| Objectives:The study taking the tissue specimens of patients with gastric cancer,human gastric cancer cell MGC803 as the research object,to explore the relationship between GNAQ and MPP8 with the prognosis of gastric cancer patient and clinicopathologic feature,to explore the function and mechanism of GNAQ and MPP8 related downstream pathway in the development of human gastric cancer.Methods:1.Immunohistochemistry was used to detect the expression of GNAQ and MPP8 in cancer tissues and adjacent normal mucosa in patients with gastric cancer.And the correlation between GNAQ and MPP8 expression with clinical features and prognosis of gastric cancer patients were also explored.2.Human gastric cancer cell MGC803 with stable knockdown of GNAQ gene and human gastric cancer cell line MGC803 with stable knockdown of MPP8 gene were established,together with human gastric cancer cell line MGC803,were used as the research object.3.MTT assay and Colony formation assay were used to assess the viabilities of cells.4.Annexin V/VAAD detection kit combined with flow cytometry was used for assessment of cell death.5.Flow cytometry was used to detect the cell cycle.6.Cell migration and invasion assay was used to detect the migration and invasion ability of the cells.7.Western blot assay was used to detect the expression of downstream apoptosis and metastasis pathway related proteins in the cells.Results:Part 1:1.The expression of GNAQ in gastric cancer tissues was significantly higher than that in normal mucosa(76% vs.51%,p<0.001***).Comparison of GNAQ and clinical features of gastric cancer indicates that GNAQ expression in patients within 60 years old was significantly lower than that of patients over 60 years old(68.3% vs.88.5%,p<0.001***),patients with low differentiation was significantly higher than that of patients with moderate differentiation(67.7% vs.83.4%,p<0.01**).There was no significant difference between the expression of GNAQ with different gender,different stages and different tumor location.2.There was no significant correlation between the expression of GNAQ and the prognosis of patients with gastric cancer.3 years DFS rate in GC patients with positive expression of GNAQ in cancer tissues and negative ones was 69.8% and 63%,respectively(log-rank p=0.496).3 years OS rate in GC patients with positive expression of GNAQ in cancer tissues and negative ones was 65.4% and 59.1%,respectively(log-rank p=0.430).In order to evaluate the relationship between the expression of GNAQ and prognosis in patients with gastric cancer,280 gastric cancer patients with curative tumorectomy was studied with DFS and OS by univariate and multivariate COX regression analysis.The results showed that the stage and differentiation could be regarded as an independent prognostic factor for gastric cancer,and other characteristics could not be an independent prognostic factor for gastric cancer.3.The growth of gastric cancer cell MGC803 was inhibited after knockdown of GNAQ.We observed the proliferative activity of the cells for 5 days.From the second day,visible cell reducing in MGC803 cells infected with Lv-sh GNAQ was observed comparing with Con and sh Con group,To the fifth day,it is more obvious,the number of cells in Lv-sh GNAQ group were 35% of sh Con group,***p <0.001.4.Reduction of MGC803 clone formation was observed after knockdown of GNAQ.Quantitative observation showed that,in the MGC803 cells,when the intracellular GNAQ was specifically reduced by sh GNAQ,the average colony number in Con group and sh Con group was 210±9.2 and 199±5.1,respectively.The average colony number in sh GNAQ group was 5.5±1.2,***p <0.001.5.MGC803 cell cycle arrest was observed in gastric cancer cells after GNAQ knockdown.Quantitative analysis showed that the proportion of cells with sh GNAQ group in G0/G1 phase was decreased significantly compared with sh Con group(19.30±0.53% vs.67.58±0.22%,***p<0.001),the proportion of cells in S phase was significantly increased(68.15±0.42% vs.19.74±0.72%,***p<0.001).No significant difference were observed between sh Con and sh GNAQ cells in the percentage of cells in G2/M phase(17.33 + 0.23%.16.62 + 1.01%,p=0.825).The proportion of sub G1 phase cells was significantly higher in the sh GNAQ group comparing with sh Con group(14.43±0.82% vs.1.23±0.09%,***p<0.001).6.With Knockdown of GNAQ in gastric cancer cell line MGC803,p53 and p21 was upregulated,Cyclin A and p-CDK2 was down regulated,p-ERK and p-MEK was also down regulated.Therefore,GNAQ can regulate the growth of gastric cancer cells by affecting p53/p21 and ERK/MEK pathway.Part 2:1.The expression of MPP8 in gastric cancer tissues was significantly higher than that of normal tissues(83% vs.52%,p<0.001***).Comparison of MPP8 and clinical features of gastric cancer indicates that MPP8 expression in male patients was significantly higher than that of female patients(86.1% vs.75.6%,p=0.035),patients with III/IV stage was significantly higher than that of patients with I/II stage(88.6% vs.79.5%,p=0.046),patients with moderate differentiation was significantly higher than that of patients with low differentiation(90.2% vs.78.6%,p=0.011),patients with adjuvant chemotherapy was significantly higher than that of patients without adjuvant chemotherapy(86.3% vs.80.1%,p<0.01).However,there was no significant difference in the expression of MPP8 in gastric cancer patients with different ages and different tumor locations.2.Patients with positive expression of MPP8 in cancer tissues tended to develop progression(recurrence or metastasis)earlier than those with negative ones,which was more pronounced in patients with poorly differentiated gastric cancer.We further evaluated the relationship between MPP8 expression and prognosis in patients with gastric cancer.3 years DFS rate in GC patients with positive expression of MPP8 in cancer tissues and negative ones was 61.9% and 74.5%,respectively(log-rank p=0.088).3 years OS rate in GC patients with positive expression of MPP8 in cancer tissues and negative ones was 66.2% and 78.4%,respectively(log-rank p=0.087).Subgroup analysis showed that this kind of situation is more significant in poorly differentiated gastric cancer patients.3 years DFS rate in poorly differentiated gastric cancer patients with positive expression of MPP8 in cancer tissues and negative ones was 51.8% and 72.2%,respectively(log-rank p=0.022).3 years OS rate in poorly differentiated GC patients with positive expression of MPP8 in cancer tissues and negative ones was 56.6% and 74.5%,respectively(log-rank p=0.017).DFS and OS by univariate and multivariate COX regression analysis were also studied.The results showed that the stage,differentiation and adjuvant therapy could be regarded as an independent prognostic factor for gastric cancer.3.Growth of gastric cancer cell MGC803 was inhibited after knockdown of MPP8.The proliferative activity of the cells was observed for 5 days.From the second day,visible cell reducing in MGC803 cells infected with Lv-sh MPP8(S1)? Lv-sh MPP8(S2)were observed comparing with Con and sh Con group,To the fifth day it is more obvious,the number of cells in Lv-sh MPP8(S1)? Lv-sh MPP8(S2)group were 28% and 20% of sh Con group,**p <0.01 and **p <0.01.4.Reduction of MGC803 clone formation was observed after knockdown of MPP8.Quantitative observation showed that,in the MGC803 cells,when the intracellular MPP8 was specifically reduced by sh MPP8(S1),the average colony number in Con group and sh Con group was similar,101±7.8 and 96±2,respectively.The average colony number in sh MPP8(S1)group was 23.7±5.7.**p<0.01.When the intracellular MPP8 was specifically reduced by sh MPP8(S2),the average colony number in Con group and sh Con group was similar,107.7±9.0 and 100.7±16.8,respectively.The average colony number in sh MPP8(S2)group was 7.0±2.6,**p<0.01.5.MGC803 cell cycle arrest was observed in gastric cancer cells after MPP8 knockdown.Quantitative analysis showed that the proportion of cells with sh MPP8(S1)and sh MPP8(S2)groups in G0/G1 phase was decreased significantly compared with sh Con group(45.10±0.79% and 38.54±0.75% vs.51.27±0.79%,**p<0.01 and **p<0.01),the proportion of cells in S phase was significantly increased(42.22±0.44% and 45.19±0.78% vs.30.80±0.82%,**p<0.01 and **p<0.01).No significant difference were observed in sh Con,sh MPP8(S1)and sh MPP8(S2)groups with the percentage of cells in G2/M phase(17.94±0.94%,16.27±1.47%,12.68±0.85%).The proportion of sub G1 phase cells was significantly higher in sh MPP8(S1)and sh MPP8(S2)group compared with sh Con group(24.33±1.17% and 36.34±0.28% vs.1.55±0.12%;**p<0.01 and **p<0.01).6.Apoptosis of gastric cancer cell MGC803 was increased after knockdown of MPP8.Annexin V / 7AAD double stains gastric cancer cell MGC803 in shcon,sh MPP8(S1)and sh MPP8(S2)group.Quantitative analysis indicates the proportion of apoptotic cells was significantly increased in sh MPP8(S1)and sh MPP8(S2)group compared with the control group(Annexin V+/7AAD-:25.55±0.56% and 24.62±0.32% vs.5.96±0.07%,**p<0.01 and **p<0.01.Annexin V+/7AAD+:40.04±0.61% and 64.18±0.23% vs.8.56±0.22%,**p<0.01 and **p<0.01).7.The ability of migration and invasion of gastric cancer cells was decreased after knockdown of MPP8.The number of migrated cells in sh MPP8(S1)and sh MPP8(S2)group was fewer than sh Con group(4.7±1.4 and 77.9±1.7 vs.109.5±1.5,**p<0.01 and **p<0.01).The results of MTT assay showed that the number of sh MPP8(S1)and sh MPP8(S2)cells was about 24% and 63% of shcon groups,**p<0.01 and **p<0.01.8.MPP8 regulates apoptosis and metastasis related pathways to influence the development of gastric cancer.With Knockdown of MPP8 in gastric cancer cell line MGC803,p53,Bax and PARP was upregulated,Bcl-2 was down regulated.N-cadherin expression was down regulated and the expression of E-cadherin is still normal.Vimentin was down regulated.Conclusions:Part 1:1.GNAQ is overexpressed in gastric tissues.2.After GNAQ knockdown,the growth and proliferation of gastric cancer cells was decreased,cell cycle arrest and apoptosis was increased.3.GNAQ can regulate the growth of gastric cancer cells by affecting p53/p21 and ERK/MEK pathway.Part 2:1.MPP8 is highly expressed in gastric cancer tissues,and the positive expression of MPP8 in the patients with cancer is prone to have poor prognosis.2.After MPP8 knockdown,the growth,proliferation and migration ability of gastric cancer cell was decreased,cell cycle arrest and apoptosis was increased.3.MPP8 regulates the growth and metastasis of gastric cancer by affecting p53/Bcl-2 and EMT pathway. |
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