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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 15  |  Issue : 1  |  Page : 32-37

Potential using of microRNA-34A in combination with paclitaxel in colorectal cancer cells


1 Department of Biology, Science and Research Branch, Islamic Azad University; Stem Cell Technology Research Center, Tehran, Iran
2 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran
3 Department of Medical Genetics, Shahid Beheshti University of Medical Sciences, Tehran, Iran
4 Department of Hematology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran

Date of Web Publication13-Mar-2019

Correspondence Address:
Masoud Soleimani
Department of Hematology, School of Medical Sciences, Tarbiat Modares University, P.O. Box: 14115-331, Tehran
Iran
Dr. Shiva Irani
Department of Biology, Science and Research Branch, Islamic Azad University, P.O. Box: 1477893855, Tehran
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_267_17

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 > Abstract 


Background: MicroRNAs are small noncoding RNAs which modulate gene expression at different levels. It has been shown that downregulation of miR-34a occurs in varieties of cancers including colorectal cancer (CRC). In this study, we investigated the potential tumor inhibitory effects of miR-34a alone or in combination with paclitaxel in CRC cells.
Materials and Methods: SW480 cells were transduced with lentiviral overexpressed miR-34a. First, using 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay, the effect of miR-34a induction alone or in combination with paclitaxel on the cell viability and cell proliferation were estimated. Then, the expression level of target genes was measured using quantitative reverse transcription-polymerase chain reaction analysis. Eventually, the role of miR-34a and paclitaxel on cell cycle were determined with flow cytometry.
Results: Gene expression analysis showed that miR-34a downregulates the expression of BCL2 and SIRT1 genes at mRNA level. Furthermore, miR-34a has a potential to reduce cell viability and cell cycle arrest at G1 phase. Combination of paclitaxel with overexpression of miR-34a significantly decreased cell viability compared to cell treated with miR-34a or paclitaxel alone. Interestingly, a combination of miR-34a and paclitaxel arrested cell cycle at two phases.
Conclusion: Our results suggested that combination therapy of miR-34a and paclitaxel could be considered as the potential treatment of CRC.

Keywords: Combination therapy, microRNA-34a, paclitaxel, SW480 cells


How to cite this article:
Soltani-Sedeh H, Irani S, Mirfakhraie R, Soleimani M. Potential using of microRNA-34A in combination with paclitaxel in colorectal cancer cells. J Can Res Ther 2019;15:32-7

How to cite this URL:
Soltani-Sedeh H, Irani S, Mirfakhraie R, Soleimani M. Potential using of microRNA-34A in combination with paclitaxel in colorectal cancer cells. J Can Res Ther [serial online] 2019 [cited 2019 Aug 20];15:32-7. Available from: http://www.cancerjournal.net/text.asp?2019/15/1/32/230442




 > Introduction Top


Colorectal cancer (CRC) is the one of the most deadly cancer in male and female with 1.2 million new cases diagnosed every year.[1] Current therapies are not capable to cure CRC because they are not targeting key genes and signaling cascades involved in the development of CRC, so its treatment remaining elusive. Approximately 50% of CRC patients with locally advanced disease (AJCC Stages II to III) show cancer's recurrence during the courses of treatment and develop resistance to chemotherapy.[2]

MicroRNAs (miRNAs or miRs) are small noncoding RNAs, 19t to 25-nt length, which play a crucial role as gene expression modulators in multiple biological and pathological conditions. MiRNAs exert their effect on gene expression by binding to the corresponded mRNA (mostly 3'UTR region) and inhibiting the mRNA translation or stability[3] or inducing gene expression through RNA activation mechanism.[4] Based on their function in cancer development, miRNAs categorized as onco-miRNAs and tumor-suppressor-miRNAs. It is common that onco-miRs located in the regions that they are amplified, whereas tumor-suppressor-miRs positioned in the regions of genome that undergo deletion or epigenetic suppression.[5] However, it has been shown that some miRNAs have dual role in different cellular content, for example miR-371-373 which act as an onco-miR in testicular germ cell tumor by neutralizing p53-mediated CDK inhibition,[6] but by arresting cell cycle in another cell has tumor-suppressor-miR role.[7]

Accumulation of evidence has shown the deregulation of different miRNAs in colorectal and their role in carcinogenesis, progression, and metastases.[8],[9],[10],[11] Some of these miRNAs could be considered as biomarkers for diagnosis and prognosis or even predict response to a certain treatment.[12] Among these miRNAs, miR-34a which is downregulated in almost all cancers has potential therapeutic value in CRC.[13],[14] P53 exerts its tumor-suppressor features such as inducing apoptosis, cell cycle arrest, senescence, and inhibiting cancer cells proliferation by upregulating miR-34a expression.[13],[14] It is also worth to note that MRX34 is the first miRNA drug in cancer has terminated phase I clinical trials in patient with liver cancer (NCT01829971).[15]

Although the role of miR-34a in different cancers has been investigated, there are few studies addressing the function of miR-34a along with chemotherapy in CRC. Hence, the aim of this study was to evaluate the role of miR-34a in combination with paclitaxel in CRC cell line. Reinforced expression of miR-34a significantly decreased cell proliferation, cell viability, and cell cycle arrest through targeting SIRT1 and BCL2 genes. In addition, combination of miR-34a overexpression with paclitaxel drug enhanced the anticancer effect of paclitaxel.


 > Materials and Methods Top


Database prediction of miRNA targets

The miR34a targets were predicted using the algorithms TargetScan version 5.1 and microRNA. org. Then, biological processes of miR-34a and its targets were analyzed using the miRegulome database.

Cell culture

Human CRC SW480 cell line and human embryonic kidney (HEK) 293T cell line were obtained from Stem Cell Technology Research Center, Iran. SW480 cells were maintained in Roswell Park Memorial Institute medium 10% fetal bovine serum (FBS, Gibco) and 1% nonessential amino acids (Invitrogen, USA), penicillin (100U/mL, Gibco), streptomycin (0.1 mg/mL, Gibco), and L-glutamine (2 mM, Gibco). HEK293T were cultured in high glucose Dulbecco Modified Eagle Medium (DMEM, Gibco BRL, Grand Island, NY, USA) supplemented with 10% (FBS, Gibco), penicillin (100U/ml, Gibco), and streptomycin (0.1 mg/mL, Gibco). Cells were cultured under standard condition in 5% CO2 at 37°C.

Recombinant lentivirus generation and transduction

Lentiviral vectors expressing miR-34a (pLEX-jRED-TurboGFP-Puro-miR-34a) as well as helper plasmids (psPAX and pMD2.G) were gifted from Stem Cell Technology Research Center, Iran. Lenti vectors containing miR-34a or control were produced as previously described,[7] in brief HEK293T were transfected at ~60%–70% confluency using calcium-phosphate method. Sixteen hours posttransfection, cell culture were replaced and 24, 48, and 72 h later supernatants were collected, filtered through 0.22-m-pore-size filters and finally concentrated using ultracentrifuge at 50,000 ×g for 2.5 h at 4°C.

For transduction, 105 of SW480 cells were seeded in 24-well plate and directly exposed to lentiviruses at a multiplicity of infection of 8 for 16 h. For enhancing transduction efficacy, 6 mg/ml of Polybrene reagent was applied along with the viral pseudotypes. Cells were evaluated for transduction by inverted fluorescent microscope.

RNA extraction and cDNA synthesis

After 24, 48, and 72 h transduction, total RNA was extracted with RNeasy Protect Cell Mini Kit (QIAGEN) according to the manufacturer's protocol. Using RNAprotect Cell Reagent, RNAs were stabilized in cells without any need for washing or trypsinization.

cDNA was synthesis using random hexamers (for mRNA genes), stem-loop RT-specific primers (for miR-34a and SNORD47), and M-MLV Reverse Transcriptase (Promega, USA).

Quantitative real-time polymerase chain reaction

Real-time polymerase chain reaction (PCR) for mRNAs was performed using on ABI 157 PRISM 7500 Real-time PCR System (Applied Biosystems, USA) with 1 μl of cDNA product, 1 × Quantitect SYBR Green PCR Master Mix (Takara, Japan), and 0.5 μl of each forward and reverse primers. Then, the reactions were incubated at 95°C for 3 min, followed by 40 cycles of 95°C for 30 s and 60°C for 30 s. Quantitative reverse transcription-PCR (QRT-PCR) reactions were run in triplicate. β-actin was used as reference gene for mRNAs and the expression of target genes was evaluated relative to reference gene using the 2−ΔΔCT method. Expression level of miR-34a was measured by universal reverse primer and specific miR-34a forward primer using SYBR Green PCR Master Mix (Takara). The relative expression level of miR-34a was evaluated and normalized to endogenous expression of SNORD47 RNA as an internal control using the 2−ΔΔCT method. Primer sequences for real-time PCR are provided in [Table 1].
Table 1: The sequence of primers used for quantitative reverse transcription-polymerase chain reaction

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Determination of paclitaxel IC50 and cell viability

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay carried out for estimating IC50 of paclitaxel and cell viability. For determining IC50 of paclitaxel in SW480 cells, 3 × 104 cell were seeded in 96-well plate and treated with 30 nM to 70 nM of paclitaxel. MTT assay was done for each well 24 after treatment with paclitaxel according to the below protocol. In addition, cell viability was monitored 24, 48, and 72 hours after transduction with either pLEX-Control or pLEX-miR-34a (with or without paclitaxel treatment) using MTT assay. At the day of MTT assay, 0.5 mg/ml MTT was added to each well of a 96-well plate and the cells were incubated at 37°c for 4 h. The MTT medium mixture was discarded and 100 μl of dimethyl sulfoxide was added to extract the dye. Absorbance was measured at 570 nm using a multi-well spectrophotometer (Bio-Tek, USA). All reactions were performed in triplicate.

Cell cycle analysis

For analyzing cell cycle, SW480 cells were grown in 24 well plates and transduced with pLEX-miR-34a or pLEX-Control with or without paclitaxel treatment. Then, cells were harvested after 72 h after transduction and washed with PBS and fixed in 70% ethanol at 4°C for 2 h. After fixation, cells were washed twice with PBS before re-suspension in a solution of 50 μg/ml propidium iodide (PI) and 100 μg/ml RNase A. Cells were incubated with PI at 37°C for 1 h. Then, cellular DNA content was evaluated by flow cytometry and obtained data were analyzed using Flowjo program.

Statistical analysis

All experiments were performed at least three times, presented as mean ± standard deviation and analyzed by Student's t-test. P <0.05 or less was considered statistically significant. Asterisks (*), (**), and (***) in the figures indicate P < 0.05, 0.01, and 0.001, respectively.


 > Result Top


Paclitaxel IC50 in SW480 cells

Hence, various doses of paclitaxel were used and cell viability of SW480 cells determined by MTT assay. As it is shown in [Figure 1], treatment of SW480 cells with 30 nM to 70 nM of drug caused a steady decrease in cell viability. At the 50 nM paclitaxel concentration, cell viability reduced 50% compared to the control group. This concentration considered as IC50 and was applied for further examines.
Figure 1: Determination of paclitaxel's IC50 in SW480 cell line. Cells were treated with 30 nM to 70 nM of paclitaxel and MTT assay carried out at the 24 h after treatment. Fifty nM considered as IC50 of paclitaxel in SW480 cell line. Data were the averages of at least three independent runs; bars, standard deviation. (*) P < 0.05 (**) P < 0.01

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The effect of miR-34a and paclitaxel on cell viability and proliferation

To found out the effect of miR-34a on cell viability and proliferation, SW480 cells were transduced with either pLEX-miR-34a or pLEX-Control lentivirus vectors and then MTT assay carried out 24, 48, and 72 h after transduction. Approximately more than 60% of cells were transduced by pLEX-miR-34a or pLEX-Control [Figure 2]. As it is shown in [Figure 3], miR-34a reduces cell viability compared with empty vector in 48 h (P ≤ 0.05) and 72 h (P ≤ 0.001) posttransduction but not in 24 after miR-34a overexpression (P > 0.05). This delay could explain by the fact that blocking translation of a gene at pretranslational level would not affect the amount of proteins that already have translated and are presented in cells. These results are in line with the previous results and confirm that miR-34a could act as tumor-suppressor-miR in cancer cells by suppressing cell proliferation and viability.
Figure 2: Transduction of SW480 cells with pLEX-miR-34a (a) or pLEX-Control (b). Almost more 60% of cells were transduced

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Figure 3: Determination of cell viability for cells received only miR-34a or combination of miR-34a and paclitaxel 24, 48, and 72 h after treatments. Data were the averages of at least three independent runs; bars, standard deviation. (*) P < 0.05 (**), P < 0.01 and (***) P < 0.001

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When transduced cells were treated with paclitaxel, cell viability reduced significantly compared to pLEX-Control group and cells that only received miR-34a (P ≤ 0.05). The group has received both miR-34a and paclitaxel showed a statically significant reduction in cell viability in two time points 24 h (P ≤ 0.05) and 72 h (P ≤ 0.01) compared with the group that only received miR-34a. These data suggested that reinforced expression of miR-34a along with paclitaxel drug could decrease proliferation and cell viability of CRC cells in vitro more than when they are used separately [Figure 3].

The effect of miR-34a upregulation on the expression of target GENES

There are accumulated data that suggest several important genes involved in carcinogenesis and tumor development are direct target of miR-34a. Among the genes that proposed by Target Scan and miRegulome databases [Supplementary Table 1], BCL2 and SIRT1 were selected for further investigations.



First, the expression level of miR-34a 24, 48, and 72 h after transduction was determined. MiR-34a was successfully overexpressed in SW480 cells by our pLEX-miR-34a lentivector [Figure 4]a. Then, the expression level of two important targets, BCL2 and SIRT1 were evaluated at mentioned time points. As it was expected, miR-34a strongly downregulates its targets probably by directly binding to the 3'UTRs of their mRNAs [Figure 4]b. These data confirm previous results that BCL2 and SIRT1 oncogenes are direct targets of tumor-suppressor-miR-34a and provide a possible mechanism for tumor inhibitory function of miR-34a in CRC.
Figure 4: Evaluation of ectopic upregulation of miR-34a on the target genes expression by QRT-PCR. First, the level of miR-34a was estimated 24 h after transduction (a). Then, 24, 48, and 72 h after transduction expression level of BCL2 and SIRT1was evaluated (b). SNORD47 and β2M were used as internal controls for miR-34a and mRNAs, respectively. Columns, mean of three different experiments; bars, standard deviation P < 0.05

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It is worth to know that paclitaxel has no effect on expression of BCL2 and SIRT1 (P > 0.05). Although paclitaxel along with of miR-34a showed small change in the expression level of BCL2 and SIRT1, but results were not statically significant (P > 0.05) (data not showed).

Cell cycle analysis of SW480 cells treated with miR-34a or paclitaxel

It has been shown that exogenous expression of miR-34a arrested cell cycle at G1/G0 phase in nonsmall cell lung cancer cells.[16] However, the effect of ectopic expression of miR-34a on cell cycle in colorectal cells has not been cleared. As shown in [Figure 5]b, miR-34a causes cell cycle arrest in the G1/G0, compared to the control group [Figure 5]a.
Figure 5: Cell cycle analysis using flow cytometry 72 h after treatment. (a) Control cells that only received pLEX-Control vector. (b) G1 arrest of cells transduced with pLEX-miR-34a. (c) G2/M arrest of cells treated with paclitaxel. (d) G1/G2 arrest of cells that upregulated miR-34a and treated with paclitaxel

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In addition, the effect of paclitaxel on cell cycle, alone or with miR-34a, were assessed in SW480 cells. Paclitaxel alone causes cell cycle arrest in G2/M phase [Figure 5]c, but its combination with miR-34a cause cell cycle arrested in two phases; G1/G0 and G2/M equally [Figure 5]d. These data suggest that miR-34a could be considered as a negative regulator of cell cycle, especially in combination with chemotherapy to suppress cell cycle progression which is one of the important cancer hallmarks.


 > Discussion Top


It is apparent that one-third of the human coding genes are regulated by miRNAs at posttranscriptional level.[17] Altered expression levels of miRNAs have been identified in many pathological conditions such as cancers, and these irregulation expression of miRNAs could be considered for therapeutic or diagnosis purposes.[3],[5],[7],[9],[18],[19] However, our knowledge about the exact role of this deregulation with in each cancer type is scanty.

A growing body of evidence indicates that miR-34a is downregulated in many cancers by deletion or epigenetic mechanisms such as promoter hypermethylation.[20],[21] In addition, restore of miR-34a expression induces cancer cells apoptosis, cell cycle arrest, and even sensitivity to chemotherapies. MiR-34a is a p53 direct miRNA that targets many important oncogenes which resulted in inhibition of cancer progression. Hence, the function of miR-34a in different cancer types makes this miRNA an interesting candidate for considering as therapeutic or prognostic factor.

In the current study, the function of forced expression of miR-34a in combination with paclitaxel drug in SW480 CRC cell line was evaluated. First of all, the effect of miR-34a on cell viability and proliferation was estimated. SW480 cells that overexpressed miR-34a have more reduced cell viability and proliferation compared with cell with normal expression of miR-34a. In addition, cells viability decreased significantly in cells treated with both miR-34a and paclitaxel compared with cells received only miR-34a [Figure 3].

For finding out the effect of miR-34a in expression of its targets, two important targets, BCL2 and SIRT1, were selected from its validated targets [Supplementary Table 1]. Analysis of the gene expression with real-time PCR showed that overexpression of miR-34a reversely downregulates BCL2 and SIRT1 [Figure 4] expression. These results are in line with the previous results that miR-34a has complementary sequences in 3'UTR of BCL2 and SIRT1 which could be an explanation for miR-34a effects on cell viability and growth as it was showed in MTT assay. BCL2 is an antiapoptotic genes which its elevated expression has been reported in many cancer types.[22],[23] On the other hand, SIRT1 promotes tumor development by inducing epithelial-to-mesenchymal transition, cell growth, and metastasis.[24],[25] The expression of miR-34a target genes were not downregulated when cells treated with paclitaxel (data not shown). This could be explained by the fact that paclitaxel only inhibits depolymerization of microtubules and it is not involved in transcriptional machinery (10).

Finally, the effect of miR-34a and paclitaxel were evaluated on cell cycle. Flow cytometry analysis showed that miR-34a arrested SW480 cells in G0/G1 phase [Figure 5]b perhaps due to targeting genes involved in cell cycle such as CCND1, CDK2, CDK4, and CCNF2.[16],[26] However, paclitaxel arrested cell cycle at G2/M phase [Figure 5]c and combination of both miR-34a and paclitaxel resulted in accumulation of cells in both G1 and G2 phases [Figure 5]d. This is an interesting result which shows both factors can exert their effect on cell cycle independent from each other. Hence, combination of miR-34a with paclitaxel will reduces the chance of appearance of cells that could escape from cell cycle checkpoints.

In summary, we tried to provide better understanding for anticancer effect of miR-34a in combination with paclitaxel in CRC cell line. This miRNA showed has potential to suppress cancer cell development by decreasing cell viability, proliferation, and cell cycle probably due to targeting important oncogenes such as BCL2 and SIRT1. However, there are few limitations in our study for instance, using one cell line and only in vitro survey which these are better to be tested in the future studies. It has been shown that processing miRNAs is much faster than mRNA translation, enabling them to modulate gene expression more quickly compared with transcriptional repressors.[27],[28] Given their vital roles in almost all biological process, our knowledge about miRNAs in cancer development is still scanty. Eventually, we propose that restore or enhanced expression of miR-34a along with chemotherapy would be an attractive strategy in CRC treatment. Although we tried to show the potential of using miR-34a in combination with paclitaxel in SW480 colorectal cell line, much works remain to be done in the path of reaching this combination therapy to the clinic.

Acknowledgments

The authors would like to thank Stem Cell Technology Research Center, Tehran, Iran, for providing technical facilities.

Financial support and sponsorship

This study was financially supported by Stem Cell Technology Research Center, Tehran, Iran.

Conflicts of interest

The authors disclose any commercial associations that might create a conflict of interest in connection with submitted manuscripts.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
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