Beta-actin was detected as the endogenous control for (ISOC1 OE) and miR-4633 stably overexpressed NSCLC cell lines, the full-length of and miR-4633 were amplified from complementary DNA (cDNA) and genomic DNA (gDNA) from the NSCLC cell line A549 by PCR and were cloned into the pCDH-CMV-MCS-EF1-GFP+Puro (CD513B) plasmid (System Biosciences, USA)
Beta-actin was detected as the endogenous control for (ISOC1 OE) and miR-4633 stably overexpressed NSCLC cell lines, the full-length of and miR-4633 were amplified from complementary DNA (cDNA) and genomic DNA (gDNA) from the NSCLC cell line A549 by PCR and were cloned into the pCDH-CMV-MCS-EF1-GFP+Puro (CD513B) plasmid (System Biosciences, USA). DNA damage lesions. RNA sequencing analysis showed that the downstream signaling pathways mediated by were mainly inflammation-related. Conclusions We demonstrated that and its intronic miR-4633, both of them could promote NSCLC cell proliferation, migration, invasion, and cell cycle progression. participates in DNA damage repair and inflammation to promote lung cancer development. gene is located on chromosome 5q23.3 and encodes a protein of 298 amino acids. Previous studies have reported that is related to eye and testis development in animal models (5,6). has been reported to be involved in neutrophil development regulated by microRNA-130A (miRNA-130A) in mice and humans (7). In addition to the above functions, was also found to play important human tumor biology roles. For instance, was identified to be deregulated in uterine leiomyoma (8) and was also proven to act as an estrogen-responsive gene, promoting breast cancer cells growth (9). Two recent studies revealed that knockdown of could impair tumorigenesis and gastrointestinal cancer progression through activating protein kinase B (Akt)/glycogen synthase kinase-3 beta (GSK-3) pathway (10,11). However, the role of in Diclofenac sodium lung carcinogenesis is still unknown. MicroRNAs (miRNAs) commonly lead to messenger RNA (mRNA) degradation and translation inhibition via binding to specific sequences. A number of miRNAs have been reported to be dysregulated in NSCLC and lead to the dysfunctions of oncogenic-related pathways (12). Intronic miRNAs, which are located on the intron of the host gene, can mediate and/or assist the host gene in performing various biological functions (13). The first intron of encodes intronic miR-4633, was reported to be involved in the lung fibrosis mediated by transforming growth factor beta (TGF-) and to contribute to the antitumor activity in sinonasal mucosal melanoma (14,15). Currently, Diclofenac sodium limited knowledge on miR-4633 exists, and its function in NSCLC remains to be explored. This study was a functional investigation of two lung cancer cell lines, A549 and H1299, with the aim to explore the roles of in the development and progression of NSCLC. Co-immunoprecipitation (Co-IP) combined with mass spectrometry (MS) and RNA sequencing were performed to uncover the potential mechanism of in lung cancer development. We present the following article in accordance with the ARRIVE reporting checklist (available at Diclofenac sodium http://dx.doi.org/10.21037/tlcr-21-219). Methods Cell culture Human NSCLC cell lines A549 and NCI-H1299, and human embryonic kidney cell line, HEK-293/HEK-293T, were preserved in the lab of Thoracic Surgery of Shanghai Pulmonary Hospital. NSCLC cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco). HEK-293/HEK-293T cells were grown in complete Dulbeccos Modified Eagles Medium (DMEM) Diclofenac sodium supplemented with 10% FBS. All cells were maintained at 37 C in a humidified incubator with 5% CO2. RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) RAD26 Total RNA was extracted from cultured cells which were harvested with TRIzol reagent (Invitrogen, USA) to quantify the expression of relevant genes. The reverse transcription reactions were carried out according to the manufacturers protocol of commercially available kits (Vazyme, China). qRT-PCR was performed on a LightCycler 96 (Roche, Switzerland) and detected with THUNDERBIRD SYBR qPCR Mix (Toyobo, Japan). The primer sequences used in this study are listed in Table S1. The qPCR conditions were 95 C for 3 min, and then 40 cycles at 95 C for 15 s and 60 C for 45 s. The differences in mRNA expression were compared using the comparative cycle threshold (Ct) method. Beta-actin was detected as the endogenous control for (ISOC1 OE) and miR-4633 stably overexpressed NSCLC cell lines, the full-length of and miR-4633 were amplified from complementary DNA (cDNA) and genomic DNA (gDNA) from the NSCLC cell line A549 by PCR and were cloned into the pCDH-CMV-MCS-EF1-GFP+Puro (CD513B) plasmid (System Biosciences, USA). To stably interfere with expression, the short hairpin RNA (shRNA) sequences Diclofenac sodium targeting were designed and cloned into the plasmid LentiLox 3.7 (pLL3.7) to knock down the expression of (ISOC1 KD). The CD513B empty vector (EV).