| The incidence rate of colibacillosis in yaks in Tibet is high.As a non-specific immunogen,bacterial lipopolysaccharide affects the function of the reproductive system.Follicular atresia is a normal physiological phenomenon in the ovary,and apoptosis of granulosa cells is the main cause of follicular atresia.In the mechanism of competing endogenous RNA,long chain non-coding RNA can competitively bind to micro RNAs to regulate the m RNA level of functional genes.Previous studies have shown that yak Smad4 is a target gene for bta-miR-146a,but its ce RNA mechanism is still unclear.This study aims to screen lncRNAs that may competitively bind with bta-miR-146a,and further investigate whether LPS affects the mechanism by which the ce RNA network regulates GCs apoptosis.This study can improve and develop the secretion regulation mechanism of animal reproductive activities,especially for the treatment of yak reproductive disorders caused by Gram negative bacteria LPS.The results are as follows:1.Collect and isolate healthy and atretic follicles of adult female yaks,and use the high-throughput sequencing platform Hi Seq Xten to conduct transcriptome sequencing to analyze their differentially expressed lncRNAs.The results showed that healthy yak follicles have 330 upregulated lncRNAs and 352downregulated lncRNAs compared with atresia follicles.Sequence analysis using RNAhybrid online software found that there were tight binding sites between lnc-MSTRG.7889 and bta-miR-146a.The RT-PCR detection results showed that the expression levels of lnc-MSTRG.7889 and Smad4 gene m RNA in healthy yak follicles were significantly higher than those in atresia follicles（P<0.05）;The expression of bta-miR-146a was opposite（P<0.01）.The TUNEL method was used to detect cell apoptosis in yak ovarian tissue,and the results showed that the apoptosis rate of atresia follicles was significantly higher than that of healthy follicles（P<0.01）,indicating a correlation between atresia follicles and GCs apoptosis.Further fluorescence in situ hybridization detection was carried out in yak ovarian tissue.The results showed that Smad4 gene m RNA,bta-miR-146a and lnc-MSTRG.7889 were co expressed in healthy and atretic follicles of yaks,and the results were basically consistent with RT-PCR detection of the expression trend of the three genes in healthy and atretic follicles,indicating that there might be a molecular regulatory mechanism of ce RNA network involved in the regulation of follicular atresia process in lnc-MSTRG.7889,bta-miR-146a and Smad4 genes,And it is related to apoptosis of follicular GCs.2.Separate and cultivate yak follicular GCs using cell sieve method,GCs were treated with LPS at a concentration of 10μg/m L,and the apoptosis rate of GCs was detected by flow cytometry.The expression of pro apoptotic proteins（CASPASE3 and BAX）and anti-apoptotic proteins（BCL-2）were detected by Western blot,respectively.The results showed that LPS could significantly promote the apoptosis of GCs（P<0.01）.Transfection of bta-miR-146a mimics and bta-miR-146a inhibitors in GCs showed that bta-miR-146a inhibited the expression of Smad4 gene in GCs.After transfection of bta-miR-146a mimics and Smad4 overexpression vectors into GCs and LPS treatment,the results showed that Smad4 could significantly inhibit the apoptosis of yak GCs（P<0.01）,while LPS weakened the inhibitory effect of Smad4 on follicular GCs apoptosis（P<0.01）;Bta miR-146a promotes apoptosis of yak GCs（P<0.01）,while LPS enhances the promoting effect of bta-miR-146a on follicular GCs apoptosis（P<0.01）;After co transfection of Smad4 overexpression vector and bta-miR-146a mimics with GCs and LPS treatment,it was found that bta-miR-146a significantly reduced its inhibitory effect on apoptosis by inhibiting Smad4 expression（P<0.01）,while overexpression of Smad4 in GCs treated with LPS inhibited the proapoptotic effect of bta-miR-146a on cells（P<0.01）.The above results indicate that LPS affects the apoptosis of GCs by targeting Smad4 with bta-miR-146a.3.The Lentivirus vector construction technology was used to construct the lnc-MSTRG.7889 Lentivirus overexpression vector,and q PCR technology was used to detect the infection efficiency of its infection with 293T cells.The test results showed that the full-length sequence of lnc-MSTRG.7889 was 2 566 bp successfully amplified from the yak follicles,and the recombinant plasmid was successfully constructed with the LV-EF1a-EGFP-2A vector,which was packaged and concentrated by the Lentivirus packaging system to construct the Lentivirus vector,The results showed that the titer of lnc-MSTRG.7889Lentivirus was 7.32×108TU·m L-1.The infection efficiency of on GCs in yak follicles was（52.93±1.12）%.（1）After infected yak follicular GCs with this lncRNA Lentivirus and treated with LPS,the results showed that lnc-MSTRG.7889 significantly inhibited the apoptosis of GCs（P<0.01）;LPS inhibited lnc-MSTRG.7889 and significantly promoted the apoptosis of GCs（P<0.01）.（2）After infecting yak GCs with lncRNA overexpression Lentivirus vector,it was found that lnc-MSTRG.7889 targeted to inhibit the expression of bta-miR-146a.After LPS treated GCs co-transfected with lncRNA Lentivirus and bta-miR-146a mimics,it was found that lnc-MSTRG.7889 targeting bta-miR-146a reduced the inhibitory effect of the former on GCs apoptosis（P<0.01）;LPS reduced lnc-MSTRG.7889 targeting bta-miR-146a and inhibited apoptosis of GCs（P<0.01）.（3）To further explore the competitive combination of lnc-MSTRG.7889 and bta-miR-146a to regulate the expression of Smad4,q PCR and Western blot were used to detect the expression of Smad4 after GCs co-transfected with bta-miR-146a mimics and lncRNA Lentivirus.The results showed that lnc-MSTRG.7889 competitively combined with bta-miR-146a to promote the expression of Smad4（P<0.01）,while LPS inhibited the expression of Smad4 after lnc-MSTRG.7889 targeting bta-miR-146a（P<0.01）.In summary,LPS affects lnc-MSTRG.7889 as ce RNA to participate in bta-miR-146a targeted Smad4 regulation of yak GCs apoptosis. |
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