Explore The Expression And Function Of Stomatin-like Protein2(STOML-2)in Cervical Cancer

BackgroundCervical cancer is the third most female cancer in the world. Although considerable improvements have been made in high-resolution imaging, surgery techniques, chemotherapy, and radiotherapy, patients with advanced cervical cancer still face poor prognosis. So, it is of great importance to find a new biomarker for the guidance of therapy.Stomatin-like protein2(STOML2), a member of the stomatin family, has been reported to be up-regulated in several types of human cancers. The clinical significance and biological role of STOML2in cervical cancer remain largely unknown.Several study indicate that SLP-2plays a role in regulating the stability of mitochondrial proteins including prohibitins and subunits of respiratory chain complexes.PurposeTo observe expression of Stomatin-like protein2(STOML2) in cervical cancer and the relationship between STOML-2expression and clinical features, as well as to investigate the possible biological function and mechanism of STOML-2in apoptsis of cervical cancer.Materials and Methods1. Cell linesThe cervical cancer cell lines, including HeLa、SiHa、C33A、Ca ski、Hcc94, were kept in our laboratory. All cell lines were maintained in DMEM or1640(Gibco) supplemented with10%fetal bovine serum (Hyclone).2. tissue specimensWe collected300paraffin-embedded tumor specimens of cervical cancer and10chronic cervicitis samples from the Sun Yat-sen University Cancer Center between1999and2005.19biopsies of cervical cancer tissues and the matched adjacent noncancerous tissues were frozen and stored in liquid nitrogen until further use.3. RT-PCR and Western blotDetect the expression of STOML-2both at transcription and translation level.4. Immunohistochemistry assayWe detected the expression of STOML-2in the specimens by immunohistochemistry of LSAB method.5. Design of siRNAApply siRNA design software for screening out of nine pairs of siRNA,and then remove the siRNA which are homologous with the other encoding genes.the final select three pairs of STOML-2-targeted siRNA.6. Transient transfectionTransfect STOML-2-siRNA into HeLa, HCC94cells by lipofection technique,and detect after48h.7. Expression of STOML-2protein was identified by Western blot after transfection8. Cell apoptosis rate was evaluated by Annexin V and TUNEL.9. Statistical methodsThe statistical analysis is performed with the SPSS13.0for Windows. The relationship between expression of STOML-2and the clinical features was analyzed by Chi-square.Factors predictive of pelvic lymph node metastasis was analyzed by Binary Logistic.Survival analysis is performed by Kaplan-Meier method. Cox proportional hazards model is used to analyze the possible factors that have influence on the survival. P<0.05(two-tailed) is considered to be of significance.Cell apoptosis was analyzed by independent-samples t test.Results1. Up-regulation of STOML-2in cervical cell linesSTOML-2is over-expressed in cervical cancer cell lines both at transcription and translation level.2. Expression of STOML-2is up-regulated in cervical cancer lesionsBy immunohistochemical analysis, the high expression of STMOL-2protein was detected in72of the300(24%) specimens of cervical cancer. STOML-2is overexpressed at both mRNA and protein levels in all19examined human primary cervical cancer samples paired with tissues adjacent to tumors from the same patients.3. STOML-2is positively correlated with SCCA,Deep Stromal Invasion, Lymphovascular space involvement,pelvic lymph node metastasis and poor prognosisSTOML-2protein expression is positively correlated with clinical characteristics,including the Squamous cell carcinoma antigen (χ2=9.014, P=0.003), Deep Stromal Invasion (χ2=5.321, P=0.021), Lymphovascular space involvement (χ2=4.050, P=0.044) and Pelvic lymph node metastasis (χ2=38.668, P<0.001) whereas it was not associated with age, gender, stage, differentiation grade, positive parametrium or histological type. In logistic-regression analysis, STOML-2protein expression(P<0.001, OR=6.810) and SCCAg≧1.5ngml(P<0.001, OR=5.361) in cervical cancer was significantly associated with the lymph nodes metastasis. The5-year survival rate, as assessed by the Kaplan-Meier method, was92%in the STOML-2negative group, while it was only78%in the STOML-2positive group. The overall survival curve of the STOML-2negative group was statistically significant better than the STOML-2positive group (Log-Rank Test, χ2=11.615,P=0.001). Furthermore, multivariate COX analysis showed that STOML-2was independent prognostic indicators of survival(P=0.001, RR=3.881).4. ApoptosisThe result of48hrs after transfection demonstrated that apoptosis rate of two experimental groups was higher than control group.Conclusions1. STOML-2is over-expressed in cervical cancer cell lines and tissues and positively correlated with SCCA,Deep Stromal Invasion, Lymphovascular space involvement,pelvic lymph node metastasis and poor prognosis.2. STOML-2protein expression was significantly associated with the lymph nodes metastasis.3. STOML-2may be a progress gene in development of cervical cancer. Over-expression of STOML-2plays an important role in the apoptsis of human cervical cancer.4. STOML-2may be a valuable marker for assessing the prognosis of cervical cancer.