Novel evidence for oncogenic piRNA‐823 as a promising prognostic biomarker and a potential therapeutic target in colorectal cancer

Abstract piRNA‐823 as a member of the piRNA family is reported to promote tumour cell proliferation in multiple myeloma and hepatocellular cancer. However, few studies on the function of piRNA‐823 in colorectal cancer (CRC). Our present study data showed that piRNA‐823 plays an oncogene role in CRC cells. Inhibition of piRNA‐823 can significantly inhibit the proliferation, invasion and apoptosis resistance of CRC cells. Mechanism studies have shown that piRNA‐823 inhibits the ubiquitination of hypoxia‐inducible factor‐1 alpha (HIF‐1α) by up‐regulating the expression of Glucose‐6‐phosphate dehydrogenase (G6PD) and ultimately up‐regulates the glucose consumption of carcinoma cells and inhibits the content of intracellular reactive oxygen species (ROS). Therefore, we speculate piRNA‐823 promotes the proliferation, invasion and apoptosis resistance of CRC cells by regulating G6PD/HIF‐1α pathway. In this study, we set up the cancer‐promoting function recovery experiment of piRNA‐823 by silencing G6PD gene to confirm the dominance of the above‐mentioned pathways. Using clinical samples, we found that overexpression of piRNA‐823 correlated with poor overall survival and predicted a poor response to adjuvant chemotherapy of patients with CRC. In a word, our research has further enriched the theory of piRNA‐823 promoting the progression of CRC, and laid a solid foundation for the development of piRNA‐823‐based gene therapy for CRC and its use as a promising prognostic biomarker in CRC patients.

in mammalian germ cells. Because of its extensive cellular regulatory functions, it has become a star molecule in non-coding RNA research. piRNA mainly exists in mammalian germ cells and stem cells. It binds with proteins belonging to Piwi subfamily to form piRNA complexes to regulate gene silencing pathways, maintain reproductive and stem cell functions and regulate the stability of translation of mRNA. 1 In recent years, many studies have found that piRNA also exists in normal somatic cells (such as heart, brain, liver and other tissues) or cancer cells other than germ line cells, and widely participates in the occurrence and development of gastric cancer, liver cancer, lung cancer, lymphoma and other cancers, which is also closely correlated with central nervous system diseases, cardiac regeneration, etc. 2,3 Nevertheless, the clinical significance and biological mechanisms of piRNAs in the progression of CRC remain largely unknown.
In our present study, piRNA-823, a family of piRNAs, was identified with high expression trends from normal colorectal tissues, adenoma and CRC by high-throughput screening using the Arraystar Human piRNAs Array. Screening data show that the expression levels of piRNA-823 are not only higher in colorectal cancer tissues than in adjacent tissues, but also positively correlated with tumour malignancy, suggesting that piRNA-823 may be closely related to the progression of colorectal cancer. Therefore, the regulatory mode and action pathway of piRNA-823 are worth studying. A group of screening data on gene expression profiles of HCT-116 cells before and after piRNA-823 intervention showed that molecule glucose-6-phosphate dehydrogenase (G6PD) gene related to glucose metabolism pathway in colorectal cancer cells changed significantly before and after piRNA-823 intervention, and positively correlated with the content of piRNA-823. Therefore, we speculated that piRNA-823 may promote the occurrence and progression of colorectal cancer by enhancing the expression of downstream key molecule G6PD, which may be a key factor in the piRNA-823 pathway to promote the progression of colorectal cancer.  Table S1. This study was approved by the hospital ethics committees, and written informed consents were obtained from patients for using their tissues and clinical information.

| Microarray analysis
Total RNA from each sample was quantified by the NanoDrop ND-1000, and RNA integrity was assessed by standard denaturing agarose gel electrophoresis. piRNA sample labelling was performed using a RNA ligase method as described in the method section. For microarray analysis, Agilent Array platform was employed. The labelled samples were hybridized onto Arraystar Human piRNA Array in Agilent's SureHyb Hybridization Chambers according to the manufacturer's standard protocols (Agilent Technologies). After having washed the slides, the arrays were scanned by the Agilent Scanner G2505C.
Agilent Feature Extraction software (version 11.0.1.1) was used to analyse acquired array images. Quantile normalization and subsequent data processing were performed using the GeneSpring GX v11. 5

| Lentivirus packaging
A siRNA sequence complementarily binding to G6PD (NM_000402, NM_001042351) was chosen. The target sequences of siRNA (5′-GGTCAAGGTGTTGAAATGC-3′) are homologous, and the oligonucleotide templates of these shRNAs were chemically synthesized and cloned into the linear pSIH1-H1-copGFP shRNA Vector (System Biosciences, CA, USA) which was obtained through digestion by BamHI and EcoRI (Takara, Dalian, China) and purification by agarose gel electrophoresis. An invalid siRNA sequence (5′-GAAGCCAGATCCAGCTTCC-3′) was used as a negative control (NC). Sequencing was used to confirm the vectors constructed (pSIH1-shRNA-G6PD and pSIH1-NC). Chemically, synthesize complementary double-stranded piRNA823 and piRNA823 sponge sequences with adding BamHI and EcoRI restriction sites at both ends. piRNA823-forward, 5′-GATCCAGCGTTGGTGGTATAGTGGTGAGCATAGCTGCG-3′; piRNA-823-reverse, 5′-AATTCGCAGCTATGCTCACC ACTATACCAC CAACGCTG-3′;piRNA-823 sponge-forward, 5′-GATCCAGCGTTGGT GGTATAGTGGTGAGCATAGCT GCTATACAGCGTTGGTGGTATAGTG G TG A G C ATA G C TG C A C ATC A G C G T TG G TG G TATA G TG G TGAGCATAGCTGCG-3′; piRNA-823 sponge-reverse, 5′-AATTCGC AG C TATG C TC ACC AC TATACC ACC A ACG C TG ATG TG C AG C   T A T G C T C A C C A C T A T A C C A C C A A C G C T G T A T A G   CAGCTATGCTCACCACTATACCACCAACGCTG-3′; Two complementary DNA annealed to form a double strand and cloned into a pcDH1 lentiviral expression vector (System Biosciences). The recombinant vector was named pcDH1-piRNA-823 and pcDH1-Sponge-piRNA-823.
All the recombinant vectors were sequenced, and plasmid DNA was prepared using an EndoFree Plasmid Kit (12362, Qiagen).
One day before transfection, 293T cells were seeded into 10-cm dishes (Corning). 2 μg of each pSIH1-shRNA-G6PD vector or pSIH1-NC or pcDH1-piRNA-823 or pcDH1-Sponge-piRNA-823 and 10 μg pPACK Packaging Plasmid Mix (System Biosciences) were co-transfected using Lipofectamine 2000 (Invitrogen) in accordance with the manufacturer's protocol. The medium was replaced with DMEM plus 1% FBS. Forty eight hours later, the supernatant was harvested, and then cleared by centrifugation at 5000 g at 4°C for 5 minutes and passed through a 0.45 µm PVDF membrane (Millipore). The titre of virus was determined by gradient dilution. The packaged lentiviruses were named as Lv-shRNA-G6PD, Lv-NC, Lv-piRNA823 and Lv-Sponge-piRNA823.

| Gene intervention via the lentiviral pathway
HCT-116 and Lovo in the logarithmic phase were seeded in 6-well plates at 2 × 10 5 cells/well. One day later, lentivirus (Lv-NC or Lv-
The cells were cultured under normal conditions, and cell viability was detected by using a cell counting kit-8 assay (CCK-8) at 24, 48, and 72 hours. Briefly, 10 µL of CCK-8 solution (CK04, Dojindo, Japan) was added, and then the cells were cultured under normal conditions for an additional 4 hours. Then, the absorbance at 450 nm was measured.

| Cell invasion assay
Cell invasion experiments were performed using the QCMTM 24-well Fluorimetric Cell Invasion Assay kit (ECM554, Chemicon International) according to the manufacturer′s instructions. The kit used an insert polycarbonate membrane with an 8-μm pore size.
The insert was coated with a thin layer of EC Matrix™ that occluded the membrane pores and blocked the migration of non-invasive cells. Culture medium (500 μL) supplemented with 10% FBS was used as a chemoattractant. Cells that migrated and invaded the underside of the membrane were fixed in 4% paraformaldehyde. The invading cells were stained with DAPI, and the number was then determined by fluorescence and reported as the relative fluorescence units (RFUs). The grouping was the same as in the proliferation assay. HCT-116 and Lovo 72 hours after being infected with the recombinant lentiviruses were seeded to transwell at 2 × 10 5 cells/well and 48 hours after seeding, cell invasion assay was performed.

| Glucose consumption assay
Glucose consumption was determined as described with some modifications. 4

| Detection of intracellular ROS
The total intracellular ROS generation was measured using H2DCFDA, as reported by a published study. 5 Briefly, HTC-116 and Lovo cells infected with Lv-Sponge-piRNA-823 or Lv-NC or without infection were seeded (5 × 104 cells/well) into black 96-well plates and maintained to attach at 37°C in 5% CO 2 for 48 hours. After that, the cells were labelled with H2DCFDA solution at 5 μmol/L in DMF.
After that, the fluorescence intensity was measured in a Synergy H1 Fluorescence Spectrophotometer (BioTek), at the excitation and emission wavelengths of 495 and 527 nm, respectively. After that, the ROS levels were measured using Cellular ROS/Superoxide Detection Assay Kit (Abcam).

| Measuring the half-life of G6PD
HTC-116 cells in logarithmic phase were seeded to 6-well plates and cultured under normal conditions overnight. Cells were divided into two groups, piRNA-823 overexpression and suppression groups (infected with Lv-piRNA-823 or Lv-Sponge-piRNA-823) and added with 50 μmol/L mg132 or 100 μg/mL CHX(sigma) and incubated under normal conditions for 0, 1, 2, 4 or 8 hours. Cells were collected and subjected to Western blotting for HIF-1α.

| Real-Time-PCR
To test the piRNA-823 levels, total RNA (2 μg) was used for cDNA prep- The cycling parameters were 40 cycles of denaturation at 95°C for 10 seconds, annealing at 60°C for 20 seconds and extension at 72°C for 20 seconds. U6 snRNA was used as a reference to normalize the hsa-miRNA-29a level using the 2 ΔΔCt method. Each RNA sample was run in triplicate.
After washing, the bands were detected by chemiluminescence and imaged with X-ray films. β-actin was used as an endogenous reference for normalization.

| Statistical analysis
The data are shown as the mean ± SD of three independent experiments. All statistical data were analysed using SPSS GradPack version 20.0 statistical software (IBM Corp.) and GraphPad Prism 7.0 (GraphPad Software, Inc). Comparisons between groups were analysed using a two-tailed Student's t test or one-way ANOVA with a post hoc Tukey's test. Differences were considered to be statistically significant when **P < .05.

| piRNA-823 expression is significantly upregulated in the CRC progression process
To search for potential piRNAs in the course of colorectal malignant transformation, we globally analysed the piRNA expression profiles of normal colorectal tissues, colorectal adenoma tissues and CRC tissues using Arraystar Human piRNAs Array ( Figure 1A). We focused on only one piRNA (piRNA-823), a family of piRNAs, its expression level with high expression trends from normal colorectal tissues, adenoma and CRC (P = .007, One-way ANOVA analysis; Figure 1B).
To confirm the altered expression of piRNA-823 in CRC, validation experiments were carried out by qRT-PCR. The similar expression pattern was also confirmed (P < .001, One-way ANOVA analysis; Figure 1C). Screening and validation data show that the expression levels of piRNA-823 are not only higher in colorectal cancer tissues than in adjacent tissues, but also positively correlated with tumour malignancy, suggesting that piRNA-823 may be closely related to the progression of colorectal cancer. Therefore, the regulatory mode and action pathway of piRNA-823 are worth studying.

| The association of the piRNA-823 with the prognosis and therapeutic outcome of CRC
To investigate the association between piRNA-823 expression and the clinicopathological characteristics of CRC patients, we exam-   Table 1). Taken together, these data demonstrated that an important role for piRNA-823 in CRC carcinogenesis and progression and might be as a potential prognostic biomarker for CRC patients.
To examine whether piRNA-823 expression can predict response to adjuvant therapy, we analysed the associations between piRNA-823 expression and the therapeutic outcomes in stage II and stage III CRC patients treated with adjuvant chemotherapy. The current results indicated that high piRNA-823 expression was associated with a poor prognosis in stage II (P < .0001) and stage III (P < .0001) patients ( Figure 1E). For individuals who received adjuvant therapy, high piRNA-823 expression was associated with a poor therapeutic outcome in the patients with stage II and III cancer (P < .0001), and patients with stage II cancer alone (P < .0001) or patients with stage III cancer alone (P = .0007, Figure 1E). A multivariate Cox regression demonstrated that high piRNA-823 expression predicted poor prognosis (HR, 12.76; 95% CI, 3.94-41.32; P < .001) and treatment with adjuvant chemotherapy was associated with beneficial survival (HR, 0.42; 95% CI, 0.15-0.69; P = .007) independent of other clinical covariates (Table 2). Therefore, piRNA-823 expression levels served as an independent predictor of the response to adjuvant chemotherapy. In the functional experiment, proliferation activity assay data showed that piRNA-823 inhibition significantly inhibited the logarithmic proliferative activity of HCT-116 and Lovo cells (P < .01, vs cell control group or NC control group, 72 hours) ( Figure 3A,B).

| Knock-down of piRNA-823 inhibits the malignant characteristics of CRC cells
Invasion assay showed that piRNA-823 inhibition significantly inhibited the invasion of HCT-116 and Lovo cells (P < .01, vs cell control group or NC control group) ( Figure 3A,B). Apoptosis assay data showed that piRNA-823 inhibition significantly up-regulated apoptosis of HCT-116 and Lovo cells (P < .01, vs cell control group or NC control group) ( Figure 3A,B).

| piRNA-823 promotes CRC progression by upregulating G6PD expression
To understand the mechanism of piRNA-823 regulation in CRC, a group of screening data on gene expression profiles of HCT-116 cells before and after piRNA-823 intervention showed that molecule glucose-6-phosphate dehydrogenase (G6PD) gene related to glucose metabolism pathway in colorectal cancer cells changed significantly before and after piRNA-823 intervention and positively correlated with the content of piRNA-823 (Table S2). Therefore, we speculated that piRNA-823 may promote the occurrence and progression of colorectal cancer by enhancing the expression of downstream key molecule G6PD, which may be a key factor in the piRNA-823 pathway to promote the progression of colorectal cancer.
In Lv-shRNA-G6PD infected group, the content of piRNA-823 did not change significantly (P > .05, vs uninfected control group or NC control group) (Figure 2A

| piRNA-823 up-regulates the expression of functional proteins CyclinD1, STAT3 and Bcl-2 by upregulating G6PD expression
Western blotting data showed that 72 hours after Lv-piRNA82 in-

| Effect of piRNA-823 on the ubiquitylation of HIF-1α in HCT-116 cells
The level of HIF-1α was increased over time when mg132 suppressed protein degradation in HTC-116 cells, and there was no difference between the groups infected with Lv-piRNA-823 or Lv-Sponge-piRNA-823, indicating piRNA-823 had no effect on protein synthesis ( Figure 6A). When CHX was used to block protein synthesis, HIF-1α decreased over time in two groups, However, the rate of change with time in Lv-Sponge-piRNA-823-infected HTC-116 cells was significantly higher than that of Lv-piRNA-823-infected HCT-116 cells ( Figure 6B). PiRNA was originally thought to play an important role in regulating reproductive system development, 3 but recent studies have confirmed that piRNA is abnormally expressed in breast cancer, pancreatic cancer, gastric cancer, liver cancer, endometrial cancer and other tumours. [9][10][11][12][13] In addition, molecular mechanism studies have revealed Increasing the content of piRNA-823 in gastric cancer cells could significantly inhibit the progress of subcutaneous tumour-bearing.

| D ISCUSS I ON
In addition, the study also found that the content of piRNA-823 in peripheral blood of patients with gastric cancer was significantly lower than that of healthy people, indicating that piRNA-823 has the function of tumour suppressor gene in the progress of gastric cancer, and the detection of piRNA-823 in peripheral blood may be an effective biomarker for gastric cancer. 16  G6PD is a key enzyme in pentose phosphate pathway (PPP), and its main function is to provide NADPH in vivo. It is also found that G6PD is highly expressed in many cancer cells and is closely related to some biological characteristics. 20 This study is the first exposition of the piR823/G6PD/HIF-α regulatory pathway in colorectal cancer and will provide strong support for the development of piRNA-823-based gene therapy for colorectal cancer. At the same time, this study also provides a theoretical basis F I G U R E 5 piRNA-823 up-regulates the expression of functional proteins CyclinD1, STAT3 and Bcl-2 through G6PD. A, HCT-116, B, Lovo. Western blotting was performed 72 h after virus infection. The protein was detected by β-actin, and the size of G6PD, CyclinD1, STAT3, Bcl-2 and β-actin was 53 kD, 33 kD, 90 kD, 26 kD and 43 kD. All data were expressed as mean ± SD, and the experiment was set to 3 biological replicates (n = 3). **P < .01, *P < .05 for piRNA-823 as a promising prognostic biomarker and a potential therapeutic target in colorectal cancer.

ACK N OWLED G EM ENTS
We would like to express our deep and sincere gratitude to the patients and clinicians from the Department of General Surgery, Shanghai Tenth People's Hospital, School of Medicine, Tongji University, for their contributions to this study.

CO N FLI C T O F I NTE R E S T
The authors declared that they have no financial competing interest.