[Hsa_circ_0000392 affects the radiation sensitivity of cervical cancer by targeting the miR-145-5p/CRKL/MAPK pathway].

Objective: To investigate the effect of hsa_circ_0000392 (circ_0000392) on the radiosensitivity of cervical cancer cells and explore its potential mechanism. Methods: Cervical cancer tissues and adjacent normal tissues of 42 patients with cervical cancer who were confirmed pathologically for the first time in Huaihe Hospital of Henan University from 2016 to 2019 were collected. According to the patients' response to radiotherapy, the cancer tissues were divided into radio-sensitive tissues and radio-resistant tissues. The expressions of circ_0000392, miR-145-5p, and CRKL in radiation-sensitive, radiation-resistant cervical cancer tissues and Hela, SiHa cells were detected by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and western blot. SiRNA circ_0000392, miR-145-5p mimic, miR-145-5p inhibitor, pcDNA 3.1-CRKL and its negative control were transfected into HeLa and Siha cells, respectively. After radiation induction, the survival fraction of cells was detected by clone formation assay, apoptosis was detected by flow cytometry, and the expressions of apoptosis-related proteins Bax and Bcl-2 and ERK pathway protein p-ERK1/2 and ERK1/2 were detected by western blot. The targeting relationship between circ_0000392, miR-145-5p and CRKL was verified by dual luciferase reporter gene assay. The effect of circ_0000392 on radiotherapy sensitivity of cervical cancer in vivo was observed in the tumor formation experiment in nude mice. Results: circ_0000392 and CRKL were upregulated in radiation-resistant tissues and cancer cells of cervical cancer, while miR-145-5p was downregulated. The clone formation numbers of Hela and SiHa cells in si-circ_0000392#1+ 6 Gy group were (78.67±10.97) and (71.00±9.54), respectively, which were lower than those in si-Ctrl+ 6 Gy group [(176.00±22.27) and (158.33±17.56), respectively]. The apoptosis rates were (41.55±3.40)% and (31.41±3.29)%, respectively, which were higher than those in si-Ctrl+ 6 Gy group [(15.91±1.37)% and (13.70±1.89)%, P<0.05]. The protein expression of Bax was higher than that of si-Ctrl+ 6 Gy group, and the protein expressions of Bcl2 was lower than those of si-Ctrl+ 6 Gy group. The clone formation numbers of Hela and SiHa cells in si-circ_0000392#1+ miR-145-5p inhibitor+ 6 Gy group were (171.33±25.01) and (137.00±21.66), higher than those in si-circ_0000392#1+ inhibitor NC+ 6 Gy group [(84.67±17.79) vs (71.00±11.00), P<0.05]. The apoptosis rates were (17.41±2.58) % and (15.96±1.25) %, lower than those of si-circ_0000392 #1+ inhibitor NC+ 6 Gy [(40.29±2.92)% and (30.82±2.34)%, respectively, P<0.05]. The expression of Bax protein was lower than that of si-circ_0000392#1+ inhibitor NC+ 6 Gy group, and the expressions of Bcl2 protein were higher than those of si-circ_0000392#1+ inhibitor NC+ 6 Gy group. Circ_0000392 can target miR-145-5p, and CRKL is the downstream target gene of miR-145-5p. The clone formation numbers of Hela and SiHa cells in miR-145-5p mimic+ 6 Gy group were (74.33±10.02) and (66.00±12.17), respectively, which were lower than those of mimic NC+ 6 Gy group [(197.67±17.21) vs (157.67±11.59), respectively, P<0.05]. The apoptosis rates were (45.58±2.16)% and (32.10±3.55)%, higher than those of mimic NC+ 6 Gy group [(15.85±2.45)% and (13.99±1.69)%, respectively, P<0.05]. The expression of Bax protein was higher than that of the mimic NC+ 6 Gy mimic group, and the expression of Bcl2 protein was lower than that of the mimic NC+ 6 Gy group. The clone formation numbers of Hela and SiHa cells in miR-145-5p mimic+ pcDNA-CRKL+ 6 Gy group were (158.00±15.88) and (122.33±13.65), respectively, which were higher than those of miR-145-5p mimic+ pcDNA+ 6 Gy group [(71.33±8.02) vs (65.67±12.22), P<0.05]. The apoptosis rates were (19.50±3.45)% and (17.04±0.94)%, respectively, which were lower than those of miR-145-5p mimic+ pcDNA+ 6 Gy group [(44.33±2.36)% and (32.05±2.76)%, respectively, P<0.05]. The expression of Bax protein was lower than that of miR-145-5p mimic+ pcDNA group+ 6 Gy group, and the expression of Bcl2 protein was higher than that of miR-145-5p mimic+ pcDNA+ 6 Gy group. Sh-circ_0000392 group had smaller tumor volume and decreased tumor weight (P<0.05). The relative mRNA expression levels of circ_0000392, miR-145-5p and CRKL and the relative protein expression levels of CRKL, Bcl-2 and p-ERK1/2 were decreased, while the relative expression level of Bax protein was increased (P<0.05). Conclusion: Circ_0000392 could enhance the radiosensitivity of cervical cancer cells, and its mechanism may be related to the regulation of CRKL/ERK signaling pathway by targeting miR-145-5p, which provides a new reference for enhancing the radiosensitivity of cervical cancer cells.

Circ_001680 aggravates the malignant process of gastric carcinoma by targeting MAP2.

OBJECTIVE: This study aims to explore the expression pattern and clinical significance of circ_001680 in gastric carcinoma (GC) process. PATIENTS AND METHODS: Circ_001680 levels in 40 pairs of GC and paracancerous ones were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The relationship between circ_001680 and GC clinicopathological parameters was analyzed. AGS and SGC-7901 cells were used for constructing circ_001680 knockdown models by shRNA transfection. Proliferative and metastatic abilities in GC cells with circ_001680 knockdown were examined by cell counting kit-8 (CCK-8) and transwell assay, respectively. Dual-Luciferase reporter assay was conducted to clarify the interaction between circ_001680 and MAP2. Their co-regulation on GC process was detected through rescue experiments. RESULTS: Circ_001680 was highly expressed in GC tissues and cell lines. High level of circ_001680 predicted high incidences of lymphatic and distant metastasis, and poor prognosis in GC patients. Knockdown of circ_001680 suppressed proliferative and metastatic abilities in AGS and SGC-7901 cells. MAP2 was the target gene binding circ_001680, which was lowly expressed in GC. In addition, MAP2 was negatively correlated to circ_001680. Knockdown of MAP2 could abolish the suppressed proliferative and metastatic abilities in GC cells with circ_001680 knockdown. CONCLUSIONS: Circ_001680 is highly expressed in GC tissues and closely related to metastasis and prognosis in GC patients, which promotes the proliferative and metastatic abilities in GC cells by negatively interacting with MAP2.

Role of H3K18ac-regulated nucleotide excision repair-related genes in arsenic-induced DNA damage and repair of HaCaT cells.

Arsenic is an environmental poison and is a grade I human carcinogen that can cause many types of damage to the body. The skin is one of the main target organs of arsenic damage, but the molecular mechanisms underlying arsenic poisoning are not clear. Arsenic is an epigenetic agent. Histone acetylation is one of the earliest covalent modifications to be discovered and is closely related to the occurrence and development of tumors. To investigate the role of acetylated histone H3K18 (H3K18 ac) in arsenic-induced DNA damage, HaCaT cells were exposed to sodium arsenite (NaAsO2) for 24 h. It was found that arsenic induced the downregulation of xeroderma pigmentosum A, D, and F (XPA, XPD, and XPF-nucleotide excision repair (NER)-related genes) expression, as well as histone H3K18 ac expression, and aggravated DNA damage. Chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR) analysis showed that H3K18 acetylation in the promoter regions of XPA, XPD, and XPF was downregulated. In addition, the use of the histone deacetylase inhibitor trichostatin A (TSA) partially inhibited arsenic-induced DNA damage, inhibited deacetylation of H3K18 ac in the promoter regions of XPA, XPD, and XPF genes, increased acetylation of H3K18, and promoted the transcriptional expression of NER-related genes. Our study revealed that NaAsO2 induces DNA damage and inhibits the expression of NER-related genes, while TSA increases the H3K18 ac enrichment level and promotes the transcriptional expression of NER, thereby inhibiting DNA damage. These findings provide new ideas for understanding the molecular mechanisms underlying arsenic-induced skin damage.

Effect of Jianpi Bushen prescription on the expression of SHP-1 and apoptosis-related genes in chemically damaged model mice.

We investigated the effect of Jianpi Bushen prescription (JBP) on the expression of the SHP-1 and apoptosis-related genes in chemically damaged model mice and a compound e-jiao slurry (EJS) group (positive control). Kunming mice received an abdominal injection of 100 mg/kg cyclophosphamide once a day for 3 consecutive days to induce chemical damage. The mice underwent lavage at a suspension of 0.1 g/kg low-dose JBP (100%), high-dose JBP (200%), and 0.2 mL/10 g EJS twice a day for 9 days. mRNA and protein expression of SHP-1 in bone marrow mononuclear cells was detected using real-time polymerase chain reaction and Western blot; mRNA expression of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X (Bax) protein was detected by in situ hybridization. Expression of SHP-1 and Bax mRNA was significantly upregulated in the model group compared to the control group (P < 0.05). Expression in the low-dose JBP, high-dose JBP, and EJS groups was significantly downregulated compared with the model group (P < 0.05). The low-dose JBP group exhibited much lower SHP-1 and Bax mRNA expression levels. Compared with controls, Bcl-2 mRNA expression was significantly reduced in the model group (P < 0.05). Expression in the low-dose JBP, high-dose JBP, and EJS groups significantly increased compared with the model group (P < 0.05). The low-dose JBP group showed much higher Bcl-2 mRNA expression. Therefore, JBP regulates the expression of the SHP- 1, Bax, and Bcl-2 genes in chemically damaged mice.

Effect of the Jianpi Bushen Prescription on the expression of SHP-1, Wnt3a, and AP-1 proteins in chemically damaged mice.

This study investigated the effect of the Jianpi Bushen Prescription (JBP) on the expression of 3 major proteins in chemically damaged model mice. The 3 proteins were the Wnt3a, the SHP-1, and the transcription factors (NF-E2, c-jun, and c-fos) of the AP-1 protein family. Kunming mice were randomly divided into chemically damaged group (N=48), which received an abdominal injection of (100 mg/kg) cyclophosphamide once a day for 3 consecutive days, and control group (N=12), which received the same amount of saline. Then, the chemically damaged mice were randomly divided into chemically damaged model group (N=12), which received 0.2 mL/10 g of saline twice a day for 9 days, positive control group (N=12), which received 0.2 mL/10 g of the e-jiao slurry (EJS) compound twice a day for 9 days, low dose JBP group (N=12), which received 0.1 g/kg suspension JBP (100% concentration) twice a day for 9 days and high dose JBP group (N=12), which received 0.1 g/kg suspension JBP (200% concentration) twice a day for 9 days. The bilateral femur and tibia bone marrow were collected from the mice in all groups. The protein expression of the specified proteins and transcription factors in the bone marrow mononuclear cells were detected by Western blot analysis. The results showed that the protein expression of Wnt3a was significantly downregulated in the chemically damaged model group compared to the control group (P<0.05). The low dose JBP, high dose JBP, and e-jiao slurry treatments significantly upregulated the protein expression of Wnt3a compared to the chemically damaged model group (P<0.05), with the low dose JBP producing the best results. Compared to the control group, the protein expressions of SHP-1, c-fos, c-jun, and NF-E2 were significantly higher in the chemically damaged model group (all P<0.05). The protein expressions of SHP-1, c-fos, c-jun, and NF-E2 were significantly lower in the chemically damaged model+the low dose JBP, chemically damaged model+high dose JBP, or chemically damaged model+EJS group compared to chemically damaged model (all P<0.05), with the low dose JBP producing the best results. These results indicate that JBP regulates the expressions of SHP-1, Wnt3a, and AP-1 proteins in chemically damaged mice.

Effect of the Jianpi Bushen Prescription on expressions of the Wnt3a and Cyclin D1 genes in radiation-damaged mice.

The effects of the traditional Chinese drug Jianpi Bushen Prescription (JBP) were investigated on expressions of Wnt3a and Cyclin D1 genes in radiation-damaged mice. The radiation damage model was induced in Kumming mice by single total body irradiation treatment for 9 days. Mice were divided into the radiation group, low-dose (100%) JBP group, high-dose (200%) JBP group, or batyl alcohol group (positive control), which were administered twice a day for 9 days. mRNA and protein expressions of Wnt3a were detected in bone marrow mononuclear cells by real-time polymerase chain reaction and Western blot, whereas Cyclin D1 mRNA was detected by in situ hybridization. Wnt3a expressions were significantly downregulated in the radiation damage model group compared with all other groups (P < 0.05). The positive cell rate of Cyclin D1 mRNA expression and the number of granulocyte macrophage colonies were significantly decreased in the radiation damage model group relative to all other groups (P < 0.05). Furthermore, mRNA and protein expressions of Wnt3a, the positive cell rate of Cyclin D1 mRNA expression in bone marrow cells, and the number of granulocyte macrophage colonies were all significantly higher in the low-dose JBP group than in the high-dose JBP group (P < 0.05). In summary, JBP plays a protective role on radiation-induced bone marrow through the activation of the Wnt3a signaling pathway, and promotes the transcription and expression of Cyclin D1.

[Yield and cytotoxicity of tumor-infiltrating lymphocyte(TIL) following immunochemotherapy]

Tumor-infiltrating lymphocyte (TIL) in the primary site of oral SCC treated with S-PVP of PVP therapy was collected respectively and its yield,that is calculating the amount of TIL per tumor tissue.The cytotoxicity of TIL against the target,Tca8113 was detected.The results showed that the yield of TIL in S-PVP group was three times over that in PVP group.At the ratio of 5 to 1 (effector to target) the cytotoxicity was 38% in S-PVP group and 32% in PVP group.There was no significant difference in its cytotoxicity between two groups.The results revealed that immunochemotherapy of S-PVP regimen for treating oral SCC had neither direct damage to tumor cell from the agents nor antitumor immune reactivity with BRM stimulating an increasing in numbers of TIL.