Roles And Regulation Mechanism Of RhoGDI2 In Lung Cancer

PurposesRhoGDI2 is one of the three human RhoGDIs. RhoGDIs are pivotal regulators of Rho GTPase function. RhoGDIs control the access of Rho GTPase to regulatory guanine nucleotide exchange factors and GTPase-activating proteins, to effector targets and to membranes where such effectors reside. Rho GTPase-RhoGDIs complexes are regulated by various proteins, lipids and enzymes that exert GDI displacement activity. RhoGDI2 has been shown to be a metastasis-related gene in bladder cancer and several cancers. But the function, effector targets, and biological roles of RhoGDI2 in health and disease are incompletely understood. As a metastasis-related gene, the expression pattern of RhoGDI2 varies in different tumors types or the tumor studies by different methods. RhoGDI2 is probably related with carcinogenesis, development and metastasis by unclear relationship.As a metastasis-related gene, RhoGDI2 has not systemic researches in lung cancer tissues and cell lines yet. In present study, we analyzed the roles and regulation mechanisms of RhoGDI2 in following points:(1) The expression and significance of RhoGDI2 in the lung cancer tissues by immunohistochemisty method and RT-PCR method.(2) The expression and location of RhoGDI2 in the lung cancer cell lines by immunofluorescent method, RT-PCR method and Western Blot method.(3) The relationship between RhoGDI2 and PI3K/Akt/mTOR signaling pathway in the lung cancer cell.Methods1. Materials112 paraffin-embedded specimens obtained from Shengjing hospital of China Medical University; while,20 frozen specimens obtained from Liaoning Province Cancer Hospital. Fresh specimens and corresponding normal tissue samples were put into liquid nitrogen immediately after resection and then stored at-80℃until the extraction of RNA and protein.Lung cancer cell lines A549, SPC-A-1,95D and NCI-H446 were purchased from Shanghai Cell Institute, Chinese Academy of Sciences. And Hela cell line is a gift of Department of Cell Biology, China Medical University.2. ImmunohistochemistyImmunostaining was performed by the SP method. They were incubated with RhoGDI2 antibody overnight. Five views were examined per slide, and 100 cells were observed per view, at 400X magnification. Labeling scores were determined by the percentage of positive cells per slide. We define results by Shimizu's standard.3. RT-PCRTotal RNA was extracted using Trizol reagent according to the manufacturer's instructions. RT-PCR was performed with the RNA PCR Kit (AMV) Version 3.0, according to the manufacturer's instructions. RT-PCR products were analyzed in 1% agarose gel.4. Cell Culture and GroupsA549, SPC-A-1,95D and NCI-H446 cells were cultured in RPMI-1640 medium, containing 10% fetal calf serum, 100IU/ml penicillin and 100μg/ml streptomycin. The cells were grown in a humidified incubator at 37℃and 5% CO2 air atmosphere. Cells were grown on sterilized culture dishes glass and were passaged every 2 to 3 days with 0.25% trypsin-EDTA.rhHGF groups:10ng/ml; 30 ng/ml。LY294002 groups:5μmol/l; 20μmol/l; 50μmol/l。Rapamycin groups:10nmol/l; 20nmol/l; 40nmol/lLY294002 and Rapamycin united groups:LY 5μmol/l+R 10nmol/1; LY 10μmol/l +R 10nmol/l5. ImmunofluorescentPrepared cell slides were fixed by 4% paraformaldehyde at room temperature for 20 min, adding RhoGDI2 antibody (1:100) in the wet box at 4℃overnight; then, adding FITC labeled goat anti-rabbit fluorescent secondary antibody in the dark room; and incubated at 37℃for 30 min. The results were observed by fluorescence microscope.6. Western Blot50μg proteins were separated by SDS-PAGE. After transferring to polyvinylidene fluoride membrane, the membrane was incubated 2 hours at room temperature with either the antibody against RhoGDI2 or GAPDH. After incubation with HRP labeled second antibody at room temperature for 1 hours, the proteins were visualized using ECL method.7. Cell viability assayCell viability was determined using the the conversion of MTT to formazan via mitochondrial oxidation.95D cells were plated in 96-well plates the day before the experiment and cells were treated with different drug groups. After drug treatment, attached cells were incubated with MTT for 4 hours and subsequently 150μl DMSO was added to each well. The absorption at 490mn was measured using an automatic microplate reader.8. Wound assay95D cells incubated in 6-well plates for 24 hours, scraped with a pipette tip, and assayed over with and without drug groups every 2 hours until the wound was completely closed.9. Invasion assayMatrigel was diluted. In the upper chambers,200μl cells were grown in serum-free medium on 8μm porous polycarbonate membranes, which were coated with Matrigel basement membrane matrix. The lower chambers were filled with RPMI-1640 medium containing 20% fetal calf serum. After incubation for 24 hours at 37℃in a humid atmosphere of 5% CO2, the cells that had migrated through the pores were fixed with ethanol for 30 minutes and stained with crystal violet. Then the number of cells counted visually using microscope in five different fields.10. Statistical analysisAll statistical data was performed by SPSS 13.0 software. P values less than 0.05 were considered statistically significant.Results 1. The expression of RhoGDI2 in the lung cancer tissues.(1) In lung cancer tissues, the expression of RhoGDI2 protein was correlated with the grades, differentiation and lymph node metastasis. The down-regulated of RhoGDI2 expression was significantly correlated with the grades (p<0.01), the degree of differentiation of tissue (p<0.01) and lymph node metastasis (p<0.01) and was not correlated with gender, pathological type, age (p>0.05).(2) The expression of RhoGDI2 mRNA was lower in lung cancer tissues than corresponding normal lung tissues.2. The expression and location of RhoGDI2 in the lung cancer cell lines.Both RhoGDI2 protein and mRNA were expressed in A549, SPC-A-1,95D and NCI-H446 lung cancer cell lines with different density. And RhoGDI2 protein was located mainly in A549 cell plasma.3. Relationship between RhoGDI2 and PI3K/Akt/mTOR signaling pathway.(1) HGF promoted the growth, motility and invasive ability of 95D cell, while LY294002 and Rapamycin inhibited the growth, motility and invasive ability of 95D cell, and combination with LY294002 and Rapamycin enhanced inhibition functions.(2) The expression of RhoGDI2 protein was decreased with the 30 ng/ml rhHGF group, while, it was increased with LY294002 groups and Rapamycin 10nmol/l and 20 nmol/1 groups. The expression of RhoGDI2 protein was decreased with Rapamycin 40nmol/l group. Combination with LY294002 and Rapamycin increased RhoGDI2 expression significantly.Conclusions(1) The Expressions of RhoGDI2 mRNA and protein are lower in lung cancer tissues than corresponding normal lung tissues. In lung cancer tissues, the expression of RhoGDI2 protein is correlated with the grades, differentiation and lymph node metastasis and not correlated with gender, pathological type and age.(2) RhoGDI2 is expressed in lung cancer cell lines, which indicates the downstream of RhoGDI2 has been blocked, and further indicates RhoGDI2 is regulated by multiple factors and signaling pathway in the malignant characters of lung cancer cells.(3) RhoGDI2 has a strong relationship with the activation of Akt by the PI3K/Akt/mTOR signaling pathway. RhoGDI2 maybe involve the invasion and metastasis of lung cancer by the interaction with Akt directly or indirectly.