The Correlation Between The Expression Of The Circadian Clock Protein Cry1 And Tumor

Objective: To investigate the expression of circadian clock protein cryptochrome1(Cry1) relationships with the development of tumors and select the cancer that have high correlation with Cry1 expression, we detect the expression of Cry1 in different tumors and analyze its relationships with clinicopathological features. By cultivating tumor cells, we detect the expression and subcellular localization of Cry1 in different tumor cells. After change the expression of Cry1 by molecular biology methods, we detect the proliferation, apoptosis and cell migration of cancer cells and study the role of Cry1 in the development of tumors.Methods:(1) Immunohistochemistry was used to detected the expression of Cry1 protein in 10 kinds of tumors( 282 clinical specimens) and statistical analysis were due to evaluate the relationship between expression of Cry1 protein and clinicopathologic features. Select the cancer that has high correlation with Cry1 expression.(2)Renal tubular epithelial cells( HK-2) and renal carcinoma cells( 786-0) were cultivated. Total RNA and total protein were extracted from the cells, Real time PCR and Western blot were used to detect the expression level of Cry1 in different cells. The eukaryon expression vector pcDNA3.1(+)-Cry1 was constructed and then transfected to kidney cancer cell 786-0 using lipofectamine 3000, after 48 h transfection, Real time PCR and Western blot were used to detect the expression Cry1 and identify the function of pcDNA3.1(+)-Cry1. CCK-8 method was used to detect cell proliferation. The apoptosis was measured with flow cytometry. Scratch test was used to detect cell migration.(3) Cultivate human gastric epithelium cells GES-1, gastric cancer cells BGC-823. Immunofluorescence assay was used to detect the subcellular localization of Cry1 protein in GES-1 and BGC-823 cells. The Cry1 protein of cytoplasm and nucleus were extracted used NE-PER Nuclear and Cytoplasmic Extraction Reagents respectively, Western blot was used to detect the expression of Cry1 in the cytoplasm and nucleus.Results:Part 1:(1) The expression level of circadian clock protein Cry1 in renal cancer and cervical cancer tissues was lower than its adjacent tissues and the difference was statistically significant( P < 0.05), but the expression intensity between carcinoma tissues and adjacent tissues, including esophageal cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, lung cancer, thyroid cancer, breast cancer, was not statistically significant( P > 0.05).(2) Cry1 was located in the nucleus and cytoplasm. In most of the adjacent tissue, Cry1 was mainly stained in nucleus. However, the proportion of cytoplasmic expression in cancer tissues, contains esophageal cancer, gastric cancer, colorectal cancer, lung cancer, thyroid cancer, breast cancer, and cervical cancer, was significantly higher than adjacent tissues(P < 0.05); the proportion of nuclear expression in cancer tissues, including esophageal cancer, colorectal cancer, lung cancer, thyroid cancer, renal cancer and cervical cancer, was significantly lower than adjacent tissues(P < 0.05); The subcellular localization of Cry1 protein in liver cancer, pancreatic cancer and renal cancer was similar and the difference of subcellular localization was not statistically significant(P > 0.05), and the proportion of cytoplasmic expression is high in both hepatic carcinoma and corresponding non-cancerous tissues.(3) The expression and subcellular localization of Cry1 in ten tumor tissues, were not correlated with age, sex, tumor size, tumor differentiation, TNM stage and lymph node status of those patients( P ï¹¥0.05).Part 2:(1)Real time PCR showed that Cry1 was expressed in 786-0 and HK-2 cells, and the expression level of Cry1 in 786-0 cells was low compared with the expression in HK-2 cells. The detection results of Western blot were similar to Real time PCR.(2)Using the eukaryotic expression vector pcDNA3.1(+)-Cry1 and empty vector transfected kidney cancer cell 786-0 and after 48 h transfection, Real time PCR showed that the expression of Cry1 mRNA in 786-0 cells was increased about 45.5 times compared with the control cells. Cell proliferation, apoptosis and migration were no significant difference between 786-0 cells tranfected with pcDNA3.1(+)-Cry1 and control cells.Part 3:(1)Cell immunofluorescence test showed that Cry1 was expressed in the cytoplasm and nucleus of BGC-823 cells, while Cry1 was found mainly in the nucleus of GES-1 cells.(2) The proteins of nucleus and cytoplasm in BGC-823 and GES-1 cells were detected by Western blot, the results were in line with cell immunofluorescence.Conclusion:(1)The expression of Cry1 in renal cancer and cervical cancer tissues is lower than the adjacent tissues.(2)In carcinoma tissue of esophageal cancer, gastric cancer, colorectal cancer, lung cancer, thyroid cancer, breast cancer, and cervical cancer, the subcellular localization of Cry1 had changed.(3)Increasing the expression of Cry1 in 786-0 cells had no influence to the proliferation, apoptosis and migration.