Retracted: Targeted Regulation of miR-26a on PTEN to Affect Proliferation and Apoptosis of Prostate Cancer Cells

Abstract

Cancer Biotherapy and Radiopharmaceuticals

officially retracts the paper entitled, “Targeted Regulation of miR-26a on PTEN to Affect Proliferation and Apoptosis of Prostate Cancer Cells” by Weilu Li, Yongjun Jiang, Xia Wu, and Fucun Yang (Cancer Biother Radiopharm 2019;34(7):480–485; doi: 10.1089/cbr.2018.2664) due to the discovery that the paper was submitted from a paper mill which is a violation of the journal's standard protocols.

The Editor and Publisher of Cancer Biotherapy and Radiopharmaceuticals are committed to preserving the scientific literature and the community it serves and does not tolerate any violations of scientific misconduct.

Introduction

Prostate cancer (PC) is one of the common malignant tumors found in male urinary system, and is the sixth most popular causing second higher mortality among all male malignant tumors.¹ With aging population, changes of lifestyle, diet, habitat, incidence of PC is increasing these years, and it has become the most common malignant tumor in male reproductive–urinary system in China. Currently, its incidence in China is 60.3 per 100,000, and the mortality rate is 26.6 per 100,000.²

Phosphatase and tensin homology deleted on chromosome 10 (PTEN) was first discovered in 1997, and is the only tumor suppressor gene with both phosphate lipase and phosphatase activities discovered so far. It can negatively regulate PI3K/AKT signal pathway, which can facilitate cell proliferation and survival, thus modulating various biological processes, including cell proliferation, apoptosis, migration, and differentiation.³ Downregulation or dysfunction of PTEN is correlated with occurrence of various tumors, including pulmonary carcinoma,⁴ gastric cancer,⁵ and esophageal carcinoma.⁶ Previous study showed significantly decreased PTEN expression in PC tissues,⁷ indicating the tumor suppressor role of PTEN in PC pathogenesis. Partial or complete deficit of PTEN function aggravates PC disease condition, leading to fetal progression.⁸

MicroRNA (miR) is one single stranded noncoding small RNA molecule with 20 ∼ 24 nucleotide length with endogenous expression. It can affect mRNA stability, and cause mRNA degradation or mRNA translation inhibition through complementary binding onto 3′-untranslated region (3′-UTR) of target gene mRNA, thus negatively regulating gene expression at transcriptional level.⁹ Increasing evidence showed that abnormal expression or function of miR might play a role of oncogene or tumor suppressor gene in PC pathogenesis.¹⁰ A study showed significantly elevated miR-26a expression in PC tumor tissues compared with normal control.¹¹ Bioinformatics analysis showed the existence of complementary binding sites between miR-26a and 3′-UTR of PTEN mRNA. This study thus investigated if miR-26a played a role in regulating PTEN expression and affecting proliferation and apoptosis of PC-3 cancer cells.

Materials and Methods

Reagent and materials

Human PC-3 cell line was purchased from Cell Bank, Chinese Academy of Science. Dulbecco's Modified Eagle Medium (DMEM) culture medium, fetal bovine serum (FBS), and penicillin-streptomycin were purchased from Gibco. Lipofectamine 2000 liposome transfection reagent was purchased from Invitrogen. ReverTra Ace qPCR RT Kit and SYBR Green dye were purchased from Toyobo (Japan). Mouse anti-PTEN and anti-p-AKT antibody were purchased from Abcam. Rabbit anti-p-FoxO3a and p27Kip1 antibody were purchased from CST. Horseradish peroxidase (HRP)-labeled goat antirabbit and rabbit antimouse secondary antibodies were all purchased from eBioscience. F Annexin V/PI apoptotic kit was purchased from Yusheng (China). Dual-luciferase reporter assay system and pGL3-promoter were purchased from Promega.

Clinical information

A total of 58 PC patients who were diagnosed and treated in Linyi Cancer Hospital (Shandong, China) from March 2015 to May 2016 were recruited. All patients were confirmed as having PC by biopsy examination. Patients aged from 61 to 76 years (average age = 62.8 years). Based on TNM (TNM Classification of Malignant Tumors) stage criteria by UICC, there were 18, 12, 12, and 16 cases of T₁, T₂, T₃, and T₄ patients. Another cohort of 32 benign prostatic hyperplasia (BPH) patients were recruited as the control group (age 63–75 years, average age = 63.3 years). The collection of sample obtained informed consents from all patients, and has been approved by the ethical committee of Linyi Cancer Hospital.

Cell culture

Human prostate carcinoma cell line PC-3 was incubated in DMEM containing 10% FBS and 1% penicillin-streptomycin, and was cultured in 37°C incubator with 5% CO₂. Culture medium was changed every 2 ∼ 3 d. Those cells at log-growth phase with good status were used in experiments.

Construction of luciferase reporter gene

Using HEK293 genome as a template, full-length fragment of 3′-UTR of PTEN gene was amplified. Polymerase chain reaction (PCR) products were purified from agarose gel, and were ligated into pGL-3M luciferase reporter plasmid after enzymatic digestion. Recombinant plasmid was then used to transform DH5α competent cells. Positive clones with primary screening were selected for further cell transfection and following experiments.

Luciferase reporter gene assay

Lipofectamine 2000 was used to transfect HEK293 cells with 500 ng pGL3-PTEN-3′UTR plasmid, 30 nmol miR-26a oligonucleotide mimic (or mimic control), and 30 ng pRL-TK. After 6 h transfection, normal DMEM containing 10% FBS and 1% streptomycin-penicillin was used. After 48 h continuous incubation, dual-luciferase assay was performed. In brief, cells were washed twice in phosphate buffered saline (PBS), with the addition of 100 μL Passive Lysis Buffer (PLB). With vortex at room temperature for 30 min, the mixture was centrifuged at 1000 rpm for 10 min. Twenty microliters cell lysate was mixed with 100 μL LAR II. Fluorescent value I was measured in a microplate reader. The enzymatic reaction was stopped in 100 μL Stop & Glo, followed by quantification of fluorescent value II. The relative expression level of reporter gene was calculated as the ratio of fluorescent value I/fluorescent value II.

Construction of overexpression plasmid and cell transfection

Using pIRES as eukaryotic expression plasmid, and Xho I and BamH I as restriction digestion enzyme, amplification primer of PTEN was synthesized using primer 6.0 based on mRNA sequence of PTEN in Gene Bank (Forward, 5′-CAGAC ATGAC AGCCA TCATC A-3′; Reverse, 5′-ATTCA GACTT TTGTA ATTTG TG-3′). PTEN gene was amplified based on cDNA template. Agarose gel electrophoresis was used to determine targeted fragments, which were extracted by gel extraction kit. After ligating with vector, recombinant plasmid was used to transfect competent cell JM109. Ampicillin-containing culture dish was used to collect positive bacterial strain, which was further amplified and extracted for recombinant plasmid containing targeted fragment. Gene sequencing was performed to confirm the correct insertion of target sequence into the plasmid. Lipofectamine 2000 was used to transfect inhibitor negative control (NC), miR-26a inhibitor, nonsense-controlled plasmid (pIRES2-Scramble), or overexpression plasmid (pIRES2-PTEN) was transfected into vascular smooth muscle cell (VSMC) in five groups: inhibitor NC, miR-26a inhibitor, pIRES2-Scramble, pIRES2-PTEN, and miR-26a inhibitor+pIRES2-PTEN group. Forty-eight hours later cells were collected for assay.

quantitative real-time PCR (qRT-PCR) for gene expression

cDNA was synthesized in a 10 μL system, including 1 μg total RNA, 2 μL RT buffer (5 × ), 0.5 μL oligo dT+random primer mix, 0.5 μL reverse transcription (RT) enzyme mix, 0.5 μL RNase inhibitor, and ddH₂O. The reaction conditions were as follows: 37°C for 15 min, followed by 98°C for 5 min. cDNA products were maintained at −20°C fridge. Using cDNA as the template, PCR amplification was performed under the direction of TaqDNA polymerase using primers (miR-26aP_F: 5′-TTGGA TCCGT CAGAA ATTCT CTCCC GAGG-3′; miR-26aP_R: 5′-GGTCT AGATG TGAAC TCTGG TGTTG GTGC-3′; U6P_F: 5′-ATTGG AACGA TACAG AGAAG ATT-3′; U6P_R: 5′-GGAAC GCTTC ACGAA TTTG-3′; PTENP_F: 5′-CTGGT CTGCC AGCTA AAGGT-3′; PTENP_R: 5′-TCACC ACACA CAGGT AACGG-3′; p27Kip1P_F: 5′-ATCAC AAACC CCTAG AGGGC A-3′; p27Kip1P_R: 5′-GGGTC TGTAG TAGAA CTCGG G-3′; β-actinP_F: 5′-GAACC CTAAG GCCAA C-3′; β-actinP_R: 5′-TGTCA CGCAC GATTT CC-3′). In a PCR system with 10 μL total volume, we added 5.0 μL 2XSYBR Green Mixture, 1.0 μL of forward/reverse primer (at 2.5 μm/L), 1 μL cDNA, and 3.0 μL ddH₂O. PCR conditions were as follows: 95°C for 15 s, 60°C for 30 s, and 74°C for 30 s. The reaction was performed on Bio-Rad CFX96 fluorescent quantitative PCR cycler for 40 cycles to collect fluorescent data.

Western blot

Cells were lysed by radioimmunoprecipitation assay buffer (RIPA) lysis buffer. Protein concentration in the supernatant was quantified. Sixty micrograms of protein samples was separated on 8% SDS-PAGE for 3 h, and was transferred to polyvinylidene difluoride membrane for 1.5 h. The membrane was blocked in 5% defatted milk powder for 60 min, followed by primary antibody (anti-PTEN at 1:300, anti-p-AKT at 1:100, anti-p-FoxO3a at 1:100, anti-p27Kip1 at 1:300, or anti-β-actin at 1:800) incubation at 4°C overnight. By phosphate buffered saline with tween 20 (PBST) washing (three times), HRP-labeled secondary antibody (1:10,000 dilution) was added for 60 min incubation. After rinsing thrice with PBST, enhanced chemiluminescence (ECL) reagent was added for 2 ∼ 3 min dark incubation. The membrane was then exposed in dark and scanned for data analysis.

Flow cytometry for cell apoptosis

Cells were collected by centrifugation, and were then washed in PBS twice. Hundred microliters binding buffer was used to resuspend cells. The mixture was added with 5 μL Annexin V-FITC and 5 μL propidium iodide (PI) staining solution. After gentle mixing, the mixture was incubated in dark for 10 min, with the addition of 400 μL 1X Binding Buffer, and was immediately loaded for online testing in Beckman FC500MCL flow cytometry apparatus.

Flow cytometry for cell cycle analysis

Cells were digested by trypsin and were washed twice in PBS. Cells were then fixed in 75% ethanol at 4°C overnight. With twice rinsing in PBS, cells were stained in dark using 100 μg/mL PI dye containing 20 μg/mL RNase A, 0.1% Triton X-100 for 20 min at 4°C, and were immediately loaded for flow cytometry assay.

Flow cytometry for Ki-67 expression

Cells were collected from all groups, and were rinsed twice in PBS containing 2% FBS. After fixation in 4% paraformaldehyde for 20 min, cells were treated using PBS containing 0.1% Triton X-100. FITC-labeled Ki-67 antibody was added for 4°C dark incubation for 30 min, followed by twice rinsing in PBS containing 2% FBS. Cells were loaded for online testing in Beckman FC500MCL flow cytometry apparatus.

Statistical analysis

SPSS18.0 software was used for data analysis. Measurement data were presented as mean ± standard deviation. Student's t-test was used to compare measurement data between groups. A statistical significance was defined when p < 0.05.

Results

miR-26a and PTEN expression in PC tissues

quantitative RT-PCR (qRT-PCR) results showed significantly lower PTEN mRNA expression in PC tumor tissues compared with BPH tissues (Fig. 1A), while miR-26a expression was significantly higher (Fig. 1A) and was related to T stage. Western blot results showed significantly lower PTEN protein expression in PC tissues compared with BPH patients (Fig. 1B). Lower expression level was observed in patients with late T stage. Results showed a possible role of abnormally elevated miR-26a expression in decreasing PTEN expression and facilitating PC occurrence.

FIG. 1.

Elevated miR-26a and lower PTEN expression in PC tissues. (A) qRT-PCR for mRNA expression of miR-26a and PTEN. (B) Western blot for PTEN protein expression. *p < 0.05 compared with BPH. BPH, benign prostatic hyperplasia; PC, prostate cancer; PTEN, chromosome 10; qRT-PCR, quantitative real-time PCR.

Targeted regulation of PTEN expression by miR-26a

Online prediction by microRNA.org for target gene showed the existence of targeted binding sites between miR-26a and 3′-UTR of PTEN mRNA (Fig. 2A). Dual-luciferase reporter gene assay showed that the transfection of miR-26a mimic significantly depressed relative luciferase activity inside HEK293 cells (Fig. 2B) and PTEN expression in PC-3 cells (Fig. 2C, D). The transfection of mimic NC, however, had no significant effects on relative luciferase activity of HEK293 cells and PTEN expression in PC-3 cells, indicating that miR-26a could target and function on 3′-UTR of PTEN mRNA and regulate its expression.

FIG. 2.

miR-26a targeted and inhibited PTEN expression. (A) Binding sites between miR-26a and 3′-UTR of PTEN mRNA; (B) dual-luciferase reporter gene assay; (C) qRT-PCR for miR-26a and PTEN expression; (D) Western blot for PTEN protein expression. *p < 0.05 compared with mimic NC. NC, negative control; 3′-UTR, 3′-untranslated region.

miR-26a downregulation facilitated PC-3 apoptosis and inhibited cell proliferation

Transfection of miR-26a inhibitor and/or overexpression of PTEN significantly depressed phosphorylation activity of AKT (Fig. 3B), and remarkably suppressed phosphorylation level of FoxO3a (Fig. 3B), accompanied with elevated p27Kip1 expression (Fig. 3A, B), more cell apoptosis (Fig. 3C), inhibition of proliferation ability (Fig. 3D), and arresting of cell cycle at G0/G1 phase (Fig. 3E).

FIG. 3.

Downregulation of miR-26a facilitated PC-3 cell apoptosis and inhibited proliferation. (A) qRT-PCR for gene expression; (B) Western blot for protein expression; (C) flow cytometry for cell apoptosis; (D) flow cytometry for Ki-67 expression; (E) flow cytometry for cell cycle. *p < 0.05 compared between miR-26a inhibitor and inhibitor NC group; #p < 0.05 compared between pIRES2-PTEN and pIRES2-Scramble group; ▵p < 0.05 compared between miR-26a inhibitor+pIRES2-PTEN and inhibitor NC group; &p < 0.05 compared between miR-26a inhibitor+pIRES2-PTEN and pIRES2-Scramble group.

Discussion

PC is one of the common urinary tumors frequently found in aged males, occurring in >90% people between 60 and 80 years of age.¹² It is estimated that 1,112,000 people were newly diagnosed with PC in 2012, with 759,000 cases in developed countries and 353,000 patients in underdeveloped countries.¹³ A total of 308,000 people died from PC (142,000 in developed countries and 166,000 in underdeveloped countries). The distribution of PC showed significant geographic difference, as developed countries had ∼25-fold higher incidence than that observed in worldwide average level. China and other Asian countries had lower incidence of PC than Western countries, but still have increasing incidence. It is reported that overall incidence of PC was 69.5 per 100,000 people in developed countries, with 10.0 per 100,000 mortality rate, and 14.5 per 100,000 incidence with 6.6 per 100,000 mortality in underdeveloped countries.¹³ PC is mostly derived from peripheral band of prostate gland with slow progression and insidious onset, thus lacking significant early symptoms. It is usually discovered during screening by abnormal elevation of prostate-specific antigen (PSA0 and/or prostate gland lesion in rectal examination).¹⁴ When symptoms are developed, patients are mostly at advanced or terminal stage. Currently surgery, hormone, and radiotherapy are major approaches in treating PC, but there is difficulty in treating late-stage cancer, recurrent disease, and hormone-independent tumors, with unsatisfactory efficacy and higher mortality.

PI3K/AKT signal transduction pathway exists widely in cells and participates in cell growth, proliferation, cycle, apoptosis, and differentiation, and plays an important role in occurrence, progression, treatment, and prognosis of malignant tumors. PI3K mainly consists of catalytic subunit P110 and regulatory subunit P85. Under activation of extracellular stimulus signals such as growth factor and hormones, PI3K can be recruited into adjacent site of plasma membrane to catalyze the phosphorylation of substrate phosphatidylinositol 4,5-diphosphate (PIP2) to generate phosphatidylinositol 3,4,5-triphosphate (PIP3), which recruits AKT from cytoplasm to the membrane. Under the function of phosphoinositide-dependent protein kinase (PDK), Ser473 and Thr308 sites of Ser/Thr protein kinase AKT were phosphorylated to further transduce signals downstream, thus modulating expression and function of critical proteins regulating cell proliferation, cycle, and apoptosis, thus exerting functions on facilitating cell growth, proliferation, and antagonizing apoptosis. FoxO3a is an important member of forkhead transcription factor (FKHR) family, and can upregulate expression of proapoptotic genes such as cyclin-dependent kinase inhibitor (CKI) p27Kip1,¹⁵ Bim,¹⁶ and PUMA,¹⁷ or downregulate antiapoptotic gene FLIP expression,¹⁸ thus playing important roles in inducing cell apoptosis. As one cell cycle regulatory factor p27Kip1 bind tightly with cell cycle protein E-CDK2, and D-CDK4 to inhibit their activity, thus impeding cell entry into S phase, and arresting cell cycle at G0/G1 phase.¹⁹ FoxO3a function is under negative regulation of PI3K/AKT signal pathway. Phosphorylated AKT further phosphorylates FoxO3a, depressing its affinity on nuclear DNA while enhancing affinity on cytoplasmic 14-3-3 protein. It can also facilitate the translocation of FoxO3a from nucleus to cytoplasm, thus weakening the regulatory effect on nuclear target genes.²⁰ With lower activity of PI3K/AKT signal pathway and inhibited FoxO3a phosphorylation, dephosphorylated FoxO3a translocated from cytoplasm into the nucleus, where it initiated target gene transcription, translation and accelerated cell apoptosis.²¹ As one tumor suppressor gene, PTEN can inhibit phosphorylated activation of AKT and transduction of downstream signal pathway through dephosphorylation of PIP3.²² Moreover, PTEN can inhibit phosphatase activity of AKT through direct dissociation of phosphate group on serine/threonine residue, thus inhibiting PI3K/AKT signal pathway activity and regulating cell proliferation, cycle, and apoptosis.²³ Study showed significantly depressed PTEN expression in PC tumor tissues.⁷ Partial or complete loss of PTEN function aggravates disease condition of PC patients and leads to fetal progression.⁸ Study showed significantly elevated miR-26a expression in tumor tissues of PC patients compared with normal control.¹¹ Bioinformatics analysis showed complementary binding sites between 3′-UTR of PTEN mRNA and miR-26a. This study investigated if miR-26a played a role in regulating PTEN expression and affecting proliferation and apoptosis of PC-3 cell.

This study showed significantly lower PTEN expression in PC tissues and elevated miR-26a expression compared with BPH tissues. With more advanced TNM stage, PTEN expression was further suppressed while miR-26a expression was elevated, indicating a possible role of abnormally elevated miR-26a expression in suppressing PTEN expression and facilitating PC pathogenesis. Noh et al. found significantly lowered PTEN expression in PC tumor tissues, with unfavorable prognosis coupled with lower expression level.²⁴ Gao et al. revealed the correlation between expressional/functional deficit and high pathology grade, high Gleason score and high recurrent rate of PC.²⁵ Ahearn et al. found complete or partial absence of PTEN function severely aggravated disease condition of PC and caused fetal progression.⁸ This study was consistent with Nodouzi et al. who showed remarkably lower PTEN expression in PC tissues compared with BPH tissues.⁷ Tian et al. found significantly elevated miR-26a expression in PC-3 cells compared with normal prostate epidermal cell PNT1B.²⁶ Mahn et al. found significantly higher miR-26a in tumor tissues and peripheral blood of PC patients compared with BPH individuals, plus lowered miR-26a expression after surgical removal of tumors.²⁷ This study found significantly higher miR-26a expression in PC tissues compared with those in BPH tissues, consistent with Nodouzi et al.⁷ Dual-luciferase reporter gene assay showed that the transfection of miR-26a mimic significantly depressed relative luciferase activity of HEK293 cells and PTEN expression in PC-3 cells, demonstrating the target regulation on PTEN expression by miR-26a. Further study found that transfection of miR-26a inhibitor and/or overexpression of PTEN significantly depressed phosphorylation activity of AKT, decreased phosphorylation degree of FoxO3a, and elevated p27Kip1 expression, enhanced cell apoptosis, weakened proliferation activity, and arrested cell cycle at G0/G1 phase.

Conclusion

PC tumor tissues had significantly elevated miR-26a expression and lowered PTEN expression. miR-26a targeted and inhibited PTEN expression, potentiated activity of PI3K/AKT signal pathway and phosphorylation degree of FoxO3a, downregulated p27Kip1 expression, and decreased cell apoptosis, and facilitated cell proliferation, probably playing a role in facilitating PC pathogenesis.

Footnotes

Disclosure Statement

No competing financial interests exist.

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