| Background:Based on histology,lung cancer can be divided into small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC).Although the SCLC patient number just account for a quarter of the NSCLC’s,but the former is the deadliest and most aggressive type of lung cancer,mainly due to the early tumor cell dissemination.so we target SCLC as our model to research the moleculer mechanism of metastasis.SPIB is a member of ETS transcription factor family,which is mainly restricted to the hematopoietic system.SPIB plays key roles in differentiation process of lymphocytes and pDCs,and sustains their normal physiological functions.It has been reported that dysregulation of SPIB is related to lymphoma or leukaemia,and the SPIB gene has also been described as gained in lung AdCs in 2005,indicating that SPIB is not limited to the hematopoietic malignancies.However,the consequences of SPIB’s ectopic expression in solid tumors are still need to be investigated.Objective:Through in vivo and vitro experiments to elucidate the effects of SPIB on lung cancer’s development and metastasis,in order to providing a novel and valuable target for clinical lung cancer therapeutics.Methods and results:Based on small cell lung cancer specific gene profile,we use Lenti-virus as genetic manipulation tool,alter innate SPIB gene expression level in SCLC cell and NSCLC cell,and then observe the cell morphology or biological behavior’s change in conjuction with the mice tumor metastasis model.1.We compared the gene expression profile of SCLC cell line H209 with NSCLC cell line A549 through RNA-Sequencing tech,found the expression restriction of SPIB,and then chose SPIB as our research target gene.2.Using chromatin-IP tech,we compared the histone 3 modification state of H209 and A549,revealed the epigenetic mechanism of different expression level of SPIB.3.Using RT-PCR detection among multiple cell lines,we found the SCLC’s specificity of SPIB;on the other hand,we did Immunohistochemistry experiment to detect the expression difference of SPIB protein among clinical patients’ tissue by using specific antibody.4.Using Lenti-virus gene transport system,we knockdown SPIB expression degree in H209,observed cells morphology change,then found the junction state between cells tend to be tighter.We used flow cytometry to detect the cell viability,found the apoptosis peak significantly rose.We detected the TJ related genes’expression level increased by QPCR.5.Using Lenti-virus gene transport system,we elevated SPIB expression level in A549,then detected the cell anchorage independent growth ability.We found SPIB facilitated tumor cell to form the softer clones.Transwell assay was used to detect the cell invasion ability in vitro,and a stronger invasion ability was detected in SPIB over-expressed A549.We detected the TJ related genes’ expression level decreased by QPCR.6.Using Lenti-virus gene transport system,we over-expressed SPIB in mice LLC1 cell line,then seeded cell to mice by hypodermic injection,after 2 weeks,we removed the situ tumor by surgery and keep raising the mice for another 2 weeks,and then sacrificed the mice and got their lung tissue.After tissue fixation,the nudules’number on the lung surface were counted.we found SPIB tend to promote LLC1 to generate more nodules,and the statistic result is significant(P<0.05).Conclusion:Hematopoietic cell-specific transcription factor SPIB ectopically expresses in SCLC,its expression level influences the Tight junction intensity between cells,and presents a negative correlation with TJ.This effect may comes from the repression role of SPIB to the TJ related proteins,such as ZO-1 and Claudins.Besides,SPIB can enhance the in vitro invasion ability of NSCLC cell,and this effect may come from the direct promotion of SPIB to MMP9 gene.Therefore,ectopic expression of SPIB can enhance the metastasis ability of lung cancer cell. |
PDF Full Text Request