RNAi Targeting-STIL Suppresses Cell Growth and Induces Apoptosis of Lung Cancer NCI-H1299 Cells via Inactivation of Akt/SAPK/TAK1 Pathways

Background: Gradually emerged studies demonstrated that SCL/TAL1 interrupting locus (STIL or SIL) is upregulated in multiple kinds of fatal tumors; at present, there is no clean understanding about the role of STIL in lung adenocarcinoma cells. This study aimed to discover the signicance of STIL in lung adenocarcinoma, so as to nd a potential gene target for diagnosis and therapy. Methods: STIL expression in lung adenocarcinoma tissue and clinical pathological characteristic was analyzed using the online databases, UALCAN and GEPIA. Lentivirus STIL-shRNA was manufactured and transducted into lung adenocarcinoma cells to seek and analyze the effects on tumor phenotype. The cell proliferation was assessed using Cellomics Array Scan imaging assay, and colony-formation assay, respectively. The apoptosis was detected by ow cytometry assay. Moreover, the antibody array of PathScan Cancer Phenotype, PathScan stress and apoptosis pathway was used to explore relevant molecular mechanisms following STIL knockdown in NCI-H1299 cells. Results: The clinical pathological characteristic assay showed that STIL is upregulated in lung adenocarcinoma tissues, and this trend was associated with cancer stage1 and histological subtypes. Silencing experiment showed that downregulation of STIL could inhibit cell growth and colony formation, induce cell apoptosis, and a G2 phase arrest effect signicantly, and antibody array detection revealed that p-Bad were upregulated, and p-Akt, p-Bad, p-HSP27, p-SAPK/JNK, p-TAK1, Vimentin, CD45, PCNA and Ki-67 were downregulated signicantly after STIL silenced in NCI-H1299 cells. Conclusions: In conclusion, STIL is overexpressed in lung adenocarcinoma tissues compared with normal lung tissue. Knockdown of STIL could inhibit cell growth and colony formation ability, promote apoptosis via Akt/SAPK/TAK1 signal pathways inactivation. China) and scanned images for quantication using ImageJ software (NIH Image for the Macintosh, USA). The western blotting experiments mentioned-above were performed in triplicate, and GAPDH (Mouse Anti-Flag, 1:2000, Santa-Cruz Biotechnology, Dallas, Texas, USA) was employed as a housekeeping control to normalize the expression of STIL protein expression by a semi-quantication method. Cell proliferation detection by Cell counting with Cellomics Array detection. The NCI-H1299 cells of both shSTIL and shCtrl groups were collected from plates using 0.25% trypsin-EDTA and suspended in standard medium until the cells grow to the logarithmic stage for cell counting with the cellomics array scan imaging detection. The specic procedures are as follows: the well prepared NCI-H1299 cells of both shSTIL and shCtrl groups were seeded in ve wells at 1000 cells/well, followed by further incubation at 37 ℃ and 50 mL/L CO 2 . A Cellomics Array Scan VT1 (Thermo Fisher Scientic) was utilized to monitor GFP expression of cells in both groups seeded in each well over 5 days continuously. In this study, statistical data of cells growth were mapped and cell proliferation curves were drawn to compare the multiplication ability of NCI-H1299 cells in both shSTIL and shCtrl groups. to analysis aberrantly after knockdown. that knockdown of STIL signicantly of phosphorylation expression of p-Akt,p-Bad p-HSP27 p-SAPK/JNK, p-TAK1, by ow cytometry showed that silencing STIL could induce lung adenocarcinoma cell apoptosis; cell cycle analysis by ow cytometry showed that silencing STIL could induce cell arrest at the G2 phase. These completed data demonstrated that STIL overexpression plays an essential role in lung adenocarcinoma tumorigenicity. It is suggested that STIL contributes to the pathogenesis of lung adenocarcinoma and STIL overexpression may be an early event in lung adenocarcinoma carcinogenesis. Therefore, STIL may represent a new promising target for gene therapy of lung adenocarcinoma. PathScan Stress and Apoptosis the of phosphorylation level of p-HSP27,p-SAPK/JNK of Phenotype assay in


Introduction
Lung cancer kept the record of the highest morbidity and mortality disease among a variety of malignant cancers in China 1 . As the most prevalent histological type of lung cancer, lung adenocarcinoma's relative survival rate of 5-year period increased with time, but not more than 21% 2 . Increasingly gene-targeted drugs against driver genes have been discovered and developed. However, gene targeted therapies against driver-gene-negative cases have not yet been fully developed. Efforts to identify a new potential target as drug for such cases are currently underway 3 . SCL/TAL1 interrupting locus (STIL or SIL), a centriolar assembly cytoplasmic protein, participate the processes of regulating the mitotic spindle checkpoint, the protein could be phosphorylated in mitosis and disappears when cell cycle enters G1 phase. It could interact with a mitotic regulatorand modulate Cdc2 kinase activity when spindle checkpoint was arrestted. Some experimental evidences come from published reports suggested that STIL is highly expressed in mutilple kinds of tumors accompanied by the features of increased mitotic activity 4 , such as prostate cancer 5 , pancreatic ductal adenocarcinoma 6 and lung cancer 7 . STIL could promote cellular proliferation, colony formation and suppress cellular apoptosis via affecting MAPK/ERK, PI3K/Akt and AMPK signaling pathways in prostate cancer 5 . SIL plays an essential role in the transition process from the G2 to the M phases of the cell cycle 4 . Silencing SIL in cancer cells could inhibited cell mitosis, decrease activation of the CDK1 (CDC2)cyclin B complex, and even led to apoptosis in a p53-independent manner 7 . Inhibition of STIL enhances the e cacy of DNA damaging chemotherapeutic drugs signi cantly in the treatment of ovarian cancer 8 .
In this current study, a lentivirus-short hairpin RNA (shRNA) containing STIL targeting sequences (Lv-shSTIL) was conceptualized and manufactured, and transduced into lung adenocarcinoma NCI-H1299 cells, to analyze the potential effects of silencing STIL on the phenotype of cancer. To be brief, a series of experiments including MTT, Cellomics Array Scan imaging, and colony formation assays were choosed to detect cell proliferation, and to analyze apoptosis and cell cycle were detected by ow cytometry assay. Besides this, the regulatory role of STIL in lung adenocarcinoma cells growth in vivo, two antibody PathScan arrays about cancer phenotype, stress and apoptosis were used to determine the underlying molecular mechanisms associated with STIL knockdown.

Materials And Methods
Extraction and analyzes of clinical data of STIL.
The clinical data about STIL expression in lung adenocarcinoma and clinical-pathological characteristic was collected using UALCAN 9 and GEPIA 10 web server (supported by TCGA database and the GTEx projects). STIL expression levels in lung adenocarcinoma tissues and adjacent tissues or Nodal Metastasis state were compared between lung adenocarcinoma tissues and adjacent tissues, and expressed as aberrant transcripts per million (TPM) values. Besides this, the correlation between STIL expression levels and prognosis in lung adenocarcinoma tissues were also analyzed by searching UALCAN, GEPIA and KM plotter 11 online database.

Cell lines and cell preparation
Lung cell line NCI-H1299 and 293T cell lines were provided by Genechem Company (Shanghai, China). All cell lines were kept in the medium composed of DMEM supplemented with 100 IU/mL Penicillin and 100 µg/mL Streptomycin, and 10% heatinactivated fetal bovine serum (BSA: Zhejiang Tianhang Biotechnology Co. Ltd.) in a humidi ed cell incubator having an atmosphere of 5% CO 2 at 37℃. All cells reaching to exponential growth stage were used for further experiments.

Lentiviral infection of lung adenocarcinoma NCI-H1299 cells.
Human lung adenocarcinoma NCI-H1299 cells were seeded in plates with six-well at 5×10 4 cells/well and incubated at 37℃in 50 mL/L CO 2 until 30% con uence was reached. The study was designed as negative control group (shCtrl, transfected with empty green uorescent protein (GFP) lentivirus) and shSTIL group (shSTIL, transfected with shSTIL GFP lentivirus). A su cient amount of lentivirus was transfected into NCI-H1299 cells according to the multiplicity of infection (MOI) appropriately. The cells were repeatedly cultured in the culture medium. GFP-tagged gene expression was monitored under a uorescence microscope at 3 d after transfection, and cells with the transfection e ciency more than 80% were selected for subsequent analysis. Cells were harvested at 48h after post-transfection to guarantee further analysis.
The 293T cells at the logarithmic growth phase were seeded in 24-well plate with 5×10 4 /mL until reached 80-90% con uence and the culture medium was changed with fresh medium by opti-MEM1 with 400 µL for RNAi plasmid transfection. Next, the successfully constructed plasmids containing shSTIL and shCtrl with 0.5µg and lipofectamine 2000 (Life Technologies, China headquarters of USA company, Shanghai, China) were dissolved in opti-MEM respectively and maintained at room temperature for 5 minutes, and then, the plasmids and lipofectamine 2000 were mixed and remained at room temperature for 20 minutes. Once nished that the above mentioned step, the mixture of plasmid DNA and Lipofectamine 2000 were added to 293T cells and cultured for 6-8 hours in incubator at 37℃, 5% CO 2 environment, and replaced with fresh complete culture medium containing 10% serum. The transfection rate was observed by uorescence microscope detection after 24 hours transfection.
RT-qPCR analysis of knockdown e ciency in NCI-H1299 cells.
To detect the e ciency of silencing STIL in lung adenocarcinoma cells, the method of qPCR analysis was utilized. To be simple, lung adenocarcinoma cells at the exponential growth stage following successful silencing of STIL with lentivirus infection were collected and lysed for extracting total RNA using RNAiso Plus regent (Takara Bio, Dalian, China). The purity and concentration of extracted RNA were measured using P100 + UV-Vis spectrophotometer (Pultton, California, Sunnyvale, United States). Next, the reverse transcription reaction from to cDNA was performed using a Prime ScriptTM RT reagent Kit (Takara Bio, Dalian, China). Finally, the ampli cationwas executed by a PCR Detection System (Roche, Light Cycle 9600, USA) using an SYBR Master Mixture (Yeasen, Shanghai, China), and the reaction of which was carried out in a nal volume of 20 µL containing 1 µl of cDNA, 0.5 mM of each primer and 1X SYBR Master Mixture. The ampli cation reaction was operated as the following programme: denaturation at 95°C for 15 sec, followed by 40 cycles of 95°C for 5 sec, 60°C for 30 sec, in which uorescence was detected to catch the ampli d DNA. After completing the ampli cation procedure, the melting curve was created to analysis the speci city of the expected PCR product of STIL. The the ampli ed DNA of STIL were normalized to GAPDH to calculate the fold change using the tradition method 2 −ΔΔCt . Each sample was run in triplicates for analysis. The primers used were as follows: STIL: forward, 5'-GAGTCAGATAATGGAATGATGGG − 3', reverse, 5'-CAGCAGTTGTCTTAGGGGAACA − 3'; GAPDH: forward, 5'-TGACTTCAACAGCGACACCCA − 3', reverse, 5'-CACCCTGTTGCTGTAGCCAAA − 3'.
Western blotting analysis of knockdown e ciency in 293T cells.
Following the transfection procedure for 36-48 hours, the 293T cells of both shSTIL and shCtrl groups were collected and washed twice using PBS solution, next, the collected 293T cells were lysed using ice-cold lysis buffer for 5 min for protein isolation. The total protein concentration of the collected 293T cells was detected with a protein assay kit (Bio-Rad Laboratories, Shanghai, China). The separation of protein samples was conducted using 10% SDS-PAGE, and the transference of which was conducted to PVDF membranes. The immunological blots were incubated with primary antibody (Mouse Anti-Flag, Sigma, 1:2000, China headquarters, Shanghai, China) in appropriate concentration at room temperature.
following washed in 5% non-fat milk containing TBST saline at room temperature for 1 h, The immunological blots were incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (Goat Anti-Mouse IgG, 1:2000, Santa-Cruz Biotechnology, Dallas, Texas, USA) for 1.5 h. Finally, the bands were evaluated with a method of chemiluminescence (ECL, Thermo Scienti c Pierce, Shanghai, China) and scanned images for quanti cation using ImageJ software (NIH Image for the Macintosh, USA). The western blotting experiments mentioned-above were performed in triplicate, and GAPDH (Mouse Anti-Flag, 1:2000, Santa-Cruz Biotechnology, Dallas, Texas, USA) was employed as a housekeeping control to normalize the expression of STIL protein expression by a semi-quanti cation method.
Cell proliferation detection by Cell counting with Cellomics Array detection.
The NCI-H1299 cells of both shSTIL and shCtrl groups were collected from plates using 0.25% trypsin-EDTA and suspended in standard medium until the cells grow to the logarithmic stage for cell counting with the cellomics array scan imaging detection. The speci c procedures are as follows: the well prepared NCI-H1299 cells of both shSTIL and shCtrl groups were seeded in ve wells at 1000 cells/well, followed by further incubation at 37 ℃ and 50 mL/L CO 2 . A Cellomics Array Scan VT1 (Thermo Fisher Scienti c) was utilized to monitor GFP expression of cells in both groups seeded in each well over 5 days continuously. In this study, statistical data of cells growth were mapped and cell proliferation curves were drawn to compare the multiplication ability of NCI-H1299 cells in both shSTIL and shCtrl groups.
The NCI-H1299 cells were harvested with 0.25% trypsin from and washed once with 4°C ice-cold D-Hanks (pH = 7.2 ~ 7.4) for centrifuge operation with the centrifuge force at 1300 rmp for 5 min. After rewashed with 1×binding buffer, the NCI-H1299 cells received another centrifuge operation at 1300 rmp for 3 min. Next, the NCI-H1299 Cells were resuspended in 200 µL binding buffer 10 6 cells/mL to execute apoptosis assay using a Annexin V-APC Apoptosis Detection Kit (MultiSciences, Hangzhou, China). The cell suspension of 100 µL volume was mixed with 10 µL Annexin V-APC for incubation in the dark for 15 min at room temperature. As a necessary step, a volume of 400-800µL 1×binding buffer was added to stained cells in proportion to the amounts of cells. The percentage of apoptotic rate was analyzed by ow cytometry in triplicate.
Human lung adenocarcinoma NCI-H1299 cells were treated as a resuspended solution and seeded in 96-well plates in triplicate with a cell density of 1,000 cells/well at 37°C under incubation environment with 5% CO 2 for caspase 3/7 activity assay. Caspase 3/7 activity was then tested using a Caspase Glo 3/7 Assay (Promega Corporation, G8091, WI, USA) kit based on the manufacturer's protocol. The Caspase-Glo3/7 buffer solution and Caspase-Glo3/7 freeze-dried powder were placed at 18-22 ℃ (room temperature) for balance. Then a volume of 10 mL Caspase-Glo3/7 buffer solution was added to the brown bottle containing the Caspase-Glo3/7 substrate, and the brown bottle was whirled or reversed repeatedly until the substrate completely dissolved to form the Caspase-Glo reaction solution. After cell counting, the cell suspension concentration was adjusted to 1 × 10 4 cells / well at room temperature, and a volume of 100 µL of non-negative control cells were added to the new 96 well plates per well. At the same time, a group of empty control group without cells was setted up (adding only 100 µL per well medium). Next, a volume of 100 µL Caspase-Glo reaction solution was added to each well. The culture plate was place with cells on the plate shaker and shake lightly for 30 minutes at 300-500 rpm speed. Then the cells were incubated at 18-22 ℃ for 2 hours. Absorbance values were measured with a microplate reader (SpectraMax i3X, MOLECULAR, California, USA) at 405 nm.
Cell cycle analysis by ow cytometry.
When the NCI-H1299 cells reached 80% con uence in the 6 cm dish of each experimental group, it will be resuspended in triplicate in full culture medium. For suspension cells, the supernatant was directly collected by centrifugation at 1300 rmp for 5 min, and then washed with the precooled D-Hanks (pH = 7.2 ~ 7.4) at 4 ℃. Next, the cells were treated with centrifuge operation at 1300 rmp for 5 min. and then treated with precooled 75% ethanol at 4 ℃ for at least 1 h. After removing the immobilization solution and the cells were washed using precooled D-Hanks, a certain volume (0.6-1 mL) of cell staining (composed with 40 × PI mother liquor (2 mg/mL), 100 × RNase mother liquor (10 mg/mL) and 1 × D-Hanks, and the proportion of which is 25: 10: 1000) solution was added to heavy suspension to stain cells with propidium iodide for cell cycle analysis according to cell quantity. The cell cycle detection was conducted using a Guava EasyCyte Plus Flow Cytometry System (Merck Millipore, Billerica, MA, USA).
Antibody Array assay of Cancer Phenotype,Stress and Apoptosis pathway.
To explore the activation of possible intracellular signaling related to the in uences in lung adenocarcinoma caused by STIL silencing, the PathScan of Cancer Phenotype Antibody Array Kit (#14821, Cell Signaling Technology, Danvers, MA, USA), Stress and Apoptosis Signaling Antibody Array Kit (#12923, Cell Signaling Technology, Danvers, MA, USA ) were used to screen and analysis potential proteins. After, till cells reaching around 85% con uence following lentivirus infection for 5 days, NCI-H1299 cells of both groups were collected and lysed to carry out detection in compliance with the manufacturer's instructions. The detection of cells between STIL silenced group and ShCtrl group was repeated in triplicate. Images were captured by exposing the slide to chemiluminescent lm according to standard protocol. Statistical analysis. ALL data were expressed as the means ± standard deviation from at least 3 independent experiments. Statistical analyses were calculated with the method of Student's two-tailed t-test. Differences with P values of < 0.05 are regarded as signi cant statistically.

Results
STIL was overexpressed in lung adenocarcinoma tissue with clinical features.
In order to understand the role of STIL lung adenocarcinoma tissues, and even nd its potential clinical signi cances, we preliminarily analyzed the aberrant expression levels of STIL between lung adenocarcinoma tissues and adjacent tissues by searching and analyzing UALCAN and GEPIA web server which was based on TCGA datasets. This result showed that STIL is upregulated in lung adenocarcinoma tissues when comparing with adjacent normal tissues ( Fig. 1(A, B)). Besides, the expression of STIL in Nodal Metastasis tissues, such as N0, N1, N2 lymph nodes was higher than that in normal tissues which embodied as Nodal Metastasis tissues having higher TPM values than normal tissues (Fig. 1C). The survival period of STIL overexpression group was less time than that in STIL lower expression group that was supported by UALCAN (Fig. 1D), GEPIA (Fig. 1E) and KM plotter online database (Fig. 1F). Summarily, STIL is upregulated in lung adenocarcinoma tissues, and which was correlated with cancer nodal metastasis statues, such as N0, N1, N2. The survival period of STIL overexpression group was associated with less survival time.

ShRNA-mediated STILsilencing e ciency in NCI-H1299 cells and 293T cells.
To investigate the loss of functional role of STIL, the lung adenocarcinoma cell NCI-H1299 was cultured and successfully infected with Lv-shSTIL or Lv-shCtrl with an infection rate greater than 80% for up to 72 hours since lentivirus infection completed( Fig. 2(A, B)). The mRNA expression level of STIL was signi cantly decreased in Lv-shSTIL groups with signi cant knockdown e ciency amounts to 59% compared with Lv-shCtrl group (P = 0.05, Fig. 2C). Human embryonic kidney 293T cells were transfected with shSTIL plasmid or shCtrl plasmid, STIL protein expression was signi cantly decreased in the Lv-shSTIL group in 293T cells, respectively (Fig. 2(D, E)). The results showed STIL-shRNA could knockdown STIL expression of the target sequence effectively.
STIL silencing inhibited lung adenocarcinoma cell proliferation by Cellomics detection.
GFP-based Cellomics Array Scan imaging assay showed that the proliferation of NCI-H1299 cells was signi cantly inhibited in STIL-shRNA group relative to that of the Lv-shCtrl group. The number of NCI-H1299 cells and the fold-change of proliferation were signi cantly reduced in the STIL-shRNA-silenced lung adenocarcinoma cells on the third, fourth and fth day following STIL silencing signi cantly completed in NCI-H1299 cells (P < 0.001, Fig. 3(A, B, C)). Consequently, the results suggested that knowdown of STIL could inhibit lung adenocarcinoma cell proliferation.

STIL silencing induced cell apoptosis.
To elucidate whether the silencing of STIL could induce apoptosis in NCI-H1299 cells further, apoptosis detection was designed to investigate the apoptotic rate between STIL silenced NCI-H1299 cells and negative control cells further. The apoptotic rate was assessed by ow cytometry using an Annexin V-APC Apoptosis Detection Kit. The proportion of apoptotic cells in NCI-H1299-silenced cells was signi cantly higher than that in the control cells (P < 0.001, Fig. 4(A, B)). These data suggested that the silencing of STIL could affect cell survival and induce apoptosis.
The caspase 3/7 activity of Lv-shSTIL group was calculated based on the Lv-shCtrl group. Compared with Lv-shCtrl group, the caspase 3/7 activity increased in the Lv-shSTIL group, indicating that the number of apoptotic cells in Lv-shSTIL group increased signi cantly (P < 0.001, Fig. 4C), re ecting STIL silencing could induce cell apoptosis in NCI-H1299 cells.
Silencing STIL induced cell cycle arrest at the G2/M stage.
To elucidate whether the silencing of STIL could arrest the cell cycle in NCI-H1299 cells further. The cell cycle was assessed between STIL silenced NCI-H1299 cells and shCtrl group cells by ow cytometry using PI (Sigma, USA). Three days after shRNA lentivirus infection, there was no signi cant difference in G1 cells in the experimental group. The number of NCI-H1299-silenced cells in the S phase decreased, and the number of cells in the G2/M phase increased than that in shCtrl group cells (P < 0.05), as shown in Fig. 4(D, E). These data suggested that the silencing of STIL could arrest the cell cycle in G2 phase.  Table 1, 2. This result indicated that STIL silencing could suppress cell proliferation and induce apoptosis re ected from the downregulation of these molecules, such as Vimentin, CD45, PCNA and Ki-67, and the process of which was probably via inactivation of p-Akt/p-SAPK//p-TAK1 pathway in lung adenocarcinoma cells. Certainly, we can conclude from the results that phosphorylation of p-Akt p-Bad p-HSP27 p-SAPK/JNK and p-TAK1 play important roles in the tumorigenesis process and maintenance of cancerous phenotype of NCI-H1299 cells. Furthermore, insightful studies are needed to clarify the mechanisms of STIL in lung adenocarcinoma progression.

Discussion
In this study, we performed clinical data analysis of STIL in lung adenocarcinoma tissues compared with normal tissues to discover its expression and clinical signi cances by searching and extracting from the data supported by UALCAN and GEPIA web server. It has been con rmed that STIL is upregulated in lung adenocarcinoma tissues, and its overexpression was associated with cancer metastasis, besides, overexpression of STIL would result in a poor prognosis with less survival time.
In summary, it is suggested that overexpression of STIL might act as a potential lung adenocarcinoma biomarker that was related to lymph node metastasis and vicious prognosis with less survival time. Therefore, it is promising if STIL would be chosen as new biomarker to attach importance to its role, function and relevant mechanisms.
In the present study, silencing STIL inhibited lung adenocarcinoma cell proliferation, reduced cell colony formation. Caspase 3/7 activity assay and apoptosis assay by ow cytometry showed that silencing STIL could induce lung adenocarcinoma cell apoptosis; cell cycle analysis by ow cytometry showed that silencing STIL could induce cell arrest at the G2 phase. These completed data demonstrated that STIL overexpression plays an essential role in lung adenocarcinoma tumorigenicity. It is suggested that STIL contributes to the pathogenesis of lung adenocarcinoma and STIL overexpression may be an early event in lung adenocarcinoma carcinogenesis. Therefore, STIL may represent a new promising target for gene therapy of lung adenocarcinoma.
The results were coincident with the reports that STIL knochdown could suppress cell growth through apoptosis and cell cycle arrest [4][5][6]8 . Apoptosis has been proved as a principal strategy, and there are multiple signaling pathways involved in the process of apoptosis. However, the mechanisms of apoptosis induced by STIL silencing still kept unclear. Therefore, the Stress and Apoptosis Signaling Antibody Array and Cancer Phenotype Antibody assay were employed to detect relevant molecular alterations about apoptosis and cancer phenotype comprehensively. The two pathways and targets included in these two arrays made it easier to screen and analyze relevant mechanisms of apoptosis. Previous reports demonstrate that these kinds of arrays have been used to detect mechanisms molecular in multiple cancers, such as in glioblastoma 12 , gastric cancer 13 and esophageal squamous carcinoma 14 . In the present study, the completed results of PathScan Stress and Apoptosis Signaling Antibody array assay showed that STIL silencing could inhibit the activation of phosphorylation level of p-Akt, p-Bad, p-HSP27,p-SAPK/JNK and p-TAK1 which could re ect molecule alterations during apoptosis. The results of the PathScan of Cancer Phenotype assay showed that STIL knockdown could block the activation of Vimentin, CD45, PCNA, and Ki-67which represent essential molecules in cancer phenotype change.
Vimentin, a major constituent of the intermediate lament family of proteins, is overexpressed in various epithelial cancers including lung cancer, and the overexpression of which in cancer was associated with tumorigenesis features, such as, accelerated tumor growth, invasion 15 TNM stage, lymph node metastasis and unfavourable prognosis 16 . Vimentin was veri ed as an independent prognostic factor regarding the overall survival time of gastric cancer 17 . CD45, also known as protein tyrosine phosphatase receptor type C, abbreviated as PTPRC, belongs to the protein tyrosine phosphatase (PTP) family. Reports showed that overexpression of CD45 was associated with weak treatment response and adverse survival pro les in elderly patients with acute myeloid leukemia 18 . Increased intraepithelial CD45RO + TILs was an independent positive prognostic factor for disease-speci c survival in all patients and squamous cell carcinoma 19 . The CD45 + erythroid progenitor cells (CD45 + EPC) played an immunosuppressive role in the responses of the impaired T cell commonly appeared in patients with advanced cancer, and the mechanism of which could be ascribed in that reactive oxygen species production contributed to CD45 + EPC-mediated immunosuppression 20 . CD45RO is a nucleus-located cofactor of DNA polymerase delta which participates in the RAD6-dependent DNA repair pathway when coming across DNA damage 21 . Antigen Ki-67, a nuclear protein, which is relevant with ribosomal RNA transcription and responsible for cellular proliferation. The activated antigen Ki-67 would inhibit the progress of ribosomal RNA synthesis, and Antigen Ki-67 could increase markedly during cell progression through S phase in the cell cycle. A report showed that the expression of Ki-67 in lung adenocarcinoma tissue is associated with poor tumor differentiation, and negatively affects patients' survival in advanced-stage lung cancer 22 . It has been proved that Ki-67 is a valuable predictive factor because overexpression of it was associated with lymph node metastasis and advanced TNM stages 23 . The overexpression of p-AKT was related to DNA aneuploidy which acts as a useful marker in a number of gastric cancer cases 24 . The survival time of PI3K and p-Akt-positive expression was signi cantly reduced in advanced NSCLC patients, and the overexpression of p-Akt was considered as an independent prognosis marker of poor outcome in advanced NSCLC 25 . AKT plays an essential role in the PI3K/Akt signaling pathway in cancer cells, and p-AKT, an activated form of AKT, plays avital role in the cell survival mechanisms and signal transduction pathways concerning tumorigenesis 26 . AKT activation which was caused by the production of PIP3 by PI3K, and PTEN dephosphorylates PIP3 at the 3' position, would give rise to the activation of downstream apoptosis signaling pathways which leads to apoptosis in the end 27 . Cell apoptosis would be induced by BAD and forkhead transcription factor (FKHR-L1) direct phosphorylation, and which could reduce the stress-activated protein kinase/c-Jun N-terminal kinase (SAP/JNK) signaling pathway that could be activated by extracellular proin ammatory cytokines or physical stress and turned into phosphorylated SAPK/JNK (p-SAPK/JNK). TAK1, also known as MAP3K7( its full name is mitogen-activated protein kinase kinase kinase 7), participates a variety of transcription regulation including apoptosis. Once, activated and turned into phosphorylated p-TAK1, it could also phosphorylate and activate MAPKKs which could bring about activation of MAPKs such as ERK, p38, and JNK 28 .
Phosphorylation of TAK1 at Ser412 site contributes to the activation of NF-κB signaling and boosts aggressiveness in ovarian cancer cells, inhibition of TAK1 activity dramatically impairs tumor growth and metastasis in ovarian cancer 29 . In this study, p-TAK1 was downregulated in STIL silenced NCI-H1299 cells compared with negative control NCI-H1299 cells.
These data suggested that the mechanisms of growth inhibition and apoptosis effects induced by STIL silencing are associated with the inactivation of phosphorylation expression of p-Akt, p-Bad, p-HSP27, p-SAPK/JNK, p-TAK1, Vimentin, CD45, PCNA and Ki-67.
Collectively, STIL is upregulated in lung adenocarcinoma tissues compared with normal lung tissue. Knockdown of STIL could inhibit lung adenocarcinoma cell growth and colony formation ability, promote apoptosis, and the mechanisms of which may block the phosphorylation of p-Akt/p-SAPK/p-TAK1 signal pathway and reverse the expressions of cancerous phenotype markers. Therefore, STIL may be considered as a promising and valuable target for gene therapy strategy in the treatment of lung adenocarcinoma.

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Consent publication
The authors all agreed for publication of this paper.
Availability of data and material Table 1 Detected proteins screened and validated by PathScan® of cancer phenotype array between shCtrl-infected cells and shSTIL-infected cells.   Figure 1 Expression of STIL in lung adenocarcinoma tissues and clinical features. Expression of STIL were upregulated in tumors than in corresponding adjacent tissues of lung, the median value of lung adenocarcinoma tissues and corresponding adjacent tissues were 73.973 and 35.160respectively, and Log2 fold change was 1.052, as shown is the value of lung adenocarcinoma in (Figure 1A, B) (GEPIA database supported by Match TCGA normal and GTEx data). Overexpression of STIL in cancer stage1, 2, 3, 4 embodies more TPM values than normal tissues ( Figure 1C) supported by both UALCAN and GEPIA web server. Overexpression of STIL in tumor stage1 embodies higher TPM value than in normal tissues ( Figure 1C).

Figures
The survival period of STIL overexpression group was less time than that in STIL lower expression group that was supported by UALCAN ( Figure 1D), GEPIA ( Figure 1E) and KM plotter online database ( Figure 1F). Summarily, STIL is upregulated in lung adenocarcinoma tissues, and which was correlated with cancer nodal metastasis statues, such as N0, N1, N2. The survival period of STIL overexpression group was associated with less survival time.  were markedly reduced in the STIL-shRNA-silenced lung adenocarcinoma cells on the third, fourth and fth day following STIL silenced signi cantly in NCI-H1299 cells compared with that of Lv-shCtrl group, as shown in Figure 3(A, B). Therefore, it is suggested that silencing of STIL could inhibit lung adenocarcinoma cell proliferation. ***P<0.001, Compared with Lv-shCtrl group. These data suggested that silencing of STIL could arrest cell cycle in G2 phase, as shown in Figure 7(A, B). ***P<0.001, Compared with Lv-shCtrl group.