Role And Mechanism Of TUSC3Gene In Small-Cell Lung Cancer

Lung cancer is one of the leading causes of cancer-related death worldwide and the morbidity is rising significantly. The overall survival rate of lung cancer patients remains low with a5-year survival rate of less than15%. The rapid development of molecular biology and molecular genetics in recent years yields deeper understand-ing for molecular mechanisms of lung cancer. As we know, tumorigenesis is essentially caused by genetic abnormalities, so do lung cancers and other tumor types. Many carcinogenic factors can induce cell transformation and carcinogenesis via activation of oncogenes and/or inactivation of tumor suppressor genes.TUSC3(tumor suppressor candidate3) was initially identified on human8p22by MacGrogan in1996. Then it was found that the gene is expressed in a variety of epithelial cells and tissues including prostate, colon, lung and liver tissue. TUSC3was identified as a potential tumor suppressor gene as its promoter CpG island methylation increased, and is closely related to the malignant transformation of cells. TUSC3shares high sequence homology with Ost3p, a subunit of the oligosaccharyltransferase(OST) complex involved in N-glycosylation of proteins. Alterations of protein N-glycosylation may lead to a large number of abnormal protein accumulation in the endoplasmic reticulum, which can not be transferred out, and then induce endoplasmic reticulum stress(ERS), thus make its effect on apoptosis which is important for tumorigenesis.Based on the above points, we detected TUSC3protein expression in lung cancer tissue slides and investigated the relationship between TUSC3expression and clinico-pathological parameters in lung cancer. As it showed a significant lower expression of TUSC3in SCLC(small-cell lung cancer) compared with normal lung tissues, we then chose SCLC cell line H446for further investigation and discussioned the role and molecular mechanism of TUSC3in regulating proli-feration and apoptosis of SCLC cells both by over-expression and silencing of TUSC3with comprehensive utilization of a series of molecular biology and cell biology methods in vitro experiments. Our research of over-expression of TUSC3gene part demonstrated that over-expression of TUSC3can significantly inhibit the growth of SCLC cells H446. SiRNA technology was used for silencing TUSC3 expression. In this section, we first used tunicamycin to build endoplasmic reticulum stress model, and then detected related protein in PERK-eIF2a-CHOP endoplasmic reticulum stress pathway in order to investigate the impact of TUSC3on endoplasmic reticulum stress pathway and apoptosis of SCLC. Finally, we injected the pcDNA3-TUSC3and TUSC3into naked mice in vivo and discuss its impact on tumor growth. We also examined serum TUSC3and serum sDR5expression levels in part of lung cancer patients, in order to provide proofs for clinical application as tumor markers for lung cancer.PART I Bioinformatics analysis of TUSC3Objective:To analyze the biological information of TUSC3gene by using bioinformatic softwares, and to predict its structure, then speculated its function.Method:Full-length gene sequence from NCBI, mRNA sequence and amino-acid fragment sequence of TUSC3were obtained by utilize Internet, and the basic nucleotide characteristics and homology of TUSC3gene were analyzed by using online ORF Finder and Blast software. The hydrophobicity/hydrophilicity, solubility of the protein presequences, transmembrane domain and signal peptide were predicted by using online tools ProtScale, ProtParam, TMpred and SignalP3.0. The secondary structure of the protein were predicted by using the online SOPMA software? The subcellular localization was predicted by using PSORTⅡ. The protein’s N-terminal were analyzed by using the NetOGlyc4.0Server process. The three-dimensional structure of the TUSC3protein was predicted by Gene3D, MMDB softwares.Results:Human mRNA sequences and protein sequences TUSC3gene are available on line. TUSC3protein is partially acidic and is mainly proposed by C,H,O,N and S. The human, mouse, rat species spare a high degree of genetic homology, indicating that the gene-coded protein is important and relatively conservative in vivo. TUSC3entire peptide chain has five putative transmembrane domains. It is considered TUSC3as hydrophobic proteins,band mainly located in endoplasmic reticulum and mitochondrion. There are three glycosylation sites and it is probably secreted proteins. TUSC3protein is an active protein which have three-dimensional structure. PART II Expression of tumor suppressor candidate3and its relationship to the clinico-pathological characteristics of lung cancerCHAPTER I TUSC3detaction in microarray slides and its relationship to the clinico-pathological parameters of lung cancerObjective To investigate the expression of TUSC3in lung cancer patients tissue on microarray slides, and determined its relationship to the clinico-pathological parameters of lung cancer.Methods Cytoplasmic TUSC3expression was evaluated by immunohistochemistry (IHC) on tissue microarray slides.The scoring of Immunoreactivity was based on cytoplasmic staining and categorized according to the immunoreactive score (IRS). The significance of correlation between clinical pathological parameters (TNM classification, T-stage, N-stage and pathological differentiation degree) and IRS of TUSC3was determined. All datas were analysized by SPSS13.0.Results(1) There was no significant difference between normal controls and lung cancer patients in TUSC3expression rate (P=0.123).Significantly reduced TUSC3expression was observed in SCLC patients compared with normal controls(P=0.001). No significant difference was observed between the normal controls and ADC or SCC patients (P=0.114; P=0.614)(2) Obvious increase of TUSC3expression in patients with III-IV stage was observed compared with lower stage (Ⅰ+Ⅱ) patients (P=0.027); Lymph node metastasis positive (LNM+) patients showed a strong decrease in TUSC3expression than lymph node metastasis negtive (LNM-) patients (P=0.011); A significant decrease of TUSC3expression was observed in patients with low-differentiation degree carcinoma than those with high-differentiation degree carcinoma (P=0.000).(3) In small-cell lung cancer (SCLC)tissue, higher TNM classificationor higher LN metastasis classification were relative to lower TUSC3expression(rs=0.4638; rs=0.3835). No such correlation was found with T statu (P-0.857)(4) In adenocarcinoma lung cancer(ADC) tissue, higher TNM classification or more LN metastasis classification were relative to lower TUSC3expression (rs=0.525; rs=0.167; rs=0.611). No such correlation was found with T status (P=0.903)(5) In squamous lung cancer(SCC) tissue,TUSC3expression was correlated with the LN metastasis classification and pathological status (rs=0.119; rs=0.329). Neither TNM classification nor T staging was correlated with TUSC3expression (P=0.173; P=0.678)Conclusion:1Significantly reduced TUSC3expression was observed in SCLC tissue compared with normal controls, and TUSC3expression was negative related with TNM classification and lymph node metastasis status, which indicate loss or reduce of TUSC3may be involved in the progression of SCLC. No significant difference was observed in TUSC3positive rates of expression between NSCLC(including ADC and SCC) and normal control.2Decreased TUSC3expression is associated with increased probability of lymph node metastasis in three types of lung cancers.3Decreased TUSC3expression is negative associated with increased pathological grade of lung cancers. CHAPTER Ⅱ Target to over-expression TUSC3effect on the biological behavior of small-cell lung cancer cells H446Objective:To investigate TUSC3over-expression and its effect on the biological behavior of small-cell lung cancer cells H446.Methods:First,we constructed the mammalian expression vector of TUSC3. pcDNA3-TUSC3was transfected into H446cells with oligofectamine. EGFP fluorescence was used for detection transfection efficiency. Levels of TUSC3protein expression were determined using Western Blot method. CCK-8test and clonogenic test were used to detect the proliferation of cells. The scratch method was used to observe TUSC3effect on cell migration.Results:(1) The eukaryotic vectors pcDNA3-TUSC3have been successfully constructed. The recombinant plasmid pcDNA3-TUSC3was confirmed by restriction enzyme digestion, PCR and DNA sequencing.(2) CCK-8test showed that compared wih pcDNA3transfected control group, the survival rate of H446cells was decreased in pcDNA3-TUSC3transfected group(P<0.05). The clonogenic test have shown that TUSC3over-expression in H446cells inhibited cell cloning growth compared with control(P<0.05). Scratch test has shown that TUSC3over-expression in H446cells inhibited cell migration compared with control(P<0.05).Conclusion:Over-expression of TUSC3can inhibit the proliferation and migration of H446cells CHAPTER III Target to silencing TUSC3gene and the effect on the biological behavior in Small-cell lung cancer cells through endoplasmic reticulum stress-induced apoptotic signal transductionObjective:To investigate the silencing of TUSC3gene expression effect on the biological behavior of the H446cells and its relationship with PEAK-eIF2a-CHOP pathway in endoplasmic reticulum stress.Methods:Using concentration-gradient method to determine the optimal concentration of tunicamycin for building cell endoplasmic reticulum stress model. The proliferation inhibition of tunicamycin on human H446cell lines was evaluated using CCK-8with the optimal inhibitory rate of50%at tunicamycin25ng/mL for24h. The experiment groups were dertermined as four and named as negative-siRNA group (N), siRNA-TUSC3silencing group(S), tunicamycin group(T) and siRNA-TUSC3silencing-tunicamycin group(S+T), respectively. The gene-silencing effect of two siRNA sequence for TUSC3was evaluated by reverse-transcript PCR and western blot. CCK-8was used to detectthe proliferation of cells in four groups; Apoptotic induction was measured by Flow cytometry using Annexin V FITC-PI method. Levels of PEAK, p-PEAK, eIF2a, p-eIF2a and CHOP protein expression were determined using western blot.Results:(1) Transiently transfected of TUSC3SiRNA (4557,50nM) to human small cell lung cancer cell line H446can effectively inhibit TUSC3gene and protein expression with the inhibiting rate of75-85%.(2) CCK-8test showed that compared with negative-siRNA transfected control group, the survival rate of H446cells was slightly increased in siRNA-TUSC3silencing transfected group,but there showed no statistic difference between groups(P>0.05). Under the circumstance of ERS induced by25ng/mL tunicamycin, CCK-8test demonstrated that H446cell proliferation increased in related to the silencing of TUSC3expression compared with H446cell with negative-siRNA transfected control group (P<0.05)(3) Annexin V FITC-PI test showed that compared with negative-siRNAtransfected control group, the apoptosis rate of H446cells was slightly decreased in siRNA- TUSC3silencing transfected group,but there showed no statistic difference between groups(P>0.05), but under the circumstance of ERS induced by25ng/mL tunicamycin, the apoptosis rate is significant lower in TUSC3silencing group compared with H446cell negative-siRNA transfected control group (P<0.05)(4) PEAK、eIF2a showed no difference among the four groups. Under the circumstance of ERS induced by25ng/mL tunicamycin, Western-Blot showed significantly reduce of the protein expression of p-PERK,p-eIF2a and CHOP in siRNA-TUSC3silencing group compared with negative transfecion group,and the apoptosis rate showed reduced in siRNA-TUSC3silencing group(P<0.05).Conclusion:Knockdown of TUSC3gene can promote the proliferation of H446cells and reduce apoptosis via the PEAK-eIF2a-CHOP pathway by reducing the protein expression of p-PERK、p-eIF2α、CHOP, which can make H446cell more resistant to apoptosis induced by ERS. CHATPER Ⅳ Studies on TUSC3inhibition of H446xenograft tumor in nude miceObjective:To investigate the inhibitory effect of TUSC3on growth of H446xenograft tumor in nude mice.Methods:The nude mice model of human small-cell lung cancer was established by subcutaneous injection with H446cell suspension. Groups of mice were injected with pcDNA3, pcDNA3-TUSC3, respectively.Growth inhibition of the xenograft tumor induced by TUSC3was observed in vivo.Results:Compared to control group, the volume and weight of xenograft tumor in pcDNA3-TUSC3injection group were significantly decreased compared with pcDNA3injected group (P<0.05).The inhibitory rate was48.0%.Conclusion:TUSC3can inhibit the growth of H446xenograft tumor in nude mice. PART Ⅲ Tumor marker detecting in lung cancer patientsCHAPTER Ⅰ Clinical significance of serum TUSC3and soluble death receptor5concentration in small-cell lung cancer patientsObjective We aimed to measure the level of TUSC3and serum soluble death receptor5(sDR5) in small-cell lung cancer patients and to evaluate its diagnostic significances in these patients.Methods The TUSC3and sDR5concentrations were evaluated by the enzyme-linked immunosorbent assay (ELISA) method in50healthy controls and82SCLC patients before and after concurrent chemoradiotherapy (CRT). All datas were analysized by SPSS13.0.Results(1) The pre-treatment TUSC3level in SCLC patients was lower than that in healthy controls (5.89±2.01pg/ml vs.8.87±2.72pg/ml, P＜0.05).(2) The pre-treatment sDR5level in SCLC patients was higher than that in healthy controls (18.95±4.80pg/ml vs.10.89±6.72pg/ml, P<0.05).(3) Patients with PS0-1had higher pre-treatment TUSC3concentration than those with PS2-3(P＜0.05); patients with limited-stage of SCLC had significantly higher pre-treatment TUSC3level compared with extend-stage of SCLC (P＜0.05). No significant difference of TUSC3level was observed between different smoking status groups, tumor size groups or lymph node metastasis group (P>0.05)(4) Patients who smoke had higher pre-treatment sDR5concentration than those non-smoker patients (P＜0.05); patients with limited-stage of SCLC had significantly lower pre-treatment sDR5level compared with extend-stage of SCLC ((P＜0.05). There were significant differences of sDR5level between different tumor size groups or lymph node metastasis group ((P＜0.05); No significant difference of sDR5level was observed between different PS status group (P>0.05)(5) There were significant differences between pre-treatment sDR5and post-treatment sDR5concentration in SCLC patient group (18.95±4.80pg/ml vs.13.54±3.75pg/ml; P=0.000), but no such difference was observed between pre-treatment TUSC3and post-treatment TUSC3concentration(5.89±2.01pg/ml vs.6.05±1.74pg/ml; P=0.061).(6) The total response rate for treating SCLC was80.5%. When patients were divided according to therapeutic response, the post-treatment sDR5level in the responder patients was significantly lower compared with non-responders (P＜0.05), No such difference was observed for TUSC3(P>0.05)Conclusion1. The pre-treatment sDR5level in SCLC patients was higher than that in healthy controls and lower after-treatment sDR5predicts good response for treatment of SCLC.2. The pre-treatment TUSC3level in SCLC patients was lower than that in healthy controls. It has no predicting effect for treatment response of SCLC. CHAPTER Ⅱ Clinical significance of serum soluble death receptor5concentration in stage Ⅲ unresectable non-small-cell lung cancer patientsObjective We aimed to measure the level of serum soluble death receptor5(sDR5) in locally advanced stage Ⅲ NSCLC patients and to evaluate its diagnostic and prognostic significances in these patientsMethods The sDR5concentrations were evaluated by the enzyme-linked immunosorbent assay (ELISA) method in50healthy controls and122locally advanced stage Ⅲ NSCLC patients before and after concurrent chemoradiotherapy (CRT). All datas were analysized by SPSS13.0.Results(1) Pre-treatment sDR5level in NSCLC patients was higher than that in healthy controls (13.72±3.61pg/ml,10.89±6.72pg/ml, P＜0.05). However, no significant difference of sDR5level was observed between adenocarcinoma (ADC) subgroup and squamous cell carcinoma (SCC) subgroup (13.67±3.89pg/ml vs.13.77±3.32pg/ml; P>0.05). (2) According to multi-clinical classifications, an obvious increase in pre-treatment serum sDR5level could be observed in ⅢB stage patients compared with IIIA stage patients (P=0.009); Patients with tumor burden>3cm had higher pre-treatment sDR5concentration than those with tumor burden<3cm (P=0.026); Additionally, patients with T4stage had significantly higher pre-treatment sDR5level compared with T1stage (P=0.000). There were no significant differences among groups divided by different N status (P>0.05)(3) There was no significant difference between pre-treatment sDR5and post-treatment sDR5concentration in all of NSCLC patient group (13.72±3.61pg/ml vs.13.39±3.39, P=0.462). Furthermore, when patients were divided according to therapeutic response, the pre-treatment sDR5level in the responder patients(CR+PR) was significantly lower compared with non-responders,((P＜0.00001), no significant differences of post-treatment sDR5concentration was found between responder and non-responder groups (P>0.05).(4) Further survival analysis showed that the patients whose pre-treatment sDR5level≤14pg/ml (cutoff value=14pg/ml) had a longer PFS time than patients with sDR5>14pg/ml. However, no correlation was observed between the post-treatment sDR5and PFS time(P>0.05).Conclusion:1The pre-treatment sDR5level in ocally advanced stage III NSCLC patients was higher than that in healthy controls.2Serum level of pre-treatment sDR5may be a useful biomarker for treatment effect and prognosis for patients with locally advanced stage III NSCLC. patients whose pre-treatment sDR5level≤14pg/ml had a longer PFS time than patients with sDR5>14pg/ml...