miR-325-3p Reduces Proliferation and Promotes Apoptosis of Gastric Cancer Cells by Inhibiting Human Antigen R

Human antigen R (HuR), also known as ELAVL1, is a widely expressed RNA-binding protein (RBP) that has a significant impact on the development and advancement of tumors. Our previous study found that 5-fluorouracil (5-FU) may impede the proliferation and increase apoptosis in gastric cancer cells by reducing the nucleocytoplasmic shuttling of HuR. However, how posttranscriptional regulation influences HuR functions in gastric cancer remains to be elucidated. Here, we demonstrated that miR-325-3p has the potential to regulate the expression level of HuR by directly binding to its 3′UTR, which in turn led to a significant reduction in proliferation and an increase in apoptosis in gastric cancer cells. In addition, xenograft experiment showed that knockdown of HuR or overexpression of miR-325-3p group exhibited smaller tumor sizes after transplant of gastric cancer cells into zebrafish larvae. Thus, our findings offer new insights into the pathogenesis of gastric cancer and may potentially assist in identifying novel targets for drug therapy.


Introduction
Patients with gastric cancer have a high mortality rate and a poor prognosis [1,2].As of 2020, gastric cancer is the ffth most commonly diagnosed cancer and the fourth leading cause of cancer-related deaths [3].Te primary treatment for gastric cancer is surgery [4].Despite undergoing gastrectomy, patients diagnosed with gastric cancer have a 5-year survival rate of only 41.6% [5].To date, gastric cancer remains a signifcant threat to human life globally.Tus, comprehending the fundamental molecular mechanisms of gastric cancer development may ofer an extensive range for discovering new drugs.
HuR binds to mRNAs 3′UTR and is extensively involved in posttranscriptional gene regulation as an RNA-binding protein [6].Troughout the human body, HuR is ubiquitously expressed due to its membership of the embryonic lethal abnormal vision-like (ELAVL) family.HuR can regulate the expression of various protooncogenes, cytokines, and growth factors which have confrmed roles in the growth, invasion, and metastasis of multiple types of tumors including colorectal cancer [7], cervical cancer [8], gastric cancer [9][10][11], and other cancers [12][13][14].Inhibition of HuR expression has been shown to reduce tumor cell proliferation and increase cell apoptosis [10,15,16].Recently, HuR has emerged as a promising target for cancer therapy [12].Various pieces of evidence point out that HuR, modulating drugs with low and manageable side efects, will be a future direction for cancer therapeutics [17,18].
MicroRNAs, approximately 18-25 nucleotides in length, are a class of small noncoding RNAs that are widely expressed in vivo and involved in many life activities [19].Abnormal expression of miRNAs have been related to multiple diseases and may even serve as biomarkers for malignant tumors [20,21].For example, miR-22 has been reported to be involved in regulating HuR and participating in the development of colorectal cancer [7].miR-519 delayed cell proliferation by decreasing the level of RNA-binding protein HuR [8] and hampers the progression of gastric cancer by targeting HuR [11].Certain miRNAs, such as miR-34 analogs and anti-miRs targeted at miR-122, have been included in clinical trials as potential therapeutic targets [22].Meanwhile, the expression level of miR-325-3p is signifcantly reduced in gastric cancer [23].In addition, miR-325-3p also plays a role in regulating resistance to chemotherapy and immunotherapy [24][25][26].Tese fndings suggest a potential association between the expression of miR-325-3p and the development of gastric cancer.
Te aim of this study is to investigate the role of miR-325-3p in regulating post-HuR transcription in gastric cancer.Tis study hypothesizes that miR-325-3p regulates intracellular expression of HuR by binding to its 3′UTR, which afects the proliferation and apoptosis of gastric cancer cells.Te hypothesis was further validated by luciferase activity assay, TUNEL assay, cell Viability assay in vitro and xenograft model in zebrafsh.

Animals.
Te zebrafsh (Danio rerio) were obtained from the National Zebrafsh Resource Center of China (Wuhan, China) and raised according to the guidelines provided in Te Zebrafsh Book [27].For mating, two males were placed with one female in a tank separated by a bafe plate.Te larvae were reared at a temperature of 28.5 °C in an E3 medium and fed three times a day with Artemia nauplii.All animal experiments were carried out by the guidelines for animal care and approved by the Ethical Commission of Wenzhou Medical University.

Cell
Culture.SGC-7901 and HGC-27 cells were purchased from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China) and cultured in an RPMI-1640 growth medium containing 10% fetal bovine serum (FBS) in a 5% CO 2 humidifed incubator at 37 °C.

2
Canadian Journal of Gastroenterology and Hepatology 2.6.TUNEL Assay.Apoptotic cells were measured via TUNEL staining, using in situ cell death detection kit (Roche, Basel, Switzerland) as described by Lv et al. [29].SGC-7901 and HGC-27 cells were fxed at room temperature for 30 minutes using 4% paraformaldehyde (PFA) solution and then treated with permeation solution (0.1% Triton X-100) for 5 minutes at 4 °C.Following washing with phosphate-bufered saline (PBS), the cells were incubated with TUNEL reagent, which contained 10% terminal deoxynucleotidyl transferase and 2% fuorescent isothiocyanate-dUTP, for 1 hour at 37 °C.Subsequently, the cellular nucleus was detected by staining the cells with diamidino-2-phenylindole (DAPI) for 30 minutes at room temperature.Finally, the cells were sequestered using an antifuorescence quencher, and the number of apoptotic bodies and TUNEL-positive SGC-7901 and HGC-27 cells were determined with fuorescence microscope.2.8.Dual-Luciferase Reporter Assay.Two conserved binding sites were identifed in the 3′UTR of HuR using the Tar-getScan database (https://www.targetscan.org/mamm_31/).Te 3′untranslated region (UTR) of HuR was cloned into the pmirGLO luciferase reporter vector, containing the predicted binding sites.miR-325-3p mimics or miR-NC and HuR 3′UTR WT or HuR 3′UTR MUT vectors were cotransfected into SGC-7901 and HGC-27 cells.After 48 hours of transfection, the luciferase activity was determined using dual-luciferase reporter analysis system.

Tumor Xenograft Model in Zebrafsh Larvae.
SGC-7901 cells were transfected with the PEGFP-N1 plasmid and selected with G418 to establish gastric cancer cell lines that stably express green fuorescent proteins (GFP).SGC-7901 cells expressing GFP were transfected with siNC, siHuR, miR-NC, and miR-325-3p mimics or inhibitor.Transfected cells were then injected into the yolk sac of zebrafsh larvae at a 2-day postfertilization stage (200 cells/fsh) [30].Tumor growth was monitored 24 hours later via fuorescence microscope (EVOS FL Auto Cell Imaging System, Termo).

Statistical
Analysis.GraphPad Prism 9.3.1 statistical software was used for data analysis.Te mean ± S.E.M. (standard error of the mean) was presented for all data.Te normal distribution of all datasets was verifed using the Kolmogorov-Smirnov test.To determine the statistical signifcance, unpaired two-tailed t-tests or ANOVA tests followed by Dunnett's multiple comparisons test were used as appropriate.Signifcance was defned as * P < 0.05, * * P < 0.01, * * * P < 0.001, and * * * * P < 0.0001.

Knockdown of HuR Inhibited Proliferation and Promoted
Apoptosis in Gastric Cancer Cells.To investigate the efect of HuR on apoptosis and proliferation in gastric cancer cells, siRNA was transfected to knockdown HuR in SGC-7901 and HGC-27 cells.Te proportion of apoptotic cells was signifcantly higher in knockdown of HuR group (Figures 1(a) and 1(d)) than that of siNC group.Supression of HuR upregulated the protein levels of BAX and Caspase3 (CASP3) (Figures 1(c) and 1(f )), and mRNA expressions of BAX, caspase3, caspase8, and caspase9 in gastric cancer cells (Figures 1(g) and 1(h)).In addition, cell growth rate of the siHuR group was remarkably lower than that of the siNC group (Figures 1(b) and 1(e)).In summary, knockdown of HuR blocks cell proliferation and promotes apoptosis in gastric cancer cells.Canadian Journal of Gastroenterology and Hepatology reduced HuR synthesis, leading to decreased proliferation and increased apoptosis of gastric cancer cells.

miR-325-3p Slowed In Vivo Tumour Growth of SGC-7901
Cells.To investigate the efects of miR-325-3p on gastric tumor growth, the SGC-7901 cells that were genetically modifed to express green fuorescent protein (SGC-7901-GFP) via plasmid transfection and cell monoclonal screening were used.After transfection with either siHuR1 or miR-325-3p mimics, the corresponding SGC-7901-GFP cells were injected into larval zebrafsh using microinjection (Figure 4(a)).Te results indicated that the fuorescence expression area was lower in the HuR knockdown group and the miR-325-3p mimics group than that in the respective control group.Furthermore, the fuorescence expression area was signifcantly higher in the miR-325-3p inhibitor group than in the control group (Figures 4(b) and 4(c)).In summary, miR-325-3p may suppress the growth of gastric cancer cells by blockng the expression of HuR in zebrafsh.

Discussion
Along with the growing number of studies examining the modulation of HuR function as a therapeutic target for anticancer drugs, the role of HuR in tumorigenesis and development has garnered increased recognition.Additionally, several studies have suggested that inhibiting HuR may prove to be a promising target for tumor therapy [31].In this study, two miR-325-3p binding sites was identifed at the 3′UTR of HuR through bioinformatics analysis.Furthermore, knockdown of HuR or overexpression of miR-325-3p led to increased apoptosis and decreased proliferation in both SGC-7901 and HGC-27 cells, as evidenced by CCK-8 and TUNEL experiments.Results of the TUNEL apoptosis detection experiments were consistent with the expression of apoptosis-related genes and proteins.Dual-Luciferase reporter assay additionally demonstrated that miR-325-3p could regulate its expression by binding to the 3′UTR of HuR.Finally, the efects of miR-325-3p on tumor growth were confrmed by in vivo zebrafsh experiments.Te results demonstrate that miR-325-3p regulates HuR expression to infuence the proliferation of gastric cancer cells.Tis discovery ofers a new strategy for developing HuRtargeting drugs for future cancer treatment.. HuR expression afects multiple tumor-related phenotypes [31].Several experiments have demonstrated increased expression of HuR in various tumor cells, with limited exploration of HuR's role in gastric cancer [17].In this study, siRNA was utilized to downregulate the expression of HuR in SGC-7901 and HGC-27 cells, resulting in decreased proliferation and increased apoptosis.Tis outcome is consistent with previous reports, providing additional evidence for the critical role played by HuR in tumor development.Importantly, it has been established that HuR also plays a signifcant role in preserving gastric cancer cells' division and proliferation.
Te elevated expression of HuR has a signifcant impact on the prognosis of diverse diseases.Currently, three primary techniques exist to suppress HuR: (1) inhibiting the expression of HuR by miRNA or small molecule RNA [32,33], (2) inhibiting the shuttling of HuR out of the nucleus by modifed molecules [34], and (3) causing the loss of its function by using drugs that competitively bind to the specifc locus of HuR [35].Each of these methods has its corresponding advantages and disadvantages.Te expression of miR-325-3p is decreased in gastric cancer patients, indicating it may regulate cisplatin resistance in this type of cancer [23].In vitro experimental results and sequence comparison analysis revealed that miR-325-3p may reduce the expression of HuR by binding to the 3′UTR of HuR, which is consistent with classic miRNA regulation.
Subsequent studies showed that a elevated expression of miR-325-3p mimics could inhibit the proliferation of gastric cancer cells.Te inhibition on cell proliferation by overexpression of miR-325-3p mimics appeared to be gentler than the direct knockdown of HuR.Similarly, for apoptosis induction, overexpression of miR-325-3p mimics showed similar yet distinct results compared to HuR knockdown.TUNEL experiment demonstrated that higher miR-325-3p expression could efectively enhance gastric cancer cell apoptosis compared to the control group.Tere have been previous clinical trials that evaluated miRNAs could be used as pharmaceutical agents [22].Tis study provides a theoretical basis for potentially utilizing miR-325-3p as a therapeutic intervention for gastric cancer.
Zebrafsh (Danio rerio) share a remarkable levle of physiological and genetic similarity with mammals.Te zebrafsh embryo is a promising model for xenograft tumors because of its transparency, which makes it convenient to observe and study tumor development and growth in vivo [36].Te experiment revealed that inhibiting miR-325-3p led to a signifcant increase in tumor volume in zebrafsh compared to the control group.Tis fnding might indicate that in animals, gastric cancer growth is more sensitive to miR-325-3p suppression.
Regarding miR-325-3p as a potential drug target, further investigation is required into its mechanistic and functional characterization.Tis study is limited by a low percentage of HuR knockdown by miR-325-3p, making it impossible to exclude the possibility that these two factors participate synergistically in the development of gastric cancer.

6
Canadian Journal of Gastroenterology and Hepatology Canadian Journal of Gastroenterology and Hepatology

Conclusions
miR-325-3p inhibited the proliferation and promoted apoptosis of gastric cancer cells by binding to HuR 3'UTR and suppress its expression.Tese results ofer valuable insights into the pathogenesis of gastric cancer and establish new potential targets for therapeutic intervention.
Levels of HuR.Bioimformatic analysis uncovered that HuR mRNA 3'UTR region contained 2 potential miR-325-3p binding sites, which is highly conserved throughout the species.To further validate the interplay between HuR and miR-325-3p, both the original (HuR 3'UTR WT) and mutated (HuR 3'UTR MUT) sequences were cloned into the dual luciferase reporter (Figures2(a) and 2(d)).Overexpression of miR-325-3p mimics led to a signifcant decrease in HuR mRNA levels in SGC-7901 cells (Figure 2(b)).Consistently, miR-325-3p mimics inhibited the relative luciferase activity of the HuR 3′UTR WT plasmid, whereas the corresponding luciferase activity of the HuR 3′UTR MUT plasmid was not afected (Figure 2(c)).

Figure 2 :
Figure 2: Overexpression of miR-325-3p reduced expression levels of HuR in SGC-7901 and HGC-27 cells.(a) Schematic diagram of potential binding sites and mutation sequence in HuR 3′UTR region.(b) Detecting relative expression levels of HuR mRNA by qRT-PCR.(c) Analyzing relative luciferase activity of each group by dual-luciferase reporter assays.(d) Te schematic of two luciferase reporter plasmids ( * P < 0.05 and * * P < 0.01, compared to siNC group).

Figure 4 :
Figure 4: Overexpression of miR-325-3p lead to slowed growth of gastric tumors in zebrafsh.(a) Schematic diagram of the fabrication of the SGC-7901-GFP monoclonal cell line and the microinjection of cells.(b) Te proliferation of siRNA knockdown SGC-7901-GFP cells in zebrafsh and the statistics of the fuorescence expression area (both groups n � 12).(c) After overexpression or inhibition of miR-325-3p, the proliferation of SGC-7901-GFP cells in zebrafsh and the statistics of the fuorescence area were compared with those in miR-NC group (n � 13), mimics group (n � 14), and inhibitor group (n � 9).