Hsa-let-7d-5p Promotes Gastric Cancer Progression by Targeting PRDM5

Gastric cancer (GC) is a common malignant tumor in the digestive system and a significant health burden worldwide. In this study, we found that hsa-let-7d-5p was upregulated in GC cells, promoted GC cell proliferation, migration, and invasion, and reduced apoptosis. Moreover, we found that the expression of PRDM5 (PR domain protein 5) was downregulated in GC cells and upregulated in GC cells treated with hsa-let-7d-5p inhibitor. Further investigation showed that hsa-let-7d-5p was the target of PRDM5, and the functions of hsa-let-7d-5p on GC progression were rescued by PRDM5 overexpression in GC cells. Collectively, our findings suggested that hsa-let-7d-5p promoted the development of GC by targeting PRDM5, indicating that hsa-let-7d-5p could be a promising therapeutic molecule for the treatment of gastric cancer.


Introduction
Gastric cancer (GC) is a highly heterogeneous disease [1]. GC is characterized by rapid progression, poor prognosis, and low survival rate. Currently, the treatment of GC is unsatisfactory because the molecular mechanism of GC progression is not clear [2,3]. Hence, it is urgent to investigate the regulating molecules in GC progression, which is beneficial to discover novel molecular targets for GC treatment [4][5][6].
MicroRNA (miRNA) was discovered in 1993 and subsequently researched in RNA biology [7]. MiRNAs are small noncoding RNAs that exert major roles in the posttranscriptional regulation of gene expression [8]. It was reported that miRNAs were the potent regulators of many cellular activities, including cell growth, proliferation, and apoptosis [7]. Moreover, they are related to the progression of multiple diseases, including neurodegenerative disorders [9], breast cancer [10], and GC [3]. Let-7 miRNA, discovered in Caenorhabditis elegans, is highly conserved in human tissues [11]. Let-7d-5p is one of the members of the 12 human let-7 family. It was known that the expression of let-7 was related to the development of aggressive cancers [12][13][14]. According to reports, the expression of let-7d is dysregulated in many types of cancer, such as HNSCC, breast cancer, renal cancer, retinoblastoma, pancreatic cancer, and prostate cancer. Let-7d plays a crucial role in cancer initiation, progression, and metastasis, where it functions as a tumor suppressor miRNA by regulating the expression of multiple oncogenes [15]. e miRNA expression of hsa-let-7d-5p was remarkedly increased in the exosomes of ovarian cancer patients compared to controls [16]. Let-7d-5p regulates the p53 signaling pathway through HMGA1 to inhibit ovarian cancer cell apoptosis [17]. Let-7d-5p can adjust HMGA1/Wnt/ β-Catenin signaling to promote the growth and metastasis of colorectal cancer [18]. However, the molecular mechanism of let-7d-5p in GC remains unclear.
It was reported that PRDM (PR (PRDI-BF1 and RIZ) domain proteins) modulated cellular processes, such as differentiation, growth, and apoptosis [19], and dysfunction of PRDM was associated with genetic and epigenetic modifications in cancers [20]. PRDM5 (PR domain protein 5), a member of the PRDM family, acted as a new transcription repressor and a tumor suppressor in various human cancers [21,22]. However, the regulatory mechanism of PRDM5 in GC needs to be investigated.
In this study, hsa-let-7d-5p was found to be significantly increased in human GC cell lines. In addition, knockdown of hsa-let-7d-5p promoted GC cell apoptosis and inhibited GC cell viability, migration, and invasion. e potential mechanism is that hsa-let-7d-5p promotes GC progression by targeting PRDM5. Our study will provide an experimental basis for the potential molecular mechanism of GC development and offer a novel therapeutic target against GC.

Colony Formation
Assay. AGS and SGC-7901 cells were seeded into 6-well plates (1 × 10 3 /well). After 2 weeks, all wells are washed with PBS three times, and the cell colonies were stained with 0.1% crystal violet at 37°C for 5 min. en, the colonies were imaged and counted.

Cell Apoptosis Analysis.
All cells were seeded in a 6-well plate (2 × 10 5 /well). After 24 h treatment, the cell apoptosis was analyzed by Annexin V-FITC apoptosis detection kit (Sigma-Aldrich, USA). AGS and SGC-7901 cells were harvested by centrifuging at 1000 rpm/min for 5 min. en, cells (1 × 10 5 ) were resuspended with 100 μl buffer containing 5 μl Annexin V-FITC and 5 μl propidium (PI) at 4°C for 15 min in darkness. At last, cells were measured by flow cytometry (BD, Franklin Lake, NJ, USA). All the data were analyzed by FlowJo.

Luciferase Reporter Assay.
We implemented luciferase experiments in AGS and SGC-7901 cells to confirm whether PRDM5 was a target of has-let-7d-5p. WT or mutated (MUT) PRDM5 3′-UTR sequences were chemically synthesized (Sangon, Shanghai, China) according to the sequence of hsa-let-7d-5p. PRDM5-WT or PRDM5-Mut was, respectively, cotransfected with has-let-7d-5p mimic or inhibitor into AGS and SGC-7901 cells. Finally, luciferase activity was detected using the luciferase reporter assay system.

Transwell Invasion
Assay. AGS and SGC-7901 cells were plated onto 24-well plates (1 × 10 4 /well) with the upper chamber with 100 μl of Matrigel. e F12K medium or RPMI-1640 medium supplemented with 1% FBS was placed into the upper chamber. e F12K medium or RPMI-1640 medium with 10% FBS was placed into the lower chamber. After 24 h of incubation at 37°C, cells that invaded through the membrane were fixed with 5% glutaraldehyde at 4°C and stained with 0.1% crystal violet for 30 min. e cells were observed under a microscope (Dianying, Shanghai, China).

Cell Scratch
Test. AGS and SGC-7901 cells were incubated in 6-well plates until 100% confluence. A sterile 10 μL pipette tip was used to make a single scratch. e culture medium was replaced by an FBS-free F12K medium or RPMI-1640. Images of the scratches were captured at 0 and 48 h. Olympus CellSens Dimension software was used to measure the width of the scratch.

Let-7d-5p Is Upregulated in GC Cells, and Knockdown of Let-7d-5p Inhibits Cell Proliferation and Promotes Cell
Apoptosis. To explore the biological function of let-7d-5p in human GC cells, we detected its expression in related cell lines. We found the upregulated expression of let-7d-5p in AGS and SGC-7901 cells compared with GES-1 by qRT-PCR analysis (Figure 1(a)). e knockdown efficiency of let-7d-5p in AGS and SGC-7901 cells was validated by qRT-PCR analysis. e results showed that let-7d-5p was downregulated by let-7d-5p inhibitor in GC cells (Figure 1(b)). Functionally, CCK-8 and colony formation assays showed that knockdown of let-7d-5p significantly inhibited the cell viability of GC cells (Figures 1(c) and 1(d)). Next, the flow cytometry assay revealed that knockdown of let-7d-5p promoted the apoptosis of AGS and SGC-7901 cells (Figure 1(e)). At the molecular level, the knockdown of let-7d-5p resulted in the downregulated expression of the antiapoptotic protein (Bcl-2). It elevated the expression levels of proapoptosis proteins (Bax, cleaved caspase-3, and cleaved caspase-9) in AGS and SGC-7901 cells (Figure 1(f )).
ese results indicated that knockdown of let-7d-5p inhibited cell proliferation and promoted the apoptosis of GC cells.

Low Expression of Let-7d-5p Inhibits the Migration and
Invasion of GC Cells. Transwell and scratch assays displayed that the migration and invasion abilities of AGS and SGC-7901 cells were remarkably restrained in the cells transfected with let-7d-5p inhibitor (Figures 2(a) and 2(b)). Also, the protein expression levels of matrix metalloproteinase-2 (MMP-2) and MMP-9, which are migration and invasionrelated proteins, were downregulated by let-7d-5p inhibitor in AGS and SGC-7901 cells (Figure 2(c)). ese results illustrated that the let-7d-5p inhibitor suppressed migration and invasion of GC cells.

PRDM5 Is a Target of Let
en, we explored the molecular mechanism of let-7d-5p in GC. Firstly, we found that the levels of PRDM5 were downregulated in AGS and SGC-7901 cells by qRT-PCR assay (Figure 3(a)). us, we used StarBase analysis to predict the potential interaction site between PRDM5 and let-7d-5p and mutated all binding sites (Figure 3(b)). To evaluate the interactions between PRDM5 and let-7d-5p, a luciferase reporter assay was performed and it revealed a significant decrease in luciferase activity in cells cotransfected with PRDM5-WT and let-7d-5p mimic (Figure 3(c)). In addition, the levels of PRDM5 were increased in AGS and SGC-7901 cells transfected with let-7d-5p inhibitor by qRT-PCR and Western blot (Figures 3(d) and 3(e)). ese results indicated that let-7d-5p functioned by targeting PRDM5 in AGS and SGC-7901 cells.

Let-7d-5p Promotes Cell Proliferation and Inhibits Apoptosis through Regulating PRDM5 in GC.
e CCK-8 assay and colony formation assay illustrated that si-PRDM5 reversed the inhibitory effect of the let-7d-5p inhibitor on cell viability and proliferation of AGS and SGC-7901 cells (Figures 4(a) and 4(b)). en, apoptosis assay was performed by flow cytometry and showed that si-PRDM5 rescued the promotive effects of the let-7d-5p inhibitor on apoptosis in AGS and SGC-7901 cells (Figure 4(c)). Moreover, Western blot analysis revealed that si-PRDM5 rescued the downregulated antiapoptotic protein (Bcl-2) and suppressed the elevation of proapoptosis proteins (Bax, cleaved caspase-3, and cleaved caspase-9) caused by let-7d-5p inhibitor in AGS and SGC-7901 cells (Figure 4(d)).
ese results demonstrated that si-PRDM5 could reverse the function of let-7d-5p inhibitor on AGS and SGC-7901 cells, suggesting that let-7d-5p promotes GC cell proliferation and inhibits cell apoptosis through PRDM5.

Let-7d-5p Promotes Migration and Invasion of GC Cells
through Regulating PRDM5. Scratch and transwell chamber assays revealed that si-PRDM5 rescued repressive effects of the let-7d-5p inhibitor on the migration and invasion abilities of AGS and SGC-7901 cells (Figures 5(a) and 5(b)).
ese results demonstrated that si-PRDM5 could reverse the suppression of let-7d-5p inhibitor on AGS and SGC-7901 cells, suggesting that let-7d-5p promotes GC cell migration and invasion through PRDM5.

Discussion
GC represents the fifth cause of cancer-related deaths worldwide [23]. MiRNAs have been found to participate in the pathogenesis of GC and could be promising therapeutic targets in cancers [24,25]. erefore, it is worth exploring the underlying regulation of miRNAs in the development of diseases and using them as therapeutic targets in clinical applications. Noncoding RNAs (ncRNAs) are associated with many human diseases [26]. MiRNAs regulate target gene expression by binding to the 3′ UTR region of the target gene and are involved in the pathogenic processes of various human diseases, including GC [27,28]. e analysis of miRNA and gene expression profiles showed that the let-7 miRNA family plays a central role in the regulation of tumorigenesis [29,30]. And the let-7 family members are the hallmarks of several cancers [31,32]. However, the downstream regulatory molecules of let-7d-5p in GC progression are rarely reported. In this study, let-7d-5p was upregulated in GC cells. Low expression of let-7d-5p suppressed cell proliferation, migration, and invasion and promoted cell apoptosis of GC cells.

Journal of Oncology
It has been shown that PRDM5 is silenced by promoter methylation in multiple cancers [36]. Few studies showed that PRDM5 was targeted by noncoding RNAs. It has been demonstrated that PRDM5 was targeted by miR-130b-5p, and BDNF-AS/miR-130b-5p/PRDM5 axis worked in the development of acute spinal cord injury [37]. In our study, we found that PRDM5 expression was negatively regulated by let-7d-5p in GC cells. Additionally, we found that the knockdown of PRDM5 reversed the suppression of let-7d-5p inhibitor in cell proliferation, migration, and invasion and the promotion of cell apoptosis in GC cells. ese results suggested that there was an interaction between let-7d-5p and PRDM5 in GC cells. We performed rescue experiments to explore the regulation mechanism of cell apoptosis, proliferation, migration, and invasion between let-7d-5p and PRDM5. It was confirmed that si-PRDM5 partially antagonized the let-7d-5p inhibitor-mediated effects on cell apoptosis, proliferation, migration, and invasion in GC cells.

Conclusion
In summary, this study demonstrated that knockdown of let-7d-5p could promote cell apoptosis and inhibited cell invasion and migration in GC cells. ere was a regulatory interaction between let-7d-5p and PRDM5 in GC cells. e complex of let-7d-5p and PRDM5 could become a promising therapeutic target for the treatment of GC.

Data Availability
All data generated or analyzed during this study are included in this published article.

Ethical Approval
Not applicable. Our study did not require an ethical board approval because it did not contain human or animal trials.  Values are mean ± SD, ## P < 0.01 vs. let-7d-5p inhibitor, * * P < 0.01 vs. NC inhibitor + si-NC, n � 3 per group.