miR-103 promotes the progression of non-Hodgkins lymphoma by inhibiting OTUD7B expression

MicroRNAs (miRNAs) are vital for regulating the malignant phenotypes of tumor cells. The purpose of this work is to investigate the function and downstream mechanism of miR-103 in the progression of non-Hodgkin lymphoma (NHL). Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to detect miR-103 and OTU deubiquitinase 7B (OTUD7B) mRNA expressions in NHL tissues and cells. Immunohistochemistry and Western blot were used to detect the expression of OTUD7B in NHL tissues and cells. CCK-8 experiment, flow cytometry analysis, and Transwell experiment were used to detect the role of NHL cell proliferation, apoptosis, migration and invasion. Bioinformatics, qRT-PCR, Western blot and dual-luciferase reporter assays were used to validate the targeting relationship between miR-103 and OTUD7B. NF-κB p65 luciferase reporter assay and Western blot were applied to determine NF-κB activity and the expression of NF-κB targeted genes.

MicroRNAs (miRNAs), about 19-25 nucleotides in size, are a class of small endogenous singlestranded non-protein-encoding RNAs, which bind to the 3' untranslated region (3' UTR) of mRNA, inducing mRNA degradation or inhibiting mRNA translation, thereby regulating target gene expression (4). So far, accumulating studies have indicated that miRNAs are implicated in a variety of physiological and pathological activities, including tumorigenesis (5,6). For example, miR-760 inhibits gastric cancer cell proliferation and induces apoptosis by negatively regulating the expression of GIT1 (7). miR-21 promotes angiogenesis, migration, and invasion of renal clear cell carcinoma by regulating the PDCD4/c-Jun (AP-1) signaling pathway (8). miR-103 plays a cancer-promoting role in various tumors, such as breast cancer, bladder cancer, and gastric cancer (9)(10)(11). However, the expression of miR-103 in NHL and its mechanism remain unclear.
Deubiquitylases are a class of proteins involved in post-translational modification of proteins, whose main function is to remove the ubiquitination modification of the target protein, thereby regulating the stability and localization of the target protein (12,13). Deubiquitylases are involved in preventing the protein from degradation, removing non-degradable ubiquitin signals, maintaining stable ubiquitin level, and mediating ubiquitin signal conversion (14). Ovarian tumor-associated protease 7B (OTU deubiquitinase 7B, OTUD7B, also known as Cezanne) belongs to the deubiquitinatylases family, which is related to the tumorigenesis (15). The role of OTUD7B in different tumors is different. For example, OTUD7B can mediate the deubiquitination of epidermal growth factor receptor in breast cancer cells to promote the proliferation and survival of breast cancer cells (16). Differently, the expression level of OTUD7B in hepatocellular carcinoma is significantly lower than that in adjacent liver tissues; patients with liver cancer with a lower expression of OTUD7B have adverse prognosis (17). However, the role of OTUD7B in NHL is unclear.
Nuclear factor-Kappa B (NF-κB) is a well known transcription factor associated with almost all aspects of life activity (18). It consists of five subunits, namely c-Rel, Rel A (p65), Rel B, NF-κB1 (p105/p50), and NF-κB2 (p100/p52) (19). NF-κB plays a very important role in the progression of tumors by regulating multiple downstream targeted genes (19). A lot of studies indicated that NF-κB signaling is abnormally activated in NHL (20)(21)(22). However, the upstream mechanisms that cause abnormal activation of NF-κB need to be further explored.
In this study, it was demonstrated that miR-103 expression was significantly up-regulated in NHL.

Cell transfection
Raji and Mino cells in the logarithmic growth phase were selected and seeded in 6-well plates with a cell density of 5 × 10 5 /well. After cultured for 23 h, transfection was performed according to the
Nanodrop-spectrophotometer was used to detect RNA concentration and purity. According to the manufacturer's protocol, the PrimeScript-RT Kit (Madison, WI, USA) was used to synthesize complementary DNA (cDNA) from 1 µg of total RNA, and then SYBR Green Premix Ex Taq II (TaKaRa, Dalian, China) and ABI 7500 system ( Applied Biosystems, Foster City, CA, USA) was applied for qRT-PCR. The total volume of the PCR system was 30 µL and each sample contained 300 ng of cDNA.
GAPDH was the internal reference for OTUD7B, and U6 was the internal reference for miR-103. The primer sequence information was shown in Table 1. NHL tissues and normal tissues were fixed in 10% formaldehyde and embedded in paraffin. The sections were dewaxed and hydrated. Before immunohistochemical staining, the dewaxed sections were baked at 37 °C for 2 h, the endogenous peroxides were blocked by 1% H 2 O 2 for 5 min and the sections were washed for 3 times, and the sections were blocked using immunostaining blocking solution for 1 h, then incubated with anti-OTUD7B antibody (1: 200 dilution, Abcam, Cambridge, UK, ab118387) overnight at 4 °C. Then the sections were washed with PBS and then incubated with biotinlinked antiserum for 1 h at room temperature. Next, the sections were washed again and stained with 3,3-diaminobenzidine (DAB) for 1 min. Then the sections were washed with double distilled water, stained with hematoxylin for 1 min, and observed under a microscope. Ultimately, the staining was scored by pathologists.

Cell proliferation assay
The cells of each group in the exponential growth phase were collected and prepared into single cell suspension (1 × 10 4 /ml), then inoculated on 96-well plates with 100 µL cell suspension per well, with 3 replicate wells in each group. Subsequently, 10 µL of CCK-8 solution (Beyotime Biotechnology, Shanghai, China) were added to the wells, and a blank control well only containing the medium and CCK-8 solution was set. After 2 h of incubation, a microplate reader at a wavelength of 450 nm was employed to determine and record the absorbance (A) values of each well. Ultimately, the plate was measured at intervals of 24 h for 5 days.

Apoptosis Experiment
Annexin V-fluoroisothiocyanate (FITC)/PI double staining kit (Sungene Biotech, TianJin, China) was used to detect the apoptosis of NHL cells. 24 h after cell transfection, the cells were collected, and the cell density was adjusted to 2 × 10 6 cells/well, and the culture was continued for 24 h. After that the cells were centrifuged and the supernatant was discarded, and the cells were washed with pre-cooled PBS twice, and the cells were resuspended with 1 × Binding buffer. Following that, 5 µL AnnexinV-FITC and 5 µL PI solution were added to the cell suspension, mixed thoroughly and incubated at room temperature for 15 min. Finally, flow cytometry analyses (Cytomics FC 500; Beckman Coulter) was employed to detect the apoptosis rate according to the instructions of the kit.

Transwell experiment
Transwell experiment was carried out using Transwell chamber (Millipore, Billerica, USA). After that, the Matrigel was added to the Transwell chamber membrane and the chambers were placed in a 24well plate for cell invasion experiments (in cell migration experiments, Matrigel was not used). NHL cells were harvested and adjusted to a cell density of 1 × 10 5 /mL with serum-free medium, and 200 µL cell suspension was added into the Transwell chamber. Meanwhile, 800 µL medium containing 10% FBS was added to the wells in the incubator, and the cells were cultured at 37 °C for 24 h. Thereafter, the chamber was removed, and the cells migrated or invaded in the wells of the 24-well plate were counted.

Western blot
Cells were lysed with a RIPA lysis (Pierce, Rockford, IL, USA), and then centrifuged to collect the supernatant. The protein samples were separated by SDS-PAGE and then electrophoretically transferred to a nitrocellulose (NC) membrane. The proteins were then blocked with 5% skim milk at room temperature for 2 h, and then the primary antibody was added to incubate the NC membrane at 4 °C overnight. After that the membrane was washed with TBST for 3 times, and the secondary antibody was added to incubate the NC membrane at room temperature for 2 h. Next, the membrane was washed with TBST for 3 times, and ECL chemiluminescence reagent (Millipore, Bedford, MA, USA) was used to develop the bands. The antibodies used in this study was purchased from Abcam

Data analysis
All experiments were repeated 3 times, and the data obtained were expressed as x ± s. SPSS17.0 software (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Independent sample t test was employed to compare the two groups. P < 0.05 indicated that the difference was statistically significant.

The characteristics of miR-103 expression NHL
In order to study the expression characteristics of miR-103 in NHL tissues, tumor tissues and normal lymph node tissues of 37 NHL patients were collected, and then miR-103 expression was measured.
qRT-PCR results showed that miR-103 expression was significantly up-regulated in NHL tissues compared to non-tumor tissues (Fig. 1A). Furthermore, compared with germinal center (GC) -B cells, it was found that the expression levels of miR-103 in four NHL cells (Raji, NK-92MI, RL, Mino cells) were also significantly increased (Fig. 1B). These research data suggested that miR-103 could probably play an oncogenic role in NHL.

miR-103 promoted the proliferation, migration and invasion of NHL cells and inhibited the apoptosis
To investigate the function of miR-103 in NHL cells, miR-103 mimics were transfected with Mino cells, and miR-103 inhibitors were transfected into Raji cells respectively (Fig. 2A). Subsequently CCK-8 experiments and flow cytometry analysis revealed that compared with the control group, miR-103 overexpression promoted Mino cell proliferation and inhibited apoptosis (Fig. 2B&C). In addition, Transwell assays confirmed that miR-103 overexpression significantly enhanced the migration and invasion of Mino cells (Fig. 2D & E). On the contrary, the inhibition of miR-103 in Raji cells showed the opposite effect in the malignant phenotypes of the cells described above (Fig. 2B-E). These studies indicated that miR-103 exerted a cancer-promoting effect in the progression of NHL.

miR-103 directly targeted OTUD7B
To probe the downstream mechanism of miR-103 in NHL cells, two online analysis tools (microRNA and miRDB) were used to search for candidate targets for miR-103. A potential binding site was found between miR-103 and the 3'UTR of OTUD7B (Fig. 3A). To verify the interaction of miR-103 with OTUD7B 3'-UTR, dual luciferase reporter assay was performed. The results suggested that miR-103 overexpression significantly reduced the luciferase activity of WT-OTUD7B 3'-UTR reporter, but did not significantly change the luciferase activity of MUT-OTUD7B 3'-UTR reporter (Fig. 3B). Additionally, Pearson correlation analysis showed a significant negative correlation between miR-103 expression and OTUD7B expression in NHL tissues (Fig. 3C). Furthermore, qRT-PCR and Western blot also demonstrated that miR-103 mimics significantly inhibited mRNA and protein expression of OTUD7B, while down-regulating miR-103 induced increased expression of OTUD7B in NHL cells (Fig. 3D & E). In summary, these studies suggested that OTUD7B was a downstream target of miR-103 in NHL cells and was negatively regulated by the latter.

OTUD7B was down-regulated NHL tissues and cells
The experiments mentioned above confirmed that OTUD7B was one of the downstream targets of miR-103, so immunohistochemistry were performed to detect the expression characteristics of OTUD7B in NHL tissues and normal tissues. Representative images of the normal and NHL immune groups were shown (Fig. 4A). The statistical results implied that compared with non-tumor tissues, the positive rate of OTUD7B low expression was significantly increased in NHL tissues (Fig. 4B). In addition, qRT-PCR results showed that OTUD7B mRNA expression levels were significantly reduced in NHL tissues compared to non-tumor tissues (Fig. 4C). Consistent with expectations, OTUD7B expression levels were significantly reduced in the four NHL cells compared to GC-B cells (Fig. 4D).
The data suggested that OTUD7B probably inhibited NHL progression.

OTUD7B inhibited the proliferation, migration and invasion of NHL cells and promoted the apoptosis
In order to explore the role of OTUD7B axis in NHL, Raji cells were transfected with OTUD7B overexpressing plasmid, and Mino cells were transfected with OTUD7B shRNA. Transfection efficiency was confirmed by qRT-PCR and Western blot (Fig. 5A & B). The results of CCK-8 experiments, flow cytometry, and Transwell experiments suggested that compared to the control group, overexpression of OUTD7B significantly inhibited Raji cell proliferation, migration, and invasion and promoted the apoptosis (Fig. 5C-F). On the contrary, knocking down OTUD7B promoted the above-mentioned malignant phenotypes of Mino cells (Fig. 5C-F). These results indicated that OUTD7B was involved in inhibiting the malignant biological behaviors of NHL cells.

OTUD7B inhibited NF-κB signaling pathway in NHL
It is worth noting that previous studies have reported that OTUD7B can inhibit the activation of NF-κB (23)(24). Therefore, we made a hypothesis that OTUD7B could inhibit NHL progression by regulating the NF-κB pathway. As we expected, luciferase reporter assay indicated that overexpression of OTUD7B significantly inhibited the p65 NF-κB luciferase reporter activity, while knockdown of OTUD7B promoted the p65 NF-κB luciferase reporter activity (Fig. 6A). In addition, OTUD7B significantly reduced the expression level of p65 protein (an indicator of NF-κB transcriptional activity) (Fig. 7B).
Consistently, the NF-κB target genes were also suppressed by OTUD7B in Raji cells, such as Bcl-2, XIAP, CIAP2, Livin and survivin (Fig. 6B). Conversely, knockdown of OTUD7B expression induced an increase in these proteins in Mino cells (Fig. 6A, B). Hence, it was concluded that OTUD7B inhibited lymphoma progression by impeding the NF-κB signaling pathway.

miR-103/OTUD7B axis was involved in regulating the malignant phenotypes of NHL cells
Subsequently, miR-103 mimics and OTUD7B overexpression plasmid or its negative control were cotransfected into Mino cells, and miR-103 inhibitors and sh-OTUD7B or its negative control were cotransfected into Raji cells (Fig. 6A & B). As shown, restoring the expression of OTUD7B reversed the effect of miR-103 overexpression on cell proliferation, migration, invasion and apoptosis (Fig. 6C-F).
Similarly, knocking down OTUD7B partially weakened the cancer-promoting effect of miR-103 inhibitors in NHL. Therefore, it was confirmed that the miR-103/OTUD7B axis was involved in promoting NHL cell proliferation, migration, and invasion.

Discussion
This study reported for the first time that miR-103 was up-regulated in NHL tissues and cells. miR-103 can promote cancer cell proliferation, invasion, migration, and inhibit apoptosis. This work also indicated that miR-103 can promote NHL progression by suppressing the expression of OTUD7B, which was a negative regulator of NF-κB signaling pathway.
Recent studies have shown that multiple miRNAs are dysregulated in malignancies, including lymphomas (25, 26). Clarifying the relationship between miRNA and lymphoma will probably provide new biomarkers and therapy targets for this diseases (27). For example, miR-374b is highly expressed in T-cell lymphoblastic lymphoma and can be used as an independent indicator of relapse and survival (28). Additionally, compared to healthy individuals, miR-21 expression levels in tissues of B-NHL patients were significantly up-regulated; compared with stage I/II B-NHL, the expression level of miR-21 in stage III/IV patients was significantly higher (29). These reports indicate that miRNA can provide a new basis for assessing lymphoma progression, monitoring treatment efficacy and predicting the prognosis. As an important member of the miRNA family, miR-103 plays a pro-cancer role in multiple tumors. For example, miR-103 promotes the stemness of colorectal cancer cells by activating the Wnt/β-catenin signaling pathway, thereby promoting tumorigenesis and metastasis (30). miR-103 is involved in regulating the AKT/mTOR signaling pathway to promote the progression of prostate cancer (31). In this study, for the first time, miR-103 expression level in NHL was investigated and it was proved to be significantly increased in NHL tissues and cells. In vitro experiments confirmed that up-regulating miR-103 significantly promoted NHL cell proliferation, migration, and invasion and inhibited the apoptosis. Conversely, miR-103 inhibitors significantly inhibited the progression of NHL cells. This suggests that miR-103 is a potential therapy target for NHL.
OTUD7B, an important deubiquitinase, specifically regulates the ubiquitination level of substrate proteins by targeting K11-linked polyubiquitin chains, and is implicated in cell proliferation, cell survival, inflammatory responses, and other physiological and pathological processes (15)(16)(17). It plays an important role in the development of malignancies including lung cancer, breast cancer and liver cancer (16,17,(32)(33)(34). OTUD7B mediates EGFR deubiquitination through its OTU domain, inhibits EGFR degradation, promotes constant activation of EGFR downstream molecules, enhances AKT signal transduction, and promotes lung cancer cell proliferation, migration and angiogenesis (32).
Interestingly, another study reports OTUD7B in non-small cell lung cancer cell line H157 inhibits nuclear translocation of NF-κB, reduces the expression of IL-8 and ICAM-1, and thus inhibits the progression of non-cellular lung cancer (24). These studies suggest that the expression pattern, function and mechanism of OTUD7B in cancer biology are controversial. In this study, it was found that compared with normal tissues, the expression level of OTUD7B in NHL tissues was significantly reduced. Gain-of-function and loss-of-function experiments confirmed that overexpression of OTUD7B significantly inhibited NHL cell proliferation, migration, and invasion. On the contrary, compared with the control group, knocking down OTUD7B significantly promoted NHL progression. Furthermore, it was also found that OTUD7B significantly inhibited the p65 NF-κB luciferase reporter activity. In addition, OUTD7B could also inhibit the expression of p65 protein and the targeted genes of NF-κB signaling. It is confirmed that OTUD7B suppresses the progression of NHL by inhibiting the NF-κB signaling pathway. Additionally, in this work, it was demonstrated that OTUD7B was a target gene of miR-103, and overexpression of OTUD7B reversed the function of miR-103 on NHL cells. These demonstrations not only partly explained the reason of OTUD7B dysregulation in NHL, but also help clarify the downstream mechanism of miR-103.     Chi-square analysis showed that the positive rate of OTUD7B in NHL increased significantly.
(C) qRT-PCR results showed that the expression of OTUD7B mRNA in NHL was significantly reduced. (D) Western blot results showed that the expression levels of the 4 NHL cell lines were significantly lower than GC-B cells. * P < 0.05, *** P < 0.001.