| Research BackgroundAs the cancer morbidity and mortality has been increasing,it is still a major problem of public health worldwide.Since 2010,cancer has become the main cause of mortality in China.According to the report of China Cancer Center in 2016,the number of newly cases and deaths in 2015 was estimated after analyzing the 72 data usable in the registration center from 2009 to 2011 in China.It was expected to have 4,292,000 new cancer cases and 2,814,000 cancer death cases,among which the incidence and mortality of lung cancer was the highest,with the total incidence of 733,300 people,including 509,300 men,accounting for the first place of malignant tumor,and 224,000 women,which was second only to the incidence of breast cancer;total death was 610,200 people,among which there are 432,400 men,177,800 women,with the mortality rate ranking at the first place.Recently,it has been reported that the highest incidence of pathological pattern of lung cancer has changed from lung squamous carcinoma to lung adenocarcinoma.The lung adenocarcinoma become a kind of lung cancer with the highest incidence rate,accounting for a half of non-small cell lung cancer,which accounts for about 85%of the total lung cancer.Lung adenocarcinoma is easy to recur and have distant metastasis.The prognosis of adenocarcinoma is worse than squamous carcinoma.Currently,the treatment for lung adenocarcinoma is the comprehensive surgical treatment combined with chemotherapy which is one of the comprehensive treatment of the lung cancer.Chemotherapy of lung cancer is mainly based on drug combination,and platinum is one of the basic drug of drug combination.Cisplatin(DDP),a front-line platinum drug,is most commonly used in lung cancer chemotherapy.DDP play an important role in the comprehensive treatment of lung cancer.Although lung adenocarcinoma is more sensitive to chemotherapy,some patients have drug resistant for various reasons,thereby reducing the effect of chemotherapy and resulting in recurrence or distant metastasis.Cisplatin is the metal complex of platinum,whose target is DNA.It acts on the inter-chain and intra-chain linkage.DDP forms DDP-DNA complexes,which interfere with the replication of DNA.DDP is a kind of non-specific cytotoxic drugs.The main mechanisms of platinum drug resistance includes:drug inactivation increase,intracellular drug accumulation decrease,DNA repair capacity enhancement,indirect regulatory factors change,involving a variety of genes and signal transduction pathways.However,the cisplatin-resistant mechanism of lung adenocarcinoma is complicated and has not been completely eluciated.Needless to say,it is of great importance to study the lung adenocarcinoma resistance mechanism of cisplatin.Therefore,this study can extend the survival time of drug resistant patient,improve the sensitivity of chemotherapy and improve the effect of chemotherapy.Galectin-1(Gal-1),one of the family members of the P-galactoside lectin,is distributed in a variety of tissues.Galectin-1 involoves many physiological processes.Gal-1 is LGALS1 gene coding located at chromosome 22q12.The methylation status of the gene promoter is a mechanism for the regulation of Gal-1 expression.Gal-1 overexpression is associated with cell transformation,conglutination,proliferation,angiogenesis and invasion and other aspects,which has a great impact on tumour evolution and prognosis.It has been found that the high expression of Gal-1 involves a variety of tumours,including nasopharyngeal carcinoma,lung cancer,liver cancer,pancreatic cancer,prostate cancer,colon cancer and gastric cancer,etc.Acculating evidence has shown that the expression of Gal-1 is positively correlated with the classification,infiltration and lymphatic metastasis of cervical cancer.It has been found that Gal-1 may be related to some tumour cisplatin-resistance based on the gene or protein screening on multidrug resistance of tumour cells aspect.Thus,Gal-1 is involved in the development of a variety of malignant tumours.It is of great significance to have in-depth study of the effect of Gal-1 on malignant tumours.At present,the effects of gal-1 on cisplatin-resistant lung adenocarcinoma has not been determined in vitro and in vivo.Therefore,we selected Gal-1 to explore its specific value and significance in cisplatin-resistant lung cancer.RNA interference(RNAi)was discovered and named by Fire and others in 1998.As one of the most important scientific and technological achievements in the scientific community in 2002,RNAi has been developed quickly in the recent years and has shown many important research results.In 2001,in the cultivation of mammalian cells,the RNAi technique was applied to inhibit the expression of genes for the first time;it made innovative application of the technique to study the gene functions of higher organism.In 2002,some researcher used gene expression silencing with RNAi technology into the study of the mammal.Since 2004,RNAi technique has demonstrated that enzymolysis was used to build whole-genome small interfering RNA(siRNA)library new technology and genome siRNA library was used to conduct the high throughput RNAi study of the gene functions of higher animal.The tumour multidrug resistance is a process involveing into polygene and multi-factors.Using RNAi technique to restrain multi-drug resistance related gene expression has become a new strategy to reverse multiple drug resistance of tumour.In this study,the effect of Gal-1 on the biological characteristics of cisplatin-resistant A549 cell was studied via observing the sensitivity of Gal-1 to cisplatin with interference with RNA interference.Moreover,the susceptibility test of cisplatin was carried out by tumorigenicity of the nude mice with A549 cell,cisplatin-resistant A549 cell(A549/DDP)and silenced Gal-1 cell line to verify the effect of Gal-1 on A549/DDP cell.Meanwhile,the patients with lung adenocarcinoma were selected and their tissues were tested for Gal-1.Clinical data were collected and analyzed statistically.This study aimed to understand the role of Gal-1 in the pathogenesis of cisplatin in lung adenocarcinoma via in vivo,in vitro and clinical studies.Our study showed that Gal-1 is a target for reversing lung resistance in cisplatin,providing experimental basis for research and development of drugs.Part ?The study of the expression of Galectin-1 in human lungadenocarcinoma A549 and A549/DDP cellObjectivesTo investigate the impact of Gal-1 on A549/DDP cell via examining the expression of Gal-1 in human lung adenocarcinoma A549 and A549/DDP cell.Methods1.The human lung adenocarcinoma A549 and A549/DDP cell were routinely resuscitated,cultured,passaged and cryopreserved.2.The two types of cells were plated on 96-well plate and cisplatin with gradient dosage were added.The gradient concentrations of cisplatin were 0,0.1,0.2,0.4,0.8,1.6,3.2,6.4,12.8,25.6 ?g/ml.Each gradient dosage had 5 wells.CCK8 was used to determine the OD value.The median inhibitory concentration(IC50)and drug resistance index was calculated in A549/DDP cell.3.The expression of Gal-1 in mRNA and protein level in two groups of cells were examined using RT-PCR and Western blot,respectively.Results1.The IC50 value of A549 and A549/DDP cell were 2.16±0.30?g/ml and 17.66±1.21?g/ml,respectively.The drug resistance index of A549/DDP cell was 8.18(P<0.05).2.Compared with A549 cell,the mRNA level of Gal-1 was significantly enhanced in A549/DDP cell(0.76±0.03 vs.0.39±0.01;P<0.05).3.Compared with A549 cell,the protein level of Gal-1 was significantly enhanced inA549/DDP cell(0.85±0.03 vs.0.61±0.02;P<0.05).Conclusions 1.The drug resistance of human lung adenocarcinoma A549/DDP cell was stable,and the drug resistance index was 8.18.2.Gal-1 may be involved into cisplatin resistance in human lung adenocarcinoma cell line A549/DDP,and can be used as a candidate drug resistance protein for further study.Part IIExperimental research on the effect of silence Galectin-1 on reversing cisplatin drug resistance for A549/DDP cell in vitro and in vivoObjectivesThis study aimed to reverse drug resistance of A549/DDP cell to cisplatin in vivo and in vivo via silencing Gal-1 expression in human lung adenocarcinoma A549/DDP cell and to invesitage the anti-cisplatin effect of Gal-1 on A549 cell.Methods1.The silencing Gal-1 in human lung adenocarcinoma A549 and A549/DDP cell was using lentivirus transduction.The cells were cultured and passaged.2.Use RT-PCR and Western blot to test the expression of Gal-1 in mRNA and protein level in the A549,A549/DDP cell and A549/C,A549/DDP/C cell in control group and A549/I,A549/DDP/I cell in experience group.3.The OD value of each group of cells was determined by CCK8,the IC50 and the drug resistance index of each group of cells was calculated.4.The A549,A549/C,A549/I,A549/DDP,A549/DDP/C,A549/DDP/I cell were injected into the oxter of nude mouse to establish nude mouse tumour model.Five nude mice from each group were randomly selected and sacrificed for experiment.Cisplatin was intraperitoneally injected into the nude mice,and the tumour's growth was observed.The nude mice were scarificed 4 weeks later and the tumour weight and diameter were measured.5.The expression of Gal-1 in the tumour of nude mouse in the A549,A549/C,A549/I,A549/DDP,A549/DDP/C,A549/DDP/I group was detected by immunohistochemistry and Western blot.Results 1.The gray value of Gal-1/gray value of internal reference mRNA expressed in cellA549,A549/C,A549/I,A549/DDP,A549/DDP/C,A549/DDP/I group were respectively(0.38±0.01),(0.39±0.03),(0.22±0.02),(0.76±0.02),(0.75±0.05),(0.23±0.03).The expression of Gal-1 mRNA level in A549/DDP cell was higher than that in A549 cell(P<0.05).The expression of Gal-1 mRNA in A549/I,A549/DDP/I cell was not statistically significant(P>0.05),which were lower than those in A549(P<0.05).2.The gray value of Gal-1/gray value of internal reference protein in the A549,A549/C,A549/I,A549/DDP,A549/DDP/C,A549/DDP/I group were respectively:(0.61±0.02),(0.59±0.03),(0.28±0.02),(0.85±0.03),(0.84±0.01),(0.31±0.04).The expression of Gal-1 protein level in A549/DDP cell was higher than that in A549 cell(P<0.05).The expression of Gal-1 protein level in A549/I,A549/DDP/I cells was not statistically significant(P>O.05),which were lower than those in A549(P<0.05).3.The IC50 value of A549 cell was(2.16±0.30)?g/ml;the IC50 value of A549/C,A549/I,A549/DDP,A549/DDP/C,A549/DDP/I was respectively(2.17±0.31),(1.88±0.27),(17.66±1.21),(17.48±1.02)and(2.97±0.18)?g/ml;the drug resistance index of cell A549/C,A549/I,A549/DDP,A549/DDP/C,A549/DDP/I was 1.00,0.87,8.18,8.09,1.38;the IC50 value and drug resistance index of A549/DDP and A549/DDP/C cell group were larger than those of A549 cell group(P<0.05);the IC50 values and drug resistance index of A549,A549/C,A549/I,A549/DDP/I cell groups had no statistical significance(P>0.05).4.No death in nude mice was observed during tumorigenicity experiment.The tumour volume of the A549,A549/C,A549/I,A549/DDP,A549/DDP/C,A549/DDP/I group was(761.19±52.13),(763.16±51.04),(725.84±67.46),(1554.61±123.35),(1523.58±98.66),(804.98±61.63)mm3.The tumour weight of the A549,A549/C,A549/I,A549/DDP,A549/DDP/C,A549/DDP/I group was(0.35±0.02),(0.35±0.02),(0.34±0.02),(0.70±0.02),(0.71±0.03),(0.37±0.03)g,respectively.The tumour weight and volume of the A549/DDP group were signicantly increased comapred with A549,A549/I,A549/DDP/I group(P<0.05).In the comparison of tumour weight and volume between A549 and A549/I,A549/DDP/I cell group,no statistical significance was shown(P>0.05).5.The expression of Gal-1 in the nude mice tumour was examined by immunohistochemical and Western blot.The result showed that the expression of Gal-1 in the A549/I,A549/DDP/I group was lower than that in A549/DDP group(P<0.05).Moreover,the comparison between A549 and A549/C nude mouse group had no statistical significance(P>0.05).The comparison between A549/DDP and A549/DDP/C nude mouse group had no statistical significance(P>0.05).Conclusions:1.Lentivirus transduction silencing Gal-1 was successfully constructed in the A549 and A549/DDP cell of human lung adenocarcinoma.2.Human lung adenocarcinoma A549 cell and A549/DDP cell were able to establish the nude mice tumourigenicity model.3.Silencing Gal-1 enhanced the sensitivity of A549/DDP cell to cisplatin in vivo and in vitro.4.Gal-1 had an impact on cisplatin-resistant of A549/DDP cell,providing experimental basis for studying resistance mechanism and exploring medicine development.Part ?The expression and clinical significance of Galectin-1 and ERCC1 in pulmonary adenocarcinomaObjectivesTo investigate the expression and clinical significance of Gal-1 and excision repair complementary gene 1(ERCC1)in lung adenocarcinoma tissue.Methods1.A total of 91 cases who had lung adenocarcinoma operation of lung adenocarcinoma confirmed by postoperative pathology in the department of thoracic surgery in the First Affiliated Hospital of Bengbu Medical College from November 2011 to December 2012 were collected.All the patients were treated with cisplatin-based adjuvant chemotherapy program.The expression of Gal-1 and ERCC1in the paraffin embedded tissue after operation were determined by immunohistochemistry.2.The relationship between Gal-1 and ERCC1 was analyzed and the relationshipbetween Gal-1/ERCC1 and the age,gender,smoking,clinical staging of patients was also analyzed.3.Through the follow-up visit,the impact of the two indicators on prognosis was analyzed to explore their relationship with prognosis.Results 1.The positive expression rate of Gal-1 was 30.77%(28/91),the positive expression rate of ERCC1 was 31.87%(29/91),there were 18 cases with positive expression and 52 cases with negative expression of Gal-1 and ERCC1 in the lung adenocarcinoma.There was no significant difference in the expression of Gal-1 and ERCC1 in lung adenocarcinoma(P=1.00,P>0.05).According to the clinical diagnostic agreement Kappa test,it showed that the expression of Gal-1 and ERCC1 was consistent in lung adenocarcinoma(Kappa=0.46,P=0.00,P<0.05).2.The expression of Gal-1 in lung adenocarcinoma was not correlated with the gender,age,smoking status and TNM staging and other clinical indexes of the patient(P>0.05).3.The expression of ERCC1 in lung adenocarcinoma was not correlated with the gender,age,smoking status and TNM staging and other clinical indexes of the patient(P>0.05).4.Comapred with the Gal-1 positive expression group,the median disease-free survival(DFS)was signicantly enhanced in the Gal-1 negative expression group(27±1.62 months vs.19±1.96 months;?2=22.48,P=0.00).Moreover,the median overall survival time(OS)in the Gal-1 negative expression group was much higher than the Gal-1 positive expression group(33±1.56 months vs.22±1.02 months;?2=36.81,P=0.00).5.Comapred with the ERCC1 positive expression group,the median DFS in the ERCC1 negative expression group was greatly increased(27±1.53 months vs.21±1.30 months;?2=7.03,P=O.01).Furthermore,the median OS in ERCC1 positive expression group was much lower than the ERCC1 negative expression group(25±1.04 months vs.33±1.72 months;?2=11.64,P=0.00).6.Comapred with the Gal-1 and ERCC1 positive expression group,the median DFS in the negative expression group was greatly increased(29±1.40 months vs.18±2.83 months;?2=12.13,P=0.00).Furthermore,the median OS in Gal-1 and ERCC1 positive expression group was much lower than the negative expression group(22±1.00 months vs.35±1.77 months;?2=21.30,P=0.00).Conclusions:1.The expression of Gal-1 and ERCC1 in lung adenocarcinoma was consistent.2.The expression of Gal-1 and ERCC1 were not correlated with the gender,age,smoking status and TNM staging and other clinical indexes of the patient.3.The expression of Gal-1 and ERCC1 were related to the postoperative chemotherapy of lung adenocarcinoma indicating Gal-1 and ERCC1 can be used as the index for the prognosis. 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