| Background:Endometrial cancer (EC) accounts for 20% to 30% in the female reproductive system tumor, it is the fourth most common female malignancy in developed countries. In developing countries, the incidence of endometrial cancer also lives the 7th of female cancer. Endometrial cancer usually occurs in women after menopause, but recent clinical studies show that women who have endometrial cancer significantly become younger and younger. Although early endometrial cancer after standard treatment,5-year survival rate increased year by year, but late, poorly differentiated endometrial cancer with poor prognosis, is an important factor leading to death in patients with endometrial cancer. Therefore, the study of the pathogenesis of endometrial cancer and effective therapeutic targets are primary subject to improve the prognosis of endometrial cancer.Most studies have shown that endometrial cancer is related to long-term exposure to estrogen without resistance, or associated with obesity, hypertension, and diabetes. But the mechanism of endometrial cancer is not clear, as abnormal gene regulation of gene expression polymorphism and others are not sufficient to fully explain the occurrence and development of endometrial cancer. Recent studies show that the development of the sub-mircroRNA and endometrial cancer are closely related, and the expression of the molecular mechanisms involved in the regulation of estrogen and progesterone endometrial cancer process.miRNA is a class of non-coding single-stranded small RNA widespread, which is a research hotspots in recent years. And it is Lee first discovered in C. elegans, a length of about 19-25 nucleotides. Lee’s team first proposed that miRNA closely is associated with tumor development and progression. Now human miRNA has been found over 1000 which regulates about 30% of human genes. Subsequent studies in many malignancies such as colorectal cancer, prostate cancer, osteosarcoma, miRNA plays tumor promoters or tumor suppressor role. MicroRNAs bind 3’UTR region of the target gene by complementary base pairing manner, to degradate the target gene mRNA or repress target gene mRNA translation. MicroRNAs negatively regulate target genes at the post-transcriptional level, thus affecting tumor proliferation, apoptosis, invasion and metastasis. Similarly, microRNAs play a dual role of tumor promoters or tumor suppressor in the progression of endometrial cancer, to participate in development and progression of endometrial cancer in each period, with the corresponding one or more of the target gene 3’UTR binding region, negatively regulate expression of target genes. Also miRNA is closely related chemosensitivity of tumor cells, it may become a judge marker of endometrial cancer prognostic after cisplatin treatment.miR-135b gene is located on the long arm of chromosome 3 1 2nd District No.1, with sub-band (1q32.1), which is overexpression in osteosarcoma, colon cancer, prostate cancer and other tumors. In non-small cell lung cancer, miR-135b promotes metastasis through the activation Hippo signaling pathway by targeting LZTS1. Arigoni found that miR-135b is upregulated by targeting estrogen receptor α, androgen receptors and hypoxia-inducible factor 1α (hypoxia-inducible factor 1α, HIF-1α) to promote proliferation of breast and prostate cancer cell line. In human head and neck squamous cell carcinoma, miR-135b promotes tumor cell proliferation, metastasis, cloning, and become oncogenic miRNA by down-regulating the expression of FIH, the active HIF pathway. Valeri and other studies have shown that over-expression of miR-135b triggers mouse and human APC deletion, abnormal PTEN/PI3K pathway, overexpression of SRC and promoting the transformation and progress. Overexpression of miR-135b and human colorectal cancer-related inflammation are common events, and is closely related with the tumor stage and poor clinical outcome. MiR-135b in feces can be a biomarker which used in colorectal cancer and high-level adenoma of non-invasive. However, the expression of miR-135b in endometrial carcinoma, and the correlation of clinical and biological characteristics, are not yet reported.Based on the status of research miR-135b, our research is to explore the biological properties of miR-135b by exploring miR-135b expression in endometrial carcinoma and 4 cell lines and vitro cell experiments. And we explored the use of miR-135b in development and progession of endometrial cancer molecular mechanisms, in order to further the search for new endometrial cancer pathogenesis and therapeutic target and lay a theoretical foundation.Methods1. Expression of miR-135b expression in endometrial carcinoma and endometrial cancer cells were detected by RT-PCRExtracted total RNA from endometrial cancer tissues and cell lines, in according to Takara Bio and fluorescence quantitative reverse transcription PCR kit instructions, we formed by reverse transcription cDNA. In accordance with SYBR Green method, we did real-time PCR. We use 2-△△Ct to express genes relative expression levels.2. Transfection efficiency of miRNA135b transient transfection was detected by RT-PCRWe use cationic liposome method for transient transfection in accordance with the instructions of LipofectamineTM 3000 reagent. The day before transfection, we use endometrial cancer cell lines Ishikawa and RL-952 in logarithmic growth phase to do digestion and centriffugation. And then we counted approximately 2× 105 cells to seed to 6-well plates and cultured. When confluent of cells reached 30 to 50%, we started further experiment. The supernatant was discarded, and then added 1.7ml DMEM high glucose medium containing 10% fetal bovine serum. 100nM siRNA purchased from RiboBio was added to the serum-free medium to prepare a mixture ①150μl, and mixed together properly.5μl LipofectamineTM 3000 reagant was added to the other serum-free medium to prepare a mixture ②150μl and mixed together properly. And we incubated two mixtures for 5 min at room temperature. And we added the mixture ①to mixture ② respectively, and incubated for 20 min at room temperature to form a transfection complex. And eventually we added the comples to 1.7ml DMEM high glucose medium containing 10% fetal bovine serum, and rocked to mix. Wherein, mimic NC and inhibitor NC is as a reference. Total RNA was extracted after 48h cells, reverse transcribed into expression cDNA, we use qRT-PCR to detect transient transfection of miR-135b.3. The effect of cell proliferation was detected by MTT assayEndometrial cancer cells were seeded 5 × 103 every well in 96-well plates, and transfected after 12h adherence. We divided cells into mimic, mimic NC, inhibitor, inhibitor NC and Negtive Control group. Every group had 6 wells. We detected respectively after transfection for Oh,24h,48h,72h,96h. We added each well 5mg/ ml solution of MTT 20μl prior to testing. And then cells were cultured for 4h in cell incubator. The supernatant was discarded, and added DMSO solution 150μl, shaker rocked for 10min fully to dissolve crystals, blank well as zero, ELISA measured at a wavelength of 490nm absorbance on instrument that represents cell proliferation.6 wells of each group were averaged to make growth curve.4. The effect of cell migration was detected by wound healing assayThe endometrial carcinoma RL-952 cell lines were seeded in six-well plates. The cells were transiently transfected 48h to confluence of 90%, and we use 200μl pipette tip along the vertical ruler wells and through the point to manufacture of "ten" word scratches. The supernatant was discarded, and we used PBS buffer to wash for 2-3 times to remove exfoliated cells and replaced by serum-free medium and then cultured. Selected scratches across the field of vision in 0,24h,48h photographed, we calculated and compared the mobility of each group.5. FOXO1 and HOXA10 expression in endometrial carcinoma were detected by RT-PCRExtracted total RNA from endometrial cancer tissues and cell lines, according to Takara Bio and fluorescence quantitative reverse transcription PCR kit instructions, we formed by reverse transcription cDNA. In accordance with SYBR Green method, we did real-time PCR. We use 2-△△Ct to express genes relative expression levels.6. The changes of FOXO1 mRNA after transient transfection of miRNA-135b were detected by RT-PCRWe extracted total RNA of endometrial cancer cell lines Ishikawa and RJL-952 after transfection for 48h, according to Takara Bio and fluorescence quantitative reversetranscription PCR kit. We used △△CT method for real-time PCR results forquantitative analysis. According to Ct value of every sample, We use 2-△△Ct to represent relative expression level of every gene.7. The changes of FOXO1 protein after transient transfection of miRNA-135b were detected by RT-PCRAfter transfection for 48h, we extracted total protein of RL-952 cell lines. Quantification is based on BCA protein quantification kit, which is measuring A562. Calculated standard average absorbance of each concentration of protein, we made standard curve and calculated the regression equation. Extraction of total protein sample was added on the 2 x SDS protein loading buffer (1:1 ratio), and boiled at 100 ℃ for 5min. They are stored 20 ℃ for use. We used estern blot to detect the expression of each group FOXO1 protein.8. Statistical analysisThe statistical software SPSS 19.0 was used for statistical analysis. Data was expressed in mean ± standard deviation format.22 cases of endometrial carcinoma and corresponding adjacent tissues were compared using paired samples t-test. Other compares between the experimental groups were compared using one-way ANOVA. MTT growth curves were compared using factorial analysis of variance. All data are the results of three independent repeat experiments, P<0.05 was considered statistically significant.Result1. Expression of miR-135b expression in endometrial carcinoma and endometrial cancer cells were detected by RT-PCRWe detected the expression of miR-135b in 22 paired endometrial carcinoma by RT-PCR, with INC as a reference value,2-△△Ct by paired samples t-test analysis. The results are shown that mean endometrial carcinoma of miR-135b expression was 8.3559 ± 10.6692, significantly higher than the average expression of miR-135b in adjacent endometrial carcinoma which was 4.1930 ± 5.1132, statistically significant (t = 2.247, P= 0.036).2. Expression of miR-135b and FOXO1 in endometrial cancer cell lines were detected by RT-PCRReferring to JEC cell lines, the expression of miR-135b in different cells were respectively Ishikawa (111.5150 ± 11.7878), HEC-1-B (20.8096 ± 5.7010), RL-952 (1.6737 ± 0.3428).There are differences in the expression level of miR-135b between the four cell lines, and the difference was statistically significant (F= 129.3, P ＜0.0001). FOXO1 expression in different cell lines were Ishikawa (0.0640 ± 0.0150), HEC-1-B (0.2994 ± 0.0592), RL-952 (0.4548 ± 0.1053). Expression levels of FOXO1 between 4 cell lines are different, and the difference was statistically significant (F= 85.36, P＜0.0001).3. Transfection efficiency of miRNA135b transient transfection was detected by RT-PCRUsing Liposome method, we transfected miR-135b mimic, inhibitor, mimic NC and inhibitor NC into endometrial carcinoma cell lines RL-952 and Ishikawa. After transfection for 48h, we extracted RNA, detected expression of miR-135b by qRT-PCR after reverse transcription. The results showed that, expression of miR-135b after transfection of mimics in RL-952, Ishikawa in miR-135b were increased by 526.39 times (t=-20.189, P= 0.002) and 32.98 times (t= 52.609, P= 0.000); and transfection after miR-135b inhibitor, expression of miR-135b was reduced by 25.28%(t=-23.539, P= 0.002) and 95%(t= 5.966, P= 0.027).4. MTT assayWe used MTT method to assay cell growth curve,which was showed that after transfection miR-135b mimic, RL-952, and Ishikawa cell growth accelerated (P= 0.0000,0.0000); after transfected miR-135b inhibitor, both cell growth rate slowed (P = 0.0000,0.0000).5. Wound healing assayAfter transfection 48h in human endometrial carcinoma cell line RL-952, changes in cell scratch was to assay cell migration. Mobility= (original pitch-pitch after migration)/original pitch × 100. After scratches 24h, mobilities between the four groups are significant different by statistical analysis (F = 24.67, P = 0.0002), after scratching 48h, mobility between the four groups are significant different by statistical analysis (F = 36.36, P<0.0001). After transfection of miR-135b mimic, RL-952 at 24h and 48h, respectively, to improve the mobility of 1.54 times (t= 12.01, P= 0.0003) and 1.54 times (t = 7.888, P = 0.0014); transfected with miR-after 135b inhibitor, RL-952 at 24h and 48h, respectively, mobility decreased 57.60%(t= 4.490, P= 0.0109) and 44.17%(t= 5.123, P= 0.0069).6. FOXOl and HOXA10 expression in endometrial carcinoma were detected by RT-PCRWe detected the expression of miR-135b in 22 paired endometrial carcinoma by RT-PCR, with INC as a reference value,2-△△Ct by paired samples t-test analysis. The results showed that the average expression of FOXOl in endometrial carcinoma was 1.6654 ± 1.6861, endometrial cancer was significantly higher than the average expression level of adjacent tissues, which was 3.2708 ± 3.4159, statistically significant (t=-2.559, P= 0.018). The average expression level of HOXA10 in endometrial carcinoma was 1.1473 ± 0.9662, significantly higher than the average expression of adjacent endometrial carcinoma,which was 1.8258 ± 1.0156, statistically significant (t=-3.039, P= 0.006).7. The changes of FOXO1 mRNA after transient transfection of miRNA-135b were detected by RT-PCRWe detected FOXO1 mRNA expression in endometrial carcinoma cell lines RL-952 and Ishikawa by qRT-PCR after miR-135b overexpression or inhibition. We found that miR-135b overexpression can significantly decrease FOXOl mRNA expression in endometrial cancer cell lines, whereas inhibitory of miR-135b can increase FOXOl mRNA expression. Namely miR-135b has a negative effect to the regulation of FOXO1 expression.8. The changes of FOXO1 protein after transient transfection of miRNA-135b were detected by RT-PCRWe extracted total protein of each group of RL-952 cell line, with the former concrete steps. We used Western blot to detect the effect of FOXO1 protein expression after different transfection. We found significantly expression of FOXO1 protein decreased after overexpression of miR-135b in endometrial cancer cell lines, and the expression of protein FOXO1 increased after downregulation of miR-135b. Namely miR-135b has a negative effect to the regulation of FOXO1 expression. Conclusion 1. miR-135b was obviously up-regulated in endometrial carcinoma tissue than para-carcinoma tissues with a significant difference. 2. miR-135b is involved in the progression, proliferation and migration of endometrial cancer, and its ectopic expression can affect proliferation as well as migration of endometrial cancer cells in vitro, which functions as a proto-oncogene.3. FOXO1 was down-regulated in endometrial carcinoma compared with its para-carcinoma tissue.4. HOXA10 was down-regulated in endometrial carcinoma compared with its para-carcmoam.5. In endometrial carcinoma, FOXO1 as a target gene of miR-135b, miR-135b suppress FOXO1 transcription by targeting it, and promote the proliferation and development of endometrial cancer. |
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