| Objective:To explore the expression, biological function and molecular mechanism of transcription factor Sox4in Xuanwei female lung cancer by clinical investigation and basic researchs in vitro and in vivo.Methods:1. Studies on the expression and clinical significance of transcription factor Sox4in Xuanwei female lung cancer patients:Comparision of Sox4expression in Xuanwei female lung cancer, non-Xuanwei female lung cancer and benign lung lesions was performed by immunohistochemical staining, after which,96cases of Xuanwei female lung cancer tissue and the corresponding distal normal lung tissue samples were freezed in liquid nitrogen and fixed with formaldehyde separately. Real-time fluorescence quantitative PCR and immunohistochemical techniques were applied to detect their Sox4mRNA and Sox4protein expression, combing with clinical pathological features to perform the Kaplan-Meier univariate survival analysis and multivariate Cox survival analysis.2. Influence of Sox4gene inhibition on biological characteristics of Xuanwei female lung cancer cell XWLC-05and its mechanism research:The relative expression level of Sox4gene in different types of lung cancer cell lines, normal cells lines and embryonic cell lines was detected by real-time quantitative PCR. On basis of effectively knocking down of Sox4gene by chemical synthetized siRNA, recombinant plasmid pGFP-V-RS-Sox4shRNA was constructed and transfected into Xuanwei female lung cancer cell line XWLC-05to stably inhibit the expression of Sox4gene. Then cell morphology, proliferation, migration, metastasis abilities and apoptosis were identified by transmission electron microscope, MTS assay, wound-healing assay, transwell assay and flow cytometry. At the same time, by using the " Function rescue experiment" to verify the specificity of RNA interference. In-depth mechanism study of Sox4gene was performed by processing pGFP-V-RS-Sox4shRNA transfected cells with or without caspase-3inhibitor Ac-DEVD-CHO and above apoptosis-correlated items were studied.3. Studies on the effects of Sox4targeted inhibition on Xuanwei female lung cancer xenografts in nude mice:XWLC-05cells stably transfected by plasmid pGFP-V-RS-Sox4shRNA were inoculated in the nude mice, taking mice inoculated with cells transfected by plasmid pGFP-V-RS-scramshRNA, without transfected cells and physiological saline as control. General observations of nude mice physiological state, tumor formation rate and tumor growth were performed. Mice were sacrificed at day25after inoculation. The tumor volume, tumor weight and tumor inhibition rate were measured. Immunohistochemical staining was applied to detect the expression of Sox4, Ki-67and caspase-3protein in the xenograft.Results:1. Studies on the expression and clinical significance of transcription factor Sox4in Xuanwei female lung cancer patients:1) The expression of Sox4protein in female lung cancer tissues was significantly higher than the expression in benign lung lesions (P<0.05); There were52.7%and51.0%positive staining of Sox4in Xuanwei and non-Xuanwei female lung cancer tissues, respectively, which had no significant difference between expression level (P>0.05) and between positive expression rate (P>0.05). But there was significant difference of Sox4expression among various lung cancer types in Xuanwei and non-Xuanwei female lung cancer,respectively (Xuanwei F=24.529, P=0.017; non-Xuanwei F=26.461, P=0.009), that is Sox4expression in SCLC was significantly lower than that in NSCLC (Xuanwei F=7.657, P=0.049; non-Xuanwei F=9.423, P=0.037),and the expression of Sox4in female adenocarcinoma was significantly higher than that in other types of NSCLC (Xuanwei F=18.510, P=0.000; non-Xuanwei F=19.518, P=0.000).2) The Sox4mRNA expression level of Xuanwei lung cancer tissues(2.53±0.35) was significant higher than that of matched normal ones(1.43±0.18),(P=0.003). Immunohistochemical staining showed that Sox4protein mainly expressed in cell nucleus. There were53.1%(51/96)positive expression cases in cancer tissues and only26.0%(25/96) in matched normal tissues (P=0.000); There were significant difference in the expression of Sox4protein among patients with different pathological grade (P=0.000),different histological types (P=0.004),whether or not had lymph node metastasis (P=0.000) and the degree of tumor differentiation (P=0.002). The survival analysis by Kaplan-Meier method showed that the median survival time of Sox4positive-expression and negative-expression patients was26months and39months, respectively (P=0.000).Patients with lymph node metastasis or high pathological grade had significant shorter median survival time than those without lymph node metastasis (P=0.012) or low pathological grade (P=0.000); Cox regression survival analysis showed that pathological grade was a significant independent factor affecting prognosis.2. Influence of Sox4gene inhibition on biological characteristics of Xuanwei female lung cancer cell XWLC-05and its mechanism research:Expression of Sox4gene in various types of lung cancer cell lines was higher than the expression in normal cell lines(P<0.05), especially highly expressed in lung adenocarcinoma cell lines(P<0.05). Sox4siRNA synthesised by chemical methods can effectively inhibit the expression of Sox4gene in XWLC-05cells and induce cell apoptosis (P<0.05), but it had no significant effects on the proliferation of cells (P>0.05). Recombinant plasmid pGFP-V-RS-A-Sox4shRNA was successfully constructed and efficiently transfected into XWLC-05cells. The Sox4expression was significantly down-regulated (P<0.01) and the caspase-3expression was significantly up-regulated (P<0.01) after transfection when compared with non-transfected cells. The apoptosis morphological changes were observed by microscope and the cell proliferation, migration and metastasis ability were significantly decreased after inhibiting the Sox4expression (P<0.01). FCM showed there had obvious sub-G1peak and higher apoptosis rate in pGFP-V-RS-A-Sox4shRNA transfected cells when compared with control(P<0.01). Rescue assay ruled out the off-target effects in RNAi and so verified the effects of Sox4gene inhibition on Xuanwei female lung cancer cells. Ac-DEVD-CHO treatment obviously inhibited caspase-3activating (P<0.01) and cell apoptosis (P <0.01) in Sox4gene knock-downed cells when compared with control.3. Studies on the effects of Sox4targeted inhibition on Xuanwei female lung cancer xenografts in nude mice:Tumor formation rate was100%in tested mice except the mice inoculated with saline. Visible tumors were observed as early as6days after transplantation. Tumor growth of xenograft was significantly slow down after inhibiting the expression of Sox4gene, while the isolated tumor weight and volume of Sox4-inhibited mice were significantly lower than the control (P<0.01).The tumor inhibition rate of Sox4-silenced mice was higher than the control(P<0.01).Immunohistochemical staining showed:Sox4,caspase-3and ki-67protein all expressed in cells nucleus, among which, the expression of Sox4and ki67were significant decreased in Sox4gene inhibited xenograft when compared with negative control and blank control (P <0.05), the expression of caspase-3had no significant difference in all group of mice (P>0.05). no tumor-bearing mice showed metastasis through imaging and pathological examination.Conclusions:1. There was abnormally up-regulated expression of Sox4gene at transcription and translation level in Xuanwei female lung cancer tissues.2. Sox4expression level combining pathological grade can be used as an assessment and prognosis-estimation reference for Xuanwei female lung cancer patients. 3. Transcription factor Sox4may promote the progression of Xuanwei female lung cancer by enhancing cell proliferation ability and inhibiting the caspase-3dependent apoptosis pathway. |
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