Hypoxia induced LUCAT1/PTBP1 axis modulates cancer cell viability and chemotherapy response

Background Hypoxic tumors are refractory to DNA damage drugs. However, the underlying mechanism has yet to be elucidated. We aimed to identify lncRNAs that upregulated under hypoxia and their effects on colorectal cancer (CRC). Methods CRC cells were treated with 1% O2 to identify lncRNAs that upregulated under hypoxia. We integrated these lncRNAs with RNA-seq of 4 paired CRC tissues and TCGA data to get candidate lncRNAs. Multiple in vitro and in vivo assays were used to explore the role of LUCAT1 in CRC. Results We identified a hypoxia-induced lncRNA LUCAT1 that facilitated the growth of CRC cells and contributed to drug resistance of CRC cells both in vitro and in vivo. Mechanically, LUCAT1 interacts with polypyrimidine tract binding protein 1 (PTBP1) in CRC cells, facilitates the association of a set of DNA damage related genes with PTBP1, thus resulting in altered alternative splicing of these genes. Moreover, ectopic expression of PTBP1 in CRC cells with knockdown of LUCAT1 abrogated the effects induced by LUCAT1 knockdown. Chemotherapeutics drug combined with LUCAT1 knockdown via antisense oligonucleotides (ASO) would get a better outcome in vivo, compared with group treated with chemotherapeutic drug only. Notably, LUCAT1 is upregulated in CRC tissues, compared to adjacent normal tissues; and CRC patients with higher LUCAT1 have a worse prognosis and poorly responded to chemotherapy in the clinic. Conclusions Our data suggested CRC cells utilizes LUCAT1 to develop resistance to DNA damage drugs, and disrupting the LUCAT1/PTBP1 axis might be a promising therapeutic strategy for refractory hypoxic tumors.


Lentivirus Production and Infection
The LUCAT1 fragment was cloned into the pCDH-Puro lentiviral vector, and the ORF region of PTBP1 was cloned into the pCDH-3×Flag lentiviral vector. shRNA sequences were synthesized and cloned into the LentiGuide-Puro lentiviral vector. The empty pCDH-Puro or LentiGuide-Puro vectors were used was control. The primers used in this study are listed in Table S8. To generate lentiviruses, the lentiviral vector, packaging plasmid (pAX2), and VSV-G envelope plasmid (pMD2.G) were cotransfected into HEK-293T cells using Lipofectamine 2000 (Invitrogen, CA, USA). The pAX2 and pMD2.G plasmids were gifts from Dr. Didier Trono (Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland). Supernatants were collected 48 h after transfection and used to infect the corresponding CRC cells.

Northern Blot Analysis
The LUCAT1 probe template was amplified by PCR (Forward Primer:

Luciferase Assay
The HIF-1α binding HRE was amplified from HCT-116 cell genomic DNA and was then subcloned into the pGL3.0-basic vector. The primers were shown in Table S8. CRC cells were seeded into 96-well plates at a density of 5000 cells per well. After 24 h, 5 ng of pRL-TK, 100 ng of pGL3.0-basic or pGL3.0-basic-HRE, and 5 pmol of NC or HIF-1α siRNA were transfected into each well of a 96-well plate. The next day, the corresponding wells were treated overnight with 100 μM CoCl2. Firefly and Renilla luciferase activities were measured using the dual-luciferase reporter assay system (Promega, WI, USA).

Chromatin Immunoprecipitation (ChIP) Assay
Cultured cells were crosslinked using 1% formaldehyde. Crosslinking was terminated by adding glycine to a final concentration of 0.125 M. Cells were scraped off the dish, collected into a fresh 1.5-mL tube, and resuspended in ChIP lysis buffer supplemented with proteinase inhibitor (Bimake, Shanghai, China). Chromatin was sheared into 200~1000-bp fragments by sonication at the proper conditions. IgG or HIF-1α antibody was added to Protein A/G magnetic beads (Bimake, Shanghai, China) and rotated at room temperature. After 30 min, the chromatin mixture was added to the beads and the sample was rotated at 4℃ overnight. Then the tube was subjected to a magnetic field to remove the supernatant, which contained nonspecific fragments. The beads were washed 4 times and then eluted using MinElute Spin Columns (Qiagen, Hilden, Germany). The primers used in the ChIP assay are listed in Table S9.

Western Blot Analysis
Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (GE, CT, USA). The membranes were blocked with nonfat milk and then probed with primary antibodies overnight at 4℃.
Membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Immune complexes were detected using LumiBest ECL Reagent Solution Kit (Share-Bio, Shanghai, China).
Antibodies used in this study are listed in Table S10.

Cell Cycle Analysis
Cells were seeded in six-well plates, deprived of serum overnight for synchronization, and then released from synchronization by adding complete medium. After 12 h, cells were collected and fixed with 70% ethanol. Prior to flow cytometric analysis, fixed cells were treated with ribonuclease A (TaKaRa, Tokyo, Japan) for 30 min at 37℃ and then stained with propidium iodide (PI) (Sangon Biotech, Shanghai, China) for 15 min at room temperature.

RNAscope Assay
The LUCAT1  To explore the sensitivity of cells to each drug, 8000 cells were seeded into each well of a 96-well plate. The appropriate concentration of drug was added to each well. After 48 h, cell viability was determined using the CCK8 assay.

Caspase 3/7 Assay
Caspase 3/7 assay was conducted using the Caspase-Glo 3/7 Assay System (Promega, WI, USA) according to the manufacturer's instructions. Briefly, 5000 cells were seeded in each well of a 96-well plate one day prior to treatment. Then, siRNAs were transfected into the cells. After 48 h, the medium was removed, and proluminescent substrates were added to each well in the dark. The luminescence of each sample was measured using a multimode reader.

RNA Pull Down
Cells in dishes were scraped and collected in tubes by centrifugation. This sample preparation was subjected to mass spectrometry analysis or western blotting.

RNA Immunoprecipitation (RIP) Assay
Cells grown in dishes were crosslinked with ultraviolet light and then collected in an RNase-free tube. An equal volume of RIP lysis buffer supplemented with RNase and proteinase inhibitors was added to the tube.
The samples were stored at -80℃ before use. The supernatant was collected by centrifugation for 20 min at 10,000×g at 4℃. Anti-IgG, PTBP1 or STAU1 antibodies were added to Protein A/G magnetic beads and rotated at room temperature for 30 min, respectively. Then, unlabeled antibodies were removed from the beads. Next, 100 μl of supernatant was                The expression level of LUCAT1 in RKO-parental and RKO-OXA was determined by qPCR. n=3 independent experiments, two-tailed Student's t-test. * p < 0.05, ** p < 0.01, and *** p < 0.001. Cohort 1 4 4 RNA-seq in Figure 1A Cohort 2 46 NA Figure S2E, Figure 4H Cohort CRC 97 97 Figure 7A and 7B