The Inhibition Of Clear Cell Renal Cell Carcinoma By RNA Binding Protein QKI And Exploration Of Its Mechanism

Renal cell carcinoma(RCC), abbreviate renal cancer, one of the malignant tumors which originate from renal tubule epithelia, which account for 80%-90% of all the malignant tumors of kidney,account for about 2%-3% of adults’ malignant tumor, the morbidity of RCC is different among areas, the incidence of developed countries are higher than development countries,and the morbidity and mortality of different areas of China are various. The mechanism of RCC is unknown. The occurrence of RCC is relate to inheritance, smoking, obesity, hypertension, hereditary or familial clear cell carcinoma is account for 2% to 4% of RCC. The VHL gene which locates in chromosome 3p25-26 closely relate to RCC. The main methods to cure RCC is surgery and subsidiarity immune and target therapy, but for metastatic or advanced RCC the effect of surgery is not very exactly,in order to diganosis and cure RCC more earlier, we urgently need new target sites which can help us predict and cure RCC, so that we can guarantee longer and better life for patients.HIF-1α is one member of hypoxia inducible factor family, is discovered by the HRE of gene which regulates erythropoietin(EPO). In hypoxia condition, HIF-1α link to HIF-1β to formate a heterodimer complex to transfer into nuclear and regulates downstream genes. Researches have proved that HIF-1α is capable of promoting cell proliferation, metastasis and angiogenesis of tumor. HIF-1α is a important compartment of VHL singal pathway, and overexpression of HIF-1α is the main mechanism of cc Rcc and lead to poor effects of treatment.RNA binding protein QKI is one member of the STAR family. QK can code diversity of proteins, there are three main subtype namely QKI-5 QKI-6 QKI-7. Researches disclose that QKI play an important role in the development of nervous system. The previous researches of our larboratory have proved that QKI involved in the differentiation of colonic epithelial cells and macrophage. QKI is also a tumor suppressor in varity of tumors, such as prostate cancer, gastric cancer and oral cancer. More recently study reveal that QKI also involve in the differentiation of muscle. Based on previous research, what is the effects of QKI in cc Rcc, whether it can be used as a independent factor for diagnosis is the main problem that need us to solve.Objectives:Using clear cell renal cell carcinoma and matched adjacent clinical samples to inspect if the expression of RNA binding protein QKI is related with cc RCC, and detect the expression of QKI in tumor cell lines and normal cell to determine whether the expression of QKI is different in tumor cell lines and normal renal cell.Overexpressing and knocking down the expression of QKI to observe the effect to the apoptosis and cycle of tumor cells.Based on the above variation, make further exploration to the mechanism of how to QKI effect on tumor cells.Using tissue array technology to collect cc Rcc samples and conduct immunohistochemical semi-quantity and statistical analysis to explore whether there are correlation between the expression of QKI and clinical index of cc RCC.Methods and Results:1) immunohistochemistry staining results indicate the expression of QKI between cc Rcc and matched adjacent normal tissues are different, and the expression of QKI in tumor tissues are less than adjacent normal tissues, and the expression of QKI is negative correlation with the tumor grade; using western Blot test tumor and matched adjacent normal tissues, and the results indicate that the expression of QKI in adjacent normal tissues are significant higher than tumor tissues. Meanwhile, the identical results in cell lines are got when using RT-PCR and Western Blot to test.2) The MTT assay shows, when overexpress QKI in 786-0 and Caki-1 cells, the proliferation of tumor cells are inhibited; the flow cytometer assay indicates that overexpress QKI in 786-0 and caki-1 cells increase the apoptosis rates of tumor cells; RT-PCR and Western Blot assay shows that overexpress QKI in 786-0 and Caki-1 cells decrease the expression of cycle relative protein cyclin D, whereas increase the expression of p27; the flow cytometer assay also indicates that overexpress QKI in 786-0 and Caki-1 cells down regalute the proportion of S-phase. Meanwhile, interference the expression of QKI makes the reverse results.3) RT-PCR and Western Blot assay shows that when overexpress QKI either in hypoxia(5%) or normoxia(20%) condition, the expression of hypoxia induced factor HIF-1αis inhibited; RT-PCR results reveal that under same conditions, the downstream target genes GLUT-1,VEGF, PGK-1 are inhibited too; also, we get the opposite results when interfere the expression of QKI. RT-PCR results show that when we simultaneously overexpress QKI and using si-RNA interfere HIF-1α, the variation trend of downstream target genes of HIF-1α is not obvious than before.4) Using 25% of immunohistochemistry semi-quantity as the cut-points to divide the expression of QKI as two groups; low expression of QKI(≤25%) and high expression of QKI(>25%). By the analysis of statistic, the high expression of QKI is negatively correlation with the tumor grade and is no apparently correlation with the TNM stage, the rate of survival patients is high in tissues of QKI high expression group.Conclusions:By researches, we primary discover that the RNA binding protein QKI play a role of tumor suppressor. We find that the expression of QKI is down regaluted in tumor tissues, and the expression of QKI is negatively correlate with the tumor grade, hower is positively correlate with the survival time of patients; and overexpress QKI can significantly inhibit the proliferation of cc Rcc cells, promote the apoptosis of cc Rcc cells; besides, we also find that overexpress QKI can suppress the expression of HIF-1α and by this way inhibits the expression of its target genes GLUT-1,VEGF, PGK-1. By the above effects, RNA binding protein QKI suppress the initialization and progression of cc Rcc. This research provides a unique direction of RNA binding protein QKI to be an independent diagnostic and treatment index.