A comprehensive search for microRNAs with expression profiles modulated by oncogenic KRAS: Potential involvement of miR-31 in lung carcinogenesis

  • Authors:
    • Koji Okudela
    • Takeshisa Suzuki
    • Shigeaki  Umeda
    • Yoko  Tateishi
    • Hideaki  Mitsui
    • Yohei  Miyagi
    • Kenichi  Ohashi
  • View Affiliations

  • Published online on: July 18, 2014     https://doi.org/10.3892/or.2014.3339
  • Pages: 1374-1384
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

Small non-protein coding RNAs that regulate messenger RNA levels, namely microRNAs (miRNAs), have been implicated in the pathogenesis of various diseases. The purpose of the present study was to identify essential miRNAs involved in lung carcinogenesis. Previous studies demonstrated that an investigation into the downstream targets of oncogenic KRAS could be used as a strategy to elucidate the molecular mechanisms involved in lung cancer; therefore, we examined the expression profiles of mRNAs modulated by oncogenic KRAS in the present study. We focused on miR-31 from the miRNAs that were differentially expressed, and evaluated its potential role in the development of lung cancer. miR-31 was upregulated not only by oncogenic KRAS, but also by oncogenic EGFR. The expression of miR-31 was markedly attenuated in some lung cancer cell lines by deleting its host gene locus. The restoration of miR-31 in lung cancer cell lines that lost its expression attenuated their growth activities. The knockdown of miR-31 expression in lung cancer cell lines retaining its expression enhanced anchorage-independent growth activity. These results suggest that miR-31 may be a suppressor that regulates an essential oncogenic pathway, the loss of which may promote lung carcinogenesis.

Introduction

Lung cancer is one of the most common causes of cancer-related mortality in the developed world (1,2). Recurrence has been reported in a large proportion of lung cancer patients in spite of successful resection of the primary tumor (1,2). Although some lung tumors are known to be sensitive to conventional chemotherapeutic agents or certain molecular targeting agents, many are not (3,4). Thus, further understanding of the molecular mechanisms involved in lung carcinogenesis is essential for developing novel therapeutic strategies.

Our previous studies, which involved a comprehensive search for the downstream targets of oncogenic KRAS, identified important molecules involved in lung carcinogenesis (3,5). Mutations in driver oncogenes, such as KRAS, EGFR, BRAF, and ALK, were found to mutually and exclusively occur, and each of these mutations has been shown to transmit the common essential oncogenic signal that promotes carcinogenesis (68). Therefore, the downstream targets of oncogenic KRAS participate not only in KRAS-mediated carcinogenesis, but also in other oncogene-mediated carcinogeneses (3,5). Thus, an investigation into the downstream targets of oncogenic KRAS is considered to be an available strategy that can identify common important molecular mechanisms involved in lung cancer.

Small non-protein coding RNAs that regulate messenger RNA levels, namely microRNAs (miRNAs), have been identified and subsequently implicated in the pathogenesis of various diseases. The present study investigated the expression profiles of miRNAs modulated by oncogenic KRAS in airway epithelial cells. We focused on miR-31 from the miRNAs that were differentially expressed, and evaluated its potential role in the development of lung cancer.

Materials and methods

Cell lines and culture

An immortalized human airway epithelial cell line [16HBE14o, simian virus 40 (SV40)-transformed human bronchial epithelial cells] described by Cozens et al (9) was kindly provided by D.C. Grunert (California Pacific Medical Center Research Institute). A sub-clone of 16HBE14o cells, described as NHBE-T in this study, was used. Human lung cancer cell lines (A549, H358, H2087, H23, EKVX, H226, H827, H1819, H441, H4006, HOP62, H1299 and H460) and a human embryonic kidney cell line (HEK293T) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). The human lung cancer cell line LC2/ad was purchased from the Riken Cell Bank (Tsukuba, Japan). The human lung cancer cell lines, PC1, PC9 and HARA were from Immuno-Biological Laboratories Co. (Gunma, Japan). The human lung cancer cell lines, TKB1, TKB2, TKB4, TKB5, TKB6, TKB7, TKB8, TKB14 and TKB20 were obtained from Dr Hiroshi Kamma via Dr Takuya Yazawa (Kyorin University School of Medicine) (10). Primary small airway epithelial cells (SAEC) were purchased from Sanko Kagaku (Tokyo, Japan).

Plasmid construction

The pro-retrovirus vector bearing KRAS [wild-type (WLD), V12, H61, V12/S35, V12/G37 and V12/C40 mutants] (11) and PIK3CA (WLD, E545K mutant, and H1047R mutant) were previously described (12). Complementary DNA (cDNA) coding EGFR (NM_001964) and BRAF (NM_004333) was PCR-amplified using cDNA from NHBE-T cells as a template and inserted into the pro-retrovirus vector pQCXIP (BD Clontech, Palo Alto, CA, USA). Mutant EGFR (deletion: E745-A750) was PCR-amplified using cDNA from the PC9 lung cancer cell line. EGFR (L858R mutant) and BRAF (V600E mutant) were generated by the method of site-directed mutagenesis. DNA fragments including the miR-31 coding region (MIMAT0000089) flanking approximately a 100 base-pair margin in both directions was PCR-amplified and inserted into the pro-retrovirus vector pLHCX (BD Clontech). Vectors bearing a sense and antisense strand of cDNA were obtained. A pNsi-based miR-31 blocking vector was purchased from Takara Bio Inc. (Kyoto, Japan).

Retroviral-mediated gene transfer

The pro-retrovirus vectors bearing the desired constructs and the pLC10A1 retrovirus-packaging vector (Imgenex, San Diego, CA, USA) were cotransfected into HEK293T cells with Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). Forty-eight hours after the transfection, conditioned medium was recovered as a viral solution. The desired genes were introduced by incubating cells with the viral solution containing 10 μg/ml of polybren (Sigma, St. Louis, MO, USA). Cells stably expressing the desired genes were selected with 250 μg/ml of hygromycin B or 1,000 μg/ml of neomycin (both from Invitrogen). The pooled clones were used for analyses as follows.

miRNA expression profiling

miRNA expression profiles in empty vector (MOCK)-, KRAS/WLD-, KRAS/V12-, and KRAS/H61-vector transduced NHBE-T cells were comprehensively evaluated with human miRNA microarray (Human miRNA Microarray kit release 16.0, 8×60K, cat. no. G4870A; Agilent Technologies, Santa Clara, CA, USA). Total RNA was extracted from the cells immediately after completing the selection (5 days post-transduction) using the RNA easy kit (Qiagen, Hilden, Germany). Labeling and hybridization was performed according to the manufacturer’s recommendations (Agilent Technologies).

Colony formation assays

Cells (appropriate count of 1.0×104 to 5.0×104) were seeded onto a 10 cm culture dish (Iwaki, Tokyo, Japan), and grown for 10 days. Cells were fixed with methanol and Giemsa-stained, and colonies visible in scanned images were counted.

Growth curve assays

Cells (2.5×105) were seeded onto a 10 cm culture dish, and were grown in DMEM (Sigma) with 10 FBS (Sigma) to a semi-confluent state for 5–7 days. Cells were counted, and 2.5×105 cells were seeded again onto a 10 cm dish. Several passages were repeated in the same manner. The sum of population doublings (PDLs) at each point was calculated by the formula SPDLn = log2 (countn/2.5×105) + SPDLn−1.

Soft agar colony formation assays

Cells (1.25×104) were cultured and grown in 1 ml of DMEM-based 0.3% agar (agar noble; Becton-Dickinson, Sparks, MD, USA) containing 10% FBS in 3.5 cm culture dishes (Iwaki) for 4 weeks. The agars were fixed with a buffered 4% paraformaldehyde solution, and colonies visible in scanned images were counted.

Treatment with an inhibitor for MEK and PI3K

Following infection of the retrovirus vector, cells stably expressing KRAS were selected for 3 days, and were then harvested. Cells were treated with an inhibitor for MEK (PD98059, 50 μM) or PI3K (LY294002, 50 μM) [both from Cell Signaling Technology (CST), Danvers, MA, USA] and their combination for the last 24 h before the harvest.

Comprehensive search for the downstream target of miR-31

Gene expression in the empty vector (MOCK)-, miR-31 sense strand (SS)-, and miR-31 antisense (AS) strand-transduced NHBE-T cells was comprehensively evaluated with a human gene chip microarray (SurePrint G3 Human Gene Expression 8×60K v2 Microarray kit, cat. no. G4851B; Agilent Technologies). Total RNA was extracted from the cells immediately after completing the selection (5 days post-transduction) using the RNA easy kit (Qiagen). Labeling and hybridization were performed according to the manufacturer’s recommendations (Agilent Technologies). Transcripts whose signal values in the miR-31 SS-transduced cells were 5-fold higher or lower than that in both the empty vector-transduced cells and the miR-31 antisense strand-transduced cells were extracted according to the same flow chart as described in a previous study (13).

Results

miRNA expression profiling modulated by oncogenic KRAS

A comprehensive evaluation of the expression of miRNA revealed that oncogenic KRAS-transduced cells had different expression profiles from empty vector- and WLD KRAS-transduced cells, as KRAS/V12- and KRAS/H61-transduced cells were classified into a distant branch from the others on a dendrogram described by hierarchical clustering analysis (Fig. 1). The miRNAs that had higher or lower levels in oncogenic KRAS (KRAS/V12 and KRAS/H61)-transduced cells than in both mock (MOCK)- and WLD KRAS (KRAS/WLD)-transduced cells are listed in Table I. Quantitative RT-PCR analysis confirmed that the transduction of oncogenic KRAS induced the largest change in let-7i and miR-31 (Fig. 2). Therefore, we focused on these two miRNAs.

Table I

MicroRNAs differentially expressed by the oncogenic KRAS.

Table I

MicroRNAs differentially expressed by the oncogenic KRAS.

NameAccessionMOCKKRAS/WLDKRAS/V12KRAS/H61
Upregulated
 hsa-miR-1238MIMAT00055934.538.4511.499.58
 hsa-miR-31MIMAT00000896.405.9616.5312.68
 hsa-miR-191*MIMAT00016185.279.3311.249.82
 hsa-let-7iMIMAT0000415109.4498.74224.70164.02
 hsa-miR-31*MIMAT000450415.0113.4422.8019.14
 hsa-miR-29b*MIMAT00045142.702.667.313.25
 hsa-miR-29bMIMAT000010095.8766.36141.79100.44
 hsa-miR-625MIMAT00032944.704.035.854.74
 hsa-miR-940MIMAT000498312.0711.9215.1112.11
Downregulated
 hsa-miR-30cMIMAT000024416.9512.5212.139.82
 hsa-miR-15bMIMAT0000417208.10154.26148.56102.32
 hsa-miR-28-5pMIMAT00000855.664.093.993.60
 hsa-miR-30a*MIMAT00000888.716.705.734.91
 hsa-miR-23bMIMAT00004188.936.365.874.78
 hsa-miR-130aMIMAT000042555.9939.1936.3126.76
 hsa-miR-210MIMAT000026741.2628.9326.1622.94
 hsa-miR-16MIMAT0000069157.08110.5796.1973.35
 hsa-miR-30eMIMAT00006926.704.474.033.68
 hsa-miR-196aMIMAT000022627.4819.7515.8511.31
 hsa-miR-4286MIMAT0016916264.56205.35150.73138.68
 hsa-miR-15aMIMAT000006857.4940.2531.7029.54
 hsa-let-7bMIMAT0000063104.4876.0654.2939.47
 hsa-miR-205MIMAT0000266116.3474.8859.4150.10
 hsa-miR-34aMIMAT000025536.3224.8316.7119.56
 hsa-miR-1246MIMAT000589852.6336.0023.6621.83
 hsa-miR-27bMIMAT000041912.858.095.494.76
 hsa-miR-429MIMAT000153611.467.444.713.73

[i] Normalized expression levels in transfectants are shown. Accession, gene bank accession no.; WLD, wild-type.

Let-7i and miR-31 expression in lung cancer cell lines

Let-7i and miR-31 expression levels were analyzed by quantitative RT-PCR analysis. Let-7i was expressed at various levels in all the cells examined. Let-7i levels were higher in cancer cell lines than in non-cancerous immortalized cell lines and primary airway epithelial cells (Fig. 3). In contrast, miR-31 expression levels were lower in cancer cell lines, and some cell lines [27.5%, 11/40, the loss of miR-31 expression was frequently detected in LCC cell lines (80.0%, 4/5)] completely lost its expression (Fig. 2). These results prompted us to further investigate the potential significance of the loss of miR-31 expression in lung carcinogenesis.

Effect of the restoration and knockdown of miR-31 on cell growth

The restoration of the miR-31 SS, but not the antisense strand, markedly suppressed the growth of cancer cell lines that lost miR-31 expression, as it decreased the formation of colonies and prolonged the doubling time (Fig. 4, TKB1 and H441 cells were examined. A representative result in TKB1 cells is shown). On the other hand, the knockdown of miR-31 expression in cell lines that retained its expression resulted in an enhancement in their growth activities in both ordinary media and soft agar (Fig. 5, HARA and A549 cells were examined. A representative result in HARA cells is shown).

Comprehensive search for potential genes modulated by miR-31

A comprehensive evaluation of gene expression profiles revealed that miR-31 SS-transduced cells had a different expression profile from empty vector (mock)- and miR-31 antisense strand (AS)-transduced cells, as miR-31SS-transduced cells were classified into the most distant branch on a dendrogram described from a hierarchical clustering analysis (Fig. 6). Transcripts with expression levels that were >5-fold higher in miR-31SS-transduced cells than in mock- and miR-31AS-transduced cells were extracted (Tables II and III).

Table II

Upregulated transcripts.

Table II

Upregulated transcripts.

MOCKmiR-31SSmiR-31AS



SymbolAccessionSignalFlagSignalFlagSignalFlagmiR-31SS/MOCKmiR-31SS/AS
NUDT10NM_1531830.0119A1.1978P0.0124A100.836096.2198
RIPK3NM_0068710.0114A1.0102P0.0121A88.431883.3022
C2CD4BNM_0010075950.0245A0.5805P0.0115A23.680950.5356
SERPINI1NM_0050250.0112A0.4145P0.0116A37.089435.6275
KRTAP10-11NM_1986920.0123A0.4065P0.0116A33.174235.1714
SLC24A4NM_1536470.0357A1.0444P0.0314A29.283333.2618
CCDC140NM_1530380.0110A0.3725P0.0115A33.964432.4076
PLA2R1NM_0073660.0103A0.2964P0.0106A28.761927.8916
HTR3DNM_1825370.0106A0.3012P0.0111A28.335327.0245
CXCR2P1NR_0027120.0128A0.2771P0.0103A21.712527.0160
STEAP2NM_0012449450.0099A0.2577P0.0102A25.908525.1678
GSG1LNM_0011097630.0148A0.2657P0.0108A17.931924.5156
PLAC9NM_0010129730.0111A0.2532P0.0109A22.731523.2710
MPPED2NM_0015840.0119A0.2784P0.0124A23.447622.5310
NKX2-8NM_0143600.0322A0.3877P0.0179A12.040921.6246
ZMAT1NM_0010116570.0112A0.2303P0.0116A20.643619.8318
CYYR1NM_052954.20.0255A0.2456P0.0128A9.635519.2358
MYL1NM_0794200.0095A0.1850P0.0098A19.393218.7914
ENTPD1NM_0017760.0106A0.2072P0.0111A19.555318.6842
OTOL1NM_0010804400.0111A0.2118P0.0115A19.151118.4960
BPIFB4NM_1825190.0106A0.2031P0.0111A19.167518.3275
STARD6NM_1391710.0104A0.1980P0.0109A18.970818.2463
FAM70ANM_0179380.0100A0.1856P0.0103A18.617918.0836
SLC34A2NM_0064240.0117A0.2084P0.0121A17.852217.1757
TCL6NR_0282880.0119A0.2186P0.0128A18.368917.0699
TMEM64NM_0010084950.0401A0.4789P0.0290A11.934316.5116
SLC25A21NM_0306310.0109A0.1860P0.0113A17.026116.4283
FABP7NM_0014460.0139A0.1728P0.0107A12.450016.2039
ADAMTSL2NM_0146940.0249A0.1737P0.0110A6.969015.7766
OR2W5NM_0010046980.0112A0.1846P0.0117A16.457115.7667
MLANANM_0055110.0094A0.1372P0.0098A14.526113.9842
LDLRAD2NM_0010136930.0119A0.1733P0.0124A14.624813.9382
OR5K1NM_0010047360.0095A0.1334P0.0098A14.098513.5742
TMPRSS4NM_0198940.0283A0.1621P0.0122A5.728713.2791
GLT25D2NM_0151010.0105A0.1410P0.0109A13.483912.8923
FMN2NM_0200660.0088A0.1484P0.0120A16.783312.4044
SLC3A1NM_0003410.0154A0.2346P0.0207A15.203811.3567
AMICA1NM_1532060.0185A0.2714P0.0247A14.632410.9989
FAM186BNM_0321300.0919P1.1598P0.1056P12.626710.9792
SNORD115-2NR_0032940.0116A0.1296P0.0123A11.162510.5393
PAX5NM_0167340.0110A0.1198P0.0114A10.932410.5101
MAGEC2NM_0162490.0137A0.1294P0.0125A9.418110.3264
IPWNR_0239150.0221A0.3631P0.0353A16.394510.2860
TNFSF11NM_0330120.0107A0.1121P0.0111A10.509510.0805
SIGLECP3NR_0028040.0627P0.3595P0.0359A5.729510.0019
WDR49AK0975560.0096A0.0992P0.0099A10.39109.9812
SATL1NM_0010129800.0120A0.1243P0.0127A10.36069.7853
SLC19A3NM_0252430.0121A0.1239P0.0127A10.23729.7655
DPP6BC0359120.0249A0.1543P0.0160A6.20279.6431
OR5H2NM_0010054820.0156A0.1053P0.0109A6.73019.6315
CIB4NM_0010298810.0117A0.1166P0.0122A9.95769.5572
WDFY4NM_0209450.0122A0.1211P0.0128A9.95579.4762
SAMD13NM_0010109710.0105A0.1029P0.0109A9.83229.4019
NBPF6NM_0011439880.0113A0.1090P0.0117A9.68559.2810
FAM135BNM_0159120.0097A0.0922P0.0100A9.49699.2206
OR4A47NM_0010055120.0105A0.1659P0.0181A15.84349.1669
CCL22NM_0029900.0117A0.1145P0.0126A9.76149.0799
SLC5A4NM_0142270.0105A0.0987P0.0109A9.42529.0167
SH3TC1NM_0189860.0838P0.6744P0.0752P8.05218.9695
MYH7NM_0002570.0119A0.1112P0.0124A9.37408.9612
TXLNG2PNR_0451290.0111A0.1045P0.0117A9.40008.9166
NUBPLNM_0251520.0104A0.0950P0.0109A9.09718.7016
CNGA3NM_0012980.0094A0.1537P0.0177A16.27208.6960
EGR4NM_0019650.0105A0.0938P0.0109A8.91988.6280
IL24NM_0011851561.4258P9.1516P1.1788P6.41877.7634
RXFP1NM_0216340.0111A0.0888P0.0115A8.00627.7382
CCDC85CNM_0011449950.0116A0.1157P0.0154A9.97297.4922
TRIM36NM_0010173970.0108A0.1024P0.0137A9.47777.4839
TACC1BC0413910.0128A0.1323P0.0177A10.30647.4633
FAM24BNM_1526440.0140A0.0911P0.0126A6.50277.2256
ITGALNM_0022090.0668P0.4498P0.0641P6.73617.0141
GMCL1P1NR_0032810.0104A0.3532P0.0506A33.90626.9811
IL24NM_00118515610.1283P66.0420P9.7012P6.52056.8076
PTPN20ANM_0010423870.0097A0.0671P0.0101A6.90396.6542
SRPXNM_0063070.0090A0.0602P0.0093A6.66226.4412
RORCNM_0050600.0096A0.0797P0.0124A8.31906.4372
HDAC11NM_0011360410.0688P0.5473P0.0856P7.95916.3925
CRHBPNM_0018820.0094A0.0623P0.0098A6.65876.3675
SPTSSBNM_0010401000.0111A0.0738P0.0117A6.65276.3147
SERPINB11NM_0804750.0110A0.1245P0.0201A11.28006.2069
CC2D2BXM_0034035280.0102A0.0656P0.0106A6.42876.2016
DNAH17NM_1736280.0097A0.1610P0.0263A16.62276.1282
DUOXA2BX5375810.0087A0.0552P0.0090A6.35246.1045
SLC35G3NM_1524620.0088A0.0557P0.0092A6.31356.0811
SLC17A4NM_0054950.0105A0.0667P0.0110A6.34936.0686
MEOX2NM_0059240.0118A0.0726P0.0123A6.17905.8922
CLDN8NM_1993280.0106A0.0630P0.0109A5.96345.7753
TMEM108NM_0239430.0179A0.2513P0.0440A13.99975.7122
DPP6NM_0010393500.0124A0.2982P0.0533A23.97895.5933
FAM164ANM_0160100.0103A0.0598P0.0107A5.80985.5618
GSTTP1NR_0030810.0118A0.0823P0.0148A6.97105.5445
OR4F6NM_0010053260.0118A0.0611P0.0113A5.18415.4305
HEATR7B1XM_0017212400.0153A0.1492P0.0277A9.73005.3768
DPYSNM_0013850.0100A0.0554P0.0104A5.53065.3504
OR10Z1NM_0010044780.0121A0.0661P0.0127A5.46145.2210
PARP10NM_0327890.1746P1.1198P0.2158P6.41495.1885
PIEZO2AK0922260.0088A0.0466P0.0091A5.29345.0979
ACCN5NM_0174190.0090A0.0478P0.0094A5.29105.0893
CAPS2AF2510560.0105A0.0636P0.0126A6.05115.0303
RBPMSAK0575330.0124A0.0900P0.0180A7.24535.0094

[i] MOCK, empty vector-transduced; miR-31SS, miR-31 sense strand-transduced; miR-31 antisense strand-tranduced NHBE-T cells; accession, gene bank accession no.; flag indicates whether gene expression was present (P) or absent (A).

Table III

Downregulated transcripts.

Table III

Downregulated transcripts.

MOCKmiR-31SSmiR-31AS



SymbolAccessionSignalFlagSignalFlagSignalFlagmiR-31SS/MOCKmiR-31SS/AS
DYTNNM_00109373058.1445P7.8986P169.4681P0.13580.0466
MGPNM_0009000.2369P0.0116A0.2249P0.04900.0516
SAMD9LNM_1527030.1643P0.0149A0.2193P0.09080.0680
SAA2NM_0011273800.3662P0.0299A0.3758P0.08170.0796
SAA4NM_0065120.6052P0.0521P0.6040P0.08610.0863
SAA1NM_00033110.6803P1.0635P10.1069P0.09960.1052
CCR1NM_0012950.0807P0.0092A0.0858P0.11440.1077
SAA2NM_0307545.8777P0.6367P5.8671P0.10830.1085
CIITANM_0002460.0657P0.0104A0.0925P0.15900.1129
SPTBN1NM_0031280.0835P0.0101A0.0893P0.12100.1132
MGPNM_0011908390.5979P0.0643P0.5636P0.10750.1141
XAF1NM_0175230.6196P0.0819P0.7122P0.13220.1150
BTBD8NM_1832420.0712P0.0083A0.0659P0.11620.1255
PLAC8NM_0011307150.0686P0.0086A0.0683P0.12590.1265
OLFM4NM_0064180.0751P0.0085A0.0667P0.11280.1269
CLCA2NM_0065360.1033P0.0105A0.0787P0.10170.1335
CCL2NM_0029820.5769P0.0771P0.5550P0.13370.1390
SAMD9LNM_1527030.7186P0.1299P0.9136P0.18070.1421
TRERF1NM_0335020.0967P0.0131A0.0889P0.13540.1472
PLEKHG4NM_0154320.0871P0.0157A0.1017P0.17970.1539
ITPK1-AS1NR_0028080.0947P0.0120A0.0760P0.12620.1572
DEFB127NM_1390740.1531P0.0088A0.0538P0.05720.1627
TFECNM_0122520.2166P0.0095A0.0576P0.04380.1648
MEOX1NM_0045270.0839P0.0131A0.0792P0.15590.1652
GGT5NM_0010997811.6842P0.2891P1.7144P0.17170.1687
RPGRNM_0010348530.0956P0.0179A0.1027P0.18690.1741
CD99L2BC0257290.0623P0.0109A0.0623P0.17490.1748
CCNG2NM_0043540.0846P0.0156A0.0878P0.18480.1781
TNFSF15 NM_0051183.6060P0.6136P3.4121P0.17020.1798
IL7RNM_0021850.7080P0.1181P0.6427P0.16680.1837
HSH2DNM_0328550.4028P0.0771P0.4128P0.19150.1869
BDKRB1NM_0007100.1140P0.0108A0.0578P0.09480.1869
SPARCNM_00311812.4315P2.4446P12.9549P0.19660.1887
HSD17B11NM_0162450.1320P0.0245A0.1294P0.18560.1894
EBI3NM_0057550.0981P0.0150A0.0789P0.15310.1904
ADRA1BNM_0006791.2211P0.2366P1.2080P0.19380.1959

[i] MOCK, empty vector-transduced; miR-31SS, miR-31 sense strand-transduced; miR-31 antisense strand-tranduced NHBE-T cells; accession, gene bank accession no.; flag indicates whether gene expression was present (P) or absent (A).

Involvement of different oncogenic pathways in the induction of miR-31

The expression of miR-31 was also induced by the transduction of oncogenic EGFR [deletion mutation (deletion: E746-A750), point mutation L858R], but not by oncogenic BRAF (V600E) or oncogenic PIK3CA (E545K, H1047R) (Fig. 7). The expression of miR-31 was consistently not induced by specific mutants in KRAS that activated MEK, RAL-GDS or PI3K (Fig. 7). Inhibitors for MEK (the BRAF-MEK-ERK pathway) or PI3Kinase (the PIK3CA-mediated pathway) interfered with the induction of miR-31 by oncogenic KRAS.

Discussion

The findings of an initial study on breast cancer suggested that miR-31 could be a tumor suppressor that especially inhibited the invasive and metastatic spread of neoplastic cells (14). Previous studies have supported this initial observation and identified the potential molecular mechanisms responsible for the inhibition of invasion and metastasis by miR-31 (15). miR-31 was also shown to be downregulated in gastric cancer and was suggested to function as a tumor suppressor (16). In contrast, previous studies reported that miR-31 was upregulated in colorectal cancer and may also promote the invasive and metastatic spread of neoplastic cells (17,18). Thus, the potential role of miR-31 in carcinogenesis may differ depending on the type of cancer. The expression of miR-31 in lung cancer has generally been reported to be higher in tumor tissue than in the corresponding non-tumorous tissue (1925). However, miR-31 levels have been shown to vary in primary lung tumors. Some tumors expressed miR-31 at lower levels than the corresponding non-tumorous tissue or did not express it at all. miR-31 expression was markedly reduced or completely lost in some lung cancer cell lines (Fig. 3). This result suggested that miR-31 may be a suppressor in lung carcinogenesis. The restoration of miR-31 in lung cancer cell lines that had lost its expression markedly attenuated their growth activities (Fig. 4). The knockdown of miR-31 expression in cell lines that retained its expression enhanced oncogenic phenotypes such as anchorage-independent growth activity (Fig. 5). These results supported miR-31 being a suppressor. However, a recent study demonstrated the oncogenic role of miR-31 in lung carcinogenesis using experiments with different cell lines from the ones used in the present study (26). miR-31 may play a pleiotropic role in the development of individual tumors. Further investigations are required to resolve this issue.

In the present study, miR-31 was upregulated not only by oncogenic KRAS, but also by oncogenic EGFR in vitro (Fig. 7). Neither oncogenic BRAF nor oncogenic PIK3CA induced its expression (Fig. 7). Similarly, the specific mutant of KRAS that activated the MEK-mediated pathway only and another specific mutant that activated the PI3K-mediated pathway only did not induce the expression of miR-31 (Fig. 7). On the other hand, the MEK inhibitor, but not PI3K inhibitor, attenuated the oncogenic KRAS-induced expression of miR-31 (Fig. 7). These results suggest that miR-31 could be a common player that regulates the KRAS/EGFR-mediated essential oncogenic pathway (Fig. 8). The MEK-mediated pathway may be necessary, but not sufficient for the induction of miR-31 expression (Fig. 8). The PI3K-mediated pathway may not be involved in its regulation (Fig. 8). A novel pathway may be inducing the expression of miR-31 in cooperation with the MEK-mediated pathway.

This comprehensive search for the potential target of miR-31 revealed that the downregulated transcripts included many molecules mediating the cytokine/chemokine signaling pathway (Tables II and III). TNFSF15, in particular, has been published on an online site as the predicted downstream target for miR-31 (http://mirdb.org/miRDB/index.html). TNFSF15, a member of the tumor necrosis factor (TNF) and TNF receptor superfamilies, regulates cell growth and apoptosis in an autocrine manner, and has been reported to suppress cancer cell growth by inhibiting angiogenesis (27). Thus, TNFSF15 could be a factor that participates in miR-31-induced growth modulations. Future studies that focus on the potential downstream targets of miR-31 may provide insight into the molecular mechanisms involved in lung cancer.

The present study comprehensively searched miRNAs, the expressions of which were regulated by oncogenic KRAS, and focused on miR-31 in order to investigate its potential involvement in lung carcinogenesis. The expression of miR-31 was markedly attenuated in lung cancer cell lines. The restoration of miR-31 in lung cancer cell lines that lost its expression attenuated their growth activities. The knockdown of miR-31 expression in cell lines that retained its expression enhanced oncogenic phenotypes. These results suggest that miR-31 may be a suppressor, the loss of which may promote lung carcinogenesis.

Acknowledgements

This study was supported by the Japanese Ministry of Education, Culture, Sports and Science (Tokyo Japan), and by a grant from the Yokohama Medical Facility (Yokohama, Japan). We especially thank Emi Honda and Misa Otara (Kanagawa Prefectural Cardiovascular and Respiratory Center Hospital, Yokohama, Japan) for their assistance.

References

1 

Hoffman PC, Cohen EE, Masters GA, et al: Carboplatin plus vinorelbine with concomitant radiation therapy in advanced non-small cell lung cancer: a phase I study. Lung Cancer. 38:65–71. 2002. View Article : Google Scholar : PubMed/NCBI

2 

Spira A and Ettinger DS: Multidisciplinary management of lung cancer. N Engl J Med. 350:379–392. 2004. View Article : Google Scholar : PubMed/NCBI

3 

Okudela K, Woo T and Kitamura H: KRAS gene mutations in lung cancer: particulars established and issues unresolved. Pathol Int. 60:651–660. 2010. View Article : Google Scholar : PubMed/NCBI

4 

Woo T, Okudela K, Yazawa T, et al: Prognostic value of KRAS mutations and Ki-67 expression in stage I lung adenocarcinomas. Lung Cancer. 65:355–362. 2009. View Article : Google Scholar : PubMed/NCBI

5 

Okudela K, Yazawa T, Ishii J, et al: Down-regulation of FXYD3 expression in human lung cancers: its mechanism and potential role in carcinogenesis. Am J Pathol. 175:2646–2656. 2009. View Article : Google Scholar : PubMed/NCBI

6 

Pao W and Girard N: New driver mutations in non-small-cell lung cancer. Lancet Oncol. 12:175–180. 2011. View Article : Google Scholar : PubMed/NCBI

7 

van Eijk R, Licht J, Schrumpf M, et al: Rapid KRAS, EGFR, BRAF and PIK3CA mutation analysis of fine needle aspirates from non-small-cell lung cancer using allele-specific qPCR. PLoS One. 6:e177912011.

8 

Xu J, He J, Yang H, et al: Somatic mutation analysis of EGFR, KRAS, BRAF and PIK3CA in 861 patients with non-small cell lung cancer. Cancer Biomark. 10:63–69. 2012.PubMed/NCBI

9 

Cozens AL, Yezzi MJ, Kunzelmann K, et al: CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells. Am J Respir Cell Mol Biol. 10:38–47. 1994. View Article : Google Scholar : PubMed/NCBI

10 

Yazawa T, Kamma H, Fujiwara M, et al: Lack of class II transactivator causes severe deficiency of HLA-DR expression in small cell lung cancer. J Pathol. 187:191–199. 1999. View Article : Google Scholar

11 

Okudela K, Yazawa T, Suzuki T, Sugimura H and Kitamura H: Role of 3′-phosphoinositides in oncogenic KRAS-induced modulation of shape and motility of airway epithelial cells. Pathol Int. 59:28–37. 2009.

12 

Okudela K, Suzuki M, Kageyama S, et al: PIK3CA mutation and amplification in human lung cancer. Pathol Int. 57:664–671. 2007. View Article : Google Scholar

13 

Okudela K, Yazawa T, Woo T, et al: Down-regulation of DUSP6 expression in lung cancer: its mechanism and potential role in carcinogenesis. Am J Pathol. 175:867–881. 2009. View Article : Google Scholar : PubMed/NCBI

14 

Valastyan S, Reinhardt F, Benaich N, et al: A pleiotropically acting microRNA, miR-31, inhibits breast cancer metastasis. Cell. 137:1032–1046. 2009. View Article : Google Scholar : PubMed/NCBI

15 

Augoff K, Das M, Bialkowska K, McCue B, Plow EF and Sossey-Alaoui K: miR-31 is a broad regulator of β1-integrin expression and function in cancer cells. Mol Cancer Res. 9:1500–1508. 2011.

16 

Zhang Y, Guo J, Li D, et al: Down-regulation of miR-31 expression in gastric cancer tissues and its clinical significance. Med Oncol. 27:685–689. 2010. View Article : Google Scholar : PubMed/NCBI

17 

Cottonham CL, Kaneko S and Xu L: miR-21 and miR-31 converge on TIAM1 to regulate migration and invasion of colon carcinoma cells. J Biol Chem. 285:35293–35302. 2010. View Article : Google Scholar : PubMed/NCBI

18 

Slaby O, Svoboda M, Fabian P, et al: Altered expression of miR-21, miR-31, miR-143 and miR-145 is related to clinicopathologic features of colorectal cancer. Oncology. 72:397–402. 2007. View Article : Google Scholar : PubMed/NCBI

19 

Guan P, Yin Z, Li X, Wu W and Zhou B: Meta-analysis of human lung cancer microRNA expression profiling studies comparing cancer tissues with normal tissues. J Exp Clin Cancer Res. 31:542012. View Article : Google Scholar : PubMed/NCBI

20 

Jang JS, Jeon HS, Sun Z, et al: Increased miR-708 expression in NSCLC and its association with poor survival in lung adenocarcinoma from never smokers. Clin Cancer Res. 18:3658–3667. 2012. View Article : Google Scholar : PubMed/NCBI

21 

Gao W, Yu Y, Cao H, et al: Deregulated expression of miR-21, miR-143 and miR-181a in non small cell lung cancer is related to clinicopathologic characteristics or patient prognosis. Biomed Pharmacother. 64:399–408. 2010. View Article : Google Scholar : PubMed/NCBI

22 

Võsa U, Vooder T, Kolde R, et al: Identification of miR-374a as a prognostic marker for survival in patients with early-stage nonsmall cell lung cancer. Genes Chromosomes Cancer. 50:812–822. 2011.PubMed/NCBI

23 

Xing L, Todd NW, Yu L, Fang H and Jiang F: Early detection of squamous cell lung cancer in sputum by a panel of microRNA markers. Mod Pathol. 23:1157–1164. 2010. View Article : Google Scholar : PubMed/NCBI

24 

Yu L, Todd NW, Xing L, et al: Early detection of lung adenocarcinoma in sputum by a panel of microRNA markers. Int J Cancer. 127:2870–2878. 2010. View Article : Google Scholar : PubMed/NCBI

25 

Tan X, Qin W, Zhang L, et al: A 5-microRNA signature for lung squamous cell carcinoma diagnosis and hsa-miR-31 for prognosis. Clin Cancer Res. 17:6802–6811. 2011. View Article : Google Scholar : PubMed/NCBI

26 

Meng W, Ye Z, Cui R, et al: MicroRNA-31 predicts the presence of lymph node metastases and survival in lung adenocarcinoma patients. Clin Cancer Res. 19:5423–5433. 2013. View Article : Google Scholar : PubMed/NCBI

27 

Duan L, Yang G, Zhang R, Feng L and Xu C: Advancement in the research on vascular endothelial growth inhibitor (VEGI). Target Oncol. 7:87–90. 2012. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

October 2014
Volume 32 Issue 4

Print ISSN: 1021-335X
Online ISSN:1791-2431

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
APA
Okudela, K., Suzuki, T., Umeda, S., Tateishi, Y., Mitsui, H., Miyagi, Y., & Ohashi, K. (2014). A comprehensive search for microRNAs with expression profiles modulated by oncogenic KRAS: Potential involvement of miR-31 in lung carcinogenesis. Oncology Reports, 32, 1374-1384. https://doi.org/10.3892/or.2014.3339
MLA
Okudela, K., Suzuki, T., Umeda, S., Tateishi, Y., Mitsui, H., Miyagi, Y., Ohashi, K."A comprehensive search for microRNAs with expression profiles modulated by oncogenic KRAS: Potential involvement of miR-31 in lung carcinogenesis". Oncology Reports 32.4 (2014): 1374-1384.
Chicago
Okudela, K., Suzuki, T., Umeda, S., Tateishi, Y., Mitsui, H., Miyagi, Y., Ohashi, K."A comprehensive search for microRNAs with expression profiles modulated by oncogenic KRAS: Potential involvement of miR-31 in lung carcinogenesis". Oncology Reports 32, no. 4 (2014): 1374-1384. https://doi.org/10.3892/or.2014.3339