CBLL1 Is Highly Expressed In Non-Small Cell Lung Cancer And Promotes Cell Proliferation And Invasion

Objective:CBLL1 is a recently discovered E3 ubiquitin ligase,and it has a RING finger domain.E-cadherin is a typical cadherins and its loss is associated with the prognosis of carcinoma.It was reported that CBLL1 protein could combinate with tyrosine phosphorylated E-cadherin and mediate the internalization and subsequent ubiquitin of E-cadherin.Studies have shown that CBLL1 plays multiple roles in tumorigenesis.First,it was reported that CBLL1 induced anchor-dependent cell growth.In addition,CBLL1 was highly expressed in cancer tissue compared to normal tissue and CBLL1 promoted cancer cell growth in the human colon cancer and stomach cancer.Secondly,expression of CBLL1 in epithelial cells which expressed E-cadherin shRNA didn’t promote the formation of pointed protrusions,suggesting that effect of CBLL1 on E-cadherin did not rely on cell phenotype.CBLL1 affects the cell adhesion of matrix and MDCK ability to attack of epithelial cells.In colorectal epithelial cells,autocrine Slit-Robo pathways promote CBLL1-mediated downregulation of E-cadherin and promotes the occurrence of cancer.These studies indicate that CBLL1 seems to participate in the important signaling pathways downstream of tumor progression.And more research is needed to clarify CBLL1 as a potential therapeutic target.In addition,CBLL1 can increase the combined of mRNA of cancer related protein coding and RNA-binding protein PSF and promote the proliferation of MDCK cells.In ER alpha dependent breast cancer MCF-7 cell,CBLL1 overexpression inhibited the estrogen dependent cell proliferation and migration.In addition,CBLL1 could inhibit HIF-1 alpha/VEGF pathway activity.In all,the potential role of CBLL1 needs further research.Based on the above reasons,we speculate that CBLL1 and the progress of cancer have a close relationship.To verify our speculation,to explore the biological effect of CBLL1 on non-small cell lung cancer,we used immunohistochemical method to detect the expression of CBLL1 in non-small cell lung cancer and adjacent lung tissue and analyze the relationship with clinical pathological features.By regulating the expression of CBLL1 we studied the effects of CBLL1 on lung cancer cell proliferation,invasion.We discussed the possible molecular mechanism to provide new targets for the early diagnosis and treatment of lung cancer.Methods:1.We selected 79 cases of lung cancer and 24 cases of lung tissue adjacent to carcinoma specimens of paraffin wax from the First affiliated hospital of China Medical University in January 2009 to December 2013.All specimens were from surgical resection of lung cancer patients.All patients did not receive radiotherapy or chemotherapy before the surgery.2.Slice after 72℃baking,dimethyl benzene dewaxing,gradient alcohol to take off the benzene,hydration.Streptomycin avidin-peroxidase(S-P method)was used to detect CBLL1 protein expression.And PBS was used as the negative control.3.Human bronchial epithelial cell line HBE,lung adenocarcinoma cells A549,H1299,SPC-A-1,H157 were cultured.4.CBLL1 expression plasmid pcDNA3.1-HA-CBLL1 was given by Professor Yasuyuki Fujita.Liposome transfection procedures were performed according to the Lipofectamine 3000 reagent instructions.According to the design principle of siRNA,human CBLL1 mRNA in specific nucleotide fragments was selected as target and constructed by the GenePharma Company(Shanghai,China).Experiment groups were divided into nonspecific siRNA transfection group and the specific siRNA transfection group,each experiment was repeated three times.Transfection of siRNA was performed according to Lipofectamine 3000 transfection reagent manual.5.Realtim RT-PCR Using RNAiso Plus reagent to extract total RNA of cells,reverse transcription reaction was used to get cDNA according to the TaKaRa RT kit manual.PCR were performed on ABI7900.6.Western blot Collecting cells and cleaning cells with 4℃precooling PBS three times,added cell lysis to the cells.60μg protein was separated on 10%polyacrylamide agarose gel electrophoresis with concentrated gum voltage 80V and separation voltage120V.Protein was transferred to PVDF under constant voltage 50V for 120 minutes.The PVDF was then blocked with 5%BSA and incubated with primary antibody at 4℃overnight.Incubate secondary antibody at room temperature for 2 hours.Bound proteins were visualized using ECL.7.Immunofluorescence staining 4%paraformaldehyde was used to fix cells for 20mins at room temperature,PBS rinsed three times.Dyeing procedure was performed as follows:5%BSA blocked for two hours,primary antibody incubation overnight at 4℃,incubation with FITC or TRITC goat anti rabbit IgG(dilution ratio of 1:100)37℃incubation for 1 hour,after staining nuclei with DAPI for 8 minutes at 37℃incubation.PBS was used as the negative control.8.CCK8 assay and colony formation experiments were used to detect cell proliferation in vitro Logarithmic growth phase cells were digested with 0.25%trypsin and adjusted to single cell suspension cell density(3 x 10~5/ml),vaccination respectively in four 96 orifice plate culture plate.20μl CCK8 was added to each well at 24,48,72hours respectively,the absorbances of 450 nm were determined on enzyme-linked immune detector after incubation with 2 hours.The experiments were repeated 3 times.Cell growth curves were drawed.Cells was planted into 6-well plate(500cells)and incubated for 12 days.Plates were washed with PBS for 2 times,each time for 3 minutes.Then cells were stained and colonies with more than 50 cells were counted.9.Flow cytometry instrument to detect the cell cycle Cells were digested after 48 h hours with no EDTA 0.25%trypsin,centrifugal,collecting cells,wash twice with precooling PBS,5 minutes,2000 RPM centrifugal supernatant,70%cold ethanol was used to fix,4℃overnight.Wash twice with PBS to remove ethanol,add 0.1 mg/ml RNaseA,37℃water bath for 30 minutes,then add 50 mg/L PI for another 30 minutes.Flow cytometry instrument was used to analyze DNA content and cell cycle.10.Matrigel matrix was diluted with 1:7 serum-free medium RPMI1640.100ul diluted Matrigel matrix was added to the upper chamber.Medium supplemented with 10%FBS was added to the lower chamber as the chemo attractant.After 24 hours,cells were fixed and stained.11.Wound Healing Assay After transfection for 24 hours processing with mitomycin for 2 hours,make straight scratch lines with 10 ul-100 ul tip.Wash the plate for 3 times with PBS,medium was added,and plate were incubated at 37℃.Wound healing with the scrape line was observed at different time points,and representative scrape lines for each cell line were photographed.12.Statistics analysis SPSS13.0 statistical software was used to analyze the relationship between CBLL1 expression and clinical pathologic factors with nonparametric test.Other experiment results were analyzed by t test,P<0.05 was considered statistically significant.Results:1.CBLL1 was expressed in the bronchial and alveolar cell nucleus,and it was high expressed in lung cancer tissues.CBLL1 high expression is associated with the high TNM staging of NSCLC patients.2.Expressions of CBLL1 in SPC,A549 and H1299 lung cancer cell lines were higher than the HBE.The expression of H157 was lower than HBE.CBLL1 was expressed in the nucleus of HBE cells.In A549 and H1299 cell lines,expressions of CBLL1 were observed both in the nucleus and cytoplasm.3.After plasmid transfection and siRNA interference of A549 and H1299,CBLL1expression levels were obvious changed accordingly.4.CBLL1 promoted lung cancer cells A549 and H1299 proliferation and cell cycle progression,CBLL1 G0/G1 phase cell percentage increased after siRNA interference.5.The results showed that invaded cells of A549 cell and H1299 cells increased significantly after transfection of pcDNA3-HA-CBLL1 than that of transfection pcDNA3-HA.Invaded A549 lung cancer cells and H1299 cells decreased significantly after transfection of CBLL1 siRNA 1 than control siRNA group.After transfection CBLL1 siRNA,the expression level of E-cadherin in A549 and H1299 cells increased significantly.Conclusion:CBLL1 was highly expressed in non-small cell lung cancer.The expression of CBLL1 was related to pTNM staging and pathological type.High TNM staging was related with CBLL1 high expression in lung cancer tissue.CBLL1 promoted cell proliferation,invasion and immigration in lung cancer.