Objective:To explore the role of long non-coding RNA (LncRNA) AC000061.1 involved in the pathogenesis of nonobstructive azoospermia (NOA) by regulating the CFTR gene expression. Methods:Bioinformatics analysis was conducted with the use of gene microarray to screen the differentially expressed LncRNAs and corresponding mRNAs in the testicular tissue of patients in three groups including obstructive azoospermia (OA) group ( n=50), NOA group ( n=50), and control group ( n=50). The expression of apoptosis-related gene Bcl-2 in the testicular tissue of patients in these three groups was detected by qRT-PCR and Western blotting. Additionally, qRT-PCR was performed to determine the expression of LncRNA AC000061.1 and CFTR mRNA in the testicular tissue, serum, and seminal plasma of patients in the three groups, and enzyme-linked immunosorbent assay (ELISA) was conducted to detect the CFTR protein level in the serum and seminal plasma. LncRNA AC000061.1 overexpression (H-LncRNA group) and silencing vectors (Si-LncRNA group) and empty vectors were constructed and transfected into the testicular cancer cell line NTERA-2. Subsequently, the expression of LncRNA AC000061.1 and CFTR mRNA was determined by qRT-PCR, and CFTR protein expression was measured by Western blotting assay. Cell proliferation was assessed using CCK-8 and cell apoptosis was evaluated by flow cytometry and TUNEL. Results:Microarray analysis and qRT-PCR showed that the expression of LncRNA AC006100.1 and CFTR decreased in NOA group compared with control group ( P=0.033 and P=0.042). But there was no difference in the expression of LncRNA AC000061.1 between OA group and control group, while CFTR in OA group was lowly expressed compared with that in control group ( P=0.039). Bcl-2 expression was sequentially upregulated in OA and NOA groups relative to normal control group ( P=0.031 and P=0.008). No significant difference was noted in the serum levels of LncRNA AC000061.1 and CFTR mRNA and protein among the three groups ( P>0.05). The results also showed sequentially decreased levels of LncRNA AC000061.1 and CFTR mRNA and protein in seminal plasma in NOA and OA groups when compared with control group ( P=0.002, P=0.038 and P=0.006, P=0.026) and a positive correlation of LncRNA AC000061.1 expression with CFTR mRNA expression ( r=0.169, P=0.039). LncRNA AC000061.1 and CFTR mRNA and protein expression decreased in Si-LncRNA group ( P=0.005, P=0.003), but significantly increased in H-LncRNA group ( P=0.002, P=0.009). Furthermore, cell proliferation was significantly repressed while apoptosis rate was elevated in Si-LncRNA group ( P=0.003 and P=0.001). On the contrary, enhanced cell proliferation and inhibited apoptosis were observed in H-LncRNA group ( P=0.017 and P=0.017). Conclusion:LncRNA AC00006.1 in the testicular tissue of NOA patients induced abnormal cell proliferation and apoptosis via mediating CFTR gene expression, thereby participating in the pathogenesis of NOA.

Objective:To explore the role of long non-coding RNA (LncRNA) AC000061.1 involved in the pathogenesis of nonobstructive azoospermia (NOA) by regulating the CFTR gene expression. Methods:Bioinformatics analysis was conducted with the use of gene microarray to screen the differentially expressed LncRNAs and corresponding mRNAs in the testicular tissue of patients in three groups including obstructive azoospermia (OA) group ( n=50), NOA group ( n=50), and control group ( n=50). The expression of apoptosis-related gene Bcl-2 in the testicular tissue of patients in these three groups was detected by qRT-PCR and Western blotting. Additionally, qRT-PCR was performed to determine the expression of LncRNA AC000061.1 and CFTR mRNA in the testicular tissue, serum, and seminal plasma of patients in the three groups, and enzyme-linked immunosorbent assay (ELISA) was conducted to detect the CFTR protein level in the serum and seminal plasma. LncRNA AC000061.1 overexpression (H-LncRNA group) and silencing vectors (Si-LncRNA group) and empty vectors were constructed and transfected into the testicular cancer cell line NTERA-2. Subsequently, the expression of LncRNA AC000061.1 and CFTR mRNA was determined by qRT-PCR, and CFTR protein expression was measured by Western blotting assay. Cell proliferation was assessed using CCK-8 and cell apoptosis was evaluated by flow cytometry and TUNEL. Results:Microarray analysis and qRT-PCR showed that the expression of LncRNA AC006100.1 and CFTR decreased in NOA group compared with control group ( P=0.033 and P=0.042). But there was no difference in the expression of LncRNA AC000061.1 between OA group and control group, while CFTR in OA group was lowly expressed compared with that in control group ( P=0.039). Bcl-2 expression was sequentially upregulated in OA and NOA groups relative to normal control group ( P=0.031 and P=0.008). No significant difference was noted in the serum levels of LncRNA AC000061.1 and CFTR mRNA and protein among the three groups ( P>0.05). The results also showed sequentially decreased levels of LncRNA AC000061.1 and CFTR mRNA and protein in seminal plasma in NOA and OA groups when compared with control group ( P=0.002, P=0.038 and P=0.006, P=0.026) and a positive correlation of LncRNA AC000061.1 expression with CFTR mRNA expression ( r=0.169, P=0.039). LncRNA AC000061.1 and CFTR mRNA and protein expression decreased in Si-LncRNA group ( P=0.005, P=0.003), but significantly increased in H-LncRNA group ( P=0.002, P=0.009). Furthermore, cell proliferation was significantly repressed while apoptosis rate was elevated in Si-LncRNA group ( P=0.003 and P=0.001). On the contrary, enhanced cell proliferation and inhibited apoptosis were observed in H-LncRNA group ( P=0.017 and P=0.017). Conclusion:LncRNA AC00006.1 in the testicular tissue of NOA patients induced abnormal cell proliferation and apoptosis via mediating CFTR gene expression, thereby participating in the pathogenesis of NOA.

Objective To explore the effects of different methods of antigen retrieval on the results of immunohistochemical staining of nuclear receptors. Methods Antigens were retrieved by using microwave oven, autoclave, enzyme and autoclave plus enzyme, respectively, in liver sections from DA.1U rats, followed by immunohistochemical staining. Results After antigen retrieval with autoclave, the positive staining intensity of nuclear receptors LXR-α, LXR-β, PPAR-γ and FXR in the liver sections from DA.1U rats was enhanced obviously, and the location of nuclear receptors was better by using this method than the others. Conclusion In the immunohistochemical staining of nuclear receptors, using autoclave to retrieve antigens is the best method.