The Caski human cervical cancer cell line was cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) (both from Gibco by Life Technologies, Grand Island, NY, USA), 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in the presence of 5% CO₂.

Ten participants were recruited at the Third Xiangya Hospital, Central South University (Changsha, Hunan, China). Consent forms were obtained from individual patients, and experimental protocols were approved by the Institutional Review Board of the Third Xiangya Hospital. All 10 participants were female with histologically-confirmed cervical cancer (Table I). All the subjects enrolled in the study were Chinese. Cervical cancer and corresponding non-tumor normal tissues were collected. Each biopsy sample was submitted to routine histological diagnosis, or quantitative polymerase chain reaction (qPCR), western blot analysis and immunohistochemistry (IHC).

Total RNA was extracted from the cervical cancer and corresponding non-tumor normal tissues with TRIzol reagent (Qiagen, Carlsbad, CA, USA). cDNA synthesis was carried out using the RevertAid First Strand cDNA Synthesis kit (CWBio, Beijing, China) according to the manufacturer’s instructions. RT-qPCR was performed with GoTaq qPCR Master Mix (CWBio). The primers used for the RT-qPCR are shown in Table II. RT-qPCR was carried out with the Bio-Rad CFK96TM Real-Time system (Bio-Rad, Hercules, CA, USA). The data were analyzed by Bio-Rad CFK manager 2.0 software. The expression of mRNA was assessed by evaluating threshold cycle (Ct) values and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control GAPDH was calculated using the 2^−ΔΔCt equation previously adopted by Livak et al (29): ΔΔCt = (Ct_Target − Ct_GAPDH) cervical cancer − (Ct_Target − Ct_GAPDH) control.

IHC was performed using the peroxidase anti-peroxidase technique following a microwave antigen retrieval procedure. Antibody for CD38 was purchased from Proteintech Biotechnology (Chicago, IL, USA). Antibody against CD38 (1:100) was overlaid on cervical cancer and corresponding non-tumor normal tissue sections and incubated overnight at 4°C. Secondary antibody incubation (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was performed at room temperature for 30 min.

The sections evaluated by two investigators in a blind manner in an effort to provide a consensus on staining patterns by light microscopy (Olympus, Tokyo, Japan). CD38 staining was assessed according to the methods described by Hara and Okayasu (28) with minor modifications. Each case was rated according to a score that added a scale of intensity of staining to the area of staining. At least 10 high-power fields were chosen randomly, and >1,000 cells were counted for each section. The intensity of staining was graded on the following scale: 0, no staining; 1+, mild staining; 2+, moderate staining; 3+, intense staining. The area of staining was evaluated as follows: 0, no staining of cells in any microscopic fields; 1+, <30% of tissue stained positive; 2+, between 30 and 60% stained positive; 3+, >60% stained positive. The minimum score when summed (extension + intensity) was, therefore, 0, and the maximum, 6. A combined staining score (extension + intensity) of ≤2 was considered to be a negative staining (low staining), while a score between 3 and 4 was considered to be a moderate staining; whereas a score between 5 and 6 was considered to be a strong staining.

Total RNA was extracted from the cervical cancer and corresponding non-tumor normal tissues with TRIzol reagent (Qiagen) according to the manufacturer’s instructions. cDNA was synthesized from 2 mg of total RNA with M-MLV Reverse Transcriptase (Promega, Fitchburg, WI, USA) in a 25 ml volume {2 mg total RNA, 400 mM reverse transcription primer [oligo(dT)18 for random primers for U6 rRNA and miR-634-, miR-664- and miR-140-5p-specific primers (Bulge-Loop™ miRNA qPCR primers from RiboBio, China) for miRNA], 4 U/ml M-MLV, 1 U/ml inhibitor, 0.4 mM dNTP mix}. qPCR was carried out with the reagents of a SYBR-Green I mix (Takara, Dalian, China) in a 20 ml reaction volume (10 ml SYBR-Green I mix, 200 mM forward and reverse primer, 2 ml cDNA template) on an MJ Opticon Monitor Chromo4 instrument (Bio-Rad) using the following protocol: 95°C for 20 sec; 40 cycles of 95°C for 10 sec, 60°C for 20 sec and 70°C for 1 sec. Data analysis was performed using the 2^−ΔΔCt method (6,29).

The coding region of CD38 gene was generated by PCR with the primer pair 5′-ATACTCGAGATGGCCAACTGCGAGTTCAG-3′ and 5′-GCGAAGCTTTCAGATCTCAGATGTGCAAG-3′. The PCR was performed under the following conditions: one cycle for 5 min at 94°C; 30 cycles for 45 sec at 94°C, 45 sec at 55°C, and 90 sec at 72°C, and ended with 10 min at 72°C. The fragments were cloned into the TA vector (Promega) and used to transform E. coli JM109 (Takara). Following selection and propagation, the pure plasmid DNA was prepared by standard methods. The DNA fragments were removed from the TA vector by restriction enzyme digestion with XhoI and HindIII (Promega) to subclone into the pEGFP-N1 vector. The fusion sequences were verified by DNA sequencing using ABI 3730.

Cell transfection was achieved by using Lipofectamine, according to the manufacturer’s instructions (Life Technologies, Grand Island, NY, USA). Cells (2×10⁵) were placed in each well of a 6-well plate 24 h prior to the transfection. For each transfection, 2 μg of pEGFP-N1-CD38 plasmid and pEGFP-N1 vector plasmid was transfected into Caski cells, respectively. The plasmids were diluted with 100 μl of serum-free media and 4 μl Lipofectamine was added into 100 μl serum-free media. The two solutions were combined, mixed gently and incubated at room temperature for 30 min. The 200 μl mixture and 200 μl of serum-free media were added into each well. The cells were then incubated at 37°C for 24 h, followed by replacing the transfection media with fresh complete culture media. After an additional 48 h culture, the cells were harvested for the western blot analysis.

The cervical cancer and corresponding non-tumor normal tissues, and Caski cells were lysed in RIPA buffer (CWBio) and total protein concentration was determined using Pierce^® BCA Protein assay kit (Thermo Scientific, Inc., Rockford, IL, USA). Extracts containing 50 μg of proteins were separated in 10% SDS-PAGE gels and electroblotted onto nitrocellulose membranes (HyClone Laboratories, Logan, UT, USA). The membranes were inhibited using Tris-buffered saline/Tween-20 (25 mM Tris-HCl, 150 mM NaCl, pH 7.5 and 0.05% Tween-20) containing 5% non-fat milk followed by overnight incubation at 4°C with primary antibodies [rabbit anti-PI3K antibody, 1:500; and rabbit anti-Akt antibody, 1:300 (Cell Signalling Technology, USA); rabbit anti-MDM2 antibody, 1:200 and rabbit anti-p53 antibody, 1:200 (Wuhan Boster, Wuhan, China)]. Following three washes, the membranes were incubated with horse-radish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc.) and the specific signals were visualized using an ECL detection system. Anti-GAPDH antibody (1:3,000; Santa Cruz Biotechnology, Inc.) was used as a loading control.

Differences of non-parametric variables were analyzed by the Mann-Whitney U test. Differences of the quantitative variables between groups were analyzed by the Student’s t-test using SPSS 11.0 program (SPSS, Chicago, IL, USA). P<0.05 was considered statistically significant.

To detect the mRNA expression levels of the CD molecules in cervical cancer and the adjacent non-cancerous tissues, 10 samples of each were selected to perform qPCR of the CD20, CD21, CD26, CD34, CD38, CD73, CD90, CD123 and CD133 genes. Data were analyzed using the 2^−ΔΔCt method. The fold-change in the expression of these genes relative to the internal control gene, GAPDH, was also analyzed. The expression of the CD38, CD34 and CD90 genes was higher in the cervical cancer samples compared with the adjacent non-cancerous tissues and the normalized CD38, CD34 and CD90 gene expression in the cervical cancer samples was upregulated by 4.40-, 2.71- and 2.64-fold, respectively (Table III). The expression levels were not significantly different for CD20, CD26, CD73 and CD133 between the cervical cancer and adjacent non-cancerous tissues (Table III). Thus, we selected to determine the funciton of CD38 and its underlying mechanism in cervical cancer.

To determine whether the CD38 gene was expressed at a higher level in the cervical cancer compared with the adjacent non-cancerous tissues, the protein expression levels of CD38 were further examined by western blot analysis (Fig. 1). In comparison with the adjacent non-cancerous tissues, the expression level was identified to be greater in cervical cancer tissues, which corresponded with the qPCR results. These results suggested that CD38 is highly expressed in cervical cancer.

IHC was carried out with antibodies against CD38 protein in the cervical cancer and adjacent non-cancerous tissues. CD38 was identified as differentially expressed between the cervical cancer and adjacent non-cancerous tissues. IHC showed a similar pattern in protein expression, which was similar to that of the western blot results. There was a 40% (12/30) high score of CD38 in the cervical cancer and 16.6% (5/30) in the adjacent non-cancerous tissues. The distribution of the low score was 23.3% (7/30) and 56.7% (17/30) in the cervical cancer and adjacent non-cancerous tissues, respectively (P=0.02<0.05) (Fig. 1) (Table IV). The IHC results corresponded with those of the qPCR results.

As CD38 is a potential miR target, the open access programs, TargetScan (http://www.targetscan.org/), PicTar (http://pictar.mdc-berlin.de/) and miRBase (http://mirbase.org/index.shtml), were used to predict the targets of miR-634, miR-664 and miR-140-5p. The endogenous expression of miR-634, miR-664 and miR-140-5p was compared between the cervical cancer and adjacent non-cancerous tissues by qPCR. The expression of miR-634, miR-664 and miR-140-5p was downregulated in the cervical cancer tissues (Fig. 3). These results suggested that CD38 was upregulated in the cervical cancer as compared with the non-cancerous tissues.

To determine the possible mechanism of CD38 in cervical cancer, we examined the expression levels of key molecules in the PI3K/Akt signaling pathway by western blot analysis. PI3K and Akt were upregulated in cervical cancer compared with the adjacent non-cancerous tissues. MDM2 had the same tendency as PI3K and Akt. However, p53 was downregulated in cervical cancer (Fig. 4). Combined with the above result showing CD38 was highly expressed in cervical cancer, the results suggested that CD38 is correlated with dysregulation of the PI3K/Akt signaling pathway in cervical cancer tissues in vitro.

To confirm whether CD38 affects the expression of PI3K, Akt, MDM2 and p53 in vivo, we constructed the plasmid of pEGFP-N1-CD38. The plasmid of pEGFP-N1-CD38 and pEGFP-N1 was transfected into the Caski cervical cancer cell line. The cells were collected after 48 h with transfection and the expression levels of PI3K, Akt, MDM2 and p53 proteins were examined in vivo. The PI3K, Akt and MDM2 were upregulated in Caski cells in which CD38 was overexpressed. At the same time, p53 was downregulated in Caski cells in which CD38 was overexpressed (Fig. 5). The results suggested that CD38 overexpression affected the expression of PI3K, Akt, MDM2 and p53 in vivo.