| Part One: expression and significance of ATG4 A in gastric carcinoma tissuesObjective: To study the expression and significance of ATG4 A in gastric carcinoma tissues.Methods: Surgical specimens involved in the study were obtained from 110 patients(67 men, 43 women) with histologically confirmed gastric cancer who accepted curative resection during 2010 at southwest Hospital(Third Military Medical University, Chongqing). these patients who received neo-adjuvant therapy prior to the surgery were not included. Formalin-fixed paraffin-embedded tissues from the 110 patients including paired normal tissues were used for immunohistochemical analysis. Tumor stage was determined according to the TNM classification system of the International Union against Cancer(UICC). Histological differentiation was determined according to the criteria of World Health Organization. In our research, we choose the Tumor tissues, lymph node, Juncitonal zone. Adjacent tumor that was free from cancer cells was used as a healthy control. All of these 110 patients gave informed consent to use excess pathological specimens for research. The protocols employed in this study were approved by the hospital’s Protection of Human Subjects Committee and conformed to the Helsinki Declaration, and to the local legislation. Juncitonal zone lymph node metastasis primary lesionsresult:1, The immunoreactive score of ATG4 A in gastric carcinoma tissues was higher than that in nontumorous(7.23±0.43 vs 3.57±0.22 p<0.05); The immunoreactive score of ATG4 A in gastric Juncitonal zone was higher than that in nontumorous(7.73±0.75 vs 3.57±0.22 p<0.05); The immunoreactive score of ATG4 A in gastric lymph node metastasis was higher than that in nontumorous 7.94±0.81 vs 3.57±0.22 p<0.05); The immunoreactive score of ATG4 A in gastric Poor-differentiated was higher than that in well-differentiated(9.78±0.83 vs6.94±0.62 p<0.05);2, The expression of ATG4 A protein was correlated with lymph node metastasis(p=0.000),but not with age gender tumor location, and depth of invasion other organ metastasis(P>0.05)Conclusion: The expression of ATG4 A in gastric carcinoma tissues was higher than that in nontumorous; the expression of ATG4 A in Juncitonal zone was higher than that in nontumorous;The expression of ATG4 A in lymph node metastasis was higher than that in nontumorous; The immunoreactive score of ATG4 A in gastric Poor-differentiated was higher than that in well-differentiated. The expression of ATG4 A protein was correlated with lymph node metastasis.Part two: Effects of ATG4 A on stem cell properties and EMT of human gastric cancer cell linesObjective: To study the Effects of ATG4 A on EMT, stem cell properties, and invasion of human gastric cancer cell lines in vitro and in vivo.Method: Experiments were divided into six groups, experimental group(MGC-803 cell line were transfected with ATG4A-sh RNNA-1 ATG4A-sh RNNA-2, ATG4 Ash RNNA-3 plasmids; SGC-7901 cell line were transfected with ATG4A-OE plasmids), negative control group(MGC-803 cell line were transfected with ATG4A-sh RNNA NC plasmids; SGC-7901 cell line were transfected with ATG4A-OE NC plasmids),Cell migration capability were evaluated by wound healing assay, Invasion capability were evaluated by transwell invasion assay; The EMT marker E-cad, N-cad, vim were detected by western blot anslysis, The stem cell properties marker SOX-2,cot-4,Bmi-1 were detected by western blot anslysis and tumor spheres formed. And cells were injected cells into the lateral tail vein for invo experiment.result:1. The expression of ATG4 A were detected by western blot in different cell lines, we found that MGC-803 with high expression and SGC7901 with low expression(6.23±0.41 vs 1.34±0.09 p<0.01).after restriction enzyme analysis and sequence analysis,three eukaryotic expression plasmids of sh RNA targeting the ATG4 A,gene and an over-expression-ATG4 A were cuccessfully constructed. And all of them were successfully transfected to MGC-803 cell line and SGC-7901 cell line. The inhibition ratio of three Sh AGT4 A gene were 39.4%±2.3%, 45.1%±1.9%, 28.2%±4.3%; p<0.01;and the expression of ATG4 A in SGC-7901 were increased 52.2%±4.3%, p<0.012. Gastric cancer cell line possess highly metastasis ability in association with ATG4 A expression. The metastasis ability of gastric cancer cell line were measured by wound healing assay, and we found that Si RNA with lower metastasis ability in vitro compared to CTRL. The ratio of metastasis ability of three Sh AGT4 A cell line were decreased 25.7%±2.4%,32.5%±4.5%,20.4%±2.3%,and the ATG4A-OE cell line were increased 27.1%±3.3%P<0.05.3, Gastric cancer cell line possess highly invasive ability in association with ATG4 A expression. The invasive ability of gastric cancer cell line were measured by transwell invasion assay and we found that Si RNA with lower invasive ability in vitro compared to CTRL. The ratio of invasive ability of three Sh AGT4 A cell line were decreased 50.2%±6.1%, 44.3%±3.8%, 46.5%±4.6%, and the ATG4A-OE cell line were increased22.5%±1.9%P<0.054,Gastric cancer cell line possess higher stem cell properties in association with ATG4 A expression. The stem cell properties of gastric cancer cell line were measured by tumor spheres formed, we found that Si RNA with lower spheres formed ability in vitro compared to CTRL. The ratio of invasive ability of three Sh AGT4 A cell line were decreased 35.3%±3.3%,42.5%±4.8%,36.5%±3.7%, and the ATG4A-OE cell line were increased 47.5%%±5.9%P<0.05.5. Expression of stem cell properties related genes in gastric cancer cells were detected by Western blot, we found that MGC-803 cell with decreased expression of SOX-2, cot-4, Bmi-1, and increased expression of them in SGC7901.( P<0.05)6. Expression of EMT related genes in gastric cancer cells were detected by Western blot, we found that MGC-803 cell with decreased expression of N-cad, VIM, and increased expression of E-cad, In SGC7901, we found that the expression of N-cad, VIM were increased and the expression of E-cad were decreased( P<0.05)7, A tail vein metastatic assay was performed in nude mice to examine the in vivo metastatic potential of SGC7901-ATG4 A and MGC803-ATG4 A sh RNAs cells compared with the control cells. the number of lung metastatic lesions derived from SGC7901-ATG4 A cells was markedly increased compared with control cells. The average number of both the micrometastases and macrometastases in mice lungs following injection of SGC7901-ATG4 A cells was >2-fold greater than when injected with control cells as determined by H&E staining. Additionally,. Collectively, these results show that ATG4 A could promote tumor metastasis in vivo.Conclusion: Down-regulation of ATG4 A could lead to a significant decrease in migration capability, invasive capability, EMT capability, and stem cell properties of MGC-803 cells; Up regulation of ATG4 A could lead to a significant increase in migration capability, invasive capability, EMT capability, and stem cell properties of SGC-7901 cells;Part three:The possible molecular mechanisms of ATG4 A in stemness maintenance, invasion, metastasis and EMT of gastric cancer cellsObjective: To explore the possible molecular mechanisms of ATG4 A in stemness maintenance, invasion, metastasis and EMT of gastric cancer cellsMethod and result: Western blot and QT-PCR showed that when down-regulation and over-expression ATG4 A mainly resulted in change of notch signaling pathway. And using the inhibitors of NOTCH signaling pathway to blocking it, The result confirmed that NOTCH inhibitor DAPT could significantly inhibited the stem cell properties, EMT, invasive and metastasis of gastric cancer cells with over-expression of ATG4 A, indicating that the function of ATG4 A may be dependent on the NOTCH signaling pathway.Conclusion: In this study, we showed that ATG4 A was frequently upregulated in human gastric cancer and the upregulated ATG4 A was significantly associated with lymph node-metastasis. Further studies showed that overexpresion of ATG4 A contributed to gastric cancer cell metastasis. In constrast, decreased ATG4 A suppressed gastric cancer cell migration and invasion in vitro and metastasis in vivo. Mechanistically, Notch was identified as a functional target of ATG4 A. The data from the current study suggest that ATG4 A is a novel indicater of invasion and metastasis in gastric cancer and as a potential candidate target for treatment of gastric cancer. |
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