ABSTRACT
【Objective】 To investigate the molecular mechanism of long non-coding RNA (lncRNA) TDRG1 in facilitating the malignant progression and poor prognosis of patients with cervical cancer. 【Methods】 Cervical cancer cell lines and normal cervical cell Ect1/E6E7 were collected. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of TDRG1. Cervical cancer cell lines were transfected with TDRG1-siRNA, and the proliferation of cancer cells was detected by CCK-8 method and cell plate cloning experiment. The invasion and migration of cancer cells were measured by Transwell experiment. The apoptosis of cancer cells was examined by flow cytometry, and the expressions of relevant proteins were tested by Western blot. 【Results】 Compared with Ect1/E6E7, cervical cancer cell lines showed relatively increased expression of TDRG1. Downregulation of TDRG1 expression inhibited the proliferation and colony formation (162±21 vs. 411±33, P<0.05), as well as the invasion and migration (invasion: 86±13 vs. 315±38, P<0.01; migration: 177±22 vs. 406±41, P<0.01) of Hela cells. Meanwhile, the apoptosis of Hela cells increased [(28±1.5)% vs. (16±1.2)%, P<0.05] and the expression of Bcl-2 protein reduced. In addition, TDRG1 knockdown also decreased the activity of autophagy in Hela cells. 【Conclusion】 TDRG1 facilitates the malignant biological progression of cervical cancer by inhibiting the apoptosis and providing a protective autophagy in cervical cells.
ABSTRACT
Objective: To investigate the expression of long non-coding RNA testis developmental related gene 1 (lncRNA-TDRG1) in cervical cancer tissues and its correlation with clinical progress and prognosis. Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to examine the relative expression of lncRNA-TDRG1 in cervical cancer tissues (n=60) and adjacent normal tissues (n=60). We also compared lncRNA-TDRG1 expression in different cervical cancer patients of different age, FIGO stages, cancer cell differentiation, and lymph node metastasis. Furthermore, four kinds of cervical cancer cells (SiHa, HeLa, CaSki, and C-33A) and a normal cervical cell (Ect1/E6E7) were used to identify the different expression of lncRNA-TDRG1 with qRT-PCR. Chi-square test was performed to analyze the relationship between lncRNA-TDRG1 expression and clinicopathological characteristics in the cervical cancer patients. Survival curves were drawn with Kaplan-Meier method and analyzed with log-rank test. In addition, we also carried out univariate and multivariate Cox regression analyses of overall survival in 60 cervical cancer patients. Results: lncRNA-TDRG1 was highly expressed in cervical cancer tissues as compared with adjacent normal tissues. And cervical cancer cells also showed a higher expression of lncRNA-TDRG1 than normal cervical cells. The expression of lncRNA-TDRG1 in cervical cancer exhibited obvious relationship with cancer cell differentiation (moderate and well vs. poor, P=0.019) and lymph node metastasis (yes vs. no, P=0.037). Meanwhile, patients with a higher expression of lncRNA-TDRG1 had a worse overall survival (P<0.05). Univariate and multivariate Cox regression analyses of overall survival indicated that lncRNA-TDRG1 was an independent risk factor for poor prognosis of cervical cancer patients (P=0.006). Conclusion: lncRNA-TDRG1 plays a very important role in the progression and prognosis of cervical cancer, and may serve as a biomarker for the prognosis of patients with cervical cancer.
ABSTRACT
Objective To construct a prokaryotic plasmid to express the testis development related gene 1 (TDRG1) recombinant protein and obtain anti-TDRG1 mAb by immunizing mice, and to identify the biological properties of the mAb. Methods The coding sequence of TDRG1 was amplified by RT-PCR from normal human testis tissue and cloned into the vector pET21, and then was expressed in the E. coli BL 21(DE3) to get TDRG1 recombinant protein. The purified TDRG1 recombinant protein was injected to immunize the BALB/C mice to develop anti-TDRG1 mAb. Splenocytes of the immunized mice were collected and fused with the mouse myeloma cell line Sp2/0 cells. The hybridoma cells that secreted anti-TDRG1 mAb were subcloned with limited dilution. Enzyme-linked immunosorbent assay (ELISA) was used to evaluate titers and subtypes of mAb. Western blot and immunohistochemistry were used to detect specificity of mAb.Results The prokaryotic plasmid expressing the recombinant protein was constructed, and the TDRG1 recombinant protein was expressed and purified. Two hybridoma cell lines that secreted anti-TDRG1 mAb were obtained. The titer of the mAb in ascites was 1∶1.6×10~6, and the subtype of the mAb was IgG_1. Westem blot and immunohistochemistry analysis indicated the mAb showed specific combination with TDRG1 protein in human testes.Conclusion The TDRG1 recombinant protein is highly purified and has strong antigenicity. The anti-TDRG1 mAb is produced successfully.