Identification And Mechanism Research Of Malignant Phenotype And Pronosis In Non-small Cell Lung Cancer Patients Of Kinesin Light Chain 2, A Novel Target Of MiR-125b

BackgroundLung cancer is estimated to be responsible for nearly one-fifth of cancer-related deaths worldwide in 2012.Metastasis is still the leading cause of failure in clinical management of lung cancer. Increasing evidence supports the involvement of microRNAs in the development of many human cancers including lung cancer. Recently, miR-125b has been demonstrated to have important roles in the invasion and migration and its reported targets differed in various cancers. For example, miR-125b could inhibited melanoma cells migration by direct regulation of C-Jun protein expression or targeting serine/threonine kinase mixed lineage kinase （MLK）3 at both the transcriptional and translational levels. MiR-125b was also reported to function as a tumor suppressor and inhibit Ewing’s sarcoma cell migration and invasion by targeting the PI3K/Akt/mTOR signaling pathway. MiR-125b exerted tumor-suppressive effects in hepatocellular carcinoma metastasis not only through the suppression of oncogene Lin28 homolog B （LIN28B） expression but also by the downregulation of suppressor of variegation 3-9 homolog 1 （SUV39H1）. In addition, matrix metallopeptidase （MMP）13 was identified as a direct target suppressed by miR-125b in cutaneous squamous cell carcinoma. Contrarily, the role of miR-125b in pro-metastasis was verified both in breast cancer cells and type I endometrial carcinoma cells by targeting tumor suppressor genes StAR-related lipid transfer domain containing （STARD）13 and Tumor Protein 53-Induced Nuclear Protein 1 （TP53INP1） separately. In lung cancer, patients with high serum miR-125b expression displayed a significantly poorer prognosis. In our previous studies[11] we identified miR-125b as a downregulated miRNA in non-small cell lung cancer （NSCLC） cell line upon metastasis-associated gene （MTA）1 depletion and verified that miR-125b reversed the stimulatous effect of MTA1 on the migration and invasion. However, the target genes elucidating the role of miR-125b in NSCLC invasion and migration have not been identified.Recently, we used bio informatics analysis to identify its target genes. Kinesin-1 light chains （KLC）2 was identified as one of the candidate targets of miR-125b based on bioinformatics analysis from three publicly available miRNA databases （Targetscan, miRanda, miRWalk）, potential binding site to miR-125b was predicted in the 3’-untranslated region （3’UTR） of KLC2.KLC2 is one of the best characterised light chain isoforms of Kinesin-1, a widely expressed molecular motor that drives transport of numerous cargoes including intracellular organelle and a variety of vesicle subtypes along microtubules. Smad2, required for transduction of TGFjJ signals, is a known KLC2 binding partner and kinesin-1 cargo. TGFβ signaling have a pleiotropic action during tumorigenesis. Another clue to KLC2 function in cancer comes from the findings that KLC2 was involved in the regulation of lemur tyrosine kinase-2 （LMTK2）, a susceptibility gene for prostate cancer, on TGFβ-induced Smad2 signalling.In the present study, we evaluated the clinical significance of KLC2 in NSCLC and its effect on the malignant phenotype of NSCLC cells. Next, we identified KLC2 as a novel target of miR-125b in NSCLC and confirmed the direct interaction between miR-125b and KLC2.MATERIALS AND METHODSTissue Samples and ChipsSixteen fresh tissues including primary NSCLC （n=8） and adjacent nontumor lung （n=8） were obtained from Cardiothoracic Surgery of Nanfang Hospital （Southern Medical University, Guangzhou, China）. None of the patients had received chemotherapy or radiotherapy before surgery. Both normal and tumor tissues were histologically confirmed in Nanfang Hospital Department of Pathology.Tissue Chips consisted of 140 pairs of human primary NSCLC and their adjacent nonneoplastic lung tissues were obtained from Outdo Biotech CO. Ltd. （Shanghai, China）.Cell CulturesHuman NSCLC cell lines （SPC-A-1,95D, A549, H460 and H1299） were purchased from Shanghai Cell Bank of Chinese Academy of Science （Shanghai, China） and maintained in complete medium with high glucose RPMI-DMEM （Hyclone） and 10% fetal bovine serum （FBS, Gibco, USA）. An immortalized bronchial epithelial cell HBE and 293 T cell were cultured in RPMI-1640 （Hyclone） with 10% fetal bovine serum （FBS, Gibco, USA）. All cells were incubated at 37℃ in a humidified atmosphere containing 5% CO2.Plasmid and Oligonucleotide ConstructionKLC2 （http://www.ncbi.nlm.nih.gov/nuccore/NM₀22822）coding sequence which don’t contain the predicted miR-125b binding sites was cloned into pReceive-M98 vector （synthesized by GeneCopoeia, USA）.Short hairpin RNA （shRNA） sequence targeting KLC2 gene of human was inserted into hU6-MCS-Ubiquitin-EGFP-IRES-puromycin vector to generate KLC2-si plasmid, and plasmid transfected with scramble siRNA was used as KLC2-si/control..Transient TransfectionCell lines were seeded （2×105/well） onto 6-well plate 18-24h before transfection. Appropriate aforementioned plasmids （3ug/well）, miR-125b inhibitor,mimics or NC （100 pmol） were added to corresponding wells using Lipofectamine 3000 reagent （Invitrogen）. Cells were then incubated under 5% CO2 at 37℃ and harvested at 48 h post transfection. Cotransfection of KLC2 with miR-125b in SPC-A-1 and 95D cells were performed according to the similar steps.RNA Extraction and Quantitative RT-PCRTotal RNAs of human tissues or cultured cells were extracted with Trizol reagent （Takara, Dalian, China） following manufacturer’s recommended protocol. cDNA was synthesized and quantitative RT-PCR undertaken according to the manufacturer’s instruction （PrimeScript RT reagent Kit, TaKaRa, Dalian, China）. Both the prime sequences of miR-125b and KLC2 were synthesized by Invitrogen. The relative expression of miR-125b and KLC2 was calculated according to the 2-ΔΔCt method.Western Blot AnalysisTotal protein from tissues and cells was extracted using RIPA lysis buffer. Western blot was performed as described previously. The anti-P-actin antibody was used as the loading control. Finally, the active bands were visualized and quantified by software QuantityOne v4.6.2.Wound-Healing AssayThe trypsinized cells （1×106/well） were plated on 6-well plates, cultured overnight in complete medium and transfected with corresponding plasmids or oligonucleotide aforementioned. Upon confluence reached approximately 90%, the mono-layers of cells were scratched using a sterile 10μL plastic pipette tip. Cells were washed with culture medium twice to remove cell debris and cultured again under 5% CO2 at 37℃ up to 48h in serum-free medium. Images were captured under an inverted microscope following wounding （Oh and 48h） to assess the rate of gap healing.Transwell Migration and Invasion AssaysTransfected cells （4×105/200μl） in serum-free medium were planted to the upper Transwell chamber （BD Bioscience, Bedford, MA, USA,24-well insert, pore size:8 μm）. Complete medium （500μl） containing 10% FBS as a Chemoattractant was placed to the lower chambers. For the invasion assay,the surface of lower chamber was covered with matrixgel （BD Biosciences, Sparks, MD, USA）. After 24-48h of incubation under standard culture conditions, tumor cells were migrated or invaded through the bottom of membrane filter. Cells which had migrated or invaded to the underside were fixed and stained. Five visual fields of each chamber were randomly chosen and photographed under the inverted microscope. The migrating or invading cells were counted offline and presented with the average value.Dual-Luciferase Reporter AssayThe fragments containing the mutant-type （MUT） or wild-type （WT） putative target site of human KLC2 3’UTR were cloned into mirGLO vector plasmids （Genepharma, Shanghai, China）.For the luciferase reporter assay,293T or SPC-A-1 cells were co-transfected using Lipofectamine 3000 reagent （Invitrogen）. Cells were harvested at 48 hours after co-transfection following the manufacturer’s instructions, and luciferase activity was assayed with Dual-Luciferase Reporter Assay System （Promega, Madison,WI, USA）. Finally, The ratio of firefly and Renilla （internal control） luciferase activities were calculated.Immunohistochemical StainingImmunohistochemical staining was carried out with immunohistochemistry kit （Zhong Shan-Golden Bridge Biological Technology, China） according to the manufacturer’s protocol. A rabbit polyclonal antibody for KLC2 （1:100; Proteintech, USA; 17668-1-AP） was used as primary antibody.Double immunohistochemical staining was performed with polymer double staining kit （Mo/HRP+Rb/AP） （Zhong Shan-Golden Bridge Biological Technology, China, Catalog number:DS-0001） according to the manufacturer’s protocol.Statistical AnalysisSPSS 13.0 software package was used for statistical analysis. All experiments were repeated three times independently at least and all data were expressed as the mean±standard deviation. The difference between groups were calculated using the two-tailed unpaired Student’s t-test （qRT-PCR, wound-healing, migrating or invading cell numbers and Dual-Luciferase Reporter Assay） and all p< 0.05 was considered statistically significant. The correlation between miR-125b and KLC2 expression was analyzed by Spearman’s correlation. Kaplan-Meier method and the log-rank test were used to estimate variable in prediction of the patient overall survival. Correlations of KLC2 expression with clinic-pathological parameters were assessed using x2 test. Hazard ratio （HR） and prognostic factors were determined by univariate and multivariate analyses using Cox regression model.RESULTSKLC2 protein is overexpressed in NSCLC and associated with poor clinical outcome of elderly NSCLCAs shown in Figure 1-1A, the protein level of KLC2 significantly increased in human NSCLC cell lines compared with normal lung epithelial cell line （HBE）. And we noticed that the level of KLC2 protein was much higher in A549, H460 and H1299 in comparison of SPC-A-1 and 95D. In addition, the capacity of cell migration and invasion is also higher in NSCLC cell lines with high KLC2 expression （Figure 1-1B and Figure 1-2）. On the tissue microarray, KLC2 expression was predominantly diffuse and cytoplasmic staining in tumor cells whereas normal lung alveoli and interstitial tissue exhibited negative or weak expression of KLC2 （Figure 1-1C, upper panel）. Double staining assays （Figure 1-1C, lower panel） further demonstrated that KLC2 was diffusely expressed in the cytoplasm （red color） of tumor cells in which Ki-67 expression was restricted to the nuclei （brown color）. KLC2 was overexpressed in 67.1% human primary NSCLC tissues but only in 2.1% adjacent non tumor lung tissues （Figure 1-1D,p<0.001）.Although KLC2 expression level was related to neither any clinicopathologic factor nor the overall survival of 140 patients （Table 1-1 and Figure 1-3）, KLC2 overexpression significantly portended inferior overall survival （Figure 1-1E, p=0.026） in elderly NSCLC patients （>60 years-of-age,81 cases）. In the multivariate model, high KLC2 expression remained to be an independent negative predictor of prognosis for elderly NSCLC patients （Table 1-2）. Notably, high KLC2 expression appeared to be associated with shorter overall survival in male patients, or in patients either with squamous cell carcinoma, or with NO disease, or with Stage I/II disease, although the correlation didn’t reach statistical significance （Figure 1-4）. Taken together, these results suggest that KLC2 may play an important role in NSCLC progression.KLC2 contributes to NSCLC cells invasion and migration in vitroTo characterize the function of KLC2 in NSCLC cells, both gain- and loss-of function models were established with a transient transfection strategy. Successful overexpression and knockdown of KLC2 was illustrated by western blotting and qRT-PCR （Figure 2-2A）. We investigated cell growth by MTT assay and found that KLC2 overexpression or knockdown produced no effects on lung cancer cells proliferation （Figure 2-1）. Next we performed wound-healing assay and transwell assays with or without matrigel to evaluate whether KLC2 can affect NSCLC cells migration and invasion. The invasion and migration ability of SPC-A-1 and 95D cells ectopically expressing KLC2 were significantly enhanced when compared with control cells. Conversely, downregulation of KLC2 impaired the invasion and migration ability of NSCLC cells （Figure 2-2B and C）. Therefore, these data further implied the biological importance of KLC2 up-regulation in lung cancer development.miR-125b regulates the expression of KLC2 at the translational levelBioinformatic analysis implied that the 3’UTR region of KLC2 contains putative binding sites for miR-125b （Figure 3A）. To determine if miR-125b directly targets the 3’UTR of KLC2, we performed luciferase reporter assay by cloning the wild-type （WT） or mutant 3’UTR of KLC2 into the downstream region of a luciferase reporter gene. The WT or mutant vector was co-transfected into HEK-293T and SPC-A-1 cells with miR-125b mimics or miR-control. We found that overexpression of miR-125b resulted in a 2-fold decrease of luciferase activity of WT KLC2 3’UTR, as compared to the miR-control. Whereas the reduction of the luciferase activity with mutant KLC23’UTR was not observed （Figure 3B）. Notably, analysis of the miR-125b expression levels in five human NSCLC cell lines showed that miR-125b was markedly suppressed in cells with high KLC2 expression （A549, H460 and H1299） compared with cells with low KLC2 expression （SPC-A-1 and 95D） （Figure 3C）.To further confirm the regulatory effect of miR-125b on KLC2 expression, we measured the mRNA and protein level of KLC2 in SPC-A-1 and 95D cells after ectopically expressing or suppressing miR-125b. Data （Figure 3D） showed that a 1-2.4-fold decrease of KLC2 protein expression in response to miR-125b up-regulation was observed. Conversely, a corresponding increment in protein expression for KLC2 was also demonstrated following transfection with the miR-125b inhibitor in SPC-A-1 and 95D cells. But miR-125b had less （no more than 40%） effect on KLC2 mRNA expression （Figure 3-3D）. Moreover, KLC2 protein and miR-125b expression were inversely correlated in our clinical NSCLC and non-tumorous lung samples （R=-0.691,p=0.003;Figure 3E）. Taken together, these data suggested that miR-125b repressed KLC2 expression at protein level by target the 3’UTR of KLC2.KLC2 has a critical role in miR-125b-mediated invasion and migration of NSCLC cellsTo determine whether KLC2 was critical mediators of miR-125b function in NSCLC invasion and migration, KLC2 was overexpressed in miR-125b-overexpressing SPC-A-1 and 95D cells. The transfection efficiency was verified by qRT-PCR and western blotting （Figure 4A）. The effect of ectopic miR-125b up-regulation on KLC2 expression were further confirmed to be consistent with above results at both mRNA and protein levels. However, overexpression of KLC2 didn’t lead to any change in miR-125b expression.Cells were then analyzed for cell invasion and migration. As expected, ectopic expression of miR-125b significantly reduced migration and invasion potential of NSCLC cells, which was contrary to the promotive effect of KLC2 on NSCLC cells. Most important, exogenously expressed KLC2 almost completely reversed the inhibitory effect of miR-125b on migration and invasion in both SPC-A-1 and 95D cells （Figure 4B and C）. Therefore, KLC2 was an important mediator responsible for the effects of miR-125b on NSCLC cell migration and invasion.Conclusions:1、KLC2 protein is overexpressed in NSCLC and associated with poor clinical outcome of elderly NSCLC.2、KLC2 contributes to NSCLC cells invasion and migration in vitro.3、Overexpression of miR-125b resulted in a decrease of luciferase activity of WT KLC2 3’UTR, as compared to the miR-control. KLC2 protein and miR-125b expression were inversely correlated in our clinical NSCLC and non-tumorous lung samples.4、KLC2 has a critical role in miR-125b-mediated invasion and migration of NSCLC cells.5、KLC2 acts as a proto-oncogene and a functional target of miR-125b in NSCLC cells.