MicroRNA-629 promotes the tumorigenesis of non-small-cell lung cancer by targeting FOXO1 and activating PI3K/AKT pathway

Abstract

OBJECTIVE:

MicroRNA-629 (miR-629) has been found to play an important role in the pathogenesis of human cancers. However, the function of miR-629 is still unknown in non-small-cell lung cancer (NSCLC). The purpose of this study is to preliminarily elucidate the regulatory mechanism of miR-629 in NSCLC.

MATERIALS AND METHODS:

The mRNA and protein expression was measured by real-time quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. The function of miR-629 was investigated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and Transwell assays. The relationship between miR-629 and FOXO1 was confirmed by dual luciferase assay.

RESULTS:

MiR-629 was upregulated in NSCLC tissues and cells. High expression of miR-629 predicted poor prognosis in patients with NSCLC. Moreover, miR-629 promoted cell proliferation, migration and invasion in NSCLC cells. In addition, FOXO1 was confirmed as a direct target of miR-629 in NSCLC. Furthermore, knockdown of FOXO1 also promoted proliferation, migration and invasion of NSCLC cells. More importantly, overexpression of FOXO1 weakened the carcinogenesis of miR-629 in NSCLC. Besides that, miR-629 promoted EMT and activated the PI3K/AKT pathway in NSCLC.

CONCLUSIONS:

MiR-629 promotes the progression of NSCLC by targeting FOXO1 and regulating EMT/PI3K/AKT pathway.

Keywords

miR-629 non-small-cell lung cancer FOXO1 PI3K/AKT pathway

1. Introduction

With the increase of the elderly population, the acceleration of industrialization and the aggravation of environmental pollution, the incidence and mortality of lung cancer have been rising in the past few decades [1]. Lung cancer has become a leading cause of cancer death in China [2]. Therefore, the prevention and early detection of lung cancer have become research focus in China. Lung cancer is mainly divided into non-small cell lung cancer (NSCLC) and small cell lung cancer. Moreover, NSCLC cells grow and differentiate more slowly than small cell cancer cells. Currently, surgical resection adjuvant radiotherapy and chemotherapy are the primary treatment for patients with NSCLC [3]. It has been reported that the survival rate is closely associated with clinical stage in NSCLC patients. In addition, many patients with NSCLC have poor prognosis due to delayed diagnosis [4]. Therefore, early diagnosis and reasonable treatment are very important to improve the survival rate of patients with NSCLC.

Previous studies have shown that microRNAs (miRNAs) are involved in tumorigenesis by binding to the 3’-UTR of target genes [5]. In addition, abnormal expression of miRNAs has been identified in various cancers that regulate tumorigenesis. In particular, many miRNAs have been reported to act as onco-miR or inhibitory miRNAs in NSCLC. For example, miR-92b was upregulated in NSCLC and promoted NSCLC progression by targeting RECK [6]. In contrast, miR-194 was found to be downregulated and suppress cell metastasis in NSCLC by regulating BMP1 and p27 expression [7]. Recently, the specific functions of miR-629 have caught our attention. It has been reported that miR-629 was upregulated and acted as an oncogene in gastric cancer [8]. Furthermore, miR-629 has been found to promote cell metastasis in ccRCC by targeting TRIM33 [9]. More importantly, it has been shown that the miR-629/NBS1 axis caused an increased risk of lung cancer in the Southern and Eastern Chinese population [10]. However, few reports have reported the biological effect of miR-629 in NSCLC, particularly on cell proliferation, migration and invasion.

In addition, previous studies have shown that forkhead box O1 (FOXO1) is an important regulator in the pathogenesis of human cancer [11]. For example, FOXO1 has been identified as a tumor suppressor in gastric cancer [12] and breast cancer [13]. In addition, FOXO1 has been reported to be regulated by several miRNAs in different cancers, such as miR-9 [14], miR-425 [15] and miR-544 [16]. Besides, FOXO1 has been found to be involved in the PI3K/AKT pathway via upstream activation of AKT [17]. Moreover, miRNA-regulated epithelial-mesenchymal transition (EMT)/PI3K/AKT pathway has been reported in NSCLC [18]. For example, miR-26a has been found to promote metastasis of lung cancer cells via the EMT/AKT pathway [19]. However, the interaction between miR-629 and FOXO1 remains unclear in the progression of NSCLC.

In this study, the functions of miR-629 and FOXO1 were investigated in NSCLC. At the same time, the effects of miR-629 and FOXO1 on the EMT/PI3K/AKT pathway were also explored in NSCLC. Further, the relationship between miR-629 and FOXO1 in NSCLC was confirmed. These findings will provide a useful therapeutic target for the treatment of NSCLC.

2. Patients and methods

2.1 Clinical tissues

NSCLC tissues and adjacent normal lung tissues were obtained from 74 patients in Shanghai Chest Hospital affiliated to Shanghai Jiaotong University. Written informed consents were obtained from all patients. All NSCLC patients did not receive any treatment prior to surgery. Theses tissues were frozen in liquid nitrogen and then stored in a $-$ 80 ${}^{\circ}$ C refrigerator. This study was approved by the Institutional Ethics Committee of Shanghai Chest Hospital affiliated to Shanghai Jiaotong University.

2.2 Cell lines culture

The human normal bronchial epithelium cell line BEAS-2B (BNCC338205) and H1299 (BNCC100859), A549 (BNCC100258), H460 (BNCC102112) NSCLC cell lines were purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China). These cell lines were then inoculated in dulbecco’s modified eagle medium (DMEM) medium (Gibco, Rockville, MD, USA) with 10% fetal bovine serum (FBS) (Gibco, Rockville, MD, USA) and cultured in an atmosphere with 5% CO ${}_{2}$ at 37 ${}^{\circ}$ C.

2.3 Cell transfection

MiR-629 mimics or inhibitor and negative control (NC) were obtained from GenePharma (Shanghai, China). FOXO1 siRNA and vector were also synthesized by GenePharma. MiR-629 mimics: 5’-UAUGGC ACUGGUAGAAUUCACU-3’, miR-629 inhibitor: 5’-AGUGAAUUCUACCAGUGCCAU C-3’, and negative control (NC): 5’-UUGUACUACACAAAAGUAAUG-3’. They were then transfected into A549 and H1299 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), respectively, based on the manufactures’ protocols.

2.4 Real-time quantitative polymerase chain reaction (RT-qPCR) analysis

Total RNA in NSCLC tissues and cells was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The cDNA was synthesized using the PrimeScript ${}^{\@setsize{\scriptsize}{8pt}{\viipt}{\@viipt}\textregistered}$ RT reagent kit (TaKaRa Biotechnology, Dalian, China). RT-qPCR was performed by using the SYBR premix real-time PCR Reagent (TaKaRa Biotechnology, Dalian, China) on ABI 7500 Fast system (Applied Biosystems, Foster City, CA, USA). U6 or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control for miR-629 or FOXO1. Their expressions were calculated using the 2 ${}^{-\Delta\Delta\text{ct}}$ method. The primers used were: miR-629 forward, 5’-TGGGTTTACGTTGGGA GAACT-3’ and reverse, 5’-GCTGTCAACGATACGCT ACGTAACG-3’; U6 forward, 5’-CTCGCTTCGGCAG CACA-3’ and reverse, 5’-AACGCTTCACGAATTTGC GT-3’; GAPDH forward, 5’-GAAGGTGAAGGTCGG AGTC-3’ and reverse, 5’-GAAGATGGTGATGGGATT TC-3’; and FOXO1 forward, 5’-AAGAGCGTGCCCTA CTTCAA-3’ and reverse, 5’-CTGTTGTTGTCCATGG ATGC-3’.

2.5 MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay

First, 6 $\times$ 10 ${}^{3}$ transfected A549 and H1299 cells were seeded into 96-well plates. The transfected cells were cultured in 100 $\mu$ L of DMEM containing 0.5 g/L MTT. Then, A549 and H1299 cells were cultured at 37 ${}^{\circ}$ C for 24, 48, 72 or 96 h, after which the medium was removed. Next, these cells were destroyed with 100 $\mu$ l DMSO at 37 ${}^{\circ}$ C for 10 min. The absorbance at 490 nm of each sample was determined using an enzyme immunoassay analyzer (Tecan Group Ltd., Männedorf, Switzerland). The method used is based on a previous study [20].

2.6 Transwell assay

Transwell chambers (8- $\mu$ m pore size membranes) were used for cell migration and invasion assay. Then, the upper surface with matrigel (BD Biosciences, Franklin Lakes, NJ, USA) was used for cell invasion. And cell migration assay was performed without matrigel. Transfected A549 and H1299 cells were cultured in the upper chamber with serum-free medium. Next, 10% FBS was added to the lower chamber. Transwell chamber was placed in a culture plate and incubated at 37 ${}^{\circ}$ C with 5% CO ${}_{2}$ for 24 h. After that, the migrated or invasive cells were fixed with methanol and stained with crystal violet. The cells on the inner layer were gently removed with a cotton swab and 3 randomly selected views were counted. Finally, the average number of cells per view was calculated.

2.7 Western blot analysis

Transfected A549 and H1299 cells were lysed using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China). The protein concentration was detected using a bicinchoninic acid (BCA) Protein Assay kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instruction. Then, 30 $\mu$ g total proteins were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred into polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk in Tris-buffered saline containing Tween-20 for 3 h at room temperature. Next, we incubated the membranes overnight at 4 ${}^{\circ}$ C with E-cadherin (Rabbit monoclonal; dilution, 1:1000; cat. no. ab1416; Abcam, Cambridge, MA, USA), N-cadherin (Rabbit polyclonal; dilution, 1:1000; cat. no. ab18203; Abcam, Cambridge, MA, USA), Vimentin (Rabbit polyclonal; dilution, 1:1000; cat. no. ab137321; Abcam, Cambridge, MA, USA), AKT (Rabbit polyclonal; dilution, 1:1000; cat. no. ab8805; Abcam, Cambridge, MA, USA), p-AKT1 (phospho S473, Rabbit monoclonal; dilution, 1:1000; cat. no. ab81283; Abcam, Cambridge, MA, USA), and GAPDH (Rabbit monoclonal; dilution, 1:1000; cat. no. ab181602; Abcam, Cambridge, MA, USA) antibodies. After washing, they were incubated with horseradish peroxidase-conjugated secondary antibodies (dilution, 1:5,000; cat no. ab190492; Abcam, Cambridge, MA, USA) for 2 h at room temperature. An enhanced chemiluminescence system (ECL, Pierce, Rockford, IL, USA) was used to detect the immunoreactive bands.

2.8 Bioinformatics analysis

Bioinformatics prediction TargetScan version 7.1 online software (www.targetscan.org) was used to predict the potential targets of miR-629, according to the manufacturer’s instructions. Briefly, human was selected as the target species and miR-629 was inserted as the investigated miRNA. MiR-629 was predicted to bind to the seeding sequences of the 3’-UTR of FOXO1.

2.9 Dual luciferase assay

The 3’-UTR of wild type or mutant FOXO1 was inserted into the pcDNA3.1 plasmid vector (Promega, Madison, WI, USA). Next, the above vector and miR-629 mimics were transfected into A549 and H1299 cells. Forty-eight hours after transfection, luciferase activity was measured by a dual luciferase assay system (Promega, Madison, WI, USA).

Figure 1.

MiR-629 was upregulated in NSCLC tissues. (A) The expression of miR-629 was detected in NSCLC tissues and normal tissues. (B) High miR-629 expression was correlated with shorter overall survival in NSCLC patients. ${}^{*}P<$ 0.05, ${}^{**}P<$ 0.01.

2.10 Statistical analysis

Data are shown as mean $\pm$ SD. These data were analyzed using Statistical Product and Service Solutions (SPSS) 19.0 (SPSS Inc., Chicago, IL, USA) or Graphpad Prism 6 (La Jolla, CA, USA). The relationship between miR-629 and clinical pathological characteristics in NSCLC patients was calculated by Chi-squared test. Pearson’s correlation analysis was performed to examine the correlation between miR-629 and FOXO1 expression in NSCLC tissues. Comparison between multiple groups was done using One-way ANOVA test followed by Post Hoc Test (Least Significant Difference). Survival curves were plotted by Kaplan-Meier analysis. Log-rank test was used to compare the survival differences. A significant difference was defined at $P<$ 0.05.

3. Results

3.1 MiR-629 was upregulated in NSCLC tissues

The expression of miR-629 was detected in NSCLC tissues by RT-qPCR assay. The expression of miR-629 was found to be increased in NSCLC tissues compared to normal tissues (Fig. 1A). Next, we found that the abnormal expression of miR-629 was associated with lymph nodes metastasis ( $P=$ 0.015) and tumor stage ( $P=$ 0.034, Table 1) in NSCLC patients. Moreover, high miR-629 expression was associated with shorter overall survival in patients with NSCLC ( $P=$ 0.0143, Fig. 1B). These results suggest that miR-629 may be involved in the pathogenesis of NSCLC.

Table 1
Relationship between miR-629 expression and clinic-pathological characteristics in NSCLC patients

Characteristics	Cases	miR-629		$P$ -value
		High	Low
Age (years)				0.101
$\geqslant$ 60	44	28	16
$<$ 60	30	13	17
Gender				0.104
Male	40	25	15
Female	34	16	18
Tumor size (mm)				0.123
$\leqslant$ 3	50	27	23
$>$ 3	24	14	10
Lymph nodes metastasis				0.015 ${}^{*}$
Yes	20	14	6
No	54	27	27
Tumor stage				0.034 ${}^{*}$
I–II	56	29	27
III–IV	18	12	6

Statistical analyses were performed by the $\chi^{2}$ test. ${}^{*}P<$ 0.05 was considered significant.

3.2 MiR-629 had carcinogenic effect on the development of NSCLC

Next, the expression of miR-629 was detected in H1299, A549, H460 and BEAS-2B cell lines. MiR-629 was found to be upregulated in H1299, A549 and H460 cell lines compared to BEAS-2B cells (Fig. 2A). The function of miR-629 in NSCLC was investigated in A549 and H1299 cells with miR-629 mimics or inhibitors. RT-qPCR assay showed that miR-629 mimics increased the expression of miR-629, while miR-629 inhibitor decreased its expression in A549 and H1299 cells (Fig. 2B). Functionally, overexpression of miR-629 promoted cell proliferation, while downregulation of miR-629 suppressed cell proliferation in A549 and H1299 cells (Fig. 2C and D). Similarly, upregulation of miR-629 was also found to promote cell migration, when downregulation of miR-629 inhibited cell migration in A549 and H1299 cells (Fig. 2E). In addition, the same effect of miR-629 on cell invasion was also identified in A549 and H1299 cells (Fig. 2F). Briefly, these results reveal that miR-629 acts as a tumor promoter in NSCLC by promoting cell proliferation, migration and invasion.

Figure 2.

MiR-629 promoted proliferation, migration and invasion of NSCLC cells. (A) MiR-629 expression was examined in H1299, A549, H460 NSCLC cells and human normal bronchial epithelium cell line BEAS-2B. (B) The expression of miR-629 was examined in A549 and H1299 cells with miR-629 mimics or inhibitor. (C) and (D) Cell proliferation was measured in A549 and H1299 cells containing miR-629 mimics or inhibitor. (E) and (F) Cell migration and invasion were detected in A549 and H1299 cells with miR-629 mimics or inhibitor (magnification: 200 $\times$ ). ${}^{*}P<$ 0.05, ${}^{**}P<$ 0.01.

Figure 3.

FOXO1 is a direct target of miR-629 in NSCLC. (A) FOXO1 has a binding site with miR-629. (B) Luciferase reporter assay. (C) MiR-629 was negatively correlated with FOXO1 expression in NSCLC tissues. (D) and (E) The expression of FOXO1 were observed in A549 and H1299 cells containing miR-629 mimics or inhibitor. ${}^{**}P<$ 0.01.

3.3 FOXO1 was a direct target of miR-629 in NSCLC.

In addition, TargetScan database (http://www. targetscan.org/) predicts that $>$ 3,000 target genes have binding sites with miR-629. However, FOXO1 was selected as previous research has revealed that FOXO1 is important in different types of cancers. The binding site between miR-629 and the 3’-UTR of FOXO1 is shown in Fig. 3A. FOXO1 has been reported to express stably and play an important role in many human cancers. But its role remains unclear in lung cancer. Luciferase reporter assay was then performed to confirm this prediction. Luciferase activity of Wt-FOXO1 was found to be significantly reduced by miR-629 mimics in A549 and H1299 cells. However, the luciferase activity of Mut-FOXO1 was not affected by miR-629 mimics (Fig. 3B). In addition, a negative correlation between FOXO1 and miR-629 expression was identified in NSCLC tissues ( $P<$ 0.001, $R^{2}=$ 0.6942; Fig. 3C). Besides that, the expression of FOXO1 was examined in A549 and H1299 cells with miR-629 mimics or inhibitor. MiR-629 mimics were found to reduce the expression of FOXO1, while miR-629 inhibitor promoted the expression of FOXO1 in A549 and H1299 cells (Fig. 3D and E). Collectively, FOXO1 is a direct target of miR-629 and negatively correlated with miR-629 expression in NSCLC.

3.4 FOXO1 was downregulated in NSCLC

Next, the alternation of FOXO1 expression was examined in NSCLC tissues and cell lines. We found that FOXO1 was downregulated in NSCLC tissues compared to adjacent normal tissues (Fig. 4A). In addition, downregulation of FOXO1 was also detected in H1299, A549 and H460 cell lines compared to BEAS-2B cells (Fig. 4B). Besides that, low FOXO1 expression was found to be associated with shorter overall survival in patients with NSCLC ( $P=$ 0.012, Fig. 4C). These findings suggest that FOXO1 may be involved in the development of NSCLC.

Figure 4.

FOXO1 was downregulated in NSCLC tissues and cells. (A) The expression of FOXO1 was measured in NSCLC tissues and normal tissues. (B) The expression of FOXO1 was observed in H1299, A549, H460 NSCLC cells and human normal bronchial epithelium cell line BEAS-2B. (C) High expression of FOXO1 was related to longer overall survival in NSCLC patients. ${}^{*}P<$ 0.05, ${}^{**}P<$ 0.01.

3.5 MiR-629 promoted the development of NSCLC through targeting FOXO1

Then, the function of FOXO1 and its interaction with miR-629 in NSCLC were investigated in A549 and H1299 cells with FOXO1 siRNA or miR-629 mimics and FOXO1 vector. FOXO1 siRNA was found to reduce its expression in A549 and H1299 cells. In addition, we found that the decreased expression of FOXO1 induced by miR-629 mimics was restored by FOXO1 vector in A549 and H1299 cells (Fig. 5A and B). Functionally, knockdown of FOXO1 was found to promote cell proliferation in A549 and H1299 cells. Moreover, the promotion of cell proliferation mediated by miR-629 mimics was hampered by upregulation of FOXO1 in A549 and H1299 cells (Fig. 5C and D). In addition, knockdown of FOXO1 also promoted cell migration and invasion in A549 and H1299 cells. Upregulation of FOXO1 attenuated the promotion of cell migration and invasion induced by miR-629 mimics (Fig. 5E and F). In conclusion, FOXO1 functions as a tumor suppressor in NSCLC. And miR-629 promoted the development of NSCLC by downregulating FOXO1.

Figure 5.

MiR-629 promoted the development of NSCLC through targeting FOXO1. (A) and (B) The expression of FOXO1 was measured in A549 and H1299 cells with FOXO1 siRNA, miR-629 mimics, or FOXO1 vector $+$ miR-629 mimics. (C) and (D) Cell proliferation was measured in A549 and H1299 cells with FOXO1 siRNA, miR-629 mimics, or FOXO1 vector $+$ miR-629 mimics. (E) and (F) Cell migration and invasion were examined in A549 cells and H1299 with FOXO1 siRNA, miR-629 mimics, or FOXO1 vector $+$ miR-629 mimics (magnification: 200 $\times$ ). ${}^{*}P<$ 0.05, ${}^{**}P<$ 0.01.

3.6 MiR-629/FOXO1 was involved in EMT/PI3K/AKT pathway in NSCLC

Finally, the effects of miR-629 and FOXO1 on EMT and PI3K/AKT pathway were investigated in NSCLC cells. We found that overexpression of miR-629 and knockdown of FOXO1 promoted the expression of N-cadherin and Vimentin and inhibited E-cadherin expression in A549 and H1299 cells (Fig. 6A). In contrast, downregulation of miR-629 and upregulation of FOXO1 had opposite effects on these markers (Fig. 6B). In addition, overexpression of miR-629 and knockdown of FOXO1 were found to promote p-AKT expression (Fig. 6C), while downregulation of miR-629 and upregulation of FOXO1 inhibited p-AKT expression in A549 and H1299 cells (Fig. 6D). However, the expression of AKT was not affected by miR-629 and FOXO1 in A549 and H1299 cells. These results imply that miR-629 and FOXO1 are involved in the EMT/PI3K/AKT pathway to regulate tumorigenesis of NSCLC.

Figure 6.

MiR-629/FOXO1 was involved in EMT/PI3K/AKT pathway in NSCLC. (A) and (B) The protein expression of E-cadherin, N-cadherin, Vimentin, AKT and p-AKT was measured in A549 cells containing miR-629 mimics or inhibitor and FOXO1 siRNA or vector. (C) and (D) The protein expression of E-cadherin, N-cadherin, Vimentin, AKT and p-AKT in H1299 cells containing miR-629 mimics or inhibitor and FOXO1 siRNA or vector.

4. Discussion

Recently, specific functions of miRNAs have been identified in the pathogenesis of human cancers. Previous studies have shown that multiple miRNAs are involved in the development of NSCLC, such as miR-150 [21], miR-195 [22] and miR-210 [23]. In this study, upregulation of miR-629 was identified in NSCLC and predicted poor prognosis in patients with NSCLC. Similarly, renal cell carcinoma patients with high miR-629 expression were also found to have poor prognosis [9]. In addition, we found that overexpression of miR-629 promoted cell proliferation, migration and invasion in NSCLC. These findings suggest that miR-629 serves as a tumor promoter in NSCLC.

In addition, miR-629 has been reported as a novel biomarker and potential therapeutic target for triple-negative breast cancer lung metastasis [24]. Consistent with our findings, upregulation of miR-629 has been reported to promote ovarian cancer cell migration and invasion and inhibited apoptosis [25]. It has been proposed that miR-629 promoted proliferation and metastasis of human pancreatic cancer cells by targeting FOXO3 [26]. In addition, we found that miR-629 promoted EMT in NSCLC, which has not been reported in previous studies. EMT is an important malignant behavior that affects the metastasis of NSCLC cells. For example, miR-186 has been shown to inhibit migration of NSCLC cells by affecting the EMT process [27]. It was also found that downregulation of miR-638 promoted cell invasion in NSCLC by inducing EMT [28]. However, the cell morphology induced by EMT has not been testified in this study due to the limitation of our lab conditions. In addition, previous studies have shown that miR-629 plays an important role in human cancers by regulating target genes. For example, miR-629 has been reported to promote the development of cervical cancer by targeting RSU1 [29]. Here, FOXO1 was confirmed as a direct target of miR-629 and was negatively correlated with miR-629 expression in NSCLC.

FOXO1, as a target gene, has been reported to be negatively correlated with several miRNAs, including miR-135a [30], miR-182 [31] and miR-222 [32]. In this study, downregulation of FOXO1 was found in NSCLC tissues and cells. Furthermore, knockdown of FOXO1 promoted the progression of NSCLC. In particular, miR-629 was found to promote the development of NSCLC by regulating FOXO1 expression. Similarly, miR-183 was found to promote tumor growth of NSCLC by inhibiting FOXO1 expression [33]. These findings confirm that FOXO1 acts as a tumor suppressor in the pathogenesis of NSCLC. Besides that, we found that miR-629/FOXO1 axis was involved in EMT/PI3K/AKT pathway in NSCLC. Moreover, TargetScan showed that AKT3, a member of PI3K/AKT pathway, is a target of miR-629. However, the effect of miR-629 on AKT3 has not been investigated in this study due to the absence of AKT3 antibody. In addition, we also found that the expression of total AKT was not affected by miR-629. The reason caused that result may be the unstable binding of AKT3 to miR-629. Similarly, miR-132 has been reported to play a carcinogenic role in laryngeal squamous cell carcinoma by targeting FOXO1 and activating the PI3K/AKT pathway [34]. This study also reveals that miR-629 functions as a tumor promoter in NSCLC by targeting FOXO1 and activating the PI3K/AKT pathway.

5. Conclusions

In conclusion, upregulation of miR-629 and downregulation of FOXO1 were detected in NSCLC tissues and cells. And upregulation of miR-629 and downregulation of FOXO1 predicted poor prognosis in patients with NSCLC. More importantly, miR-629 promotes the progression of NSCLC by targeting FOXO1 and activating the EMT/PI3K/AKT pathway. These findings will provide new avenues for the diagnosis and treatment of NSCLC.

Footnotes

Conflict of interest

The authors declared no conflict of interest.

References

Siegel

Naishadham

and Jemal

, Cancer statistics, 2012, CA Cancer J Clin 62(1) (2012), 10–29.

Xie

X.W.

Wang

H.Y.

L.Y.

and Wen

Z.G.

, Clinical evaluation of tumor markers for diagnosis in patients with non-small cell lung cancer in China, Asian Pac J Cancer Prev 16(12) (2015), 4891–4894.

Ramalingam

S.S.

Owonikoko

T.K.

and Khuri

F.R.

, Lung cancer: New biological insights and recent therapeutic advances, CA Cancer J Clin 61(2) (2011), 91–112.

Schabath

M.B.

Thompson

Z.J.

and Gray

J.E.

, Temporal trends in demographics and overall survival of non-small-cell lung cancer patients at Moffitt Cancer Center from 1986 to 2008, Cancer Control 21(1) (2014), 51–56.

Huang

and Yang

, Circulating miRNAs: Promising biomarkers of human cancer, Asian Pac J Cancer Prev 12(5) (2011), 1117–1125.

Lei

Huang

and Gong

, Inhibition of miR-92b suppresses nonsmall cell lung cancer cells growth and motility by targeting RECK, Molecular and Cellular Biochemistry 387(1–2) (2014), 171–176.

Liu

Fang

Leach

L.J.

and Luo

, miR-194 suppresses metastasis of non-small cell lung cancer through regulating expression of BMP1 and p27 (kip1), Oncogene 33(12) (2014), 1506–1514.

Shin

V.Y.

E.K.

Chan

V.W.

Kwong

and Chu

K.M.

, A three-miRNA signature as promising non-invasive diagnostic marker for gastric cancer, Molecular Cancer 14 (2015), 202.

Jingushi

Ueda

Kitae

Hase

Egawa

Ohshio

et al., miR-629 targets TRIM33 to promote TGFbeta/smad signaling and metastatic phenotypes in ccRCC, Molecular Cancer Research 13(3) (2015), 565–574.

10.

Yang

Cheng

Huang

Zheng

Liu

et al., A functional polymorphism at microRNA-629-binding site in the 3’-untranslated region of NBS1 gene confers an increased risk of lung cancer in Southern and Eastern Chinese population, Carcinogenesis 33(2) (2012), 338–347.

11.

Zhao

Tindall

D.J.

and Huang

, Modulation of androgen receptor by FOXA1 and FOXO1 factors in prostate cancer, International Journal of Biological Sciences 10(6) (2014), 614–619.

12.

Choi

Park

Choi

Y.S.

D.A.

Kim

et al., c-Jun N-terminal kinase activation has a prognostic implication and is negatively associated with FOXO1 activation in gastric cancer, Bmc Gastroenterology 16(1) (2016), 59.

13.

Elshimali

Sarkissyan

Mohamed

Clayton

and Vadgama

J.V.

, Expression of FOXO1 is associated with GATA3 and Annexin-1 and predicts disease-free survival in breast cancer, American Journal of Cancer Research 2(1) (2012), 104–115.

14.

Aishanjiang

Rouzi

Jiao

Wang

Wusainahong

Wumanjiang

et al., MicroRNA-9 enhances invasion and migration of cervical carcinomas by directly targeting FOXO1, Eur Rev Med Pharmacol Sci 22(8) (2018), 2253–2260.

15.

Yang

Guo

Xiong

Pan

et al., MicroRNA-425 facilitates pathogenic Th17 cell differentiation by targeting forkhead box O1 (Foxo1) and is associated with inflammatory bowel disease, Biochem Biophys Res Commun 496(2) (2018), 352–358.

16.

Yao

G.D.

Zhang

Y.F.

Chen

and Ren

X.B.

, MicroRNA-544 promotes colorectal cancer progression by targeting forkhead box O1, Oncology Letters 15(1) (2018), 991–997.

17.

Zaballos

M.A.

and Santisteban

, FOXO1 controls thyroid cell proliferation in response to TSH and IGF-I and is involved in thyroid tumorigenesis, Molecular Endocrinology 27(1) (2013), 50–62.

18.

Chen

Q.Y.

Jiao

D.M.

Y.Q.

Chen

Wang

Tang

X.L.

et al., MiR-206 inhibits HGF-induced epithelial-mesenchymal transition and angiogenesis in non-small cell lung cancer via c-Met/PI3k/Akt/mTOR pathway, Oncotarget 7(14) (2016), 18247–18261.

19.

Liu

Wang

Liu

Zhou

et al., MiR-26a enhances metastasis potential of lung cancer cells via AKT pathway by targeting PTEN, Biochim Biophys Acta 1822(11) (2012), 1692–1704.

20.

Jiang

Wang

Zhang

and Lai

, MicroRNA-599 suppresses glioma progression by targeting RAB27B, Oncology Letters 16(1) (2018), 1243–1252.

21.

Zhang

Wei

and Xu

, miR-150 promotes the proliferation of lung cancer cells by targeting P53, Febs Letters 587(15) (2013), 2346–2351.

22.

Liu

Guo

Wang

et al., MiR-195 suppresses non-small cell lung cancer by targeting CHEK1, Oncotarget 6(11) (2015), 9445–9456.

23.

Eilertsen

Andersen

Al-Saad

Richardsen

Stenvold

Hald

S.M.

et al., Positive prognostic impact of miR-210 in non-small cell lung cancer, Lung Cancer 83(2) (2014), 272–278.

24.

Wang

Song

Tang

Zhang

Tang

and Li

et al., miR-629-3p may serve as a novel biomarker and potential therapeutic target for lung metastases of triple-negative breast cancer, Breast Cancer Research 19(1) (2017), 72.

25.

Shao

Shen

Qian

Zhou

and Chen

, Knockdown of miR-629 inhibits ovarian cancer malignant behaviors by targeting testis-specific y-like protein 5, Dna and Cell Biology 36(12) (2017), 1108–1116.

26.

Yan

Jiang

Shi

et al., MiR-629 promotes human pancreatic cancer progression by targeting FOXO3, Cell Death & Disease 8(10) (2017), e3154.

27.

Dong

Jin

Sun

Zhao

and Song

, MiR-186 inhibited migration of NSCLC via targeting cdc42 and effecting EMT process, Molecules and Cells 40(3) (2017), 195–201.

28.

Xia

Liu

Wang

and Chen

, Downregulation of miR-638 promotes invasion and proliferation by regulating SOX2 and induces EMT in NSCLC, Febs Letters 588(14) (2014), 2238–2245.

29.

Phuah

N.H.

Azmi

M.N.

Awang

and Nagoor

N.H.

, Suppression of microRNA-629 enhances sensitivity of cervical cancer cells to 1’S-1’-acetoxychavicol acetate via regulating RSU1, Onco Targets Ther 10 (2017), 1695–1705.

30.

Mao

X.P.

Zhang

L.S.

Huang

Zhou

S.Y.

Liao

Chen

L.W.

et al., Mir-135a enhances cellular proliferation through post-transcriptionally regulating PHLPP2 and FOXO1 in human bladder cancer, Journal of Translational Medicine 13 (2015), 86.

31.

Huang

Fan

Tao

Song

Wang

Zhang

et al., Effect of miR-182 on hepatic fibrosis induced by Schistosomiasis japonica by targeting FOXO1 through PI3K/AKT signaling pathway, Journal of Cellular Physiology 233(10) (2018), 6693–6704.

32.

Shen

Wang

Yang

Chen

Zhou

et al., MiR-222 promotes drug-resistance of breast cancer cells to adriamycin via modulation of PTEN/Akt/FOXO1 pathway, Gene 596 (2017), 110–118.

33.

Zhang

Quan

Wang

and Che

, MiR-183 promotes growth of non-small cell lung cancer cells through FoxO1 inhibition, Tumour Biol 36(10) (2015), 8121–8126.

34.

Lian

Jiao

Wang

Miao

et al., MiR-132 plays an oncogenic role in laryngeal squamous cell carcinoma by targeting FOXO1 and activating the PI3K/AKT pathway, European Journal of Pharmacology 792 (2016), 1–6.

MicroRNA-629 promotes the tumorigenesis of non-small-cell lung cancer by targeting FOXO1 and activating PI3K/AKT pathway

Abstract

OBJECTIVE:

MATERIALS AND METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Introduction

2. Patients and methods

2.1 Clinical tissues

2.2 Cell lines culture

2.3 Cell transfection

2.4 Real-time quantitative polymerase chain reaction (RT-qPCR) analysis

2.5 MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay

2.6 Transwell assay

2.7 Western blot analysis

2.8 Bioinformatics analysis

2.9 Dual luciferase assay

3. Results

3.1 MiR-629 was upregulated in NSCLC tissues

Table 1 Relationship between miR-629 expression and clinic-pathological characteristics in NSCLC patients

3.4 FOXO1 was downregulated in NSCLC

5. Conclusions

Footnotes

Conflict of interest

References

Table 1
Relationship between miR-629 expression and clinic-pathological characteristics in NSCLC patients