| Part One The construction and identification of human gastric subcutaneous tumor model and orthotopic transplantation tumor model which stable si RNA CLIC1 of nude miceObjectivesTo take advantage of stable and monoclonal si RNA CLIC1 gastric cancer cell linesfrom early stage research and to constructhuman gastric subcutaneous tumor model and orthotopic transplantation tumor model in nude mice.Protein Simple Wes technology(WES)was used to identify models.MethodsWe selected the cell lines which from the early stage research and cultured,the cell lines contain 2 control group(CON group)include SGC-7901 and MGC-803,2 negative control groups(NC group)include SGC-7901-NC and MGC-803-NC1,2 interference silence groups(Knock Down,KD group)include SGC-7901-CLIC1-KD and MGC-803-CLIC1-KD.The cell lines in logarithmic growth phase were obtained by conventional digested and made into single cell suspension,six BALB/C nude mice which were randomly selected of each groupwere injected 0.2ml single cell suspension(cells above 1×10~7)in forelimb armpit,7 days after the mice appear around6mm diameter subcutaneous nodules,then subcutaneous tumor model was successfully established.The morphology of subcutaneous tumor was observed by HE staining,the expression of CLIC1 in subcutaneous tumor was detected by WES.The tumor mass of each group was passaged and cultured in six nude mice which were randomly selected(generally 2nd generation).The tumor mass was planted under nude mice stomach serosaby surgical operation,then constructingorthotopic transplantation tumor model in nude mice.Each group contains six mice which randomly selected.he expression of CLIC1inorthotopic transplantation tumor detected by WES.ResultsSuccessfully constructed the subcutaneous tumor models of SGC-7901,SGC-7901-NC,SGC-7901-CLIC1-KD,MGC-803 and MGC-803-NC1,but the MGC-803-CLIC1-KD subcutaneous tumor model fails to build,probably due to after silencing CLIC1 MGC-803-CLIC1-KD in vivo tumorigenicity capacity was significantly decline.The research group decided to use targeted gene silencing CLIC1 specially chemically modified si RNA(chol-si RNA)by novo synthesis,and applied to multiple local multiple injections of subcutaneous tumors(MGC-803),then obtained corresponding subcutaneous tumor MGC-803-CLIC1-chol whichtested the inhibition rate of CLIC1 was 79%vs MGC-803-NC,could be used for subsequent experiments.HE staining confirmed subcutaneous tumor was gastric cancer cell xenografts.The tumor mass of six groups was accomplishedpassaged and cultured(generally 2nd generation).The speed of tumor formation of primary subcutaneous tumors and2 generations subcutaneouswas significantly slower(P<0.05 KD vs CON and NC).Finally,the tumor mass was planted under nude mice stomach serosaby surgical operation,then successfully constructing orthotopic transplantation tumor model in nude mice.The inhibition rate of CLIC1 of orthotopic transplantation tumor mass of SGC-7901-CLIC1-KD and MGC-803-CLIC1-chol was 67%and 87%,respectively.ConclusionEach group was successfully constructed human gastric orthotopic transplantation tumor model in nude mice.Part Two Roles of si RNA-mediated inhibition of CLIC1 expression on the biological behaviors of human gastric orthotopic transplantation tumor of nude miceObjectivesTo explore the effect of silencing CLIC1 by si RNA interfence on the the biological behaviors(growth,metabolism,apoptosis,invasion and metastasis)of human gastric orthotopic transplantation tumor.MethodsOrthotopic transplantation tumor volume and weight was measured in each group;apoptosis was detected with Tunel assay;PET-CT was used for the detection of the changes of tumor metabolism and the metastases of each group of orthotopic transplantation tumor;Organs and tissues(intestine and lymph nodes)invasion and metastasis of orthotopic xenograft tumor in nude mice in each group was observed by HE staining.ResultsThe volume of gastric orthotopic transplanted tumor of silencing CLIC1expression group(SGC-7901-CLIC1-kd and MGC-803-CLIC1-chol)were(0.071±0.021)cm~3,(0.004±0.002)cm~3,respectively,was lower than the CON group and NC groups(F=37.27 and 4.740,respectively,P<0.05),the difference between the CON and NC groupwas not statistically significant(P>0.05).Tumor weights of orthotopic transplanted tumor of SGC-7901-CLIC1-kd and MGC-803-CLIC1-chol were(0.387±0.108)g,(0.0350±0.0105)g,respectively,were significantly lower than CON and NC group(F=19.93 and4.331,espectively,P<0.05),the difference between the CON group and NC group was not statistically significant(P>0.05).The apoptosis condition of orthotopic transplantation tumor of SGC-7901-CLIC1-kd and MGC-803-CLIC1-chol were significantly higher than CON and NC group(P<0.05),the number of fluorescent cells were(102±15.26),(100±19.53),respectively,were significantly higher than CON and NC group(F=67.50 and41.82,respectively,P<0.001),the difference between the CON and NC group was not statistically significant(P>0.05).The number of positive cells of normal staining were(96.6±18.49),(96.5±14.27),respectively,were significantly more than CON and NC group(F=48.89 and 33.66,respectively,P<0.001),the difference between the CON and NC group was not statistically significant(P>0.05).The metabolism(%ID/gvalues)orthotopic xenograftstumor of SGC-7901-CLIC1-kd and MGC-803-CLIC1-chol were(1.283±0.172),(0.6915±0.1344),respectively,was significantly weaker than the CON group and NC groups(F=25.65 and 22.33,respectively,P<0.001).The number of lymph node metastasis of orthotopic transplantation tumor model of SGC-7901-CLIC1-kd and MGC-803-CLIC1-chol was 0,1,respectively,were significantly less than the CON group and NC group(X~2=7.255 and 7.467,respectively,P<0.05).Finally CON and NC group observed intestinal invasion infiltration,silencing CLIC1expression group was not observed.ConclusionThe inhibition of CLIC1 expressioneffectivelysuppresses tumor growth,metabolism and lymphatic infiltration capacity in vivo.Inhibiting CLIC1expressionpromoted orthotopic xenografts tumor apoptosis in vivo.Part Three The mechanisms of si RNA-mediated inhibition of CLIC1influencing the tumor progression of human gastric orthotopic xenografts tumor of nude miceObjectiveTo evaluate the possible molecular mechanism of influencing the tumor progression of human gastric orthotopic xenografts tumor of nude mice by inhibiting CLIC1.MethodsFluorescence quantitative PCR were used to test the expression of apoptosis(Bcl-2,Caspase-3)and integrin(α1,α3,αv,β1)genes.Western blot were used to test the expression of apoptosis(Bcl-2,Caspase-3,Bax)and integrin(α1,α3,αv,β1,β3)proteins.Immunohistochemistrywere used to test the expression of CLIC1and integrin(α1,α3,αv,β1,β3,β5)proteins.ResultsThe m RNA relative expression of Bcl-2 and Caspase-3 of gastric orthotopic transplanted tumor of SGC-7901-CLIC1-kd was(0.659±0.009)and(0.973±0.063),respectively.And MGC-803-CLIC1-chol was(0.790±0.008)and(0.960±0.113),respectively.The expression of Bcl-2 was lower than the CON group and NC groups(P<0.01),its expression inhibition rate was 34.90%and22%,respectively,and the expression of Caspase-3 did not change significantly(P>0.05).The m RNA relative expression of integrin gene(α1,α3,αv,β1)of gastric orthotopic transplanted tumor of SGC-7901-CLIC1-kd was(1.518±0.132),(0.633±0.045),(0.558±0.089)and(0.603±0.081),respectively.Integrinα1 was 50.20%higher than CON group,integrinα3,αv,β1was 36.67%,44.27%,39.77%lower than the CON group(P<0.05).And MGC-803-CLIC1-chol was(1.417±0.053),(0.705±0.031),(0.861±0.024)and(0.616±0.062),respectively.Integrinα1 was 41.73%higher than CON group,integrinα3,αv,β1was 30.83%,13.97%,38.47%lower than the CON group(P<0.05).The proteins relative expression of Bcl-2,Bax and Caspase-3 of gastric orthotopic transplanted tumor of SGC-7901-CLIC1-kd was(0.062±0.003),(0.264±0.006)and(0.061±0.010),respectively.And MGC-803-CLIC1-chol was(0.048±0.002)(0.175±0.009)and(0.061±0.010),respectively.Bcl-2and Caspase-3wasdown-regulated than the CON group and NC groups(P<0.05),and Baxwasup-regulated than the CON group and NC groups(P<0.05).The proteins relative expression of Bcl-2 of KD group was 41.50%and 54.71%lower than the CON group,respectively,and Caspase-3 was 62.58%and 71.18%,respectively.But Bax was 120%and 200%higher than CON group,respectively.The proteins relative expression of integrinsα1,α3,αv,β1,β3of gastric orthotopic transplanted tumor of SGC-7901-CLIC1-kd was(0.582±0.080),(0.076±0.017),(0.082±0.012),(0.077±0.002)and(0.051±0.014),MGC-803-CLIC1-chol group were(0.607±0.109),(0.049±0.016),(0.067±0.010),(0.049±0.007)and(0.038±0.002).Compared with the CON and NC group,α1 expression was up-regulated,α3,αv,β1 andβ3 expression was down-regulated(P<0.05).The proteins relative expression of Integrinα1 of KD group was 1.16 times and 1.13 times higher than the CON group,respectively.But integrinα3 was 55.29%and 66.89%lower than the CON group,integrinαv was 86.03%and 86.43%,integrinβ1 was 33.62%and 43.02%,and integrinβ3was 48.49%and 42.22%,respectively(P<0.05).The proteins relative expression of p-AKT,AKT,p-p38,p38,p-ERK,ERK of gastric orthotopic transplanted tumor of SGC-7901-CLIC1-kd was(0.058±0.018),(0.163±0.016),(0.120±0.004),(0.131±0.027),(0.073±0.013)and(0.159±0.019),MGC-803-CLIC1-chol was(0.041±0.008),(0.164±0.006),(0.211±0.015),(0.133±0.009),(0.062±0.011)and(0.134±0.016).Compared with the CON and NC group,p-AKT p-ERK expression was down-regulated,p-p38expression was up-regulated(P<0.05).and the expression of AKT,p38 and ERK did not change significantly(P>0.05).The expression inhibition rate of p-AKT of KD group was 36.96%and 47.44%,respectively.p-ERK was 46.32%and 53.03%,But the proteins relative expression of p-p38was 88.89%and 151%higher than the CON group.Immunohistochemistry showed:the proteins expression of CLIC1 and integrinsα1,α3,αv,β1,β3andβ5 of gastric orthotopic transplanted tumor was visible,integrinβ5 which detected by RT-PCR and WESwere no expressionwas also visibly expressed.Through observation,compared with CON and NC group,the proteins expression of integrinsα1of gastric orthotopic transplanted tumor was up-regulated trend,CLIC1,αv,β1,β3 andβ5 was down-regulated trend.ConclusionAfter the silenced CLIC1 expression,the expression of gene and protein of integrins family and apoptotic family werechanged significantly,and also found that Akt and MAPK pathways werechanged significantly.Therefore,we concluded:the possible molecular mechanism of influencing the tumor progression(growth,metabolism and invasion diminished capacity was decreased,apoptosis was increased and)of human gastric orthotopic xenografts tumor of nude mice by inhibiting CLIC1may be related to integrins family changed significantly,influenced Akt,MAPK pathway then occured sequential reactions that ultimately affected the progression of gastric cancer. |
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