MiR-181a confers resistance of cervical cancer to radiation therapy through targeting the pro-apoptotic PRKCD gene

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Abstract

The purpose of this study was to define the roles of miR-181a in determining sensitivity of cervical cancer to radiation therapy, to explore the underlying mechanism and to evaluate the potential of miR-181a as a biomarker for predicting radio-sensitivity. Tumor specimens from 18 patients with a histological diagnosis of squamous cervical carcinoma (stage IIIB) were used in the micro-RNA profiling and comparison. These patients never received any chemotherapy before radiation therapy. Human cervical cancer cell lines, SiHa and Me180, were used in vitro (cell culture) and in vivo (animal) studies. Transfection of tumor cells with the mimic or inhibitor of miR-181a, and reporter gene assay, were performed to investigate the role of miR-181a in determining radio-sensitivity and the target gene. Higher expression of miR-181a was observed in human cervical cancer specimens and cell lines that were insensitive to radiation therapy, as compared with sensitive cancer specimens and the cell lines. We also found that miR-181a negatively regulated the expression of PRKCD, a pro-apoptotic protein kinase, via targeting its 3′-untranslated region (UTR), thereby inhibiting irradiation-induced apoptosis and decreasing G2/M block. The role of miR-181a in conferring cellular resistance to radiation treatment was validated both in cell culture models and in mouse tumor xenograft models. The effect of miR-181a on radio-resistance was mediated through targeting the 3′-UTR of PRKCD gene. Thus, the expression level of miR-181a in cervical cancer may serve as a biomarker for sensitivity to radiation therapy, and targeting miR-181a may represent a new approach to sensitizing cervical cancer to radiation treatment.

Introduction

Cervical cancer, the third most common malignancy in women, is often diagnosed at an advanced clinical stage.1 At present, radiotherapy remains the most common therapeutic intervention for cervical cancer either as pre-operative adjuvant therapy or as primary treatment. Nevertheless, 30–50% of patients with stage IIB–IV will ultimately fail after radiotherapy, and the main cause of the treatment failure in patients with local cervical cancer is resistance to radiotherapy.2 Clinically, identification and treatment of radio-resistant cervical cancer remains an unsolved problem.

Micro-RNAs (miRNAs) are a class of endogenous, 20–22 nucleotides, non-coding RNA molecules that can induce mRNA degradation, translational repression or both, via pairing with partially complementary sites in the 3′-untranslated region (UTR) of the targeted genes.3, 4, 5 It is estimated that 30% of all genes are regulated by miRNAs.6 Because miRNAs have the ability to target numerous mRNAs, these small RNA molecules can operate highly complex regulatory networks and regulate the expression of genes in many pathways that are associated with tumor initiation, development and progression, and response to treatment. For example, downregulation of miRNA, let-7, in lung cancer was found to be associated with shortened post-operative survival.7 Overexpression of miR-200c contributed to chemo-resistance in esophageal cancers.8 In human cervical cancer, miR-200a was believed to regulate the metastatic potential of tumor cells.9 In this study, we found that miR-181a was upregulated in tumor specimens from cervical cancer patients who were insensitive to radiation therapy. We further demonstrated that the critical role of miR-181a in modulating radio-resistance in vitro and in vivo, and revealed that the effect of miR-181a on radio-resistance was mediated through its negative regulation of the expression of PRKCD, a pro-apoptotic protein kinase, by targeting the 3′-UTR of PRKCD gene. Our study implied that expression level of miR-181a in cervical cancer may serve as a biomarker for sensitivity to radiation therapy, and targeting miR-181a may represent a novel therapeutic approach to sensitizing cervical cancer to radiation treatment.

Results

Increased expression of miR-181a in radio-resistant human cervical cancer specimens and cell lines

To explore whether miRNAs were associated with resistance of cervical cancer to radiation therapy, we first compared the expression of miRNAs in 7 radio-resistant tumor specimens with 11 radio-sensitive tumor specimens using microarrays. All of these specimens were from the patients with stage IIIb cervical cancer who received radiation therapy. The clinical and pathological characteristics of these 18 patients are shown in Table 1. Analysis of microarray data showed that expressions of miR-181a and miR-21 were significantly increased, and expressions of miR-30*, miR-23a, miR-16–2*, miR-378, miR-18a and miR-221 were significantly decreased, in the radio-resistant tumor samples as compared with that in the radio-sensitive tumor samples (Figure 1a and Table 2). After excluding two miRNAs*, the preliminary screening in cell lines proceeded for the remaining six miRNAs. The results showed miR-181a probably had the most significant role in the radio-sensitivity of cervical cancer (Supplementary Figure 1a). In addition, miR181a demonstrated high fold change and significance in the microarray assay (Table 2). With regard to all of the points stated, we chose miR-181a for further validation of its probable relationship with radio-sensitivity. The significant higher expression of miRNAs in radio-resistant tumors was confirmed by qRT–PCR analysis (Figure 1b and Supplementary Figure 1b). The association of miR-181a with radiation sensitivity was also observed in the two cervical cancer cell lines having different degrees of sensitivity to radiation. As shown in Figure 1c, Me180 cells were more sensitive to radiation treatment than SiHa cells, and showed a 65% lower expression of miR-181a than SiHa cells (Figure 1d). These results suggest a possible role for miR-181a in modulating resistance of cervical cancer to radiotherapy.

Table 1 Clinicopathologic parameters of 18 cervical cancer patients
Figure 1
figure1

Expression of miR-181a is increased in radio-resistant cervical cancer specimens and cell lines. (a) MiRNA expression profiles in 11 radio-sensitive specimens and 7 radio-resistant specimens. The green color represents elevated expression. (b) Quantitative real-time PCR analysis of miR-181a expression in radio-sensitive and radio-resistant tumor specimens. P=0.035. (c) SiHa and Me180 cells (3 × 105) were seeded into six-well plates. The plates were irradiated with 137Cs with doses of 0, 2, 4, 6 and 8 Gy given in a single fraction. Various tumor cells (for SiHa, the number were 200, 500, 1500, 4000 and 8000; for Me180, the number were 200, 500, 1500, 10 000 and 15 000) were plated into six-well plates. After 10–13 days, the cells were fixed with 10% paraformaldehyde and stained with 1% crystal violet in 70% ethanol. Colonies containing 50 cells or more were counted. Surviving fraction was the number of colonies/(cells inoculated × plating efficiency). Survival curve was derive from multi-target single-hit model: SF=1−1−exp (−D/D0)n. SiHa cell was radio-resistant compared with Me180 cell. Each bar represents the mean±s.d. of three experiments. **P<0.01. (d) Expression of miR-181a in Siha and Me180 cell lines was examined by quantitative real-time PCR.

Table 2 Statistical results of different expression of miRNAs in radio-sensitive and radio-resistant cervical cancer tissue biopsies by microarrays

Expression of miR-181a confers resistance of cancer cells to radiation treatment both in vitro and in vivo

To determine the effect of miR-181a expression on sensitivity of tumor cells to radiation treatment, we transfected SiHa and Me180 cells with a miR-181a mimic or a control miRNA. As shown in Figure 2a, treatment with the miR-181a mimic decreased sensitivity of tumor cells to radiation treatment. In contrast, treatment with the miR-181a inhibitor enhanced radio-sensitivity in the tumor cells. To further verify the effect of miR-181a on sensitivity of tumor cells to radiation treatment, we generated two cell lines stably expressing miR-181a. In these cells, expression of miR-181a was confirmed by real-time qRT–PCR (Supplementary Figure 1b). Figure 2a showed that tumor cells expressing higher levels of miR-181a had a decreased sensitivity to radiation treatment.

Figure 2
figure2

Effects of miR-181a on radio-sensitivity of cervical cancer cells in vitro and in vivo. (a) SiHa and Me180 cells (1 × 105) were seeded into six-well plates and subjected to transfection with a miR-181a mimic or inhibitor the next day. Forty-eight hour after transfection, the plates were irradiated with 137Cs with doses of 0, 2, 4, 6, 8 Gy given in a single fraction. Various tumor cells (for SiHa, the number were 200, 500, 1500, 4000 and 8000; for Me180, the number were 200, 500, 1500, 10 000 and 15 000) were plated into six-wellplates. After 10–13 days, the cells were fixed with 10% paraformaldehyde and stained with 1% crystal violet in 70% ethanol. Colonies containing 50 cells or more were counted. Surviving fraction was the number of colonies/(cells inoculated × plating efficiency). Survival curve was derive from multi-target single-hit model: SF=1−1−exp (−D/D0)n. The pre-miR-181a sequence was cloned into pWPXL vector to constructing stable transfection cell lines. The miR-181a mimic decreased sensitivity of tumor cells to radiation. In contrast, treatment with the miR-181a inhibitor enhanced radio-sensitivity in the tumor cells, moreover stable expression of miR-181a decreased sensitivity of tumor cells to radiation. Each bar represents the mean±s.d. of three experiments. *P<0.05; **P<0.01. (b) Me180 cells (2 × 106/mouse) stably transfected with a miR-181a expression vector or a control empty vector were injected into the right thigh of 6-week-old BALB/C nude mice. When tumors reached 8 mm in diameter, the mice bearing either miR-181a-expressing tumors or control vector harboring tumors were randomly divided into two groups. One group received irradiation treatment (16 Gy with 6 MV X ray), and the other group served as controls. Tumor sizes were measured twice a week. There no difference in tumor weights and volumes between the group transfected with miR-181a and the group transfected with vector control, but after receiving irradiation, the tumor weights and volumes of stably expressing miR-181a group were significantly higher than that of vector control group.

The inhibitory effect of miR-181a on radio-sensitivity was also observed in animal tumor models. As shown in Figure 2b, without γ-ray irradiation treatment, the tumors with or without overexpression of miR-181a grew similarly, and had approximately same weights and volumes of the tumors. In contrast, following γ-ray irradiation treatment, the weights and volumes of the tumors that did not overexpress miR-181a were significantly decreased as compared with that of the tumors overexpressing miR-181a, suggesting that over-expression of miR-181a confers resistance of cervical cancer to radiation therapy.

miR-181a has no effects on cell proliferation, but inhibits irradiation-induced apoptosis and decreases G2/M block

We next wanted to explore the mechanism by which miR-181a modulated sensitivity of tumor cells to radiation treatment. We first determined whether or not the inhibitory effect of miR-181a on tumor sensitivity to radiation was associated with its effects on tumor cell proliferation. Our experiments did not find any difference in proliferation between the cells with and without overexpression of miR-181a (Figure 3a). However, miR-181a inhibited induction of apoptosis in tumor cells treated with irradiation, as demonstrated by decreased caspase 3/7 activity in the cells transfected with a miR-181a mimic, as compared with the cells transfected with a control RNA (Figure 3b). Western blot analysis showed that 24 h after exposure to γ-radiation, caspase-3 protein expression in SiHa and Me180 cells transfected with miR-181a were lower than that in cells transfected with a control vector (Figure 3c). These results indicated that miR-181a may protect cells from radiation-induced damage by inhibiting apoptosis. We also observed that after irradiation, although there was a decrease in the proportion of cells in the G1/S phase and an increase in the proportion of cells in the G2/M phase, overexpression of miR-181a decreased the proportion of cells in the G2/M phase (Figure 4). As cells are usually most sensitive to radiation in the late G2/M phase and are most resistant to radiation in the mid- to late S and early G1 phases, miR-181 may render tumor cells insensitivity to radiation through blocking transition of cells to the G2/M (Figure 4).

Figure 3
figure3

Effects of miR-181a on proliferation, apoptosis and cell cycle of cervical cancer cells. (a) MiR-181a had no effect on cervical cancer cell growth. (b) Overexpression of miR-181a did not change the activity of caspase3/7, but after receiving irradiation, weaker activity was detected in the cells transfected with miR-181a. Underexpression of miR-181a did not change the activity of caspase3/7, but after, stronger activity was detected in the cells transfected with miR-181a inhibitor. Weaker activity of caspase-3/7 was observed in SiHa and Me180 cells receiving irradiation with stable expression of miR-181a. (c) MiR-181a inhibited the apoptosis induced by radiotherapy. The expression of miR-181a did not change the expression of caspase-3-cleaved protein. But, after cells receiving irradiation, the overexpression of miR-181a inhibited the expression of caspase-3-cleaved protein.

Figure 4
figure4

miR-181a did not change cell cycle distribution, but miR-181a decreased G2 block induced by irradiation. Forty-eight hours after cells exposed to ray, the proportion of cells in the G1/S phase decreased and the proportion of cells in the G2 phase increased. Overexpression of miR-181a inhibited the G2/M block, underexpression of miR-181a facilitated the G2/M block. Cells were harvested and fixed with 70% ethanol at −20 °C for 24 h, and then were stained with 50 μg/ml of propidiumiodide. DNA content was analyzed on FACS Caliber. The results were analyzed using ModFit software. *P>0.05; **P<0.05; ***P<0.001.

miR-181a negatively regulates the expression of PRKCD via targeting its 3′-UTR

To understand how miR-181a modulates sensitivity of cancer cells to radiation, we utilized two prediction algorithms, PicTar and TargetScan, to analyze the possible target gene. We found that PRKCD, whose gene product is known to have a role in promoting apoptosis, was one of the possible target genes of miR-181a. Thus, we performed qRT–PCR to test whether expression of PRKCD could be altered by miR-181a. Figure 5a shows that transfection with the miR-181a mimic decreased the expression of PRKCD mRNA, and the miR-181a inhibitor increased the expression of PRKCD mRNA. Western blot showed that the expression of PRKCD protein was similarly altered by miR-181a mimic and inhibitor (Figure 5a).

Figure 5
figure5

PRKCD is a target for miR-181a. (a) Overexpression of miR-181a inhibited the expression of PRKCD mRNA and protein, and underexpression of miR-181a facilitated the expression of PRKCD mRNA and protein. The expression of PRKCD mRNA and protein were suppressed by the stable miR-181a expression. (b) Potential binding site of miR-181a on PRKCD 3′-UTR in different species. (c) Sketch of the construction of wild-type or mutant PRKCD 3′-UTR vectors. (d) The pLUC-wild-PRKCD 3′-UTR or pLUC-mutant-PRKCD 3′-UTR was transfected into 293T cells with pWPXL or pWPXL-miR-181a. Renilla luciferase vector was used as an internal control. The relative luciferase activity of pWPXL group was set as 1. (e) Quantitative real-time PCR analysis of PRKCD expression in radio-sensitive and radio-resistant tumor specimens.

TargetScan analysis informed that there was a binding site on the 3′-UTR of the PRKCD for miR-181a, which was highly conserved in mammals (Figure 5b). To validate the predicted consensus sequences for miR-181a in the PRKCD-3′ UTR, and determine whether these miR-181a-binding sequences directly contributed to the negative regulation of PRKCD expression, we tested the effects of miR-181a on activity of a reporter gene using the vectors that either contained wild-type or mutant miR-181a-targeting sequences (Figure 5c). As shown in Figure 5d, co-transfection of HEK-293T cells with the pWPXL-miR-181a resulted in a 52% reduction in the activity of the reporter gene vector containing wild-type miR-181a targeting sequences; in contrast, there was only 11% decrease in the activity of the reporter gene vector containing mutant 3′-UTR sequences. The significant higher expression of PRKCD in radio-sensitivity tumors was confirmed by qRT–PCR analysis (Figure 5e). These results demonstrated that the miR-181a-binding sequences in the PRKCD-3′-UTR was the region required for the miR-181a-mediated inhibition of PRKCD expression.

Functional role of PRKCD in the miR-181a-modulated radio-sensitivity

To demonstrate the role of PRKCD in modulating radio-sensitivity, we silenced the expression of PRKCD in Me180 cells using siRNA, and then measured sensitivity of the cells to radiation. Figures 6a and e show that silencing of PRKCD expression by siRNA decreased the sensitivity of the tumor cells to radiation treatment, as compared with the control. We also tested the effect of overexpression of PRKCD on radio-sensitivity. In these experiments, Siha cells were infected with a lentivirus expression vector of PRKCD without the 3′-UTR; expression of PRKCD mRNA and protein were confirmed by qRT–PCR and western blot, respectively (Figure 6b). As shown in Figure 6f, sensitivity to radiation treatment was enhanced in the cells overexpressing PRKCD. We further demonstrated that transfection of the Me180 cells stably expressing miR-181a with the PRKCD expression vector containing the 3′-UTR could abrogate the effect of miR-181a and restore radio-sensitivity in the tumor cells (Figures 6c and g), and knockdown of PRKCD expression in SiHa cells treated by a miR-181a inhibitor rendered radio-resistance (Figures 6d and h). The over-expression of PRKCD in the cells stably expressing miR-181a could abolish the inhibitory effects of miR-181a on apoptosis (Figure 6i), but it could not reverse the effects on cell cycle (Figure 6j). These results further corroborated the role and mechanism of miR-181a in conferring radio-resistance to cancer cells.

Figure 6
figure6

Effect of PRKCD on sensitivity of cervical cancer cells to radiation treatment. (a, e) SiRNA inhibited the expression of PRKCD mRNA and protein in Me180 cell. Underexpression of PRKCD decreased the radio-sensitivity. (b, f) The stable expression of PRKCD mRNA and protein in Siha cells. Overexpression of PRKCD increased the radio-sensitivity. (c, g) Me180 cells stably expressing miR-181a were re-introduced PRKCD without its 3′-UTR, which resulted in the constitutive expression of PRKCD without the potential for miR-181a-mediated inhibition. The results showed that the restoration of PRKCD in this stable cell line was confirmed by western blot analysis and qRT–PCR. Forced expression of PRKCD restores miR-181a-regulated cervical cancer cell radio-sensitivity. (d, h) Decreased expression of miR-181a facilitated the expression of PRKCD mRNA; SiRNA inhibited the expression of PRKCD mRNA and protein in SiHa cells transfected by a miR-181a inhibitor. Knockdown of PRKCD in SiHa cells transfected with a miR-181a inhibitor could restore radio-resistance. There were significant differences in the radio-sensitivity between SiHa cells transfected with miR-181a inhibitor and SiHa cells transfected with miR-18a inhibitor and siRNA (*P<0.05). (i) Overexpression of PRKCD in Me180 cell transfected with stable miR-181a restored the depression effect in apoptosis regulation by miR-181a. (j) Overexpression of PRKCD in Me180 cell transfected with stable miR-181a did not restore the effect in cell cycle regulation by miR-181a. **P<0.001, ***P>0.05.

Discussion

In the current study, we identified miR-181a as a critical contributor to radio-resistance in cervical cancer. Our conclusion is supported by both of in vitro and in vivo experiments, and clinical investigation. Moreover, we demonstrate that miR-181a confers resistance to radiation therapy by repressing the expression of PRKCD, a pro- apoptotic protein kinase, through targeting the 3′-UTR of the PRKCD gene.

Radiation therapy is one of the main therapeutic interventions for a variety of malignancies including cervical cancer, and sensitivity of tumor cells to this treatment may directly determine the prognosis of cancer patients. How to improve the effectiveness of this therapy and to reduce the radiation dose to avoid damaging normal tissue has been one of the challenges for radiation oncologists. Sensitivity of cancer cells to radiotherapy can be affected by various factors, such as cell proliferation, cell cycle, DNA damage repair, apoptosis and so on.10 In recent years, miRNA has been found to have critical roles in cancer development, progression and response of tumor cells to treatment. For example, Weidhaas et al.11 reported that expression of the miRNAs, let-7b and miR-521, are associated with radio-sensitivity in several types of cancer cells. Although the role of miR-181a in radio-sensitivity has been reported in glioma cell line U87MG,12 the study were conducted in in vitro cell models. Our study not only shows that miR-181a is a major player in modulating resistance of cervical cancer cells to radiation therapy both in vitro and in vivo (Figure 2), but most importantly also provides the first clinical evidence implying the importance of miR-181a in determining resistance of cancer cells to radiation therapy (Figure 1). Furthermore, we reveal the molecular mechanism of how miR-181a alters sensitivity of cancer cells to radiotherapy, that is, miR-181a renders radio-insensitivity to cancer cells through targeting the 3′-UTR of the PRKCD gene, thereby suppressing the expression of the pro-apoptotic PRKCD protein (Figure 5). Indeed, the tumor cells with higher level of miR-181a underwent less apoptosis following irradiation (Figures 3b and c). To our knowledge, this is the first study reporting the mechanism of miR-181a regulation of tumor cell resistance to radiation.

PRKCD, also known as protein kinase Cδ, was a protein kinase C isozyme that does not require calcium for its activity.13 Activation of PPKCD was known to be associated with inhibition of cell cycle progression,14 and the decrease of PRKCD was linked to tumor progression,15 suggesting that PRKCD had a negative effect on cell survival. Proteolytic activation of PRKCD had been reported to be associated with apoptosis induced by various DNA damaging agents, including UV radiation, ionizing radiation, cisplatin, etoposide, cytosine arabinoside, mitomycin C and doxorubicin.16, 17, 18, 19, 20, 21 Our results that demonstrate the role of PRKCD in radio-sensitivity and apoptosis are consistent with those observations, and provide additional evidence for the importance of PRKCD in determining cellular fate under various stresses. Because miRNAs can regulate multiple target genes, PRKCD identified in the present study might be only one of them and there might be other target genes regulating apoptosis and cell cycle to have a role in the radio-sensitivity of cervical cancer, which warrants further investigations. As shown in our experiment, we could not restore the effects of miR-181a on cell cycle by overexpression of PRKCD but we indeed restored the inhibitory effects on apoptosis caused by miR-181a that probably mediated by PRKCD.

Taken together, we report here that there is a causal relationship between high expression of miR-181a and resistance to radiation therapy in cervical cancer, and that miR-181a contributes to radio-resistance by targeting the pro-apoptotic gene, PRKCD, leading to inhibition of radiation-induced apoptosis. The results of our study suggest that expression of miR-181a might be a biomarker for sensitivity of cervical cancer to radiation treatment, and that targeting miR-181a may be utilized as a new approach to sensitizing radio-resistant cancers.

Materials and methods

Patients and tumor samples

Tumor specimens from 18 patients with a histological diagnosis of squamous cervical carcinoma (stage IIIB) and treated at Fudan University Shanghai Cancer Center (Shanghai, China) between 2007 and 2009 were selected and utilized. The tumor samples were obtained from the patients who never received any chemotherapy before radiation therapy. All these patients received the standard, whole pelvic irradiation (45 Gy) in 25 fractions and an additional para-metrium boost of 10–14 Gy in 5–7 fractions. Following the external beam radiation therapy to the whole pelvic, patients received intra-cavitary radiation therapy (20–25 Gy, twice a week) administered as 4–5 fractions to point A using an 192Ir high-dose-rate brachytherapy unit. The patients underwent concurrent chemotherapy of weekly cisplatin 40 mg/m2. The radio-sensitive and radio-resistant cases were distinguished based on histological finding of residual tumor cells in the cervical biopsies sampled 6 months after completion of radiotherapy.

All tumor specimens were stored at −80 °C in the tissue bank. All patients gave their informed consent before gaining the specimens. That was approved by Fudan University Shanghai Cancer Center Institutional Review Board. Histological assessment informed that all of the samples contained at least 70% tumor cells. Among the total of 75 patients who met the criterion, seven cases were radio-resistant. According to the clinical and pathological characteristics of these seven patients, we selected 11 radio-sensitive patients with similar characteristics.

miRNA microarrays

Microarray experiments were performed at the National Engineering Center for Biochip at Shanghai. Briefly, total RNAs were prepared from the tissues using the mirVana kit from Ambion (Austin, TX, USA), and then labeled and hybridized using Agilent’s miRNA Complete Labeling and Hyb Kit. Arrays were scanned on an Agilent Microarray Scanner; data were extracted using Agilent Feature Extraction Software. (Agilent, Santa Clara, CA, USA)

Cell culture

Human cervical cancer cell line SiHa was cultured in Eagle's minimum essential medium supplemented with 10% of fetal bovine serum and antibiotics at 37 °C with 5% CO2. Human cervical cancer cell line Me180 was cultured in McCoy’s 5a medium supplemented with 10% of fetal bovine serum and antibiotics under same condition.

RNA extraction and quantitative real-time PCR

Total RNAs were extracted using TRIzol reagent (Invitrogen, Shanghai, China), and cDNAs were synthesized using the PrimeScript RT reagent Kit (TaKaRa, Dalian, China). Real-time PCR analyses were performed with SYBR Premix Ex Taq (TaKaRa). For miRNA, RNA was reverse-transcribed using a specific reverse-transcription primer. The expression of miRNA was quantified using a TaqMan probe and was normalized by U6 small nuclear RNA using TaqMan miRNA assays (Applied Biosystems, Foster City, CA, USA).

Transfection

miRNA mimics or inhibitors, or siRNAs were transfected into cell using Lipofectamine 2000 reagent (invitrogen) according to the manufacturer’s instruction.

Vector constructs

The pre-miR-181a sequence and the open reading frame of PRKCD were amplified from human genomic DNA and then cloned into pWPXL vector (a gift from Dr T Didier) to replacing the green fluorescence protein fragment. To generate the luciferase reporters, the wild-type and mutant 3′-UTR sequences of PRKCD were amplified from human genomic DNA and cloned into the region downstream of the stop codon in the luciferase gene in the luciferase reporter vector.

Lentivirus production and cell transduction

Viruses were harvested 48 h after the co-transfection with the pWPXL-miR-181a vector or pWPXL-PRKCD with the packaging plasmid psPAX2 and envelope plasmid pMD2.G (provided by Dr T Didier) into HEK 293T cells. SiHa and Me180 cells were infected with 1 × 106 recombinant lentivirus-transducing units in the presence of 6 μg/ml polybrene (Sigma). Assays were carried out 48 h after transfection.

Cell proliferation assay

Cell proliferation was measured using the Cell Counting Kit-8 (CCK-8) assay kit (Dojindo, Shanghai, China).

Flow cytometric analysis of cell cycle distribution

Cells were harvested and fixed with 70% ethanol at −20 °C for 24 h, and then were stained with 50 μg/ml of propidium iodide (Kaiji, Nanjing, China).DNA content was analyzed on FACSCaliber (BD Bioscience, San Diego, CA, USA). The results were analyzed using ModFit software (BD Bioscience).

Colony-formation assay

SiHa and Me180 cells (1 × 105) were seeded into six-well plates and subjected to transfection the next day. Forty-eight hour after transfection, the plates were irradiated with 137Cs (Nordion, Ottawa, ON, Canada). The dose rate was 1.25 Gy/min, with doses of 0, 2, 4, 6, 8 Gy given in a single fraction. After incubation at 37 °C/5% CO2 for 10–13 days, the cells were fixed with 10% paraformaldehyde and stained with 1% crystal violet in 70% ethanol. Colonies containing 50 cells or more were counted. Surviving fraction was the number of colonies/(cells inoculated × plating efficiency). Survival curve was derived from multi-target single-hit model: SF=1−1−exp (−D/D0)n. D0 was defined as the dose that gave an average of one hit per target.

Apoptosis assay

Apoptosis was determined by measuring activation of caspase 3 and 7 using the caspase Glo-3/7 assay kit (Promega, Shanghai, China), and by detecting cleaved caspase-3 protein using western blot.

Western blot

Cell lysates were resolved by SDS–polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane and blocked in phosphate-buffered saline/Tween-20 containing 5% non-fat milk. The membrane was incubated with antibodies for PRKCD (CST) or β-actin. Protein signals were detected using enhanced chemiluminescence (Pierce, Rockford, IL, USA).

In vivo radio-sensitivity assay

Me180 cells (2 × 106/mouse) stably transfected with a miR-181a expression vector or a control empty vector were injected into the right thigh of 6-week-old BALB/c nude mice. When tumors reached 8 mm in diameter, the mice bearing either miR-181a-expressing tumors or control vector harboring tumors were randomly divided into two groups. One group received irradiation treatment (16 Gy with 6 MV X ray), and the other group served as controls. Tumor sizes were measured twice a week.

Luciferase reporter assay

A mixture of 50 ng pLUC-UTR, 100 ng pWPXL-miR-181a and 10 ng Renilla were transfected into HEK293T cells, and firefly and renilla luciferase activities were measured using a dual-luciferase reporter system (Promega).

The entire 3′ UTR of PRKCD was used in the luciferase assays as follows:

PRKCD-UTR-FTR: 5′-IndexTermCGGGGTACCTGGGACTGTGGTGACTTCTG-3′

PRKCD-UTR-RTR: 5′-IndexTermGCTCTAGACCTGGTGGTTCAAACAGCTT-3′

Statistical analysis

Student’s t-test was performed to determine statistical difference. P<0.05 was considered statistically significant.

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Acknowledgements

We are most grateful for the pWPXL, psPAX2 and pMD2.Glentivirus plasmids that were provided by Professor Didier Trono from the School of Life Sciences, EcolePoly technique Fédérale de Lausanne, 1015 Lausanne, Switzerland. This work was supported by National Natural Science Foundation of China (No. 81072128), and Science and Technology Commission of Shanghai Municipality (Academic leader foundation of Shanghai, 09XD1401100).

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Correspondence to X He or X Wu.

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Supplementary Information accompanies the paper on the Oncogene website

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Keywords

  • miR-181a
  • PRKCD
  • apoptosis
  • cervical cancer
  • radiation therapy

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