{'fraction_non_alphanumeric': 0.0477226048085055, 'fraction_numerical': 0.036929644396117754, 'mean_word_length': 4.715074798619103, 'pattern_counts': {'":': 0, '<': 12, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 6, 'xml': 0}, 'pii_count': 1, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Long non-coding RNA small nucleolar RNA host gene 6 aggravates pancreatic cancer through upregulation of far upstream element binding protein 1 by sponging microRNA-26a-5p

Abstract Background Pancreatic cancer (PC) is a highly deadly malignancy with few effective therapies. We aimed to unmask the role that long non-coding RNA small nucleolar RNA host gene 6 (SNHG6) plays in PC cells by targeting far upstream element binding protein 1 (FUBP1) via microRNA-26a-5p (miR-26a-5p). Methods SNHG6 expression was predicted by bioinformatics, followed by verification via reverse transcription quantitative polymerase chain reaction. Then, the interactions among SNHG6, miR-26a-5p, and FUBP1 were detected through online software analysis, dual luciferase reporter assay and RNA pull-down. After that, cells were treated with different small interfering RNAs and/or mimic to determine the interactions among SNHG6, miR-26a-5p, and FUBP1 and their roles in PC cells. Finally, the role of SNHG6 in tumor growth in vivo was evaluated by measuring the growth and weight of transplanted tumors in nude mice. A t-test, one-way and two-way analysis of variance were used for data analysis. Results Compared with that in normal tissues, SNHG6 was highly expressed in PC tissues (1.00 ± 0.05 vs. 1.56 ± 0.06, t = 16.03, P < 0.001). Compared with that in human pancreatic duct epithelial cells (HPDE6-C7), SNHG6 showed the highest expression in PANC-1 cells (1.00 ± 0.06 vs. 3.87 ± 0.13, t = 34.72, P < 0.001) and the lowest expression in human pancreatic cancer cells (MIAPaCa-2) (1.00 ± 0.06 vs. 1.41 ± 0.07, t = 7.70, P = 0.0015). Compared with the levels in the si-negative control group, SNHG6 (0.97 ± 0.05 vs. 0.21 ± 0.06, t = 16.85, P < 0.001), N-cadherin (0.74 ± 0.05 vs. 0.41 ± 0.04, t = 8.93, P < 0.001), Vimentin (0.55 ± 0.04 vs. 0.25 ± 0.03, t = 10.39, P < 0.001), and β-catenin (0.62 ± 0.05 vs. 0.32 ± 0.03, t = 8.91, P < 0.001) were decreased, while E-cadherin (0.65 ± 0.06 vs. 1.36 ± 0.07, t = 13.34, P < 0.001) was increased after SNHG6 knockdown or miR-26a-5p overexpression, accompanied by inhibited cell proliferation, migration, and invasion. SNHG6 overexpression exerted the opposite effects. SNHG6 upregulated FUBP1 expression by sponging miR-26a-5p. Silencing SNHG6 blocked the growth of PC in vivo. Conclusion Silencing SNHG6 might ameliorate PC through inhibition of FUBP1 by sponging miR-26a-5p, thus providing further supporting evidence for its use in PC treatment.


Introduction
Pancreatic cancer (PC) is a severe malignancy in the digestive system characterized by aggressive clinical behavior, rapid expansion, and poor prognosis. [1] PC may be caused by an increased serum concentration of free insulin-like growth factor, stimulating the growth of pancreatic cells in the long term. [2] PC has been demonstrated to be one of the most deadly diseases with few available interventions and with less than 5% 5-year overall survival. [3] PC is generally diagnosed at an early stage and is resistant to known anti-cancer drugs. [4] Despite some diagnostic and therapeutic improvements, the mortality of PC has remained almost the same over the past several decades, partly due to a lack of screening methods and markers for early diagnosis. [5] Therefore, it is of great importance to identify the underlying molecular mechanisms of the carcinogenesis and progression of PC and to explore novel therapeutic and diagnostic targets for PC treatment.
Long non-coding RNAs (lncRNAs) have been reported to be involved in many biological processes and their deregulation is associated with cancer development. [6] As one of the housekeeping genes in the 5 0 -terminal oligopyrimidine tract family, lncRNA small nucleolar RNA host gene 6 (SNHG6) is associated with ribosomes. [7] Different cancers, such as breast cancer, colorectal cancer, osteosarcoma, and PC, have been reported to be correlated with SNHG6. [8][9][10][11] A previous study demonstrated that microRNA (miR) expression can affect normal biological processes in pancreatic cells, resulting in tumor occurrence and progression. [12] microRNA-26a-5p (miR-26a-5p) plays a role in the occurrence and development of several tumors, including papillary thyroid carcinoma, osteosarcoma, and breast cancer. [11,13,14] SNHG6 has also been found to affect cancer cells through regulation of miR-26a-5p in breast cancer and lung adenocarcinoma. [15,16] Interestingly, miR-26a has been revealed to participate in PC in different studies. [17,18] However, the specific roles of SNHG6 and miR-26a-5p in PC progression remain to be elucidated.
Bioinformatics analysis and dual luciferase reporter assays in our study showed that there are interactions among SNHG6, miR-26a-5p and far upstream element binding protein 1 (FUBP1) in PC. FUBP1 could control mRNA transcription of its target genes via the single-stranded DNA element far upstream element and interactions with the basal transcriptional machinery. [19] A previous study confirmed that a high level of FUBP1 is associated with a significant reduction in PC patient survival. [20] Based on these studies, we aimed to investigate the relationships among SNHG6, miR-26a-5p, and FUBP1 in the course of PC development. Moreover, we mainly discuss the effects of SNHG6 on PC cell progression.

Ethics statement
All participants signed informed consent forms. The study was officially approved by the Ethics Committee of Shanghai University of Medicine & Health Sciences Affiliated Sixth People's Hospital South Campus (No. 2017-Ethical review-KY-05). The animal experiments were conducted based on a minimal animal number and the least pain on experimental animals.

Tissue samples
From September 2017 to September 2018, patients diagnosed with PC at the Shanghai University of Medicine & Health Sciences Affiliated Sixth People's Hospital South Campus were enrolled in this experiment. Five pairs of PC and adjacent normal tissues (at least 3 cm away from the margin of PC tissues) were resected and collected. None of the patients received radiotherapy or chemotherapy before the operation and all were diagnosed with PC by pathological examination after the operation. Patients with chronic system diseases or other malignant tumors were excluded.

RT-qPCR
A TRIzol extraction kit (Invitrogen) was used to extract overall RNA from tissues and cells. Primers were designed and synthesized by Takara (Kyoto, Japan) [ Table 1]. Then,  Chinese Medical Journal 2020;133(10) www.cmj.org using the Rever Tra Ace qPCR RT Master Mix kit (TOYOBO, Osaka, Japan), RNA was reverse transcribed into complementary DNA. As instructed by the SYBR ® Premix Ex Taq TM II kit (Takara, Dalian, Liaoning, China), quantitative fluorescence PCR was performed in the ABI PRISM ® 7300 system (ABI, Inc., Foster City, CA, USA). U6 served as an internal reference for miR-26a-5p, while glyceraldehyde-3-phosphate dehydrogenase served as an internal reference for FUBP1 and SNHG6, with the relative expression measured using the 2 -DDCt method.

Western blotting analysis
Proteins were extracted from tissues and cells, and the protein concentration of each sample was determined. The 10% sodium dodecyl sulfate (Solarbio) separating and concentrating gels were prepared. The samples were mixed with the loading buffer and boiled for 5 min at 100°C. After an ice bath and centrifugation, equal amounts of sample were added into the lanes with a pipette for electrophoresis separation. Then, proteins in the gel were transferred to cellulose nitrate membranes and blocked with 5% skimmed milk powder at 4°C overnight. Afterward, the membranes were incubated with primary antibodies (Abcam, Cambridge, UK) against FUBP1 (ab181111, 1:2000), E-cadherin (ab40772, 1:10,000), N-cadherin (ab18203, 1 mg/mL), b-catenin (ab32572, 1:10,000), and Vimentin (ab92547, 1:1000), at 4°C overnight. After that, the membranes were washed with phosphate-buffered saline (PBS, Wuhan Servicebio Co., Ltd., Wuhan, Hubei, China) at room temperature three times for 5 min each time. Subsequently, the membranes were incubated with horseradish peroxidase-labeled secondary antibody immunoglobulin G (1:1000, Boster Biological Technology Co., Ltd, Wuhan, Hubei, China) for 1 h at 37°C, followed by three PBS washes for 5 min each time. The membranes were submerged in enhanced chemiluminescence reaction solution (Pierce Company, now part of Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 1 min. After liquid removal, the membranes were covered with food preservation film and exposed. After developing and fixing, the images were observed. The protein marker was purchased from Pierce (#84785, Thermo Fisher Scientific) with b-actin as an internal control, and the protein band images were analyzed using ImageJ2x V2.1.4.7 (Rawak Software, Inc., Germany).

Cell counting kit-8 (CCK-8) assay
A PC cell suspension was diluted to a certain concentration and then seeded at a density of 2 Â 10 3 cells/100 mL per well into 96-well plates. Each group had 12 parallel wells. PC cells were cultured for different durations (12,24,48, and 72 h, retrospectively), and three replicates were arranged for each time point. CCK-8 solution (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) was added to the cell-free medium as the blank control. The cells were cultured at 37°C with 5% CO 2 . At each time point, 10 mL CCK-8 solution was added to the corresponding wells and incubated in an incubator for 4 h. Finally, the optical density value at 450 nm was measured using a microplate reader and recorded as A 450nm .
Colony formation assay PC cells were detached using trypsin (Solarbio) to fully disperse the suspension in each group. Then, 200 cells were seeded into six-well plates, which were shaken gently to evenly disperse the cells. The cells were cultured for 2 to 3 weeks. When cell colonies were visible to the naked eye, the culture was terminated, and the culture medium was discarded followed by PBS (Wuhan Servicebio) washing. After that, the cells were fixed for 30 min in 4% paraformaldehyde (Solarbio) and washed in PBS three times. Then, the cells were stained in Giemsa staining solution (Shanghai Regal Biology Technology Co, Ltd, Shanghai, China) for 60 min, washed under running water, and air-dried. The plates were inverted and covered with transparent film with a grid. After images were acquired with a Sony HX350 camera (Japan), the cell colony number was directly counted using the naked eye.

Flow cytometry
The staining was carried out according to the instructions of the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit (BestBio, Shanghai, China). The cells were centrifuged after detachment to remove the supernatant. The cells were resuspended in PBS to adjust the concentration to 1 Â 10 6 cells/mL. Then, 200 mL cells were washed twice using 1 mL pre-cooled PBS followed by centrifugation. The cells were mixed with 2 mL Annexin-V-FITC (20 mg/mL) after being resuspended in 100 mL binding buffer and were incubated on ice, avoiding light exposure, for 15 min. Subsequently, the cells were transferred to a flow cytometry tube, and 300 mL PBS was added. Each sample was mixed with 1 mL PI (50 mg/mL) and detected on a flow cytometer within 30 min.
Hoechst 33258 staining PC cells (1 Â 10 5 cells/mL) in the logarithmic phase were seeded into six-well plates (3 mL per well) with cover glasses. The cells were incubated (37°C, 5% CO 2 ) for 24 h, and then the culture solution was discarded. Then, the cells were fixed with fixation solution for 10 min and stained with Hoechst 33258 dye solution (Beyotime Biotechnology Co., Ltd., Shanghai, China) for 5 min away from light at room temperature. The slides with cells attached were placed on the anti-fluorescence quencher droplets. A fluorescence microscope (BX60, Olympus Optical Co., Ltd, Tokyo, Japan) was used to observe the slides, and images were collected.

Transwell assay
When PC cells reached the logarithmic phase, they were cultured at 37°C for 24 h in six-well plates before transfection. After 48 h of culture, 0.25% trypsin was added to the cells, and the cells were resuspended in DMEM to adjust the concentration to 5 Â 10 5 cells/mL. The basolateral chamber was filled with 500 mL DMEM containing 10% FBS (Solarbio), while the apical chamber was filled with 200 mL cell suspension followed by 24 h incubation at 37°C. The chambers were transferred to a clean 24-well plate, and 4% polyformaldehyde (500 mL, Chinese Medical Journal 2020;133 (10) www.cmj.org Solarbio) was added to each well. The cells were fixed for 30 min at room temperature, stained with crystal violet (Solarbio) for 60 min, and washed with PBS (Servicebio) two or three times. Five images were collected from each well and analyzed.
The invasion chamber was coated with 50 mL Matrigel at 500 g/mL (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). The Matrigel was solidified at 37°C for 4 h. The next steps were similar to those in the cell migration experiment mentioned above.

Dual luciferase reporter gene assay
Bioinformatics software (http://starbase.sysu.edu.cn/pan GeneDiffExp.php) was used to predict the target relationship between miR-26a-5p and SNHG6 as well as the binding site. The SNHG6 sequence containing the binding site of miR-26a-5p was synthesized and inserted into the pMIR-REPORT TM Luciferase vector (Ambion, Austin, TX, USA) to construct the SNHG6 wild-type plasmid (SNHG6-WT). In this plasmid, the binding site was mutated to construct the SNHG6 mutant plasmid (SNHG6-MUT). Plasmid extraction was carried out according to the instructions of the plasmid extraction kit (Promega, Madison, Wisconsin, USA). SNHG6-WT and SNHG6-MUT were mixed with mimic NC and miR-26a-5p mimic (Shanghai GenePharma Biological Co., Ltd., Shanghai, China), respectively, followed by co-transfection into 293T cells, which were obtained from ATCC, using Lipofectamine TM 2000. Luciferase activity was measured by a luciferase detection kit and a GloMax 20/20luminometer (Promega). As described above, the target relationship between miR-26a-5p and FUBP1 as well as the binding site was predicted by http://www. targetscan.org, and the plasmids FUBP1-WT and FUBP1-MUT were constructed.

RNA pull-down assay
The experimental procedure was carried out according to the instructions of the biotin RNA labeling mix kit (Roche Diagnostics, Indianapolis, IN, USA). The biotin-labeled miR-26a-5p WT and miR-26a-5p MUT were incubated with MIAPaCa-2 cytoplasmic extract. Cells were resuspended in lysis buffer, placed on ice for 10 min, and centrifuged for 10 min at 10,000 Â g. The magnetic beads coated with streptavidin (Thermo Fisher Scientific) were blocked with lysis buffer containing yeast tRNA and bovine serum albumin at 4°C for 2 h and washed twice with 1 mL lysis buffer. The lysate was added to the magnetic beads coated with streptavidin and incubated at 4°C for 4 h. Then, the cells were washed twice with 1 mL lysis buffer, three times with low salt buffer, and one time with high salt buffer. RNA bound to magnetic beads was separated by TRIzol reagent (Thermo Fisher Scientific). SNHG6 expression was detected by RT-qPCR.

Xenograft tumor model in nude mice
si-NC or si-SNHG6-1 was used to treat nude mice (five mice in each group, 6 weeks old) (Cancer Research Institute of Chinese Academy of Medical Sciences, Beijing, China). Panc-1 cells that were stably growing in the logarithmic phase were collected and resuspended in PBS, the supernatant was then removed, and the cell concentration was adjusted to 1 Â 10 7 cells/mL. Each nude mouse was injected subcutaneously with 200 mL cell suspension via the right hind leg and housed under the same conditions for 4 weeks. Tumor volume was measured with Vernier calipers once every 7 days. After 28 days, nude mice were killed by cervical dislocation, and the tumors were weighed. The obtained tumors were made into tissue homogenate. The levels of SNHG6, miR-26a-5p, and FUBP1 were detected by RT-qPCR and western blotting analysis.

Statistical analysis
SPSS21.0 (IBM, Armonk, NY, USA) was used for data analysis. The Kolmogorov-Smirnov test was performed to test whether the data were normally distributed. Data are expressed as the mean ± standard deviation. The t test was conducted for comparisons between two groups. One-way or two-way analysis of variance was performed to compare data among multiple groups, followed by Tukey multiple comparison test. The P value was calculated by a bilateral test, and P < 0.05 indicated a statistically significant difference.

SNHG6 was upregulated in PC
SNHG6 expression was predicted by bioinformatics analysis and verified by RT-qPCR. Through the http:// starbase.sysu.edu.cn/panGeneDiffExp.php website, we found that SNHG6 was overexpressed in PC tissues compared to adjacent normal tissues in 178 PC patients [ Figure 1A]. To verify the results, five pairs of PC and adjacent normal tissues were tested by RT-qPCR. The results were consistent with the database results [ Figure 1B]. The RT-qPCR data also showed that among four PC cell lines (MIAPaCa-2, BxPC-3, Capan-1, and Panc-1), SNHG6 expression was the highest in Panc-1 cells, followed by Capan-1 cells and BxPC-3 cells, with the lowest expression in MIAPaCa-2 cells (P < 0.01) [ Figure 1C].

Silencing SNHG6 promoted apoptosis but inhibited the proliferation, invasion, and migration of PC cells
Next, the changes in PC cell biology were surveyed after transfection of the Panc-1 cell line with si-SNHG6. si-SNHG6 decreased SNHG6 expression in Panc-1 cells [ Figure 2A], downregulated b-catenin, Vimentin, and Ncadherin levels, upregulated E-cadherin levels [ Figure 2B], inhibited cell proliferation in a time-dependent manner [ Figure 2C], reduced the cell colony formation rate [ Figure 2D], accelerated cell apoptosis [ Figure 2E], induced highly aggregated nuclear chromatin and nuclear rupture [ Figure 2F], and inhibited cell migration and invasion [ Figure 2G]. The MIAPaCa-2 cell line treated with a plasmid overexpressing SNHG6 showed the opposite results.
Overexpressed miR-26a-5p abolished the effect of overexpressed SNHG6 on PC cells Compared with MIAPaCa-2 cells overexpressing miR-26a-5p, the MIAPaCa-2 cell line treated with the SNHG6 overexpression plasmid and mimic NC showed decreased miR-26a-5p expression [ Figure 4A], increased cell proliferation [ Figure 4B], a higher cell colony formation rate [ Figure 4C], reduced cell apoptosis [ Figure 4D and 4E], and increased cell migration and invasion (all P < 0.01) [ Figure 4F]. However, when the miR-26a-5p mimic was transfected into MIAPaCa-2 cells treated with the SNHG6 overexpression plasmid, it was found that overexpression of miR-26a-5p inhibited the growth and metastasis of MIAPaCa-2 cells with SNHG6 upregulation compared with the mimic NC transfection.

Silencing SNHG6 inhibited tumor formation in nude mice
After silencing SNHG6, the expression of FUBP1 and SNHG6 in tumor tissues was significantly decreased, while miR-26a-5p expression was increased (all p < 0.01) [ Figure 5A and 5B], and the growth rate and weight of tumors in nude mice were reduced when compared with those in nude mice treated with si-NC [ Figure 5C and 5E], which further confirmed that SNHG6 promoted tumor growth.

Discussion
PC is related to a poor prognosis, evidenced by the close link between disease occurrence and mortality. [21] LncRNAs have been indicated to be modulators of cellular development and human diseases via their effects on gene expression. [22] They are also competing endogenous RNAs that regulate miRs and the expression of their target genes, thus influencing cancer development. [23] Herein, we discussed the underlying mechanisms of SNHG6, FUBP1, and miR-26a-5p in PC progression. The evidence obtained from both PC tissues and cell lines demonstrated that silencing SNHG6 could ameliorate PC by sponging miR-26a-5p.
Initially, SNHG6 was overexpressed in PC tissues, and silencing SNHG6 inhibited epithelial-mesenchymal transition (EMT) and ameliorated cell activities in PC. LncRNA SNHG6, a recently identified cancer-related lncRNA, was found to be upregulated in Wilms' tumor. [24] Another study proved that SNHG6 was upregulated in ovarian  Chinese Medical Journal 2020;133 (10) www.cmj.org clear cell carcinoma and that upregulated SNHG6 contributed to the tumorigenesis and progression of ovarian clear cell carcinoma. [25] Zhang et al [26] found that knockdown of SNHG6 inhibited colorectal cancer cell activities and EMT by regulating EZH2 expression by sponging miR-26a. Similar to our study, SNHG6 was reported by Yan et al [10] to be overexpressed in PC, which predicted poor prognosis as well.
Another study suggested that FUBP1 was the target gene of miR-16-1 and that miR-16-1 could inhibit FUBP1 directly and specifically in gastric cancer. [28] A study also identified FUBP1 as a novel miR-16 target, and miR-16 overexpression decreased FUBP1 levels in human breast cancer and gastric cancer cells. [29] Figure 3: SNHG6 inhibited miR-26a-5p expression to upregulate FUBP1 expression. (A) Prediction of binding sites between the SNHG6 sequence and miR-26a-5p sequence by the StarBase online database; (B) miR-26a-5p expression in the Panc-1 cell line treated with si-SNHG6 and in the MIAPaCa-2 cell line overexpressing SNHG6 detected by RT-qPCR; (C) the relationship between SNHG6 and miR-26a-5p verified by dual luciferase reporter gene assay; (D) RNA pull-down between SNHG6 and miR-26a-5p, and expression in MIAPaCa-2 cells was detected by RT-qPCR, which showed that SNHG6 expression in the miR-26a-5p NC group was decreased; (E) prediction of the binding sites between the FUBP1 sequence and miR-26a-5p sequence by the TargetScan online database; (F and G) FUBP1 expression in the MIAPaCa-2 cell line overexpressing miR-26a-5p detected by RT-qPCR and western blot analysis; (H) the relationship between FUBP1 and miR-26a-5p verified by dual luciferase reporter gene assay; (I and J) FUBP1 expression in the Panc-1 cell line treated with si-SNHG6 detected by RT-qPCR and western blot analysis. One-way ANOVA was used in panels B, D, F, G, I, and J, and two-way ANOVA was used in panels C and H, followed by Tukey multiple comparisons test. * P < 0.01. n = 3. ANOVA: Analysis of variance; FUBP1: Far upstream element binding protein1; miR-26a-5p: MicroRNA-26a-5p; mimic NC: Mimic negative control; MUT: Mutant; RT-qPCR: Reverse transcription quantitative polymerase chain reaction; si-NC: Small interfering RNA negative control; SNHG6: Small nucleolar RNA host gene 6; si-SNHG6: Small interfering RNA-SNHG6; WT: Wild type. Chinese Medical Journal 2020;133 (10) www.cmj.org Furthermore, we also concluded that overexpressed miR-26a-5p inhibited EMT in PC. miR-26a-5p was found to block cell migration in assorted malignant tumors. For instance, miR-26a-5p was indicated to be downregulated in malignant melanoma, and forced expression of miR-26a blocked melanoma cell invasiveness and growth through mediation of microphthalmia-associated transcription factors. [30] An obvious downregulation of miR-26a-5p was also observed in bladder cancer and hepatocellular carcinoma, and downregulated miR-26a-5p accelerated cell invasion, migration, and EMT processes. [31,32] One study discovered that SNHG5 triggered melanoma cell aggressiveness while hindering apoptosis by sponging miR-26a-5p. [33] FUBP1 was found to be involved in the regulation of diverse cellular actions and correlated with oncogenesis. [34] Upregulated FUBP1 was observed, and FUBP1 was suggested to be an indicator and a possible target in sacral chordomas and human clear cell renal cell carcinoma. [35,36] A recent study elucidated that FUBP1 was an important transactivator of the c-Myc protooncogene overexpressed in PC and inhibited cancer cell activities by increasing PD-L1 expression modulated by Myc. [37] We found that overexpressed miR-26a-5p could inhibit the expression of FUBP1, thereby ameliorating PC progression.
Consistent with previous studies, we confirmed that SNHG6 is an oncogenic lncRNA in PC. Through our investigation in vitro and in vivo, we proved that SNHG6 could accelerate PC cell activities by regulating FUBP1 to a higher level by sponging miR-26a-5p. Furthermore, our study revealed encouraging data that suggest the value of miR-26a-5p as a tumor inhibitor and a potential target for PC. In the future, we will investigate and discuss the potential mechanism of the miR-26a-5p target gene FUBP1 and deeply estimate the underlying effects of other miRs regulated by SNHG6 in PC progression. Further studies on tissues from PC patients are required to fully understand the detailed mechanisms of SNHG6, miR-26a-5p, and FUBP1 in PC.  Chinese Medical Journal 2020;133 (10) www.cmj.org