With the patient's informed consent, from 2008 and 2018, 388 samples of human cervical tissues were gathered from the Gynecology and Obstetrics Department, Shandong First Medical University Affiliated Provincial Hospital, including 248 cervical cancer, 80 CIN (cervical intraepithelial neoplasia), as well as 60 healthy cervical tissues. All cervical cancer patients were diagnosed as per the modified FIGO (International Federation of Gynecology and Obstetrics) staging system. The present work was approved by the Shandong First Medical University’s Medical Ethics Committee, and all procedures were implemented following the relevant regulations and guidelines.
Human cervical carcinoma (Ca Ski, C-33A, HT-3, HeLa and SiHa) cells were acquired from the CAS Shanghai Institute for Biological Sciences. Ca Ski, HeLa and SiHa cells were cultured with complete cell culture RPMI-1640 (Gibco) involving 10% FBS (fetal bovine serum; Gibco) plus 1% penicillin-streptomycin solution. The HT-3 cells were cultivated using McCOY's 5A medium (Gibco) involving 10% FBS plus 1% penicillin-streptomycin, while cultivation of the C-33A cells was accomplished with minimum Eagle's medium (MEM, Gibco) also involving 10% FBS plus 1% penicillin-streptomycin. The foregoing cells were all cultivated in a 37°C incubator including 5% CO2 and subcultured with 0.25% trypsin (Gibco) digestion.
Immunohistochemistry (Ihc) And Immunocytochemistry (Icc)
A standard SP (streptavidin-biotin-peroxidase) staining procedure was adopted for IHC and ICC, with which the STEAP2 expressions in human cervical tissues and carcinoma cells were examined. Sequential baking, deparaffinization and PBS immersion were accomplished on the paraffin-embedded tissue sections, and finally antigen retrieval was carried out by high pressure 2 min in antigen repair solution. The prepared cell coverslips were subjected to a 30-min fixation in paraformaldehyde (4%) and subsequently washed using PBS. After goat serum closed, tissue sections and cell coverslips were subjected to an overnight incubation using polyclonal rabbit anti-human STEAP2 antibody (1:100 dilution, PA5-25495, Invitrogen) under a 4℃ condition. The specific steps were referred to the instruction of SP staining kit (ZSGB-BIO, China). The paraffin-embedded human prostate carcinoma section (STEAP2 positive) served as the positive control, while the negative control was prepared by substituting PBS for the primary antibody. Every tissue section/cellular coverslip was prepared in triplicates.
The Analysis Of Ihc And Icc
Given the location of STEAP2 on the cellular membrane, the cytoplasmic and membrane brown granules were considered positive. The expression intensity of STEAP2 was evaluated according to a semi-quantitative system of scoring created by Soumaoro et al.16 The total score of expression was a sum of staining intensity and positive cell proportion scores. Rating of staining intensity was accomplished under 4 grades: total negative scored 0; light yellow slightly higher than background scored 1; brown yellow obviously higher than background scored 2; brown meaning strongly positive scored 3. Furthermore, the positive cells were divided into 5 grades: according to the number of positive cells 0%, 0%-10%, 10%-25%, 25%-75% and ≥ 75%, the score 0, 1, 2, 3 and 4 was recorded in turn. The expression was considered low when the overall score lay between 0–3, whereas high when the score range was 4–7. Each tissue section or cell coverslip was evaluated by two pathologists independently. Any inconsistency was resolved by consulting a third pathologist.
Quantitative Real-time Polymerase Chain Reaction (Qrt-pcr)
After collecting of log-growth cells, sampling of total RNA proceeded by the trizol- chloroform-isopropanol method, followed by reverse transcription to cDNA (complementary DNA) via the RT reagent Kit (TaKaRa Bio, China). Furthermore, preparation of the reaction system was accomplished as per the guidelines of SYBR Primescrip real time PCR kit (TaKaRa), containing 10 µl of Power SYBR Green PCR master mix, 7.2 µl of DNAse/RNAse-free water, 2 µl of cDNA, and each 0.4 µl of upstream and downstream primers. PCR amplification was implemented in accordance with kit instructions for comparing the relative STEAP2 mRNA levels in five cervical carcinoma cells against the β-actin internal reference. The results were analyzed by the convenient 2-ΔΔCt method.17 Primers design and synthesis were undertaken by TaKaRa, the specific forward or reverse sequences were shown in Table 1. qRT-PCR experiments were repeated 3 times.
The sequence of primer in real time RT-qPCR
Logarithmic growth cells were collected and added with appropriate amount of protein lysate (RIPA:PMSF = 100:1), after centrifugation, extraction of total protein proceeded. The sample amount of protein in each lane was 40µg, SDS-PAGE (10% gel) was performed for isolation of proteins with different molecular weights, and then the electrophoretically isolated proteins were shifted onto PVDF (polyvinylidene difluoride) membranes. Next step was placement of the membranes in a shaker and a 1-h blockage using 5% non-fat milk. After thrice washing in TBST (tris-buffered saline containing Tween 20), an overnight incubation was accomplished using primary antibodies (1:1000 diluents) at 4°C, followed by a further 1-h incubation at ambient temperature using corresponding secondary antibodies on the next day. The enhanced chemiluminescence (ECL) working solution (Solarbio) was used to develop the target protein blot, whose gray value was then examined with the aid of ImageJ program.
Lentiviral Transfection For Steap2 Up-regulation Or Down-regulation
Vector construction and virus packaging for STEAP2 up-/down-regulation were accomplished by the Shanghai Genechem. For construction of the lentiviral over-expression vector of STEAP2, the Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin vector was recombined with STEAP2 (NM_001040665). Meanwhile, the lentiviral knockdown vector of STEAP2 was constructed by recombining the hU6-MCS-CBh-gcGFP-IRES-puromycin vector with the STEAP2 cloning small hairpin RNAs (shRNAs). The cervical cancer cell line Ca Ski was selected for RNA interference (RNAi) to down-regulate the expression of STEAP2, another cancer cell line HT-3 was selected for cDNA transfection to achieve the STEAP2 up-regulation. Negative controls of lentiviral transfections were also designed. Logarithmic phase cells were gathered, seeded onto the 24-well microplate and subjected to a 24-h cultivation in a 37°C incubator including 5% CO2. According to the pre-test results, the MOI (multiplicity of infection value; quantitative ratio of viruses to cells) was assigned as 50 for RNAi, whereas as 100 for over-expression. To reduce cytotoxicity, complete fresh medium was replenished 12 h later to substitute for the viral mixture. For the transfection efficiency assessment, a fluorescence microscope was utilized to observe the green fluorescence following 72 h of transfection, while for the expression evaluations of STEAP2 mRNA and protein in STEAP2 RNAi, over-expression and negative control groups, the qRT-PCR in conjunction with Western-blot were employed to validate the transfection performance.
Growth Curve Assay
Following digestion and centrifugation, the logarithmic phase cells were quantified, and subsequently each 1×104 cells were seeded per well of the 24-well microplate. From the next day, the mean cell counts in 3 wells were regarded as the cell quantity in that day, and the growth curve experiment lasted for one week. Curve plotting was accomplished by taking the cultivation duration as the horizontal axis, and the cell counts derived from daily statistics as the longitudinal axis.
Detection Of Cell Cycle By Flow Cytometry
The logarithmic growth cells were gathered, washed thrice in pre-cooled PBS, and then subjected to overnight fixation at − 20°C in 75% ethanol. A 30-min treatment of the fixed cells proceeded using RNase at 37°C, followed by a 30-min PI (propidium iodide) staining under ambient temperature and dark conditions. Finally, the cellular cycle was assessed with a Muse Cell Analyzer (Merck-Millipore, US).
Cell Plate Cloning Formation Assay
After gathering and PBS washing, each 500 logarithmic growth cells were seeded per well of a 6-well microplate, and subsequently incubated for 10 days, during which cell growth was observed. Ten days later, residual cells and the medium were rinsed in PBS, and then the cells were subjected to a 20-min fixation in inparaformaldehyde (4%) reagent until full coverage, and further subjected to a 10-min staining using hematoxylin dye, followed by removal of the dye solution. Thereafter, the 6-well microplate was continuously washed for about 5 min under the slow water flow of tap water and dried at room temperature. Three repeat wells were established for each group of cells and the experiment was triplicated.
Transwell Invasion And Migration Assay
The transwell chamber is divided into two parts: upper and lower by the microporous membrane 8.0 µm in pore dimension, when chemokines act, the upper chamber cells can penetrate into the lower chamber. Counting the cells crossing the microporous membrane can compare the migration ability of different cells. At ambient temperature, BD Matrigel can be polymerized to form a 3D matrix with biological activity, simulating the basal membrane in terms of composition, architecture, functionality and physical properties. In transwell invasion assay, Matrigel was diluted with serum-free cold medium at 1:5 ratio, 50 µl mixture were taken out and covered evenly and carefully on the microporous membrane, and then subjected to a 1-h incubation at 37℃ until thorough solidification of the Matrigel matrix glue. Serum-free medium was utilized to adjust the density of gathered logarithmic cells to 1×106 per ml, 200 µl of which was dispensed to the upper chamber. At the same time, the lower chamber was dispensed with 600 µl of 20% serum-containing media as Chemokines. After culturing for 12 h at 37℃ and 5% CO2, erasing residual Metrigel and not penetrated upper chamber cells and immersing the chamber for 30 min within paraformaldehyde (4%), the penetrated cells at the bottom of the membrane was fixed and stained by hematoxylin dye. Under microscopy, 5 visual fields were stochastically picked to quantify the cells crossing the membrane and calculate the average value. The experimentation was triplicated. The operational steps of transwell migration assay were fully identical to the invasion experiment in addition to the absence of Matrigel.
Nude Mice Orthotopic Transplantation Tumor Model In Vivo
Female 4–6 weeks of age BALB/C/nu/nu mice (body masses: 18–22 g) were kept in SPF (specific pathogen free) sterile facility and fed sterile food and water on a regular basis. We randomized these mice into Ca Ski, Ca Ski-sh1, Ca Ski-sh2, HT-3 and HT-3-exSTEAP2 5 groups with 5 mice per group. After gathering the logarithmic phase cells and suspending them in sterile saline, subcutaneous administration of 300 µl cell suspension containing 1×107 cells proceeded into the mice at back and neck. After injection, the tumor formation and changes were regular observed and the size of the nodule (tumor) was measured and recorded weekly with vernier calipers exploiting a formula: Volume = Length×Width2×0.5, tumor volume of nude mice per group were calculated. After observing for 8 weeks, mice were all euthanized with CO2 (carbon dioxide) for dissecting and assessing subcutaneous tumors per group. The approval of all animal experiments were obtained from the Shandong First Medical University’s Institutional Animal Care and Use Committee and implemented following the regulatory guidelines.
Processing of all the data was accomplished via the SPSS 24.0. The inter-group rates were evaluated for the results of immunohistochemistry through Chi-square or Fisher exact test. Pairwise comparisons of independent samples were accomplished through t-test, while the comparisons among multiple groups were made via single-factor ANOVA, and the SNK method was used to make pair comparison between multiple samples. Survival analysis was accomplished by Kaplan-Meier combined with Log Rank procedures. P values of < 0.05 were regarded to be significant.