Protection Of Ring Finger Protein 146 For DNA Damage Of Mouse Spermatogonia After Chemotherapy

Testis is highly sensitive to chemotherapeutic agent of organs. More and more patients with different degree of testicular function injury after chemotherapy, seriously affect the male reproductive health. Pirarubicin(THP) is blood system and malignant tumor of urinary system [3,4] commonly used chemotherapy drug therapy. Against the abnormal proliferation of tumor cells, THP can cause DNA damage and induce tumor cell apoptosis, however, the cells in the body, such as germ cells, show a high sensitivity to chemotherapeutic drugs.Ring finger protein 146 is a newly discovered with E3 ubiquitin ligase a ctivity of DNA damage repair proteins. Pre experiment, we found that the in adult male mice testis highly expressed.RNF146 in neonatal mouse testis in expression is very low, with the developmental expression level increased rapidly. This phenomenon caused our attention. However, there are still some problems need to be explored. What is the meaning of rnf146 in adult mouse testis spermatogonial cells with high expression? Is there a link between DNA damage and RNF146 after chemotherapy in mice, and can RNF146 be used as a target for improving the DNA damage of sperm cells after chemotherapy?To the end, we conducted the following two parts of the research work.Part? Expression and localization of RNF146 in mice testisObjective To identify the expression and cell location characteristics of DNA damage repair protein RNF146 in mouse testis.Methods Tissue specificity of 8 adult male C57 mice, were extracted total protein of testis and other major organs, to evaluate expression of RNF146 by Western blot test. From newborn, 7 days of age, 1 month of age, 3 months and a year of age male C57 mice, extraction of total protein from testis by Western blot for the observation of the expression rule of C57 Mouse Testis in RNF146. Preparation of the adult mouse testis tissue paraffin sections and by immunohistochemistry and immunofluorescence staining confirm cell fixed position characteristics RNF146 adult mouse testis.Results Western blot test showed that RNF146 was mainly expressed in the lungs, testis, brain and spleen in adult male C57 mice, and the expression level was very low in liver, kidney, stomach and heart tissue; With the development of mouse, the expression of RNF146 in testis tissue increased rapidly and reduced in old rat testis. RNF146 immunohistochemistry and immune fluorescent staining tips in adult male mice testicular tissue RNF146 mainly located in spermatogenic cells on, vascularity and supporting cells almost no expression. RNF146 immunohistochemistry and immunofluorescence staining indicated that RNF146 was mainly localized in the testis of adult male mice, and the interstitial blood vessels and supporting cells were almost not expressed.Conclusion Expression of RNF146 in normal adult male C57 mice in each organ is tissue-specific, lung, brain and testis is the main expression of organs. Iduna in Testis mainly located in seminiferous tubules spermatogonia region, suggesting that it may be related to the function of spermatogonial cells.Part? Protection of RNF146 for DNA injury of mouse spermatogonia after pirarubicinObjective To identify the protection of RNF146 for DNA injury of GC-1 cells with pirarubicin, a lentivirus system which used to upregulate RNF146 expression in GC-1 cells was constructed.Methods Basing on the sequence of mouse rnf146 c DNA, its cloning primers were designed and synthesized. c DNA from the reverse transcription of total RNA in mice testis as the template, the c DNA containing the entire open reading frame of rnf146 was amplified using a Polymerase Chain Reaction and inserted into a overexpressing vector of a lentivirus, p CDH. The overexpressing recombinant-plasmid(p CDH-rnf146), a packaging plasmid ps PAX2 and an envelope plasmid p MD2.G were co-transfected into HEK293 T cells with Lipofectamine?3000 to package the overexpressing lentivirus of RNF146. The expression effeciency of target gene in GC-1 cells with Lenti-RNF146 infecting was dected using a western blot assay. In vitro expriments were divided into three groups: Blank, Lenti-EGFP and Lenti-RNF146. The cellular model of DNA injury of mice spermatogonia(GC-1 cells) was established using a chemotherapy drug, pirarubicin. Brd U assay was used to assess GC-1 cell proliferation ability and determine an adaptive concentration of THP. DNA integrity and cell apoptosis were respectively identified using a comet assay and a tunel staining.Results The overexpressing plasmid of the lentivirus-RNF146, p CDH-rnf146, was constructed successfully. We gained an effective lentivirus which signifincantly upregulated the expression of RNF146 in GC-1 cells. Brd U assay showed that RNF146 alleviated inhibition of GC-1 cell proliferation from pirarubicin. Tunel staining and comet assay suggested that RNF146 was helpful for relief GC-1 cell apoptosis and DNA injury from pirarubicin.Conclusion RNF146 acts a protective role in DNA injury of mouse spermatogonia with chemotherapy pirarubicin.