The Study On Expression Of Migration And Invasion Inhibitory Protein In Renal Carcinoma And The Effect On Renal Carcinoma Cells

Background:Renal cell carcinoma(RCC) arises from the tubular epithelial cells of renal parenchyma. The main pathological type is renal clear cell carcinoma(80%-90%). At present, the incidence of renal carcinoma is only lower than bladder cancer in urologic neoplasms and it is on the rising. Especially, quite a number of patients already suffered from invasion and metastasis when pain, neoplasm and hematuresis occurred. For the particularity of pathological types, renal cancer is not sensitive to radiotherapy and chemotherapy, then the main therapeutic methods are surgery and targeted cancer therapy. But, there was no effective method for the tumors that in the phase T3/T4, even for the prevention of tumors’ invasion and metastasis. Migration and invasion inhibitory protein(MIIP, also IIP45)was being found in recent years that could prevent the proliferation, invasion and metastasis of tumor cells. It’s encoding gene on chromosome 1p36.22. Multiple system malignant tumors occurred when lack of genetic haploid dose or allelic loss, e.g.: prostatic cancer, neuroglioma, colorectal cancer, breast cancer, endometrial cancer. Foreign research has proved that as a upstream gene of signaling pathways, MIIP is related to tumor cell’s biological behavior, such as proliferation, invasion and metastasis. At the same time, its function could be inhibited effectively when we increase the MIIP expression. As so far, there is no report about renal carcinoma. Objective:1. To investigate the expression of migration and invasion inhibitory protein(MIIP) in renal cell carcinoma(RCC) and normal tissue adjacent to carcinoma, to clear the relationship between the expression level and RCC’s clinical stages.2. To prepare renal carcinoma cell line 786-0/MIIP via plasmid transfection technique that could express MIIP at a higher level,then investigate the change of renal carcinoma cell’s proliferation, invasion and migration ability. Methods:1. The study of MIIP expression level on different pathological tissuesThe pathological tissue group of 84 cases that have been diagnosed as renal cell carcinoma were collected. Renal cell carcinoma tissues were classified as experimental group, and normal tissues adjacent to carcinoma were classified as control group. Paraffin sections that contain the two groups, then SP immunohistochemical staining was performed. Semi-quantitative analysis helps us to get the difference of MIIP’s expression level between the two groups, even more, the differences between different clinical stages in RCC. Simultaneously, we detected the MIIP expression level in the two groups by Western-blot technology and then did further analysis.2. The biological behavior study of MIIP on RCCThe renal carcinoma cell strain 786-0 transfected by MIIP gene plasmid was experiment group, the 786-0 transfected by P-Flag-CMV4 plasmid was control group, and the 786-0 cell strain without anything was blank group. The MIIP protein and mRNA expression level of each cell strain were detected by Real-time PCR and Western-blot. The proliferation ability of each cell strain was detected by cell cycle analysis and MTT assay. The invasion and metastasis ability of each cell strain was analyzed by Transwell assay and Wound-healing assay. Results:1. The study of MIIP expression level on different pathological tissuesThe result of SP was that the rate of MIIP positive expression in RCC tissue specimens was 30.95%, which in normal tissue adjacent to carcinoma was 85.71%. In RCC tissue, there has a different expression between the different clinical stages. The rate of phaseⅠ-Ⅲ was 54.55%, 40.54% and 13.89%, respectively. From the result of Chi-square test, the MIIP expression level in phase Ⅲ was different from the other two phases and was the lowest one(P<0.00625).By Western-blot technology, we knew that the expression level of MIIP in RCC was lower than that in normal tissues adjacent to carcinoma. This result is consistent with SP immunohistochemical staining’s.2. The biological behavior study of MIIP on RCCThe renal carcinoma cell strain 786-0 transfected by MIIP gene plasmid. By the Western-bolt technique and Real-time PCR technique, we found that the expression levels of MIIP protein and mRNA in 786-0/MIIP cell strain were higher than that in control group and blank group. The cell multiplication index of 786-0/MIIP group was 53.69, lower than that in blank group(PI: 56.33)(P<0.05). Meanwhile, the growth curve showed that the proliferation ability of 786-0/MIIP group was weaker than the other two groups(P<0.01). The number of 786-0/MIIP cell(15.23±3.21) that had through the membrane was lower than that in control group(39.03±7.49) and blank group(40.17±4.24)(P<0.01). Similarly, the migration distance of 786-0/MIIP cell was shortest in Wound- healing assay(P<0.01). All of these showed that 786-0 cell’s ability of proliferation, migration and invasion ability could be weakened when we raised the MIIP’s expression level. Conclusion:The MIIP are expressed in RCC and in normal tissue adjacent to carcinoma, but there has a different expression level. The expression quantity in normal tissue was higher than that in RCC. About RCC, the level is connected with tumor’s clinical stages, in other words, that of phase Ⅲ are lower than that of phaseⅠand Ⅱ. Through the plasmid transfection technique we prepared RCC cell lines that could over expressed MIIP, meanwhile, that suppresses the renal cancer cell proliferation, invasion and metastasis.