miRNA-338-3p suppresses cell growth of human colorectal carcinoma by targeting smoothened

AIM:To investigate the regulative effect of miRNA-3383p(miR-338-3p) on cell growth in colorectal carcinoma(CRC).METHODS:The lentiviral vector pLV-THM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed.The recombinant viral vector encoding the pre-miR338-3p or miR-338-3p-inhibitor and the two packaging plasmids psPAX2 and pMD2.G were cotransfected into human embryonic kidney 293T cells to package lentivirus.The supernatant containing the lentivirus particles was harvested to determine the viral titer,and this supernatant was then used to transduce CRCderived cell line,SW-620.Flow cytometry was utilized for sorting the green fluorescent protein(GFP) + cells to establish the SW-620 cell line stably expressing premiR-338-3p or miR-338-3p-inhibitor.Moreover,the expression of miR-338-3p was determined by real-time reverse transcriptase polymerase chain reaction,andWestern blotting was used to detect the expression of the smoothened(SMO,the possible target of miR-3383p) protein in SW-620 cells.Furthermore,the status of CRC cell proliferation and apoptosis were detected by 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay and flow cytometry,respectively.RESULTS:Restriction enzyme digestion and DNA sequencing demonstrated that the lentiviral vector pLVTHM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed successfully.GFP was expressed after the SW-620 cells were transduced by the lentivirus.Expression of miR-338-3p in SW-620 cells transduced with the lentivirus pLV-THM-miR-338-3p was significantly increased(relative expression 3.91 ± 0.51 vs 2.36 ± 0.44,P < 0.01).Furthermore,overexpression of miR-338-3p inhibited the expression of SMO protein in SW-620 cells,which showed obviously suppressed proliferation ability [cellular proliferation inhibition rate(CPIR) 61.9% ± 5.2% vs 41.6% ± 4.8%,P < 0.01].Expression of miR-338-3p in SW-620 cells transduced with the lentivirus pLV-THM-miR-338-3p-inhibitor was significantly decreased(relative expression 0.92 ± 0.29 vs 2.36 ± 0.44,P < 0.01).Moreover,the downregulated expression of miR-338-3p caused upregulated expression of the SMO protein in SW-620 cells,which showed significantly enhanced proliferation ability(CPIR 19.2% ± 3.8% vs 41.6% ± 4.8%,P < 0.01).However,anti-SMO-siRNA largely,but not completely,reversed the effects induced by blockage of miR-338-3p,suggesting that the regulative effect of miR-338-3p on CRC cell growth was indeed mediated by SMO.CONCLUSION:miR-338-3p could suppress CRC growth by inhibiting SMO protein expression.

Anti-miRNA-221 sensitizes human colorectal carcinoma cells to radiation by upregulating PTEN

AIM:To investigate the regulative effect of miRNA(miR)-221 on colorectal carcinoma(CRC)cell radiosensitivity and the underlying mechanisms.METHODS:A human CRC-derived cell line was cultured conventionally and exposed to different doses of X-rays(0,2,4,6 and 8 Gy).The total RNA and protein of the cells were extracted 24 h after irradiation,and the alteration of miR-221 and phosphatase and tensin homolog deleted on chromosome 10(PTEN)gene mRNA expression was detected by real-time reverse transcriptase polymerase chain reaction(PCR).The protein alteration of PTEN in the cells was detected by Western blotting.Caco2 cells were pretreated with or without anti-PTEN-siRNA prior to the addition of premiR-221 or anti-miR-221 using Lipofectamine 2000.Colony formation assay and flow cytometry analysis were used to measure the surviving cell fraction and the sensitizing enhancement ratio after irradiation.Ad-ditionally,PTEN 3′-untranslated region fragment was PCR amplified and inserted into a luciferase reporter plasmid.The luciferase reporter plasmid construct was then transfected into CRC cells together with premiR-221 or anti-miR-221,and the luciferase activity in the transfected cells was detected.RESULTS:The X-ray radiation dose had a significant effect on the expression of miR-221 and PTEN protein in human Caco2 cells in a dose-dependent manner.The miR-221 expression level improved gradually with the increase in irradiation dose,while the PTEN protein expression level reduced gradually.miR-221 expression was significantly reduced in the anti-miR-221 group compared with the pre-miR-221 and negative control groups(P<0.01).Anti-miR-221 upregulated expression of PTEN protein and enhanced the radiosensitivity of Caco2 cells(P<0.01).Moreover,the inhibitory effect was dramatically abolished by pretreatment with anti-PTEN-siRNA,suggesting that the enhancement of radiosensitivity was indeed mediated by PTEN.A significant increase of luciferase activity was detected in CRC cells that were cotransfected with the luciferase reporter plasmid construct and anti-miR-221(P<0.01).CONCLUSION:Anti-miR-221 can enhance the radiosensitivity of CRC cells by upregulating PTEN.

Techniques and feasibility of laparoscopic extended right hemicolectomy with D3 lymphadenectomy

AIM: To illustrate the critical techniques and feasibility of laparoscopic extended right hemicolectomy (LERH), according to our previous experience.

Real-time in vivo distal margin selection using confocal laser endomicroscopy in transanal total mesorectal excision for rectal cancer

BACKGROUND Transanal total mesorectal excision(TaTME)allows patients with ultralow rectal cancer to be treated with sphincter-saving surgery.However,accurate delineation of the distal resection margin(DRM),which is essential to achieve R0 resection for low rectal cancer in TaTME,is technically demanding.AIM To assess the feasibility of optical biopsy using probe-based confocal laser endomicroscopy(pCLE)to select the DRM during TaTME for low rectal cancer.METHODS A total of 43 consecutive patients who were diagnosed with low rectal cancer and scheduled for TaTME were prospectively enrolled from January 2019 to January 2021.pCLE was used to determine the distal edge of the tumor as well as the DRM during surgery.The final pathological report was used as the gold standard.The diagnostic accuracy of pCLE examination was calculated.RESULTS A total of 86 pCLE videos of 43 patients were included in the analyses.The sensitivity,specificity and accuracy of real-time pCLE examination were 90.00%[95%confidence interval(CI):76.34%-97.21%],86.96%(95%CI:73.74%-95.06%)and 88.37%(95%CI:79.65%-94.28%),respectively.The accuracy of blinded pCLE reinterpretation was 86.05%(95%CI:76.89%-92.58%).Furthermore,our results show satisfactory interobserver agreement(κ=0.767,standard error=0.069)for the detection of cancer tissue by pCLE.There were no positive DRMs(≤1 mm)in this study.The median DRM was 7 mm[interquartile range(IQR)=5-10 mm].The median Wexner score was 5(IQR=3-6)at 6 mo after stoma closure.CONCLUSION Real-time in vivo pCLE examination is feasible and safe for selecting the DRM during TaTME for low rectal cancer(clinical trial registration number:NCT04016948).

Changes in the humoral immunity response in SARS-CoV-2 convalescent patients over 8 months

Many countries around the world have seen a sharp rise in COVID-19 cases since the beginning of October due to the second wave of the pandemic.A decline in the antibody response to severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),which was reported exclusively in the early month,increases the risk of reinfection for convalescent individuals.There is a current need to follow the maintenance of specific antibodies against SARS-CoV-2.

The clinical and immunological features of pediatric COVID-19 patients in China

In December 2019,the corona virus disease 2019(COVID-19)caused by novel coronavirus(SARS-CoV-2)emerged in Wuhan,China and rapidly spread worldwide.Few information on clinical features and immunological profile of COVID-19 in paediatrics.The clinical features and treatment outcomes of twelve paediatric patients confirmed as COVID-19 were analyzed.The immunological features of children patients was investigated and compared with twenty adult patients.The median age was 14.5-years(range from 0.64 to 17),and six of the patients were male.The average incubation period was 8 days.Clinically,cough(9/12,75%)and fever(7/12,58.3%)were the most common symptoms.Four patients(33.3%)had diarrhea during the disease.As to the immune profile,children had higher amount of total T cell,CD8t T cell and B cell but lower CRP levels than adults(P<0.05).Ground-glass opacity(GGO)and local patchy shadowing were the typical radiological findings on chest CT scan.All patients received antiviral and symptomatic treatment and the symptom relieved in 3e4 days after admitted to hospital.The paediatric patients showed mild symptom but with longer incubation period.Children infected with SARS-CoV-2 had different immune profile with higher T cell amount and low inflammatory factors level,which might ascribed to the mild clinical symptom.We advise that nucleic acid test or examination of serum IgM/IgG antibodies against SARS-CoV-2 should be taken for children with exposure history regardless of clinical symptom.

Survival outcomes following laparoscopic vs open surgery for non-metastatic rectal cancer:a two-center cohort study with propensity score matching

Background:Laparoscopic surgery for rectal cancer is commonly performed in China.However,compared with open surgery,the effectiveness of laparoscopic surgery,especially the long-term survival,has not been sufficiently proved.Methods:Data of eligible patients with non-metastatic rectal cancer at Nanfang Hospital of Southern Medical University and Guangdong Provincial Hospital of Chinese Medicine between 2012 and 2014 were retrospectively reviewed.Long-termsurvival outcomes and short-termsurgical safety were analysed with propensity score matching between groups.Results:Of 430 cases collated from two institutes,103 matched pairs were analysed after propensity score matching.The estimated blood loss during laparoscopic surgerywas significantly less than that during open surgery(P=0.019)and the operative time and hospital staywere shorter in the laparoscopic group(both P<0.001).The post-operative complications ratewas 9.7%in the laparoscopic group and 10.7%in the open group(P=0.818).No significant differencewas observed between the laparoscopic group and the open group in the 5-year overall survival rate(75.7%vs 80.6%,P=0.346),5-year relapse-free survival rate(74.8%vs 76.7%,P=0.527),or 5-year cancer-specific survival rate(79.6%vs 87.4%,P=0.219).An elevated carcinoembryonic antigen,<12 harvested lymph nodes,and perineural invasion were independent prognostic factors affecting overall survival and relapse-free survival.Conclusions:Our findings suggest that open surgery should still be the priority recommendation,but laparoscopic surgery is also an acceptable treatment for non-metastatic rectal cancer.

Correction: Changes in the humoral immunity response in SARS-CoV-2 convalescent patients over 8 months

Correction to:Cellular&Molecular Immunology https:/doi.org/10.1038/s41423-020-00605-4,published online 08 January 2021 The licence information was missing from this article and should havebeen CC-BY.The original article has been corrected.