Expression Of Cyclin G1 And Cyclin G2 In Laryngeal Squamous Cell Carcinoma And Its Significance

The laryngeal carcinoma is one of the most common malignancies in North China, accounting for 5.7-7.6% of all kinds of human malignant tumors of whole body. Ninety-three to ninety-nine percent of the laryngeal carcinomas are squamous cell carcinoma (LSCC) in pathological type. The occurrence of the LSCC has been greatly increasing with the development of modern industry and aggravation of the air pollution. The distribution of LSCC cases is largely varied in different countries. It is reported that the first three areas with the highest incidence of LSCC are Varese of Italy, St Paulo of Brazil and Bombay of India. The occurrence rate of laryngeal carcinomas in China also has great variance regionally with the highest incidence in Northeast China. The incidence is higher in cities than that in countryside, and higher in cities with severe air pollution than those with lightly polluted cities. The majority of LSCC present during the sixth to eighth decades in age, and less occur younger than 35 years old. It is more common in males than in females. Several factors have been related to LSCC. There is emerging evidence that the lifestyle, in particular, smoking, is associated with an increased risk.Along with the continuous development in diagnosis and treatment technic, there is obviously progress in the reservation of laryngeal function, the post-treatment living time of the patient has been longer than before. But the therapeutic efficacy of advanced stage cancer is still not as good as what we hoped. In order to improve the therapeutic efficiency, the research about the correlation factors of this disease has become one of the major tasks of early discovery and early treatment.Cyclin is the major modulators in cell cycle of eucaryotic cell, it control the course of cell cycle, and is one of the most important proteins in cell activities. It has been found there are aberrant expressions of many kinds of Cyclin in human neoplasms. Recent researches show that the abnormal expression of Cyclin G is closely related to various tumors.At present, the reports about Cyclin G expression in laryngeal squamous cell carcinoma were absence and the correlation between its expression and clinical pathology was not clear. There were no reports about the relationship between Cyclin G expression and angiogenesis of ISCC. The expression of Cyclin G1 and Cyclin G2 protein in 81 cases of laryngeal squamous cell carcinoma, 20 cases of benign tumors and 20 cases of laryngeal normal tissues was detected by the immunohistochemistry method. The relationship between clinical data including age, gender, tumor stage, pathological type, and prognosis were analyzed using software SPSS 12.0. Forty cases of fresh LSCC tissue samples. 10 cases each of normal laryngeal mucosa tissue and polyp of vocal cord were collected, respectively, and the mRNA expression level of cyclin Gl and cyclin G2 were examined by using reverse transcription polymerase chain reaction (RT-PCR). The relationship was statistically analyzed between expression levels of cyclin Gl, cyclin G2 and clinicopathological characteristics of the patients. To explore the mRNA expression of cyclin G1 and cyclin G2 in laryngeal squamous cell carcinoma (LSCC) and its clinical significance. Angiogenesis is essential for growth, development, invasion, metastasis and recurrence of malignant tumours.The process of angiogenesis play an important role in the aspect of diagnosis, treatment and prognosis in malignant tumours. The vascular endothelial cells were marked by CD34 to evaluate the clinical significance of micro vessel density(MVD)and investigate the relationship of CD34, Cyclin G1 and Cyclin G2 protein and their significance of clinical pathology, we studied the expression of CD34, Cyclin G1 and Cyclin G2 protein in laryngeal carcinoma and compared the relevance among them in order to provide theoretic basis and experimental evidence for application of Cyclin Gl and Cyclin G2 in the aspect of diagnosis, treatment and prognosis and research about anti-angiogenic and genic therapy in laryngeal carcinoma.1 .Materials1)Cases of partâ… 81 cases of laryngeal squamous cell carcinoma (LSCC) treated at Shenyang 463 Hospital and the General Hospital of Chinese PLA in between Jan., 2000 to Dec, 2000 was collected. 71 cases were male and 10 cases were female. The patient age ranged from 36 to 82 years old with the average of 54.8. According to the tumor position, 61 cases of glottic carcinoma , 18 cases of supraglottic carcinoma and 2 cases of infraglottic carcinoma; According to the differentiating degree, the cases were classified as high differentiated (42 cases), moderately differentiated (30 cases) and poorly differentiated (9 cases); According to 1997 UICC stating criteria, 15 cases were classified as stageâ… , 24 casesâ…¡, 29 casesâ…¢and 13 cases IV; The cervical lymph node metastasis napped in 13 cases and 68 cases were free. None of the patients received radiotherapy or chemiotherapy before operation. 20 cases of normal laryngeal mucosa tissues adjacent to the malignant tumors and 20 cases of laryngeal benign diseases (10 case of laryngeal papilloma and 10 cases of polyp of vocal cord) were obtained in the same time.2)Cases of partâ…¡ 40 cases of fresh laryngeal squamous cell carcinoma (LSCC) treated at Shenyang 463 Hospital between Jan., 2000 to Dec, 2000 was collected. The patient age ranged from 39 to 82 years old with the average of 60.8. As to the tumor position, 28 cases were of glottic, 10 of supraglottic and 2 of infraglottic carcinoma; According to the differentiating degree, the cases were classified as high differentiated (11 cases), moderately differentiated(19 cases) and poorly differentiated(10 cases); According to 1997 UICC stating criteria, 5 cases were classified as stageâ… ,11 casesâ…¡, 18 casesâ…¢and 6 casesâ…£; The cervical lymph node metastasis napped in 13 cases and 27 cases were free. None of the patients received radiotherapy or chemiotherapy before operation. 10 cases of normal tissues adjacent to the carcinomas and 10 cases of laryngeal benign lesions (1 cases of laryngeal papilloma and 9 cases of polyp of vocal cord) were obtained in the same time. All fresh tissues were snap frozen in liquid nitrogen and stored at -70℃.2 methods1) Immunohistochemistry for Cyclin G1 and Cyclin G2 protein and microvascular density labled by CD₃₄Immunohistochemical stainings were performed by SP method. The SP Kit was produced by BeiJing ZhongShan Biological Corp China. The primary antibodies were mouse monoclonal anti-human antigen Cyclin G1, rabbit polyclonal anti-human antigen Cyclin G2, mouse monoclonal anti-human antigen CD₃₄ and all the procedures were followed by the instruction. The negative control was prepared by replacing first antibodies with PBS, the positive control for staining was obtained from the company. The positive were defined as brown to yellow staining in cell nuclear (Cyclin G1) , nuclear and cytoplasm (Cyclin G2) or cytoplasm (CD34). The MVD values were calculated by Weidner's method.2) RT-PCR for cyclin G1 mRNA and cyclin G2 mRNA Total RNA from each sample (contain 50-100mg tissue) were isolated by using Trizol Solution, then the concentration and purity of RNA were detected by DNA/RNA detector.RT-PCR was done for cyclin G1, cyclin G2 and 6-actin (the primers and PCR product are listed in the following table).The RT solution contained 25mmol /L MgCl₂ 4μl, 10×RNA PCR Buffer 2μl, RNase Free dH20 3.75μl, 10 mmol/L dNTP 2μl, RNase inhibitor 1μL AMV reverse tanscriptaselμl, Oligo dT-Adaptor Primer1μl, RNA 10μg and add RNase Free ddH₂O to 20μl. Followed by the condition: 60min at 42℃,5min at 99℃, 5 min at 4℃.PCR amplification was performed on a PCR thermal cycler. 3μl of cDNA mixture was subject to amplification in 25ul solution containing the following: 10×PCR buffer 2.5μl, 2.5 mmol/L dNTPs 2μl, Taq DNA polymerase (5U/μl) 0.2μl, each of 5'and 3'primers 2μl. Add sterile purified water to25μl. PCR conditions were initial denaturation for 3 min at 94℃, followed by 30 cycles of denaturation of 94℃for 30sec and annealed at 55℃for1 min, then extended at 72℃for 4min. PCR productswere resolved on a 1.5% agarose gel and the bands were visualized by ethidiumbromide staining. Leader Ver 3.0 software was used to analysis electrophoresisstrip.the relative values were obtained by calculating the values between cyclinG andβ-actin.3. Statistical analysisThe SPSS software 12.0 analyzed the data. Chi-squared test and Fisher (Fisher's exact test) test was used in data of quantity. Independent-Samples T and One-way ANOVA test was used in data of quality. Kaplan-Meier method was used in cumulative survival rate of patients with CyclinG 1 or CyclinG2 in laryngeal squamous cell carcinoma.4.Results1) The positive rates of Cyclin G1, laryngeal benign diseases and the normal laryngeal mucosa were 61.7%(50/81), 5%(1/20), 0%(0/20) respectively. The expression of Cyclin Gl was higher in LSCC than that in laryngeal benign diseases and normal tissues(P=0.000).2) The overexpression of Cyclin G1 in LSCC was related to the histological grade, cervical lymph node metastases and recurrence of tumor (P value is 0.007, 0.013 and 0.035, respectively).3) The expression of Cyclin G2 protein: 67.9% (55/81) in LSCC, 90% (18/20) in the laryngeal benign diseases and 100% (20/20) in the normal tissues. The difference is significant (P=0.001).4) The expression of Cyclin G2 in LSCC was related to the histoiogical grade and cervical lymph node metastases (P value is 0.033 and 0.013).5) The mRNA expression of cyclin Gl was higher in LSCC than that in normal tissues and laryngeal benign disease (P=0.000). The middle-poor differentiation LSCCS had significantly higher cyclin G1 level than that in well differentiation ones(P=0.001).6) The mRNA expression of cyclin Gl in the LSCC with lymph node metastasis was higher than that without lymph node metastasis (P=0.013). cyclin G1 mRNA expression in LSCC not correlated with age, gender, clinical stage, and clinical type.7) The expression of cyclin G2 mRNA was (0.65±0.13) in LSCC, (0.94±0.11) in normal tissue and (0.91±0.14) in laryngeal benign disease. The well differentiation LSCCS had significantly higher cyclin G2 level than that in middle-poor differentiation ones (P=0.000).8) The expression of cyclin G2 mRNA was (0.69±0.11)in those without lymph node metastasis and(0.57±0.12) in those with lymph node metastasis (P=0.004). Cyclin G2 mRNA expression in LSCC not correlated with age, gender, clinical stage, and clinical type.9) The expression of MVD was (44.88±15.11) mv in LSCC, (5.30±1.03)mv in the laryngeal benign diseases and (4.70±1.30) mv in the normal tissues. The difference is statistically significant (P=0.000).10) The mean value of MVD was (53.34±12.53)mv in Cyclin G1 positive group, and (30.74±3.49) mv in negative ones. The difference is significant (P=0.000).11) The mean value of MVD was (39.53±9.93) mv in Cyclin G2 positive group, and (55.62±17.82) mv in negative ones. The difference is statistically significant (P=0.000).5 Conclusions1) Cyclin Gl is highly expressed in laryngeal squamous cell carcinoma, and is associated with differentiation degree and tumor angiogenesis. It may play an important role in carcinogenesis and metastasis in LSCC, and may be one of prognostic index for LSCC patients.2) Cyclin G2 protein highly expressed in LSCC and had correlation with the cervical lymph node metastasis and MVD.The expression level of Cyclin G2 negatively correlates with the malignant degree and development tendency of LSCC. Cyclin G2 may thus further restrain tumor angiogenesis, invasion and metastasis of the laryngeal carcinoma.3) The high expression of cyclin G1mRNA is correlated with the pathogenesis of laryngeal carcinoma and may be one of the indexes for the poor prognosis of LSCC patients.4) The expression level of cyclin G2mRNA negatively correlates with the malignant degree and development tendency of LSCC. cyclin G2mRNA is an important biological factor affecting the prognosis.5) There was concordance between the methods of immunohistochemistry and RT-PCR, the test rigs for immunohistochemistry technic was simpler and the cost was lower, it can be applied in initial detection of gene target.