Regulation On UPA Expression With RNA Interference In Testis Of Male Mice

PartⅠSuppression of the uPA expression by siRNA in TM4 cellsObjective:To screen the effective sequences of siRNA targeting uPA gene, with analysis of the supperssive effects of uPA expression after transfection of different sequences of siRNA into mousetesticular cell line (TM4 cells).Methods:After investigating endogenous expression of uPA in TM4 cells and identifying the proper concentration of transfection with fluorescently labeled siRNA (Cy3-siRNA), three siRNA targeting uPA mRNA (si-uPA1, si-uPA2 and si-uPA3)were synthesized chemically and transfected into TM4 cells with the mediation of Lipofectamine2000, and those TM4 cells without transfection, supplemented with only Lipofectamine2000 and transfected with siRNA-scramble (siRNA-sc) were taken as blank control group, reagent control group and negative control group, respectively. The expression of uPA mRNA and protein were detected using real time fluorescence quantitative real-time PCR technology (qRT-PCR) and Western Blot. After comparison and analysis of the different expression of uPA, the effective sequence of siRNA targeting uPA gene was identified. Then, three diffenrent concentration of that si-uPA (30 nM,50 nM and 100 nM) were transfected into TM4 cells, uPA mRNA were detected with qRT-PCR at various time points (24 h,48 h and 72 h) after transfection to observe the suppressive effects of siRNA interfering uPA expression.Results:Endogenous expression of uPA was detected in TM4 cells. When TM4 cells were transfected with different concentration of Cy3-siRNA, the fluorescence intensity was observed to be increased progressively with the increased concentration of transfection. TM4 cells transfected with 20 nM Cy3-siRNA only emitted slight red fluorescence while those with 50 nM and 100 nM emitted brightter fluorescence. So 50 nM was regarded as the proper concentration of transfection. The expression of uPA mRNA and protein were significantly lower in TM4 cells transfected with three si-uPA than those in blank control group, especially with si-uPA1. However, the expression level did not change much in reagent control group. Wih the respect of transfection expriments of si-uPAl, the decrease of uPA mRNA expression were observed from 24 h after transfection in all three si-uPAl groups, and the suppresive rate reached 70% in 100 nM group, which was much obvious than those in 30 nM and 50 nM groups (P<0.05). The relative expression of uPA reduced steadily with the extension of transfection. At 72 h after transfection, the uPA mRNA in those three groups were 53.9%,35.3% and 27.7%, respectively, compared with those in blank control.Conclusions:The effective sequence of siRNA targeting uPA gene was screened from three candidates. After transfected into TM4 cells, it could decreased the expression of uPA from 24 h and retain the effects till 72 h. The suppresive effects of the same concentration of siRNA showed no significant differences when detected at different time points. While variant concentration of siRNA decreased the expression of uPA gene in a dose-dependent manner. PartⅡEstablishment of inducible lentiviral RNAi systemObjective:To construct a lentiviral RNAi vector containing elements required for tetracycline regulation. After being packaged and titrated, the viral constructs were transduced into TM4 cells, and a dual stable cell line was selected using antibiotics.Methods:According to the sequence of si-uPA1 screened in partⅠ, two complementary oligonucleotides encoding uPA-shRNA were designed and synthesized. And they were annealed to create a double-stand oligoncleotide, which was then cloned into pENTR/H1/TO entry vector using ligation procedure. After transforming competent E. coli, the entry clones were generated. Next, using Gateway technology, the LR recombination reaction was performed to transfer the H1/TO RNAi cassette in the entry clone into the lentiviral expression plasmid pLenti4-BLOCK-iT-DEST and generate the lentiviral vector pLenti-sh which contained both uPA-shRNA casstte and elements allowed packaging of the construct into virion. And then, the pLenti4-BLOCK-iT-DEST expression construct and pLenti6/TR vector, which contained tetracycline repressor (TetR) gene, were transfected into the 293FT cell line, respectively, to produced lentiviral stocks. The viral titers of two lentiviral stocks were determined using qRT-PCR. Before transduction of TM4 cell line, the antibiotic sensitivity and the optimal MOI were determined by using kill curve expriments and Lenti-EGFP transfection, respectively. After that, a TetR-expressing host stable cell line (TM4-Lenti6/TR cell line) was generated with transduction of Lenti6/TR construct into TM4 cells and Blasticidin selection. While in the same way, a dual stable cell line (TM4-Lenti6/TR & Lenti4-sh) was generated with transduction of pLenti-sh construct into TM4-Lenti6/TR cell line and antibiotic selection using Blasticidin and Zeocin. The expression of TetR and antibiotic gene were detected in these two constructs with qRT-PCR.Results:The sequences of lentiviral RNAi vector pLenti4-sh were verified to be correct using DNA sequencing. After being packaged in 293FT cell line, the viral titers of two lentiviral stocks were determined to be 9.8 X 109vp/ml for Lenti6/TR and 1.72 X 1010vp/ml for Lenti4-sh, respectively. With kill curve expriments, the antibiotic sensitivity of Blasticidin and Zeocin for TM4 cell was 1μg/ml and 600μg/ml, respectively. And the optimal MOI for TM4 cell line transduction of lentivirus was determined to be 100 using a range of MOI of Lenti-EGFP transfection. According to results from 2-ΔΔCt method using in qRT-PCR, the relative expressions of TetR gene in TM4-Lenti6/TR and TM4-Lenti6/TR & Lenti4-sh cell line were 27301.01 and 406623.29 folds, respectively, compared with those in TM4 cell line. While similar high expression of the antibiotic gene was also observed in the dual stable cell line.Conclusions:A lentiviral RNAi vector containing elements required for tetracycline regulation was constructed. And after transduction into TM4 cell line and antibiotic selection, a TetR-expressing stable cell line (TM4-Lenti6/TR) and a dual stable cell line (TM4-Lenti6/TR & Lenti4-sh) were generated.PartⅢRegulation of uPA expression with inducible lentiviral RNAiObjective:With the dual stable cell line and the lentiviral stock expressing inducible RNAi cassette, we constucted the cell culture system and mouse model with lentiviral vetor. After Dox administration, the expression of uPA mRNA and protein and the uPA activity were observed in both cells and testis, respectively, to evaluate the effects of Dox on uPA regulation.Methods:The effects of different concentration of Dox on viability of TM4 cells were detected by MTT assay to determine the optimal Dox dose on TM4. After that, with TetR-expressing stable cell line (TM4-TetR) and TM4 cell line taken as control, the dual stable cell line (TM4-TetR&shuPA) was cultured. The expression of uPA mRNA and protein and the uPA activity were observed in cells at 24 h to 72 h after Dox induction. Then, Dox was removed from the culture media, and the uPA expression was also detected at different time points after removal. For gene regulation in vivo, the two lentiviral stocks, both Lenti4-sh and Lenti6/TR were injected intratesticularly to construct the mouse model (TetR&shuPA), and those mice injected with Lenti6/TR and normal saline, respectively, were taken as control. The expression of uPA mRNA and protein in testis and uPA activity in homogenate were compared at 24 h,48 h,72 h and lweek after lavage with Dox.Results:The optimal Dox dose of Dox on TM4 cells was determined to be below 5μg/ml by MTT assay, and 1μg/ml was used in induction for TM4-TetR&shuPA, TM4-TetR and TM4 cells. No alterations of uPA expression were observed in the latter two groups, when Dox was supplemented in culture. While the expression of uPA mRNA decreased in TM4-TetR&shuPA cell after Dox induction for 24 h. And the suppressive effects were apparent with the extension of induction. The expression of uPA mRNA in Dox group went down to 35% of that in non-inducible group after 72 h. When it comes to uPA protein comparison, it was not until to 72 h that the Dox group showed much lower expression than non-inducible group. Also the chromogenic substrate assay showed silimilar results that uPA activity in TM4-TetR&shuPA group was much lower by Dox induction. On the other hand, the expression of uPA mRNA and protein recovered to normal level after Dox removal for 48 h.After being injected intratesticularly with lentiviral stocks, the male mice were lavage with Dox of 0.75 mg/ml. At 24 h after induction, the expression of uPA mRNA in TetR&shuPA mice was much lower than that in control. And the suppressive effect was most obvious after induction for 1 week. As for uPA protein expression, it showed a little difference with mRNA that the expression in Dox-inducible group of TetR&shuPA mice was similar to that in non-inducible one within 72 h. While after 1 week, uPA protein expression in Dox groups was significantly lower than that in non-inducible group. Conclusions:Dox could regulate the expression of uPA both in dual stable cell line and the mouse model. The suppressive effects in vitro could maintain to 72 h after induction. While after removal of Dox, the uPA expression could recover to normal level. As for regulation in vivo, the uPA expression and uPA activity could be reduced by Dox and the suppressive effect was most obvious after induction for 1 week.Part IV Effects of uPA suppression on fertility in male miceObjective:With the animal model in which uPA expression was regulated by inducible RNAi system in vivo, the alteration of male mice and sperm fiunction were observed and the mechanism of uPA regulation on male fertiltiy was analyzed.Methods:After constructing an animal model expressing inducible RNAi system (TetR&shuPA) with injection of lentiviral vectors intratesticularly in male mice, Dox was administrated for 1 week to induce the expression of shRNA targeting uPA. With those mice injected with TetR vector and normal saline, respectively, being taken as control, all the male mice were copulated with adult female mice. The mating rates of male mice and the pregnancy rates, litter sizes and number of corpus luteum of female mice were compared among the groups. Also the sperm motility and viability were compared and the sperm function was evaluated with hypo-osmotic swelling (HOS) test. Moreover, the histology structure and ultrastructure of testis and epididymis were observed. All the male mice were copulated with female mice again after Dox withdrawl for 2,4 and 8 week, respectively, to observe the restoration of male fertilty and sperm function.Results:There were no obvious differences in the mating rates and average number of corpus luteum among all the groups. While the pregnancy rate and litter size in Dox-inducible mice of TetR&shuPA group were significantly lower than those in non-inducible and blank control (P<0.05) after Dox treatment for 1 week. However, those figures recovered when Dox was discontinued for 2 weeks and the no differences were observed among all groups after withdrawl for 8 weeks.Compared with non-inducible group and blank control, TetR&shuPA mice had a reduced percentage for motile sperms after Dox treatment for 1 week (P<0.05), although similar figure were observed in the sperm viability and positive sperms detected with HOS test among all groups. Similar to the results obtained in mating experiment, the sperm motility recovered when Dox was discontinued, and it rised to normal level after 8 weeks.No obvious damage was observed in both testis and epididymis of all groups after Dox treatment, and all the tissues including heart, liver, spleen, lung and kidney were normal morphologically.Conclusions:The down-regulation of uPA in testis induced by Dox had no effects on mating rate of male mice and corpus luteum number of female mice. But the pregnancy rate and litter size were reduce after induction. Also it affected the sperm motility but no sperm viability, nor sperm membrane function. However, the male fertility and sperm motility could restore after Dox withdraw1 for 8 week. Suppression of uPA expression had no obvious negative impacts on structural changes in both testis and epididymis, even tissues including heart, liver, spleen, lung and kidney remained normal.