Expression of Smo in pancreatic cancer CD44+CD24+cells and construction of a lentiviral expression vector to silence Smo.

The present study focused on the roles of members of the Hedgehog (Hh) signaling pathway in the maintenance of malignant biological characteristics, such as tumorigenesis, similar to that of pancreatic tumor cells. Cluster of differentiation (CD)44+CD24+/CD44-CD24- cells were isolated from three different pancreatic cancer cell lines by flow cytometry. Among the three pancreatic cancer cell lines, the SW1990 cell line exhibited the highest percentage of CD44+CD24+ cells, which accounted for 39.9% of the total. The expression of members of the Hh signaling pathway in CD44+CD24+/CD44-CD24- cells was detected using reverse transcription-polymerase chain reaction and western blot analysis. The results demonstrated that members of the Hh signaling pathway were differentially expressed in CD44+CD24+ cells compared with CD44-CD24-, normal pancreatic duct cells and unsorted SW1990 cells. In addition, lentiviral expression vectors expressing Smoothened (Smo) small interfering RNA (siRNA) were constructed. Following transfection with the lentiviral expression vectors, Smo expression was markedly reduced in CD44+CD24+ cells. The present study represents a preliminary investigation into the biological characteristics of CD44+CD24+ pancreatic cancer cells.

Introduction

The incidence of pancreatic cancer is increasing globally (1). The latest data from the American Cancer Society for the USA reveals that the incidence of pancreatic cancer is 11 per 100,000 people, ranking it tenth of the known malignant tumors; the mortality rate has been ranked fourth out of the known malignant tumors (2). In addition, epidemiological data from China demonstrates an increasing incidence in pancreatic cancer (3–6).

Pancreatic cancer stem cells (CSCs) are considered to influence the early metastasis of pancreatic cancer, are insensitive to radiation and chemotherapy and are the main cause of rapid disease progression (7). CSCs cannot be eradicated, and as such drive invasion, metastasis and recurrence, and are the source radiation and chemotherapy resistance (8). In 2007, Lee et al (9) using flow cytometry to isolate cluster of differentiation (CD)44+CD24+, epithelial surface antigen+ pancreatic cancer cells from surgically removed specimens. These cells have the abilities of self-renewal and multi-directional differentiation, and may adapt to alterations in circumstances. Lee et al (9) hypothesized that this group of pancreatic cancer cells were pancreatic CSCs. In recent years, one study has validated that the Hedgehog (Hh) signaling pathway exhibits a function in the process of embryonic development (10). The abnormal activation of growth and development is associated with the development of tumors (1,2). In 2003, Berman et al (11) and Thayer et al (12) demonstrated an association between the Hh signaling pathway and pancreatic cancer. Thayer et al (12) used 26 cancer cell lines, including pancreatic cancer (either from a human primary source or a metastatic tumor), to screen the expression of members of the Hh signaling pathway. Thayer et al (12) demonstrated that expression of members of the Hh signaling pathway, smoothened (Smo), zinc finger protein Gli1 (GLI-3) and Patch-1, was observed in each pancreatic cell line. The occurrence of Hh signaling pathway activation in pancreatic cancer serves an important role in its development (11,12). Smo a typical G protein coupled receptor, is a positive regulator of cellular proliferation and differentiation in insects and vertebrates (13,14). Abnormal activation of Smo leads to development of a number of types of cancer, which makes Smo an attractive therapeutic target. At present, vismodegib, an inhibitor of Smo, is approved by the food and drug administration for the treatment of cancer; however, cancer cells may acquire resistance (15,16). A number of resistance mechanisms have been demonstrated, including mutations in Smo, the negative control receiver, suppressor of fused homolog (Sufu) and the mitogen-activated protein kinase signaling pathway (13). Therefore, an improved understanding of the Smo regulating mechanism to develop effective therapies to treat cancer caused by Smo mutations is required.

Small interfering RNA (siRNA) is an RNA molecule that can efficiently and specifically degrade target gene mRNA to inhibit the excessive expression of genes. In addition, they may be used as a treatment in patients and are a useful tool for studying gene function in the future. The occurrence and development of pancreatic cancer is a multi-factor and multi-stage process (17,18). In the present study, experimental siRNA stability infection in pancreatic cancer cells of CD44+ CD24+ cells was used to determine whether or not RNA and protein expression was affected.

Patients and methods

Patients

A total of 19 cases (11 males, 8 females; mean age, 59.89 years; range, between 40 and 78 years) of pancreatic cancer were included in the present study. There were 9 tumors of the head of the pancreas, 9 tumors of the body and tail of the pancreas, and 1 pancreatic neck tumor. Pathological results according to the pathology classification system of the Cancer Hospital Affiliated to Xinjiang Medical University (Urumqi, China), included the following: 2 cases of highly differentiated carcinoma, 4 cases of moderately/highly differentiated carcinoma, 8 cases of moderately differentiated carcinoma, 4 cases of moderately/poorly differentiated carcinoma and 1 case of poorly differentiated carcinoma. Local lymph node metastasis was identified in 7 cases. The patients were all treated at the Cancer Hospital Affiliated to Xinjiang Medical University between January 2014 and December 2015. The Ethics committee of the Affiliated Cancer Hospital to Xinjiang Medical University approved the present study and all patients provided written informed consent.

Materials

SDS-PAGE gel preparation kit, dNTPs and Taq DNA polymerase, 2 kb plus DNA Marker and PCR primers were obtained from Shanghai Shenggong Biology Engineering Technology Service, Ltd. (Shanghai, China). The PCR reagent, primers and dsDNA oligos were obtained from Shanghai Jikai Gene Chemical Technology Co., Ltd. (Shanghai, China). Taq polymerase was obtained from Clontech Laboratories, Inc. (Mountainview, CA, USA) and the Qiagen plasmid Maxi kit was obtained from Qiagen, Inc. (Valencia, CA, USA). Bovine serum albumin (BSA) was obtained from Shanghai JIEBEISI Gene Technology Co., Ltd. (Shanghai, China). Lysogeny broth (LB), super optimal broth (SOB) and super optimal broth with catabolite repression (SOC) were obtained from American Type Culture Collection (Manassas, VA, USA). T4 DNA ligase, T4 DNA ligase buffer, and the restriction enzymes AgeI, EcoRI, HpaI and XhoI were obtained from New England BioLabs, Inc. (Ipswich, MA, USA). MgSO4 was obtained from Wuhua Chemical Industry Co., Ltd. (Shanghai, China). Agarose was obtained from Saibaisheng Biochemical Co., Ltd. (Beijing, China). The 250 bp DNA ladder marker was obtained from Beijing Huada Jierui Biotechnology Co., Ltd. (Beijing, China). Gene sequencing for the positive clone was performed by Shanghai Genechem Co., Ltd. (Shanghai, China).

The intermediate clone vector pUC57, pLenti6/V5-D-TOPO recombinant lentiviral vector, ViraPower™ Packaging Mix, viral quantitative primers and the TaKaRaMiniBEST Viral RNA/DNA Extraction kit were obtained from Baiao Maike Bio-Technology Co., Ltd. (Nantong, China). Lipofectamine® 2000 transfection reagent kit was obtained from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). TOP10 chemical competent cells and 293T virus packaging cells were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), trypsin, and OptiMEM medium were obtained from Gibco; Thermo Fisher Scientific, Inc.

Sorting of CD44+CD24+ cells

BXPC-3, PANC-1 and SW1990 (all from Shanghai Genechem Co., Ltd., Shanghai, China) pancreatic cancer cell lines were cultured at 37°C in an atmosphere containing 5% CO2 in RMPI-1640 (Hyclone; Thermo Fisher Scientific, Inc.) or DMEM, supplemented with 10% FBS. Subsequently, CD44+CD24+ cells were sorted from the three cell lines using flow cytometry as previously described (19,20).

RT-PCR

TRIzol® (Thermo Fisher Scientific, Inc.) was used to extract total RNA from SW1990 cellsof each experimental group, according to the manufacturers protocol, and the RNA concentration and purity were detected using a spectrophotometer. The RNA was reverse transcribed into complementary DNA according to the manufacturers protocol of the two-step M-MuLV First Strand cDNA Synthesis kit (cat no. B532435; Sangon Biotech Co., Ltd., Shanghai, China) according to the manufacturers protocol. For the PCR reaction, sonic hedgehog protein (Shh), Smo, protein patched homolog 1 (Ptch1), zinc finger protein Gli1 (Gli1), zinc finger protein Gli2 (Gli2), Sufu and apoptosis regulator Bcl-2 (Bcl-2) primers (Table I) were designed and synthesized according to the sequences in GeneBank, as previously described (21). Primer sequences and their annealing temperatures, including for the reference gene β-actin, are provided in Table I. PCR was performed with the Bio-Rad MyiQ™2 two-color real-time PCR detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using SYBR Green I as the fluorophore. The components and their volumes in the polymerase chain reaction are shown in Table II. The reaction conditions used were: 95°C for 5 min, followed by 40 cycles of 95°C for 10 sec, 60°C for 30 sec. Quantitative analysis of Shh, Smo, Ptch1, Gli1 and Gli2, Sufu and Bcl2 mRNA expression was performed with the 2−∆∆Cq method (22). Data analysis was performed using Microsoft Excel 2016.

Table I.

Primers used for reverse transcription-polymerase chain reaction.

Gene	Primer sequences	Amplification fragment size, bp	Annealing temperature, °C
Sonic hedgehog	Forward: 5′-GTCTCCTCGCTGCTGGTATG-3′	150	56
	Reverse: 5′-TTGGGGATAAACTGCTTGTAGG-3′
Protein patched homolog 1	Forward: 5′-CTCCTTTGCGGTGGACAA-3′	109	54
	Reverse: 5′-CCTCAGCCTTATTCAGCATTTC-3′
Smoothened	Forward: 5′-CTCCTACTTCCACCTGCTCAC-3′	105	57
	Reverse: 5′-CAAAACAAATCCCACTCACAGA-3′
Zinc finger protein GLI1	Forward: 5′-ATCCTTACCTCCCAACCTCTGT-3′	84	55
	Reverse: 5′-AACTTCTGGCTCTTCCTGTAGC-3′
Zinc finger protein GLI2	Forward: 5′-GCGGAATTCGCAACGGAATG-3′	472	55
	Reverse: 5′-GCTGGATCCTTAGTCACA-3′
Suppressor of fused	Forward: 5′-CGGACCCACCAGAAGCGG-3′	398	52
homolog	Reverse: 5′-GGAGGCGTCCTTCCGAC-3′
B-cell lymphoma	Forward: 5′-ACCTTAGCCCCATGCATTCTG-3′	287	54
	Reverse: 5′-CTAATCGGCTAGCTTCGAAAT-3′
β-actin	Forward: 5′-GGGACCTGACTGACTACCTC-3′	543	56
	Reverse: 5′-CGTCATACTCCTGCTTGCTG-3′

Table II.

Polymerase chain reaction components and volumes.

Reagent	Volume per reaction/µl
2X ES Tap Master Mix	12.5
Upstream primer (10 µmol/l)	1
Downstream primer (10 µmol/l)	1
RNase-free water	8.5
Complementary DNA template	2
Total volume	25

Western blot analysis

A total protein extraction kit (Trizma® base Vetec reagent grade ≥99%; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was used to extract the total protein of the cells and the quantity of protein was determined using a BCA protein concentration detection kit. A total of 125 µg of protein was used for 12% SDS-PAGE electrophoresis. Proteins were transferred to polyvinylidene fluoride membranes and blocked with 5% bovine serum albumin (Gibco; Thermo Fisher Scientific, Inc.) at room temperature for 2 h. The membranes were washed three times with TBS-Tween-20 (TBST) and were incubated with the following primary antibodies overnight at 4°C with gentle agitation: Bcl-2 (1:1,000; cat no. ab32124), Gli1 (1:500; cat no. ab92611), SMO (1:1,000; cat no. ab38686), PTCH1 (1:500; cat no. ab53715), Gli2 (1:1,000; cat no. ab26056) and SHH (1:2,000; cat no. ab53281; Abcam, Cambridge, MA, USA). The membranes were washed three times with TBST prior incubation with the alkaline phosphatase-conjugated anti-rabbit secondary antibody (1:2,000; cat no. WB7105; WesternBreeze; Thermo Fisher Scientific, Inc.) for 30 min at room temperature. Membranes were washed three times with TBST and the blots were developed using enhanced chemiluminescence kit (Western Breeze Chemiluminescence Reagent kit; cat. no. WB7105; Thermo Fisher Scientific, Inc.). Using β-actin as a reference, the relative protein expression was calculated. The Gel Doc XR+ system (Bio-Rad Laboratories, Inc.) was used to capture and analyze results of gels.

Construction of the lentiviral expression vector expressing Smo siRNA

For the design and synthesis of Smo siRNA fragments, the information and the full-length nucleotide sequence of the human Smo gene were obtained by searching the NCBI GenBank database (https://www.ncbi.nlm.nih.gov/genbank/; no. NM_005631) and used to design three siRNA targets (Table III). Three siRNAs were used in the current study: siRNA1 (TGATGGACACAGAACTCAT); siRNA2 (GGAGAAGATCAACCTGTTT) and siRNA3 (TGACTGTGAGATCAAGAAT) (Shanghai Genechem CO., Ltd.).

Table III.

siRNA targets designed using the NCBI GenBank database.

siRNA target	Target sequence information	GC content, %	Start position
SMO-RNAi(37303–1)[a]	TGATGGACACAGAACTCAT	42.1	2604
SMO-RNAi(37304–1)[b]	GGAGAAGATCAACCTGTTT	42.1	1831
SMO-RNAi(37305–1)[c]	TGACTGTGAGATCAAGAAT	36.8	1795

siRNA1

siRNA2

siRNA3. siRNA, small interfering RNA.

293T cells were cultured in DMEM, containing 10% FBS and seeded into 24-well culture plates (3.5×10−4 cells/ml) one day prior to transfection using carrier GV plasmid (20 µg;), PHelper1.0 carrier plasmid (15 µg) and PHelper 2.0 carrier plasmid, 10 µg) (Shanghai Genechem, Co., Ltd.), with Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) After 24 h, the expression of the intracellular fluorescent-labeled gene (GFP) was observed under a fluorescence microscope, and then the cells were collected for western blot analysis 36 h following transfection. The viral titer was determined by Virus drops degree detection; fluorescence/absolute quantitative method (23).

The CD44+CD24+ cells that were sorted as previously mentioned were cultured to the logarithmic growth phase and then digested into single cell suspension with trypsin. 293T cells were cultured for 24 h to 90% confluence, washed three times, then incubated in a 5% CO2 incubator at 37°C for 20 min. Cells were transfected with the Lipofectamine® 2000 transfection kit, according to the manufacturer's protocol. Following incubation at 37°C for 72 h, the SMO gene expression in the cells of each group was determined by the RT-PCR method, the Smo siRNA with the highest transfection efficiency was screened for the follow-up study.

Statistical analysis

Analysis of expression of the genes was performed using the SASJMP10.0 software (SAS Institute, Cary, NC, USA). Data are expressed as the mean ± standard deviation. Differences among >2 groups were assessed using one-way analysis of variance with Bonferroni post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

Results

Three lines of CD44+CD24+pancreatic cancer cells were isolated using flow cytometry (BXPC-3, 10.6% CD44+CD24+ cells; PANC-1, 12.5% CD44+CD24+ cells; SW1990, 39.9% CD44+CD24+ cells; Fig. 1). CD44+CD24+ SW1990 pancreatic cancer cells were selected for further experiments.

Figure 1.

CD44+CD24+ cell proportion in three pancreatic cancer cell lines. Flow cytometry analysis of the proportion of CD44+CD24+ cells in BXPC-3, PANC-1 and SW1990 cells. CD, cluster of differentiation.

Expression of Hedgehog (Hh) signaling pathway members in pancreatic cancer cells

The expression of the Hh signaling pathway members Shh, Smo, Ptch1, Gli1, Gli2, Sufu and Bcl2 (24) was investigated in unsorted SW1990 pancreatic cancer cells, sorted CD44+CD24+ cells, sorted CD44−CD24− cells and normal pancreatic duct cells obtained from patients using RT-PCR (Fig. 2) and western blot analysis (Fig. 3). The expression of Gli1 and Bcl2 in pancreatic ductal cells and cancer cells was increased compared with that in SW1990 pancreatic cancer cells (Fig. 4); however, no significant differences were observed between the expression of the other proteins in pancreatic cancer cells compared with that in the pancreatic duct cells. The expression of Shh and Smo in CD44+CD24+ cells was markedly increased compared with that in pancreatic cancer cells; however, no significant differences were observed between the expression of the other proteins in the two groups. In addition, no significant differences in expression were observed between the CD44−CD24− cells and pancreatic cancer cells.

Figure 2.

The electrophoresis maps of reverse transcription-polymerase chain reaction products of members in the Hedgehog signaling pathway in pancreatic cancer tissue, CD44+CD24+ SW1990 cells, CD44-CD24- SW1990 cells, normal pancreatic tissue adjacent to carcinoma cell search group. Shh, sonic hedgehog; Smo, smoothened; Ptch1, protein patched homolog 1; Gli1, zinc finger protein GLI1; Gli2, zinc finger protein GLI2; Bcl-2, B-cell lymphoma 2; Sufu, suppressor of fused homolog; CD, cluster of differentiation.

Figure 3.

Western blot analysis of members of the Hedgehog signaling pathway in pancreatic cancer tissue cells, CD44+CD24+ SW1990 cells, CD44-CD24- SW1990 cells, normal pancreatic tissue adjacent to carcinoma cells. Shh, sonic hedgehog; Smo, smoothened; Ptch1, protein patched homolog 1; Gli1, zinc finger protein GLI1; Gli2, zinc finger protein GLI2; Bcl-2, B-cell lymphoma 2; Sufu, suppressor of fused homolog.

Figure 4.

Relative expression level of members of the Hedgehog signaling pathway in the cells of each group. The polymerase chain reaction Shh, sonic hedgehog; Smo, smoothened; Ptch1, protein patched homolog 1; Gli1, zinc finger protein GLI1; Gli2, zinc finger protein GLI2; Bcl-2, B-cell lymphoma 2; Sufu, suppressor of fused homolog; CD, cluster of differentiation. *P<0.05, **P<0.01.

Construction of the siRNA expression vector carrying Smo

Sequencing of the KL15570-Smo siRNA sequencing demonstrated that the sequence was consistent with the designed fragment (data not shown). The electrophoresis map of the PCR products of the KL15570-Smo siRNA vector digested by SacII B restriction endonucleases confirmed that the fragment had been inserted (Fig. 5). Fluorescence microscopy confirmed that Three groups of interfering lentivirus cells had been successfully transfected with the plasmid (Fig. 6). The viral titers for the siRNA1, siRNA2 and siRNA3 plasmids were 5×108, 6×108 and 5×108 viral TU/ml, respectively.

Figure 5.

Electrophoresis maps of digestion products of the smoothened siRNA plasmid. Lane 1, blank; 2, control; 3, overexpression; 5–7, siRNA. SiRNA, small interfering RNA.

Figure 6.

Images of HEK-293T cells 72 h following transfection with lentiviral vector plasmid (SMO-RNAi 37304-1). Magnification, ×200.

Detection of Smo gene expression in CD44+CD24+ cells infected with three Smo- siRNA lentiviral expression vectors

RT-PCR analysis demonstrated that Smo expression was markedly decreased in CD44+CD24+ cells following infection with the constructed Smo siRNA lentiviral expression vectors (Fig. 7). The blocking efficiency of the three siRNAs were 54.293, 32.188 and 62.531%, respectively (data not shown). The efficiency of siRNA3 was the highest; therefore the lentiviral expression vector carrying Smo-siRNA3 was used in the follow-up study to block the Hh signaling pathway.

Figure 7.

Expression of the Smo gene in CD44+CD24+ cells transfected with the siRNA lentiviral vector. (A) Smo gene expression in cells transfected with siRNA1; (B) Smo gene expression in cells transfected with siRNA2; (C) Smo gene expression in cells transfected with siRNA3. Smo, smoothened; siRNA, small interfering RNA.

Discussion

The Hh signaling pathway serves key roles in the proliferation of epidermal stem cells, the development of embryonic cells with stem cell characteristics, and the development of mouse cerebral cortex cells following birth (25). Its abnormal activation is associated with the occurrence and development of a number of types of malignant tumor, multiple drug resistance and other characteristics. The presence of CD44+ CD24− cells was detected by Tao et al (8) using the RT-PCR method; the results demonstrated that the expression of Smo and GLI-1 genes was significantly increased between CD44+ CD24− cells and non CD44+ CD24− cells, and indicated that associated factors of the Hh signal pathway were overexpressed in breast cancer stem cells (CSCs). The Hh signal pathway serves important roles in the proliferation, differentiation and maintenance of malignant biological characteristics of CSCs (26). Therefore, multiple studies have focused on the association between the Hh signaling pathway and the development of pancreatic cancer. Onishi et al (27) confirmed in vivo and in vitro that cyclopamine, a specific blocking agent for the Hh signaling pathway, can significantly improve the sensitivity of pancreatic cancer cells to chemotherapeutic medicines, including fluorouracil and gemcitabine. Hao et al (28) demonstrated that the invasion and metastasis of pancreatic cancer cells could be significantly inhibited by blocking the Hh signaling pathway. In addition, gene expression studies have demonstrated that expression of Hh signaling pathway-associated molecules are significantly higher in pancreatic cancer tissues compared with normal tissues (29).

Gene therapy has been developed using DNA recombination technology to correct mutated or defective genes by introducing normal genes or therapeutic genes into cells, including tumor and immune cells, in order to modify the biological behavior of tumor cells (30).

MicroRNAs are endogenous non-coding small RNA molecules, which serve important roles in tissue inflammation, cell proliferation and apoptosis, tissue differentiation, and malignant tumor occurrence and development (31). siRNA viral vector transfection is a primary method used for studying signal transduction pathways at present. In addition, it is possible to use these molecules in clinical applications for tumor-targeted therapy due to low cost and high efficiency (32).

The tumorous characteristics, including cell morphology and growth characteristics, of three pancreatic cancer cell lines (BXPC-3, PANC-1 and SW1990) and the percentage of CD44+CD24+/CD44−CD24− cell subsets of pancreatic cancer cells isolated by flow cytometry.

In the present study, three siRNA fragments were designed against the Smo gene in the signal pathway, and the lentiviral expression vectors carrying the three Smo fragments were successfully constructed. Through the transfection of CD44+CD24+ cells, the Smo siRNA lentiviral expression vector that was the most suitable for the follow-up study was selected according to the inhibition rate of Smo expression. This not only laid a good experimental basis for the smooth development of the follow-up study of this research, but also provided reliable experimental evidence for the gene therapy of targeting interference of the Hh signaling pathway. The Hh signaling pathway serves an important role in the maintenance of pancreatic CSC malignant biological characteristics, but further studies are required to elucidate the underlying molecular mechanisms for this association.