br in SCLC progression HDLBP was originally reported
in SCLC progression. HDLBP was originally reported to be involved in cholesterol-level regulation, but its relationship with cancer has been recently paid attention (e.g. it was found to be overexpressed in liver cancer tissue but downregulated in breast cancer tissue) with unclear mechanism [13,14]. Here we found HDLBP was overexpressed in SCLC tissues for the first time. Functional and mechanistic studies revealed that inhibiting the expression of HDLBP could arrest tumor Vaborbactam in G0/ G1 phase and suppress SCLC progression in vitro and in vivo. Therefore, the new function of HDLBP in SCLC progression is demonstrated. Since the effective HDLBP protein inhibitor has also been reported , we expect HDLBP could be a druggable SCLC biomarker. This work pro-vided a novel and promising strategy for the development of SCLC targeted therapy.
2.1. Target protein identification and mass spectrometry analysis
Four aptamers (C1, C3, C7 and C12) generated by cell-SELEX with high binding specificity to SCLC cells were employed for tumor marker discovery . To enrich the protein targets of these aptamers, bioti-nylated aptamers were incubated with the lysate of a typic SCLC cell, NCI-H446. The aptamer-protein complexes were extracted using streptavidin-coated beads. After thorough washing, the binding pro-teins were denatured and subjected to SDS-PAGE gel electrophoresis and Coomassie Blue staining,.Then the specific protein bands were applied for further MS analysis, a general method used for aptamer targets identification [9–12,16]. To rule out possible nonspecific pro-teins in MS analysis of this aptamer based affinity enrichment, we in-troduced two ssDNA oligomers as negative controls, that are RTT, a thymine rich ssDNA (AATTTTTTAATTATTTATATTA) [17,18], and a Random sequence (24nt ssDNA). Then we gave preference to those protein bands that only presented with one or two aptamers for further MS identification. Such a design ensures the protein band used for MS was specific for the aptamer. Compared with control ssDNA sequences, a total of 6 bands specifically enriched by SCLC aptamers were dis-covered and then treated with trypsin digestion for further mass spec-trometry analysis (Fig. 1A). While several different proteins were de-tected by MS in one specific protein band, we checked those top 5 proteins ranked in the list generated according to the MS score and protein coverage since this could reflect the protein abundance in the band.
Among the MS-produced protein hits, HDLBP in band 2 received substantial attention for a number of reasons. First of all, the molecular weight of HDLBP (130 kDa) was in well accordance with the SDS-PAGE result. Second, the MS score of HDLBP ranked the first in the MS protein hits of band 2. Third, the expression of HDLBP have been previously reported to elevate in highly proliferating ovarian cancer cell line TOV-112D and gastric carcinoma cell, suggesting its possible role in tumor.
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However, the abnormal expression of HDLBP was only found in liver cancer and breast cancer with clinical samples [13,14]. The expression pattern and clinical significance of HDLBP in lung cancer, including SCLC remains unknown. Last, Tashiro’s group recently reported that Aralin, a natural polypeptides isolated from Aralia elata, was a selective cytotoxic lectin against some cancer cells, including SV40-transformed human lung fibroblast cells (VA-13), cells from cervical cancer cell line (Hela), leukemia cell line (HL-60), liver cancer line (Huh7) in vitro, and Hela cell derived mice tumor in vivo [19,20]. Further target identifi-cation worksuggested the cell membrane located HDLBP was the binding target of Aralin . In this respect, HDLBP would be a pro-mising therapeutic target in tumor therapy. All these facts led us to concentrate our further verification work on HDLBP in SCLC.