Downregulation of HNRNPK in human cancer cells inhibits lung metastasis

Abstract Background Lung cancer frequently occurs in the clinic, leading to poor prognosis and high mortality. Markers for early diagnosis of lung cancer are scarce, and further potential therapeutic targets are also urgently needed. Method We established a new mouse model in which the specific gene HNRNPK (heterogeneous nuclear ribonucleoprotein K) was downregulated after administration of doxycycline. The lung metastatic nodules were investigated using bioluminescence imaging, micro‐CT, and autopsy quantification. Results Compared with the short hairpin negative control group, less lung metastatic nodules were formed in the short hairpin RNA group. Conclusion Downregulation of HNRNPK in cancer cells can inhibit lung metastasis.

DNA damage enables HNRNPK to be recruited to the promoter of the p53 downstream gene, thereby promoting the expression of p21, HDM2, C/EBPα, and C/EBPβ. Downregulation of HNRNPK reduces p53 transcription, leading to DNA damage-induced cell cycle arrest. 11 In addition, HNRNPK can bind to the promoter region and to the 3′ untranslated region of p21 messenger ribonucleic acid (mRNA), which inhibits translation of p21 and increases translation initiation, cell division, and tumor formation. 14,15 Although previous research has revealed some effects of HNRNPK in cancer and normal cells, the exact role of HNRNPK in lung cancers is still unknown and requires further investigation. We established a new human tumor cell line with reduced expression of HNRNPK. In vivo data showed that downregulation of HNRNPK significantly reduced the formation of metastatic lung tumor nodules. Notably, downregulation of HNRNPK was performed 1 week after tumor implantation by induction with doxycycline (DOX). This research paves the way for clinical applications targeting HNRNPK and improved therapies. Gibco), 1% penicillin-streptomycin (15140-122; Gibco), and 3 μg/mL puromycin in a cell culture incubator with 5% CO 2 at 37°C. To specifically downregulate the expression of HNRNPK, 5 μg/mL DOX (REVG1004; GeneChem Co. Ltd.) was used.

| Animals
Animals had free access to food and water and were maintained under controlled temperature, humidity, and light conditions. All procedures were approved by the Biomedical Ethical Committee of the Chinese Academy of Medical Sciences (GR17001). Six-to eight-week-old female BALB/C-nu mice were purchased from Beijing Huafukang Biotechnology. Six mice were used for each group. 1 × 10 6 A549-luc-H1TetO-shRNA-HNRNPK or A549-luc-H1TetO-shRNA-NC (negative control) cells were injected through the mouse tail vein. One week later, the mice were fed 200 ppm DOX (C11300-200i; Research Diets). In addition, mouse body weight was recorded weekly. Lung tumor masses were detected by bioluminescence imaging every week.
Six weeks later, all mice were killed, and the lungs were harvested to quantify the number of metastatic nodules.

| Reverse trancription polymerase chain reaction
Total RNA was extracted from cultured A549 cells in accordance with the in-  Table 1.
To avoid bias, each assay was performed in triplicate, and the relative gene expression was calculated by the comparative cycle threshold (2 −ΔΔC t ) method following the manufacturer's instructions.

| Western blot assay
First, cells were homogenized in radio-immunoprecipitation assay lysis buffer (R0010; Solarbio) containing 1% phenylmethanesulfonyl fluoride on ice for 30 minutes. Total protein concentrations were estimated using the Pierce bicinchoninic acid protein assay kit (23225; Thermo).
Then, equal amounts of cell lysates were loaded and separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Then, proteins were transferred to nitrocellulose membranes for 1 hour with a 300 mA current. Next, the membranes were incubated with a 1:1000 dilution of anti-HNRNPK antibody (ab52600; Abcam) and a 1:10 000 dilution of anti-β-actin antibody (ab49900; Abcam) overnight at 4°C with 5% nonfat milk, washed three times with tris-buffered saline tween 20 buffer, and then incubated with a 1:10 000 dilution of horseradish peroxidase-conjugated goat anti-rabbit Immunoglobulin G (ZDR-5307; ZSGB-Bio) for 1 hour at room temperature. The membranes were visualized with an emitter coupled logic detection system (Thermo). Ten minutes later, the mice were anesthetized using 1.5% isoflurane. To maintain body temperature, mice were placed on a thermostatically controlled heating pad (37°C) during imaging. Acquisition binning and duration were set according to tumor activity. Signal intensity was quantified as the total flux (photons/s) within regions of interest drawn manually around the tumor area using Living Image 4.0 software (Perkin Elmer).

| Bioluminescence imaging
Signals from both prone and supine positions were obtained.

| Micro-CT
Micro-CT scanning was performed using a FLE computed tomography system (InSyTe TM ; TriFoil Imaging) with 45 kVp X-ray voltage, 1100 ms exposure time, and a pitch of 1. The total projection of 240 slices over 360° of rotation was acquired. Projection data were rebinned in a ratio of 1:4 and reconstructed with a Butterworth filter.

| Statistical analysis
GraphPad Prism 5.0 was used for statistical analysis. All the data were expressed as the mean ± SEMs. Statistical differences between two groups were determined by Student's t test, while one-way ANOVA was used for multiple groups. P < .05 was considered statistically significant.

| Establishment of lentiviral vector for the short hairpin RNA (sh-HNRNPK) sequence
A PCR product with artificial XhoI and EcoRI enzyme restriction sites at the 5′ and 3′ ends containing the full-length coding sequence of HNRNPK-RNAi (69976-1) was amplified and subsequently cloned into the multiple cloning site of the lentiviral expression vector GV553. The resulting construct was verified by restriction enzyme digest and direct sequencing (Figure 1).

| HNRNPK is downregulated in a DOX-dependent manner
To investigate whether HNRNPK expression was regulated by the siRNA, an in vitro assay was performed in lentiviral vector-transfected A549 lung cancer cells. As Figure 2 shows, HNRNPK was downregulated F I G U R E 1 Construction of a lentiviral vector for the sh-HNRNPK sequence. The sh-HNRNPK sequence was cloned into the multiple cloning site of the GV553 lentiviral vector F I G U R E 2 Doxycycline (DOX) decreased HNRNPK expression in A549 cells. A, Western blot results indicated that HNRNPK expression was downregulated after DOX administration in a dosedependent manner. B, HNRNPK mRNA was significantly decreased after DOX induction, and a higher dose of DOX did not lead to further HNRNPK downregulation at both the mRNA and protein levels after DOX induction. As shown in Figure 2A, Western blot results indicated that HNRNPK was downregulated in a DOX-dependent manner. HNRNPK expression dramatically decreased after 5 and 10 μg/mL DOX treatments compared with the levels seen in the negative control and with lower DOX concentrations. This result was confirmed by RT-PCR, as shown in Figure 2B: 5 and 10 μg/mL DOX significantly reduced the mRNA expression of HNRNPK. Although 1 and 2 μg/mL DOX also decreased HNRNPK expression, the extent of reduction was not as high as was seen with 5 μg/ mL. Surprisingly, 10 μg/mL DOX did not further reduce HNRNPK levels.

| Downregulation of HNRNPK decreased lung metastasis
To reveal the role of HNRNPK, a mouse lung metastasis model was used ( Figure 3). Figure 3A illustrates the time course of the experiment. A549 tumor cells were intravenously injected through the tail vein, and then 1 week later, DOX treatment was started to induce the downregulation of HNRNPK expression. Simultaneously, metastatic foci in the lung were followed by bioluminescence imaging and micro-CT in vivo. Figure 3D,F illustrates that a lower signal was obtained in the sh-HN-RNPK group than in the control group 6 weeks after tumor implantation. At the end of the experiment, mice were killed, and the lungs were harvested ( Figure 3A). Additionally, the metastatic foci were calculated ( Figure 3B), and the quantification results indicated that in the sh-HN-RNPK group, the number of metastatic nodules was significantly decreased. However, this did not lead to bodyweight changes ( Figure 3E).

| D ISCUSS I ON
Clinical data show that HNRNPK is overexpressed in lung cancer tissues, 16 and high expression of HNRNPK is usually associated with poor prognosis. 15,17  Although our model reveals some mechanisms surrounding the role of HNRNPK in lung cancer metastasis, the mechanism and factors involved in the regulation of HNRNPK expression are still unknown and need to be investigated in the future.
Overall, we established a new model in which HNRNPK expression in cancer cells was specifically downregulated and downregulation of HNRNPK significantly inhibited lung metastasis. This indicates that HNRNPK may serve as a target for the development of new therapeutic strategies for the treatment of lung metastasis.

ACK N OWLED G M ENT
This work was supported by grants from the National Natural Science Foundation of China (no. 81602460).

CO N FLI C T O F I NTE R E S T
None.

AUTH O R CO NTR I B UTI O N S
RG and ML designed the experiment. XY, HL, LC, WL, LW, and GZ performed the experiment. ML and WZ carried out the data analysis.
RG, LM, and WZ prepared the manuscript. All authors read and approved the final manuscript.