Genome-Wide Identification Of LincRNA And Functional Analysis Of PLA2G16-AS In Longissimus Dorsi Between High-And Low-Altitude Tibetan Pigs

Tibetan pigs are important traditional Chinese pig breeds,which are known for its strong survivability and resistance to stress.In addition,Tibetan pigs are also good materials for studying the adaptability of mammals living at high altitudes.Previous research has drawn a relatively complete molecular genetic landscape related to the environmental adaptation of Tibetan pigs at the DNA,protein-coding genes and the miRNA level.LncRNA are a kind of non-coding RNAs with important gene regulatory functions that play important roles in a variety of biological processes.However,the regulatory role of lncRNA in the adaptation of Tibetan pigs remains to be further explored.In this study,two population of adult female pigs（2 years）,which living in high altitude areas and low altitude areas were studied,and the longissimus dorsi muscles of the two groups were sequenced by lncRNA-seq.Differentially expressed lincRNAs were identified by comparing the sequencing data of the two groups.After functional analysis of differentially expressed lincRNAs,one antisense lncRNA（TCONS₀0039686）with potentially important functions were screened for subsequent regulatory function analysis.The results of this study are as follows:（1）LncRNA analysis showed that 7 lincRNAs were differentially expressed,with 4 lincRNAs were up-regulated at high altitude and 3 lincRNAs were up-regulated in lowaltitude groups.Cis and trans analysis showed that the lincRNA up-regulated in the highaltitude group was mainly associated with transmembrane transport and ion transport,and the lincRNA up-regulated in the low-altitude group was mainly associated with immune response.（2）Comparing the results of functional analysis of each differentially expressed lincRNA,the trans results showed that TCONS₀0039686 had the highest proportion of differentially expressed genes among the co-expressed protein-coding genes.Cis analysis showed that TCONS₀0039686 also had cis-regulated basis,and complemented with the energy metabolism-related gene（PLA2G16）,which indicated that TCONS₀0039686 was a potential antisense lncRNA.Considering this,choose the candidate lincRNA as a follow-up study.（3）The result of strand-specific reverse transcription followed with PCR analysis showed that TCONS₀0039686 was an reliable antisense lncRNA of PLA2G16 gene,so we named TCONS₀0039686 as PLA2G16-AS.In addition,sequence conservation analysis of PLA2G16-AS revealed that the sequence of porcine PLA2G16 AS was less conservative.（4）Quantification of PLA2G16-AS and PLA2G16 showed that the expression pattern of PLA2G16-AS and PLA2G16 were similar,and the correlation analysis showed that the expression levels of the two were highly positively correlated.（5）Knockdown or overexpression of PLA2G16-AS in PK-15 cells showed that the expression of PLA2G16 mRNA was significantly up-regulated after overexpression of PLA2G16-AS,and significantly down-regulated after interfere the expression of PLA2G16-AS.（6）Endogenous ribonuclease protection assay showed that PLA2G16 and PLA2G16-AS could form RNA duplexes.In addition,mRNA stability analysis showed that overexpression of PLA2G16-AS increased the stability of PLA2G16 mRNA,indicating that PLA2G16 AS may increase the stability of PLA2G16 mRNA by form a duplex with it,thereby regulating the expression level of PLA2G16 in pigs.In summary,there were differences in the expression levels of whole-genome lincRNA between the two groups of Tibetan pigs,and these changes were related to the environmental adaptation of Tibetan pigs.PLA2G16-AS may affect the energy metabolism of the longissimus dorsi muscle by regulating the expression of PLA2G16 gene,and help Tibetan pigs to better adapt to the environment.