Objective:To investigate the effectiveness of new cryoprotective agents in preserving and transplanting human adipose tissue.Methods:The adipose tissue samples were obtained from healthy adult females who underwent liposuction at the Department of Plastic Surgery of Peking University Third Hospital from January to March 2022. The adipose tissue samples were centrifuged and then randomly divided into 9 groups. These groups were cryopreserved in liquid nitrogen using different cryoprotective agents [group A, group B, and dimethyl sulfoxide (DMSO) group] and cryopreservation times (1-month, 2-month, and 3-month groups), respectively. The cryoprotective agent formulation in group A was dextrose glycoside 40 (DEX), amino acids, vitamins, and inorganic salts. In group B, the formulation included DMSO and DEX. The ratio of cryoprotective agent in the DMSO group was 10% DMSO, 20% fetal bovine serum (FBS), and 70% DMEM-12. For cryopreservation, 5 ml cryogenic tubes were used with a fat to cryoprotective agent ratio of 3∶2, and each group contains 6 tubes for cryopreservation. After thawing the adipose tissue, HE staining was used to observe the histological morphology. Immunohistochemical staining was employed for the quantitative analysis of lipid droplet-encapsulated protein (Perilipin), and the Perilipin positivity rate was calculated by the ratio of the number of positive cells to the total number of cells. Adipocyte viability was assessed using the CCK-8 method. Thirty-eight healthy, clean nude mice were selected and divided into 3 groups of 12 mice each according to the use of different cryoprotective agents (groups A, B, and DMSO), while the other 2 mice were used as the day 0 control group. The mean fat freezing duration for all groups was 3 months. After nude mice were anesthetized intraperitoneally, 0.9 ml of thawed cryopreserved fat was injected into the dorsum bilaterally. The rate of adipose tissue retention was calculated by MRI scanning and three-dimensional software at 1, 2, and 3 months after transplantation, and compared between the groups. The fat grafts were explanted from the mice after they were sacrificed, and then subjected to histological morphology and quantitative analysis of Perilipin by using HE staining and immunohistochemical staining. GraphPad Prism 8.0 software was used for statistical analysis of the data. The data that conformed to a normal distribution were expressed as Mean ± SD. The overall comparison between multiple groups used analysis of variance for repeated measures. The comparison of data between groups at the same time point used Tukey’s multiple comparison test.Results:The morphology of adipose tissue in different cryoprotective agent groups closely resembled that of normal fresh adipose tissue after being cryopreserved in liquid nitrogen for 1-3 months. The difference in the proportion of Perilipin-stained positive cells in each group was not statistically significant ( P>0.05). The CCK-8 method indicated that the effect of the DMSO group was superior to groups A and B at 1 and 3 months of cryopreservation ( P<0.01), and that the DMSO group and group B were superior to group A at 2 months of cryopreservation ( P<0.01). In the animal experiments, there was no statistically significant difference between the groups in the volume retention rate 1-3 months after cryopreserved fat transplantation ( P>0.05). Additionally, the adipose tissues in each group exhibited varying degrees of localized necrosis accompanied by an inflammatory reaction 1-3 months after transplantation. There was no statistically significant difference in the Perilipin staining positivity between the groups ( P>0.05). Conclusion:The use of new cryoprotective agents for cryopreserving adipose tissue does not show a significant difference compared to the traditional cryoprotective agent. However, it is theoretically safer as it avoids the potential toxic effects of using DMSO or FBS on the human body.

Objective:To investigate the effectiveness of new cryoprotective agents in preserving and transplanting human adipose tissue.Methods:The adipose tissue samples were obtained from healthy adult females who underwent liposuction at the Department of Plastic Surgery of Peking University Third Hospital from January to March 2022. The adipose tissue samples were centrifuged and then randomly divided into 9 groups. These groups were cryopreserved in liquid nitrogen using different cryoprotective agents [group A, group B, and dimethyl sulfoxide (DMSO) group] and cryopreservation times (1-month, 2-month, and 3-month groups), respectively. The cryoprotective agent formulation in group A was dextrose glycoside 40 (DEX), amino acids, vitamins, and inorganic salts. In group B, the formulation included DMSO and DEX. The ratio of cryoprotective agent in the DMSO group was 10% DMSO, 20% fetal bovine serum (FBS), and 70% DMEM-12. For cryopreservation, 5 ml cryogenic tubes were used with a fat to cryoprotective agent ratio of 3∶2, and each group contains 6 tubes for cryopreservation. After thawing the adipose tissue, HE staining was used to observe the histological morphology. Immunohistochemical staining was employed for the quantitative analysis of lipid droplet-encapsulated protein (Perilipin), and the Perilipin positivity rate was calculated by the ratio of the number of positive cells to the total number of cells. Adipocyte viability was assessed using the CCK-8 method. Thirty-eight healthy, clean nude mice were selected and divided into 3 groups of 12 mice each according to the use of different cryoprotective agents (groups A, B, and DMSO), while the other 2 mice were used as the day 0 control group. The mean fat freezing duration for all groups was 3 months. After nude mice were anesthetized intraperitoneally, 0.9 ml of thawed cryopreserved fat was injected into the dorsum bilaterally. The rate of adipose tissue retention was calculated by MRI scanning and three-dimensional software at 1, 2, and 3 months after transplantation, and compared between the groups. The fat grafts were explanted from the mice after they were sacrificed, and then subjected to histological morphology and quantitative analysis of Perilipin by using HE staining and immunohistochemical staining. GraphPad Prism 8.0 software was used for statistical analysis of the data. The data that conformed to a normal distribution were expressed as Mean ± SD. The overall comparison between multiple groups used analysis of variance for repeated measures. The comparison of data between groups at the same time point used Tukey’s multiple comparison test.Results:The morphology of adipose tissue in different cryoprotective agent groups closely resembled that of normal fresh adipose tissue after being cryopreserved in liquid nitrogen for 1-3 months. The difference in the proportion of Perilipin-stained positive cells in each group was not statistically significant ( P>0.05). The CCK-8 method indicated that the effect of the DMSO group was superior to groups A and B at 1 and 3 months of cryopreservation ( P<0.01), and that the DMSO group and group B were superior to group A at 2 months of cryopreservation ( P<0.01). In the animal experiments, there was no statistically significant difference between the groups in the volume retention rate 1-3 months after cryopreserved fat transplantation ( P>0.05). Additionally, the adipose tissues in each group exhibited varying degrees of localized necrosis accompanied by an inflammatory reaction 1-3 months after transplantation. There was no statistically significant difference in the Perilipin staining positivity between the groups ( P>0.05). Conclusion:The use of new cryoprotective agents for cryopreserving adipose tissue does not show a significant difference compared to the traditional cryoprotective agent. However, it is theoretically safer as it avoids the potential toxic effects of using DMSO or FBS on the human body.

New daily persistent headache(NDPH)is a kind of persistent headache that patients can i-dentify the exact date of the sudden onset.It is one of the rare primary headaches difficult to be cured and may lead to disability,seriously affecting the daily life and work.The exact pathogenesis of NDPH remains unclear,which makes the treatment difficult.Here we report a case of refractory NDPH treated by intravenous injection of esketamine at a sub-anesthetic dose.

Auditory neuropathy spectrum disorder (ANSD) represents a variety of sensorineural deafness conditions characterized by abnormal inner hair cells and/or auditory nerve function, but with the preservation of outer hair cell function. ANSD represents up to 15% of individuals with hearing impairments. Through mutation screening, bioinformatic analysis and expression studies, we have previously identified several apoptosis-inducing factor (AIF) mitochondria-associated 1 (AIFM1) variants in ANSD families and in some other sporadic cases. Here, to elucidate the pathogenic mechanisms underlying each AIFM1 variant, we generated AIF-null cells using the clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system and constructed AIF-wild type (WT) and AIF-mutant (mut) (p.‍T260A, p.‍R422W, and p.‍R451Q) stable transfection cell lines. We then analyzed AIF structure, coenzyme-binding affinity, apoptosis, and other aspects. Results revealed that these variants resulted in impaired dimerization, compromising AIF function. The reduction reaction of AIF variants had proceeded slower than that of AIF-WT. The average levels of AIF dimerization in AIF variant cells were only 34.5%‍‒‍49.7% of that of AIF-WT cells, resulting in caspase-independent apoptosis. The average percentage of apoptotic cells in the variants was 12.3%‍‒‍17.9%, which was significantly higher than that (6.9%‍‒‍7.4%) in controls. However, nicotinamide adenine dinucleotide (NADH) treatment promoted the reduction of apoptosis by rescuing AIF dimerization in AIF variant cells. Our findings show that the impairment of AIF dimerization by AIFM1 variants causes apoptosis contributing to ANSD, and introduce NADH as a potential drug for ANSD treatment. Our results help elucidate the mechanisms of ANSD and may lead to the provision of novel therapies.
Humans ; Apoptosis Inducing Factor/metabolism* ; NAD/metabolism* ; Dimerization ; Apoptosis

Objective: To explore the association between dietary inflammatory index (DII) and metabolic syndrome (MetS) and its components among children aged 6-14 years in Beijing, so as to provide a reference for preventing MetS. Methods: A cross sectional study was carried out in 2 086 records of 1 832 children from the 2017 and 2019 Nutrition and Health Surveillance in Primary and Secondary school students of Beijing (NHSPSB). Three day consecutive 24 hour dietary recalls combined with weighing household cooking oils and condiments were used to collect dietary intake and calculate DII. MetS was diagnosed according to "Definition and Suggestion on the Metabolic Syndrome of Chinese Children and Adolescent". The Generalized estimating equations (GEEs) models were used to analyze the association between DII and the presence of MetS and its components (abdominal obesity, high triglyceride, low high density lipoprotein cholesterol, hypertension, and hyperglycemia). Results: The mean DII score was (1.64±1.07) for the included children. No significant association was found between DII scores and the likelihood of MetS (per 1 point increment: OR =1.16, 95% CI =0.92-1.48, P >0.05). In terms of the components of MetS, DII scores were positively associated with the odds of high triglyceride (per 1 point increment: OR =1.17, 95% CI =1.01-1.36, P <0.05). There was no statistically significant difference in the association among different age groups ( P >0.05). No significant associations were observed between DII and other MetS components( P >0.05). Conclusion DII scores may not be correlated with the risk of MetS, but proinflammatory diet might increase the risk of high triglyceride. DII score in childhood should be emphasized to identify and prevent MetS as soon as possible.

Objective:By studying the gait changes of patients with knee osteoarthritis (KOA), the study provided a theoretical basis for the quantitative indicators obtained from gait analysis to the diagnosis of KOA. And it provided a gait reference for the clinical diagnosis, treatment, rehabilitation, prevention and efficacy evaluation of KOA.Methods:A total of 30 patients (KOA group) with KOA hospitalized in our hospital from May 2021 to October 2021 and 30 healthy people (control group) were compared for gait changes. The t test, Mann-Whitney U test and Fisher′s exact test were used to compare differences between groups. Results:The KOA group were greater than the control group in terms of step time [(642±81) ms and (548±62) ms, t=-5.01, P<0.001], gait cycle [(1 284±168) ms and (1 076±114) ms, t=-5.61, P<0.001], double support time [(531±125) ms and (331±51) ms, t=-8.10, P<0.001], double support time period proportion (0.417±0.063 and 0.309±0.023, t=-8.50, P<0.001), total support time [(914±135) ms and (678±107) ms, t=-7.52, P<0.001], total support time period proportion (0.711±0.027 and 0.627±0.044, t=-8.87, P<0.001), and left static standing time (55.7±8.4 and 51.5±2.2, t=-2.65, P=0.012), for which the differences were statistically significant. The KOA group were lower than the control group in terms of single support time period proportion (0.287±0.030 and 0.334±0.013, t=7.80, P<0.001), right static standing time (44.3±8.4 and 48.5±2.3, t=2.65, P=0.012), step length [(36±8) cm and (52±5) cm, t=9.97, P<0.001], stride length [(70±16) cm and (103±8) cm, t=10.00, P<0.001], velocity [(0.60±0.18) m/s and (1.05±0.19) m/s, t=9.54, P<0.001], left knee range of motion [(42±17)° and (63±4) °, t=6.49, P<0.001], and right knee range of motion [(37±18) ° and (62±3)°, t=7.54, P<0.001], for which the differences were statistically significant. Conclusion:Gait analysis can quantitatively evaluate the condition of patients with KOA, making it possible to transform the diagnostic criteria of KOA from qualitative to quantitative.

Auditory neuropathy spectrum disorder (ANSD) represents a variety of sensorineural deafness conditions characterized by abnormal inner hair cells and/or auditory nerve function, but with the preservation of outer hair cell function. ANSD represents up to 15％ of individuals with hearing impairments. Through mutation screening, bioinformatic analysis and expression studies, we have previously identified several apoptosis-inducing factor (AIF) mitochondria-associated 1 (AIFM1) variants in ANSD families and in some other sporadic cases. Here, to elucidate the pathogenic mechanisms underlying each AIFM1 variant, we generated AIF-null cells using the clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system and constructed AIF-wild type (WT) and AIF-mutant (mut) (p.T260A, p.R422W, and p.R451Q) stable transfection cell lines. We then analyzed AIF structure, coenzyme-binding affinity, apoptosis, and other aspects. Results revealed that these variants resulted in impaired dimerization, compromising AIF function. The reduction reaction of AIF variants had proceeded slower than that of AIF-WT. The average levels of AIF dimerization in AIF variant cells were only 34.5％?49.7％ of that of AIF-WT cells, resulting in caspase-independent apoptosis. The average percentage of apoptotic cells in the variants was 12.3％?17.9％, which was significantly higher than that (6.9％?7.4％) in controls. However, nicotinamide adenine dinucleotide (NADH) treatment promoted the reduction of apoptosis by rescuing AIF dimerization in AIF variant cells. Our findings show that the impairment of AIF dimerization by AIFM1 variants causes apoptosis contributing to ANSD, and introduce NADH as a potential drug for ANSD treatment. Our results help elucidate the mechanisms of ANSD and may lead to the provision of novel therapies.

Objective:To investigate the epidemiological characteristics, diagnosis, treat-ment and prognosis of gallbladder cancer in China from 2010 to 2017.Methods:The single disease retrospective registration cohort study was conducted. Based on the concept of the real world study, the clinicopathological data, from multicenter retrospective clinical data database of gallbladder cancer of Chinese Research Group of Gallbladder Cancer (CRGGC), of 6 159 patients with gallbladder cancer who were admitted to 42 hospitals from January 2010 to December 2017 were collected. Observation indicators: (1) case resources; (2) age and sex distribution; (3) diagnosis; (4) surgical treatment and prognosis; (5) multimodality therapy and prognosis. The follow-up data of the 42 hospitals were collected and analyzed by the CRGGC. The main outcome indicator was the overall survival time from date of operation for surgical patients or date of diagnosis for non-surgical patients to the end of outcome event or the last follow-up. Measurement data with normal distribu-tion were represented as Mean±SD, and comparison between groups was conducted using the t test. Measurement data with skewed distribution were represented as M( Q1, Q3) or M(range), and com-parison between groups was conducted using the U test. Count data were described as absolute numbers or percentages, and comparison between groups was conducted using the chi-square test. Univariate analysis was performed using the Logistic forced regression model, and variables with P<0.1 in the univariate analysis were included for multivariate analysis. Multivariate analysis was performed using the Logistic stepwise regression model. The life table method was used to calculate survival rates and the Kaplan-Meier method was used to draw survival curves. Log-rank test was used for survival analysis. Results:(1) Case resources: of the 42 hospitals, there were 35 class A of tertiary hospitals and 7 class B of tertiary hospitals, 16 hospitals with high admission of gallbladder cancer and 26 hospitals with low admission of gallbladder cancer, respectively. Geographical distribution of the 42 hospitals: there were 9 hospitals in central China, 5 hospitals in northeast China, 22 hospitals in eastern China and 6 hospitals in western China. Geographical distribution of the 6 159 patients: there were 2 154 cases(34.973%) from central China, 705 cases(11.447%) from northeast China, 1 969 cases(31.969%) from eastern China and 1 331 cases(21.611%) from western China. The total average number of cases undergoing diagnosis and treatment in hospitals of the 6 159 patients was 18.3±4.5 per year, in which the average number of cases undergoing diagnosis and treatment in hospitals of 4 974 patients(80.760%) from hospitals with high admission of gallbladder cancer was 38.8±8.9 per year and the average number of cases undergoing diagnosis and treatment in hospitals of 1 185 patients(19.240%) from hospitals with low admission of gallbladder cancer was 5.7±1.9 per year. (2) Age and sex distribution: the age of 6 159 patients diagnosed as gallbladder cancer was 64(56,71) years, in which the age of 2 247 male patients(36.483%) diagnosed as gallbladder cancer was 64(58,71)years and the age of 3 912 female patients(63.517%) diagnosed as gallbladder cancer was 63(55,71)years. The sex ratio of female to male was 1.74:1. Of 6 159 patients, 3 886 cases(63.095%) were diagnosed as gallbladder cancer at 56 to 75 years old. There was a significant difference on age at diagnosis between male and female patients ( Z=-3.99, P<0.001). (3) Diagnosis: of 6 159 patients, 2 503 cases(40.640%) were initially diagnosed as gallbladder cancer and 3 656 cases(59.360%) were initially diagnosed as non-gallbladder cancer. There were 2 110 patients(34.259%) not undergoing surgical treatment, of which 200 cases(9.479%) were initially diagnosed as gallbladder cancer and 1 910 cases(90.521%) were initially diagnosed as non-gallbladder cancer. There were 4 049 patients(65.741%) undergoing surgical treatment, of which 2 303 cases(56.878%) were initially diagnosed as gallbladder cancer and 1 746 cases(43.122%) were initial diagnosed as non-gallbladder cancer. Of the 1 746 patients who were initially diagnosed as non-gallbladder cancer, there were 774 cases(19.116%) diagnosed as gallbladder cancer during operation and 972 cases(24.006%) diagnosed as gallbladder cancer after operation. Of 6 159 patients, there were 2 521 cases(40.932%), 2 335 cases(37.912%) and 1 114 cases(18.087%) undergoing ultrasound, computed tomography (CT) or magnetic resonance imaging (MRI) examination before initial diagnosis, respec-tively, and there were 3 259 cases(52.914%), 3 172 cases(51.502%) and 4 016 cases(65.205%) undergoing serum carcinoembryonic antigen, CA19-9 or CA125 examination before initially diagnosis, respectively. One patient may underwent multiple examinations. Results of univariate analysis showed that geographical distribution of hospitals (eastern China or western China), age ≥72 years, gallbladder cancer annual admission of hospitals, whether undergoing ultrasound, CT, MRI, serum carcinoembryonic antigen, CA19-9 or CA125 examination before initially diagnosis were related factors influencing initial diagnosis of gallbladder cancer patients ( odds ratio=1.45, 1.98, 0.69, 0.68, 2.43, 0.41, 1.63, 0.41, 0.39, 0.42, 95% confidence interval as 1.21-1.74, 1.64-2.40, 0.59-0.80, 0.60-0.78, 2.19-2.70, 0.37-0.45, 1.43-1.86, 0.37-0.45, 0.35-0.43, 0.38-0.47, P<0.05). Results of multivariate analysis showed that geographical distribution of hospitals (eastern China or western China), sex, age ≥72 years, gallbladder cancer annual admission of hospitals and cases undergoing ultrasound, CT, serum CA19-9 examination before initially diagnosis were indepen-dent influencing factors influencing initial diagnosis of gallbladder cancer patients ( odds ratio=1.36, 1.42, 0.89, 0.67, 1.85, 1.56, 1.57, 0.39, 95% confidence interval as 1.13-1.64, 1.16-1.73, 0.79-0.99, 0.57-0.78, 1.60-2.14, 1.38-1.77, 1.38-1.79, 0.35-0.43, P<0.05). (4) Surgical treatment and prognosis. Of the 4 049 patients undergoing surgical treatment, there were 2 447 cases(60.435%) with complete pathological staging data and follow-up data. Cases with pathological staging as stage 0, stage Ⅰ, stage Ⅱ, stage Ⅲa, stage Ⅲb, stage Ⅳa and stage Ⅳb were 85(3.474%), 201(8.214%), 71(2.902%), 890(36.371%), 382(15.611%), 33(1.348%) and 785(32.080%), respectively. The median follow-up time and median postoperative overall survival time of the 2 447 cases were 55.75 months (95% confidence interval as 52.78-58.35) and 23.46 months (95% confidence interval as 21.23-25.71), respectively. There was a significant difference in the overall survival between cases with pathological staging as stage 0, stage Ⅰ, stage Ⅱ, stage Ⅲa, stage Ⅲb, stage Ⅳa and stage Ⅳb ( χ2=512.47, P<0.001). Of the 4 049 patients undergoing surgical treatment, there were 2 988 cases(73.796%) with resectable tumor, 177 cases(4.371%) with unresectable tumor and 884 cases(21.833%) with tumor unassessable for resectabi-lity. Of the 2 988 cases with resectable tumor, there were 2 036 cases(68.139%) undergoing radical resection, 504 cases(16.867%) undergoing non-radical resection and 448 cases(14.994%) with operation unassessable for curative effect. Of the 2 447 cases with complete pathological staging data and follow-up data who underwent surgical treatment, there were 53 cases(2.166%) with unresectable tumor, 300 cases(12.260%) with resectable tumor and receiving non-radical resection, 1 441 cases(58.888%) with resectable tumor and receiving radical resection, 653 cases(26.686%) with resectable tumor and receiving operation unassessable for curative effect. There were 733 cases not undergoing surgical treatment with complete pathological staging data and follow-up data. There was a significant difference in the overall survival between cases not undergoing surgical treatment, cases undergoing surgical treatment for unresectable tumor, cases undergoing non-radical resection for resectable tumor and cases undergoing radical resection for resectable tumor ( χ2=121.04, P<0.001). (5) Multimodality therapy and prognosis: of 6 159 patients, there were 541 cases(8.784%) under-going postoperative adjuvant chemotherapy and advanced chemotherapy, 76 cases(1.234%) under-going radiotherapy. There were 1 170 advanced gallbladder cancer (pathological staging ≥stage Ⅲa) patients undergoing radical resection, including 126 cases(10.769%) with post-operative adjuvant chemotherapy and 1 044 cases(89.231%) without postoperative adjuvant chemo-therapy. There was no significant difference in the overall survival between cases with post-operative adjuvant chemotherapy and cases without postoperative adjuvant chemotherapy ( χ2=0.23, P=0.629). There were 658 patients with pathological staging as stage Ⅲa who underwent radical resection, including 66 cases(10.030%) with postoperative adjuvant chemotherapy and 592 cases(89.970%) without postoperative adjuvant chemotherapy. There was no significant difference in the overall survival between cases with postoperative adjuvant chemotherapy and cases without postoperative adjuvant chemotherapy ( χ2=0.05, P=0.817). There were 512 patients with pathological staging ≥stage Ⅲb who underwent radical resection, including 60 cases(11.719%) with postoperative adjuvant chemotherapy and 452 cases(88.281%) without postoperative adjuvant chemotherapy. There was no significant difference in the overall survival between cases with postoperative adjuvant chemo-therapy and cases without post-operative adjuvant chemo-therapy ( χ2=1.50, P=0.220). Conclusions:There are more women than men with gallbladder cancer in China and more than half of patients are diagnosed at the age of 56 to 75 years. Cases undergoing ultrasound, CT, serum CA19-9 examination before initial diagnosis are independent influencing factors influencing initial diagnosis of gallbladder cancer patients. Preoperative resectability evaluation can improve the therapy strategy and patient prognosis. Adjuvant chemotherapy for gallbladder cancer is not standardized and in low proportion in China.

OBJECTIVE: To explore the genetic basis for a Chinese pedigree affected with non-syndromic cleft lip and cleft palate (NSCLP). METHODS: With informed consent obtained, members of the pedigree were subjected to clinical examination and history taking to exclude syndromic cleft lip and palate. One affected member was subjected to whole-exome sequencing and bioinformatics analysis. Candidate variant was verified by Sanger sequencing and co-segregation analysis of her family members and 100 unrelated healthy individuals. RESULTS: Whole-exome sequencing and co-segregation analysis showed that all affected members of this pedigree have carried a heterozygous missense c.253A>G (p.Cys85Arg) variant in exon 4 of the IRF6 gene, which has co-segregated with the phenotype and was not found among the 100 unrelated healthy individuals. CONCLUSION The missense c.253A>G variant in exon 4 of the IRF6 gene probably underlay the NSCLP in this pedigree.
Brain/abnormalities* ; China ; Cleft Lip/genetics* ; Cleft Palate/genetics* ; Female ; Humans ; Interferon Regulatory Factors/genetics* ; Mutation, Missense ; Pedigree ; Whole Exome Sequencing

Objective:To examine the expression of T-box5 (TBX5) in colorectal cancer tissues and its clinical significance, and explore the effects of TBX5 on the invasion and metastasis of colorectal cancer cells and its mechanism.Methods:The expressions of TBX5 in cancer and adjacent normal tissues were tested by immunohistochemistry (IHC), and the relationship between TBX5 and clinicopathological features and prognosis of colorectal cancer was analyzed. Real-time quantitative PCR (RT-qPCR) and western blot were used to detect the expressions of TBX5 in different colorectal cancer cell lines. TBX5 overexpression plasmid was constructed and transfected into human colorectal cancer cell line HT-29, and cell counting kit-8 (CCK-8) was used to detect the activities of transfection HT-29 cells. Cell scratch test and Transwell assay were used to detect the migration and invasion abilities of cells, while RT-qPCR and western blot were used to detect the mRNA and protein expressions of PCNA, p21, p16, p27, MMP-2, MMP-7 and TIMP-1.Results:The positive rate of TBX5 protein in colorectal cancer tissues was 24.44% (22/90), significantly lower than 65.56% of adjacent normal tissues ( P<0.001). The expression of TBX5 was significantly related to lymph node metastasis, depth of invasion and nerve invasion ( P<0.05). The survival period of 22 patients with positive TBX5 expression was (60.2±2.4) months, better than (44.3±2.8) months of 68 patients with negative TBX5 expression ( P<0.05). Among human colon cancer cell lines of HT29, SW620, SW480, LOVO and HCT116, the expression of TBX5 in HT29 cells was the weakest. After transfection, the expression of TBX5 in transfection group was significantly higher than those in control group and blank group ( P=0.043 and P<0.001). Cell viability in transfection group was significantly lower than those in control group and blank group (both P<0.001). The ratio of cells in G 0/G 1 phase was increased ( P=0.009), while in G 2/M phase was decreased ( P<0.001). Cells′ abilities of migration and invasion in transfection group were also significantly decreased (both P<0.001). Overexpression of TBX5 downregulated the expressions of PCNA, MMP-2 and MMP-7, while upregulated the expressions of p21, p16, p27 ( P<0.05 for all). TBX5 had marginal effect on the expression of TIMP-1 ( P>0.05). Conclusions:Downregulation of TBX5 is a marker of poor prognosis in patients with colorectal cancer. TBX5 may inhibit the progression of colorectal cancer by inhibiting proliferation, invasion and metastasis related genes.