| Background and ObjectiveRenal cell carcinoma(RCC)is a malignant tumor originating from the urinary epithelial system of the renal parenchyma.It is a common malignant tumor in the urinary system.About 30%of the patients have been diagnosed with renal cancer.The prognosis and prognosis of these RCC patients Poor survival,advanced renal cell carcinoma is generally targeted for drug therapy,even so,some patients will appear on the treatment of congenital resistance or secondary resistance,resulting in poor treatment.In recent years,with the deepening of the study of Gab2,a large number of studies have shown that Gab2protein in a variety of malignant tumors in the abnormal expression of known malignant tumors include breast cancer,leukemia,ovarian cancer,melanoma and lung cancer,Gab2may be a new oncogene.With the study of microRNAs found that:in the cell differentiation and disease development process plays an important regulatory role.This study aims to study the expression and biological characteristics of Gab2(Grb2-associated connexin 2)in renal cell carcinoma(RCC)cells;to explore the microRNA-302c-3p targeting Control the role of Gab2.So as to provide a new idea for clinical treatment of renal cell carcinoma.Materials and Methods1.Materials1.1 RCC cell line culture Human RCC cell lines(786-O and A489)and HK-2 renal tubular epithelial cells were obtained from the Shanghai Institute of Biosciences cell banks.1.2 patients with primary RCC cell culture from two cases of primary RCC patients with diseased tumor tissue(patients 1,male,54 years;patients 2,male,43 years old),patients were received by the Second Affiliated Hospital of Nantong University,in No treatment before surgery.The minced RCC tumor tissue was digested by collagenase I(Sigma,0.05%w/v).Thereby establishing human primary RCC cell lines and dividing them into RCC(P1)and RCC(P2).1.3 11 cases of renal tumor patients(2011-2014 treatment,aged 45-73 years,including 5 women,6 males,tumor size 4.3-7.6cm),patients were admitted to the Second Affiliated Hospital of Nantong University,before surgery Did not receive any treatment.1.4 nude mice tumor model nude mice 20,male and female 10,4-5 weeks old,16-18g,purchased from Nantong University Animal Laboratory.1.5 Gab2 shRNA:Purchase two different Gab2 shRNA lentiviruses from Santa Cruz Biotech and Genepharm.1.6 Gab2 siRNA:Gab2 siRNA synthesized by Genechem.2.Methods2.1 The expression of Gab2 mRNA and microRNA-302c-3p in 11 normal human renal tissues and RCC tissues were detected by real-time quantitative PCR(qRT-PCR).The expression of Gab2 mRNA and microRNA-302c-3p in RCC tissues were detected by immunohistochemistry.Proteins were detected by Western Blotting(WB).2.2 In addition,qRT-PCR and WB were also used in the human RCC cell lines(786-O and A498)and primary human RCC cells[RCC(P1)and RCC(P2)]and HK-2 tubular epithelial cells The expression of Gab2 mRNA and microRNA-302c-3p and the expression of protein and the activation of Akt were measured respectively.2.3 shRNA interference in Gaba 2 in 786-O RCC cell line The expression of Gab2mRNA and Akt activation were measured by qRT-PCR and WB method,and cell proliferation was detected by MTT assay,Clonogenicity assay and BrdU ELISA.2.4 Overexpression of Gab2 in 786-O RCC cell lines The expression and cell proliferation of Gab2 and Akt were detected by the method described above.2.5 siRNA disrupts the expression of Gab2 in human primary cultured RCC cells and detects cell proliferation.The expression of Gab2 and cell proliferation were detected in exogenous supernatant of microRNA-302c-3p in 786-O RCC cell line.The expression of Gab2 and Akt activation and cell proliferation were detected in Antigosi-302c overexpression in 786-O RCC cell line The2.7 The nude mice tumor model,the same number(5×10 ~6mice per mouse)miR-302c overexpression,miR-C overexpressing 786-O cells injected into the left lobe of the nude mice,The tumor volume was about 100mm3,and the nude mice were closely observed and recorded every day.The mice were weighed every two days and the two-way tumor was measured.The determination of the end point of the experimental animals was rapid weight loss(>10%),severe fever,vomiting or skin problems(signs of wound or inflammation).When the animals had the above symptoms,euthanasia was performed by bloodletting with 2,2,2-tribromoethanol anesthesia(4 mg/10 g body weight,Sigma).Results:1.First,the specific binding of miR-302c-3p and Gab2 m RNA 3’untranslated region(3’-UTR)(position 125-131)was found by target sequencing.Secondly,compared with normal renal tissue,The expression of miR-302c-3p was significantly up-regulated and the expression of miR-302c-3p was negatively correlated with the expression of Gab2.2.In the RCC tissue,the use of RNA interference method can effectively reduce the expression of Gab2,and inhibit Akt activity,thereby inhibiting cell proliferation.On the contrary,the use of plasmid transfection method can effectively upregulate the expression of Gab2,and increase the Akt activity,thereby increasing cell value.The expression of Gab2 mRNA was significantly down-regulated,Akt activity and cell proliferation were inhibited after exogenous overexpression of microRNA-302c-3p in786-O RCC cell line.Antisense microRNA-302c(AntagomiR-302c)The expression of microRNA-302c-3p was down-regulated in the 786-O RCC cell line,and the expression of Gab2 mRNA was increased,the activity of Akt was up-regulated and the cell proliferation was increased.In addition,shRNA was used to interfere with the expression of Gab2 in 786-O RCC cell lines,and the exogenous overexpression of microRNA-302c-3p,Akt activity and cell proliferation were not inhibited.In addition,nude mice tumorigenesis experiments showed that exogenous overexpression of miR-302c-3p inhibited 786-O tumor in vivo growthConclusions1.The expression of mi R-302c-3p was significantly down-regulated in RCC tissues and cells,and the expression of miR-302c-3p was negatively correlated with the expression of Gab2.2.Down regulation of Gab2 expression in RCC tissue can inhibit Akt activity and inhibit cell proliferation.Conversely,upregulation of Gab2 expression can increase Akt activity and increase cell proliferation.3.In human RCC cells,Gab2 is the target protein of microRNA-302c-3p,and microRNA-302c-3p can reverse regulate Gab2.Exogenous overexpression of miR-302c-3p inhibits 786-O tumor growth in vivo. |
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