temperature. e dilution ratio was determined according to the instructions.

Cell Counting Kit-8 (CCK-8).
Cell proliferation analysis was established using the CCK-8 reagent (FC101-03, Beijing, China). OCI-LY1 cells were seeded into 96-well plates at 5 * 10 3 cells/well. At the indicated time points, 10 μl CCK-8 was supplemented, and the cells were incubated for 1 hour at 37°C. e optical density was identified at 450 nm with a microplate spectrophotometer.

Flow Cytometry Assays.
Cell cycle analysis was performed using flow cytometry (LSRFortessa, BD, USA). e cells were resuspended in 500 µl of PBS (ZLI-9062, Beijing, China) and 3.5 ml of anhydrous ethanol for fixation overnight. Cells (1 * 10 6 ) were separated by centrifugation at 2,000 rpm for 5 minutes and accumulated and washed twice with cold PBS. en, 500 µl of PI/RNase (550825, BD, USA) was added to resuspend the cells. After passing through a 200 Mesh Nylon screen, single-cell suspensions were prepared, and the sample was incubated at 4°C in the dark for 30 minutes. Flow cytometry was used to detect red fluorescence at 488 nm and light scattering. DNA content analysis and light scattering analysis were carried out with analysis software. For apoptosis analysis, cells were collected 48 hours after transfection. Cells were doublestained using a FITC Annexin V PE/7AAD Kit (559763, BD, USA). en, cells were detected by flow cytometry after 30 minutes.
2.11. Statistical Analysis. SPSS 23.0 statistical software and GraphPad Prism 8.0 software were applied for data analysis. All data are presented as the means plus or minus standard deviation. Student's t-test, one-way ANOVA, and Fisher's exact test were used to perform statistical analysis. p < 0.05 was considered statistically significant.

hsa-miR-28-5p Is Downregulated and YWHAZ Is Upregulated in Human DLBCL Tissues.
e expression levels of hsa-miR-28-5p and YWHAZ in DLBCL cell lines were detected by qRT-PCR, and the DLBCL cell lines for subsequent cell function tests were identified. To study the roles of hsa-miR-28-5p in the development of DLBCL, the expression patterns of hsa-miR-28-5p were analyzed by qRT-PCR. We chose reactive hyperplasia of lymph nodes for our experiment. As shown in Figures 1(a)-1(d), hsa-miR-28-5p was downregulated, and YWHAZ was upregulated in DLBCL tissues. Figure 1(d) shows that the ABC group had a higher expression of YWHAZ than the other groups in fresh and frozen DLBCL tissues. YWHAZ immunoreactivity was not observed in reactive hyperplasia of lymph nodes tissue (Figure 1(e)). Heavy and diffuse YWHAZ immunoreactivity was observed in the cytoplasm of DLBCL cells (Figure 1(f )). e relative expression of hsa-miR-28-5p was the highest in the IM-9 cell line and clearly decreased in DLBCL cell lines; the difference was statistically significant (Figure 1(g)). YWHAZ was significantly negatively correlated with hsa-miR-28-5p (Figure 1(h)). e relative expression of YWHAZ was the lowest in IM-9 cells and clearly increased in DLBCL cell lines; the difference between the OCI-LY1 group and the OCI-LY3 group was statistically significant (Figure 1(i)). Based on the above results, we identified OCI-LY1 cells for subsequent cell function studies. Figure 1(j) shows the growth of OCI-LY1 cells.

YWHAZ Is a Direct Target Gene of hsa-miR-28-5p.
To ascertain the detailed regulatory mechanism of hsa-miR-28-5p in DLBCL, we searched the TargetScan and StarBase databases and observed that YWHAZ was predicted to be a downstream target of hsa-miR-28-5p (Figure 2(a)). e luciferase activity in the YWHAZ-WT + hsa-miR-28-5p group was significantly lower than that in the other three groups (Figure 2(b)). e 3'-UTR of the YWHAZ gene and hsa-miR-28-5p complemented each other, and their binding caused the fluorescence expression of the plasmid to decrease; however, the fluorescent expression of the plasmid did not change after cotransfection with the reporter plasmid of hsa-miR-28-5p and YWHAZ's 3'-UTR mutant sequence.
e results showed that hsa-miR-28-5p could interact with YWHAZ by binding to the 3'-UTR of YWHAZ (Figure 2(b)), indicating that YWHAZ is a target gene regulated directly by hsa-miR-28-5p. e mutation points for YWHAZ are shown in Figure 2(c).

Screening the Best Appropriate siRNA and Verifying the
Effect of Silencing YWHAZ. qRT-PCR was used to choose the most proper siRNA-YWHAZ (Figures 3(a)-3(c)). e concentration, 260/280, and quality of total RNA extracted from experimental cell samples were measured. We selected a 0.1 μm siRNA transfection concentration, and 48 hours was used as the best transfection conditions for follow-up experiments. Figure 3(a) shows that the siRNA-YWHAZ (Y319) group could effectively inhibit the gene expression of YWHAZ. Western blotting analysis was used to confirm the effect of silencing YWHAZ (Figures 3(b) and 3(c)). e concentration and quality of the protein extracted from the experimental cell samples were both satisfactory. e protein level of YWHAZ in the siRNA-YWHAZ group was lower than that in the other two groups. SiRNA-YWHAZ suppressed the protein level of YWHAZ (Figures 3(b) and 3(c)).

Mechanism by Which hsa-miR-28-5p Targets YWHAZ and Affects DLBCL Cell Proliferation, Apoptosis, and Cell
Cycle Progression. Compared with the miRNA mimic NC group and control group, overexpression of hsa-miR-28-5p (Figures 4(a) and 4(b)) in OCI-LY1 cells led to lower As shown in our experiments, apoptosis-related protein and gene expression levels were determined by Western blotting and qRT-PCR, respectively. e results indicated that the gene and protein levels of YWHAZ were higher in the siRNA-YWHAZ + hsa-miR-28-5p inhibitor group than in the siRNA-YWHAZ group (Figures 6(a) and 6(b)). Compared with the control group, miRNA mimic NC group, miRNA inhibitor NC group, and hsa-miR-28-5p inhibitor group, overexpression of hsa-miR-28-5p significantly increased the protein levels of the cleaved proapoptotic proteins BAD and BAX (Figures 6(c) and 6(d)) and decreased levels of the antiapoptotic protein BCL-2 in OCI-LY1 cells (Figure 6(e)). A subsequent comparison with the control group, siRNA-NC group, siRNA-YWHAZ + hsa-miR-28-5p inhibitor group, and siRNA-YWHAZ group obtained the same result (Figures 6(c)-6(e))). In conclusion, siRNA-YWHAZ inhibits the transcription and translation of YWHAZ, suppresses proliferation, promotes apoptosis, and inhibits proliferation by regulating the cell cycle of DLBCL cells.

Effect of siRNA-YWHAZ + hsa-miR-28-5p Inhibitor on DLBCL Cells.
e CCK-8 results showed that the survival rate of DLBCL cells in the siRNA-YWHAZ group was 82.973%, which was significantly lower than that in the siRNA-NC group (96.708%). However, the cell survival rate     of the siRNA-YWHAZ + hsa-miR-28-5p inhibitor group was 88.302%, which was significantly higher than that in the siRNA-YWHAZ group, although there was no clear difference. e results showed that silencing YWHAZ could inhibit the proliferation of DLBCL cells, but the hsa-miR-28-5p inhibitor could reverse the effect induced by YWHAZ silencing on the proliferation inhibition of DLBCL cells (Figures 5(a) and 5(b)). e total apoptosis rate in the siRNA-YWHAZ group was 13.820 ± 0.788, which was significantly higher than that in the siRNA-NC group (8.690 ± 0.847%), and the difference was statistically significant. e total apoptosis rate of the siRNA-YWHAZ + hsa-miR-28-5P inhibitor group was 11.150 ± 1.447%, which was significantly lower than that of the siRNA-YWHAZ group.
ese results suggested that silencing YWHAZ can promote apoptosis of DLBCL cells, but an hsa-miR-28-5p inhibitor can reverse this effect in DLBCL cells (Figures 5(c) and 5(d)). e percentage of S phase cells in the siRNA-YWHAZ + hsa-miR-28-5p inhibitor group (27.627 ± 0.599%) was lower than that in the siRNA-YWHAZ group (36.893 ± 0.602%). ese results suggested that the hsa-miR-28-5p inhibitor can reverse the regulatory effect of siRNA-YWHAZ on the DLBCL cell cycle, and YWHAZ can inhibit the proliferation of DLBCL cells by regulating the cell cycle. Inhibiting hsa-miR-28-5p on this basis could reverse this regulatory effect and promote DLBCL cell proliferation (Figures 5(e) and 5(f)).
e results indicated that the gene and protein levels of YWHAZ are higher in the siRNA-YWHAZ + hsa-miR-28-5p inhibitor group than in the siRNA-YWHAZ group (Figures 6(a) and 6(b)). We concluded that the hsa-miR-28-5p inhibitor can reverse the inhibition of YWHAZ transcription and translation induced by siRNA-YWHAZ. erefore, the hsa-miR-28-5p inhibitor could partially reverse the effect of siRNA-YWHAZ. ese results suggested that siRNA-YWHAZ can inhibit the activation of DLBCL. hsa-miR-28-5p affects DLBCL cell proliferation, apoptosis, and cell cycle progression by targeting YWHAZ.  influence comprehensive biological processes by causing targeted degradation or translation inhibition through binding the 3' UTR of a target mRNA [12]. miRNAs are involved in the basic pathways of B-cell development, such as B-cell receptor signaling (BCR) and B-cell migration/adhesion, and affect B-cell maturation and the production of marginal zone, follicular, plasma, and memory B cells [13]. miRNAs may function as oncogenes, tumor suppressors, or both, depending on the tumor environment [14]. hsa-miR-28 expression can downregulate downstream effectors of BCR signaling, downstream effectors have great effects on B lymphocyte proliferation and survival, and their expression is upregulated in germinal center-derived malignant tumor cells [8]. e expression of hsa-miR-28-5p inhibits the proliferation of B-cell lymphoma and renal cell carcinoma cells by regulating the expression of BAG1 [7] and RAP1B, respectively [15]. hsa-miR-28-5p also restrains the migration and invasion of gastric cancer cells by inhibiting AKT [16]. Although the expression of hsa-miR-28-5p is downregulated in B-cell lymphoma, renal cell carcinoma, hepatocellular carcinoma, and colorectal cancer [7,[17][18][19], the expression level of hsa-miR-28-5p is increased in ovarian, esophageal, and cervical cancer [6,20,21]. Most articles regarding the role of hsa-miR-28-5p in tumors indicate that miRNA has universal inhibitory activity in vitro, consistent with a previous article [7], and the finding that reexpression of hsa-miR-28 can damage tumor growth in several lymphoma models [8] verified that the reexpression of hsa-miR-28-5p in a DLBCL xenotransplantation model hampers tumor growth [8]. ese findings opened the way for hsa-miR-28-5p-based replacement therapy as a new treatment strategy for DLBCL. e above conclusion is inconsistent with another prior report [9]. According to the other theory, hsa-miR-28 expression could decrease the proliferation and survival of both primary and tumor B lymphocytes, likely by inhibiting BCR signaling [8]. Considering that YWHAZ is located downstream of the BCR signaling pathway and that YWHAZ is the target gene of hsa-miR-28-5p as predicted from the database, we can study the mechanism of hsa-miR-28 in DLBCL by analyzing its regulation of YWHAZ.

Discussion
Accumulated research has revealed the mechanism of hsa-miR-28-5p in the initiation and development of cancers, including ovarian cancer, glioma, and hepatocellular carcinoma [6,15,18], but there are no reports on DLBCL. Functional experiments have shown that the hsa-miR-28-5p inhibitor promotes DLBCL cell proliferation, restrains cell apoptosis, and leads to an abnormal cell cycle. In the present work, we found that YWHAZ acted as the target of hsa-miR-28-5p. In addition, hsa-miR-28-5p was expressed at low levels in DLBCL cell lines. We found that hsa-miR-28-5p could partially reverse the YWHAZ-induced oncogenic effects on DLBCL cells, which was consistent with the previously discovered tumor suppressive roles of hsa-miR-28-5p in other tumors [7,[17][18][19]].
14-3-3 is a family of acidic and dimeric proteins that are profoundly conserved and broadly expressed in eukaryotic cells. Seven 14-3-3 isoforms (β, c, ζ, σ, є, t, and η) have been discovered in human cells. YWHAZ, which is part of the 14-3-3 protein family, can perform primarily by binding to clarified phosphoserine/phosphothreonine-containing motifs in protein targets to mediate signaling pathways and regulate many biological processes, including protein transport, metabolism, cell proliferation, migration, apoptosis, and cell cycle regulation. YWHAZ was shown to be upregulated frequently and to function as an oncogene by regulating multiple signaling pathways in tumors [22]. Overexpression of YWHAZ is regulated by miRNAs or long noncoding RNAs. Overexpression of YWHAZ activates downstream molecules, including protein kinases, apoptotic proteins, and metastasis-related molecules, and ultimately promotes the malignant potential of cancer cells [22]. e increased expression of YWHAZ promotes the proliferation, migration, and resistance to apoptosis of prostate cancer cells, while the downregulation of YWHAZ significantly affects the invasiveness of tumor cells [23,24]. Overexpression of YWHAZ can also be found in adenocarcinoma of the esophagogastric junction, lung cancer, and intrahepatic cholangiocarcinoma [25][26][27]; thus, overexpression of YWHAZ is related to invasiveness and drug resistance in multiple types of tumors. Our data showed that YWHAZ is a direct target gene of hsa-miR-28-5p and is overexpressed in DLBCL cell lines, which was in line with its oncogenic role in other tumors [23,25,27]. Furthermore, the expression of siRNA-YWHAZ inhibited proliferation, promoted apoptosis, and caused the cell cycle to remain in the S phase in DLBCL cells, all of which were reversed by the hsa-miR-28-5p inhibitor. Our investigation demonstrates the underlying mechanism of hsa-miR-28-5p in the initiation and development of DLBCL. BV02 is a nonpeptide inhibitor of the 14-3-3/c-Abl protein-protein interaction, its bioactive form is phthalimide derivative 9, and inhibitors of 14-3-3 proteinprotein interaction derived from BV02 are chemically stable [28]. Inosine monophosphate (IMP), pyridoxal phosphate (PLP), and the derivatives show inhibitory action of the 14-3-3/c-Abl PPI poorly [29]. So far, 14-3-3 inhibitors have mono-and bivalent forms [30].

Conclusions
In summary, we found an oncogenic role for YWHAZ and an antioncogenic role for hsa-miR-28-5p in the proliferation and apoptosis of DLBCL cells. Moreover, our research suggests that hsa-miR-28-5p may suppress the growth of DLBCL cells by inhibiting YWHAZ expression. ese findings could provide novel targets for DLBCL diagnosis and therapy.