Fig. 4. Hypoxia inhibits LINC00671 expression and activates LDHA expression largely through STAT3 transcription.

A qRT-PCR and Western blot analysis of TPC-1 and BCPAP cells exposed to normoxia (0 h) or hypoxia at different times (0.5, 2, 6 h). The representative immunoblot shows the expression of LDHA and HIF-1α. β-actin was used as a loading control. The expression level of LINC00671 was determined by qRT-PCR. B JASPAR (http://jaspar.genereg.net/) predicts that the STAT3 binding element on the LINC00671 promoter is conserved. TPC-1 cells transfected with different LINC00671 constructs or empty vector and exposed to normoxia or hypoxia, the luciferase activity of the different LINC00671 promoter-reporter genes was measured. The solid circle shows the position of the putative STAT3 binding site, and the “X” shows the mutated STAT3 binding site. The red letters in each binding region indicate a putative STAT3 binding sequence or a mutated STAT3 binding sequence. C qRT-PCR analysis of LINC0071 expression in TPC-1 cells transfected with EV, STAT3, and STAT3 (Y705F), and exposed to normoxia or hypoxia condition. A representative immunoblot shows the expression of LDHA and HIF-1α. β-actin was used as an internal control. D qRT-PCR analysis of LINC00671 expression in TPC-1 cells treated with Niclocide with or without hypoxia. E ChIP analysis of LINC00671 promoter or STAT3 occupancy rate upstream of the promoter in TPC-1 cells under normoxia or hypoxia. F qRT-PCR analysis of LINC00671 expression in TPC-1 cells transfected with LINC00671 siRNAs, treated with Niclocide, and exposed to hypoxia or not. The representative immunoblot shows the expression of LDHA, HIF-1α, and pSTAT3 (Y705). β-actin was used as a loading control. The displayed values are the mean ± standard deviation. The triplicate measurement results were repeated 3 times and the results were similar. *P < 0.05, **P < 0.01, relative to the corresponding empty vector. ^##P < 0.01 versus the corresponding empty carriers under hypoxic conditions.