Lentivirus-mediated disintegrin and metalloproteinase 17 RNA interference reversed the acquired resistance to gefitinib in lung adenocarcinoma cells in vitro.

OBJECTIVE: The aim of the study is to evaluate the effects of silencing a disintegrin and metalloproteinase 17 (ADAM17) gene expression by lentivirus-mediated RNA interference (RNAi) in the gefitinib-resistant lung adenocarcinoma cells, and then to explore whether the recombinant lentivirus mediated ADAM17 RNAi reversed the acquired resistance of lung adenocarcinoma to gefitinib in vitro. METHODS: The gefitinib-resistant RPC-9 cells were established and the mutations of EGFR were detected by gene sequencing. The ADAM17 shRNA expression vectors were constructed and packaged to recombinant lentivirus. The cell proliferation viability was detected by MTT, and cellular apotosis was analyzed by flow cytometry assay. The expression levels of ADAM17, EGFR and the phosphorylated EGFR were respectively detected by reverse transcription polymerase chain reaction and western blot. TGF-α production in the supernatant was detected by enzyme-linked immunosorbent assay. RESULTS: The gefitinib-resistant RPC-9 cells in which mutated EGFR (exon 20) carried 790T > T/M mutation were established. When the concentrations of gefitinib were less than 10µmol/L, there were no significant changes in the apoptosis and cellular proliferation of RPC-9 with the dose-escalation of gefitinib. The cell proliferation viability of RPC-9 was significantly decreased by lentivirus mediated ADAM17 RNAi (P < 0.05). Gefitinib did not inhibit ADAM17 expression in both the gefitinib-sensitive PC-9 and gefitinib-resistant RPC-9 cells (P > 0.05). Gefitinib had no significant effects on TGF alpha production in the supernatants (P > 0.05). Gefitinib did not inhibit EGFR expression in gefitinib-sensitive PC-9 and gefitinib-resistant RPC-9 cells (P > 0.05). The phosphorylation of EGFR in gefitinib-sensitive PC-9 cells was significantly inhibited by gefitinib (P < 0.05), but that in gefitinib-resistant RPC-9 could not be inhibited by gefitinib (P > 0.05). Lentivirus mediated ADAM17 RNAi significantly inhibited the mRNA and protein expression of ADAM17 in gefitinib-resistant RPC-9 cells (P < 0.05), as well as TGF alpha production in the supernatants (P < 0.05). Also, the phosphorylation of EGFR was significantly reduced in gefitinib-resistant RPC-9 cells by lentivirus mediated ADAM17 RNAi (P < 0.05); however, the mRNA and protein expression of EGFR could not be inhibited. CONCLUSION: Lentivirus mediated ADAM17 RNAi may reverse the acquired resistance of lung adenocarcinoma to gefitinib via inhibiting the upstream of EGFR signal pathway, which may provide a new therapeutic target to solve the acquired resistance to EGFR tyrosine kinase inhibitors in lung adenocarcinoma. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:196-205, 2018.

Overexpression of eukaryotic translation initiation factor 4E-binding protein 1 induces the alteration of immune status in H1299 lung cancer cells.

BACKGROUND: Eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) is an important factor regulating protein translation. It also impacts proliferation, apoptosis, invasion, and the cell cycle of cancer cells. The aim of this study was to investigate the relationship between 4E-BP1 and human immune status, recognizing immunomodulatory molecules involved in the overexpression of 4E-BP1. METHODS: A lentivirus expression system was used to overexpress 4E-BP1 in the H1299 cell line. Western blot was performed to investigate the expression level of 4E-BP1 and P-4E-BP1, and quantitative polymerase chain reaction was used to quantify gene expression of immunomodulatory molecules. RESULTS: The expression level of 4E-BP1 increased significantly after lentivirus infection (P < 0.05). Overexpression of 4E-BP1 upregulated the expression of interleukin (IL)-1ß (P < 0.05), IL-5 (P < 0.001), IL-23 (P < 0.001), macrophage inflammatory protein-1ß (P < 0.001), Eota-3 (P < 0.05), and MCP-4 (P < 0.05). Most of the increases were observed at the seventh day. The variation trend of IL-10, cell division cycle protein 2, proliferating cell nuclear antigen, and phosphatase and tensin homolog was not clear. CONCLUSION: Overexpression of 4E-BP1 altered immune status by upregulating the expression of a series of immunomodulatory molecules, indicating that 4E-BP1 could serve as a potential therapeutic target against cancer.

Genetic analysis of 17 Y-chromosomal STR loci of Chinese Tujia ethnic group residing in Youyang Region of Southern China.

Y-STR haplotype data were obtained in a population sample of 197 unrelated healthy male individuals of Chinese Tujia ethnic minority group residing in an autonomous county of Southern China using 17 Y-chromosome STR markers. A total of 197 haplotypes were identified in the set of Y-STR loci. The overall haplotype diversity for the Tujia population at 17 Y-STR loci was 1.0000±0.0005. Genetic distance was estimated between this population and other 14 Chinese populations including Paiwan and Atayal populations of Taiwan, and Southern Han, Dong, Jing, Miao, Yao, Zhuang, Yi, Maonan, She, Hui, Sala, and Tibetan ethnic groups. The results demonstrated that the 17 Y-STR loci analyzed were highly polymorphic in Tujia ethnic group examined and hence useful for forensic cases, paternity testing, and population genetic studies.