| The significanceLung cancer is a highly malignant neoplastic disease,the mortality rate ranks first in all kinds of malignant tumors,the incidence rate increased year by year,among which small cell lung cancer?SCLC?accounts for about 15%to 20%of primary lung cancer.Due to short doubling time,rapid development,accompanied by endocrine abnormalities or carcinoid syndrome and hematogenous spread earlier of SCLC cell,so the main treatment of SCLC should be systemic chemotherapy combined with radiotherapy and surgery.Although SCLC cells are very sensitive to chemotherapy and radiation in the beginning,but decades of clinical trials and programs did not find an effective way to completely cure SCLC,the multi-drug resistance in majority of patients occurs rapidly in combination therapy with cisplatin-based treatment plan,and lead to treatment failure and poor clinical prognosis.Thus,drug resistance of SCLC has become an urgent problem in clinical treatment.Resistance is one of the main cause resulted in cancer treatment failure.Although we still do not get an overall understanding about targeted cancer drugs,there are many new and more creative methods emerging continually to overcome drug resistance.Substantial progress in the field of drug resistance research showed that drug resistance of tumor cells emerges mainly through the following several mechanisms,including changes in drug efflux,drug metabolism increasing,drug target mutations,changes in DNA repair and cell cycle,apoptosis and autophagy changes induced epithelial-mesenchymal transition?EMT?,tumor stem cell generation and so on.EMT refers to epithelial cells lose cell polarity,tight junctions and adhesion connection,get the ability to invasive and migration,and obtain a mesenchymal cell morphology and cell character:istics under the influence of a number of factors.Recent studies have found a close relationship between EMT and tumor resistance.In addition,cell signaling pathway has been shown to play a critical role in the physiologieal,abnormal activation of the signal transduction pathway may lead to drug resistance.Recently,non-coding RNAs?ncRNAs?has become an important regulatory molecule for gene expression and alternative splicing,which is a kind of nucleic acid sequence in eukaryotes aceounting for 90%of the human genome in reeent years,they include both microRNAs?miRNAs?with transcripts long for 19-25 nucleotides and long non-coding RNAs?lncRNAs?up to hundreds of nucleotides.They can be adjusted at the level of transcription and post.transcriptional expression of protein-coding genes,and be widely involved in cell differentiation and ontogeny and other important life processes by providing an alternative control methods that greatly increase the complexity of regulation,fine-tuning transcriptome and quick adjustment of protein group under stress.Thus,a variety of biological functions has been confirmed to achieve optimal coordination of the interaction between physiological outcomes depending on ncRNAs and cellular signaling networks,while ncRNA mutations and disorders have been shown to closely related with many human diseases,including cancer-resistant medicine.Studies have found that IncRNA with miRNA effect element also promote the expression of miRNA target gene binding miRNA target gene reduces free functional miRNA through competition in addition to the regulation of gene expression in the proximal end.Such lncRNA was called a competitive endogenous RNA?ceRNAs?,"lncRNAs-miRNAs-target genes" regulation axis may be an important form of ncRNAs involved in regulation of gene expression,which is confirmed by more and more recent studies,the gene regulation pattern of IncRNA-miRNA interaction significantly enriched RNA-RNA interaction networks.However,whether HOTTIP participates in the important life proeesses by eeRNA mechanisms,such as the development of tumors,has not been reported.incRNA HOTTIP?HOXA distal transcript antisense RNA?is a HOX family member without encoding protein up to 4665 bp,chromosome cycle brings it to the proximal end of HOXA gene,then assists the activation of HOXA gene expression with transcription initiation at HOXA gene 5'-UTR,the related HOXA genes are called HOTTIP coordinating genes.Studies have demonstrated,HOTTIP may participate the development of tumors and chemotherapy drug resistance by upregulating expression of HOXA13.Our previous study found that,HOTTIP may be involved in small-cell lung cancer drug resistance by positive regulation of gene expression HOXA 13 and HOXA11,and induced EMT process as a ceRNA,the specific mechanism is still unclear.Our team has been engaged MDR mechanisms of small cell lung cancer over a long period of time,the EMT epithelial and mesenchymal markers?E-cadherin,Snail,Twist,Vimentin,p-catenin?being involved in the regulation of small cell lung cancer drug was found in the pre-funding from the National Natural Science Foundation,while the specific mechanism needs further study.Next,ncRNA microarray analysis were carried out under the situation of small cell lung cancer multidrug resistance cell model H69AR and its parent cell?chemotherapy sensitive cell line?H69 cell,46 kinds of miRNAs including miR-574-5p and multiple HOXA family members including IncRNA HOTTIP were found to be related closely to drug resistance of small cell lung cancer,the results were then verified by qRT-PCR technology.The results showed a relative high levels of expression of HOTTIP and a relative low levels of miR-574-5p expression in small cell lung cancer H69AR cells and paraffin-embedded tissues,and HOTTIP may be used as prognostic indicator of disease stage and survival time prediction.Bioinformatics website microRNA seq and microRNA.org found,miR-574-5p has binding sites in HOTTIP and EZH1 promoter region,and then we verified that miR-574-5p silencing can promote the expression of HOTTIP and EZHlby RT-PCR and Western blot.On the basis of preliminary studies,Our team intends to use molecular biology,cell biology techniques,small cell lung cancer cell line H69/H69AR and H446/H446DDP to explore HOTTIP as ceRNA to induce EMT process involved in small cell lung cancer drug molecule mechanism.The study includes the following aspects:?to discuss further the correlation of HOTTIP expression with clinical staging and prognosis on paraffin tissues of small cell lung carcinoma and para-carcinoma;?loss and gain of functions on HOTTIP expression in cell cycle migration,drug sensitivity,apoptosis,cell proliferation and tumor capacity;?using dual luciferase reporter gene experiments to confirm HOTTIP and EZH1 influence respectively on miR-574-5p activities;?H69 and H69AR cells were raised or lowered in miR-574-5p expression as well as H446 and H446DDP cells,then we observe the influence on cell proliferation,cell cycle migration,drug sensitivity,apoptosis,and tumorigenesis ability;?HOTTIP can down-regulate the expression of miR-574-5p and then up-regulate EZH1 expression,which results in the changed expression of EMT-related markers,which is consistent with the conclusion of our previous research about EMT function on small cell lung cancer multidrug resistance.We believe,HOTTIP may induce EMT and is involved in the regulation of small cell lung cancer multidrug resistance process as ceRNA.LncRNA chip and miRNA chip were used in this study as high-throughput screening techniques,and was supplemented by bioinformatics software and website prediction,together with classical molecular biology experimental techniques and other means,small cell lung cancer clinical paraffin,small cell lung cancer multiple levels of multi-drug resistant cell model H69AR and in vivo in nude mice models are used to explore the biological functions of HOTTIP development and multi-drug resistance occurs in small cell lung cancer,which will further enrich the the molecular mechanism of resistance of small cell lung cancer,and provide new targets for the treatment of small cell lung cancer..Research purposesBased on the results of previous studies,using bioinformatics,cell biology,molecular biology techniques and small cell lung cancer cells model to explore HOTTIP as ceRNA to induce EMT and is involved in the regulation of small cell lung cancer resistance.This research project will enrich the moleeular mechanisms of small cell lung cancer drug resistance and provide a theoretical basis for drug screening and prognosis of small cell lung cancer biomarkers..The Materials and Methods1.Non-coding RNA chipUse Arraystar group's human IncRNA chip technology to analyze the IncRNA differential expression profiles of small cell lung cancer multidrug resistance cell line?H69AR?and chemotherapy sensitive cell line?H69?,combined with the miRNA differential expression profile results from Beijing Capital Biochip Co.,Ltd.,using the bioinformatics to find relevant non-coding RNA molecules with small cell lung cancer drug.2.Clinical SamplesOne hundred and fifteen clinical cases of small cell lung cancer paraffin-embedded tissues diagnosed were collected from January 2008 to January 2015 from Shunde People's Hospital of Southern Medical University.This study was approved by the Ethics Committee of the People's Hospital of Shunde and all patients were consent.3.Cell CultureHuman small cell lung cancer sensitive cell line NCI-H69,NCI-H446,NCI-H146 and NCI-H69AR multi drug resistant cell lines were purchased from ATCC,H446DDP cell line was obtained by culturing the NCI-H446 cells in gradually increasing doses of CDDP.After 8 months,cells which grew in 0.4 uM CDDP were obtained.After a further 6 months,cells which grew in 0.8 uM CDDP were obtained.This cell line has been designated H446DDP and has been maintained by alternate feedings with drug-free medium or medium containing 0.8 uM CDDP.The stability of the resistant phenotype was determined by culturing continuously in medium with either 0.8 uM CDDP or no drug and assessing relative resistance after various periods of time up to 5months.The process is established by referring establishment of H69AR?CANCER RESEARCH 47,2594-2598,May 15,1987?.4.Cell transfection?1?Transient transfection:by LipofectamineTM 2000,the miR-574-5p mimics,inhibitors and negative control?miR-NC?sequence,small interfering RNA sequence and pcDNA3.1 or PEX-2 expression vector and negative control?NC?of HOTTIP,HOXAll,HOXA13,EZH1 and EMT related markers were transfected into four small cell lung cancer cell to up-and-down regulate the instantaneous miR-574-5p,HOTTIP,HOXA11,HOXA13 and EZH1 expression.?2?Stably transfected:?by LipofectamineTM 2000,the pcDNA3.1 HOTTIP expression vector,the corresponding pcDNA3.1 mock vector,the HOXA11,HOXA13 and EMT related markers' PEX-3 expression vectors or PEX-2 mock vector were transfected into NCI-H69 and H446 cells by G418 selection,stable and high expression H69 and H446 cell lines with genes above were established.?lentiviral virus method,the small interfering sequence of HOTTIP was packaged by LV3 lentiviral vector and then performed experimental infection of target cells,with the establishment of LV3-NC control transfected in the same time,thus stable and silent HOTTIP of H69AR H446DDP cell lines were established.5.Total RNA extraction and qRT-PCR?Real time fluorescence quantitative PCR??1?The total cellular RNA was extracted with reagent TRIZOL according to the operating instructions.Paraffin-embedded tissue RNA was extracted using the Qiagen RNA extraction products paraffin sample kit?miRNeasy FFPE Kit?.?2?The RNA above was then used to operate reverse transcription and real-time quantitative PCR referring AMV reverse transcription reaction kit instructions in real-time quantitative PCR reaction instrunent.miRNA sequence and specific retrovirus were carried out according to the reference of the internal reference U6 miRNAs qRT-PCR kit instructions.GAJPDH and U6 snRNA were used as internal reference,data were treated using the formula of three independent experiments obtained by RQ=2-??Ct method of analysis.6.Western blotTotal protein was extracted from cells and tissues and detect the protein concentration taking the BCA kit,preparing 10%to 12%separating gel electrophoresis by SDS-PAGE,transferring to a membrane,immunoblotting,developing and fixing using Image J software for protein expression relative intensity quantified.7.CCK8 analysis ceil sensitivity to chemotherapeutic drugsCells were plated in 96-well plates at 5×103 cells per well.After transient transfection or adherence of stable transfected cells,cells were treated with drugs for 24h.A total of three chemotherapy drugs[Cisplatin?DDP;Shandong,China?,Etoposide?VP-16;Jiangshu,China?,Adriamycin?ADM;Jiangshu,China?]were used.The absorbance at 450nm was measured after incubation with 10(1 of CCK-8 reagent?Dojindo,Kumamato,Japan?for 4h.The cells incubated without drugs were set at 100%survival and were used to calculate the concentration of each chemotherapeutic drug IC50.The assay was conducted in five replicate wells for each sample and three parallel experiments were performed.8.Flow cytometric analysisCells were treated with drugs for 24h after transfection,and then collected for apoptosis and cell-cycle assay.Cell apoptosis assay was performed by using AnnexinV/propidium iodide detection kit?Keygene,Nanjing,China?.For cell-cycle assay,the cells were collected and fixed in 70%ethanol at 4oC for 16h and then stained with propidium iodide.9.Colony formationPlate clone formation assay:the stable transfected cells were seeded?500 cells/well?in six-well plates overnight and chemotherapy drugs[Cisplatin?DDP;Shandong,China?,Etoposide?VP-16;Jiangshu,China?,Adriamycin?ADM;Jiangshu,China?]?4 ?M?or PBS?100 ?L?was added to the cultured cells on the second day.After 14 days,the culture medium was removed,and cells were briefly rinsed with PBS.The cells were then fixed with 4%paraformaldehyde and stained with 0.1%crystal violet,and colonies were counted by visual inspection.Soft agar clone formation assay was carried out according to procedures in the text of the paper.10.In vivo tumor formation and chemo-sensitivity assaysTumor formation experiment in nude mice was carried out according to the institutional guidelines of Guangdong Province and being approved by the Use Committee for Animal Care.Twenty-four BALB/c nude mice?male,5-6 weeks old,18.0±0.5g?were got from the Guangdong Medical Animal Center,This experiment was carried out at the Animal Experimental Department of Sun Yat-sen University North District.They were randomly divided into the following groups?n=6 mice per group?:?a?H69AR/lentivirus-NC,PBS;?b?H69AR/lentivirus-siHOTTIP,PBS;?c?H69AR/lentivirus-NC,CDDP+VP-16;?d?H69AR/lentivirus-siHOTTIP,CDDP+VP-16.Each mouse was injected subcutaneously in the flanks with cells above injection,the mice received chemotherapy drugs[Cisplatin?CDDP;Shandong,China?5mg/kg and Etoposide?VP-16;Jiangshu,China?2.5 mg/kg]or PBS?100 ?l?intraperitoneally?i.p.?injection after the tumor volume over 100 mm3 twice a week for five times totally.The tumors were measured every 3-4 days,and tumor volume was calculated using the following formula:volume=?L×W2?/2,and L and W are the longest and shortest diameters,respectively.The mice were sacrificed when the average L of any group reached approximately 1 cm.11.Luciferase reporter assayPSICHECK2.0 plasmid encoding a luciferase reporter gene was purchased from Promega.Recombinant plasmid of PSICHECK2.0-H-HOTTIP-3'-UTR,PSICHECK2.0-H-Bcl2-3'-UTR?wildtype?or corresponding mutant type was constructed in GenePharma?Sunzhou,China?.H69AR cells?1-2×105 cells/welz?were plated in a 12-well plate and cotransfected with 40nM of either hsa-miR-Z16a-5p or miRNA negative control of either recombinant plasmids or corresponding mutants,and 1 ng of PSICHECK2.0?Promega?by using LipofectamineTM 2000.The PSICHECK2.0 vector was used as an internal control to correct the differences in both transfection and harvest efficiency.H69AR cells were collected 48 h after transfection and analyzed using the Dual-Luciferase Reporter Assay System?Beyotime Biotechnology?.12.Immunohistochemistry staining?IHC?and scoringFFPE tissues were sectioned at 4 ?m thickness,and were analyzed by IHC for protein expression.Visualization was achieved by the EnVision peroxidase system?Dako?.For evaluation and grading of staining results,a seoring criterion previously described by Ohara et al.was used.Briefly,a sample was considered positive if more than 50%of the tumor cells retained nuclear staining,and 5 fields were randomly selected according to semi-quantitative scales.The intensity of staining was scored manually?high,3;medium,2;low,1;no staining,0?by 2 independent experienced pathologists,and only tumor cells were scored.Negative controls were performed by replacing the primary antibodies stated above with PBS.Positively stained was mainly located in the cytoplasm of SCLC cells and appeared as light brown and brown particles.13.Statistical analysisAll results were treated by SPSS 18.0 statistical software.The experiments were repeated independently for at least three times.The selected charts were a result of repeated experiments.Data was showed as mean±standard deviation.Difference between samples in quantitative real-time PCR,western blotting,apoptosis and cycle experiment,luciferase reporter gene assay was used t test or one-way ANOVA analysis if data homogenity of variance,and F test/Welch method if data heterogeneity of variance.LSD/Dunnett's T3 methods for multiple compansons.Difference in CCK8 by two factors factorial design of variance and SNK method for multiple comparisons.Before comparison,data homogenity of variance was first examined using F test.If the case of heterogeneity of variance,the approximate variance F test/Welch method was used.Fisher 's accurate analysis was used to evaluate the relations between HOTTIP expression and the clinical and pathological features.Kaplain-Meier analysis was used to evaluate the relationship between HOTTIP expression and survival rate.The relationship between multiple genes expression was explored by Spearman's correlation.The result of immunohistochemistry was analyzed by Mann-Whitney U test.All experiments were done at least in triplicate and the level of statistical significance was set at P value<0.05 for all tests.Research contents1.The relationship of HOTTIP and small cell lung cancer clinicopathological parameters and patient survival was verified in clinical samplesTo clarify the clinical expression of HOTTIP in small cell lung cancer patients in vivo as well as clinical efficacy,patient prognosis and drug resistance relationship,we continue to collect clinical biopsies of patients for the following research:?using immunohistochemical analysis on clinical biopsies to explore the relations of HOTTIP patients and lung cancer chemotherapeutic effect and prognosis;?real-time PCR were used to detect the changes in tissues of chemotherapy sensitive and resistant patients and analyzed the correlation;? biometric software was used to analyze HOTTIP expression level in clinically proven chemotherapy sensitive and resistant patients and analyze the relationship of HOTTIP expression with clinicopathological factors and survival rate.Through the above research,HOTTIP was further clarified to guide the treatment of patients with small cell lung cancer and prognosis as molecular markers.2.The in vivo experiments confirmed the relationship of HOTTIP in small cell lung cancer drug resistance,proliferation,migration,apoptosis,cell cycle and tumorlgenicityHOTTIP protein may act as a key molecules leading to a new cancer drug,in order to validate our preliminary findings and further confirm HOTTIP being involved in small cell lung cancer drug resistance,we will conduct the following studies:?gene transfection and RNA interference technology were used to increase the expression level of HOTTIP in respectively H69,H446 cells and reduce that in H69AR,H446DDP cells,CCK8 experiment was used to analyze the relationship between HOTTIP and cell chemosensitivity;?CCK8 experiment was used to analyze the relationship between HOTTIP and cell proliferation;?Transwell chamber experiments were used to analyze the relationship between HOTTIP and cell migration;?flow cytometry analysis was used to detect the influence of HoTTIP on cell cycle and apoptosis rate;?plate clone forming experiment was used to detected the influence of HOTTIP on cell proliferation and chemoresistance.?using small cell lung cancer nude mice model,after tumor volume over 100 mm3,chemotherapy drugs were intermittently administered via tail vein injection,the changes in tumor survival,tumor growth and drug resistance of nude mice in vivo was observed and analyzed.The above research will further clarify the relationship between the expression HOTTIP in small cell lung cancer and the development of drug resistance.3.Clarify the molecular mechanism of HOTTIP involved in chemoresistance of small cell lung cancer as ceRNABioinformatics website microRNA seq?https://cm.jefferson.edu/rna22/Precomputed/ImputController?viewtype=Text&transcr ipt=ENST00000421733&version=MB21E78v2&species=HomoSapiens&type=lncR NA?and microRNA.org?http://www.microrna.org/microma/home.do?found,miR-574-5p has binding sites in HOTTIP and EZH1 promoter region,and then we verified that miR-574-5p silencing can promote the expression of HOTTIP and EZH1by RT-PCR and Western blot.In order to clarify the relationship between miR-574-5p between the two genes,we will perform the following studies:? by respectively increasing and decreasing the expression of miR-574-5p in cells,RT-PCR and Western blot were used respectively from the mRNA and protein levels,then apoptosis rate,cell cycle and changes of-chemosensitivity were analyzed of the HOTTIP and EZH1 expression by flow cytometry analysis and CCK8;?after a steady increase or decrease of miR-574-5p expression,and then using RNA interference or gene transfection to decrease or increase expression of HOTTIP and EZH1 to detect whether effects of miR-574-5p on cell sensitivity to chemotherapy were reversed;?luciferase reporter gene assays were used to detect whether miR-574-5p could bind to HOTTIP and EZH1 promoter specifically.The above research would further clarify the molecular mechanism of HOTTIP involved in drug resistance of small cell lung cancer as ceRNA.After transfecting miR-574-5p antagomirs into H146 cells with HOTTIP stable down-regulated expression,the cell morphology changed was observed by inverted microscope,compared with the control group,cells morphology of H146/si-HOTTIP+miR-574-5p antagomirs group change to be fusiform and fibroblast morphology like cells,combined with our previous study results that EMT process is closely related to small cell lung cancer drug,we speculate whether HOTTIP induced EMT involved in small-cell lung cancer drug generation by adsorption miR-574-5p.To verify our hypothesis,the following studies will be carried out:? to verify our previous study that EMT process is related closly with small cell lung cancer drug resistance;?to clarify whether HOTTIP may be involved in small-cell lung cancer drug resistance mediated by EMT;?to verify HOTTIP induced EMT by adsorption niR-574-5p;?to verify whether the EMT of HOTTIP induced could be reversed by miR-574-5p up regulation.Through the above research,the molecular mechanisms of HOTTIP involved in small cell lung cancer drug resistance will be further clarify.Results1.LncRNA and miRNA microarray chip results show that HOTTIP and other HOXA family member expression are abnormally high in human small cell lung cancer multidrug resistant cell line H69AR,whereas multiple miRNAs expression including miR-574-5p is abnormally low.1.1 IncRNA chip results showed that HOTTIP and other HOXA family members'expression are abnormally high in H69AR cellsWe use Arraystar company IncRNA chip to analyze IncRNA expression profiles difference between human small cell lung cancer cells H69AR?multidrug-resistant cell line?and its parent cell H69?chemotherapy sensitive cell line?and 1443 lncRNAs in total were found with the difference statistically significant?2-fold above,P<0.05?,which accounts for 4.37 percent of all lncRNAs?1443/33045?.Biological function of 1443 IncRNAs are differentially involved in apoptosis,regulation of enzyme activity,cell cycle regulation,metabolism,protein binding,signal transduction and so on.Among them,985 IncRNAs express higher and 458 IncRNAs express lower,microarray results were verified by qRT-PCR later on.Wherein,IncRNA chip results contains 39 individual HOX genes and 231 ncRNAs,55 HOX genes are significantly upregulated and only one down-regulated?P<0.05?.Wherein,HOX family members have high expression in the SCLC drug resistant cell lines:HOTTIP is 14.9-fold,HOXA13 and HOXAllare 13.5 and 2.2-fold.Next,by qRT-PCR,relative expression of HOTTIP and its coordinating role genes HOXA13,HOXA11 in H69AR were 15.1433±0.6501,12.99±0.5411,8.23±0.2706?P<0.001?,respectively.1.2 miRNA chip results show,miR-574-5p and other 37 miRNAs in cells abnormally low expression H69AROur previous miRNA microarray analysis results revealed a significant differences in expression profiles between H69AR and H69 cells,61 miRNAs expressed significantly differentially,24 miRNAs including including miR-375 in H69AR cell was significantly higher than H69 cell;37 miRNAs including miR-574-5p in H69 cells was significantly higher than H69AR.We next verify the accuracy of the above results by qRT-PCR technology,which suggests that miR-574-5p may be involved in regulating small cell lung cancer multidrug resistance.1.3 MiR-574-5p may target and regulate HOTTIP expressionBy using bioinformatics website microRNA seq?https://cm.jefferson.edu/ma22/Precomputed/InputController?viewtype=Text&transcr ipt=ENST00000421733&version=MB21E78v2&species=HomoSapiens&type=lncR NA?,miR-574-5p has mutual binding sites with HOTTIP promoter region,which suggests that miR-5745p may be involved in the small cell lung cancer multidrug resistance by targeting HOTTIP.2.The relationship of HOTTIP and its coordinating role genes HOXA11,HOXA13's expression and clinical pathology in small cell lung cancer tissues2.1 QRT-PCR detection found that HOTTIP and its coordinating role genes HOXA11,HOXA13's expression significantly higher in tissues of chemotherapy-resistant patients clinically provenQRT-PCR detection found that HOTTIP and its coordinating role genes HOXA11,HOXA13's expression significantly higher in 115 cases of chemotherapy-resistant patients elinically proven,which are 2.03 ± 0.18-fold,6.34±0.22,and 12.34 ±0.84-fold respectively eompared with para-cancer tissues.Further analysis revealed that HOTTIP expression?0.7825 ± 0.1764?in 50 chemoresistant cases clinically proven is is higher than other 50 cases with good effect of chemotherapy?0.3904±0.1665??P<0.001?,the difference was statistically significant;expression of HOXA13 and HOXA11 are also relatively high in chemoresistant group?P<0.001?.The above results suggest that HOTTIP and HOXA111 HOXA13 may be associated with small cell lung cancer drug resistance.2.2 HOXA13 and HOXA11 protein express higher in tissues from patients resistant to chemotherapyBy immunohistochemistory technique,HOXA13and HOXA11 protein were detected in 100 cases of small cell lung cancer?chemotherapy group and chemotherapy drug sensitivity group is both 50 cases?and noncancerous lung tissue and found that expression localized in the nucleus in cancer tissue.Combining with the clinical effect of chemotherapy,the results of unsensitive to chemotherapy patients was about 3 points,while weaker expression of patients sensitive to chemotherapy,the score was about 1,while the adjacent lung tissue is 0 point with negative expression.In 50 patients with small cell lung eancer,HOXA13 is positive expression in 39 cases,the positive rate was 78%?39/50?;HOXA11 is positive expression in 31 cases,the positive rate was 62%?31/50?.2.3 Using Kaplan-Meier method to analyze the relationship of HOTTIP expression and clinical pathological data of the patients,and found it is irrelevant between HOTTIP expression and gender differences??2=0.4183,P=0.5178?,HOTTIP expression has nothing to do with the age of patients??2=5957,P=0.4402?;and HOTTIP positive expression rate of extensive stage patients was significantly higher than the limited stage patients,the difference was statistically significant??2=9.6084,P=0.0019?,positive HOTTIP expression of clinical effect ratio of chemotherapy appears increased resistant than HOTTIP negative expression patients,the difference was statistically signifieant??2=9.6976,P<0.0015?,the median survival time?f positive HOTTIP expression patients is short with a higher mortality rate??2=23.5898,P<0.001?.The results above suggest that HOXA11 and HOXA13 protein may act as a molecular marker to predict the prognosis and chemotherapy of patients with small cell lung cancer.2.4 The analysis on median survival time of patients with small cell lung cancerUsing the Kaplan Meier method to analyze the survival time of the patients.The results showed that there is no significant correlation between the survival time and the patients' sex?P=0.8306?.There is also no significant correlation between the survival time and the patient's age?P=0.8198?.However,there is statistically significant difference between patients survival time and cancer stage?P<0.001?.There 1s also statistically significant difference between patients' survival time and HOTTIP positive or negative expression?P<0.001?.3.HOTTIP can regulate expression of HOXA11 and HOXA13 genes positively3.1 QRT-PCR was used to detect the expression of HOTTIP,HOXA11 and HOXA13 in H69AR and H446DDP cells and found they were higher than inH69 and H446 cells,the difference was statistically significant?P<0.001?.3.2 HOTTIP siRNA sequences?siHOTTIP-1,-2,-3?and a negative control vector were transfected into H69AR and H446DDP cells,using RT-PCR to detect the silencing effect and found that of siHOTTIP-1 sequence is the best,it will be used for the follow-up experiments.Next,the siHOTTIP-1 sequences was packaged by LV3 lentiviral vector and transfected into cells to obtain HOTTIP stable lower expression cell lines for the following plate clone formation experiments and in vivo nude mice experiments.3.3 Expression plasmids of pcDNA3.1-HOTTIP gifted from Wang,Professor KC of Stanford University,together with pcDNA3.1-NC empty vector were transfected into H69 and H446 cells,RT-PCR method was used to detect the over-expression effect of HOTTIP and got a significant effect,then stable overexpressing cell line was obtained by G418 selection for clone formation experiments and in vivo nude mice experiments.3.4 The HOTTIP expression was silenced by RNA interference method,total RNA and total cellular protein were extracted and detected using qRT-PCR and Western Blot technique,the results show that expression of HOXA11 and HOXA13 are correspondingly reduced at both mRNA and protein levels;furtherly,increasing the expression of HOXA11 and HOXA13 in HOTTIP-silenced cells,we detected a significant increase of HOXA11 and HOXA13 expression in both mRNA and protein levels,which suggested that HOTTIP may play a positive role in regulating the expression of HOXA11 and HOXA13.4.HOTTIP is closely related with small cell lung cancer cells chemoresistance,apoptosis,proliferation,cell cycle,colony formation,migratio and tumorigenesis4.1 CCK8 and flow cytometry detection found that silencing HOTTIP may significantly increase the chemosensitivity and apoptosis rate,reduce survival rate and IC50 values of cells;otherwise not.4.2 CCK8 and flow cytometry detection found that silence HOTTIP made an arrest of S phase cell cycle and got a decreased cell proliferation;otherwise not.4.3 Colony formation assay and soft agar eolony formation assay found that silenced HOTTIP expression may get a decreased colony forming ability and a reduced number of clones;otherwise not.4.4 Transwell cell migration detection found silenced HOTTIP expression can significantly reduce the migration of cells;otherwise not.4.5 Silenced HOTTIP expression can significantly reduce the ability of tumor formation in nude mice with a decreased tumor volume and weight,the survival of mice was signifieantly prolonged;otherwise not.5.HOTTIP promotes the expression of miR-574-5p target genes EZH1 by adsorption miR-574-5p5.1 According to the results of miRNA microarray,miR-216a and miR-574-5p were screened out for being related to the drug resistance of small cell lung cancer.Next,the over-expression of miR-5... |
PDF Full Text Request