Role Of Rsf-1/HBX In The Progression Of Cervical Cancer And The Effect On Chemoradiotherapy Sensitivity

BackgroundCervical cancer is one of the most common gynecological malignancies and the fourth prevalent cause of cancer-related death in women.It is a heterogeneous multifocal disease and the incidence rate is increasing.The mechanisms that influence the oncogenesis and progression of cervical cancer include multi-step processes,which involve both gene tampering in cervical epithelial cells and infection of HPV.Although several relatively effective therapies,such as platinum-based chemodrugs and concurrent chemoradiotherapy(CCRT),are available for treating patients,locoregional recurrence-free survival(LRFS),disease-free survival(DFS),median survival and overall survival(OS)are still low and need to be improved.Due to such limitations,it is necessary to identify other molecular effectors that influence oncogenesis of cervical cancer in order to find more effective and safe strategies or therapeutic avenues for preventing and curing cervical cancer.It has been known that genomic DNA existing in eukaryotic nuclei is closely packaged and organized with histone proteins into the basic frame of chromatin,nucleosomes.Chromatin remodeling factors are found to participate in regulation of chromatin structure dynamics through DNA packaging and chromatin winding/unwinding,providing the access for other nuclear proteins,which subsequently controls DNA synthesis,gene transcription and DNA damage repair.Thus,chromatin remodeling factors play important roles in many cellular processes,which determine tissue development and differentiation,as well as the pathogenesis of various diseases including cancer.Based on previous findings,there are at least three main families related to chromatin remodeling complexes,including the ISWI,SWI/SNF,and CHD/Mi-2 families.These chromatin remodelers possess differential binding affinities toward distinct specific nucleosome positions and establish unique chromatin structures.Genetic alterations or mutations in chromatin remodeling factors have been frequently identified in various cancers by the cancer genome project and recognized as one of main driving forces for cancer development.Rsf-1/HBXAP(also called HBXAP)was previously found as the key cancer-driving gene within the defined 11q13.5 genetic amplicon in ovarian cancers,which can be supported by several other studies in other cancer types.Results from these studies came to a similar conclusion that the expression levels of Rsf-1/HBXAP in human tissues correlated with advanced cancer progression and can serve as a prognostic marker for poor clinical outcomes and shorter survival rate.Rsf-1/HBXAP overexpression was also found to contribute to paclitaxol resistance,and recurrent tumors after chemo-or radio-therapy showed higher Rsf-1/HBXAP levels.By contrast,Rsf-1/HBXAP knockdown by mi RNA treatment or functional competition with deletion mutants reduced cell growth,increased drug and radiation sensitivity and triggered cell death in cancer cells with Rsf-1/HBXAP-overexpression.In addition,the RSF complex(Rsf-1/HBXAP with hSNF2H)can collaborate with cyclin E,another well-known cancer-driver,to trigger more aggressive cancer behaviors through promoting G1-S transition.Recent studies further identified the involvement of Rsf-1/HBXAP in DNA recombination and unequal chromosome segregation that may result in genome instability.With solid evidences showing the involvement of Rsf-1/HBXAP in cancer development,however,there are few reports regarding its roles in cervical cancer.Our study therefore aims to identify potential effects of Rsf-1/HBXAP overexpression in cervical cancer by immunohistochemistry.The association between Rsf-1/HBXAP levels and clinical pathological characteristics were analyzed to know its prognostic potential.Moreover,changes in Rsf-1/HBXAP expression after chemoradiotherapy were also investigated.Besides,Rsf-1/HBXAP levels in Hela,Siha and 293 T cells were detected by fluorescence real-time quantitative PCR and Western blotting.Hela cells were used as an experimental model to verify the efficacy of radiation for treating cervical cancer,which was researched by RNA interference.Finally,Kaplan–Meier survival analysis was applied to evaluate the difference of median survival rate among patients that suffered cervical cancer.This study will investigate the mechanism of Rsf-1/HBXAP gene expression in cervical cancer and the sensitivity of tumor to chemoradiotherapy.ObjectivesExplore the role of remodeling spacing factor 1(Rsf-1/HBXAP)expression in cervical cancer and mechanism of resistance to chemoradiotherapyMethodsImmunohistochemical staining was performed to detect Rsf-1/HBXAP expression on 14 cases of normal cervical tissue,35 cases of CIN(cervical intraepithelial lesion)and 50 cases of cervical cancer.The different scores of cervical tissues were analyzed by ?2 test.The difference of Rsf-1/HBXAP protein content between cervical cancer tissues with different clinical and pathological features was analyzed.At the same time,20 tissue cases from patients who had been treated with chemoradiotherapy including IMRT(intensity-modulated radiotherapy),brachytherapy and Taxol+ Cisplatin chemotherapy were detected via immunohistochemical staining.Rsf-1/HBXAP expression levels in Hela,Siha and 293 T cell lines were detected by fluorescence real-time quantitative PCR and Western blotting.After ionizing radiation,Rsf-1/HBXAP expression levels in Hela will be discovered by fluorescence real-time quantitative PCR and Western blotting.The Rsf-1/HBXAP gene in cervical cancer cells was silenced by miRNA interference technique while the sensitivity of cervical cancer cell lines to ionizing radiation after silencing was detected by MTT colorimetric method and flow cytometry.Kaplan–Meier survival analysis was applied to evaluate the difference of median survival rate among patients that suffered cervical cancer.ResultsImmunohistochemical staining showed that the positive expression rate of Rsf-1/HBXAP in cervical cancer tissues was higher than that in CIN tissues and normal cervical tissues(P <0.05).Rsf-1/HBXAP staining rate increased with the improving of CIN tissue level,the difference among them was statistically significant(P <0.05).The differences of Rsf-1/HBXAP expression were statistically significant in the general type,tumor diameter,HPV infection,histological grade,depth of myometrial invasion,lymphatic vessel invasion and clinical stage(P <0.05).The differences of expression of Rsf-1/HBXAP was not statistically significant in the aspect of age(P> 0.05).There was a significant difference between the expression of Rsf-1/HBXAP gene in cervical cancer before and after concurrent chemoradiotherapy(P <0.05).The expression of Rsf-1/HBXAP mRNA and protein in different cervical cancer cell lines were analyzed and the levels in cervical cancer cell line was higher than that in 293 T cell line.The expression of Rsf-1/HBXAP mRNA and protein in Hela cells decreased after ionizing radiation,which was lower than that before ionizing radiation(P<0.05).After transfection,the copy number of Rsf-1/HBXAP mRNA was significantly decreased in Hela cells.MTT colorimetric method showed that there was no significant difference related to survival rate between the transfected mimics group and the NC group after ionizing radiation.Meanwhile,the survival rate between the inhibitor group and the inhibitor NC group after ionizing radiation was also not statistically significant(P> 0.05).Result of flow cytometry showed that,the apoptotic rate of mimic group was increased compared with NC group after transfection of mimics and the difference was not statistically significant(P>0.05).However,the apoptotic rate of the inhibitor group was lower than that of the mimics group(P<0.05).All of patients with cervical cancer received concurrent radiotherapy and chemotherapy,including 27 cases in Rsf-1/HBXAP high expression group and 23 cases in Rsf-1/HBXAP low expression group.The differences of median survival rate of Rsf-1/HBXAP high expression group and low expression group was statistically significant(P <0.05).Conclusions1.Rsf-1/HBXAP gene overexpresses in cervical cancer tissue and its cell line,suggesting that it may amplificate in cervical cancer tissue,thus affecting the progress of cervical cancer.2.The expression of Rsf-1/HBXAP in cervical cancer tissues decreased significantly after chemoradiotherapy,indicating that the gene may affect the sensitivity of cervical cancer to chemoradiotherapy3.Silencing of the cervical cancer cell line Rsf-1/HBXAP gene can lead to an increase in the sensitivity of cervical cancer cell lines to ionizing radiation,suggesting that Rsf-1/HBXAP gene may be one of the causes leading to resistance to radiation therapy4.The survival rate of high Rsf-1/HBXAP expression was significantly lower than those of low expression group,suggesting that Rsf-1/HBXAP gene may play a critical role in the recurrence and metastasis of cervical cancer.5.This study provides the experimental basis for the early diagnosis of cervical cancer,the prognosis of patients and the clinical development and implementation of individualized cervical cancer treatment program.