Organic anion transporting polypeptides (OATPs) have been extensively recognized as key determinants of absorption, distribution, metabolism and excretion (ADME) of various drugs, xenobiotics and toxins. Putative N-glycosylation sites located in the extracellular loops 2 and 5 is considered a common feature of all OATPs and some members have been demonstrated to be glycosylated proteins. However, experimental evidence is still lacking on how such a post-translational modification affect the transport activity of OATPs and which of the putative glycosylation sites are utilized in these transporter proteins. In the present study, we substituted asparagine residues that are possibly involved in N-glycosylation with glutamine residues and identified three glycosylation sites (Asn134, Asn503 and Asn516) within the structure of OATP1B1, an OATP member that is mainly expressed in the human liver. Our results showed that Asn134 and Asn516 are used for glycosylation under normal conditions; however, when Asn134 was mutagenized, an additional asparagine at position 503 is involved in the glycosylation process. Simultaneously replacement of all three asparagines with glutamines led to significantly reduced protein level as well as loss of transport activity. Further studies revealed that glycosylation affected stability of the transporter protein and the unglycosylated mutant was retained within endoplasmic reticulum.

Resistance to therapeutic drugs is a major challenge in the treatment of cancers, including breast cancer. Long noncoding RNAs (lncRNA) are known to have diverse physiologic and pathophysiologic functions, including in cancer. In searching for lncRNA responsible for cancer drug resistance, we identified an intergenic lncRNA ERINA (estrogen inducible lncRNA) as a novel lncRNA highly expressed in multiple cancer types, especially in estrogen receptor–positive (ER+) breast cancers. Expression of ERINA was inversely correlated with survival of patients with ER+ breast cancer and sensitivity to CDK inhibitor in breast cancer cell lines. Functional characterization established ERINA as an oncogenic lncRNA, as knockdown of ERINA in breast cancer cells inhibited cell-cycle progression and tumor cell proliferation in vitro and xenograft tumor growth in vivo. In contrast, overexpression of ERINA promoted cell growth and cell-cycle progression. ERINA promoted cell-cycle progression by interacting with the E2F transcription factor 1 (E2F1), which prevents the binding of E2F1 to the tumor suppressor retinoblastoma protein 1 (RB1). ERINA also functioned as an estrogen and ER-responsive gene, and an intronic ER-binding site was identified as an enhancer that mediates the transactivation of ERINA. In summary, ERINA is an estrogen-responsive oncogenic lncRNA that may serve as a novel biomarker and potential therapeutic target in breast cancer. Significance: These findings identify ERINA as an estrogen-responsive, oncogenic lncRNA, whose elevated expression may contribute to drug resistance and poor survival of patients with ER+ breast cancer.

As an important structure in membrane proteins, transmembrane domains have been found to be crucial for properly targeting the protein to cell membrane as well as carrying out transport functions in transporters. Computer analysis of OATP sequences revealed transmembrane domain 2 (TM2) is among those transmembrane domains that have high amino acid identities within different family members. In the present study, we identify four amino acids (Asp70, Phe73, Glu74, and Gly76) that are essential for the transport function of OATP1B1, an OATP member that is specifically expressed in the human liver. A substitution of these four amino acids with alanine resulted in significantly reduced transport activity. Further mutagenesis showed the charged property of Asp70 and Glu74 is critical for proper function of the transporter protein. Comparison of the kinetic parameters indicated that Asp70 is likely to interact with the substrate while Glu74 may be involved in stabilizing the binding site through formation of a salt-bridge. The aromatic ring structure of Phe73 seems to play an important role because substitution of Phe73 with tyrosine, another amino acid with a similar structure, led to partially restored transport function. On the other hand, replacement of Gly76 with either alanine or valine could not recover the function of the transporter. Considering the nature of a transmembrane helix, we proposed that Gly76 may be important for maintaining the proper structure of the protein. Interestingly, when subjected to transport function analysis of higher concentration of esteone-3-sulfate (50 µM) that corresponds to the low affinity binding site of OATP1B1, mutants of Phe73, Glu74, and Gly76 all showed a transport function that is comparable to that of the wild-type, suggesting these amino acids may have less impact on the low affinity component of esteone-3-sulfate within OATP1B1, while Asp 70 seems to be involved in the interaction of both sites.

Inhibiting apoptosis of type II alveolar epithelial cells (AEC II) is an effective way to decrease hyperoxic acute lung injury (HALI); however, the specific underlying molecular mechanisms have not yet been fully elucidated. Although miRNA‑21‑5p has previously been reported to decrease H2O2‑induced AEC II apoptosis by targeting PTEN in vitro, whether miR‑21‑5p can decrease HALI in vivo and the downstream molecular mechanisms remain unclear. In the present study, rats were endotracheally administered with an miR‑21‑5p‑encoding (AAV‑6‑miR‑21‑5p) or a negative control adenovirus vector, and then a HALI model was established by exposure to hyperoxia. At 3 weeks following the administration of AAV‑6‑miR‑21‑5p, the severity of HALI was decreased, as evidenced by the improved outcome of the oxygenation index, respiratory index, wet/dry weight ratio and pathological scores of the HALI lungs. To further investigate the underlying mechanisms, AEC II cells were isolated from the lungs of the experimental rats and cultured. The expression levels of miR‑21‑5p and its target gene, PTEN, were detected, as well as the levels of phosphorylated and total AKT. In addition, the apoptosis rate of AEC II was detected by flow cytometry. The results demonstrated that AAV‑6‑miR‑21‑5p administration increased the miR‑21‑5p levels in primary AEC II cells, while it decreased the expression levels of PTEN. miR‑21‑5p overexpression also increased AKT phosphorylation in AEC II cells from the HALI lungs compared with that of the HALI alone group and the control virus group. The present study indicated that miR‑21‑5p ameliorated HALI in vivo, which may have resulted from the inhibition of PTEN/AKT‑induced apoptosis of AEC II cells. These findings suggest that miR‑21‑5p and PTEN/AKT signaling might serve as potential targets for HALI treatment.

The organic anion-transporting polypeptides (OATPs, gene symbol SLCO) are a family of transporters that play important roles in the absorption, distribution, metabolism, and excretion of various drugs. Although substrate specificity of transporter proteins is under extensive study, the underlying mechanisms for substrate binding and/or recognition remain largely unknown. Transmembrane domain 6 (TM6) is a relatively conserved region within OATP family members, and several amino acid residues on its extracellular half are part of the OATP family signature sequence D-X-RW-(I,V)-GAWWX-G-(F,L)-L. In the present study, two adjacent tryptophan residues (Trp258 and Trp259) within TM6 were identified as critical amino acids for the transport function of OATP1B1. Kinetic studies showed that substitution of Trp258 with alanine resulted in monophasic kinetics for estrone-3-sulfate uptake, with a significantly higher K_m value (K_m = 12.0 ± 2.8 μM) than the high-affinity component of wild-type OATP1B1 (K_m = 0.38 ± 0.06 μM). On the other hand, W259A retained the biphasic characteristic of the transporter. K_m values of the high- and low-affinity components for estrone-3-sulfate of W259A are 1.93 ± 0.76 μM and 30.8 ± 4.4 μM, respectively. Further studies revealed that W258A retained transport function of another prototypic substrate, taurocholate, while W259A displayed a dramatically reduced uptake of the substrate and exhibited an 8-fold increase in the K_m value compared with that of the wild-type and W258A. Our results suggest that Trp258 and Trp259 may play different roles in the uptake of different substrates by OATP1B1.