Let-7b Inhibits Human Cancer Phenotype by Targeting Cytochrome P450 Epoxygenase 2J2

Background MicroRNAs (miRNAs) are small, noncoding RNA molecules of 20 to 22 nucleotides that regulate gene expression by binding to their 3′ untranslated region (3′UTR). Increasing data implicate altered miRNA participation in the progress of cancer. We previously reported that CYP2J2 epoxygenase promotes human cancer phenotypes. But whether and how CYP2J2 is regulated by miRNA is not understood. Methods and Results Using bioinformatics analysis, we found potential target sites for miRNA let-7b in 3′UTR of human CYP2J2. Luciferase and western blot assays revealed that CYP2J2 was regulated by let-7b. In addition, let-7b decreased the enzymatic activity of endogenous CYP2J2. Furthermore, let-7b may diminish cell proliferation and promote cell apoptosis of tumor cells via posttranscriptional repression of CYP2J2. Tumor xenografts were induced in nude mice by subcutaneous injection of MDA-MB-435 cells. The let-7b expression vector, pSilencer-let-7b, was injected through tail vein every 3 weeks. Let-7b significantly inhibited the tumor phenotype by targeting CYP2J2. Moreover, quantitative real-time polymerase chain reaction and western blotting were used to determine the expression levels of let-7b and CYP2J2 protein from 18 matched lung squamous cell cancer and adjacent normal lung tissues; the expression level of CYP2J2 was inversely proportional to that of let-7b. Conclusions Our results demonstrated that the decreased expression of let-7b could lead to the high expression of CYP2J2 protein in cancerous tissues. These findings suggest that miRNA let-7b reduces CYP2J2 expression, which may contribute to inhibiting tumor phenotypes.

Several studies have reported that EETs have diverse biological effects within the cardiovascular system. The EETs released from the endothelium activated calcium-sensitive potassium channels and resulted in hyperpolarization of smooth muscle cells and vascular relaxation [7]. Moreover, physiological concentrations of EETs or overexpression of CYP2J2 reduced vascular cell adhesion molecule-1 (VCAM-1) expression and prevented leukocyte adhe-sion to the vascular wall [8]. The inhibitory effects of EETs show that they have anti-inflammatory effects in vascular system independent of their membrane-hyperpolarizing effects [8]. In addition, EETs also promoted endothelial cell proliferation, migration, and angiogenesis by activating both mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 (PI3)-kinase/ Akt pathways [9].
On the other hand, other evidences indicate that epoxygenase overexpression or EETs treatment have potentially deleterious effects. In recent publications, CYP2J2-derived EETs were found to play important roles in a host of processes related to cancer cell behavior and tumor pathogenesis. We found high expression of CYP2J2 in human tumors, as well as in eight human-derived carcinoma cell lines, but not in adjacent normal tissues and nontumoral human cell lines [10]. Overexpression of CYP2J2 or addition of exogenous EETs markedly accelerated proliferation and metastasis of cancer cells in vitro and in vivo [10,11]. CYP2J2 overexpression or addition of exogenous EETs protected human carcinoma cells from apoptosis by upregulating the antiapoptotic proteins, Bcl-2 and Bcl-xL, and by downregulating the proapoptotic protein, Bax [10]. In contrast, selective inhibitors of CYP2J2 had significant antitumor effects in vitro and in vivo and were associated with reduced EET biosynthesis [12]. Collectively, all the findings demonstrated the important and previously unrecognized roles of CYP2J2 and its EET products in carcinogenesis.
Increasing evidences indicate that miRNAs play important roles in diverse biological processes, such as proliferation, differentiation, and apoptosis during development [13,14,15]. Several studies have indeed described the aberrant expression in human tumors of miRNAs and their function controlling the expression of certain oncogenes and tumor suppressor genes [16,17,18]. For example, miR-15a and miR-16 are frequently deleted or downregulated in squamous cell carcinomas and adenocarcinomas of the lung [19]. MicroRNA-101 is downregulated in bladder transitional cell carcinoma (TCC) tissues and inhibits cell proliferation and colony formation in TCC cell lines by directly repressing oncogene EZH2 [20]. A recent study indicated that miRNA-let-7a inhibits the expression of MYC and reverses MYC-induced growth in Burkitt lymphoma cells [21]. Previous reports indicate that let-7 is poorly expressed in a variety of human tumors and reduced let-7 level results in over-expression (cyclinD, RAS, MYC) of let-7-responsive genes in tumors [22,23,24,25]. However, the exact role of let-7 in cancer is not yet fully understood. Our preliminary data show that let-7b is down-regulated in human lung squamous tumors, while levels of CYP2J2 protein is up-regulated, suggesting that human CYP2J2 might be post-transcriptionally regulated by let-7b. Hence, the purpose of the present study was to investigate this hypothesis that let-7b might act as a tumor suppressor through targeting CYP2J2.

Let-7b Targets the 39UTR of CYP2J2
To investigate if CYP2J2 is regulated directly by let-7b, we constructed a luciferase reporter plasmid containing the 39UTR of CYP2J2 cloned downstream of the luciferase reporter gene ( Figure 1A). We transfected the luciferase construct into HepG2 cells together with let-7b or random let-7b. We found that transfection of pMIR/CYP2J2-39UTR along with let-7b resulted in a significant reduction in reporter activity than did those of control and random transfections. On the other hand, no significant downregulation of the pMIR reporter activity could be determined when we transfected the pMIR reporter (empty vector) along with let-7b or random let-7b into HepG2 cells ( Figure 1B). To further confirm that CYP2J2 is a target of let-7b, we constructed seven mutants (pMIR/CYP2J2-3?UTR mutant, mutant-1, mutant-2 … and mutant-6, respectively) based on pMIR/CYP2J2-3?UTR. Then we transfected these constructs into the cells (MDA-MB-435 and SK-MES-1) and analyzed luciferase reporter activity. The assays showed that luciferase activity of pMIR/CYP2J2-3?UTR mutant was not repressed by let-7b, compared with the wild type pMIR/ CYP2J2-3?UTR ( Figure 1C and D). Among the remaining six mutants (mutant-1, mutant-2…mutant-6), luciferase activity of mutant-3 was repressed by let-7b, comparing with random and control (P,0.05) ( Figure 1E). Furthermore, binding site III completely matched the seed sequence of let-7b if wobble base pairing was allowed. These data indicate that CYP2J2 is one of the targets of let-7b. To investigate the effects of exogenous let-7b on the protein  level and enzymatic activity of endogenous CYP2J2 proteins, we  examined the protein levels of CYP2J2 in HeLa, Tca-8113, SK-MES-1, and MDA-MB-435 cells after treatment with let-7b for  48 h (100 nM). Western blot analysis showed that overexpression of let-7b significantly downregulated the expression of CYP2J2 in four cancer cell lines (Figure 2A). Relative CYP2J2 expression was quantified by densitometry ( Figure 2B). As in Figure S1A and B, western blots showed dose-dependent effect of let-7b and let-7b inhibitor on CYP2J2 protein expression. Given the instability of EETs, we determined concentration of the stable metabolite 14,15-DHET in cell culture media to confirm inhibition of let-7b on the enzymatic activity of CYP2J2. The results showed that 14,15-DHET levels were significantly decreased by the transfection of let-7b ( Figure 2C). These data showed that human CYP2J2 is directly downregulated by let-7b.

Let-7b Reduces Cancer Cell Growth and Induces Apoptosis by Directly Downregulating CYP2J2
We have previously reported that CYP2J2 stimulates proliferation of carcinoma cells and protects human carcinoma cells from apoptosis [10]; so it was of interest to evaluate the effects of overexpression of let-7b on cell proliferation and apoptosis when endogenous CYP2J2 was inhibited by let-7b. We treated HeLa, Tca-8113, SK-MES-1, and MDA-MB-435 cells with exogenous let-7b or random let-7b for 48 h. The proliferation rate was determined via Cell-Light TM EDU DNA Cell Proliferation Kit (Ribobio, China). The results showed a significant inhibition of cell proliferation by let-7b. For example, in MDA-MB-435 and SK-MES-1 cells, let-7b reduced cell proliferation by 50% compared with negative control and random let-7b ( Figure 3A). Apoptosis, measured by Annexin V and propidium iodide staining, significantly increased in cells transfected with let-7b ( Figure 3B). In addition, exogenous let-7b decreased the percentage of EDU-positive cells and increased the apoptotic cells in HeLa and Tca-8113 cells ( Figure 3C and D). To provide further evidence that the effects of let-7b expression on cell proliferation and apoptosis were related to CYP2J2, cells transfected with let-7b were treated with C26 (10 mM) (specific CYP2J2 inhibitor [12] ) and 14,15-EET (250 nM) for 48 h. To minimize reduction in levels of 14,15-EET due to autooxidation, cells were stimulated with 14,15-EET or a comparable volume of vehicle (DMSO) every 6 h. As expected, the effects of let-7b expression on cell proliferation and apoptosis were enhanced by C26 and abolished by 14,15-EET ( Figure 3E and F). In addition, to determine whether let-7b treatment affect cell growth of H9c2 (H9c2 cells do not have CYP2J2), we used Cell-Light TM EDU DNA Cell Proliferation Kit and Annexin V and propidium iodide staining to measure the cell proliferation and apoptosis of H9c2 cells. As shown in Figure S2A and B, not only cell proliferation but also cell apoptosis of H9c2 cells weren't affected by let-7b or let-7b inhibitor treatment. These data suggested that overexpression of let-7b resulted in decreased proliferation and activated apoptosis of carcinoma cell lines.
We also investigated the influences of let-7b on the expression of proapoptotic protein Bax and antimetastatic protein nm-23 and the activation of the PI3K/Akt and MAPK signaling pathways, which play important roles in P450 epoxygenase-and EETmediated tumorigenesis and metastasis [10,11,12]. We found that let-7b overexpression in MDA-MB-435 cells significantly downregulated the PI3K, pAkt, pERK pathways but increased Bax and nm-23 expression ( Figure 3G). Similar results were also observed in MDA-MB-435 cell which treated with let-7b agomir ( Figure  S1C and D). However, let-7b did not affect PI3K, pAkt, and Bax of H9c2 cells ( Figure S2C). We speculated that this is because H9c2 cells do not have CYP2J2. These results indicated that the enhancing effect of CYP2J2 on tumor formation could be attenuated by let-7b.

Expression Level of CYP2J2 Protein and let-7b in Human Lung Cancer and in Adjacent Normal Tissues are Inversely Correlated
To investigate an association between let-7b level and the expression of CYP2J2 in human carcinomas, the expression of CYP2J2 protein and let-7b in 18 paired human lung squamous cancer tissues and adjacent nontumor tissues was examined by western blot analysis and real-time RT-PCR, respectively. Results showed that CYP2J2 was highly expressed in the majority of Figure 1. Identification of CYP2J2 as a direct target of let-7b. A, schematic representation of the predicted target sites of let-7b in the 39UTR of CYP2J2. The 39UTR of CYP2J2 was cloned into a luciferase reporter plasmid, termed pMIR/CYP2J2-39UTR. A series of mutants carried mutated nucleotides in six potential binding sites for the let-7b seed region were generated based on wild type pMIR/CYP2J2-39UTR. Mutants of pMIR/CYP2J2-39UTR are constructed by mutating the complementary site (labeled by underline) in the let-7b seed region to their complementary bases. B, luciferase activity was analyzed in HepG2 cells 24 h after transfection with reporter plasmid pMIR/CYP2J2-39UTR or pMIR (empty vector). C-D, fulllength sequence CYP2J2-39UTR containing mutant binding sites for the let-7b seed region were generated based on wild type pMIR/CYP2J2-39UTR. We transfected these constructs into the cells (MDA-MB-435 and SK-MES-1) and analysed luciferase reporter activity. As we expected, let-7b did not affect luciferase activity of mutants compared with wild type. E, the six mutants were transfected into SK-MES-1 cells, in addition to let-7b or random let-7b. The luciferase activity of the six mutants was not repressed by let-7b. Renilla luciferase activities were used to normalize firefly luciferase activity. Columns, mean of three experiments; bars, SD. *, P,0.05. doi:10.1371/journal.pone.0039197.g001 cancer tissue samples compared with adjacent normal tissues ( Figure 4A). Real-time RT-PCR was used to determine the mature let-7b levels in 18 paired human lung squamous cancer tissues and adjacent nontumor tissues. Although changes of 2DDCT values [2(DCT non-tumor tissue 2DCT cancer tissue )] were relatively mild (from 21.85 to 5.4, 2.661.27), the fold change (2 2DDCT ) of let-7b expression between 18 paired human lung squamous cancer tissues and adjacent nontumor tissues was significant (from 0.277 to 42.22, 6.0662.43). Consequently, the results showed that levels of mature let-7b were significantly decreased in 14 lung tumors compared with their matched controls among 18 samples analyzed ( Figure 4B). We next investigated a correlation between let-7b expression level and CYP2J2 protein level in human lung squamous cancer tissues. And there is statistically significant inverse correlation between let-7b expression level and CYP2J2 protein level in 18 sets of lung squamous tumors cancer and paired adjacent nontumor tissues ( Figure 4C). Furthermore, we found that CYP2J2 had inverse expression levels to let-7b in four paired human breast cancer and adjacent nontumor tissues ( Figure 4D and E). And let-7b expression in SK-MES-1 and MDA-MB-435 cells was shown in Figure S3.

Let-7b-mediated Knockdown of CYP2J2 Inhibits Tumor Growth and Metastasis
Furthermore, we investigated the influence of let-7b on tumor growth in an in vivo model. To generate a tumor xenograft model, 2610 6 MDA-MB-435 cells were injected subcutaneously into the right flank of nude mice. Two weeks after injection, when tumors had grown to approximately 40 mm 3 , the let-7b expression vector pSilencer-let-7b was injected into mice at a dose of 4 mg/kg body weight through tail vein every 3 weeks. As expected, mice injected with pSilencer-let-7b via tail vein showed a significant reduction in tumor volume compared to the controls ( Figure 5A). During the treatment period, we determined the 14,15-DHET level in the urine of nude mice and found a significant reduction in 14,15-DHET in the let-7b treatment group compared with the controls (Figure 5B). At the end of the treatment period, let-7b resulted in significantly decreased tumor weight, but body weight had not changed ( Figure 5C). We also treated MDA-MB-435 cell with let-7b agomir (150 nM) or agomir control for 48 h, and then injected these cells into the right flank of nude mice. After 4 weeks, necropsies were performed, and all tumors per mouse were weighed. Let-7b agomir resulted in significantly decreased tumor weight ( Figure  S4). Furthermore, the level of mature let-7b was validated by real-time RT-PCR; results showed that pSilencer-let-7b treatment resulted in a significant increase in both tumor and organs ( Figure 5D and E). Western blot analysis showed a markedly decreased expression level of CYP2J2 protein and a significantly increased expression level of proapoptotic protein Bax and antimetastatic protein nm-23 in the let-7b-treated mice compared with controls ( Figure 5F). These results suggest that let-7b can inhibit the expression and tumor-promoting functions of CYP2J2.
Furthermore, we evaluated whether overexpression of let-7b affected metastasis. At the end of the experiment, spleens were removed and incised vertically to count metastatic tumor colonies. Metastases in longitudinal section of the spleen were visible to the naked eye as nodules. We observed a significant decrease in the average number of spleen metastases in mice injected with pSilencer-let-7b compared with controls ( Figure 6A). We also determined the extent of lymph node metastasis by measuring the weight of lymph nodes. We found that injection of pSilencer-let-7b through tail vein reduced the average weight of axillary lymph nodes ( Figure 6B). Further histological analysis after hematoxylin and eosin staining of paraffin sections showed a marked difference between athymic mice treated with let-7b and controls ( Figure 6C and D): the let-7b treatment caused a significant increase in the incidence of necrotic regions in tumor and spleen. Compared with the control group, the metastases focis were smaller and fewer in sections of spleen of the let-7b treatment group. Furthermore, TUNEL staining of sections showed that let-7b treatment resulted in a significant increase in TUNEL-positive cells in tumors and in mice spleen ( Figure 6E and F). These data indicate that let-7b treatment can inhibit tumor metastasis.

Discussion
In the present study, we have shown a new mechanism for controlling the tumor-promotion function of CYP2J2. Bioinformatics analysis predicted that let-7b is a potential regulator of CYP2J2 gene. Using CYP2J2-expression cancer cell lines and a tumor xenografs model, we have shown that human CYP2J2 is posttranscriptionally regulated by let-7b and that the posttranscriptional regulation is responsible for the tumor-promotion function of CYP2J2. Furthermore, in human lung squamous cancer and adjacent nontumor tissues, we observed a reverse relationship between let-7b and CYP2J2 expression levels.
It has become apparent that microRNAs regulate the expression of target genes by binding to the complementary regions of the 39UTR of their target genes [26], but whether hsa-let-7b regulates human CYP2J2 expression is not known. Previous study has shown that let-7 regulates RAS through its 39UTR [27]. In the present study, luciferase assays showed that let-7b repressed the activity of the reporter construct containing the 39UTR region of CYP2J2 mRNA. Transfection of carcinoma cell lines with let-7b and injection of pSilencer-let-7b producing mature let-7b in vivo decreased CYP2J2 protein expression and enzymatic activity, suggesting that CYP2J2 is posttranscriptionally negatively regulated by let-7b.
Recent studies have demonstrated that let-7 might act as a tumor suppressor and that reduced let-7 level results in let-7responsive gene (cyclinD, RAS, MYC, etc.) overexpression in tumors [22,23,24,25]. With western blot analysis and real-time RT-PCR, we showed higher expression of CYP2J2 protein and lower expression of let-7b in 18 paired human lung squamous cancer tissues compared with the adjacent nontumor tissues. Thus, the high expression of CYP2J2 protein in cancer tissues may result from the decreased expression of let-7b, at least in part. Of course, it is likely that another mechanism(s) is involved in regulating CYP2J2 expression. Our previous study showed that overexpression of CYP2J2 or additing exogenous EETs decreased apoptosis and increased cell proliferation in cancer cell lines and increased tumor growth and lung metastasis in a murine xenograft model [10]. Furthermore, we recently found that Compound 26, the selective inhibitor of CYP2J2, significantly repressed the tumorpromotion function of CYP2J2 [12].
In the present study, we described a new mechanism for suppression of CYP2J2. We treated carcinoma cell lines with let-7b and found that overexpression of let-7b resulted in decreased proliferation and activated apoptosis of cancer cells. We also found that pSilencer-let-7b treatment significantly inhibited tumor growth in a tumor xenograft model. Although downregulation of CYP2J2 by pSilencer-let-7b did not result in the eradication of tumors, the reverse correlation between the expression level of CYP2J2 and let-7b in human cancer tissue and the effectiveness of inhibiting the tumor-promoting functions of CYP2J2 revealed the potential therapeutic benefit of let-7b in human cancers.
In our previous study, we reported that EETs protect endothelial cells from apoptosis by activating the phosphatidylinositol 3-kinase (PI3K)/Akt and MAPK signaling pathways [25]. In the current study, overexpression of let-7b suppressed the expression of CYP2J2 protein and its EET products in vitro and in vivo. We also found that let-7b could inhibit the proliferation, and enhance the apoptosis, of human tumor cells by inhibiting the PI3K/Akt and MAPK signaling pathways and by activating the proapoptotic protein Bax and antimetastatic protein nm-23, both of which are involved in P450 epoxygenase-and EET-mediated tumorigenesis and metastasis [10]. Our data suggest that the effect of CYP2J2-derived EETs on tumor formation was mediated by let-7b. The 11 members of the let-7 family have similar target genes and functions on cell proliferation because of the high similarity among their sequences [23,28]. In the present study, we focused on the effect of let-7b on the expression of CYP2J2. Effects of the other members of let-7 family on CYP2J2 expression and cancer cell proliferation need further study.
In conclusion, our data confirmed that let-7b expression is frequently decreased in lung squamous cell cancers. Our work also indicated that human CYP2J2 is posttranscriptionally regulated by let-7b, which results in the high expression of CYP2J2 protein in human carcinomas. Our study demonstrated a new mechanism whereby CYP2J2-and EET-mediated tumorigenesis and metastasis are associated with let-7b. Besides MYC and RAS, let-7b can function as a tumor suppressor by blocking CYP2J2.  , the fold changes of let-7b expression between 18 paired human lung squamous cancer and adjacent nontumor tissues are significant (from 0.277 to 42.22, 6.0662.43). C, relationship between CYP2J2 protein and let-7b expression in lung cancer and paired adjacent nontumor tissues. Expression level of CYP2J2 protein was increased in 13 of 18 sets of lung cancer compared with the adjacent normal tissues (the fold change.1). As expected, let-7b levels were commonly reduced in these tumors compared with the adjacent normal tissues (the fold change,1). D-E, expression levels of CYP2J2 and let-7b in breast cancerous and adjacent nontumor tissues (n = 4). Real-time RT-PCR was used to determine mature let-7b levels. The U6 snRNA expression level was used to normalize the relative let-7b level. Columns, mean of three experiments; bars, SD. *, P,0.05. doi:10.1371/journal.pone.0039197.g004

Cell Lines
The human cervix adenocarcinoma cell line HeLa, human hepatoma liver cell line HepG2, human tongue squamous cell carcinoma Tca-8113, and human lung squamous cancer cell line SK-MES-1 were obtained from the American Type Culture Collection (Manassas, Virginia). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Carlsbad, California) with 10% fetal bovine serum (FBS) at 37uC in the presence of 5% CO 2 at constant humidity. The MDA-MB-435 human breast carcinoma cell line was also obtained from the American Type Culture Collection and was grown in RPMI 1640 medium supplemented with 10% FBS and maintained at 37uC in 95% air/5% CO 2 .

MicroRNA Target Prediction
RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/cgi-bin/ rnahybrid_submit) was used for miRNA target prediction. We found four potential target sites (site I, site III, site IV, and site VI) in 39UTR of human CYP2J2 for let-7b. In addition, we also compared the let-7b sequence to the full length sequence of CYP2J2-39UTR with DNAMAN software (LynnonBiosoft, Quebec, Canada). We also found another two potential bingding sites (site II and site V), besides the four potential target sites previously described ( Figure 1A).

Plasmid Construction
The full length sequence of human CYP2J2-3?UTR was amplified by PCR using the primers 59-GCGACTAGTGTAT-CACCATTTCCCCAGTCAGTCA-39 (CYP2J2-39UTR-former primer) and 59-GCGAAGCTTCATGGGAATAAGTGTCT-GATGGAGG-39 (CYP2J2-39UTR-reverse primer). To construct luciferase reporter plasmids, the 39UTR sequence of CYP2J2 was inserted into the HindIII and SacI sites, downstream of the luciferase reporter gene in the pMIR-REPORT TM Luciferase Vector (Ambion, Carlsbad, California). This plasmid was termed pMIR/ CYP2J2-39UTR. Correspondingly, constructs containing CYP2J2-39UTR with point mutations in the seed sequence were constructed using the Fast Mutagenesis System kit (TransGen, Biotech, China) according to the manufacturer's protocol. PMIR/CYP2J2-39UTR mutant, which carried mutated sequence in the complementary site for the let-7b seed region in all the six potential binding sites, was generated based on the wild type plasmid pMIR/CYP2J2-39UTR. Furthermore, the other six plasmids, in which one of the six potential binding sites was wild and the other five putative binding sites were mutated, were termed mutant-1, mutant-2, mutant-3, mutant-4, mutant-5, and mutant-6, respectively. For example, the construct, in which the seed-matching sequence of site I was not mutated and the other five were mutated, was named mutant-1. Mutant-2 carried the mutated seed-matching sequence of five binding sites except site II. The remaining mutants were named by analogy ( Figure 1A).

Luciferase Assay
HepG2, MDA-MB-435, and SK-MES-1 cells were transfected in 24-well plates by Lipofectamine 2000 (lipo2000) (Invitrogen, Carlsbad, California) in accordance with the manufacturer's instructions. Cells were transiently transfected with 200 ng of the luciferase constructs pMIR/CYP2J2-39UTR or the empty vector and 10 ng of pRL-TK plasmid (Promega, Madison, Wisconsin). In some cases, let-7b (mature let-7b) or random let-7b (Ribobio, China) was cotransfected with reporter plasmids in a final concentration of 100 nM. Random let-7b was the scrambled oligonucleotide and used as negative control. Luciferase activity was analyzed 24 h after transfection by using the Dual-Luciferase Reporter Assay System (Promega, Madison, Wisconsin). Renilla luciferase activities were used to normalize the transfection efficiency and the internally controlled firefly luciferase activity.

Western Blotting
Cells grown in six-well plates were transfected with let-7b or random let-7b to a final concentration of 100 nM. 48 h after transfection, cells were harvested and homogenized with lysis solution (50 mM Tris-Cl, pH 8.0; 150 mM NaCl; 0.02% sodium azide; 0.1% SDS; 1 mg/ml aprotinin; 1% Nonidet P-40; and 0.5% sodium deoxycholate) containing protease inhibitors (100 mg/ml phenylmethylsulfonyl fluoride, 2 mg/ml aprotinin, 2 mg/ml leupeptin). Supernatant was collected after centrifuging at 12,0006g for 20 min at 4uC. The protein concentration was measured using the BCA protein assay reagent kit (Boster, China). Lysates were resolved by 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% nonfat milk, blots were probed with a specific antibody and incubated with a peroxidase-conjugated secondary antibody. Bands were visualized by enhanced chemiluminescence reagents (Pierce Chemical, Rockford, Illinois) and quantified by densitometry.

Cell Proliferation and Apoptosis Assays
Cell proliferation was monitored using Cell-Light EDU DNA Cell Proliferation Kit (Ribobio, China). Cells grown in 96-well plates were transfected with miRNA as described. Proliferation assays were done 48 h after transfection following the manufacturer's protocol. The blue-stained cells were stained by Hoechst, and the red were EdU add-in cells. EdU add-in cells and Hoechststained cells were counted in six randomly selected microscopic fields per well. EdU-positive cell was calculated as (EdU add-in cells/Hoechst stained cells) 6100%.
To measure the effects on cell apoptosis rates, cells were harvested 48 h posttransfection and resuspended in binding buffer.

RNA Extraction and Real-time PCR
Total RNA was extracted from frozen tissue samples by using Trizol Reagent Kit (Invitrogen, Carlsbad, California) according to the manufacturer's instructions. Two micrograms of total RNA were reverse-transcribed using a reverse transcription kit (Takara, Japan). Quantification of miRNA was done by quantitative realtime PCR according to the manufacturer's protocol (Ribobio, China). Expression of U6 small nuclear RNA (snRNA) was used as an internal standard.
The specific primers were purchased from Ribobio Company (Ribobio, China). The PCR profiles for human let-7b and human U6 snRNA were performed as follows: after an initial denaturation at 95uC for 20 s, amplification was finished by denaturing at 95uC for 10 s, annealing at 60uC for 20 s, and extending at 70uC for 1 s through 40 cycles.

Measuring Let-7b and CYP2J2 Levels in Human Lung Cancerous and Adjacent Noncancerous Tissues
Lung cancerous and adjacent noncancerous tissues were collected during surgical procedures at Tongji Hospital from 18 patients with lung squamous cell cancer. Informed consents were obtained. In addition, breast cancer and adjacent noncancerous tissues were also collected from four patients. The samples were immediately frozen in liquid nitrogen and stored at 280uC. Paired nontumor and tumor tissues were further examined by quantitative real-time reverse transcription-PCR (qRT-PCR) for the expression levels of let-7b and by Western blot analysis for CYP2J2 as described above. This study was approved by the Clinical Research Committee of Tongji Medical College and was carried out according to the guidelines of the NIH.

Xenograft Model of Tumor Growth
All animal studies were approved by the Animal Research Committee of Tongji Medical College and were carried out in accordance with the guidelines of the NIH (approval ID: S215). Male athymic BALB/C nude mice 4 weeks old were housed in specific pathogen-free conditions on a daily 12-h light/12-h dark cycle. MDA-MB-435 cells were harvested on the day of use and injected subcutaneously into the right flank of the nude mice. Approximately 2 weeks after implantation, when tumors had grown to approximately 40 mm 3 , mice were randomly divided into control and let-7b precursor expression vector treatment groups (n = 16). Mice received either let-7b precursor expression plasmid (pSilencer-let-7b) at a dose of 4 mg/kg body weight or a comparable dose of empty plasmid pSilencer (dissolved in 100 ml deionized water) as control through tail vein injection every 3 weeks. The mice were monitored daily and weighed weekly. Tumor volume was calculated weekly according to the formula (TV (mm 3 ) = length6width 2 60.5236). At the end of the experiment, tumor xenografts were harvested, weighed, and snap-frozen. Tissues were fixed in 3.7% formalin, embedded in paraffin, and serially cut into 4-mm thick sections for hematoxylin and eosin staining and TUNEL assay.

Terminal Deoxynucleotidyl Transferase-mediated dUTP Labeling
Apoptosis in tissues was detected by using the TACSH 2 TdT DAB In Situ Apoptosis Detection Kit according to the manufacturer's instruction (R&D Systems, Minneapolis, Minnesota).
Sections were incubated with 20 mg/ml proteinase K for 15 min and then immersed in 3% H 2 O 2 for 5 min to quench endogenous peroxidase. After being immersed in 16TdT Labeling Buffer for 5 min, the sections were incubated in TdT Labeling Reaction Mix for 60 min at 37uC in a humidity chamber and then incubated with 16TdT Stop Buffer for 5 min to stop the labeling reaction. After deionized water washes, sections were incubated with Strep-HRP solution for 10 min at 37uC, washed twice with 16PBS, and immersed in DAB solution for 2 to 7 min. Sections were then counterstained with methyl green, and the TUNEL-positive cells were counted in five fields per tissue section.

Statistical Analysis
Data are presented as mean 6 SD from at least three separate experiments. The data were analyzed by Student's t-test. A P value ,0.05 was considered as significant for all tests.