Study On Expression Of Nuclear Matrix Protein LaminB1,NuMA And Relationship Between Unclear Matrix And Telomere In Esophageal Squamous Cell Carcinoma

Objective Atypia is morphologic characterized in tumor, especially in malignant nucleus. Nuclear atypia means that morphology of tumor nucleus is different from normal nucleus. The morphology of nucleus was effected by compose of nuclear envelope and inner nuclear substance (included chromatin, RNA, nuclear matrix and protein) and those substance distributed. Nuclear matrix is a nuclear framework structure in eukaryotic nucleus. It is important for nuclear structural formation. To investigate the nuclear matrix effect on tumor nuclear morphology and its significance, we investigated follow as: (1) to examine the expression of the common nuclear matrix protein laminBl and NuMA in normal esophageal epithelia and in esophageal squamous cell carcinoma (ESCC). (2) To examine interaction between telomeric DNA and nuclear matrix in normal epithelia and in ESCC.Materials and methods (1) materials: (1)30 cases of fresh ESCC specimens surgically resected were collected, which have pathologically proved to be of esophageal squamous cell carcinoma nature. 2 samples ( mucosae of surgical up-edge and cancer lesions) were taken from each specimen. Each sample was divided into two parts. One half was made continuously frozen section for preparing nuclear matrix in situ and for immunohistochemical detecting. The other half was stored at -70℃ and for detecting Western blot, Southern blot, RT-PCR, and telomerase activity. (2) EC109 esophageal cells were cultured for immunofluorescence-confocal examination.(2) Prepared frozen section nuclear matrix in situ using the modified ammonium sulfate method.(3) Detected the expression of laminBl and NuMA proteins before and after prepared nuclear matrix in situ in normal epithelia and ESCC using immunohistochemical-method.(4) Detected the expression of laminBl and NuMA proteins before and after prepared nuclear matrix in situ in EC 109 cultured cells using immunofluorescence-confocal method.(5) Prepared nuclear matrix protein using ammonium sulfate method and detected the expression of laminBl, NuMA proteins in normal mucosa and ESCC using Western blot.(6) Prepared telomeric DNA-nuclear matrix complex, extracted DNA from nuclear matrix, and detected telomeric DNA fragments attached to the nuclear matrix using Southern blot with (TTAGGG)n probe and semi-quantitative analyzed.(7) Extracted ESCC tissue genomic DNA, digested with restriction enzyme Hinf I and Rsa I (never cut telomere repeated fragments), and detected telomere Terminal Restriction Fragment (TRF) using Southern blot with (TTAGGG)n probe. Image analysized and calculated the mean TRF length(8) Detected telomerase activity in ESCC using TRAP (telomeric repeat amplification protocal)-silver staining assay.(9) Detected hTERT mRNA transcription in ESCC using RT-PCR assay.(10) Detected the expression of hTERT protein in ESCC using immunohistochemical-method.Results (1) The architecture of tissue was retained in nuclear matrix of frozen sections in situ. Nuclear matrix showed less intense staining material (Hematoxylin stained) with reticular-like. Feulgen stained DNA was lightly positive signal and histone HI was negative staining.(2) Immunohistochemical observation of laminBl protein: laminBl positive signal was brown, located in nucleus. The positive rate of normal epithelia, nuclear matrix of normal epithelia, cancer, and nuclear matrix of cancer were 93.3%, 86.7%, 96.7% and86.7% respectively. The difference of positive rate was found no significant (x2=2.702, P>0.05) among normal epithelia, nuclear matrix of normal epithelia, cancer and nuclear matrix of cancer. In normal epithelia, the expression level of laminBl diminished from basal cells to granular cells. In nuclear matrix of normal epithelia, the expression level decreased and most positive cells located in basal cells. Most positive cells showed whole nuclear staining. The different of expression level between normal epithelia and nuclear matrix of normal epithelia was highly significant ( x 2=9.041, PO.01). In ESCC, expression of laminBl showed no regular staining patterns. In nuclear matrix of cancer tissue, the expression level of laminBl reduced and showed a rim at the nuclear periphery positive signal. The difference of expression level between cancer and nuclear matrix of cancer was significant ( x 2=4.176, PO.05). The difference of expression level between normal epithelia and cancer was no significant ( x 2=1.707, P>0.05). the expression level in nuclear matrix of cancer was higher than in nuclear matrix of normal epithelia, and the difference between them was significant (x2=5.042, PO.05).(3) Immunohistochemical observation of NuMA protein: NuMA positive signal was brown, located in nucleus. The positive rate of normal epithelia, nuclear matrix of normal epithelia, cancer, and nuclear matrix of cancer were 96.7%, 90.0%, 93.3% and 86.7% respectively. The difference of positive rate was found no significant (x2=2.182, P>0.05) among normal epithelia, nuclear matrix of normal epithelia, cancer and nuclear matrix of cancer. NuMA protein expressed in most epithelial cells and cancer cells. The expression level was reduced in nuclear matrix of normal epithelia and nuclear matrix of cancer. The difference of expression level between normal epithelia and nuclear matrix of epithelia was significant ( x 2=6.103, P<0.05), while the difference between cancer and nuclear matrix of cancer was significant ( x 2=5.417, P>0.05). The difference of expression level between normal epithelia and cancer was no significant (x2=3.766, P>0.05). The difference of expression level between nuclear matrix of normal epithelia and nuclear matrix of cancer was highly significant (x2=6.839, PO.01).(4) Localization of laminBl and NuMA protein revealed byimmunofluorescence-confocal microscope: laminBl protein positive signal was green fluorescence, located in EC 109 nucleus. The positive signal of laminBl in nuclear matrix was decreased and showed a rim at nuclear periphery. NuMA protein positive signal was green fluorescence, located in EC 109 nucleus. The positive signal of NuMA in nuclear matrix was decreased and most nuclear showed blue fluorescence.(5) Western blot analysis: LaminBl protein positive staining showed a 70kd band. NuMA protein positive staining showed a 228kd band. The positive rate of laminBl in normal mucosa nuclear matrix and cancer nuclear matrix was 93.3%, 90% respectively. The difference of laminB 1 positive rate between normal and cancer was no significant ( x 2=0.185, P>0.05). The positive rate of NuMA in normal mucosa nulear matrix and cancer nuclear matrix were same of 90%. But the bands of laminBl and NuMA in normal mucosae nuclear matrix were weaker staining than in cancer nuclear matrix.(6) Semi-quantitative analysis of telomeric DNA associated with nuclear matrix:The relative quantity of telomeric DNA associated with nuclear matrix was 2.046 + 0.505 ( x±s) in normal mucosae, and 3.414 + 0.544 ( x + s) in cancer. The difference between normal mucosae and cancer was highly significant (t=l0.093, P<0.01).The mean length of TRF in 30 cases of ESCC was 7.02Kb ±1.62 ( x + s) .The relative quantity of telomere-nuclear matrix associated fragment had no correlation with TRF in cancerous lesion (t=0.543, P>0.05).(7) Relationship between telomere DNA-nuclear matrix association and telomerase:The telomerase activity detected rate was 80% in ESCC. The relative quantity of telomeric DNA-nuclear matrix association fragment in cases of telomerase activity was higher than in cases of no telomerase activity. The difference between them was significant (t=2.679, P<0.05).The positive rate of hTERT mRNA expression was 65.0% in ESCC. The relative quantity of telomeric DNA-nuclear matrix association fragment in cases of positive expression was higher than in cases of negative expression. The difference between them was significant (t=2.941, PO.05).The positive rate of hTERT protein expression was 70.0% in ESCC. The relative quantity of telomeric DNA-nuclear matrix association fragment in cases of positive staining was higher than in cases of negative staining. The difference between them was significant (t=2.941, PO.05). Conclusions:(1) laminBl and NuMA proteins were expressed in most normal esophageal epithelia and esophageal squamous cell carcinoma.(2) In nuclear matrix of normal epithelia and cancerous lesions, laminB 1 and NuMA proteins were decreased. It suggested that some laminB K NuMA protein were soluble and some were insoluble. Soluble protein was extracted in the process of prepared nuclear matrix.(3) The expression level of laminBl > NuMA protein in nuclear matrix of cancerour lesion was higher than in nuclear matrix of normal epithelia. The expression of laminBl protein showed a rim at nuclear periphery in most nuclear matrix of cancer(4) Telomeric DNA-nuclear matrix association fragment in ESCC was more than in normal mucosae.(5) In ESCC, telomere was short, and the detected rate of telomerase activity was high. The telomeric DNA attached to nuclear matrix had correlation with telomerase activity, hTERT mRNA transcription, and hTERT protein expression.