MicroRNA-665 Regulated Cell Proliferation, Migration and Apoptosis by Targeting Nemo-like Kinase in Non-small Cell Lung Cancer


 Background: Many evidences proved important roles of microRNAs in the occurrence and development of non-small cell lung cancer (NSCLC). This work aimed to explore the effects and regulating mechanism of microRNA-665 (miR-665) in NSCLC. Methods: Here, quantitative real time PCR was used to evaluate the expression level of miR-665 in NSCLC tissues and several cell lines (H520, H1299, PC9, H358, and A549). Then, MTT, apoptosis assays and wound-healing assays were performed to study miR-665 functions on cell proliferation, apoptosis and migration. Based on bioinformatics analysis, nemo-like kinase (NLK) was predicted as a potential target of miR-665 and then a series of experiments were performed to explore miR-665 mechanism.Results: In NSCLC tissues and several cell lines, miR-665 was up-regulated and cell experiments displayed that it had abilities to promote cell proliferation and migration, and inhibit cell apoptosis. MiR-665 over-expression could reduce NLK mRNA and protein level by binding to 3′-untranslated region of NLK, which suggested that miR-665 could regulate NLK expression. Further analysis found that there was an inverse correlation f between miR-665 and NLK in NSCLC. Moreover, low-level NLK possessed same effects on cells as miR-665 over-expression, and its knockdown could partly recovery the miR-665-mediated cell proliferation, migration and anti-apoptosis, indicating that miR-665 might regulate biological behaviors of NSCLC cells via targeting NLK. Conclusion: MiR-665 probably promoted NSCLC occurrence and progression via targeting NLK, which might be a hopeful biomarker for NSCLC diagnosis or a target for treatment.

and diagnosis of lung cancer. In 2004, carcinoembryonic antigen as a protein biomarker has been approved for lung cancer diagnosis. Unfortunately, it has poor effect and it has better to be combined with other biomarkers (such as cytokeratin fragment-21) [7,8]. Thus, more biomarkers were necessary for more accurate detection of lung cancer, particularly early stage.
In clinic, the biomarkers used for lung cancer detection, include tumor in ltrating lymphocytes, cell-free DNA (cfDNA), autoantibodies, microRNAs (miRNAs) and so on [9][10][11][12]. Among them, miRNAs are considered as important players in many biological processes and linked with pathological parameters of various cancers, such as ovarian cancer, liver cancer and colorectal cancer, implying their potential as cancer biomarkers [13][14][15]. In lung cancer, some studies disclosed that many miRNAs displayed 92-100% speci city and 75-85% sensitivity in differentiation between lung cancer and normal samples [2,16]. Moreover, expression regulation of miRNAs probably was linked with the occurrence and progression of lung cancer. For instance, miR-4735-3p and miR-183-5p were over-expressed in NSCLC tissues and cell lines, while miR-143 and miR-520a-3p were down-regulated in lung cancer [17][18][19][20]. Nevertheless, the regulatory mechanism of miRNAs is still exploring in lung cancer and other cancers. It was generally accepted that the miRNAs possibly regulated expression of target genes in tumors by blocking or degrading mRNA molecules of target genes [21,22]. More works need to be done to deeply understand their mechanism, especially NSCLC.
MicroRNA-665 (miR-665), an exosomal miRNA, has been found that it is associated with different diseases, like congestive heart failure [23], anesthesia-related cognitive dysfunction [24], in ammatory bowel disease [25], and cancer [26]. Interestingly, miR-665 showed different functions for different diseases. For examples, miR-665 upregulation promoted cell apoptosis and colitis in in ammatory bowel disease through suppressing the endoplasmic reticulum stress components XBP1 and ORMDL3; while its high-expression could promote tumor growth and cell proliferation in hepatocellular carcinoma by increasing the expression of proteins in the MAPK/ERK pathway [25,26]. However, its effects on NSCLC have not been reported. The key purpose of this work was to explore miR-665 roles and possible mechanism in NSCLC. Firstly, quantitative real time PCR (q RT-PCR) was performed to measure the expression level of miR-665 in NSCLC tissues and cell lines. Then, we constructed cells that could stably over-express and low-express miR-665 by cell transfection, and then studied its functions on cell proliferation, migration and apoptosis. Finally, prediction software was used to obtain a potential target of miR-665 and further experiments were carried out to verify it.

Specimen collection
This research has been approved by the Ethics Committee of Qilu Hospital, Cheeloo College of Medicine, Shandong University, and each patient participating in this study has signed the written informed consent. We collected 38 pairs of NSCLC tissues and corresponding normal lung tissues from patients who were diagnosed with NSCLC based on histopathological evaluation and undergone surgery at this hospital during 2015-2017. In addition, none patients had treated by radiotherapy or chemotherapy before surgery. All tissue specimens were ash-frozen in liquid nitrogen and preserved at -80°C.

QRT-PCR
Total RNA of cells or tissues was obtained using the TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Then, the RNA was used to synthesize cDNA by reverse transcriptase M-MLV (Takara, Dalian, China) following the manufacturer's instruction to synthesize cDNA. Q RT-PCR tests were performed using the standard SYBR Green Assay protocol and the ABI PRISM 7500 Sequence Detection System (ABI). The reaction conditions were as below: pre-denaturation at 95 °C for 10 min, denaturation at 95 °C for 10 s, renaturation at 60 °C for 60 s, and extension at 70 °C for 10 s. Q RT-PCR had a total of 40 cycles. The primers were as follows: miR-665, forward: 5'-ACCAGGAGGCTGAGGCCCCTAA-3', reverse: 5'-GCTGTCAACGATACGCTACCTA-3'; NLK, forward: 5'-ATCATCAGCACTCGCATCATC-3', reverse: 5'-GACCAGACAACACCAAAGGC-3'. U6 snRNA was used as a normalization control of miR-665. Lactate dehydrogenase A (LDHA) was also analyzed by q RT-PCR using SYBR Premix Ex Taq (TaKaRa) and GAPDH acted as internal control. Cycle threshold (Ct) of each gene was recorded and applied to calculate the relative expression of genes as per 2 − ΔΔCt method.

Cell proliferation assay
Cell proliferation assay was performed according to MTT method. H358 cells (1 × 10 3 cells per well) were plated into 96-well plates after transfection. At the indicated time points (1, 2, 3, and 4 day), 50 µL MTT (2 mg/mL; Keygen, China) solution was added into each well and the cells were incubated for 4 h at 37 °C. After that, the culture medium was removed and 150 µL DMSO was added into wells. The optical density (OD) of each well at 550 nm was measured in a Tecan In nite Multiskan (Tecan, Swiss).

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2.5 Cell apoptosis assay H358 cells (1 × 10 5 cells) were seeded into 6-well plates and transfected with miR-665 mimic/inhibitor or mimic NC/inhibitor NC. After 48 h transfection, cells were harvested and resuspended in 500 µL binding buffer. Then cell suspension was incubated with 5 µL Annexin V-FITC for 15 min in the dark, following 5 µL of PI (50 µg/mL) was added to each sample. Finally, ow cytometry (C6; BD Biosciences, Franklin Lakes, NJ, USA) was used to determine apoptosis of the H358 cells.

Wound healing assays
The migration ability of NSCLC cells was investigated by wound healing assays. After 48 h cell transfection, H358 cells were plated in 24 well plates at a density of 8 × 10 4 cells/well and cultured until they formed a con uent monolayer. Wounds were scratched by 10 µL pipette tips. Then, each well was washed 3 times with phosphate-buffered saline (PBS) and added serum-free medium. QImagine Software was used to photograph each well every 12 h.

Luciferase reporter assay
The 3′-UTR of nemo-like kinase (NLK) mRNA, containing a putative target region for miR-665, was ampli ed from genomic DNA by PCR. The mutant of NLK 3′-UTR was constructed by overlap extension PCR. Then, fragments obtained were inserted between the XhoI and NotI sites in the psiCHECK™-2 Dual Luciferase miRNA target expression vector (Promega, USA) to produce the recombinant vectors of NLKwide type (NLK-WT) and NLK-mutant type (NLK-MT). Both recombinant vectors were detected by sequencing (Sangon, China). The reporter vectors and miR-665 mimic/mimic NC was co-transfected into H358 cells using Lipofectamine 2000. After 48 h co-transfection, luciferase activity of each group was detected using the Dual Luciferase Reporter Assay Kit (Promega, USA) based on the direction.

Western blotting
Cells were rstly washed twice using Hanks' balanced salt solution and lysed in RIPA lysis buffer (50 mM Tirs-Cl, pH 7.4, 120 mM NaCl, 1% NP-40, 0.2% SDS, 1 mM EDTA and complete protease inhibitor), following centrifuged at 4 °C for 20 min with 13,000 g. Subsequently, the protein concentration was tested by a BCA Protein Assay Kit (Beyotime, China) based on the instructions. Next, 20 µg of protein were denatured using 4 × loading buffer (Takara, Japan) at 95 °C for 5 min. Equal quantities of protein were separated via electrophoresis using 10% SDS polyacrylamide gel, and then transferred onto polyvinylidene di uoride (Life Technologies, USA) membranes. The membranes were rstly washed by PBS and blocked with 5% non-fat milk. Then, the membranes were incubated with anti-NLK (1:500; Abcam, UK, ab97642) and anti-GAPDH (1:2000; Abcam, UK, ab9485) at 4 °C overnight. After the membranes were washed three times with PBS containing 5% Tween (Sigma-Aldrich), they were incubated with horseradish peroxidase-conjugated rabbit anti-mouse secondary antibody at room temperature for 2 h. An ECL kit was used to visualize the protein bands according to the manufacturer's instructions. The relative protein expression levels were analyzed using Image-ProPlus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

Statistical analysis
All data were analyzed via SPSS 17.0 software and expressed as mean ± standard deviation. The independent-samples t-test was used for the comparison between two groups, and the analysis among more than two groups was performed using the one-way ANOVA test, followed by Bonferroni's post-hoc test. If P < 0.05, the results were statistically signi cant.

Results
3.1 MiR-665 was greatly up-regulated in NSCLC tissues and cell lines To con rm whether miR-665 had abnormal expression in NSCLC, we rstly performed q RT-PCR to detect the expression level of miR-665 in 38 pairs of NSCLC tissues and adjacent normal lung tissues. Figure 1A showed that the level miR-665 was signi cantly higher in an independent panel of 38 primary lung tumors than that in normal matched lung tissue (P < 0.01). Subsequently, we determined an average of 2.0546 based on the relative miR-665 expression level. NSCLC patients were divided into high expression group (≥ 2.0546, n = 18) and low expression group (< 2.0546, n = 20) based on the average miR-665 expression in 38 samples. Moreover, to assess the clinical signi cance of miR-665, we evaluated the correlation between expression level and clinicopathological parameters. Table 1 displayed that miR-665 expression was closely associated with smoking history, while it was not linked to other clinical risk factors including age, gender, tumor type and differentiation, T-classi cation, and lymphatic metastasis (*P < 0.05). We then explored miR-665 expression levels in ve NSCLC cell lines (H520, H1299, PC9, H358, and A549). Figure 1B revealed that miR-665 had higher level in four NSCLC cell lines (A549, H358, H1299 and PC9) than that in BEAS-2B (*P < 0.05, **P < 0.01), while there was no signi cant difference between H520 and BEAS-2B (*P > 0.05). These results indicated that the expression of miR-665 was up-regulated in NSCLC, which might have roles on NSCLC progression.

MiR-665 enhanced proliferation and migration ability of NSCLC cells and inhibited cell apoptosis
In order to explore the underlying functions of miR-665 in NSCLC development and progression, H358 cells were transfected by miR-665 mimic, inhibitor and their corresponding negative control oligonucleotides to regulate miR-665 expression, and q RT-PCR results ( Fig. 2A, B) displayed that miR-665 mimic signi cantly increased the level of miR-665 while its inhibitor reduced its expression, suggesting cell transfection was successfully done (**P < 0.01). Then, MTT, wound-healing assay and FACS analysis were respectively performed to study the roles of miR-665 on cell proliferation, migration and apoptosis in H358 cells. As shown in Fig. 2C, cells transfected with miR-665 mimic showed signi cantly higher OD values at 550 nm from day 2 to day 4 in a time dependent manner (**P < 0.01). However, the miR-665 inhibitor group displayed lower OD values (Fig. 2D) implying that cell proliferation was suppressed (*P < 0.05, **P < 0.01). Figure 3A and B showed that overexpression of miR-665 signi cantly promoted cell migration (*P < 0.05), while its down-regulation had opposite effect (**P < 0.01). As shown in Fig. 4A and B, cell apoptosis was signi cantly inhibited in miR-665 mimic group compared with NC group (**P < 0.01) and miR-665 inhibitor group had higher apoptosis rate (**P < 0.01), which suggested that miR-665 may affect apoptotic pathways in regulating tumorigenicity. The above results suggested that miR-665 upregulation could promote the cell proliferation and migration, and inhibit cell apoptosis for NSCLC cells in vitro.

NLK was a novel target gene of miR-665 in H358 cells
To nd the underlying mechanisms of miR-665 in NSCLC, we investigated potential targets of miR-665 using prediction software (TargetScan, PicTa, miRanda, the miRBase, DIANA TarBase). Bioinformatic analyses predicted that NLK was a potential target of miR-665 (Fig. 5A). To validate whether miR-665 might mediate the decay of NLK mRNA via the 3′-UTR, we generated NLK-WT and NLK-MT vectors, and these recombinant vectors were transfected into H358 cells with miR-665 mimic or NC miRNA. Figure 5B showed that miR-665 overexpression remarkably down-regulated the luciferase activity of the NLK-WT group (**P < 0.01), while it was not in uenced the luciferase activity in NLK-MT group (*P > 0.05). As shown in Fig. 5C and D, mRNA and protein levels of NLK were signi cantly decreased in miR-665 mimic group compared to NC group (**P < 0.01). Furthermore, NLK was low-regulated in NSCLC tissues and there was a negative correlation between NLK and miR-665 ( Fig. 5E and F). These data demonstrated that miR-665 could down-regulate NLK expression by directly targeting NLK 3′-UTR.

MiR-665 regulated cell growth, migration and apoptosis of NSCLC cells by targeting NLK
H358 cells were transfected with NLK shRNA or NC shRNA to further investigate whether the effect of miR-665 on NSCLC cells was via targeting NLK. Figure 6A showed that the three kinds of NLK shRNA could obviously decrease the relative mRNA level of NLK, implying successful transfection (**P < 0.01). As shown in Fig. 6B, the OD value in H358 cells transfected with NLK shRNA was signi cantly higher than those transfected with NC shRNA after transfection 2 days (*P < 0.05, **P < 0.01). Figure 6C and D indicated that suppressing NLK expression by shRNA could promote cell migration and inhibit cell apoptosis (**P < 0.01). From the above, our ndings suggested that miR-665 promoted cell growth, migration and inhibited apoptosis of NSCLC cells, at least in part by targeting NLK.

Interference of NLK partly restored miR-665-mediated proliferation and migration of NSCLC cell
Finally, to deeply verify the mechanism of miR-665 in NSCLC cells, the NLK-targeting shRNA oligo were employed to deplete endogenous NLK in NSCLC cells. NC, miR-665 inhibitor and NLK shRNA oligo were transfected independently or simultaneously into H358 cells. As shown in Fig. 7A and B, depletion of NLK induced by transfection of NLK shRNA could obviously promote cell proliferation and migration (*P < 0.05, **P < 0.01), and miR-665 inhibitor weakened them, compared with NC group. However, cell proliferation and migration abilities were partly improved in cells co-transfected with miR-665 inhibitor and NLK shRNA, compared to the cells only transfected with miR-665 inhibitor (**P < 0.01).In Fig. 7C, the result of FACS analysis exhibited that the transfection of NLK shRNA could obviously reduce cell apoptotic (**P < 0.01), and the NLK shRNA weakened the roles on apoptotic caused by miR-665 inhibitor in some degree, indicating that depletion of NLK could partly recovery the miR-665-mediated antiapoptotic effect. Based on all data, we inferred that miR-665 probably regulated cell proliferation, migration and apoptosis in NSCLC by manipulating NLK expression.

Discussions
As one of the most common malignancy, lung cancer has become a leading cause of cancer-related death worldwide, and its morbidity and mortality come out in front in various malignant tumors [27]. NSCLC account for nearly 80% cases and every year more than thousands of patients were diagnosed with NSCLC. In clinical, the delay of detection and diagnosis for NSCLC patients are a serious problem. At present, most patients were diagnosed in locally advanced or metastatic stages, while only 16% of them were detected before malignancy [28]. In addition, the survival time of patients with NSCLC at early stages was much longer than those at late stage [29]. Moreover, because NSCLC showed insensitivity to chemotherapy, therapeutic outcomes were worse and the patients had poorer prognosis [5]. Thus, to identify potential biomarkers for NSCLC is quite essential, which will contribute to improve detection and therapeutic strategy in clinical management. MiRNAs, containing 19 to 25 nucleotides, play important roles in cell growth, proliferation and apoptosis, which makes it exert as oncogenes or anti-oncogenes in different tumors. Various miRNAs have been considered as potential speci c biomarker for cancers, for example, miR-21 showed 100% specificity and 70% sensitivity in NSCLC cases and it was related with aggressive clinicopathologic characteristics and poor prognosis of NSCLC patients [31,32]. Thus, searching potential miRNA in NSCLC was good strategy.
Previous papers reported that miR-665 possessed different functions in different diseases. For examples, it had neuroprotective effect against sevo urane anesthesia-induced cognitive dysfunction, and it promoted apoptosis and colitis in in ammatory bowel disease [24,25]. However, the expression level and effects of miR-665 in NSCLC were still unclear. In this work, we mainly studied the effects and possible mechanism of miR-665 in NSCLC. The results of q RT-PCR showed that it was up-regulated in NSCLC tissues and four cell lines (A549, H358, H1299 and PC9), implying that it might be a candidate gene used for NSCLC diagnosis. The further experiments exposed that miR-665 over-expression could promote cell proliferation and migration and inhibit apoptosis for NSCLC cells in vitro. Recent paper reported that miR-665 was increased in hepatocellular carcinoma and it possessed promoting ability on tumor growth and cell proliferation [26]. Our results were consistent with their, suggested that miR-665 might act as oncogenes in cancers and have close relationships with occurrence and progression of cancers.
Many studies have proved that there were close relationships between miRNAs and cancers, but the mechanism needed more researches. Some experiments displayed that miRNAs could acts as posttranscriptional regulators to regulate gene expression via binding to 3′-untranslated region (3′-UTR) of their target mRNAs [23,30]. Then, we used prediction software to obtain a potential target of miR-665-NLK. Luciferase reporter assay displayed that miR-665 might promote decay of NLK mRNA by basepairing to the 3′-UTR of NLK. The results of q RT-PCR and western blot showed that miR-665 up-regulation could signi cantly reduce the mRNA and protein level of NLK, proving that miR-665 might regulate NLK expression by targeting 3′-UTR. Moreover, suppression NLK could enhance proliferation and migration ability, while weaken cell apoptosis. To verify whether miR-665 played roles on cancer cells by regulating NLK, MTT, wound healing and ow cytometry assays were performed after H358 cells were transfected with NLK shRNA, miR-665 inhibitor, or miR-665 inhibitor + NLK shRNA. The data showed that depletion of NLK could restore miR-665-mediated proliferation, migration and anti-apoptotic functions for NSCLC cells in part.

Conclusions
This work found that miR-665 was up-regulated in NSCLC tissues and cell lines (A549, H358, H1299 and PC9), and it mediated proliferation, migration and anti-apoptotic in NSCLC probably by targeting NLK, suggesting that miR-665 might exert as an oncogene in NSCLC. In addition, miR-665 was hopeful to be a potential biomarker for NSCLC diagnosis or a new target for NSCLC treatment.

Declarations
Ethics approval and consent to participate The present study was approved by the Ethics Committee of Xinxiang Central Hospital. The research has been carried out in accordance with the World Medical Association Declaration of Helsinki. All patients and healthy volunteers provided written informed consent prior to their inclusion within the study.

Consent for publication
All authors have read and approved the nal manuscript.
Availability of data and materials The analyzed data sets generated during the study are available from the corresponding author on reasonable request.

Competing interests
There was no any con ict of interest. Authors contribution CH and HT concept, study design, experiments, manuscript preparation. CH, WMY, LL, SHL, CG, LQ, CLC, ML and LBS experiments, data analysis, manuscript preparation. All authors have read and approved the manuscript.