| Background Due to causing a critical threat to human health, Lung cancer hasbeen recognized as one of the most serious disease, classifying Non-small Cell Lung carcinoma(NSCLC) and Small Cell Lung Cancer(SCLC) in accordance with the pathology. In the last ten years, molecular biology of lung cancer has beenstudied further comprehensively. The survival time of advanced non-small cancer patients is obviously extended, and the life quality is remarkably enhanced, with the clinical application of Epidermal Growth Factor Receptor(EGFR) gene and Anaplastic Lymphoma Kinase(ALK) fusion protein molecular targeted drug, which has opened a new treatment for NSCLC. However, in nearly 30 years small cell lung cancer treatment progress almost stagnate with the evidence of 5-year survival rate less than 5%. The biological characteristics of SCLC include that the time of tumor cell doubles shortly; distant metastasis occurs easily in the early stage, the sensitivity of first-line chemotherapy is high, etc. At present, the main treatments measures for SCLC include joint local radiotherapy and systemic chemotherapy.The efficacy of first-line chemotherapy to SCLC gets 60-80%, but it is extremely easily to cause drug resistance after chemotherapy and give rise to relapse and metastasis. Meanwhile, the second-line chemotherapy is lack of effective treatment at present. Some studies have found that SCLC has a large number of somatic mutation and heterogeneity. Although the clinical trials has been conducted many times, but the validity is proved very low, and so far the effective molecular target treatment has not yet been found. All the time it is a big challenge in the clinical research of SCLC, so the effective treatment is urgently needed.The popular researches mainly focused on improving the chemotherapy drug resistance and the different treatments, such as immune therapy and biological therapy, etc. Becauseapoptosis is the active and programmed death by multiple gene regulation in order to maintain stable internal environment, which play a significant role in the incidence and development of malignant tumor.Inhibitor of Apoptosis Proteins(IAPs) is a family of apoptosis inhibiting factors containing eight kinds of protein factor, and Survivin is an important member of the family. Survivin has the character of obvious selective distribution of tissue, and its main function is to promote cell growth and proliferation, as well as inhibit cell apoptosis. Survivin doesn't express in adults' differentiated tissues except thymus gland and gonad, but could be detected expression in malignant tumor tissues. It is found that Survivin is closely related to the drug resistance of tumor cells, which might be one of the mechanisms of tumor resistance to chemotherapy drug.In recent years, Survivin is researched increasingly in Lung cancer. In the cases of NSCLC patients,it is found that Survivin expresses highly in tumor tissues, and is strongly linked to clinical stage, disease-free survival and overall survival time. So Survivin is able to be as a kind of NSCLC patients' diagnosis sensitive indicator and therapeutic target. At present a majority of tests concentrates in NSCLC, not pay equal attention to SCLC. The research about SCLC is in the exploration phrase,especially in the aspects of the First-line chemotherapy sensitivityand the characteristic of drug resistance SCLC will be easy to relapse and metastasis. This study explores the mechanism of SCLC's occurrence and development and the characteristic of SCLC's reverse transformation of drug resistance, in order to find the effective treatments for SCLC patients.Method1.With the immunohistochemical method(SP method), this study detected comparatively the expression of Suvivin protein in the 120 SCLC patients' cancer tissues and 13 adjacent normal lung specimens, and analyzed the results combining with the basic clinical data and follow-up result of SCLC patients.2.With specific si RNA liposome wrapping Survivin sequences, this study transferred humen small cell lung cancer cell lines NCI-H446. The test is divided into three groups: Survin si RNA transfection group(Si-Sur group), no sense si RNA transfection group(Si-NS group) and pure liposome transfection group( normal control group). It detected Survivin m RNA and its protein expression by RT-PCR and Western blot technology, tumor cell proliferation with the MTT colorimetry, and the invasion ability of tumor cells with Transwell Chambers combined with crystal violet staining. 3. In vitro experiments with liposome wrapping Survivin si RNA,it transferred human small cell lung cancer drug resistant cell lines NCI-H446/VP, the experiment is divided into three groups: Survivin si RNA transferction group(Si-Sur group),no sense si RNA transfection group(Si-NS group) and pure liposome transfection group( normal control group). The purpose is to observe the change of the biological characteristics of cancer cell. It detected cell apoptosis with the double staining of flowing cytometry instrument Annexin V/PI, and test protein expression of Suvivin, Bcl-2, Bax, and Cleaved Caspase-3 with Western blot, as well as analyzed the possible mechanism of its reverse resistance.Results 1.Thepositive expression rate is 62.5%(75/120) in Suvivin protein in SCLC patients, in contrast, there is one case of adjacent normal lung tissue has weak expression in 13 cases of normal lung tissue, the proportion is 7.7%. The two groups have obvious difference(p<0.05). In 18 cases of patients with mixed SCLC, 5 cases(27.8%) have weak expression of Survivin, and in the 120 cases of pure SCLC patients, 70 cases(68.6%)have high expression of Survivin, which has remarkable statistical significance(p<0.05). In the cases of patients within the limited stage of small cell lung cancer(T1-2N0M0), Survivin weakly expresses with the percentage of 16.7(2/12). While in the cases of patient in other limited stage and extensive stage, the expression ratio of Suvivin protein is respectively 58.5%(24/41), and 73.1%(49/67). The expression of Survivin increase with the clinical staging in SCLC, and so is its positive rate, the difference is statisticallysignificant(p<0.05). In the brain metastases of SCLC, Suvivin protein expression proportion is about 91.5%, while the positive expression rate is only 20.4% in the no-brain metastases of SCLC, the difference between them has statistical significance. And Suvivin protein expression is no obvious correlation with sex, age, and smoking condition. It is found that Suvivin protein in cancer tissue is the independent risk factor affecting the prognosis of SCLC patients. The median survival time ratio of high expression of group and the low expression group is 8:17(month), the progression-free survival time ratio of them is 4:8(month).2. Comparing the transfection group with the control group through the detection from RT-PCR and Western blot, Survivin m RNA and protein expression rate significantly decreased in NCI-H466 cells. Within a week observing the proliferation of NCI-H466 cells after the process from each group, MTT experiment result show suggest that cell proliferation significantly suppressed in Si-Sur liposome transfection group. The proliferation rate of NCI-H466 cell has no obvious difference between the Si-NC group and the control group. There is obvious difference in the three groups above, with statistical significance. Meanwhile, it is observed that the size and arrangement of NCI-H466 cell have also been obviously changed through Si-Sur liposome transfection. With tested by Transwell Chambers combined with crystal violet staining,the results of the invasion ability of cancer cell suggest that the OD value in Si-Sur group is visibly lower than the value in the control group, there is significant difference(F168.460,P<0.05). The difference of the OD value between negative control group and normal control group is narrowed at some level, there is no statistical significant(P>0.05). Compared all the result, it is found that the invasion ability of cancer cell in Si-Sur group have been obviously inhibited.3.Comparing the response of NCI-H446/VP cells of different groups in different concentration of etoposide, it is found that the cell mortality in Si-Sur group is obviously higher than those in Si-NC group and blank control group in the same concentration of etoposide, In Si-Sur group, the resistant index of NCI-H466/VP cells to etoposide is 7.62.,the index respectively is 11.17 and 11.39 in Si-NC group and blank control group, there is statistically significant(p<0.05), while the difference between Si-NC group and control group is not statistical significance(P>0.05). Theresults suggest that Si RNA Survivin could partly reverse the resistance of NCI-H466/VP cell to etoposide. To know its reverse drug-resistant mechanism, this experiment used flow cytometry instrument to analysis apoptosis level, the index respectively is49.512±0.248 in Si-Sur group,4.890±0.854 in Si-NC group, and 4.890±0.854 in blank control group. By Western blot to detect Survivin, the index in three groups(Si-Survivin ? Si-NC and Blank) is 0.032±0.012, 0.081±0.007,0.123±0.004; the index of Bcl-2 protein in order is 0.045±0.013,0.147±0.012,0.381±0.017; the index for Bax protein in order:0.334±0.015, 0.077±0.005, 0.046±0.005; the index for Cleaved Caspase-3 in order :1.374±0.015,0.240±0.016,0.232±0.033. Compared with normal control group and negative control group,there is statistical significant(P<0.05). The results suggest that the stripe grey value of Survivin in Si-Sur group is weaker than those in normal cell control group and negative control group, it is statistical significant(P<0.05). Using Western blot to detect the Bcl-2, Bax and Cleaved Caspase-3 proteins expression, the results suggest that Si-Sur liposomes could cut down Bcl-2 protein expression, while raise up Bax and Cleaved Caspase-3 protein expression. In Si RNA Survivin group, compared with normal cell control group and negative control group, there is statistical significant for Bax and Cleaved Caspase-3 protein(P<0.05).Conclusion1. Suvivin protein expression in SCLC tissues is closely related to patients' clinical stage, brain metastases and the chemotherapy curative effect. In addition, if Survivin expresses high, that the patients with carcinoma tissues has poor prognosis.2. Liposome-encapsulated Survivin-specific si RNA could inhibit Survivin transcription and expression in translational level, cell proliferation, and promote apoptosis.3. Si-Sur could reverse drug resistance to etoposide in NCI H466/VP cell lines. The possible mechanism is that Si-Sur promotes apoptosis through cutting down Bcl-2 protein expression and raising up Bax and Cleaved Caspase-3 protein expression. So we could control apoptosis molecules(Bax, Bcl-2, and Cleaved Caspase-3) to achieve the induction of apoptosis. |
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