| Background: Periostin(POSTN),originally identified as an osteoblastspecific factor,is a multifunctional extracellular matrix protein that secreted by osteoblasts.It has been found to be overexpressed in various types of human cancers such as pancreatic cancer,ovarian cancer,and prostate cancer.Then it is considered as one candidate gene of malignant tumors metastasis.Studies showed that high expression of periostin correlates with poor prognosis of non-small cell lung cancer(NSCLC).Meanwhile,periostin can alter the chemosensitivity of the tumor cells.Periostin can activate some signal pathways and result in chemoresistance in ovarian cancer cells and colon cancer cells.But there are no discussion and research about the relationship between POSTN and cisplatin resistance of non-small cell lung cancer and the possible mechanism.Based on these backgrounds,in this study,we aimed to explore the possible role of periostin in the development of cisplatin resistance in NSCLC cells and its mechanisms.In the first part,we build human non-small cell lung cancer cisplatin-resistant cell line A549/CDDP by stepwise increasing the dosage of cisplatin;and analyze the expression of periostin in cisplatin-resistant cell line A549/CDDP and the parental cell line A549.Preliminary explored the relationship between periostin and cisplatin--resistance in human non-small cell lung cancer.In the second part,we systematically analyzed the effect of periostin on a series of biological behavior of human non-small cell lung cancer cell line A549 cells such as proliferation,cell cycle and apoptosis;and to study the role of Stat3/survivin signal pathway known as inhibition of apoptosis,as to further explore the possible mechanism involved.In the third part,we explored whether silencing POSTN could reverse cisplatin resistance of non-small cell lung cancer in vitro and the related mechanism.Then we established nude mouse xenograft tumor model of non-small cell lung cancer,and further explored effect of POSTN knockdown on cisplatin resistance in vivo.Part one An experimental study on the correlation between periostin and cisplatin resistance in non-small cell lung cancerObjective: The purpose of this study was to build human non-small cell lung cancer cisplatin-resistant cell line A549/CDDP by stepwise increasing the dosage of cisplatin;and analyze the expression of periostin in cisplatinresistant cell line A549/CDDP and the parental cell line A549.And explore the relationship between periostin and cisplatin-resistance in human non-small cell lung cancer.Methods: A549 cells were incubated with different concentrations of cisplatin and calculate the 50% inhibitory rate(IC50)by MTT assay,and then build human non-small cell lung cancer cisplatin-resistant cell line A549/ CDDP by stepwise increasing the dosage of cisplatin.By comparing the m RNA and protein expression of periostin in these two cell line or in A549 cells incubated with different concentrations of cisplatin;we explored the relationship between periostin and cisplatin-resistance in human non-small cell lung cancer.Data were analyzed with ANOVA test.Results: 1 By stepwise increasing the dosage of cisplatin,ultimately,we obtained continues proliferation cells in 20μM cisplatin-containing medium,namely A549/CDDP.IC50 of A549/CDDP and A549 were 25μM and 10μM,A549/CDDP obtained resistance to cisplatin.2 Cells were incubated with 0~30μM CDDP for 48 h,and then detected the expression of POSTN.It was found that m RNA and protein expression levels of POSTN were increased in a CDDP concentration-dependent manner.The difference was statistically significant(P<0.01).3 m RNA expression level of POSTN in A549/CDDP cells was about 6 times of A549 cells;and protein expression level of POSTN in A549/CDDP cells was about 5.1 times of A549 cells.The differences were statistically significant(P<0.05).Conclusion: 1 We successfully constructed human non-small cell lung cancer CDDP-resistant cell line A549/CDDP by stepwise increasing the dosage of cisplatin.2 By analysis the relationship between periostin expression and resistance to CDDP of these two cell lines,periostin was found to have a positive correlation with CDDP-resistance in non-small cell lung cancer.Part two Effect of periostin on the biological behavior of A549 cells and its mechanismObjective: To systematically analysis effect of periostin on a series of biological behavior of human non-small cell lung cancer cell line A549 cells such as proliferation,cell cycle and apoptosis;and to study the role of Stat3/survivin signal pathway known as inhibition of apoptosis,as to further explore the possible mechanism involved.Methods: 1 First constructed POSTN overexpression plasmid vector,and the over-expression vector was transiently transfected into A 549 cells.The transfection efficiency was detected by western blot assay.2 MTT,flow cytometry,Western blot and other detection methods were used to analysis effect of POSTN overexpression on cisplatin-induced cell survival,cell cycle,apoptosis and the activity of Stat3/survivin signal pathway.3 By co-transfection of POSTN overexpressing plasmid vector and survivin silencing sh RNA,we explored the possible mechanism involved in the regulation of POSTN on cisplatin resistance.Results: 1 Transfection the POSTN plasmid into A549 cells could significantly increase the periostin expression.2 The cell viability was measured by the MTT assay.Data showed that compared with the control group,the cell survival rate in POSTN overexperssion group after incubation of 10~ 30μM CDDP increased to 1.56 ±0.37,1.62±0.17,3.43±1.82-fold(P<0.05).It demonstrates that exogenous overexpression of POSTN could decrease cell sensitivity of A549 cells to cisplatin.3 Cell cycle distribution analysis using flow cytometry showed that cell population in G0/G1 phrase in control group,10μM CDDP treatment group,CDDP+pc DNA3.1(+)vector treatment group was 50.04±3.27%,90.30± 4.25%,88.2±3.78%,and cell population in S phrase was 46.44±2.31%,8.69±2.79%,8.92±2.81%,suggesting that transfection of pc DNA3.1(+)vector has no significant effect on CDDP-induced cell cycle inhibition.Compared to CDDP treatment group,cell population in G0/G1 phase in CDDP+periostin overexpression group decreased to 67.49±5.3% from 90.3±4.25%,and cell population in S phase increased to 28.84±2.11% from 8.69±2.79%(P<0.05 vs.CDDP).4 Quantitative analysis of cell apoptosis by flow cytometry Annexin V/PI double staining showed that early apoptotic rate in control group,10μM CDDP treatment group,CDDP+pc DNA3.1(+)vector treatment group was 2.45±0.56%,26.32±2.21%,27.93±3.71%;and the late apoptotic rate was 2.57±0.46%,13.02±2.01%,12.33±2.35%.Compared to CDDP treatment group,early cell apoptosis in CDDP+periostin overexpression group decreased to 11.91±2.74% from 26.32±2.21%,and the late cell apoptosis in CDDP+periostin overexpression group decreased to 7.03±2.05% from 13.02±2.01%(P<0.05 vs CDDP).It demonstrated that exogenous overexpression of POSTN significantly inhibited CDDP-induced apoptosis in A549 cells.5 Western Blot assay was used to investigate Caspase-3 activation in A549 cells.Data showed that with the incubation of 10μM CDDP for 48 h,remarkably increased cleaved caspase-3 protein expression,but overexpression of POSTN can significantly reverse this activation effect of CDDP.6 We tested the involvement of Stat3/surviving signal pathway in periostin-mediated CDDP resistance.Western blot analysis showed that compared with the control group,incubation with 10μM CDDP for 48 h could decrease the phosphoration of Stat3 to 0.43±0.05-fold,and after overexperssing POSTN,the phosphoration of Stat3 increased to 0.87±0.09(P<0.05 vs.CDDP);the expression of survivin reduced to 0.38±0.03-fold by incubation with 10μM CDDP for 48 h,while POSTN overexpression increased survivin protein expression to 0.79±0.06-fold from 0.38±0.03(P<0.05 vs.CDDP).7 After silencing survivin in periostin overexpressing A549 cells,cell apoptosis was detected by flow cytometry Annexin V/PI double staining.Data showed that early apoptotic rate in 10μM CDDP treatment group,periostin overexperssion group was 26.89±2.24%,13.13±1.54%;and late apoptotic rate was 13.24±2.63%,8.42±2.57%.Compared to periostin overexperssion group,the early apoptotic rate in POSTN+S-sh RNA group increased to19.97±2.14% from 13.13±1.54%(P<0.05 vs.POSTN).More than that,the Caspase-3 activation was also significantly enhanced(P<0.05 vs.CDDP).Conclusion: We successfully constructed periostin overexpression vector,and the study indicated that exogenous overexpression of periostin could promote cell cycle transition,promote cell proliferation,while inhibit cell apoptosis and reverse pro-apoptotic effect of cisplatin in A549 cells.The mechanism may be related to the activation of Stat3/survivin signal pathway,leading to tumor cells apoptotic resistance.Part three Down-regulation of periostin reverses cisplatin resistance of human non-small cell lung cancer in vitro and in vivoObjective: To explore whether silencing POSTN could reverse cisplatin resistance of non-small cell lung cancer in vitro and the related mechanism.Then we established nude mouse xenograft tumor model of non-small cell lung cancer,and further explored effect of POSTN knockdown on cisplatin resistance in vivo.Methods:1 Periostin-targeted sh RNA was used to silence POSTN expression in A549/CDDP cells.And MTT,flow cytometry and Western blot assays were used to analysis effect of POSTN depletion on cisplatin-induced cell survival and cell apoptosis.2 We established nude mouse xenograft tumor model of non-small cell lung cancer,and further explored effect of POSTN knockdown on anti-tumor efficiency of cisplatin resistance,and analyzed the activation of Stat3/survivin signal pathway in tumor tissues.Results: 1 Western blot assay was used to detect the transfection efficiency after transfected with P-sh RNA and selected by G418.Data showed that the expression of POSTN in P-sh RNA group decreased by 0.42±0.09 fold in the right position,compared with the control group.It demonstrated that transfection with P-sh RNA could impressively inhibit the endogenous periostin expression.2 MTT assay was used to detect cell survival rate.Results showed that compared with the control group,the cell survival rate in P-sh RNA group after incubation of 0~ 30μM CDDP reduced 1.0±0.05,0.7±0.04,0.64±0.09 fold(P<0.05).3 Flow cytometry apoptosis detection showed that: the early apoptotic rate in control group,30μM CDDP-treatment group,CDDP+C-sh RNA treatment group were 2.15±0.41%,13.41±2.43%,15.14±2.54%,and the late apoptotic were 1.92±0.32%,8.83±2.01%,10.01±2.37%.Compared with CDDP treatment group,the early apoptotic rate in CDDP+P-sh RNA group increased to 29.87±3.21% from 13.41±2.43%,and the late apoptotic rate increased to 20.13±3.14% from 8.83±2.01%(P<0.05 vs.CDDP).4 Mouse studies showed that mean volume of xengraft tumor in each group has no significant difference before treatment with PBS or CDDP(P>0.05),but after that mean volume of xengraft tumor in each group increased slowly and the difference emerged in 20 days later.At the end of experiment,mean volume of xengraft tumor in C-sh RNA+PBS group,P-sh RNA+PBS group,C-sh RNA+CDDP group,P-sh RNA+CDDP group were(2900±150.28),(2100±135.16),(1630.75±109.60),(445±41.04)mm~3.All were much bigger(P<0.01),and the xengraft tumor volume in P-sh RNA+CDDP group was the least.5 Western blot analysis of resected xenograft tumors showed that compared with C-sh RNA+PBS group,the phosphoration of Stat3 in C-sh RNA+CDDP and P-sh RNA+CDDP group reduced to 0.63±0.06 and 0.32±0.03-fold(P<0.05 vs.C-sh RNA);the expression of survivin in C-sh RNA+CDDP and P-sh RNA+CDDP group reduced to 0.58±0.07 and 0.21±0.02-fold(P<0.05 vs.C-sh RNA).Conclusion: 1 Silence POSTN could reverse cisplatin resistance of non-small cell lung cancer A549/CDDP and promote cell apoptosis.2 POSTN depletion re-sensitizes CDDP-resistant A549 cells to CDDP treatment and suppresses the activation of Stat3/survivin signal pathway in vivo. |
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