The Preliminary Research In The Role And Mechanism Of Spindle And Kinetochore Associated Complex Subunit 1 Gene In Salivary Adenoid Cystic Carcinoma

Background:The salivary adenoid cystic carcinoma(SACC),with a reported yearly annual incidence of 3 cases per million,is an important head and neck epithelial cancer.It occurs mainly in the minor salivary glands and parotid gland,followed by the submandibular gland,accounting for 21-24%of the overall incidence of salivary gland cancers,and about 1%of all head and neck malignant tumors.Salivary gland adenoid cystic carcinoma can happen at any age,in which middle-aged and elderly patients,50 and 60 years old age group most common.Although SACC has been described as having apparently indolent course,the invasive ability of the tumors is very strong,easy to invade the peripheral nerve and vascular.As the distant metastasis rate is as high as 40%,the salivary adenoid cystic carcinoma has been regarded as one of the highest transferring rate of tumor in oral and maxillofacial malignant tumors.Together with the aggressive long-term behavior,the persistent and recurrent growth pattern and late onset of distant metastases result in frequent eventual death.Although complete surgical resection and adjuvant chemotherapy have been shown to improve long-term survival of patients,the prognosis of SACC remains poor.It has been described as "one of the most biologically destructive and unpredictable tumors of the head and neck".Mitosis is a major proliferation of eukaryotic cells.Cell division cycle is a tightly regulated,highly ordered process by intracellular intrinsic cell cycle checkpoint to check,in order to ensure faithful replication of cells.Checkpoint defect may cause genetic mutation and chromosomal structural abnormalities,which make the cell to obtain proliferation,leading to tumorigenesis.Abnormal mitosis is a common characteristic of most malignant tumors.Mitotic chromosome segregation requires kinetochores to generate physical connections between chromosomes and spindle microtubule polymers.The spindle and kinetochore-associated complex subunit 1(SKA1),a newly discovered gene,associating with mitosis,has been found to lead to silence the spindle checkpoint.SKA1,along with its coworkers SKA2 and SKA3,constitutes the SKA complex(spindle and kinetochore associated complex).Notably,spindle and kinetochore-associated protein complexes have several key properties,and play an important role in coupling chromosome movement to microtubule depolymerization.SKA1 was found to play a pivotal role during the prometaphase of chromosome segregation interacting with SKA2.Overexpression of SKA1 has been found in malignant progression of several human cancers,such as hepatocellular carcinoma,gastric cancer,bladder cancer,non-small cell lung cancer,papillary thyroid carcinoma,neuronal glioblastoma and prostate cancer.There existed a significant correlation among a higher SKA1 expression in papillary thyroid carcinoma(PTC)tissues,including the lymphoid node,clinical stage,and extrathyroid invasion.Survival analysis showed high SKA1 expression in PTC patients more likely to relapse after surgery.Studies revealed that knockdown of SKA1 expression significantly inhibited cell proliferation and colony formation capacity.The distribution of the cell cycle may also be influenced.Other studies had found that,the expression of SKA1 in tumor cells was also related to p21,ERK2,Bcl-2,Bax,Akt and so on.SKA1,therefore,may influence the malignant transformation and development of tumors through a variety of mechanisms,suggesting that SKA1 may be a novel,promising prognostic factor and a new target for cancer gene therapy.There is no study demonstrating the role of SKA1 in salivary adenoid cystic carcinoma.This study aimed to investigate the expression of SKA1 in SACC tissues and analyse the relationship with the clinical significance.This study also aimed to explore the effect of SKA1 in the process of malignant proliferation of adenoid cystic cancer cells by silencing SKA1 gene through a technique called RNA interference.Part I The study about the expression of SKA1 in the salivary gland adenoid cystic carcinoma and its clinical significanceObjective:This study aimed to investigate the expression of SKA1 in ACC tissues and analyse the clinical significance.Materials and Methods:1.Patients selection:All of the 42 paraffin embedding tissue specimens analyzed were selected from patients with the complete clinical data and 40 with the complete follow-up data in the Department of pathology,Liaocheng People's Hospital(Liaocheng Clinical School of Taishan Medical University)of China,between December 2005 and March 2013.The criteria for study enrollment were as follows:patients(25 females,17 males)with head and neck adenoid cystic carcinoma underwent resection without pre-operative chemotherapy,hormone therapy,radiotherapy or other anti-cancer treatment.The results were evaluated independently by two pathologists blinded to all clinical data.2.Evaluation of immunohistochemical staining:SKA1 expression in samples from 42 cases of SACC and 20 subjects with normal tissue adjacent to carcinoma was detected by immunohistochemical analysis.Immunohistochemical staining for SKA1 was evaluated in compliance with intensity and proportion.Immunopositivity was scored according to the percentage of positive cells in four distinct categories:0 for 0-5%,1 for 6-20%,2 for 20-60%,and 3 for 60-100%.The staining intensity was then scored where 0 stands for bright yellow staining,1 for yellow staining,2 for brown yellow staining,and 3 for red brown staining.Both scores were multiplied,resulting in the final scores:0-1,negative(-);2-4,positive(+);and>5,strong positive(++).3.Statistical methods:Statistical analysis was performed using the SPSS software package(version 16.0;SPSS,USA).A P value of less than 0.05 was considered to be significant.Association was evaluated according to the chi-square test or Fisher exact test(n?1)between several clinicopathological variables.Results:1.The expression of SKA1 in adenoid cystic carcinoma tissuesSKA1 positive expression was mainly distributed in tumor cells,including duct cells and variant myoepithelial cells,while the tumor stroma had no obvious coloring.SKA1 positive expression was mainly found in the cytoplasm and/or nucleus of tumor cells.Most of the positive cases showed that both the nucleus and cytoplasm were dyed,while the nucleus tinting strength was more obvious.A few cases showed only cytoplasm were colored.There was a significantly higher SKA1 expression in SACC tissues compared with adjacent tissues.2.The correlation between the expression of SKA1 the clinical pathological characteristics and prognosis of patientsImmunostaining results suggested SKA1 expression was significantly correlated with tumor-node-metastasis(TNM)stage and histological growth.The expression of SKA1 in ??? stage was significantly higher than that in???? stage.SKA1 expression was not significantly associated with age,gender,lymph node involvement,perineural invasion or 5-year survival time.The 5-year survival rate of 40 patients was 52.5%.Conclusions:SKA1 is closely related to the occurrence,development and prognosis of the head and neck adenoid cystic carcinoma.Detecting SKA1 expression is not only useful to the early diagnosis and treatment of the head and neck adenoid cystic carcinoma,but also to the objective judgement of the prognosis.Part ? The mechanism research of lentivirus mediated SKA1 gene silencing to cell proliferation and metastasis in salivary gland adenoid cystic carcinomaObjective:To verify whether lentivirus mediated shRNA can effectively silence SKA1 gene in salivary gland adenoid cystic cancer cells,explore the molecular mechanism of SKA 1 in the process of malignant proliferation and cell cycle of adenoid cystic cancer cells.Materials and Methods:1.Lentivirus infection and the evaluation of the efficiency of infection:SACC-83 cells were transduced with lentivirus-mediated SKAl shRNA(Lv-shSKA1)or control shRNA(Lv-shCon)at a multiplicity of infection(MOI)of 10.As the lentivirus carries a GFP reporter,the infection rate was determined by counting GFP-positive cells using fluorescence microscopy 3 days after infection.2.Real time PCR:Total RNA was extracted with TRIzol reagent from SACC-83 cell lines according to the manufacturer's instructions.The compositions of the reverse transcription reaction system were as follows,4.0 ?l 5×PrimeScript@Buffer,1.0 ?l PrimeScript@RT Enzyme Mix,1.0 ?l RT Primer Mix,10 ?l RNA aqua,and 4.0 ?l RNase Free dH2O.Realtime PCR reaction system was composed by 10 ?l 2× SYBR(?)Premix Ex TaqTM,0.8 ?l primers(2.5?M),5 ?l cDNA and 4.2 ?l double-distilled H2O.The relative expression levels of mRNA were calculated using the 2-??Ct method.3.Western blot:SACC-83 cells were collected 5 days after lentivirus infection when the cell fusion rate was above 80%and lysed in 2×lysis buffer.After determination of protein sample concentration,the proteins were separated using SDS-PAGE and transferred to PVDF membranes.The membranes were then incubated with primary antibodies and followed by secondary antibodies.Then,the bands were exposed to films using ECL method.4.MTT assay:SACC-83 cells in logarithmic phase were collected and dispensedinto 96-well plates at a final concentration of 2×103 cells per well.The plates were incubated at 37? for 1-5 days in a humidified CO2 incubator.After the treatment time,20 ?l of MTT(5 mg/ml)was added to each well and the plates were incubated at 37? for 4 h.The absorption values were measured at 595 nm(optical density(OD))using a microplate reader.5.Flow cytometer assay:Upon reaching 80%confluence,cells were released by digestion with trypsin and harvested and then dyed with PI,filtered,computer tested in turn as required.6.Wound healing assay:The wound healing assay was used to evaluate the cancer cell motility capacity.The three groups of SACC-83 cells were seeded in 24-well plates after being counted.When the culture had reached nearly 90%confluency,the cell layer was wounded.Cells were then cultured for up to 24 h with serum-free medium.Photographic images of the plates were acquired under a microscope and the migration distance was assessed.7.Transwell assay:Cell invasion activity was assessed using 24-well Chemicon Matrigel invasion chambers(8-?m pores)according to the manufacturer's instructions.Transfected SACC-83 cells were loaded into the top chamber with serum-free medium.After being cultured for 24 h,the migrated cells were fxed in 4%methyl alcohol,stained with crystal violet solution and counted under a microscope.Results:1.Lentivirus infection and the evaluation of the efficiency of infection:GFP positive cells count results:The efficiencies of infection of both the lentivirus-mediated SKA1 shRNA(Lv-shSKA1)and control shRNA(Lv-shCon)groups were more than 80%as shown by fluorescence microscopy.Real time PCR and Western blot analysis confirmed that SKA1 mRNA and protein expression were obviously down regulated in salivary gland adenoid cystic carcinoma cells in the Lv-shSKA1 group.2.Western blot:Compared with the control group,the expressions of SKA1,Ndc80,CDK4,Cyclin D1,Cyclin E1,Cyclin B1 and N-cadherin,MMP-9 decreased after silencing SKA1 gene expression.While the expressions of p27 and E-cadherin increased significantly.3.MTT array:Compared with the control group,the viability of the SACC-83 cells infected by Lv-shSKAl has been inhibited.4.Flow cytometer:After infected by Lv-shSKAl,the cell cycle of the SACC-83 cells has been led to an arrest in S phase.5.Wound healing assay:The migration distance of the Lv-shSKA1 group was significantly less than the other two groups.6.Transwell assay:The cells through the Matrigel in the Lv-shSKA1 group were significantly less than the other two groups.Conclusions:1.Knock down of SKA 1 gene expression in head and neck adenoid cystic carcinoma induced cell cycle arrest in the S phase and then effectively inhibited tumor cell proliferation.2.SKA1 gene silence could block the cell cycle progression and cell growth by down-regulated the expression of cell cycle related genes Ndc80,CDK4,Cyclin Dl,Cyclin E1,Cyclin B1 and up-regulated p27 protein expression.3.Knockdown of SKA 1 gene could promote E-cadherin protein expression but inhibit the N-cadherin expression,inhibited cell epithelial mesenchymal transition(EMT),reduce the matrix metalloproteinase(MMP-9)expression to suppress adenoid cystic carcinoma cell invasion and metastasis.4.SKA1 gene is a potential new target for gene therapy of head and neck adenoid cystic carcinoma.Innovation:1.The present study found that the SKA1 protein expression characteristics,the correlation between SKA1 expression level and clinicopathological factors or outcomes in adenoid cystic carcinoma.Our research suggested a significantly higher SKA1 expression in SACC tissues compared with adjacent tissues and the positive expression was related to the advanced stage and solid pathological types,suggesting that SKA1 could predict poor prognosis of patients.2.SKA1 could change the cell cycle related proteins,such as Ndc80,CDK4,Cyclin D1,Cyclin E1,Cyclin B1 and p27 expression,promote the adenoid cystic carcinoma cell malignant proliferation,and regulate the expression of MMP-9,E-cadherin and N-cadherin expression,promote the migration and invasion.The presen study certificated that SKA1 gene is a potential new target for gene therapy of head and neck adenoid cystic carcinoma.