Knockdown of LINC01614 inhibits lung adenocarcinoma cell progression by up‐regulating miR‐217 and down‐regulating FOXP1

Abstract We tried to identify the function of LINC01614 in lung adenocarcinoma (LUAD) and reveal its underlying mechanisms. qRT‐PCR was applied to assess the expression of LINC016014 in LUAD tissues, noncancerous tissues and cells. Through colony formation assay, MTT assay and apoptosis analysis, we examined the variation of cell proliferation and apoptosis ability after silencing LINC01614. Moreover, the targeting interactions among LINC01614, miR‐217 and FOXP1 were validated via luciferase reporter assay, and then, we regulated the expression of miR‐217 and FOXP1 to ascertain their importance in cell proliferation and apoptosis. LINC01614 and FOXP1 were found to be up‐regulated in LUAD tumours and cells, whereas miR‐217 was down‐regulated. The experiment showed that target‐specific selectivity exists between LINC01614‐miR‐217 and miR‐217‐FOXP1 3′UTR. Furthermore, we disclosed that inhibition of LINC01614 could activate miR‐217, which subsequently restrained FOXP1. It was proved that LINC01614 promoted FOXP1 by inhibiting miR‐217, which ultimately stimulated the development of LUAD.


Introduction
Lung cancer, which consisted of non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), maintains a high mortality rate among a variety of cancers [1]. NSCLC includes lung squamous cell carcinomas, LUADs and large cell carcinomas [1]. LUAD is the most prevalent subtype of NSCLC, with an increasing morbidity and mortality rate over these years worldwide. Platinum-based regimens are standard chemotherapies, but its side effects usually weakened the therapeutic effects. Therefore, LUAD patients still have very low survival rate in spite of multiple therapies [2]. It is urgent for researchers to identify more efficient and effective therapeutic targets for LUAD [3].
Long non-coding RNAs (lncRNAs) represent an emerging and fascinating class of transcripts, which play a significant role in many different bioprocesses [4]. For instance, by binding to miRNA-binding sites, lncRNA functions as the regulator of gene expressions and this kind of regulation has been detected in various diseases [5][6][7][8][9]. LncRNA could be effective biomarkers for early diagnosis and screening of cancer in specific tissues and organisms [10]. Increasing number of studies about lncRNA relating to various cancers, including colon cancer, oesophageal squamous cell carcinoma and gastric cancer, have been intensively conducted [11][12][13]. Several lncRNAs related to lung cancer have been discovered, for example MALAT1 [14], LINC01186 [15] and LINC01207 [16]. With the rapid development of cutting-edge techniques like RNA sequencing and microarray, more and more lncRNAs in lung cancer have been characterized [17,18]. Located in the genomic 2q35 locus, LINC01614 has two exons and could be transcribed into an lncRNA of 2904 nucleotides (nt). In the current research, LINC01614 expressions and functions were closely investigated.
MicroRNAs (miRNAs) are small interference RNAs with about 20nt which could regulate gene expressions via binding to mRNA and silencing target genes [19]. Mounting evidence has indicated that miRNAs participate in many important bioprocesses, including cell proliferation, differentiation and apoptosis [20,21]. Down-regulation of miR-217 is frequently discovered in various cancers, suggesting its important role in tumorigenesis [22][23][24]. However, the potential role of miR-217 in LUAD is elusive. In this study, we studied miR-217 expressions in LUAD tissues and cell lines and its effects on morphologic changes in LUAD.
The forkhead box P subfamily is a group of transcription factors with highly conserved forkhead or winged helix DNA-binding domain [25,26], which take part in a wide range of developmental events, including cellular differentiation and proliferation, pattern formation, immune regulation, signal transduction and oncogenesis [27]. Recent researches have revealed that some members of this protein family are relevant to tumorigenesis and cancer progression, and an increasing amount of evidence suggests that FOX can act as direct targets and indirect effectors of therapeutic intervention. For example, FOXP1 dysregulation is related to poor prognosis in B-cell lymphomas and NSCLC [28,29]. However, when it comes to other cancers like neuroblastoma or prostate cancer, FOXP1 can inhibit cell growth and attenuate tumorigenesis as a tumour suppressor [30,31]. Hence, the role of FOXP1 in tumour progression is uncertain.
Here, we demonstrated LINC01614 played an important role in LUAD. The results showed that LINC01614 strongly promoted tumorigenesis and progression through miR-217/FOXP1 axis. This study may offer new knowledge for understanding the occurrence and development of LUAD.

Clinical samples
A total of 23 pairs of LUAD tissues and matched normal tissues were obtained from the Affiliated Yantai Yuhuangding Hospital of Qingdao University Medical College; all of them were independently diagnosed by at least three experienced pathologists. All patients never receive chemoradiotherapy or other treatments before operation. All samples were immediately transferred to frozen liquid nitrogen and stored in À80°C refrigerator before further analysis. Each participant signed informed consent, and this study went through the ethic approval of Ethics Committee of the Affiliated Yantai Yuhuangding Hospital of Qingdao University Medical College.

Quantitative RT-PCR
After being isolated by TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and quantified by NanoDrop 2000 (Thermo Fisher Scientific Inc, Rockford, IL, USA), 20 ng of total RNA underwent reverse transcription by ReverTra Ace qRT-PCR Kit (Toyobo, Osaka, Japan) on the basis of the manufacturer's instruction, and the product was then used for real-time PCR by THUNDERBIRD SYBR â qPCR Mix (Toyobo). PCR conducted as follows: predegeneration at 94°C for 2 min.; denaturation at 94°C for 30 sec.; annealing at 56°C for 30 sec.; extension at 72°C for 1 min.; repeating the previous process for 30 times; continuous extension at 72°C for 10 min. With GRAPDH and U6 as the internal references, experiment of each group was repeated three times, respectively, and quantification of the relative expression levels of lncRNA, mRNA and miRNA by 2 ÀDD Ct method. Primers sequences were present in Table 1.

Western blot
Total protein was lysed with RIPA lysate (Beyotime, Shanghai, China). After being quantified by Pierce BCA Protein Assay Kit (Pierce, Rockford, IL, USA), 10 lg of proteins was electrophoresed in SDS-PAGE and blotted on a PVDF membrane for 120 min. under 200 mA constant current. The membrane was blocked in Tris buffer saline-Tween-20 (TBST) with 5% fat-free milk at room temperature for 1 hr. Then, primary antibody (anti-FOXP1, ab16645, 1:5000; anti-GAPDH, ab9485, 1:2500; Abcam, Cambridge, MA, USA) was incubated overnight under 4°C. Then, the membrane was rinsed in TBST for 3 times. Afterwards, the secondary antibody (goat anti-rabbit IgG, ab6721, 1:5000; Abcam) was added and then incubated for 1 hr and rinsed in TBST for 3 times. After visualization with Enhanced Chemiluminescence Plus (ECL Plus) Detection System (Thermo Scientific), the immunoreactive proteins were processed by Lab Works 4.5 software to detect the integral optical density value. GAPDH was used to compare with the proteins in grey density value.

Colony formation assay
Cells in the period of logarithmic phase were isolated by pancreatic enzyme digestion and then incubated in RPMI1640 medium. Afterwards, cells were inoculated in 6-well plates at a density of 1 9 10 3 cells/well. Cells were shaken evenly and then put into an incubator supplemented with 5% CO 2 at 37°C. Cultivation was terminated at the sight of visible cell clones. After being washed with FBS and dehydrated, cells were fixed with methanol and stained with violet. Last, staining fluid was removed and cells were dried. Five fields were randomly selected, and cells were counted with a microscope. All experiments were practised three times.

MTT assay
To prepare a single cell suspension, cells were cultured in a medium with 10% FBS and then plated in 96-well plates at a density of 2 9 10 3 cells/well. Then, 10 ll of MTT reagent (5 mg/ml, Sigma-Aldrich) was added to each well and incubated for 4 hrs at 37°C. After culturing termination, supernatant in each well was separated and discarded (suspension cell need to be centrifuged first). Samples in each well were dissolved in 100 ll DMSO, and the absorbance was recorded at 490 nm using ELISA plate reader.

Statistical analysis
Data comparison between two groups was carried out using Student's two-tailed t-test, while comparison among three or more groups was employed with one-way analysis of variance (ANOVA). All experiments were repeated at least three times. All statistical calculations were performed by the software package GraphPad Prism version 6.0 (GraphPad Prism, La Jolla, CA, USA). A value of P < 0.05 was considered statistically significant. All data are present as mean AE S.D.

Down-regulated LINC01614 inhibited LUAD progression
Twenty-three cancerous and adjacent noncancerous tissues were used to identify the expression level of LINC01614 in LUAD via qRT-PCR. The analysis showed that LINC01614 was highly expressed in LUAD tissues compared to noncancerous tissues ( Fig. 1A, P < 0.01). Additionally, the expression levels of LINC01614 in two LUAD cell lines (NCI-H1395, NCI-H1975) and normal pulmonary epithelial cells (BEAS-2B) were detected (Fig. 1B), and qRT-PCR results showed the LINC01614 were significantly up-regulated in two LUAD cell lines compared to BEAS-2B cells (P < 0.01). After being transfected with siRNA1 or siRNA2, LINC01614 expression was significantly downregulated and siRNA1 displayed a strong inhibition effect (Fig. 1C, P < 0.01). Figure S1 showed that the most suitable transfection concentration for siRNAs was 50 nM considering transfection and economy efficiency. Then, we observed the variation of cell proliferation through colony formation assay and MTT assay, the results of which indicated that the cell proliferation was attenuated when the LINC01614 was inhibited ( Fig. 1D and E, P < 0.01). Apoptosis   analysis suggested that apoptosis rate was higher in siRNA1-and siRNA2-transfected LINC01614 groups than in the mock groups ( Fig. 1F, P < 0.01). Taken together, we revealed that LINC01614 knockdown inhibited cell proliferation and promoted cell apoptosis in LUAD cells. Therefore, we verified that the LINC01614 contributed to growth and metastasis of LUAD.

Inhibition of LINC01614 restrained the expression of FOXP1
By qRT-PCR and Western blot, we proved FOXP1 was overexpressed in NCI-H1395 and NCI-H1975 cell lines compared to BEAS-2B ( Fig. 2A and B, P < 0.01). In both tumour cell lines, LINC01614 knockdown significantly inhibited FOXP1 expression ( Fig. 2C and D, P < 0.01). Therefore, we supposed that LINC01614 may impact cancer development by regulating FOXP1.
were dramatically increased in miR-217 mimics groups in both NCI-H1395 and NCI-H1975 cells. Collectively, these data showed LINC01614, which suppressed miR-217 and activated FOXP1, and then promote cell proliferation.

Discussion
Mammalian genomes are confirmed to encode numerous lncRNAs, which used to be regarded as 'junk' or 'transcription noise'. At present, there is growing evidence confirming that many lncRNAs are playing a very important role in bioprocesses [32,33]. Accommodative dysfunction of lncRNAs has been certified to exist in various diseases [34][35][36]. Those lncRNAs could also provide more particular knowledge of cancer mechanism [37]. Meanwhile, the majority of those cancer-associated lncRNAs have been proved to be effective biomarkers [37,38]. However, deeper functions of tumour-related lncRNAs remain to be unveiled. White et al. [4] has reported various lncRNAs related to LUAD via RNA-seq data analysis. In the current study, we found a novel lncRNA, LINC01614, was up-regulated among all LUAD tissues and cells. Moreover, inhibition of LINC01614 in NCI-H1395 and NCI-H1975 cells significantly decreased the cell proliferation, migration and invasion while markedly enhanced cell apoptosis. Thus, we came to the conclusion that down-regulated LINC01614 inhibited the cell proliferation in LUAD.
It was approved by dozens of researches that FOXP1 may be a very valuable marker for cancer patients' prognosis. However, the expression patterns of FOXP1 vary in different types of cancers. It was confirmed by Banham et al. [39] that complete loss of FOXP1 protein was inhibited the progression of both six SCLCs and six NSCLCs. Simultaneously, Kyoko et al. have reported that reduced expressions of FOXP1 may play key a role in the development of LUADs in rats [40]. Intriguingly, by qRT-PCR and Western blot, we identified that FOXP1 was overexpressed in LUAD, which was in accordance with the research conducted by Feng et al. [28]. We further discovered that down-regulation of FOXP1 inhibited cell progression of LUAD. Moreover, a positive correlation between FOXP1 and LINC01614 expression can be drawn based on our results.  These results clued the underlying molecular mechanism in the carcinogenesis and metastasis of LUAD. According to the ceRNA hypothesis, lncRNAs may elicit their biological effects through their ability to act as endogenous decoys for miRNAs. Such activity would affect the ability of miRNAs to bind their targets [41]. The present study showed that LINC01614 could regulate FOXP1 via miRNA. Through in silico analysis, we found miR-217 could be a promising target of LINC01614 and FOXP1, which was then confirmed via luciferase assay. To further confirm the relationship among LINC01614, miR-217 and FOXP1, we conducted the qRT-PCR and Western blot experiments and verified that si-LINC01614 suppresses FOXP1 via the promotion of miR-217. In the end, cell proliferation assay revealed that LINC01614 knockdown significantly inhibited cell proliferation through miR-217/FOXP1 axis and vice versa. Apoptosis assay also showed that LINC01614 functioned as an inhibitor of miR-217 and that it could increase the expression levels of the miR-217 target gene, FOXP1, to promote cell proliferation.
Our study provides novel insights into the role of LINC01614 in cancer and may aid in the development of diagnostic and therapeutic tools for LUAD treatment. As no research about the LINC01614 function in diseases has been performed so far, our study is the first one that demonstrates the biological function of LINC01614 in LUAD. Further studies with a larger pool of samples should be conducted to verify the prognostic value of LINC01614 in LUAD. Meanwhile, further experiments such as whether FOXP1 does indeed act as a tumour suppressor in LUAD are in need.

Funding source
None.

Conflict of interests
The authors declare no conflict of interests.

Supporting information
Additional Supporting Information may be found online in the supporting information tab for this article: Figure S1 The most suitable transfection concentration for si-LINC01614 and si-FOXP1 in NCI-H1395 and NCI-H1975 cells.