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1.
Cancer Research and Clinic ; (6): 191-198, 2024.
Article in Chinese | WPRIM | ID: wpr-1030434

ABSTRACT

Objective:To explore the expression of long non-coding RNA (lncRNA) RP13-349O20.2 in cervical cancer tissues and its impact on the migration, invasion abilities and chemotherapy sensitivity of cervical cancer cells in vitro and the possible mechanisms.Methods:The GEPIA.CANCER website (the data was updated in June 2023) was used to analyze the relationship between the expression level of RP13-349O20.2 and the overall survival of 253 cervical cancer patients. From January 2020 to August 2022, cancer tissues and paracancerous tissues (>2 cm from the tumor edge) from 40 cervical cancer patients in the Affiliated Tengzhou Central People's Hospital of Xuzhou Medical University were retrospectively collected. Human normal cervical epithelial cells H8 and human cervical cancer cell lines HCC94, C33A, Hela, HCC1106 and SiHa were used for cell experiments in vitro. Real-time fluorescence polymerase chain reaction (qRT-PCR) was used to detect the relative expression of RP13-349O20.2 in cervical cancer tissues, paracancerous tissues and each cell line. The C33A cells with the highest relative expression level of RP13-349O20.2 were transfected with small interfering RNA (siRNA) of RP13-349O20.2 and siRNA of its negative control sequence, and they were si-RP13-349O20.2 group and si-Con group, respectively. The scratch healing assay was used to detect the migration ability of C33A cells in the two groups, the Transwell assay was used to detect the invasion ability of C33A cells, and the CCK-8 method was used to detect the sensitivity of C33A cells to 5-fluorouracil. The absorbance value indicated the cell proliferation ability, the lower the absorbance value, the weaker the proliferation ability, the more sensitive to the drug. Dual-luciferase reporter gene assay was used to verify the targeting relationship between RP13-349O20.2 and miRNA-493-5p (miR-493-5p), miR-493-5p and Nectin-4. qRT-PCR was used to detect the relative expression of miR-493-5p and Nectin-4 mRNA in two groups of C33A cells, and Western blotting was used to detect the expressions of Nectin-4 protein and PI3K-AKT signaling pathway proteins in two groups of cells.Results:Analysis based on data from GEPIA.CANCER website shows that patients with low expression of RP13-349O20.2 had better overall survival than patients with high expression ( P < 0.01). The relative expression levels of RP13-349O20.2 in cervical cancer tissues and paracancerous tissues of 40 patients were 4.04±0.32 and 1.18±0.14, and the difference was statistically significant ( t = 8.29, P < 0.01). Compared with H8 cells, the expressions of RP13-349O20.2 in human cervical cancer cell lines HCC94, C33A, Hela, HCC1106 and SiHa were higher (all P < 0.01). The relative expression levels of RP13-349O20.2 in C33A cells in the si-Con group and si-RP13-349O20.2 group were 7.30±0.30 and 1.01±0.27, and the difference was statistically significant ( t = 15.62, P < 0.01). The scratch healing rates of C33A cells in the si-Con group and si-RP13-349O20.2 group were (32±9)% and (75±6)% ( t = 3.97, P < 0.01), and the numbers of invasive cells were (106±12) cells and (36±8) cells ( t = 4.79, P < 0.01). After the action of 5, 10, 20, 40 and 80 μmol/L 5-fluorouracil for 24 h, the absorbance value of C33A cells in the si-RP13-349O20.2 group was lower than that in the si-Con group. Dual-luciferase reporter gene assay confirmed that there was a targeting relationship between P13-349O20.2 and miR-493-5p ( P < 0.01), and there was a targeting relationship between miR-493-5p and Nectin-4 ( P < 0.01) . The relative expression levels of miR-493-5p in C33A cells in the si-Con group and si-RP13-349O20.2 group was 1.02±0.13 and 5.48±0.85 ( t = 5.21, P < 0.01). The relative expression levels of Nectin-4 mRNA were 5.65±0.33 and 0.99±0.34 ( t = 9.87, P < 0.01). The expression of Nectin-4 protein in C33A cells in the si-RP13-349O20.2 group was lower than that in the si-Con group ( t = 9.21, P = 0.001), and the expressions of PI3K-AKT signaling pathway proteins p-STAT3, p-PI3K, p-AKT and p-mTOR were lower than those in the si-Con group (all P < 0.01). Conclusions:The level of RP13-349O20.2 in cervical cancer tissues is high, and its high expression may indicate the poor prognosis of patients. Interfering with the expression of RP13-349O20.2 in vitro can inhibit the migration and invasion abilities of cervical cancer cells and promote the sensitivity of cervical cancer cells to 5-fluorouracil. The mechanism may be related to the miR-493-5p/Nectin-4 signaling pathway and the PI3K-AKT signaling pathway.

2.
Article in Chinese | WPRIM | ID: wpr-885631

ABSTRACT

Objective:To analyze the characteristics of immune cross-reaction between herpes simplex virus type 1 (HSV-1) and HSV-2 in terms of serology and clinical protection aiming to provide data for the control and prevention of diseases caused by the two viruses.Methods:An attenuated HSV-1 strain M3 was used to immunize BALB/c mice. Specific immune responses indicated by the production of neutralizing antibodies were detected. Wild-type HSV-1 and HSV-2 strains were respectively used to infect the mice through different ways 28 d after the immunization to observe the protective immunity in the M3-immunized mice against HSV-1/2 infection.Results:M3 strain could not induce specific neutralizing antibodies against HSV-2. Therefore, viral loads in tissues of the immunized mice increased significantly following different modes of HSV-2 exposure. However, no obvious abnormal clinical manifestations were found and the histopathological damage was only slight inflammatory reaction. In contrast, HSV-1-specific neutralizing antibodies were elicited in the M3-immunizaed mice with significant protective effects against HSV-1 infection.Conclusions:The immune response induced by attenuated HSV-1 strain M3 in mice exhibited immune-protective effects characterized by production of neutralizing antibodies and inhibition of virus proliferation in vivo against wild-type HSV-1 infection. For HSV-2, instead of neutralizing virus in form of antibodies, it featured by more of clinical cross-immunoprotective abilities to control virus growth.

3.
Article in Chinese | WPRIM | ID: wpr-756225

ABSTRACT

Objective To observe and analyze the pathological changes in BALB/c mice infected with herpes simplex virus typeⅡ (HSV-2) through nasal and genital inoculation. Methods Six-week old female BALB/c mice were divided into two groups, experimental and control groups. In the experimental group, the mice were infected with HSV-2 (104 CCID50/20μl per mouse) through nasal and genital tract in-oculation. Accordingly, the mice in the control group were injected with equal volume of PBS. Tissue speci-mens were collected from lung, nervous system and reproductive system for pathological analysis and viral load detection at different time points after infection. Lat gene expression in mouse trigeminal and sacral gan-glia was detected through in situ hybridization. In addition, the proliferation of viruses isolated form trigemi-nal and sacral ganglia of the infected mice was observed in vitro. Results Weight loss and histopathological lesions were observed in the mice of the experimental group 6 d after infection. Major pathological changes in the HSV-2-infected mice through nasal tract inoculation involved the lung and central nervous system( CNS) , including alveolar wall congestion, cerebrovascular cuff response and lymphocyte infiltration. How-ever, the major lesions in the infected mice through genital tract inoculation were found in the reproductive ducts, such as sacral ganglion necrosis, eosinophilia in the vagina and uterus, and ovarian congestion. Re-sults of the viral load detection in tissues and organs of the infected mice were consistent with the pathological changes. The mice infected through nasal tract inoculation had significantly higher viral loads in the nerves and lungs than those by genital tract inoculation, but lower viral loads in the genital tracts and sacral ganglia. Positive expression of lat gene at mRNA level was detected in the trigeminal and sacral ganglia of mice with HSV-2 latency 28 d after infection. In addition, both of the tissue fragments from trigeminal and sacral ganglia had cytopathic effects ( CPEs) on Vero cells. Enhanced expression of lat gene at mRNA level and much severer CPEs were induced by genital tract inoculation than by nasal tract inoculation. Conclu-sions HSV-2 could infect and cause histopathological damages in BALB/c mice through both nasal and genital tracts. In addition, the locations of the pathological lesions were closely related to the mode of infection.

4.
Article in Chinese | WPRIM | ID: wpr-513563

ABSTRACT

Objective To provide a comprehensive reference index for different mouse models of herpes simplex virus 1 (HSV-1) infection by investigating the related clinical manifestations, pathological features and characteristics of viral distribution in tissues and organs of BALB/c mice infected with different HSV-1 strains by using different strategies.Methods Acute infection models were established by challenging BALB/c mice at age three or six weeks with HSV-1 17+ and McKrae strains via intranasal and corneal administrations.Correspondingly, chronic infection models were established with BALB/c mice through subcutaneous and foot pad injections.Results Although all experimental mice showed trichiasis and roachback, there were differences in weight and fatality rate among different groups.Results of the quantitative PCR detection indicated that the proliferation of HSV-1 in the nervous tissues (brain, spinal cord, trigeminal ganglion) varied among different groups.The pathological examination indicated that in the acute infection groups, significant pathological changes only occurred in the brain tissues, while in the chronic infection groups, pathological injuries only occurred in the trigeminal ganglia.Although a key index latency-associated transcript (LAT) was not detected in the trigeminal nerve tissues of mice in the chronic infection groups, co-culturing the tissues with Vero cells resulted in infectious lesions in the cells.Conclusion This study indicates that there are significant differences in weight and fatality rate among different BALB/c mouse models of HSV-1 infection.Varied replication dynamics of HSV-1 were observed in different tissues or organs of the BALB/c mice in different groups.Therefore, different indexes should be adopted to evaluate different HSV-1 infection models.

5.
Article in Chinese | WPRIM | ID: wpr-486639

ABSTRACT

Objective To express and purify the recombinant UL7 protein of herpes simplex virus 1 (HSV-1), to prepare the corresponding UL7-specific polyclonal antibody and to preliminarily analyze the expression of UL7 protein during the proliferation of HSV-1. Methods The UL7 gene was amplified by PCR and then cloned into the pGEX-5X-1 vector for expression of UL7 protein in the prokaryotic expression system. The constructed expression plasmid, pGEX-5X-1-UL7, was transformed into E. coli BL21 (DE3) to induce the expression of UL7 protein by IPTG. The purified GST-UL7 fusion protein was used as antigen to inject the ICR mouse for the preparation of polyclonal antibody specific for UL7 protein. The titer and speci-ficity of the polyclonal antibody were analyzed by using indirect ELISA and Western blot assay, respectively. The UL7 protein-specific polyclonal antibody was used to detect the expression of UL7 protein at different time points after infecting Vero cells with HSV-1. Results The GST-UL7 fusion protein was efficiently ex-pressed in E. coli BL21 (DE3). The UL7 protein-specific polyclonal antibody was prepared with high titer (1 ∶ 105) and high specificity as indicated by the indirect ELISA and Western blot assay. The expression of UL7 protein was detected at different time points after infecting Vero cells with HSV-1. Conclusion The GST-UL7 fusion protein was obtained successfully and the UL7 protein-specific polyclonal antibody was pre-pared. Accompany with the proliferation of HSV-1, the expression of UL7 protein was detected at different time points by using the polyclonal antibody.

6.
Article in Chinese | WPRIM | ID: wpr-492789

ABSTRACT

The viral genome editing technologies have been shown to largely contribute to the stud-ies of viral gene functions and to the treatment of infectious diseases. With the development of genetic engi-neering technologies, a variety of viral genome mutation technologies have been established. The homologous recombination is traditionally used to edit the viral genome, but the low frequency of homologous recombina-tion limits its application. Large genome DNA viruses including herpes simplex virus 1 can be edited by bac-terial artificial chromosome system. However, the cloning of viral genome to bacterial artificial chromosome plasmid is both laborious and time-consuming, and parts of the plasmid genes or drug selection markers may remain in the genome of virus and then affect the function of virus. Fortunately, the CRISPR-cas9 system which was discovered in 2013 can be used to edit the viral genome efficiently and accurately. Furthermore, the experiment is simple and will not leave any trail of the viral genome when the genetic mutation is per-formed. Here, we briefly review the application of homologous recombination, bacterial artificial chromosome system and CRISPR-cas9 system in researches on herpes simplex virus 1.

7.
Article in Chinese | WPRIM | ID: wpr-464028

ABSTRACT

Objective To investigate the epidemic patterns and the characteristics of influenza in Chi-na through a Meta-analysis based on the studies published in domestic literatures.Methods Related articles published during 2005 to 2012 were screened out from domestic databases and analyzed through a Meta-analysis with Review Manager 5.0 software.Results Twenty-two articles covering 957 901 patients with influenza-like-illness (ILI) and 148 233 pathogen samples were screened out according to the inclusion criteria.No significant difference with the ILI diagnosis rate was found between subjects at age 0-4 years and those at age 15-59 years. Higher ILI diagnosis rates were observed in those two groups as compared with subjects elder than 60 years old. Most of the pathogen samples were carried by subjects aged 25-59 years.More influenza virus strains were isola-ted in 2009 as compared with those of the seven other years (OR=2.25, 95%CI=1.27-3.70).There was sta-tistical difference between the numbers of influenza A H1N1 and seasonal influenza A strains (OR=2.25, 95%CI=1.30-3.91) .Significant difference was also observed between the numbers of influenza A and influenza B strains (OR=4.05, 95%CI=2.53-6.47).Conclusion There was significant difference with the diagnosis rate between subjects aged 0-4 years and those aged≥60 years.More attention should be paid to people at high risk of infection (0-4 years old and≥60 years old) and those at 25-29 years with high mobility and social inter-course for the timely prevention and control of pandemic influenza.The detection rate of influenza virus strains was increased during the outbreak of novel influenza A H1N1 infection in 2009.After that outbreak, the detec-tion rate of novel influenza A H1N1 strains was 2.25 times the rate of seasonal influenza strains.The detection rate of influenza A was 4.05 times the rate of influenza B virus strains.Therefore, it is necessary to strengthen the surveillance for influenza A virus and other epidemic influenza virus strains.

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