Differential Gene Expression Screening Of Cisplatin-resistant Human Lung Adenocarcinoma Cell And Initial Function Research

BackgroundLung adenocarcinoma is one of the most common malignant tumors of respiratory system.Due to the increase of environmental pollution, numbers of the smokers and their smoking frequencies,the incidence and mortality of lung adenocarcinoma are progressively increasing all over the world these years.The global incidence of lung adenocarcinoma is as high as seventy every hundred thousands,which is the idiopathic reason of malignant tumors.In China the incidence of lung cancer has been in the first place of malignant tumors in many major cities,According to the data of American Cancer Society,nearly among 500,000 patients died of cancer each year,61%are related to idiopathic drug resistance and 33% are related to insecondary drug resistance.Most patients died of cancer have something to do with the developmeng of drug resistance,whether indirectly or directly.Among lung cancer and other tumors,Cisplatin is the most wide-used drug for chemotherapy.While with the development of idiopathic or insecondary drug resistance in tumor cells their clinical therapeutic effects are reduced,which brings a great deal trouble to the treatment.The mechanism of drug resistance exhibited in lung cancer is a complex biological process with multiple factors and causes.There is no single mechanism that can explain this phenomenon completely. Therefore,it is necessary to research the drug resistance mechanism of lung cancer.The process of cells growing,differentiation,and death are based on specific gene expressions.Differential gene expression produce different cell biological behaviors,in which the phenotype of a cell is determined by type,numbers,and orders of the expression of gene.It has been reported by many researchers on lung cancers that the gene expression profiling of various pathological types,differentiation levels,clinical stages,and potentiality of metastasis in cells are different.By a comparison of the gene expression differences between tumor cells with different drug resistance levels but similar genetic background,the tumors' relative drug resistance genes can be screened out.The two cell strains of human lung adenocarcinoma cell strain(A 549)and cisplatin-resistant human lung adenocarcinoma cell strain(A 549/CDDP), which come from the same parental origin,with identical genetic background have a primary difference on the drug sensitivity and resistant level,that they are suitable for this comparison research.In this way,the histological differences during sampling can be avoided, relationship among each screened genes can be reflected completely and accurately.As a result,the cell strains of A 549 and A 549/CDDP are selected to establish the gene expression profiling,and screen out the differential gene expression,so that the cisplatin resistance gene in lung adenocarcinoma cells will be found,and the resistance mechanism will be discussed.While,the screening on tumor cells of different phenotypes,either sensitive or resistant to cisplatin,can only serve as an initial insight into the possible differential gene expressions.To make sure their genetic function,further investigation must be conducted on the relationship between differential gene expressions and drug resistance.RNA interference,which is one of the newest techniques employed in biological science research recent years,was discovered by Fir and his colleagues in 1998.It describes a process performed by a double-stranded RNA molecule,which shuts down its corresponding sequences of gene expression and makes a specific post-transcriptional silence at certain sequence to generate a similar effect with gene knock-out.This technique offers a unique advantage over traditional method,because of its high specificity and efficacy,which is easier to carry out.As a result,RNA interference has become an important approach in gene function research. Genes with obvious up-regulating expressions from the screening of differential gene expression in drug resistance of lung cancer are selected. RNA interference is used to seal off the expression of these specific genes in cisplatin-resistant lung cancer tumor cells(A 549/CDDP).The in vitro experiment could prove that these genes do participate in the development of cisplatin-resistance in lung cancer cells.By reversaling this kind of cisplatin resistance better and effective approaches in improving chemotherapeutic effects of cisplatin drugs for treating lung cancer could be carried out.In this study,DNA chips were employed to construct gene expression profiling of human lung adenocarcinoma cell strains,A 549 and A 549/CDDP(with drug resistance)of same parental origin.In this way the differential gene expression was confirmed initially which was relative with cisplatin-resistance in the lung adenocarcinoma cells.The mechanism of drug resistance was discussed to provide clues for future researches.By gene chip technology,several genes with differential expression were selected.The effect of genes involved in cisplatin-resistance lung cancer cells was analyzed with helps from relative reported references. Among them,a particular gene CDK7,with obvious up-regulating gene expression,was targeted to conduct the later parts of the experiment in this study.Gene CDK7 silenced by siRNA interference technique was observed for its effect on the bionomics of the cisplatin-resistance lung adenocarcinoma cell strains A 549/CDDP.Further research on the effect of CDK 7 on the development of cisplatin resistance was carried out, which could provide effective evidences on developing drugs deteriorating tumors with CDK 7 as a target.Chapter 1:Using DNA Chips to screen Gene Expression Variation in Cisplatin-Resistant Lung Adenocarcinoma CellsObjective:To establish differential expression profiling of human lung adenocarcinoma cells(A 549)and cisplatin-resistant human lung adenocarcinoma cells(A 549/CDDP),screen the gene expression variation to provide an initial confirmation on the relationship between gene expression and the drug resistance.Method:The mRNA of cell strains of regular human lung adenocarcinoma(A 549)and cisplatin-resistant human lung adenocarcinoma(A 549/CDDP)are first extracted and underwent reverse transcription to create cDNA.The cDNA will then be labeled by cy5-dUTP and cy3-dUTP before hybridization on DNA chips.The chips will then be scanned by Scan Array 4000 for its fluorescent signals,to create a gene image,where it is further digitalized and studied by gene pix analyzing software,GerePix 3.0.Results:232 genes with differential expression associated with cisplatin-resistant lung adenocarcinoma are,screened and 18 different genes show up-regulation in gene expression while 50 are down-regulated.Further analysis on these genes shows that they can be classified into different categories: oncogene and,anti-oncogene,gene regulating DNA binding/transcription and transcription factors,gene for cellular signaling and transducing, gene for cell receptors,gene regulating protein translation synthesis,gene involving in cellular cytoskeleton and motility,gene for metabolism and growth,and lastly genes with unknown functions.Conclusion:1)there exhibits differential gene expression between the tumor cells of lung adenocarcinoma and cisplatin-resistant ones.2)The number of genes with differential gene expression is 232,and the classifications are as follows:oncogene and,anti-oncogene,gene regulating DNA binding/transcription and transcription factors,gene for cellular signaling and transducing,gene for cell receptors,gene regulating protein translation synthesis,gene involving in cellular cytoskeleton and motility, gene for metabolism and growth,and lastly genes with unknown functions.It suggests that the cisplatin-resistance mechanism in lung cancer is a complex biological process involving multiple genes and factors.3)The screened genes have the greatest chance of being the ones involved in developing the drug-resistance ability.Related references have suggested that CDK7 has the greatest possibility of possessing the function of drug-resistance for cisplatin in lung cancer.4)DNA chips is a highly effective,accurate and high-throughput research method,,and can be used to study the gene expression level in lung cancer.Chapter 2:The Verification of CDK7's Role,with Differential Expression,in Development of Cisplatin-resistant ability in Human Lung Adenocarcinoma CellsObjective:To verify the reliability of the established differential expression profiling of both cell strains,of lung adenocarcinoma(A 549) and cisplatin-resistant ones(A 549/CDDP).Method:Real-time Fluorescence Quantitative PCR is employed to detect CDK₇ mRNA and Western blot is used to detect CDK₇ protein level;the expression levels of the two cell strains(A 549 and A 549/CDDP)are measured.Results: Agarose gel electrophoresis shows that there is a statistical significance(p＜0.01),where the expression level of CDK7 mRNA is much higher in the cell strain A 549/CDDP than that in the cell strain of A 549.Western blot analysis further demonstrates that the resulting protein expression from CDK7 mRNA is indeed much greater in the cell strain A 549/CDDP. Conclusion:The mRNA and protein level in the cell strain A 549/CDDP are much higher than the A 549 strain,suggesting CDK7 gene is related to the cisplatin resistance exhibited by A 549 tumor cells.This result provides strong evidence for our selection of CDK7 gene as target for future research on treatment of cisplatin-resistant lung cancer and the reversal of such resistance.Chapter 3:Initial Research on the Function of CDK₇ GeneObjective:To observe the effect of interference of CDK₇ gene on the biological characteristics of cisplatin-resistant human lung adenocarcinoma tumor cells A 549/CDDP.To further understand CDK₇ gene's role in the drug resistance development,and provide evidence for ensuring CDK₇ gene as a target for reversal of such resistance in the future.Method:With CDK₇ as target,siRNA eukaryotic expression vector of CDK7 gene is constructed,DNA plasmids are extracted and sequenced,as well as siRNA-CDK7 is used to transfect A 549/CDDP cells.The screening of the siRNA sequence is then conducted in the target transfected cells A 549/CDDP by RT-PCR technique.The cells are then classified as:(1)A 549 Group:A 549 cells with lipofectmaine²⁰⁰⁰and 2μg/mL of CDK₇-siRNA2 plasmid DNA(2)Control Group:A 549/CDDP cells with equal amount of media added(3)Liposome Group:A 549/CDDP cells with lipofectmaine²⁰⁰⁰added(4)siRNA group:A 549/CDDP cells with lipofectmaine²⁰⁰⁰and 2μg/mL of CDK₇-siRNA2 plasmid DNA.After the inhibition of gene expression of CDK₇ by siRNA, the indicator of cisplatin resistance is measured by MTT method;the influence on cell cycles of both cell strains A 549 and A 549/CDDP is detected by flow cytometry;as well as the effects on LRP mRNA and protein expression levels of A 549/CDDP is detected by RT-PCR and immunocytochemistry techniques.Results:(1)With the MTT test and calculation:(a)siRNA group:the 50%inhibitory concentration(IC₅₀)is measured at 66.69±8.746μmol/L and the cisplatin-resistance index(RI) is 3.79;(b)A 549/CDDP Control group:the IC₅₀and RI are 185.48±10.754μmol/L and 10.51,respectively;(c)Liposome group of A 549/CDDP:IC₅₀and RI are 182.53±9.625μmol/L and 10.39,respectively; (d)For the A 549 group,the 50%inhibitory concentration is 16.83±7.562μmol/L and the RI value is 0.95.As from these results,it can be seen that the sensitivity for cisplatin drug has increased in the siRNA group of A 549/CDDP cells,and its resistance index,in comparison to the Control group and Liposome group,is dramatically lower(p＜0.01).The IC₅₀and RI value for A 549/CDDP cells in the siRNA group are several times higher than that in the A 549 group(p＜0.01).In comparison to the Control group,the IC₅₀and RI for the Liposome group shows no significant differences(p＞0.05).(2)For the flow cytometry,the percentage of cells in G₀/G₁ cell period shows dramatic increase in the siRNA and A 549 groups,while their cell number in the S-period and G₂/M-period has decreased.This result has statistical significance(p＜0.01).On the other hand,the Control group's cells in G₀/G₁-period show the opposite and decrease while the cells in S and G₂/M-periods increase. The cytometric cell cycle distribution of the siRNA and A 549 groups is similar,while the distribution mapping of the cell cycle of Liposome group matches with the Control group.(3)As the results of RT-PCR and Immunocytochemistry techniques:the LRP mRNA expression level starts to decline 24 hours after the inhibition of CDK₇ gene by siRNA in the A 549/CDDP cells,reaching the minimum level at 48 hours,and this result is significantly lower than the value seen in the Control group(p＜0.01). At 72 hours,the expression level starts to increase,but still at a lower level when compared to the Control group(p＜0.05).The comparison of LRP mRNA expressions at 24^th,48^th,and 72^ndhour periods does show statistical significance(p＜0.05).The Liposome group and the Control group comparison have no statistical significance(p＞0.05).As the results of Immunocytochemistry techniques:the changes in LRP mRNA and protein expression are identical in A 549/CDDP cells of the siRNA group,in which the levels start to decrease at 24 hours with minimum at 48 hours,apparently lower than the Control group(p＜0.01).Although the level starts to increase at 72^ndhour,the level is still lower than the Control group(p＜0.05).The results of imunnocytochemistry analysis at 24^th,48^th,and 72^ndhours have statistical significance(p＜0.05).There is no statistical significance between the Liposome and the Control groups (p＞0.05).Conclusion:1)CDK₇ gene plays an important role in the development of cisplatin resistance in the lung adenocarcinoma,and is possibly one of the related genes of this resistance 2)CDK₇ gene participates in the mechanism of drug resistance to cisplatin in lung cancer,and not only influences it through regulation of cell cycle,but also through the LRP pathway to achieve this 3)Through RNA interference in this study,evidences are provided to suggest that a gene therapy,utilizing reversal of the cisplatin drug resistance in lung cancer by induction of CDK₇ gene,is possible.Final Conclusion1.The gene expression is obviously different between the tumor cells of the human lung adenocarcinoma and the cisplatin-resistant ones.2.The number of genes with differential gene expression is 232,and the classifications are as follows:oncogene and anti-oncogene,gene regulating DNA binding/transcription and transcription factors,gene for cellular signaling and transducing,gene for cell receptors,gene regulating protein translation synthesis,gene involving in cellular cytoskeleton and motility,gene for metabolism and growth,and lastly genes with unknown functions.It suggests that the cisplatin-resistance mechanism in lung cancer is a complex biological process involving multiple genes and factors.3.CDK₇ gene is highly expressed in the tumor cells of cisplatin-resistant lung adenocarcinoma,suggesting a possible relationship between them.4.CDK₇ gene participates in the mechanism of drug resistance to cisplatin in lung cancer,and not only influences it through regulation of cell cycle,but also through the LRP pathway to achieve this. 5.Through RNA interference in this study,evidences are provided to suggest that a gene therapy,utilizing reversal of the cisplatin drug resistance in lung cancer by induction of CDK₇ gene,is possible.6.DNA chips is a highly effective,accurate and high-throughput research method,and can be used to study the gene expression level in lung cancer.