The Effects Of NONRATT008453.2 On Autophagy Of Genital Tubercle Fibroblast Of Hypospadias Rat Induced By DBP

Part1 Analyzing the Differential Expression Profile of Long non-coding RNA of Genital Tubercle of Hypospadias rats induced by DBPObjective: To study the differential expression of long non-coding RNA in genital tubercle of hypospadias rats induced by DBP and carry out bioinformatics analysis.Method: Female rats mated successfully were randomly divided into DBP group and corn oil group.At GD13-18,pregnant rats in DBP group were treated with 800mg/kg DBP dissolved in 1ml corn oil by gavage,the pregnant rats of corn oil group were administrated 1ml corn oil daily.On the day GD19,fetal rats were brought out by cesarean section and hypospadiac genital tubercles of the male offsprings in DBP group were identified and harvested under anatomical microscope.Genital tubercles of the male offsprings in the corn oil group were harvested as control.Then the genital tubercles were morphologically studied after H&E staining in paraffin section and total RNA of the genital tubercles in hypospadias and control groups were extracted to construct RNASeq library and identify differential expression of m RNA and lnc RNAs(Fold Change≥2 or≤0.5)by high-throughput whole transcriptome sequencing.q RT-PCR was used to detect the expression level of 8 Lnc RNAs to verify the reliability of sequencing results.Finally,we analyze the differential expression m RNA by gene oncology(GO)and KEGG and then find gene co-expression with Lnc RNA to predict the function of Lnc RNA.Results: The incidence of successfully induced hypospadias by DBP in male fetuses was 48.81%,and the urethral opening was observed on the ventral side of the genital tubercle with absence of surrounding tissue around on H&E staining sections in the hypospadias group.598 differential expression m RNAs(269 up-regulated and 329 down-regulated m RNAs)and 427 differential expression Lnc RNAs(253 up-regulated,174 down-regulated Lnc RNAs)were detected through RNAseq.The function of Lnc RNA was studied through bioinformatics analysis to find the maximum fold change and the highestexpression lnc RNA called NONRATT008453.2 from 103 lnc RNA related to autophagy.Finally,the expression trend of 8 lnc RNAs was verified by q RT-PCR to be consistent with the sequencing results confirming no different trend between the q Rt-PCR and the sequencing results.Finally,the expression trend of 8 lnc RNAs was verified by q RT-PCR to be consistent with the sequencing results confirming no different trend between the q Rt-PCR and the sequencing results.Conclusion: Our study reveals abundant amount of significant differential expression of lnc RNA and m RNA in fetal genital tubercles between hypospadias and control group and in-depth study are needed for future exploration of potential target genes relevant to the diagnosis or treatment of hypospadias.Part2 Effects of NONRATT008453.2 overexpression on autophagy of fibroblasts in the developing genital tubercle of hypospadias fetal rat induced by DBPObjective: To study the effect of overexpressed NONRATT008453.2 on autophagy in genital tubercle fibroblasts of hypospadiac male offsprings when mother rats were exposed to DBP during pregnancy and knowhow of NONRATT008453.2 regulating autophagy.Methods: The genital tubercles of hypospadiac male offspring rats were obtained on GD19 in DBP group.Genital tubercle was cut into(1-2)×(1-2)×(1-2)mm3 tissue in serum free DMEM/F12 medium and planted with 10 pieces per culture bottle.The bottle was incubated at 37°C with 5%CO2 for 30 min,and then the tissue were cultured in DMEM/F12 with 10% fetal bovine serum.The tissue blocks were removed when surrounding cells proliferated to occupy 90 percent bottom surface area of the bottle.The cells were digested with 0.125% trypsin for 2~3min then kept culturing in DMEM/F12 medium containing 10% fetal bovine serum.Purified froblasts were obtained utilizing natural purification of medium to suppress epithelial cell growth,and then identified by immunofluorescence to detect Vimentin protein.Target NONRATT008453.2 overexpressing adenovirus was built and NONRATT008453.2 expression level was detected by q RT-PCR;Cell proliferation ability was detected by CCK-8;Autophagosome and autolysosomes in fibroblast were examined under transmission electron microscope.Finally,the expression level of proteins associated with autophagy such as Atg5,Atg7,Beclin-1,LC3A/B-II:LC3A/B-I was detected by western blot.Results: The morphology of fibroblasts showed elongated fusiform or polygonal cells arranged in a vortex,or palisade under the inverted microscope.Immunofluorescence results showed that positive expression of vimentin in fibroblast cell’s cytoplasm.q RTPCR results showed that the expression of NONRATT008453.2 in overexpression group was significantly higher than the other groups,CCK-8 result showed that overexpression of NONRATT008453.2 suppresses hypospadias rat genital tubercle fibroblast proliferation.The number of autophagosome in fibroblast of overexpression group was decreased though observing 10 cell slices each group.Compared with the control group,expression level of Atg5 and Beclin-1 and LC3A/B-II:LC3A/B-I was decreased while Atg7 was significantly increased on Western blot in the overexpression group.Conclusion: NONRATT008453.2 regulate autophagy by decreasing Atg5、Beclin1 and LC3A/B-II:LC3A/B-I expression while increasing Atg7 expression.