ABSTRACT
Objective:To analyze the expression of circular RNA circ_0008274 in cetuximab-resistant colorectal cancer cells using bioinformatics technology and to explore its involvement in the development of cetuximab resistance.Methods:Five concentrations of cetuximab (10, 50, 100, 150, 200 nmol/L) were set. Cetuximab-resistant cells DiFi-R and Caco-2-R were screened out and established by concentration increasing method using colorectal cancer cells DiFi and Caco-2. The expression of circ_0008274 in DiFi-R and Caco-2-R cells was detected by reverse transcription-polymerase chain reaction(RT-PCR). The interaction and regulation between circ_0008274 and microRNA(miR)-140-3p were analyzed by double-luciferase reporter assay. The highly expressed gene SMARCC1 related to cetuximab resistance was determined by Western blotting. Circ_0008274 in DiFi-R and Caco-2-R cells were knocked out with small interfering RNA si-circ_0008274 transfection. After knock out, the differences in the colony formation and cell proliferation in DiFi-R and Caco-2-R cells were compared. MiR-140-3p mimic and blank control miR were transfected into DiFi-R and Caco-2-R cells. After transfection the difference in cell proliferation between transfected with miR-140-3p mimic and blank control miR in DiFi-R and Caco-2-R cells were analyzed. After Caco-2-R cell was knocked out with si-circ_0008274, the changes of SMARCC1 protein expression rescued by pcDNA3.1 SMARCC1 and cell viability were analyzed. The tumor specimens of 15 colorectal cancer patients hospitalized in Renmin Hospital of Wuhan University from March 2019 to August 2020 were included. According to the treatment effect, the patients were divided into sensitive group (11 cases) and drug-resistant group (4 cases). The relative expression levels of circ_0008274, downstream SMARCC1and miR-140-3p in colorectal cancer tissues in the two groups were detected by RT-PCR. Independent sample t test was used for statistical analysis. Results:The level of circ_0008274 in DiFi-R cells was 2.33±0.12 times of that of DiFi cells, while the level in Caco-2-R was (2.92±0.42) times of that of Caco-2 cells, and the differences were statistically significant ( t=19.97 and 7.80, both P<0.05). The results of double-luciferase reporter showed that after miR-140-3p mimic combined with wild-type circ_0008274, the relative fluorescence intensity was lower than before (0.28±0.04 vs. 1.00±0.00), and the difference was statistically significant ( t=-30.71, P=0.001). The expression of SMARCC1 protein in DiFi-R and Caco-2-R cells was significantly increased, the expression at protein level was higher than that of DiFi and Caco-2 cells (2.22±0.36 vs. 0.61±0.17, 0.85±0.11 vs. 0.35±0.08), and the differences were statistically significant ( t=6.23 and 6.32, both P<0.01). After circ_0008274 was knocked out, the numbers of colony formation of DiFi-R and Caco-2-R cells were both lower than those of before knockout (36.67±4.04 vs. 66.00±9.54, 17.35±4.04 vs. 52.33±8.02), the relative active cell ratios after interventing by 10, 50, 100, 150 and 200 nmol/L cetuximab were also lower than those of before knockout (DiFi-R cells: (73.75±2.75)% vs. (88.10±2.48)%, (56.50±6.66)% vs. (75.15±6.03)%, (35.75±5.32)% vs. (59.63±6.67)%, (24.25±3.30)% vs. (52.40±6.71)%, (6.25±2.75)% vs. (48.60±5.38)%; Caco-2-R cells: (63.74±5.25)% vs. (85.76±4.79)%, (56.50±4.20)% vs.(83.50±3.90)%, (46.00±2.94)% vs. (80.00±6.05)%, (35.30±5.56)% vs. (68.30±4.57)%, (12.25±7.37)% vs. (62.40±7.51)%), and the differences were statistically significant ( t=4.90, 6.71, -7.75, -4.16, -5.60, -7.53, -14.02, -6.19, -8.33, -10.10, -9.17 and -9.56, all P<0.01). After transfecting with miR-140-3p mimic, the relative active cell ratios of DiFi-R and Caco-2-R cells interventing by 10, 50, 100, 150 and 200 nmol/L cetuximab were both lower than those transfected with blank control miR (DiFi-R cells: (71.55±4.97)% vs. (85.90±2.66)%, (51.58±3.91)% vs. (74.95±6.35)%, (41.23±8.84)% vs. (58.43±7.05)%, (28.60±5.26)% vs. (53.75±5.65)%, (18.90±5.13)% vs. (51.30±3.30)%; Caco-2-R cells: (61.75±2.22)% vs. (90.10±1.41)%, (53.25±4.17)% vs. (86.18±2.69)%, (46.38±4.55)% vs. (77.75±6.70)%, (36.10±8.76)% vs. (70.15±4.18)%, (24.25±2.63)% vs. (65.10±7.62)%), and the differences were statistically significant ( t=-5.09, -6.47, -3.05, -6.28, -10.30, -21.48, -12.83, -8.01, -6.79 and -10.12, all P<0.01). After circ_0008274 was knocked out, the SMARCC1 protein level of Caco-2-R cells rescued by pcDNA3.1 SMARCC1 was higher than that of before rescue (0.63±0.19 vs. 0.09±0.03), and the relative active cell ratios after interventing by 10, 50, 100, 150 and 200 nmol/L cetuximab were also higher than that of before rescue ((93.10±3.56)% vs. (83.83±3.97)%, (83.28±4.26)% vs. (60.90±7.02)%, (61.83±2.12)% vs. (50.10±5.59)%, (53.20±3.74)% vs. (40.50±3.42)%, (46.20±4.08)% vs. (30.80±4.82)%), and the differences were statistically significant( t=3.55, 3.52, 5.44, 3.87, 4.64 and 4.88, all P<0.01). The relative expression levels of circ_0008274 and downstream SMARCC1 of colon cancer tissues in the drug-resistant group were higher than those in the sensitive group (6.45±1.32 vs. 2.26±1.39, 12.53±1.60 vs. 3.82±1.56), and the relative expression level of miR-140-3p was lower than that in the sensitive group (3.91±1.25 vs. 7.43±2.23), and the differences were statistically significant ( t=5.22, 9.51, -2.93, all P<0.01). Conclusions:Circular RNA circ_0008274 is highly expressed in colorectal cancer tissues and cetuximab resistant cells, interacts and inhibits miR-140-3p expression, up-regulates SMARCC1, and participates in the occurrence of cetuximab resistance. PcDNA3.1 SMARCC1 rescue can block the sensitization effect of si-circ_0008274 on cetuximab, and can significantly increase cetuximab resistance of colorectal cancer cells.
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Objective To investigate the efficacy and safety of full?thickness peroral endoscopic my?otomy( POEM) for achalasia in a follow?up period of 1 year. Methods Data of 28 patients who underwent full?thickness POEM in our hospital from July 2013 to June 2014 were retrospectively studied. Procedure completion and complications were assessed at the end of follow?up of 12 months. Results All patients were treated successfully by full?thickness POEM with the mean operation time of (61?7±15?6) minutes. Intraop?erative mucosal perforation occurred in 2( 7?1%) patients. Symptoms were relieved in 28 patients, but ster?num pain occurred in 6(21?4%) patients. POEM failed in one case in the first 3 months, and was successful in 27 other cases.treatment success rate (Eckardt≤3) was 89?3%(25/28). The incidences of postoperative reflux and esophagitis were 25?9%( 7/27 ) and 22?2%( 6/27 ) , respectively. Conclusion The full?thickness POEM is safe and effective with less complications, but further study is needed to evaluate long?term efficacy and complications.
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Objective To investigate mutations of S protein gene in positive HBsAg and anti-HBs patients with chronic hepatitis B virus (HBV) infection.Methods Fifteen HBsAg(+) and anti-HBs(+) patients and 22 HBsAg(+) and anti-HBs (-) patients (control group) admitted in Renmin Hospital of Wuhan University during January and December 2011 were enrolled in the study.The S protein gene was amplified and sequenced, and the amino acid sequences were translated from the obtained DNA sequences and compared with the reference sequences.Results Compared with the control group, HBsAg (+) and anti-HBs(+) patients showed a higher variability in amino acid within major hydrophilic region (2.95 vs.0.78,x2 =18.059, P＜0.01) and the a determinant (4.44 vs.1.52, x2 =6.985, P＜0.01).The mutations in a determinant at positions P127T, G130E, G130N, M133S, F134I, T140I and G145R were detected only in HBsAg(+) and anti-HBs (+) patients.Conclusion Co-existence of HBsAg and anti-HBs in patients with chronic HBV infection might be associated with the increased amino acid mutations in and around the a determinant of protein S.
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Objective To investigate the safety and effectiveness of endoscopic submucosal dissection(ESD)for elderly patients(≥60 years old)with colorectal lesions.Methods Data of 31 elderly patients(≥60 years old)and 23 non-elderly(0.05 ). Thirty-two lesions in elderly group and twenty-five lesions in non-elderly group were all curative resection.En bloc resection rates were 96.9%(31 /32)and 96.0%(24 /25)in the elderly group and non-elderly group respectively;the rates of bleeding during ESD procedure were 3.2%(1 /31 )and 4.3%(1 /23);delayed bleeding rates were 12.9%(4 /31)and 13.0%(3 /23);the rates of perforation was 12.9%(4 /31)and 0;postoperative infection rates were 3.2%(1 /31)and 4.3%(1 /23)respectively.There were no statistical differences between the two groups in any of these data (P >0.05 ).The mean time of follow-up were (14.8 ±1.7)months in elderly group and (14.7 ±1.8)months in non-elderly group,and there was no significant difference between two groups.No residual lesion or recurrent lesion was found in the follow-up period.Conclusion ESD is a safe and effective treatment for the elderly patients with colorectal lesion.
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BacKground:Human pancreatic cancer is a highIy maIignant tumor of digestive system. CurrentIy,gemcitabine based conventionaI chemotherapy has onIy very Iimited efficacy on metastasis of pancreatic cancer. Studies have shown that thymoquinone has remarkabIe effect of inhibiting proIiferation and enhancing apoptosis on a variety of cancer ceIIs. Aims:To investigate the effect and mechanism of thymoquinone on inhibiting the migration and invasion of human pancreatic cancer BxPC-3 ceIIs in vitro. Methods:Human pancreatic cancer BxPC-3 ceIIs were conventionaIIy cuItured and treated with different concentrations of thymoquinone. The migration and invasion of BxPC-3 ceIIs were determined by Boyden chamber assay. The expressions of FAK,Akt and phosphoryIation of Akt were measured by Western bIotting,and immunofIuorescence was used to detect expression of FAK,focaI adhesions and F-actin. Results:The inhibitory rates of 10,25μmoI/L thymoquinone on migration of BxPC-3 ceIIs were 43. 4% and 73. 8%,respectiveIy,and the inhibitory rates of invasion were 60. 5% and 75. 6%,respectiveIy. The reduction of migration and invasion of pancreatic cancer BxPC-3 ceIIs by thymoquinone was in a dose-dependent manner( P < 0. 05 ). Thymoquinone obviousIy down-reguIated the expression of FAK and suppressed the phosphoryIation of Akt in BxPC-3 ceIIs. Thymoquinone induced the dispersed distribution of FAK in cytopIasm and inhibited the formation of focaI adhesions and assembIy of F-actin. Conclusions:Thymoquinone inhibits the migration and invasion of human pancreatic cancer BxPC-3 ceIIs in a dose-dependent manner in vitro through suppression of FAK/PI3K/Akt signaIing pathway and activity of kinase.