Background
As we all know, lung cancer results in the most deaths among cancers in the worldwide. Based on previous studies, lung cancer mainly includes two types: non-small-cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Specially, NSCLS accounts for 85% of all lung cancer cases [
1]. In addition, NSCLS can be divided into lung squamous carcinoma (LUSC), lung adenocarcinoma (LUAD) and large cell carcinoma (LCC) [
2,
3]. The role of many lncRNAs in LUSC and LUAD has been studied in previous researches [
4,
5]. Here, we mainly focus on probing molecules involved in LUAD in this investigation.
Long non-coding RNAs (lncRNAs) are non-coding RNAs longer than 200 nucleotides and play a vital role in the progression of human diseases [
6,
7]. Besides, reports also demonstrated that lncRNAs can work in certain cancer types [
8,
9]. For instance, lncRNA OIP5-AS1 is apt to aggravate LUAD progression through regulating miR-448/Bcl-2 pathway [
10]. Dysregulation of lncRNA-ATB has been shown to contribute to cell proliferation, migration and invasion, as well as EMT in cancers [
11]. LncRNA LOC389641 promotes the progression of pancreatic ductal adenocarcinoma by regulating E-cadherin [
12]. LncRNA XLOC_009167 becomes a novel biomarker owing to its overexpression in lung cancer [
13]. There are countless examples about the lncRNAs that we can’t give them one by one. As for long intergenic non-protein coding RNA 1194 (LINC01194) that is mentioned in this article, it has been recently reported to have an important influence on colorectal carcinoma development [
14]. However, the function of LINC01194 in LUAD still keeps unknown by the public, so there is urgent need to explore it.
In this research, we chiefly utilized diverse assays to verify that whether LINC01194 could act on the proliferation, migration, invasion of LUAD cells, such as colony formation assay, EdU assay, transwell assay, etc. Moreover, we further studied the interaction among LINC01194, miR-641 and SET domain containing 7, histone lysine methyltransferase (SETD7).
Methods
Clinical tissues
This study was implemented with the approval of the Ethics Committee of Dongying District People’s Hospital. Total of 64 pairs of LUAD tissues and adjacent non-tumor tissues were acquired from LUAD patients in the hospital of Dongying District People’s Hospital. Before surgery, the patients involved in this research received no any treatment and all of them signed the informed consents. Samples were processed with liquid nitrogen immediately after excision, and then maintained at -80℃ until subsequent use.
Cell lines
The normal human bronchial epithelioid cell line (16HBE) and LUAD cell lines (A549, PC-9, HCC827, NCI-H1975 and NCI-H1299) in this study were attained from ATCC (Manassas, VA). In a humidified incubator with 5% CO2 at 37 °C, cells were cultivated in DMEM (Gibco, Rockville, MD) with additional 1% penicillin/streptavidin (Gibco) and 10% FBS (Gibco).
Total RNA extraction and qRT-PCR
Total RNA extraction was performed by use of TRIzol Reagent (Invitrogen, Carlsbad CA), and then subjected to cDNA synthesis as per the protocol of PrimeScript Reverse Transcriptase Kit (Takara, Shiga, Japan). SYBR Green PCR Kit (Takara) was acquired for quantitative analyses. Relative expression of all target genes was processed based on 2−ΔΔCt method and standardized to U6 snRNA or GAPDH mRNA.
Western blot
Proteins extracted from LUAD cells were subjected to separation via 12% SDS-PAGE, followed by transferring onto PVDF membranes. After treating with 5% nonfat milk, the membranes were processed at 4℃ with the overnight incubation of primary antibodies (Abcam, Cambridge, MA) specific to SETD7 or GAPDH (loading control), which were further probed by the corresponding secondary antibodies at room temperature for 2 h. Finally, bands in the membranes were visualized with the enhanced chemiluminescence (ECL) detection system (Bio-Rad lab, Hercules, CA).
Cell transfection
The shRNAs specific to LINC01194 or SETD7, and relative control-shRNAs, were all bought from GenePharma (Shanghai, China). The pcDNA3.1 vector (Invitrogen) was loaded with the cDNA sequence of SETD7 to acquire pcDNA3.1/SETD7, and the empty vector was exploited as the negative control. Besides, miR-641 mimics/inhibitor or NC mimics/inhibitor were produced by Ribobio (Guangzhou, China). A549 and HCC827 cells were transfected with indicated plasmids for 48 h with the help of Lipofectamine 3000 (Invitrogen).
After 48 h of transfection, the processed LUAD cells were added to 6-well plates with 1000 cells per well, for the 14-day of cell culture purposes. Following fixation by formaldehyde, the colonies were stained by 0.5% crystal violet solution for counting manually.
EdU assay
EdU assay was performed in processed LUAD cells in light of the protocol of BeyoClick™ EdU Cell Proliferation Kit (Beyotime, Shanghai, China). The EdU medium diluent was added to cell samples for 2 h, and then washed in PBS and subjected to DAPI staining. All cells were studied by an inverted microscope (Olympus, Tokyo, Japan).
Transwell assay
Transwell chamber was pre-coated with Matrigel (BD Biosciences, Franklin Lakes, NJ) for invasion assay, and migration assay was performed similarly without Matrigel coating. 1 × 105 LUAD cells were collected after transfection and cultured in serum-free medium after adding into the upper chamber. The complete medium was added to the lower chamber. 24 h later, cells in the lower chamber were fixed and stained in crystal violet solution for observation using microscope (Olympus).
TUNEL assay
The transfected LUAD cell samples were rinsed utilizing PBS for fixing. Then, the TUNEL assay reagent (Merck KGaA, Darmstadt, Germany) was used for staining the apoptotic cells. After DAPI staining of cell nuclei, the optical microscopy (Olympus) was applied for analysis.
Flow cytometry analysis
The apoptosis of LUAD cells was also determined using flow cytometry (BD Biosciences, Franklin Lakes, NJ). The double Annexin V/PI staining method was employed as required by the provider (Invitrogen). After double-staining in Binding Buffer, samples were analysis via flow cytometry.
Subcellular fractionation
Subcellular fractionation assay in LUAD cells was accomplished with the application of Cytoplasmic & Nuclear RNA Purification Kit as per user manual (Norgen, Belmont, CA). LINC01194 content in cell nucleus and cell cytoplasm was separately detected using qRT-PCR, with U6 as the nuclear index and GAPDH as the cytoplasmic index.
FISH
LUAD cells were washed in PBS after fixing, and then digested and air-dried. After culturing with LINC01194-specific FISH probe (Ribobio) in hybridization buffer, cell samples were treated with Hoechst staining and finally imaged under Olympus fluorescence microscope.
RNA immunoprecipitation (RIP)
By applying Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA), RIP assay was conducted with anti-Ago2 antibody and anti-IgG antibody (negative control). The cultured cells were lysed in RIP lysis buffer, and then the lysates were treated with RIP buffer covering antibody-bound magnetic beads, followed by analysis of the precipitated RNAs by qRT-PCR.
RNA pull down assay
The miR-641 sequences covering LINC01194 or SETD7 target sites (wild-type and mutant-type) were biotin-tagged into Bio-miR-641-WT/Mut probes. Cell lysates were mixed with probes and beads overnight, and then the retrieved RNA mixture was monitored by qRT-PCR.
Luciferase reporter assay
The LINC01194 or SETD7 3′UTR fragments containing miR-641 target sites (wild-type and mutant-type) were inserted to pmirGLO dual-luciferase reporter vectors (Promega, Madison, WI), which were then named as LINC01194-WT/Mut and SETD7 3′UTR-WT/Mut. The vectors were co-transfected with miR-641 mimics or NC mimics into LUAD cells. Their luciferase intensity was individually estimated using dual-luciferase reporter assay system (Promega).
Statistical analyses
Data were exhibited as means ± SD of more than two independently conducted assays. All group differences were compared in form of Student’s t-test or one-way analysis of variance (ANOVA), by applying GraphPad Prism 7 (La Jolla, CA). The experimental results were thought as statistically significant when p < 0.05.
Discussion
It has been reported that some lncRNAs are chosen to be candidate targets because of their functional roles in LUAD. For example, since the smaller tumor size is connecting to DGCR5 down-regulation [
15]. LncRNA RCC2 promotes significantly LUAD cell migration, invasion, and proliferation [
16]. SBF2-AS1 contributes to the tumorigenesis of LUAD by sponging miR-338-3p and miR-362-3p to regulate E2F1 expression [
17]. It is reported that HOXA11 functions as an oncogene in LUAD, and LUAD patients have great overall survival rates with down-regulated HOXA11 [
18]. LPCAT1 has been reported to be involved in the progression, metastasis and recurrence of LUAD as well [
19]. HCP5 can serve as a potential therapeutic target in LUAD because overexpression of HCP5 is positively correlated with the poor prognosis of LUAD patients [
20]. Taken together, we can see it clearly that these lncRNAs make great differences to LUAD development.
As for this study, we aimed to analyze the expression level and role of LINC01194 and its molecular mechanism in LUAD. Previously, LINC01194 has been suggested to play oncogenic parts in several different cancer types, such as colorectal cancer [
14] and laryngeal squamous cell carcinoma [
21]. Importantly, a recent work also indicated LINC01194 as a carcinogene in NSCLC [
22]. Consistent with these findings, here we discovered the upregulation of LINC01194 in LUAD tissues and cells, and that silencing LINC01194 hampered LUAD cell proliferation, migration and invasion. Based on these observations, we believe that LINC01194 may also be a promising therapeutic target for LUAD.
In addition, it is still necessary for us to probe the downstream regulation mode of LINC01194 in LUAD. Recently, a cytoplasmic lncRNA usually functions as a ceRNA to exert its post-transcriptional regulation on gene expression [
23]. For instance, TTN-AS1 boosts CDK5 via sequestering miR-142-5p to facilitate malignancy in LUAD [
24]. In the present work, we unveiled miR-641 as the downstream sponged by LINC01194. MiR-641 has been previously supported as a tumor suppressor in human malignancies including lung cancer [
25,
26]. Here, we also validated that miR-641 was a tumor-inhibitor which was lowly-expressed in LUAD.
Furthermore, SETD7 was then recognized as the target of miR-641. SETD7 has been reported to play a tumor-contributing role in breast cancer [
27] and hepatocellular carcinoma [
28]. However, with respect to the role of SETD7 in lung cancer, there is a dispute over this matter in the current literatures. Cao et al. unveiled that SETD7 is downregulated in LUAD and has restraining effects on LUAD cell migration and invasion [
29]. In contrast, Fu et al. argue that SETD7 activates Hedgehog pathway to aggravate the tumorigenesis of NSCLC [
8], and Lezina et al. believe that SEDT7 contributes to cell proliferation in lung tumors [
30]. In this work, we disclosed that SETD7 was upregulated in LUAD tissues and cells, and that the absence of SETD7 led to impaired proliferative, migratory and invasive capacities of LUAD cells. In other word, SETD7 was unveiled as a tumor-promoter in LUAD by the current research. Moreover, we certified that LINC01194 contributed to LUAD progression through miR-641/SETD7 signaling.
Conclusion
In this investigation, LINC01194 is highly expressed in LUAD and is recognized as a carcinogenic gene to push LUAD cell proliferation, migration and invasion. In addition, our study also presents that miR-641 could combine with LINC01194 and down-regulation of miR-641 also elevates cell proliferation. In this work, we further identify that SETD7 is downstream gene of miR-641, and SETD7 increasing tends to promote LUAD cell progression as well. In conclusion, all data verify that LINC01194/miR-641/SETD7 regulatory axis contributes to LUAD progression, which implies that LINC01194 may serve as a biomarker and potential therapeutic target for LUAD.
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