MiR-186 serves as a tumor suppressor in lung adenocarcinoma cells by down-regulation of Shp2 gene and inhibiting PI3K/Akt/mTOR signaling pathway

Background The study aimed to investigate the effect and mechanism of miR-186, which targets protein tyrosine phosphatase (Shp2) PI3K/Akt/mTOR signaling pathway, on the biological characteristics of lung adenocarcinoma cells. Methods In this experimental study, Human lung adenocarcinoma cell line SPC-A-1 was grouped as Blank group, negative control (NC) group, miR-186 mimic group, miR-186 inhibitor group, si-Shp2 group and miR-186 inhibitor+si-Shp2 group. Results The results showed that miR-186 can target and down-regulate the expression of Shp2 gene. Compared with the Blank group, levels of Shp2, N-cadherin and Bcl-2 and level of PI3K/p-PI3K, Akt/P-Akt, mTOR/p-mTOR as well as cell proliferation, migration and invasion ability and the proportion of cells in S phase significantly decreased in the miR-186 mimic group and the si-Shp2 group, while the levels of E-cadherin and Bax as well as the proportion of cells in G1 phase and cell gene and mediates apoptosis rate increased significantly (all P < 0.05). Compared with the miR-186 inhibitor group, the miR-186 inhibitor + si-Shp2 group showed similar trend in all parameters with the comparison above (all P < 0.05). Conclusions The overexpression of miR-186 can down-regulate Shp2 gene expression, further inhibit the proliferation, invasion and migration and promote apoptosis of lung adenocarcinoma cells by inhibiting the activation of PI3K/Akt/mTOR signaling pathway.


Abstract
Background The study aimed to investigate the effect and mechanism of miR-186, which targets protein tyrosine phosphatase (Shp2) PI3K/Akt/mTOR signaling pathway, on the biological characteristics of lung adenocarcinoma cells. Methods In this experimental study, Human lung adenocarcinoma cell line SPC-A-1 was grouped as Blank group, negative control (NC) group, miR-186 mimic group, miR-186 inhibitor group, si-Shp2 group

Background
With the occurrence and development of cancer, the life quality and health of patients have been greatly impacted. In China, lung adenocarcinoma accounts for a large part of cancer cases as the risk of lung cancer as well as the morbidity and mortality of lung cancer patients is gradually increasing, which seriously impacts human life safety [1,2]. Therefore, it's extremely important to explore the molecular mechanism of lung adenocarcinoma for the clinical treatment of lung cancer.
As a proto-oncogene, the expression of Shp2 up-regulated in lung cancer, which may affect the biological characteristics, such as migration, apoptosis, proliferation and invasion, of lung adenocarcinoma cells [3][4][5][6]. Shp2 gene can also promote the activation of Ras pathway, which further activates PI3K through direct interaction with p110 catalyst.
Afterwards, it facilitates the transformation of PIP2 to PIP3. PIP3 then integrates with Akt to promote the phosphorylation of Akt, so as to affect the cell proliferation, migration, invasion and apoptosis of lung adenocarcinoma [7][8][9][10]. mTOR, a kind of serine-threonine protein kinase, is a member of PI3K protein kinase family. When p-mTOR is activated, it can form a complex with downstream receptors to promote Akt phosphorylation, which can facilitate the expression of Bcl-2 (anti-apoptotic protein) and inhibit the expression of Bax (pro-apoptotic protein). E-cadherin protein can inhibit the invasion of cancer cells, while N-cadherin has an opposite effect. Activation of Akt inhibits the expression of E-cadherin, but facilitates the expression of N-cadherin, thus promoting the invasion of lung adenocarcinoma cells [11][12][13][14]. MicroRNA has been a hot topic in recent years, especially in the field of cancer research. Via bioinformatics prediction, we found that there is a targeting relationship between miR-186 and Shp2. Previous studies have reported that miR-186 is downregulated in non-small lung cancer, colon cancer, cervical cancer and gastric cancer, and miR-186 can inhibit the migration and invasion of these cancers [15][16][17][18][19]. Therefore, we hypothesized that miR-186 may down-regulate the expression of Shp2, in turn inhibiting the activation of PI3K/Akt/mTOR signaling pathway, so as to inhibit the proliferation, invasion and migration and promote the cell apoptosis of lung adenocarcinoma. Herein, we cultured SPC-A-1, a lung adenocarcinoma cell lines, and transfected the cells with miR-186 mimic, miR-186 inhibitor, si-Shp2 and miR-186 inhibitor + si-Shp2, respectively, to detect the proliferation, migration, invasion and apoptosis of each group, so as to verify our hypothesis.

Dual-luciferase reporter assay
The analysis of miR-186 target gene was performed by using a biological prediction website (http://www.microrna.org/microrna/home.do), and the dual luciferase reporter assay was used to verify whether Shp2 is a direct target gene of miR-186. The 3'-UTR gene fragment of the Shp2 gene was cloned and amplified, and the PCR product was cloned into the multiple cloning site in downstream of the Luciferase gene of pmirGLO (Cat. E1330, Promega, USA), which were named Wt-Shp2. The predicted specific binding site of miR-186 and Shp2 was mutated to construct the Mut-Shp2 vector. The Renilla luciferase pRL-TK vector (Cat. E2241, Promega, USA) was used as an internal reference.
The luciferase reporter vector was co-transfected with miR-186 mimic and NC mimic into ∆C t = CT (target gene) -CT (GAPDH) , ∆∆Ct = ∆Ct (experim ental group) -∆Ct (control group) . miR-186 used U6 as internal reference and other genes adopted GAPDH as internal reference, and 2 − ΔΔCt represents the relative expression level of target gene. The sequence of primers is shown in Table 1. Table 1 The sequence of primers Gene Sequence Shp2 Upstream

Statistical analysis
All data were processed by using SPSS 21.0 statistical software. The measurement data were expressed as mean ± standard deviation. One-way ANOVA were adopted for the comparison among groups and Tukey post-hoc test was used for pairwise comparison between groups. P < 0.05 indicated a significant difference.

miR-186 targeted and negatively regulated the expression of Shp2 gene
The biological prediction website microrna.org (http://www.microrna.org/microrna/home.do) predicted that miR-186 had a specific binding site with Shp2 (also known as PTPN11, Fig. 1a). According to the result of dual luciferase reporter assay (Fig. 1b), the miR-186 mimic transfected group showed a lower luciferase activity of Wt-Shp2 group, compared with the NC mimic group (P < 0.05). The luciferase activity of Mut-Shp2 group was not significant different between the two groups (P > 0.05). Therefore, miR-186 could target and negatively regulate the expression of the Shp2 gene.

Expression of Shp2, PI3K, Akt and mTOR mRNA in each group
To investigate how miR-186 targeted the Shp2 gene and mediated mTOR signaling pathway to affect the biological activity of lung adenocarcinoma cells, we detected the mRNA expression of miR-186, Shp2, PI3K, Akt and mTOR by qRT-PCR (Fig. 1c). The apoptosis rate was also detected by flow cytometry (Fig. 3). The results showed that compared with the Blank group, there was no significant difference in apoptosis rate in the NC group and the miR-186 inhibitor+si-Shp2 group (P > 0.05), however, the apoptosis rate of miR-186 mimic group (P < 0.001) and si-Shp2 group (P < 0.001) was significantly increased, which was obviously decreased in the miR-186 inhibitor group when compared with miR-186 mimic group (P < 0.001).

Detection of cell migration in each group by wound healing assay
The cell migration ability was observed by the wound healing assay (Fig. 4a, b). The results showed that compared with the Blank group, there was no significant difference in cell migration ability in the NC group and the miR-186 inhibitor+si-Shp2 group (P > 0.05), however, the migration ability of miR-186 mimic group (P = 0.006) and si-Shp2 group (P = 0.029) was significantly decreased, which was obviously increased in the miR-186 inhibitor group when compared with miR-186 mimic group (P < 0.001). Compared with the miR-186 inhibitor group, the migration ability of miR-186 inhibitor+si-Shp2 group was significantly decreased (P = 0.019).

Transwell assay to detect cell invasion in each group
The Transwell assay was used to detect the invasive ability of each group (Fig. 4c, d). The results showed compared with the Blank group that there was no significant difference in cell invasion in the NC group and the miR-186 inhibitor+si-Shp2 group (P > 0.05), however, the number of invasive cells in miR-186 mimic group (P < 0.001) and si-Shp2 group (P < 0.001) was significantly decreased, which was obviously increased in the miR-

Discussion
In China, patients with lung adenocarcinoma account for a large proportion of people suffering from cancer, moreover, the current death rate of lung adenocarcinoma patients is extremely high, of which the occurrence and development is seriously affecting human health [20][21][22][23]. However, the molecular mechanisms involved in adenocarcinoma cells have not yet been fully elucidated now, exploring which therefore has far-reaching implications for the treatment of patients with lung adenocarcinoma.
Previous study has confirmed that the expression of Shp2 is elevated in lung cancer, which can promote the expression of the downstream molecular, PI3K, by activating Ras pathway, leading to the activation of the PI3K/Akt/mTOR signaling pathway, which can further facilitate the expression of Bcl-2 and N-cadherin, while inhibit the expression of Bax and E-cadherin, thus to affect the cell biological characteristics such as migration, apoptosis, proliferation and invasion in lung adenocarcinoma cells [24][25][26]. In the study, we used the SPC-A-1, a lung adenocarcinoma cell line, to silence the expression of Shp2 and our results showed that silencing of Shp2 inhibited the expression of PI3K/Akt/mTOR signaling pathway, thereby inhibiting the proliferation, migration and invasion, blocking the cell cycle progression and promoting apoptosis of lung adenocarcinoma cells. This is consistent with the previously reported literature results.
To further explore the mechanism of upstream signaling of Shp2, we predicted the targeting relationship between miR-186 and Shp2 through the website. It's reported that miR-186 inhibits the development of non-small lung cancer, colon cancer, cervical cancer and gastric cancer [27][28][29][30]. In our study, dual luciferase reporter assay confirmed that

Conclusions
We demonstrated that miR-186 can achieve targeted inhibition of Shp2 expression, thereby inhibiting the expression of PI3K/Akt/mTOR signaling pathway, to inhibit the proliferation, invasion and migration and promote apoptosis of lung adenocarcinoma cells.
This study further clarified the development mechanism of lung adenocarcinoma and laid a theoretical foundation for the treatment of clinical lung adenocarcinoma. In the further, we need to perform in vivo experiments to further verify the above results. Additionally, the exact mechanisms for inhibition of Shp2 by miR-186 and the targeted regulatory network of miR-186 in lung adenocarcinoma is still unclear.

Ethics approval and consent to participate
Not applicable.

Consent for publication
Not applicable.

Availability of data and material
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.    compared with miR-186 inhibitor group, @P < 0.05; compared with si-Shp2 group, $P < 0.05. NC, negative control; Shp2, protein tyrosine phosphatase.