Fanlu Meng, Yanbo Wang and Qiang Sun*

Department of Gastroenterology, Shandong Provincial Third Hospital, Shandong University, Jinan, Shandong 250031, China

*Corresponding Author:
Q. Sun
Department of Gastroenterology, Shandong Provincial Third Hospital, Shandong University, Jinan, Shandong 250031, China
E-mail: sunqiang20231214@126.com
Date of Received 05 January 2023
Date of Revision 11 July 2023
Date of Acceptance 10 November 2023
Indian J Pharm Sci 2023;85(6):1685-1691  

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DOI: 10.36468/pharmaceutical-sciences.1224

Abstract

This research explored the possible mechanism of Tripterygium wilfordii in suppressing cancer progression. Cancer coli-2 cells were exposed to Tripterygium wilfordii treatment (0, 20, 40, 80 μmol/l) for 24 h. Cell proliferation, clone formation, and apoptosis were monitored using cell counting kit-8 reagent, plate clone formation, and flow cytometry experiments. PRR34-antisense ribonucleic acid 1, miR-3614-5p, and cleaved-caspase-3 contents were assessed using real-time quantitative reverse transcription assay or Western blot. Dual-luciferase reporter system confirmed the targeting between PRR34-antisense ribonucleic acid 1 and miR-3614-5p. After treatment with various doses of Tripterygium wilfordii, cancer coli-2 cell proliferation, inhibition rate, apoptosis rate, cleaved-caspase-3 protein level and miR-3614-5p expression were increased, and clone formation number as well as PRR34-antisense ribonucleic acid 1 expression were decreased in a concentration-dependent manner. PRR34-antisense ribonucleic acid 1 directly targeted miR-3614-5p. Upregulated PRR34-antisense ribonucleic acid 1 partly abolished Tripterygium wilfordii-induced cancer coli-2 cell proliferation repression and apoptosis promotion via targeting miR-3614-5p. Tripterygium wilfordii might hinder cancer coli-2 cell proliferation by regulating PRR34-antisense ribonucleic acid 1/miR-3614-5p.

Keywords

Colorectal cancer, Tripterygium wilfordii, long noncoding ribonucleic acid, PRR34-antisense ribonucleic acid 1, miR-3614-5p, cell proliferation, apoptosis

As a commonly diagnosed malignancy of the digestive system in China, Colorectal   Cancer (CRC) has been recognized as a major challenge that seriously threatens the life of patients[1]. Despite significant advances   in   surgery   and classic adjuvant   chemotherapy,   patients   continue to experience recurrence, metastatic disease, and ultimately experience death[2]. During the past decades, Traditional Chinese medicine (TCM), especially natural products derived from Chinese herbal medicines, have been identified with emerging anti-cancer activities in different human cancers[3]. In fact, TCM has been reported to prevent and treat CRC progression through modulating multiple targets or pathways[4,5]. As a natural anti-inflammatory phytomedicine, Tripterygium wilfordii   has   the   functions   of   activating   blood circulation, clearing collaterals, reducing swelling, and relieving pain. Interestingly, convincing evidence has indicated that multi-glycoside of Tripterygium wilfordii (GTW), extracted from the toot of Tripterygium wilfordii, might promote the apoptosis of epithelial ovarian cancer cells and enhance their sensitivity to cisplatin[6].   However, the specific mechanism of GTW on CRC cell biological behaviors has not been clarified.

Currently, some researchers have suggested that Long   Non-Coding   Ribonucleic   Acids   (lncRNAs), a class of evolutionarily conserved non-coding RNAs with lengths exceeding 200   nucleotides, were   aberrantly   expressed   in    various    tumors, and acted as the competitive endogenous RNA (ceRNA) of microRNAs (miRNAs) to control the occurrence and development of tumors[7]. Notably, some recent literature has implied that LncRNA expression, PRR34-Antisense RNA 1   (PRR34- AS1) was obviously upregulated in Hepatocellular Carcinoma (HCC) cell lines, and its upregulation might boost tumor cell growth and metastasis via regulating several miRNAs[8,9]. Here, based on StarBase analysis, there are some binding sites between PRR34-AS1 and microRNA (miR)-3614- 5p. Beyond that, miR-3614-5p has been confirmed as an underlying novel biomarker for CRC and participates in the modulation of cell migration[10,11]. Therefore, the purpose of this study is to validate the role of GTW block CRC progression via the PRR34-AS1/miR-3614-5p axis.

Materials and Methods

Reagents:

Human CRC cell line Cancer coli-2 (Caco-2) was provided by HonSun Biological (Shanghai, China). GTW (10 mg×100 tablets/bottle) was purchased from Meitong Pharmaceutical (Jiangsu, China). Invitrogen (Carlsbad, California,   USA)   provided the Total RNA Isolation Reagent (TRIzol) reagent and Lipofectamine™ 2000. Reverse   transcription and fluorescent quantitative Polymerase Chain Reaction (PCR) reagents were acquired from Tiangen (Beijing, China). GenePharma (Shanghai, China) offered miR-Negative   Control   (NC), mimics of miR-3614-5p, small interfering (si) NC, si-PRR34-AS1, plasmid cloning Deoxyribonucleic Acid (pcDNA), and pcDNA-PRR34-AS1. Solarbio (Beijing, China) supplied Cell Counting Kit-8 (CCK-8) reagent, cell apoptosis detection kit, and dual-luciferase activity detection reagent. Promega Corporation (Madison, WI, USA) provided dual- luciferase technology (pmirGLO)   gene   vectors. Cell Signaling Technology (CST) (Danvers, Massachusetts, USA) offered rabbit anti-human cleaved-caspase-3 antibody and Horseradish Peroxidase (HRP) labeled goat anti-rabbit Immunoglobulin G (IgG) secondary antibodies.

Method:

Cell treatment: According to the previous description[12], 1×104 Caco-2   cells   were   exposed to GTW at various doses for 24 h, recorded at 0, 20, 40 and 80 μmol/l and was considered to be GTW group. For cell transfection, which included lipofectamine method, si-NC or   si-PRR34-AS1 were introduced   into   Caco-2   cells   respectively and was named   as   si-NC   or   si-PRR34-AS1 group. Simultaneously, the research conducted transfection   of    pcDNA   or    pcDNA-PRR34-AS1 in Caco-2 cells, followed by treatment with 80 μmol/l GTW for 24 h, generating GTW+pcDNA or GTW+pcDNA-PRR34-AS1 group.

CCK-8 assay:

After being treated and collected in 96-well plates, 3×103 Caco-2 cells were mixed with 10 μl CCK-8 solution at 37° for 2 h. Finally, the samples from different groups were monitored under a microplate reader at 450 nm.

Clone formation assay:

500 Caco-2 cells were cultured in 6-well plates in an incubator for 14 d. Then, cells were subjected to 400 μl of 1 % paraformaldehyde fixing agent. The sample number from each group was assessed using a microscope by staining with 1 % crystal violet staining solution (≥50 cells were regarded as 1 clone).

Flow cytometry:

After being processed with 0.25 % trypsin, Caco- 2 cells were subjected to centrifugation at 3 000 r/min for 6 min, re-suspending in binding buffer, and dual-staining with AnnexinV Fluorescein Isothiocyante (FITC) and Propidium Iodide (PI). After 15 min, apoptotic cells were determined using FACS Calibur flow cytometry within an hour.

Real-Time quantitative Reverse Transcription (qRT)-PCR assay:

Using TRIzol reagent, the total RNA from Caco-2 cells was extracted. Then, 2 μg RNA was reverse transcribed to   complementary   DNA   (cDNA), which was used to carry out qRT-PCR reaction. Finally, 2-??Ct method analyzed the obtained data. Dual-luciferase reporter assay: At first, based on StarBase analysis, base complementarity between PRR34-AS1 and miR-3614-5p was acquired. Subsequently,   Wild-Type   (WT)-PRR34-AS1   and its corresponding Mutant (MUT) were synthesized and respectively inserted into pmirGLO vectors. Then, Caco-2 cell transfection was implemented with these vectors and miR-Negative Control (NC) or miR-3614-5p for 48 h, followed by analysis of cellular luciferase activity.

Western   blotting   analysis: After   the   addition   of 400 μl Radioimmunoprecipitation Assay (RIPA) buffer, Caco-2 cell proteins were acquired and denatured in boiling water for 10   min.   Then, these samples were subjected to Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE),   transmembrane,    and    blocked    for 2 h. After incubation with primary antibodies of cleaved-caspase3 (1:1000) and Glyceraldehyde 3-Phosphate Dehydrogenase   (GAPDH)   (1:3000), the gray value was analyzed using Image J software before adding secondary antibodies (1:5000) to membranes.

Statistical analysis:

In this research, results were analyzed according to Statistical Package for Social Sciences (SPSS) 21.0 version. p<0.05 was considered to be the significant difference. Meanwhile, measured data were presented as (x? ±s) and compared with t-test and One-Way Analysis of Variance (ANOVA).

Results and Discussion

The effects of GTW treatment on Caco-2 cell proliferation and apoptosis was studied. Based on the data displayed in fig. 1A, fig. 1B and Table 1, cell proliferation,   inhibition   rates,   apoptosis rate, and cleaved-caspase-3 protein level were obviously improved with increased GTW dose (p<0.05), and cell colony number was apparently reduced (p<0.05).

GTW concentration (µmol/l) Inhibition rate Apoptosis rate Colony number Cleaved-caspase-3
0 0.00±0.00 7.81±0.57 121.78±7.16 0.15±0.02
20 15.35±1.28* 12.90±0.84* 100.11±4.15* 0.28±0.03*
40 37.25±2.01*# 18.11±0.93*# 78.22±4.34*# 0.51±0.05*#
80 56.31±1.72*#& 24.02±1.60*#& 51.78±3.88*#& 0.74±0.05*#&
F 2540.633 390.867 315.92 386.667
p <0.05 <0.05 <0.05 <0.05

Table 1: Effect of GTW on Caco-2 Cell Proliferation, Inhibition Rates And Apoptosis (X?±s, n=9)

apoptosis

Fig. 1: Effect of GTW on (A): Caco-2 cell apoptosis and (B): Cleaved-caspase-3 protein expression

Further, the effects of GTW treatment on PRR34- AS1   and   miR-3614-5p   expression   in   Caco-2 cells were also studied. The results from Table 2 exhibited that PRR34-AS1 content gradually decreased in Caco-2   cells   after   GTW   treatment in   concentration-dependent   ways    (p<0.05), whereas miR-3614-5p expression was found to be reinforced (p<0.05).

GTW concentration (μmol/l) PRR34-AS1 miR-3614-5p
0 1.00±0.00 1.00±0.00
20 0.76±0.06* 1.53±0.08*
40 0.51±0.05*# 2.23±0.10*#
80 0.26±0.03*#& 3.62±0.16*#&
F 522.985 1103.457
p <0.05 <0.05

Table 2: Effect of GTW on PRR34-AS1 expression and miR-3614-5p expression (X?±s, n=9)

It can be observed that PRR34-AS1 directly targeted miR-3614-5p   as   it   has   binding   sites with miR-3614-5p (fig. 2). In the experiment of cells co-transfected with WT-PRR34-AS1, cell luciferase activity in miR-3614-5p group was clearly reduced (p<0.05), as shown in Table 3. These results indicated that PRR34-AS1 had a targeted regulation with miR-3614-5p.

Groups WT-PRR34-AS1 MUT-PRR34-AS1
miR-NC 0.93±0.08 0.96±0.09
miR-3614-5p 0.36±0.04* 1.01±0.12
t 19.118 1
p <0.05 0.332

Table 3: Dual Luciferase Experimental Activity (X?±s, n=9)

Complementary

Fig. 2: Complementary sequences of PRR34-AS1 and miR-3614-5p

Effects of PRR34-AS1 downregulation on Caco- 2 cell proliferation and apoptosis was explored. According   to   the   data   illustrated   in   fig.   3A, fig. 3B and Table 4, miR-3614-5p expression, proliferation inhibition rates, apoptosis rate, and cleaved-caspase-3 protein were increased in the si- PRR34-AS1 group, and cell colony number was decreased (p<0.05).

Groups PRR34-AS1 miR-3614-5p Proliferation inhibition rate Apoptosis rate The number of cell colonies Cleaved-caspase3
si-NC 1.00±0.00 1.00±0.00 0.00±0.00 7.79±0.57 122.33±7.09 0.16±0.02
si-PRR34-AS1 0.36±0.03* 3.31±0.08* 52.29±2.72* 21.57±1.03* 65.56±2.27* 0.58±0.05*
t 64 86.625 57.673 35.117 22.877 23.398
p <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

Table 4: Effect of PRR34-AS1 Downregulation on Caco-2 Cell Proliferation and Apoptosis (X?±s, n=9)

Accelerated

Fig. 3: Effect of PRR34-AS1 on (A): Accelerated Caco-2 cell apoptosis and (B): Cleaved-caspase-3 protein

PRR34-AS1 regulated GTW-mediated Caco-2 cell proliferation and apoptosis was analyzed.

Based on the results displayed in fig. 4A, fig. 4B and Table 5, relative to the GTW+pcDNA group, miR- 3614-5p content, proliferation inhibition rates, apoptosis rate, and cleaved-caspase-3 level were elevated in the si-PRR34-AS1 group (p<0.05), cell colony number was reduced (p<0.05).

Groups PRR34-AS1 miR-3614-5p Inhibition rate Apoptosis rate Cell colony number Cleaved-caspase3
GTW+pcDNA 1.00±0.00 1.00±0.00 56.07±2.88 24.15±1.49 53.44±1.95 0.73±0.07
GTW+pcDNA-PRR34-AS1 2.76±0.11* 0.39±0.04* 23.42±1.45* 13.79±1.08* 91.78±4.78* 0.34±0.04*
t 48 45.75 30.378 16.889 22.28 14.512
p <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

Table 5: PRR34-AS1 Regulated GTW-Mediated Caco-2 Cell Proliferation and Apoptosis (X?±s, n=9)

Cleaved

Fig. 4: Regulation of PRR34-AS1, (A): Reversed the promoting effect of GTW on Caco-2 cell apoptosis and (B): Cleaved-caspase-3 protein expression

With the advancement of medical technology, molecular targeted therapy has become the focus of research, but the morbidity and mortality of CRC are still increasing annually[13]. Convincing evidence shows that the active ingredients extracted from TCM can inhibit tumor cell proliferation and metastasis at different stages of tumorigenesis[14]. Interestingly, many laboratory works have found that lncRNAs-miRNA-messenger RNA (mRNA) regulatory networks could be involved of the progression and development in different human tumors, containing CRC[15]. However, whether lncRNAs might be a potential target of TCM for CRC treatment needs to be further explored.

Notably, recent literature has suggested that GTW, as a Chinese traditional patent   medicine,   that might enhance the cisplatin sensitivity of ovarian cancer cells by inhibiting the Phosphatidylinositol 3-Kinase/Protein   Kinase   B/Nuclear   Factor   kappa B (PI3K/Akt/NF-κB) signaling pathway and thus inducing apoptosis[12]. Herein, our data identified that the CRC   cell   proliferation   inhibition   rate was increased and clone formation number was reduced   with   the   increase   in   the   concentration of GTW, implying that GTW might inhibit CRC cell proliferation   ability.   Beyond   that,   it   has been reported that caspase-3 belongs to the apoptotic execution factor, which might facilitate apoptosis when activated[16,17]. In this work, CRC cell apoptosis rate and cleaved-caspase 3   level were upregulated in GTW-induced CRC cells in a concentration-dependent manner, verifying the promotion of GTW on CRC cell apoptosis. These above findings discovered the anti-tumor role of GTW on CRC development.

Previous literature has revealed that dysregulated PRR34-AS1 might be correlated with many tumor processes. For   example,   PRR34-AS1   expression is   upregulated   in   acute   myeloid   leukemia   and its expression is associated with poor patient prognosis[18]. Meanwhile, PRR34-AS1 has been reported as an aging-related lncRNA, which might perform the early breast cancer diagnosis and therapeutic target[19]. Furthermore, it has been widely accepted that PRR34-AS1 mainly serves as miRNA sponges to exert diverse biological roles[8,9]. Here, we preliminarily demonstrated that PRR34- AS1 can target binding miR-3614-5p. Some studies have indicated that abnormally expressed miR- 3614-5p might suppress the growth and metastasis of multiple tumors, containing CRC cells[10,11]. Herein, our study’s first step documented that PRR34-AS1 expression was   significantly   reduced in GTW-triggered CRC cells, and miR-3614-5p content was increased. Simultaneously, functional analysis discovered that down-regulation of PRR34-AS1 might nullify GTW-mediated proliferation inhibition and   apoptosis   promotion in CRC cells, whereas the phenomenon was partly counteracted by interacting with miR-3614-5p. It is suggested that the anti-cancer effect of GTW on CRC was mediated by PRR34-AS1/miR-3614-5p axis, which provided potential targets of GTW in the treatment of CRC.

In summary, GTW might block CRC cell proliferation and boost apoptosis by inhibiting PRR34-AS1 expression and promoting miR-3614- 5p level. These results suggested that PRR34-AS1 and miR-3614-5p may be potential targets for molecular targeting of CRC, which may lay the experimental foundation for further revealing the molecular mechanism of GTW in CRC.

Acknowledgements:

Fanlu Meng and Yanbo Wang contribute same to this work.

Conflict of interests:

The authors declared no conflict of interests.

References