| Objective: Oral squamous cell carcinoma(OSCC) is one of the common malignant tumors in head and neck, accounting for above 80% of malignant tumors in the maxillofacial region[1]. Over the recent years its occurance has been on the rise. At present, the clinical therapeutic methods are predominated by excision, supplemented with radiotherapy or chemotherapy. However, the curative effect is unsatisfactory, the prognosis is poor, and the postoperative 5-year survival rate is only about 60%[2]. Therefore, it appears quite urgent to have a thorough understanding into the early diagnosis and prognostic analysis of OSCC. E2 F is a new cell factor Rovesdi et al. discovered in 1986 when conducting a study on activation of adenovirus E2 promoter[3]. The E2 F family has 8 members, including E2F1 ~ E2F8, among which E2F1 is most studied. It plays a vital role with regards to cell cycle control, apoptosis and proliferation. Studies have been available at present that substantiate the high expression of E2F1 in a multitude of human tumors, which evinces that E2F1 acts the function of oncogene. However, some other studies suggest that E2F1 may also have the function of anti-oncogene. Whether E2F1 acts as oncogene or anti-oncogene in the formation of tumors may depend on the approach to tumor inhibition, but its concrete mechanism remains to be fully recognized. Survivin is one of the members of the anti-apoptosis protein family and the strongest anti-apoptosis factor discovered at the moment[4]. Its main functions are: 1 inhibiting cell apoptosis; 2 inducing cell differentiation; 3 getting involved in mitosis, and; 4 promoting angiogenesis. The current research indicates, Survivin is highly expressed in a majority of malignant tumors, while its expression is beyond test in a majority of grown-up tissues. This characteristic has made it a new focus of oncotherapy for the recent years. The P53 gene was discovered by Lane et al. in SV40 infected mouse cells in 1979. It is the tumor suppressor gene in human tumors which most frequently mutates and plays a vital role in cell cycle control. The objective of this experiment is to conduct detection of E2F1, Survivin and P53 through the pathological specimens of OSCC tissue, understand the condition of their expression in OSCC tissues and normal mucosal tissues, make a preliminary explorative discussion on its relation with the clinical features of OSCC and prognosis, so as to provide reference for early diagnosis, therapy and prognosis of OSCC.Methodology : 52 pairs of specimens of excised OSCC and their corresponding mucosal tissues with normal incisal edge were collected from the Department of Stomatology, the Fourth Hospital of Hebei Medical University from January 2013 to December 2014. All the collected patients with OSCC had not undergone preoperative radiotherapy or chemotherapy. All the specimens are immediate postoperative drawn materials, divided into two parts- one fixed in 4% formaldehyde solution, embedded in paraffin; the other fixed in 70% alcohol, saved at 4℃. 1 Immunohistochemistry(IHC) for the expression of E2F1, Survivin and P53 in OSCC tissues and normal oral mucosal tissuesThe paraffin blocks of all groups of embedding tissues were taken. The paraffin slicing machine was used for serial section with a thickness of 4 μm. Conventional dewaxing was performed till water. Operation was conducted according to the S-P kit operating instructions. The protein expression of E2F1, Survivin and P53 between groups through microscopic observation was compared to decide whether there is statistically significant difference. 2 Flow cytometry(FCM) for the expression of E2F1, Survivin and P53 in OSCC tissues and normal oral mucosal tissues.The specimens fixed by 70% alcohol was taken, and made into single-cell suspension, which was then added with working solution of primary antibodies with E2F1, Survivin and P53, and incubated for 30 min at indoor temperature; and then the working solution of secondary antibodies with goat-anti-mouse FITC-Ig G was added, placed for 1 hour at indoor temperature; and then machine detection was conducted. 3 Results criteria 3.1 ImmunohistochemistryThe positively stained E2F1 and P53 were the particles rendering tan in the nucleus; The positively stained Survivin was the particle rendering brown in the cytoplasm or nucleus. The IHC semi-quantitative method offers a criterion on their expression. 3.2 Qualitative analysis of protein expression through FCMData were collected in logarithmic forms. And mean fluorescence intensity of the protein of experimental specimens was used to express the content of proteins of all groups. 4 Statistical methodsSPSS13.0 was used for statistical processing. The χ2 test and rank sum test were adopted for rate comparison between groups. The Spearman correlation analysis was adopted for grade correlation comparison. P<0.05 was indicative of statistically significant difference.Results:1 Immunohistochemistry(IHC) results on the expression of E2F1, Survivin and P53 in OSCC tissues and normal oral mucosal tissuesThe positive expression rate of E2F1 was 75%(39/52) in OSCC tissues, higher than the counterpart 36.6%(18/52) in normal oral mucosal tissues(P<0.05); the positive expression rate of Survivin was 88.5%(46/52) in OSCC tissues, higher than the counterpart 3.8%(2/52) in normal oral mucosal tissues(P<0.05); and the positive expression rate of P53 was 82.7%(43/52) in OSCC tissues, higher than the counterpart 11.5%(6/52) in normal oral mucosal tissues(P<0.05).2 Flow cytometry(FCM) results on the expression of E2F1, Survivin and P53 in OSCC tissues and normal oral mucosal tissuesThe mean channel value of E2F1 was 393.47±10.48 in OSCC tissues and 280.35±22.38 in normal oral mucosal tissues, and there was statistically significant difference between both groups(P<0.01). The mean channel value of Survivin was 412.38±18.80 in OSCC tissues and 78.69±29.66 in normal oral mucosal tissues, and there was statistically significant difference between both groups(P<0.01). The mean channel value of P53 was 400.98±10.45 in OSCC tissues and 289.19±19.70 in normal oral mucosal tissues, and there was statistically significant difference between both groups(P<0.01). The result of FCM revealed a significant difference among E2F1, Survivin and P53 in their expression in OSCC tissues and normal oral mucosal tissues, in line with the result of IHC.3 The relation between expression of E2F1, Survivin and P53 in OSCC tissues and the pathological and clinical featuresIHC indicated there was no correlation between the expression of E2F1 and cell differentiation, lymphatic metastasis and TNM staging(P>0.05); but there was correlation between the expression of Survivin and P53, and cell differentiation, lymphatic metastasis and TNM staging(P<0.05). The result of FCM was in line with that of IHC. The detection by both methods drawn the conclusion that there was insignificant correlation between the expression of E2F1, Survivin and P53 and patients’ age, gender(P>0.05).4 The correlation of expression of E2F1, Survivin and P53 in OSCC tissuesIHC revealed the intensity of expression of E2F1 protein was strongly positively correlated to the intensity of expression of Survivin and P53 proteins, respectively, in OSCC tissues(rs1=0.446, P1<0.01; rs2=0.386, P2<0.01). The mean channel value of proteins detected through FCM was in line with the result of expression of protein through IHC. The E2F1 protein was strongly positively correlated to the mean channel value of Survivin and P53 proteins, respectively.Conclusions:1 The expressions of E2F1, Survivin and P53 in oral squamous cell carcinoma tissues is high.2 The expression of E2F1 is not related to gender, age, cell differentiation, lymphatic metastasis and TNM staging; the expression of Survivin and P53 is related to cell differentiation, lymphatic metastasis and TNM staging, but bears no relation to age and gender.3 The expression of E2F1 in OSCC tissues is strongly positively correlated to the expression of Survivin and P53, respectively. It is speculated that E2F1 plays a vital role along with Survivin and P53 in the generation and development of OSCC. |
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