MicroRNA-559 restrains gastric cancer progression via activating AKT signaling pathway by targeting TRIM14 CURRENT STATUS: REVIEW

Background: MicroRNAs (miRNAs) act as pivotal functions in gastric cancer (GC) carcinogenesis and progression. MiR-559 has been defined as a potential cancer suppressor gene in a few cancers. Nevertheless, the biological effect of miR-559 in human GC and the underlying molecular mechanism still unclear and need to be further illuminated. Methods: Quantitative real-time PCR (qRT-PCR) was fulfilled for measuring the miR-559 expression level in GC. The dual-luciferase reporter was used to verify that tripartite motif-containing 14 (TRIM14) is a target gene of miR-559. The expression levels of TRIM14 were examined by qRT-PCR and Western blot in GC tissue specimens and cell lines. The effects of miR-559 on GC cell growth were detected with MTT and cell counting assays. Cell cycle and apoptosis were examined by using flow cytometry. Overexpression and siRNA further demonstrated the role of TRIM14 in GC. Results: Our results revealed that miR-559 expression was dramatically downregulated in human GC tissue specimens and cell lines. MiR-559 overexpression restrained GC cell growth, and induced cell cycle G1-S phase arrest and apoptosis. MiR-559 inhibitors promoted cell multiplication and cell cycle G1-S transition, and inhibited apoptosis. MiR-559 level was negatively related to TRIM14 mRNA level in GC. The results showed that TRIM14 was affirmed to be a target gene of miR-559. MiR-559 overexpression downregulated the TRIM14 expression, and miR-559 inhibitors upregulated the TRIM14 expression. Particularly, knockdown of TRIM14 gave rise to the semblable cellular effects observed upon miR-559 overexpression. TRIM14 overexpression recapitulated the revulsive cellular and molecular effects by suppression of miR-559. Furthermore, both miR-559 overexpression and TRIM14 silencing led to the inhibition of the AKT signaling pathway. On the contrary, miR-559 suppression and TRIM14 overexpression activated the AKT signaling pathway, and inhibited Bax/Bcl-2 pathway. Conclusions: The findings illustrate that miR-559 restrains cell multiplication via suppressing the AKT signaling pathway in human GC and induces cell apoptosis through the suppression of Bax/Bcl-2 signaling pathway via targeting TRIM14. The findings imply that miR-559 acts as a pivotal function in human GC and represents a latent novel target in GC therapy. Our results showed that low miR-559 expression levels were observably associated with histologic classification, cancer magnitude and lymph node metastasis in GC patients, indicating that miR-559 may act a crucial role in GC diagnosis. The experiments further illustrated that miR-559 overexpression significantly suppressed GC cell proliferation by inhibiting G1-S phase transition and accelerated cell apoptosis, the suppression of miR-559 facilitated cell growth and G1-S transition, and restrained apoptosis. Our findings suggest cells

domain, and a coiled coil (CC) region [18]. These domains may exist or miss in diverse TRIM protein members, such as TRIM14 including B1-CC domains but lacking the R domain (an order of R-B1-B2-CC) [19]. It is reported that TRIM protein members may play oncogene or anti-oncogene to modulate different tumorigenesis and progression [20]. Accumulating evidences imply that TRIM14 acts as an oncogene role in colorectal cancer and breast cancer [21]. Nevertheless, the precise functions and the molecular mechanisms of TRIM14 in GC are still largely unknown.
In our current study, we explored the precise biological effect and mechanism of miR-559 in GC. It was discovered that the miR-559 expression level was remarkably reduced in GC tissue specimens and correlated with clinicopathological features, such as histologic classification, cancer magnitude and lymph node metastasis. Moreover, miR-559 dramatically suppressed MKN-45 and BGC-823 cell multiplication and cell cycle progression by the AKT signaling pathway by targeting TRIM14, and induced apoptosis through stimulating Bcl-2/Bax signalling. Knockdown of TRIM14 gave rise to the semblable biological effects observed upon miR-559 overexpression. TRIM14 overexpression promoted GC growth and inhibited apoptosis. Our results illustrate an anti-oncogenic function of miR-559 in GC, implying that miR-559 may represent a latent novel target in GC therapy.

Materials And Methods Human GC tissue preparation
In the Department of Oncological Surgery, First Affiliated Hospital, Xi'an Jiaotong University, China, we collected seventy GC tissue specimens and matched adjacent normal tissue samples from sufferer who were diagnosed, between August 2017 and November 2018. We gained the consent form from patients before tissue collection. The obtained tissue specimens were promptly refrigerated and 10% fetal bovine serum (Gibco), streptomycin (100 µg/mL) and penicillin (100 U/mL), and were cultured at 37 °C in a thermotank under 5% CO 2 and 95% air.
Hsa-miR-559 vector construction Hsa-miR-559 precursor expression vector (called after miR-559) and control empty vector (called after control) were constructed with chemosynthetic oligonucleotides and incorporated into the pcDNA6.2-GW/EmGFPmiR plasmid on the basis of the manufacturer's instructions. Full-length human TRIM14 gene DNA was cloned into the pCMV2-GV146 vector (Genechem Co. Ltd, Shanghai, China).
Transfection was fulfiled through using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer's instructions.

Dual-luciferase reporter assay
The binding site for miR-559 in the 3′-UTR of TRIM14 was constructed with synthetic oligonucleotides (Beijing AuGCT DNA-SYN Biotechnology Company, China) and inserted to the pmirGLO Dual-Luciferase expression vector (called after TRIM14-WT). The mutated 3′-UTR sequences of TRIM14 were also cloned and named TRIM14-MT. The pre-miR-559 plasmids and reporter plasmids (WT or MT) were cotransfected into HEK293T cells. The cells were collected and examined 24 hours after transfection.
We measured the reporter activity with the Dual-Luciferase Assay System (Promega, Madison, USA).
Quantitative real-time PCR (qRT-PCR) RNA was extracted from GC tissue specimens and cultured cells with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The SYBR Premix Ex Taq II Kit (Takara, China) was used to measure miR-559 expression and TRIM14 mRNA expression. qRT-PCR was performed. The data were normalized to RNU6B (U6) or GAPDH gene expression. The primer sequences contained the miR-559 reverse-

Western blot
Western blot was performed according to standard methods. Human GC tissue specimens and cultivated cells were lysed using RIPA lysis buffer (Invitrogen, Carlsbad, CA, USA) and centrifuged at 12,000 g. Next, we detected the protein concentration with the bicinchoninic acid (BCA) assay. The

Statistical analysis
All experiments were fulfilled minimally in triplicate independently. The data were analyzed with SPSS 25.0 software (SPSS, Inc., Chicago, IL, USA). The data were represented as the means ± SEM from at least 3 experiments. One-way ANOVA and Student's t-test were used to analyze the statistical significance of differences between groups. Correlation analysis between miR-559 and TRIM14 in human GC tissues was performed with Pearson's correlation analysis. Values of p < 0.05 were deemed to indicate statistically significant differences.

MiR-559 is dramatically downregulated in human GC and related to the clinicopathological features
To analyze the miR-559 level in human GC, we fulfilled qRT-PCR to detect miR-559 expression in clinical specimens (70 primary GC tissues and matched adjacent normal tissues) and GC cell lines (AGS, MKN-45 and BGC-823). The qRT-PCR assays revealed that miR-559 expression was observably reduced in 84.29% (59/70) of human GC tissues as compared to that in normal tissues (Fig. 1a, Table 1; p < 0.01). Further evaluation showed the relationship between miR-559 expression level and certain pathological characters of GC. It was discovered that lower miR-559 expression was correlated with histologic classification, cancer magnitude and lymph node metastasis (Table 1; p < 0.01). Nevertheless, miR-559 level was not related to age, venous invasion, gender, lymphatic invasion, T stage and TNM stage. Moreover, miR-559 expression was remarkably fewer in GC cell lines (AGS, MKN-45 and BGC-823) than in normal GES-1 cell ( Fig. 1b; p < 0.01). The findings indicated that miR-559 might be an effective marker and play as a cancer suppressor gene in human GC. Table 1 Clinical significance of miR-559 expression in gastric cancer (n = 70). MiR-559 restrains GC cell proliferation, induces cell cycle arrest and apoptosis To discover the potential function of miR-559 in GC, MKN-45 and BGC-823 cells were transfected with miR-559 vector, control vector, miR-559 inhibitor or negative control, respectively. qRT-PCR was performed to examine the miR-559 level after transfection. The results revealed that the miR-559 expression level was dramatically raised after transfection with miR-559 vector ( Fig. 2a; p < 0.01).
Whereas, there were no remarkable variations between cells transfected with anti-miR-559 and those with anti-miR-Control (Fig. 2b). MTT assay showed that miR-559 overexpression suppressed the Similar results were also discovered in the cell counting assay. MiR-559 overexpression inhibited MKN-45 and BGC-823 cell multiplication, but anti-miR-559 promoted cell multiplication (Fig. 2e, f; p < 0.01).
Since cell cycle is related to the modulation of cell multiplication, we determined the processe by using a flow cytometer. Our findings revealed that miR-559 overexpression gave rise to a prominent amassing of the G0/G1 phase cells and a decreasing of the S and G2/M phase cells in MKN-45 and BGC-823 cells ( Fig. 2g; p < 0.01); anti-miR-559 dramatically reduced the G0/G1 phase cells and enhanced the S and G2/M phase cells ( Fig. 2h; p < 0.01). Analysis of cell apoptosis verified that the percentage of early apoptosis and late apoptosis cells was markedly added when miR-559 was overexpressed, and significantly reduced when anti-miR-559 was transfected (Fig. 2i, j; p < 0.01).
Taken together, our results illuminated that miR-559 suppressed human GC cell growth, and caused G1-S cell cycle arrest and apoptosis.

MiR-559 inhibits GC cell growth and induces apoptosis via the AKT and Bcl-2/Bax signaling pathways through targeting TRIM14
MiR-559 vector observably decreased the mRNA expression of TRIM14 in MKN-45 and BGC-823 cells, while anti-miR-559 memorably promoted TRIM14 mRNA expression (Fig. 4a, b; p < 0.01). Similar results were also found in protein expression (Fig. 4c, d). For exploring the possible mechanisms of miR-559 in GC progression, we detected the related protein expressions in the AKT signaling pathway and Bcl-2/Bax expressions. The findings displayed that miR-559 overexpression decreased the protein expressions of p-AKT, CDK2, Cyclin D1 and Bcl-2 in MKN-45 and BGC-823 cells, and upregulated Bax protein level in GC cells (Fig. 4c). Nevertheless, anti-miR-561 promoted the protein expressions of p-AKT, CDK2, Cyclin D1 and Bcl-2, and suppressed Bax protein level (Fig. 4d). Furthermore, the total AKT protein expression remained unchanged. Our findings suggested that miR-559 could regulate human GC cell growth, cell cycle transition and apoptosis via modulating the AKT and Bcl-2/Bax signaling pathways. showed that the protein expression of TRIM14 dramatically reduced in the siRNA group. TRIM14 siRNA reduced p-AKT, CDK2, Cyclin D1 and Bcl-2 protein expressions, and increased Bax protein expression in MKN-45 and BGC-823 cells (Fig. 6f). The findings were similar to the results after miR-559 overexpression, implying a same effect of TRIM14 knockdown and miR-559 overexpression.

Discussion
Growing evidence showed that the disorder expression of miRNAs plays pivotal roles in the processes of various tumorigenesis and progression [22,23]. MiRNAs have been discovered to play either tumor suppressor genes or carcinogenic genes, according to the peculiar functions of the targeted gene.
Increased expression of oncogenic miRNAs results in decreased anti-oncogene translation, which suppresses to the development and progression of cancer. Semblable effects are caused by reduced expression of tumor suppressor miRNAs leads to increased oncogene expression [24]. GC is a highly malignant tumor with complex pathogenesis. GC development is a typical multiple factors and multistep process. More and more researchers reported that miRNAs participated in GC occurrence and development [25]. Abnormity of miR-559 has been reported for several different cancers. Even though recent evidences demonstrated the inhibitory effect of miR-559 on human cancers, such as breast cancer, papillary thyroid carcinoma and glioblastoma [16,17], the functions of miR-559 in GC and the underlying mechanisms are still unknown. In this study, it was found that the expression of miR-559 was remarkably downregulated in both GC tissues and cell lines. The clinicopathological significance of miR-559 expression was analyzed. Our results showed that low miR-559 expression levels were observably associated with histologic classification, cancer magnitude and lymph node metastasis in GC patients, indicating that miR-559 may act a crucial role in GC diagnosis. The experiments further illustrated that miR-559 overexpression significantly suppressed GC cell proliferation by inhibiting G1-S phase transition and accelerated cell apoptosis, the suppression of miR-559 facilitated cell growth and G1-S transition, and restrained apoptosis. Our findings suggest that miR-559 acts as an anti-oncogene in GC and has the potency for being a novel diagnostic marker and a therapeutic target molecule.
Moreover, the miR-559 target analysis identified TRIM14 as a direct target of miR-559. TRIM14 belongs to the TRIM protein family, which includes more than 70 members. Dysregulation of these proteins are linked to different biological processes, such as cell proliferation, differentiation, apoptosis, angiogenesis, migration and invasion [26][27][28]. Previous studies have reported that TRIM14 is dramatically increased in lung cancer cells and promotes the retinoic acid-inducible gene-I-like receptor-mediated innate immune response against viral infections [29]. Valentina et al. found that TRIM14 overexpression promoted the increasing of a few innate immunity-related genes that participated in the regulation of NF-κB and Wnt/β-catenin signaling pathways in human HEK293T cells [30]. Su et al. discovered that TRIM14 was increased in tongue squamous cell carcinoma (TSCC), and promoted the progression of TSCC by regulating the NF-κB signaling pathway [31]. Additionally, TRIM14 has been shown to promote cancer cell proliferation, chemoresistance, migration and invasion in breast cancer, colorectal cancer, osteosarcoma and gliomas [32][33][34][35]. Wang et al. reported that TRIM14 promote the migration and invasion of GC through regulating epithelialtomesenchymal transition [36]. However, the role of TRIM14 on GC cell proliferation and the underlying mechanisms remain unclear. In this study, we discovered that TRIM14 was increased in GC tissues, which showed an inverse correlation between its mRNA level and miR-559 expression in GC tissues. Our findings The AKT signaling pathway is one of the most common proliferation pathways in many cancers [37]. It is well known that dysregulation of the AKT signaling pathway is refered to tumorigenesis and progression, such as liver, lung, breast, thyroid, prostate, bladder, pancreatic, gastric, colorectal and cervical cancers [38,39]. It is reported that the various clinicopathological characteristics of cancers are closely related to the activation of AKT signaling pathway [40]. AKT can modulate the function of plentiful substrates associated with cell cycle progression through direct phosphorylation of target molecules or indirectly regulating protein expressions [38,41]. Previous studies show that the AKT downstream regulators, such as CDK2 and Cyclin D1, are momentous transcriptional factors in the G0/G1-S phase [42]. As we know, Cyclin A-CDK2 and Cyclin D-CDK4/6 protein kinase complexes are pivotal cell cycle regulators and may regulate cell cycle transition of the G1/G0-S phase [43]. It is reported that Cyclin D1 and CDK2 regulate cell proliferation and cell cycle in many human cancers [44,45]. Our results demonstrated that miR-559 overexpression and TRIM14 siRNA downregulated The tumor growth is refered to cell proliferation and apoptosis. Apoptosis is an important indicator for anticancer therapy. A dysregulation between Bcl-2 and Bax can cause disorder of apoptosis, which result in oncogenesis and cancer progression. Bcl-2, a anti-apoptotic member, play protectors of the outer membrane and preserve its integrity by inhibiting the release of cytochrome c. On the other hand, Bax, a pro-apoptotic member, causes the release of cytochrome c and lead to mitochondrial dysfunction [46]. Therefore, a pivotal determinant of the intrinsic apoptosis pathway is the balance between Bax and Bcl-2 protein expressions [47]. Our findings demonstrated that miR-559 overexpression or TRIM14 siRNA induced GC cell apoptosis by modulating Bax/Bcl-2 signaling pathway; anti-miR-559 and TRIM14 overexpression suppressed apoptosis through controlling Bax/Bcl-Bax/Bcl-2 signaling pathway via TRIM14.

Conclusions
In summary, our study demonstrates that miR-559 plays as an anti-oncogene in GC. We find that miR-559 is markedly downregulated in human GC tissues and and associated with the clinicopathologic features of GC sufferers. MiR-559 suppresses GC cell growth by restraining the AKT signaling pathway  showed the ratios of early and late apoptosis after miR-559 overexpression. j The data revealed the proportions of early apoptosis and late apoptosis after anti-miR-559 treatment.

Figure 3
MiR-559 directly targets the TRIM14 gene. a Bioinformatics predicted reciprocities of miR-559 and its binding sites in the 3′-UTR of TRIM14. b Luciferase activity was measured by using the dual-luciferase assay. c TRIM14 mRNA expression was detected in GC tissues. d TRIM14 protein levels were examined by Western blotting. e MiR-559 and TRIM14 expressions were negatively correlated. The 2−ΔΔCt values of miR-559 and TRIM14 were subjected to a Pearson correlation analysis (n = 70, r = -0.4710, p < 0.01). *p < 0.01.