MiR-376a inhibits non-small cell lung cancer cell progression by regulating Rab1A

Post transcriptional gene regulation of microRNA-376a (miR-376a) plays a crucial role for tumorigenesis and cancer development. However, the potential role of miR-367a in non-small cell lung cancer (NSCLC) remains unclear. In this study, we investigated the crucial role of miR-376a in NSCLC by analyzing miR-376a expression as well as its target genes. Through overexpression strategies, we uncovered the molecular mechanisms underlying miR-376a-mediated tumorigenesis. Quantitative real-time PCR analysis demonstrated miR-376a levels to be significantly decreased in NSCLC cells compared with non-tumorigenic counterparts. Interestingly, miR-376a overexpression potentially repressed NSCLC cell proliferation, migration, and invasion, but increased apoptosis in A549 cells. Using bioinformatic approaches, we predicted that miR-376a targets Rab1A, and further luciferase fusion assay demonstrated Rab1A was a direct target of miR-376a and miR-376a inhibited cell proliferation by regulating the mRNA and protein levels of Rab1A in NSCLC cells. Overall, our findings uncover the miR-376a could suppress NSCLC cells progression via directly targeting Rab1A. transfection, the transfection efficiency was examined using quantitative reverse transcription polymerase chain reaction (qRT-PCR).


Introduction
Lung cancer is a common malignant tumor that accounts for nearly 10% of cancer cases and is the most deadly malignant tumor, posing a significant threat to human health worldwide, both in males and females. Lung cancer is generally classified based on histological type into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). NSCLC has emerged as the most common type of lung cancer, and it accounts for nearly 85% of total cases. Despite the efficacy of surgical therapy for almost 50% of cases, many NSCLC patients still have a poor prognosis [1][2].Thus, the findings of novel diagnosis and therapeutic target for NSCLC patients are extremely urgent. Congruently, miR-15a and miR-16, which are expressed from the intron region of the prostate cancer gene, have been shown to act as tumor suppressor genes [4][5]. An increase in miR-216a and miR-217 levels in hepatocellular cancer cells leads to activation of the TGF-b and PI3K/Akt pathways, conferring drug resistance to sorafenib and causing neoplasm recurrence [6]. MiR-376a also regulates cell processes in several cancers via targeting of various genes, and low levels of miR-376a induce apoptosis in retinoblastoma cells by targeting caspase-3. Furthermore, Liu et al. reported that the transcript level of miR-376a was increased in murine malignant lung cancer compared to adjacent normal murine lung [7]. Overexpression of miR-376a has been reported to attenuate the proliferation and migration of melanoma cells by targeting IGF1R, a tyrosine kinase receptor associated with tumorigenesis and metastasis [8]. MiR-376a also regulates proliferation, apoptosis, migration and invasion in metastatic prostate cancer and hepatocellular carcinoma [9][10], and functions as a tumor suppressor in nasopharyngeal carcinoma [11]. Nevertheless, few studies have focused on the function of miR-376a in lung cancer, especially NSCLC, despite its significance to human health. In this study, we aimed to uncover the relation between miR-376a and NSCLC tumorigenesis.

Cell viability assay
The viability of the cell was performed by using Cell Counting Kit-8 (CCK-8; Dojindo, Japan) assay. Briefly, transfected cells were cultured for 72 h in RPMI 1640 medium containing 10% FBS (HyClone, USA) and then seeded into 96-well plates at a density of 5×10 3 cells per well, 10 μL of fresh CCK-8 solution was added to each well and then co-incubated for 4 h at 37 °C. The optical density (OD) of each well was measured the absorbance at 450 nm using a microplate reader (Bio-Rad, USA).

Migration and invasion assays
Cell migration was determined using the scratch assay. After transfection, a 2-mL (1×10 5 6 cells/mL) sample of cells was seeded in 6-well plates and incubated for 24 h to allow attachment. The cell layer was scratched, down the middle of the plate well, and the medium was removed; the cells were then washed three times with PBS. Fresh medium containing 3% FBS was added, and the cells were visualized under microscopy after 24 h.
To minimize deviation, at least triple views were visualized and captured, and the average number was used.
For invasion assay, dissolved ECM gel (Sigma, 100 mg/well) was added to a 24-Transwell Boyden chamber (Corning, USA) and incubated for 4-8 h at 37 °C. Post-transfection cells in logarithmic phase after trypsinization and suspension were seeded into the Transwell Boyden chamber using 100 μL per well at 5×10 5 cells/mL, after which 500 μL RPMI1640 medium containing 10% FBS was added to the lower chamber. The cells that did not invade the upper chamber were removed and fixed with methanol. Cells were stained with Giemsa solution and observed by optical microscopy (ECLIPSE Ti2, Nikon, Japan).

Dual-luciferase reporter assay
The luciferase reporter assay was performed according to the methods described previously [10]. In brief, the 3'-UTR of Rab1A with wild-type or mutant binding sites for miR-376a were cloned into pmiR-GLO-Dual-Luciferase miRNA target expression vector plasmid (E1330; Promega, USA) naming pmiR-GLO-Rab1A-WT and pmiR-GLO-Rab1A-Mut, respectively. The A549 cells were co-transfected with pmiR-GLO-Rab1A-WT or pmiR-GLO-Basic-Rab1A-MUT and miR-376a or miR-376a mimic using Lipofectamine TM 2000 Transfection Reagent (Invitrogen, USA), according to the manufacturer's protocol. After incubation for 72 hours, the firefly and Renilla luciferase activities were measured using the Dual-Glo Luciferase System (Promega) according to the manufacturer's protocol. The firefly luciferase activity was normalized with the Renilla luciferase activity.

Hoechst 33342 staining assay
Cell apoptosis was determined using the Hoechst 33342 staining assay. In brief, 2 mL (1×10 5 cells/mL) sample of cells was seeded in 12-well plates and incubated for 24 h to allow attachment. After transfection, 10 μL of Hoechst 33342 live cell staining solution (100X) (Beyotime Biotechnology, Shanghai, China) was evenly added to each well. and then co-incubated for 10 minutes at 37 °C. The dye-containing medium was removed and the cells were washed three times with PBS and observed by fluorescence microscope.

Western blot assay
The total proteins were extracted from treated the cells using RIPA lysis buffer (Beyotime

Statistical analysis
Experiments in this study were repeated independently at least three times, and the results are expressed as the mean ± SD (standard deviation of mean). All data were analyzed using the SPSS statistical package. Univariate sample means were compared using one-way analysis of variance (ANOVA). Least significant difference (LSD) was used to compare multivariate sample means pairwise. P<0.05 (*) or P<0.01 (**) was considered statistically significant.

MiR-376a is downregulated in NSCLC cells
To evaluate the expression of miR-376a in NSCLC cells and normal lung cells, quantitative real-time PCR (qRT-PCR) assay was performed. The results showed that miR-376a expression level was markedly lower in A549 cells, with the reduction of around 71.1%, compared with that in the NuLi-1 cells (P<0.01, Fig. 1a). The above data revealed that the miR-376a might play a protective role in NSCLC pathogenesis.

The overexpression of miR-376a inhibits cell proliferation, migration, invasion and promotes cell apoptosis
To further investigate whether the protective role of miR-376a on in NSCLC pathogenesis, we performed that A549 cells were transiently transfected with miR-376a mimics or inhibitor to study gain-and loss-of-function of miR-376a. The transfection efficiency of miR-376a mimics and inhibitor was confirmed by qRT-PCR (Online Resource 1). The viability of cells was detected using CCK-8 assay. As shown in Fig. 1b, miR-376a mimics inhibited cell proliferation ability compared with the miR-NC in A549 cells, while miR-376a inhibitor increased cell proliferation ability. To further assess the effect of miR-376a on 9 cell migration by scratch wound healing assay, the results showed that miR-376a mimics in A549 cells could strongly suppress cell migration compared with the miR-NC group, while miR-376a inhibitor increased cell migration (Fig. 1c). Furthermore, transwell assay was performed to detect the cell invasive ability and the results showed that miR-376a mimics significantly inhibited cell invasion compared with control, while miR-376a inhibitor increased cell invasion (Fig. 1d). In addition, Hoechst 33342 staining was taken to detect the effect of miR-376a mimics on apoptosis in A549 cells. The results revealed that the percentage of apoptotic cells was markedly increased in A549 cells with miR-376a mimics (Fig. 1e). In brief, the above results revealed that miR-376a could inhibit cell proliferation, migration and invasion and promote cell apoptosis in A549 cells.

Rab1A is a target of miR-376a in NSCLC cells
To predict the potential target genes regulated by miR-376a in NSCLC cells, the predicted software targetscan and miRanda were performed in the study. We found that 3'-UTR of Rab1A had complementary sites with miR-376a. We further determined the abundance of Rab1A in A549 cells. Both the level of mRNA and protein were measured. The level of Rab1A was higher in A549 cells than that in NuLi-1 cells (Fig. 2a), which was in good agreement with the Western blot analysis (Fig. 2b). To further verify the interaction between miR-376a and Rab1A, we determined the relative transcript abundance of Rab1A under different miR-376a levels. As shown in Fig. 2c, miR-376a mimics markedly downregulated the expression of Rab1A, whereas miR-376a inhibitor upregulated the expression of Rab1A. Western blot analysis was also performed to confirm the qPCR results, showing the same trends (Fig. 2d). To validate whether miR-376a regulates the expression of Rab1A, 3'-UTR of Rab1A with wild-type (Rab1A-WT) or mutant (Rab1A-Mut) binding sites for miR-376a were cloned into pmiR-GLO-Dual-Luciferase plasmid, respectively. Relative luciferase activity was determined after plasmids co-transfection 10 with miR-376a mimics. As shown in Fig. 2e and f, miR-376a mimics significantly reduced the luciferase activity of the Rab1A-WT-transfected A549 cells, while no significant change was found in A549 cells transfected with Rab1A-Mut. Taken together, these findings suggested that Rab1A is a target of miR-376a.

Rab1A regulates tumorigenesis in NSCLC cells
To gain further insight into the mechanistic role of Rab1A in NSCLC tumorigenesis, we knocked down Rab1A or overexpressed Rab1A in A549 cells. The mRNA level and the protein expression of Rab1A were determined to verify the effects of different treatments.
As shown in Fig. 3a and b, both mRNA and protein levels were reduced in A549 cells transfected with si-Rab1A, but increased in A549 cells transfected with Rab1A, indicating that Rab1A were successfully silenced and overexpressed in the transfected cells.
To determine the role of Rab1A in the life cycle of NSCLC cells, cell proliferation, migration, invasion and apoptosis were measured under different levels of Rab1A.
Knockdown of Rab1A (si-Rab1A) significantly decreased the growth rate of cells, but overexpression (Rab1A) accelerated cell proliferation (Fig. 3c). si-Rab1A inhibited cell migration and invasion but enhanced apoptosis, whereas overexpressing Rab1A had the opposite effects (Fig. 3d, e and f). These results demonstrate that Rab1A participates in NSCLC pathogenesis.

MiR-376a inhibits the NSCLC cells proliferation by targeting Rab1A
To evaluate whether the effects of miR-376a on NSCLC proliferation via mediating Rab1A, we performed the CCK-8 cell proliferation assays (Fig. 4). The results showed that si-Rab1A inhibited the cell proliferation, but co-transfection of si-Rab1A and miR-376a inhibitor dismissed the effects. The Rab1A increased the cell proliferation, but cotransfection of Rab1A and miR-376a mimics dismissed the effects. The above results suggested that miR-376a inhibits cell proliferation by targeting Rab1A in NSCLC cells.

Discussion
Owing to the functional significance of miRNA on post-transcriptional regulation, governing crucial cellular mechanisms, investigations on miRNAs have garnered a great deal of research attention. Recent studies have explored the potential of miRNAs to regulate expression of crucial proteins involved in cancer and have demonstrated their efficacy as molecular markers for diagnosis of various cancers [12], including NSCLC, a kind of deadly lung cancer with few and unsatisfied therapeutic treatments. A diversity of miRNAs is involved in NSCLC cells, and their functions differ. For example, miR-33a targets the mRNA of Methyltransferase like 3 (METTL3), a key protein to upregulate several crucial oncoproteins in human lung cancer cells, leading to attenuate NSCLC cells proliferation [13]. MiR-142-3p overexpression in NSCLC cells is effective to inhibit anticancer drug-induced autophagy as well as increase chemo-sensitivity of NSCLC [14].
MiR-376a is considered a meaningful prognostic biomarker and a potential therapeutic target in various types of cancers. For example, miR-376a directly targets specificity protein 1, markedly decreasing the proliferation and invasion of globlastoma multiforme cells in vitro [15]. In hepatocellular carcinoma tissues and cell lines, miR-376a was decreased by targeting p85α [10]. Furthermore, it is reported that miR-376a regulates human erythropoiesis by targeting CDK2 and Ago2 [16]. Nonetheless, the molecular mechanisms underlying the inhibition of tumorigenesis in NSCLC remain unknown, and the interaction and relationship between miR-376a and its target genes in NSCLC have not yet been explored.
In an effort to investigate the biological function of miR-376a in NSCLC, A549 cells were used as a model NSCLC cell, and we verified miR-376a to be downregulated in NSCLC cell lines using qRT-PCR, which is consistent with previous research in hepatocellular carcinoma, melanoma tissue and colorectal cancer [8,10,17]. It is reported that arsenic trioxide-mediated apoptosis of retinoblastoma cells was regulated by miR-376a, which targets caspase-3, a major effector of cell apoptosis pathway [18]. The effect of miR-376a expression on NSCLC cell proliferation and invasion may be associated with its target gene. Bioinformatic analysis predicted that miR-376a targets Rab1A. To the best of our knowledge, this study provides the first evidence that miR-376a regulates NSCLC by targeting Rab1A. The proteins of the Rab family are small GTPases regulating vesicle trafficking from the ER to Golgi apparatus, which are crucial for cell survival and growth [19]. Using an unbiased knockdown gene, Rab1a was identified as a novel candidate in cell migration [20]. Rab1A was also found to be overexpressed in HCC compared with normal livers, and Rab1A increased cell growth and migration, cell cycle progression and tumor formation in vivo and in vitro [21]. It is proved that Rab1A promotes mTORC1 signaling and oncogenic growth and enhances tumor progression and invasion in colorectal cancer [12]. In our study, experimental evidences corroborate that overexpression of miR-376a inhibited that of Rab1A, as indicated by qPCR and western blotting. These data provide valuable insight into the role of miR-376a through its ability to inhibit Rab1A expression, in turn leading to inhibiting NSCLC cell processes. Thus, targeting Rab1A by miR-376a are potential diagnostic and treatment approaches for NSCLC cells.
In summary, miR-376a regulates cell proliferation, migration, invasion and apoptosis in A549 cells by targeting Rab1A. These data were corroborated by overexpression and

Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.