MiRNA-219a-1-3p inhibits the malignant progression of gastric cancer and is regulated by DNA methylation

Objective: Worldwide, gastric cancer (GC) is one of primary reasons for cancer-related deaths. However, the pathogenic mechanism underlying GC remains to be fully understood. MicroRNAs are momentous regulators of diverse biological progression in cancer. Even though the ability of miR-219a-1-3p to inhibit malignant progression in pancreatic cancer have been previously reported, its role in GC remains to be elucidated. Methods: Quantitative real-time PCR (qRT-PCR) was performed to measure miR-219a-1-3p expression levels in collected GC samples (n=98) and paired nearby non-tumor tissues. Cell proliferation, migration, and invasion assays were then conducted to explain the biological in ﬂ uences of miR-219a-1-3p in vitro . In vivo e ﬀ ects were con ﬁ rmed by subcutaneously injecting miR-219a-1-3p overexpressing MGC-803 cells into nude mice. Methylation-speci ﬁ c PCR was employed to evaluate the CpG island upstream methylation condition of miR-219a-1-3p in collected clinical tissues (n=22), GC cell lines and GES-1 cells. GC cells were supplemented with 5-aza-2 ′ -deoxycytidine to identify the miR-219a-1-3p expression changes using qRT-PCR. Results: The miR-219a-1-3p expression was obviously suppressed in GC tissues relation to nearby non-tumor tissues, along with in GC cell lines in comparison to GES-1. Moreover, in vivo and in vitro functional evaluations indicated the function of miR-219a-1-3p in inhibiting the malignant characteristics of GC cells. Mechanistically, MiR-219a-1-3p expression was partly regulated utilizing DNA hyper-methylation in GCs. In addition, overexpression of miR-219a-1-3p inhibited PI3K/AKT signaling. Conclusions: MiR-219a-1-3p might function as a tumor suppressor in GC, and our investigation creates a foundation to diagnose of GC.


Introduction
Human health is severely threatened by gastric cancer (GC), a malignancy that arises from the stomach mucosal epithelium.In 2021, the latest statistics indicated that GC has no less than 1 million new cases and 768 thousand deaths, ranking fifth in incidence rate and third in death rate due to malignant tumors [1] Early stage of GC is often without symptoms or manifests with no evident symptoms, resulting in delayed identification and lost chances for radical operation.The individuals with survival rate of five years with early GC was 90 %, but it was less than 30 % for those with progressed GC [2].Although recent breakthroughs in treatment strategies including surgery coupled with chemotherapy and radiation, have improved the prognosis for those who have advanced GC, the prognosis is still bad [3].Hence, it is crucial to discover an efficient, innovative, and noninvasive diagnostic marker for earlier GC screening.
More recently, the possible importance of circulating microRNAs (miRNAs) as molecular markers of many cancers, including GC, has been gradually explored [4,5].microRNAs are single-chain, non-coding, small-molecule RNAs with an average length of 22 nucleotides [6].Emerging data demonstrates that cancer cells express miRNAs differently than healthy tissues and cells [7,8].Numerous investigations have shown that microRNAs were crucial regulators of several biological progressions in cancer, such as cell growth, migration, and invasion [8,9].Moreover, miRNAs might flow in the blood with a stable extracellular type, and the identification of miRNAs that circulate might be utilized to determine the tumor state [10].Thus, circulating miRNAs might be used as blood-based biomarkers for cancer detection in the early stages.
Therefore, in this study, we tested the biology impacts of miR-219a-1-3p in vitro and in vivo.Moreover, both the reason affecting the miR-219a-1-3p expression and the influence of its were discussed.
All clinical tissues were collected from the First People's Hospital of Chengdu, China, and informed consent was obtained from all patients.

qRT-PCR reaction
Regarding the recommendations of manufacturer, total RNA was sorted from all cell lines and clinical tissues employing TRIzol Reagent (Invitrogen, Rockford, IL, USA) and opposite transcription was conducted utilizing the Hifair ® III 1st Strand cDNA Synthesis Kit (gDNA digester plus) (Yesen, Shanghai, China).With the 2× SYBR Green qPCR Master Mix (TaKaRa, Tokyo, Japan) and CFX Manager v2.1, RT-qPCR was applied Bio-Rad (Hercules, CA, USA).The relative target genes expression was assessed utilizing the 2 −ΔΔCt , and all qRT-PCR primers are given in Supplemental Table S1 (Table S1).

Cell proliferation assay
GC cells were treated with control-mimic or miR-219a-1-3p mimics, and properly transfected cells were coated at a concentration of 5,000 cells for every well in 96-well plates.Employing Cell Counting Kit-8 (ABP Biosciences, Rockville, MD, USA), cell viability was detected at 0, one, two, three, and four days, and 10 L cck8 was supplemented to every well and preserved at 37 °C for 1 h.Using a microplate reader (Thermo scientific, Rockford, IL, USA) with the absorbance set at 450 nm, the optical density was measured.

Cell migration assay
GC cells were cultivated in 6-well plastic plates to up to 95 % confluence and then supplemented with control or miR-219-2-3p mimics.Utilizing a 200 µL pipe tip, linear scratch wounds were generated on syncretic cell monolayers 24 h following transfection.To prevent effect of cell growth on the experimental outcomes, serum-free media was employed to sustain the cells.At 0, 24, 36, and 48 h, the migrating cells and healing of wound were observed and photographs were captured (50×; Olympus, Tokyo, Japan).

Cell invasion assays
Roughly 4×10 5 cells in media free from serum were planted into transwell migration chambers (Millipore, Switzerland) that were covered with Matrigel (Sigma-Aldrich, St. Louis, MO, USA) in the top one 24 h following transfection.Typically, 10 % FBS-containing media was administered to the bottom chamber.Following one day, non-invading cells were excluded employing cotton wool, and invasive cells were subjected to crystal violet stain (Sigma Diagnostics, St. Louis, MO, USA) and quantified with an Olympus microscope (200×; Olympus, Tokyo, Japan).

Flow cytometry (FCM)
Before examining the cell cycle, the indicated cells treated with the control mimic or miR-219a-1-3p mimic were preserved overnight with 70 % alcohol.The cell cycle was then studied employing FCM after the cells were treated with Cell Cycle Kit stain (Yesen, Shanghai, China) in agreement with the recommendations of the manufacturer.

Subcutaneous transplanted tumor model in nude mice
For the xenograft trials, ten immunocompromised male nude rats aged 4 weeks were obtained from Yuanmin Biological Company (Shanghai, China).Single-cell suspensions of MGC-803 cells treated with the control mimic or miR-219a-1-3p mimic were obtained by digesting the cells.Each rat was administered subcutaneously with 5 × 10 6 cells in 100 µL of PBS.After measuring cancer growth every three days for 31 days, beginning seven days following injection, all rats were euthanized.The cancer volume (mm 3 ) was computed utilizing formula volume=length × width 2 × π/6.

The model of the liver metastasis of gastric cancer cells in nude mice
To generate single-cell suspensions, MGC-803 cells treated with the control mimic or the miR-219a-1-3p mimic were digested.Each mouse received an intravenous tail injection of 100 µL phosphate-buffered saline containing 1 × 10 7 cells.After 8 weeks, all of the mice were euthanized, and the amount of liver metastasis of cancer cells was counted.

Statistical analysis
The data are illustrated as mean ± standard deviation (SD).All data were examined for normality and variance equivalency to detect the most suitable statistical test.Employing a one-way analysis of variance, variations between different groups that were statistically significant were examined (ANOVA).The two cohorts were examined utilizing Student's t-test with two tails.p<0.05 was judged as a statistically significant.All quantitative tests were conducted in duplicate and were performed three times.

MiR-219a-1-3p expression was downregulated in a GC tissues
To investigate the difference of miR-219a-1-3p expression in collected clinical GC samples and paracancerous tissues, the miR-219a-1-3p expression levels in 98 clinical GC specimens and corresponding nearby non-tumor tissues were distinguished employing qRT-PCR.We discovered that miR-219a-1-3p expression was decreased in 54 out of 98 (55.10 %) GC tissues compared to that in paracancerous samples (p<0.05; Figure 1A, B).Furthermore, lower miR-219a-1-3p expression in GC tissues was positively linked with metastasis of lymph node, distant metastasis, and higher medical phase (refer to TNM stage) (p<0.05).Whereas, the miR-219a-1-3p expression has no significance in T stage (p=0.717)(Table 1).In addition, paired GC specimens and neighboring non-tumor samples were evaluated by H&E staining, which showed that peri-tumor tissues had an obvious gastric mucosal structure compared to GC tissues (Figure 1C).Collectively, this data indicated that the miR-219a-1-3p expression was reduced in GC tissues compared to that in paracancerous tissues, and the decreased miR-219a-1-3p level was linked to unfavorable prognosis.

Overexpression of miR-219a-1-3p inhibited the cell proliferation, migration, and invasion of GC cells in vitro
To discover miR-219a-1-3p actions in GC cells, we initial measured the miR-219a-1-3p expression in five cell lines of GC (HGC-27, MGC-803, SGC7901, K562 and MKN-45) and gastric mucosa GES-1 cells with qRT-PCR.The outcomes revealed that the miR-219a-1-3p expression was substantially downregulated in all five GC cell lines contrasted with that in GES-1 cells (p<0.01)(Figure 2A).The HGC-27 and MGC-803 cell lines were randomly chosen for further experiments.Next, we engineered a miRNA mimic (miR-219a-1-3p) to overexpress miR-219a-1-3p in the two chosen GC cell lines.The miR-219a-1-3p expression levels were confirmed in HGC-27 and MGC-803 cells by qRT-PCR (p<0.01; Figure 2B).The CCK8 test revealed that the miR-219a-1-3p overexpression observably suppressed cell growth in both HGC-27 and MGC-803 cells in comparison to that in the untreated cells and the control mimic cells (HGC-27 at 72, 96 h and MGC-803 cells at 48, 72, 96 h, p<0.05; Figure 2C, D).Moreover, the wound healing evaluation indicated that miR-219a-1-3p overexpression diminished the cell migration ability in comparison to that in the untreated cells and the control mimic cells (p<0.05; Figure 2E-H).Additionally, the transwell tests revealed that the miR-219a-1-3p overexpression noticeably weakened the cell invasion ability in vitro in comparison to that in the untreated cells and the control mimic cells (p<0.05) (Figure 3).These outcomes illustrated that the overexpression of miR-219a-1-3p suppressed the aggressiveness of GC cells in vitro.

Overexpression of miR-219a-1-3p inhibited cell growth and liver metastasis in vivo
To additional validate the impact of miR-219a-1-3p overexpression on cell growth in vivo, MGC-803 cells infected with the miR-219a-1-3p mimic were supplemented subcutaneously into athymic nude mice.After one week, the size of tumors was evaluated once every 3 days.We observed that the freshly retrieved tumor samples from the miR-219a-1-3p overexpression group inhibited cell growth in vivo compared to those of the control mimic cohort (p<0.05; Figure 4A, B).The retrieved subcutaneous tumors did not show any obvious histological changes in the miR-219a-1-3p overexpressed and control mimic groups by H&E.IHC staining of the retrieved cancer samples showed that Ki-67 was reduced in the miR-219a-1-3p overexpressed cohort in comparing with that of the control mimic group (p<0.05; Figure 4C).Furthermore, we recognized that the number of liver metastasis nodes was inhibited in the miR-219a-1-3p overexpressed group in comparison to that in the control mimic group (Figure 4D).In summary, our findings revealed that in vivo miR-219a-1-3p overexpression inhibited cell growth and liver metastasis.

The expression of miR-219a-1-3p was partly regulated by DNA methylation
To elucidate the reasons for the downregulated expression of miR-219a-1-3p in GCs, we first conducted bioinformatics analyses, and the CpG island of the miR-219a-1-3p upstream region was predicted using Meth Primer software (Figure 5A).The miR-219a-1-3p methylation status of 22 GC samples and nearby healthy tissues was evaluated employing MSP.Compared with neighboring normal tissues, upstream region hypermethylation was found in seven out of 22 (31.82%) GC tissues (Figure 5B).Additionally, the methylation levels of miR-219a-1-3p in GC cells MGC-803 and HGC-27 were also evaluated using MSP, and the outcomes illustrated that the methylation levels of miR-219a-1-3p in these cells were higher than those in GES-1 cells (Figure 5C).To further confirm that DNA methylation was among the reasons of miR-219a-1-3p suppression, HGC-27 and MGC-803 cells were supplemented with 5-aza-2′-deoxycytidine (5′ AZa-CDR, a methyltransferase inhibitor).The current outcomes indicated that the miR-219a-1-3p expression was markedly raised in HGC-27 and MGC-803 cells following supplementation with 5′ AZa-CDR (p<0.05; Figure 5D).Collectively, the present outcomes indicated that miR-219a-1-3p was inhibited in GC and that this is partly due to upstream region hypermethylation.

Overexpression of miR-219a-1-3p inhibits the proliferation of GC cells in vitro but has no connection with cell cycle arrest
Cell cycle arrest is one of the reasons for the suppression of cancer cell development [30].To define whether miR-219a-1-3p overexpression inhibits the malignant progression of GC cells owing to cell cycle arrest, we additional investigated the influence of miR-219a-1-3p on the cell cycle.FCM investigation revealed that miR-219a-1-3p overexpression did not considerably alter the cell cycle in GC cells (Figure S1).This result suggested that the overexpression of miR-219a-1-3p, which inhibited cell overgrowth in vitro, and was not associated with cell cycle arrest.

Inhibition of the malignant progression of GC as a result of miR-219a-1-3p overexpression was linked with the PI3K/AKT pathway
For more understand the pathway of miR-219a-1-3p, we analyzed the potential target genes of miR-219a-1-3p utilizing the online prediction software Targetscan7.2(Table S2), and miRDB (Table S3).The overlap after crosslinking contained 262 genes (Figure 6A).KEGG pathway analysis of crosslinking genes was performed using DAVID, and the results predicted pathways that might be related to the miR-219a-1-3p mechanism (Figure 6B).In addition, we selected the predicted downstream PI3K/AKT pathway for preliminary  verification.The expression of p-PI3K and p-AKT were downregulated in the miR-219a-1-3p overexpression group by western blotting (Figure 6C).Altogether, these outcomes indicated that miR-219a-1-3p overexpression of reduced the malignant progression of GC and this effect was related to the PI3K/AKT pathway.
inhibit cell growth and relocation in pancreatic cancer [26].Furthermore, a new investigation indicated that miR-219a-1 could noticeably suppress the overgrowth and invasion of colon cancer cells [25].In brief, our outcomes reveal that miR-219a-1-3p could as a tumor suppressor in GC.
Some miRNAs are normally expressed in the stomach.However, aberrant levels of these miRNAs can promote the growth and progression of GC [8,31].Epigenetic modulation of gene expression is before, during, and after transcription, and many studies have suggested that abnormal DNA methylation was a common phenomenon in human tumors [32,33].As epigenetic modulators, miRNAs complicate epigenetic regulation [34,35].A previous study reported that hypermethylated miR-219a-1 may be involved in glioblastoma multiforme pathogenesis [36].Strikingly, epigenetic alterations were identified in both early and advanced stages of GC [37].A previous finding discovered that miR-219a-2-3p is regulated by DNA methylation in GC [28].Based on the result, we hypothesized that miR-219a-1-3p is modulated by DNA methylation in GC.In recent investigation, we discovered that the upstream region of the genome spanned by miR-219a-1-3p was hypermethylated and that miR-219a-1-3p mRNA levels were decreased in tumor samples and cancer cell lines.Furthermore, the miR-219a-1-3p expression in GC cells was substantially upregulated after supplemented with 5-AZA-CdR, indicating that miR-219a-1-3p hypermethylation resulted in the reduction of the miR-219a-1-3p expression in GC cells.These data suggest that the upstream region of the genome spanned by miR-219a-1-3p results in a decrease of miR-219a-1-3p expression in GC.
MiRNA could affect many signal pathways and target genes, such as, miR-219a-5p represses the malignancy of GC cells via Wnt signaling [38].Another study reported that miR219a-2-3p dramatically inhibited the overgrowth, relocation, and invasion of GC cells by ERK pathway [28].Most importantly, in pancreatic cancer, miR-219-1-3p inhibiting malignant progression was corelative to AKT and ERK pathway [26].In our study, the signaling pathways that miR219a-2-3p may affect were predicted.We further selected the predicted downstream PI3K/AKT pathway for Figure 6: Inhibition of the malignant progression of GC as a result of miR-219a-1-3p overexpression was associated with the PI3K/ AKT signaling pathway.(A) Venn diagram of the number of genes identified as potential targets of miR219a-1-3p by TargenScan and miRanda.(B) KEGG pathway analysis of miR-219a-1-3p potential target genes by DAVID bioinformatics resources.(C) The effect of miR-219a-1-3p overexpression on the expression of the components of the PI3K/AKT pathway was detected using western blotting analysis.The cell lysate was collected and subjected to western blotting analysis with antibodies against members of the PI3K/AKT pathway.Levels of β-actin were used as a loading control.
preliminary validation.The expression of p-PI3K and p-AKT were downregulated in the miR-219a-1-3p overexpression group by western blotting.These outcomes indicated that miR-219a-1-3p overexpression suppressed the malignant progression of GC and this effect is related to the PI3K/AKT pathway.The specific regulatory mechanism underlying this effect requires further investigation.

Conclusions
Our investigation indicated that miR-219a-1-3p expression was suppressed in GC tissues contrasted with that in nearby healthy tissues.The miR-219a-1-3p expression was correspondingly reduced in GC cell lines contrasted with that in GES-1 cells.Additionally, the miR-219a-1-3p overexpression substantially repressed the aggressiveness of GC cells in vitro.MiR-219a-1-3p overexpression notably inhibited the growth of subcutaneous cancers and liver metastasis in nude mice.Mechanistically, we discovered that miR-219a-1-3p expression was partly modulated by DNA hypermethylation in GCs.In addition, miR-219a-1-3p overexpression inhibited the PI3K/AKT signaling pathway.Our outcomes demonstrate that miR-219a-1-3p may have a predominant function in regulating GC-associated biological mechanisms.This study suggests the possibility of developing novel therapeutics to target miR-219a-1-3p in GC.

Figure 1 :
Figure1: The miR-219a-1-3p was downregulated in gastric cancer (GC) tissues compared to that in paired adjacent non-tumor tissues, and its downregulated expression was correlated with a poor clinical prognosis.(A) The mRNA expression of miR-219a-1-3p was detected in 98 GC tissues (C) and paired non-tumor tissues (N).(B) The expression of miR-219a-1-3p is presented as normalized data, *p<0.05.(C) The paraffin sections of adjacent non-tumor tissues and tumor tissues in GC patients were subjected to H&E staining and observed using a bright field microscope (100×).Scale bar: 50 μm.

Table  :
Correlation between the expression of miR-a--p and clinical features in  GC patients.
a Mean of Log.(C/N); b Standard definition of Log.(C/N).C, normalized expression of cancer tissues; N, normalized expression of non-cancer tissues, c p<..