Effects Of Cistanche Deserticola On Proliferation, Apoptosis And Migration Of Breast Cancer Cell Line T-47D By Regulating LncRNA LIFR-AS1
Apr 24, 2023
[Abstract] Objective To investigate the effects of Cistanche deserticola on the proliferation, apoptosis, and migration of breast cancer cell line T-47D and its regulation on long chain noncoding (Lnc) RNA leukemia inhibitory factor receptor antisense RNA-1 (LIFR-AS1). Method T-47D cells were cultured in different concentrations (100, 200, 400 μ Cultivate the water extract of Cistanche deserticola in a culture medium of g/ml for 24 hours, divided into low, medium, and high dose groups of Cistanche deserticola. At the same time, normal cultured cells were used as the control group. Transfect si-NC and si-LIFR-AS1 into T-47D cells and add 400 μ Cultivate with g/ml Cistanche deserticola culture medium for 24 hours, including the Cistanche deserticola high-dose+si-NC group and the Cistanche deserticola high-dose+si-LIFR-AS1 group. Real-time fluorescence quantitative polymerase chain reaction (qRT PCR) was used to detect the expression level of LIFR-AS1; Detection cell proliferation ability using plate cloning assay; Using flow cytometry to detect cell cycle and apoptosis rate; The ability of cell migration was tested by scratch test. Results Compared with the control group, the expression level and apoptosis rate of LIFR-AS1 in the middle and high dose groups of Cistanche deserticola were significantly increased, the number of clones was significantly reduced, the proportion of G0-G1 cells was significantly increased, and the proportion of S cells and scratch healing rate was significantly reduced (P<0 05); Compared with Cistanche deserticola high-dose+si NC group, the number of clones formed in Cistanche deserticola high-dose+si LIFR-AS1 group was significantly increased, the proportion of G0-G1 cells and the apoptosis rate were significantly reduced, and the proportion of S cells and the scratch healing rate were significantly increased (P<0 05)。 Conclusion Cistanche deserticola can induce cell cycle arrest by up-regulating the expression of LIFR-AS1, thereby reducing the proliferation of breast cancer cells, inducing apoptosis, and inhibiting cell migration.
[Keywords] Cistanche deserticola; Long chain noncoding (Lnc) RNA leukemia inhibitory factor receptor antisense RNA-1 (LIFR-AS1); Breast cancer; Proliferation; Apoptosis; transfer
Breast cancer is one of the common malignant tumors in clinics. The incidence rate of breast cancer in China is as high as 12. 5% 2%, the cure rate of early breast cancer is as high as more than 86%, but there is still no effective marker for early diagnosis of breast cancer. Radiochemical · 624 · Chinese Journal of Gerontology Vol.43, January 2023, and other comprehensive treatments can improve the treatment effect of patients, but the prognosis of patients is poor. At present, the pathogenesis of breast cancer has not been clarified. Previous studies have shown that hyperoside, apigenin, and other drugs have anti-breast cancer effects, However, the specific mechanism of its action has not yet been elucidated [1-4].
Effects Of Cistanche-Antitumor
Cistanche deserticola belongs to the perennial vine plant of the Rubiaceae family, which has the effects of tonifying kidney yang and dispelling wind and dampness. Studies have shown that Cistanche deserticola has pharmacological effects such as anti-tumor and antioxidant effects [5]. However, the therapeutic effect of Cistanche deserticola on breast cancer and its possible mechanism has not been clarified. Long chain noncoding (Lnc) RNA leukemia inhibitory factor receptor antisense RNA-1 (LIFR-AS1) is low expressed in breast cancer and can regulate cell proliferation and migration [6]. However, whether LIFR-AS1 can be used as the therapeutic target of Cistanche deserticola against breast cancer is unknown. This study was to explore whether Cistanche deserticola could affect the proliferation, apoptosis, and migration of breast cancer cell T-47D by regulating LIFR-AS1.
1 Material and Methods
1.1 Materials and reagents
Cistanche deserticola (20190203) was purchased from Bozhou Baichuan Pharmaceutical Co., Ltd; Breast cancer cell T-47D was purchased from Guangzhou Saiku Biotechnology Co., Ltd; Lipofectamine2000 (20190412) and apoptosis detection kit (20181223) were purchased from Beijing Solaybao Technology Co., Ltd; Trizol reagent (20181203) was purchased from Beijing Quanshijin Biotechnology Co., Ltd; Reverse transcription (20181103) and fluorescence quantitative polymerase chain reaction (PCR) detection reagents (20181116) were purchased from Beijing Tiangen Biochemical Technology Co., Ltd; Si-NC and si-LIFR-AS1 were purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd; Rabbit anti-human B-cell lymphoma (Bcl) -2 and Bcl-2 related X protein (Bax) antibodies were purchased from Santa Cruz Company in the United States; The goat anti-rabbit IgG secondary antibody labeled with horseradish peroxidase (HRP) was purchased from Wuhan Doctor Bioengineering Co., Ltd. The StepOnePlus real-time fluorescence quantitative PCR instrument was purchased from ABI Corporation in the United States; The FACS Calibur flow cytometry was purchased from Beckman Kurt Company in the United States.
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1.2 Experimental grouping
Water extract of Cistanche deserticola [7]: Weigh 100 g of Morinda officinalis medicine, soak in water for 30 minutes, and then decoct for 60 minutes. Filter the water decoction and decoct again for 45 minutes. Collect the filtrate twice and concentrate under reduced pressure to a volume of 160 ml. Cool it and incubate it in a refrigerator for 24 hours. After centrifugation at 3000 r/min, take the supernatant with a drug concentration of 2 g/ml. Dilute the culture solution to the required concentration of 100, 200, and 400 μ g /ml。 T-47D cells were cultured in different concentrations (100, 200, 400 μ Cultivate the water extract of Cistanche deserticola in a culture medium of g/ml for 24 hours, divided into low, medium, and high dose groups of Cistanche deserticola. At the same time, normal cultured cells were used as the control group. Transfect si-NC and si-LIFR-AS1 into T-47D cells and add 400 μ The culture medium of g/ml Cistanche deserticola was cultured for 24 hours, including the high-dose Cistanche deserticola+si-NC group and the high-dose Cistanche deserticola+si-LIFR-AS1 group.
1.3 Detection of LIFR AS1 expression level using real-time fluorescence quantification (qRT) - PCR
The total RNA of T-47D cells in each group was extracted using the Trizol method and reverse transcribed to synthesize cDNA. DeRNA enzyme (DEPC) was added and diluted 20 times in water before being stored in a -20 ℃ refrigerator. The qRT-PCR system and reaction procedure were operated according to the instructions of the fluorescence quantitative detection kit, and the relative expression of LIFR-AS1 (internal reference GAPDH) in cells was detected.
1.4 Plate cloning experiment
Inoculate T-47D cells in each group onto a 6-well plate (1 × 103 cells/well) and then placed them in the incubator for further cultivation for 14 days. Wash with pre-cooled phosphate buffer (PBS) and add methanol (500 μ l) Place it in a -20 ℃ refrigerator for 20 minutes, discard methanol, and add 1% crystal violet staining solution (400 μ l) Incubate at room temperature for 15 minutes and wash with distilled water. After drying, observe the number of clones formed.
1.5 Cell cycle detection experiment
T-47D cells in each group (1 × Inoculate 105 pieces/ml onto 96 well plates (100 μ L/well), centrifuge at a speed of 3000 r/min for 5 minutes, discard the supernatant and add pre-cooled PBS resuspended cells (500 μ l) Add pre-cooled ethanol (70%) to the cell suspension, discard the supernatant, and add RNaseA (50 μ l) Add propidium iodide (PI) staining solution (450) after a 30-minute water bath at 37 ℃ μ l) Incubate it in a 4 ℃ refrigerator for 30 minutes and analyze the proportion of cell cycles in each group using FACS Calibur flow cytometry and Flowjoy software.
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1.6 Flow cytometry detection of cell apoptosis rate
Add pre-cooled PBS to T-47D cells for washing, discard the supernatant, and add 500 to the cell precipitate μ Resuspension cells with buffer solution, operate according to apoptosis detection kit, and detect cell apoptosis rate.
1.7 Scratch test
Inoculate T-47D cells onto a 6-well plate (1 × 103/well), after each group is processed, draw a line in the horizontal direction at the bottom of the culture plate as a reference. After 48 hours of cultivation, draw the line again and observe the scratch width at two-time points to calculate the scratch healing rate [(scratch width 0 h - scratch width 48 h)/scratch width 0 h × 100%〕。
1.8 Western blot detection of Bax and Bcl-2 protein expression
After extracting the total protein of T-47D cells, detect the protein concentration and take an appropriate amount of protein samples for the SDS PAGE reaction. After membrane transfer and sealing, add the first antibody diluent (1:1000) and the second antibody diluent (1:5000), and use the automatic gel imaging system to analyze the gray value of each protein band. 1.9 Statistical processing using SPSS 21 Perform t-test and one-way ANOVA using software 0.
Experimental Study on Cistanche deserticola
2 Results
2.1 Effects of Cistanche deserticola on T-47D proliferation and LIFR-AS1 Expression
Compared with the control group, the expression level of LIFR-AS1 in the middle and high dose groups of Cistanche deserticola was significantly higher, the number of clones was significantly reduced, the proportion of G0-G1 phase cells was significantly higher, and the proportion of S phase cells was significantly lower, and the differences of each index between the middle and high dose groups of Cistanche deserticola deserticola were statistically significant (all P<0. 01) 05), but there was no statistically significant difference in the proportion of G2-M phase cells among the groups (P>0 05), see Table 1.
2. 2 The effect of Cistanche deserticola on T-47D apoptosis
Compared with the control group, the apoptosis rate and Bax protein level in the medium and high-dose groups of Cistanche deserticola significantly increased, while the Bcl-2 protein level significantly decreased. The differences in various indicators between the medium and high-dose groups of Cistanche deserticola were statistically significant (all P<0. 05) 05), as shown in Table 1, Figure 1, and Figure 2
Table 1 Effects of Cistanche deserticola on T-47D cell cycle clone formation, LIFR-AS1 expression, and cell apoptosis (x ± s, n=3)
Compared with the control group: 1) P<0 05; Compared with the low-dose group of Cistanche deserticola: 2) P<0 05; Compared with the medium and low dose group of Cistanche deserticola: 3) P<
Figure 1: Effect of Cistanche deserticola on apoptosis of T-47D in each group
1-4: Control group, low dose group of Cistanche deserticola, medium dose group of Cistanche deserticola, high dose group of Cistanche deserticola
Figure 2 Effect of Cistanche deserticola on the expression of T-47D apoptotic protein
2.3 Effect of Morinda officinalis on T-47D migration
Compared to the control group [(67 16 ± 1. 39)% comparison, medium-dose group of Cistanche deserticola [(43 56 ± 1.00%, and the scratch healing rate of the high-dose group of Cistanche deserticola [(23 87 ± 0.72%] significantly decreased, and the high-dose group of Cistanche deserticola was significantly lower than the middle-dose group of Cistanche deserticola (P<0 05)。
2.4 The effect of interference with LIFR-AS1 on the proliferation of T-47D treated with Cistanche deserticola
Compared with the Cistanche deserticola high-dose+si NC group, the number of clones formed in the Cistanche deserticola high-dose+si LIFR-AS1 group significantly increased, the proportion of G0-G1 cells significantly decreased, and the proportion of S cells increased (P<0 There was no statistically significant difference in the proportion of G2-M phase cells between the two groups (P>0. 05) 05), see Table 2.
Table 2 Effects of Interfering LIFR-AS1 on T-47D Cell Cycle, Clonogenesis, Apoptosis, and Migration in Cistanche deserticola Treatments (x ± s, n=3)
2. 5 Effect of Interfering LIFR-AS1 on T-47D Apoptosis Induced by Cistanche Deserticola Treatment
Compared with the high-dose+si-NC group of Cistanche deserticola, the apoptosis rate, Bax protein level, and Bcl-2 protein level of the high-dose+si-LIFR-AS1 group of Cistanche deserticola were significantly reduced (P<0 05), see Table 2, Figure 3, and Figure 4.
Effect of 2.6 interference with LIFR-AS1 on T-47D migration and LIFR-AS1 expression in Cistanche deserticola treated cells
Compared with the high-dose+si-NC group of Cistanche deserticola, the scratch healing rate of the high-dose+si-LIFR-AS1 group of Cistanche deserticola was significantly increased, and the expression of LIFR-AS1 was significantly reduced (P<0 05), see Table 2.
Figure 3 Effect of Interfering LIFR-AS1 on T-47D Apoptosis in Cistanche deserticola Treatment
1-2, Cistanche deserticola high-dose+si-NC group, Cistanche deserticola high-dose+si-LIFR-AS1 group
Figure 4: Effect of Interfering LIFR-AS1 on the Expression of T-47D Apoptosis Protein in Cistanche deserticola Treatment
3 Discussion
Chinese herbal medicine has an anti infection, anti-tumor and other effects. It is mild and can effectively alleviate some symptoms in patients, but the pathogenesis of breast cancer is more complex. The molecular mechanism of Chinese herbal medicine against breast cancer has not yet been clarified [8~10]. Cistanche deserticola has the effects of improving immunity, antioxidation, and effective treatment of osteoporosis, but its specific mechanism of action is not yet known [11,12]. The results of this study suggest that Cistanche deserticola can reduce the proliferation of breast cancer cells by inducing cell cycle arrest, and it can promote the apoptosis of breast cancer cells and inhibit the cell migration of breast cancer cells.
Benefits of cistanche tubulosa-Antitumor
The downregulation of LIFR-AS1 expression is associated with poor survival in gastric cancer [13]. LIFR-AS1 inhibits invasion and metastasis of non-small cell lung cancer through the miR-942-5p/zinc finger protein (ZNF) 471 axis [14]. These results suggest that interfering with the expression of LIFR-AS1 can significantly reverse the effects of Cistanche deserticola on the proliferation, apoptosis, and migration of breast cancer cells.
To sum up, Cistanche deserticola can induce cell cycle arrest, weaken the proliferation ability of breast cancer cells, promote cell apoptosis, and inhibit cell migration. Its mechanism is related to the upregulation of LIFR-AS1 expression.
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4 References
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