Study On The Expression And Biological Function Of KIF-15 In Esophageal Squamous Cell Cancer

The new incidence of esophageal cancer in 2015,one of the most common malignant tumors of the digestive tract,is expected to rank third,including male incidence rate of 320.8/100000,women 157.2/100000,and the expected death rate is fourth,including male 253.8/100000,female 121.3/100000[1-2],the clinical prevention and treatment of esophageal cancer is still a long way to go.With the cooperation of clinical model combined such as oncology,radiotherapy,biotherapy and other multidisciplinary treatment,the comprehensive treatment of esophageal cancer has also made great progress,but its cure rate is still disappointing[3].The main reason leading to the poor curative effect of esophageal cancer is the abnormal cell proliferation and apoptosis of esophageal cancer cell,promoting recurrence and metastasis.Thus elucidating the mechanism of proliferation and apoptosis of esophageal cancer,screening and identification of effective targets and inhibitors,is the hot spots of basic research and clinical trail in esophageal cancer.Recently,with the development of tumor research,the skeletal dynamic protein in tumor pathogenesis has aroused people’s attention,especially Kinesin superfamily,the kind of microtubule-dependent molecular motor protein.The increasing number of students suggest that the Kinesin family is involved in the regulation of cell membrane organelles transport,mitosis,gene translation and protein transport,cell signaling pathway,affecting the cell cycle and apoptosis including the astrocyte and tumor cell,promoting the progression of tumor[4-5].Therefore,to explore the mechanism of esophageal cancer proliferation,definite the role of Kinesin family in the development of esophageal cancer,screening and identify some effective targets,will provide new ideas and basis for further improving the curative effect of esophageal cancer.Tumor cell growth and metastasis are dependent on cell mitosis,cytoskeletal proteins play a key role in mitosis,and cell biofilm system,genetic system and said tumor cells,"three control system"[6].Cytoskeleton protein system mainly includes microfilm protein,tubulin,fibrin,micro-beam network,which is almost involved in all life activities of tumor cells,can maintain tumor cell morphology,regulate cell polarity and movement,participate in cell material transport,Tumor signal transduction and so on.Studies have shown that tubulin is a key protein that regulates the transport of cellular material and provides the necessary molecular signals and related proteins for tumor cell growth and metastasis,which regulates the movement of organelles in cell mitosis and equilibrates the tension of cell microfilaments and promotes chromosome Positioning and separation[7-10].Kinesin family compose two parts of amino acid sequence and microtubule binding domain composed,according to the difference of microtubule binding domain sequence,the Kinesin family can be divided into 14 subfamilies,respectively Kinesin-1~14,according to the different function of subfamilies,the Kinesin family are divided into two categories:One is the conservative and functional subfamily,mainly involving in the transport of organelles and vesicles.The other is mainly closely related to mitosis,especially playing an important role in the maintenance of cell mitotic spindle function[11-13].Kinesin-1 mainly participates in the separation of chromosomes and assembly of bipolar spindles,playing the key regulatory role in early mitosis,whereas cytoplasmic isolation depends on the involvement of Kinesin-12 phosphorylation(KIF-15),thus affecting the process of mitosis,cell proliferation and apoptosis[14-15].Studies have shown that KIF-15 plays an important role in the proliferation of astrocytes[16-17].Silencing expression of KIF-15 can significantly inhibit cell proliferation and promote apoptosis[18-19].KIF-15 as the important oncogene has proposed,but the expression of KIF-15 in esophageal squamous cell carcinoma and its mechanism in the development of esophageal cancer cells has not been reported.Our preliminary study shows that KIF-15 is highly expressed in esophageal squamous cell carcinoma and may be involved in the development of esophageal squamous cell carcinoma.The aim of this study is to further explore the role of KIF-15 in the development and progression of esophageal squamous cell carcinoma,providing new ideas to elucidate the pathogenesis of esophageal squamous cell carcinoma.This study is designed to further explore whether KIF-15 as a potential new therapeutic target for esophageal squamous cell carcinoma.In our study,firstly,the expression of KIF-15 protein and mRNA were detected in 50 cases of esophageal squamous cell carcinoma and matched para-cancerous tissues detected by immunohistochemistry,Western blot and Real time-PCR.Acording to the result of expression of KIF-15 immunohistochemistry,the relationship of the expression of KIF-15 and clinical pathological parameters was analyzed,and then the relationship between KIF-15 expression and the prognosis of esophageal carcinoma was also elucidated by Kaplan-Meier survival curve.It was elucidated whether KIF-15mediating the development of esophageal squamous cell carcinoma and is a potential target for poor prognosis of esophageal cancer.The recombinant lentiviral vector GV115/KIF-15 shRNA and the recombinant lentivirus GV115/KIF-15 shRNA were constructed,evaluate the infection efficiency of the recombinant lentivirus GV115/KIF-15 shRNA in Eca-109 and the function of recombinant lentivirus GV115/KIF-15 shRNA,and establish the Eca109/KIF15~-cells,which were used to further elucidate the biological function of KIF-15 in esophageal cancer cells.Additionally,the Eca109/KIF15~-cells,as the study object,were used to further study that KIF-15 has an effect on growth,proliferation,apoptosis,plate cloning,invasion and metastasis in Eca109 cell in vitro and the growth of nude mice transplanted tumor in vivo with silencing KIF-15 gene expression.The experiments in vitro and in vivo further explore the biological function of KIF-15 in esophageal squamous cell carcinoma.This study is aimed to explore the role KIF-15,as the research target,in the development and progression of esophageal squamous cell carcinoma,mainly divided into three parts:Part I:The expression of KIF-15 in human esophageal squamous cell carcinoma and adjacent tissues and its clinicopathological significancePart II:Preparation and functional determination of recombinant lentivirus GV115/KIF-15 shRNAPart 3:The effect of recombinant Lentiviral GV115/KIF-15 shRNA on biological function in esophageal carcinoma cell Eca-109Part 1:The expression of KIF-15 in human esophageal squamous cell carcinoma and adjacent tissues and its clinicopathological significanceMaterials and methods1.Immunohistochemistry method was used to detect the expression of KIF-15protein in 50 cases of esophageal carcinoma and adjacent tissues2.Real time-PCR was used to detect the expression of KIF-15 mRNA in 50cases of esophageal carcinoma and adjacent tissues3.Western blot was used to detect the expression of KIF-15 mRNA in 50 cases of esophageal carcinoma and adjacent tissues4.Kaplan-Meier survival curve was used to analyze the relationship between KIF-15 protein expression and PFS of patients with esophageal cancer.5.Statistical analysis:the data were evaluated using GraphPad Prism 6.0software.The Pearson’sχ~2 test was used to compare the categorical variables.The Spearman rank correlation coefficient analysis was used to assess the correlation between two ordinal variables.Student’s t-test was used to determine differences between the two groups.P value<0.05 was considered as statistical significance.Results1.The results of immunohistochemistry showed that the positive expression rates of KIF-15 protein in esophageal carcinoma tissues and adjacent tissues were66.00%and 24.00%(P<0.05),respectively.2.The results of Real time-PCR showed that the positive expression rates of KIF-15 protein in esophageal carcinoma tissues and adjacent tissues were 72.00%and 26.00%(P<0.05),respectively.3.The results of Western blot showed that the positive expression rates of KIF-15 protein in esophageal carcinoma tissues and adjacent tissues were 58.00%and 18.00%(P<0.05),respectively.4.The expression of KIF-15 protein in esophageal carcinoma tissues was not correlated with age,sex,diameter of lesion,clinical grade(P>0.05).The positive correlation between KIF-15 expression and TNM staging,as well as lymph node metastasis(P<0.05).5.The Kaplan-Meier survival curve showed that the median PFS of KIF-15 protein positive expression group and negative expression group were 9.5 months and14.2 months(P<0.05),respectively,and the difference was statistically significant,suggesting that the high expression of KIF-15 may be a biomarker of poor prognosis of esophageal cancer.Part 2 Preparation and functional determination of recombinant lentivirus GV115/KIF-15 shRNAMaterials and methods1.The KIF-15 oligonucleotide fragment was annealed and the product was inserted into the downstream of the pCMV promoter in GV115 lentiviral expression vector.Screen the positive colony and construct the GV115/KIF-15 shRNA lentivirus expression vector.2.The lentivirus GV115/KIF-15 was packaged in 293T cells using the second generation self-inactivating lentivirus packaging system of Gekai Company.The supernatant was collected and concentrated and purified by ultracentrifugation.The Infection efficiency of lentivirus GV115/KIF-15 shRNA on esophageal cancer cell line Eca-109 was evaluated.3.The expression of KIF-15 mRNA was detected by Real time-PCR,evaluating the silencing efficiency4.The expression of KIF-15 protein was detected by Western blot,evaluating the silencing efficiency.Results1.The results of Enzyme digestion and sequencing showed successful construction of GV115/KIF-15 shRNA lentivirus expression vector2.The lentivirus GV115/KIF-15 shRNA was packaged successfully,and infected effectively the esophageal cancer cells3.The recombinant lentiviral GV115/KIF-15 shRNA cells could inhibit the expression of KIF-15 mRNA.4.The recombinant lentiviral GV115/KIF-15 shRNA cells could inhibit the expression of KIF-15 proteinPart 3 The effect of recombinant Lentiviral GV115/KIF-15 shRNA on biological function in esophageal carcinoma cell Eca-109Materials and methods1.The recombinant lentivirus GV115/KIF-15 shRNA with the effect on the proliferation of Eca-109 cells was detected by MTT assay.2.The recombinant lentivirus GV115/KIF-15 shRNA with the effect on the growth of Eca-109 cells was detected by Celigo cell counting3.The recombinant lentivirus GV115/KIF-15 shRNA with the effect on the apoptosis of Eca-109 cells was detected by Annexin V-APC single staining4.The recombinant lentivirus GV115/KIF-15 shRNA with the effect on the clonal ability of Eca-109 cells was detected by plate cloning assay.5.The recombinant lentivirus GV115/KIF-15 shRNA with the effect on the invasive ability of Eca-109 cells was detected by transwell method.6.The recombinant lentivirus GV115/KIF-15 shRNA with the effect on the growth of nude mice transplanted tumor was detected in vivoResults1.Compared with the control group,recombinant lentivirus GV115/KIF-15shRNA can effectively inhibit the proliferation of Eca-109 cells.2.Compared with the control group,recombinant lentivirus GV115/KIF-15shRNA can effectively the growth of Eca-109 cells.3.Compared with the control group,recombinant lentivirus GV115/KIF-15shRNA can effectively promote the apoptosis of Eca-109 cells.4.Compared with the control group,recombinant lentivirus GV115/KIF-15shRNA can effectively inhibit the clonal formation of Eca-109 cells.5.Compared with the control group,recombinant lentivirus GV115/KIF-15shRNA can effectively inhibit the invasion of Eca-109 cells.6.Compared with the control group,recombinant lentivirus GV115/KIF-15shRNA can effectively inhibit the growth of nude mice transplanted tumor in vivoConclusion1.The expression of KIF-15 protein and mRNA in human esophageal squamous cell carcinoma was significantly higher than that in adjacent tissues.The expression of KIF-15 protein in esophageal squamous cell carcinoma was significantly correlated with TNM staging and lymph node metastasis.The high expression of KIF-15 was close relations with PFS of ESCC patients,suggesting that KIF-15 may be as a potential predictive biomarker for poor prognosis in ESCC patients.2.The GV115/KIF-15 shRNA lentivirus expression vector and recombinant lentivirus LV115/KIF-15 shRNA was successfully constructed,which could successfully infected the human esophageal cancer cell line Eca-109 and then could inhibit the expression of KIF-15 mRNA and protein.3.Recombinant lentiviral GV115/KIF-15 shRNA can effectively inhibit the proliferation,growth,clonal formation and invasion of Eca-109 cells in vitro and the growth of transplanted tumor in vivo,and promote the apoptosis of Eca109.Collectively,these results suggest that KIF-15 may be as a potential therapeutic target for esophageal squamous cell carcinoma...