Ageing threatens sustainability of smallholder farming in China.

Rapid demographic ageing substantially affects socioeconomic development1-4 and presents considerable challenges for food security and agricultural sustainability5-8, which have so far not been well understood. Here, by using data from more than 15,000 rural households with crops but no livestock across China, we show that rural population ageing reduced farm size by 4% through transferring cropland ownership and land abandonment (approximately 4 million hectares) in 2019, taking the population age structure in 1990 as a benchmark. These changes led to a reduction of agricultural inputs, including chemical fertilizers, manure and machinery, which decreased agricultural output and labour productivity by 5% and 4%, respectively, further lowering farmers' income by 15%. Meanwhile, fertilizer loss increased by 3%, resulting in higher pollutant emissions to the environment. In new farming models, such as cooperative farming, farms tend to be larger and operated by younger farmers, who have a higher average education level, hence improving agricultural management. By encouraging the transition to new farming models, the negative consequences of ageing can be reversed. Agricultural input, farm size and farmer's income would grow by approximately 14%, 20% and 26%, respectively, and fertilizer loss would reduce by 4% in 2100 compared with that in 2020. This suggests that management of rural ageing will contribute to a comprehensive transformation of smallholder farming to sustainable agriculture in China.

Cost-effective mitigation of nitrogen pollution from global croplands.

Cropland is a main source of global nitrogen pollution1,2. Mitigating nitrogen pollution from global croplands is a grand challenge because of the nature of non-point-source pollution from millions of farms and the constraints to implementing pollution-reduction measures, such as lack of financial resources and limited nitrogen-management knowledge of farmers3. Here we synthesize 1,521 field observations worldwide and identify 11 key measures that can reduce nitrogen losses from croplands to air and water by 30-70%, while increasing crop yield and nitrogen use efficiency (NUE) by 10-30% and 10-80%, respectively. Overall, adoption of this package of measures on global croplands would allow the production of 17 ± 3 Tg (1012 g) more crop nitrogen (20% increase) with 22 ± 4 Tg less nitrogen fertilizer used (21% reduction) and 26 ± 5 Tg less nitrogen pollution (32% reduction) to the environment for the considered base year of 2015. These changes could gain a global societal benefit of 476 ± 123 billion US dollars (USD) for food supply, human health, ecosystems and climate, with net mitigation costs of only 19 ± 5 billion USD, of which 15 ± 4 billion USD fertilizer saving offsets 44% of the gross mitigation cost. To mitigate nitrogen pollution from croplands in the future, innovative policies such as a nitrogen credit system (NCS) could be implemented to select, incentivize and, where necessary, subsidize the adoption of these measures.

m6A methyltransferase METTL3 inhibits endometriosis by regulating alternative splicing of MIR17HG.

In brief: Inflammation and abnormal immune response are the key processes in the development of endometriosis (EMs), and m6A modification can regulate the inflammatory response. This study reveals that METTL3-mediated N6-methyladenosine (m6A) modification plays an important role in EMs. Abstract: m6A modification is largely involved in the development of different diseases. This study intended to investigate the implication of m6A methylation transferase methyltransferase like 3 (METTL3) in EMs. EMs- and m6A-related mRNAs and long non-coding RNAs were identified through bioinformatics analysis. Next, EM mouse models established by endometrial autotransplantation and mouse endometrial stromal cell (mESC) were prepared and treated with oe-METTL3 or sh-MIR17HG for pinpointing the in vitro and in vivo effects of METTL3 on EMs in relation to MIR17HG through the determination of mESC biological processes as well as estradiol (E2) and related lipoprotein levels. We demonstrated that METTL3 and MIR17HG were downregulated in the EMs mouse model. Overexpression of METTL3 suppressed the proliferation, migration, and invasion of mESCs. In addition, METTL3 enhanced the expression of MIR17HG through m6A modification. Moreover, METTL3 could inhibit the E2 level and alter related lipoprotein levels in EMs mice through the upregulation of MIR17HG. The present study highlighted that the m6A methylation transferase METTL3 prevents EMs progression by upregulating MIR17HG expression.

Identification and validation of the high expression of pseudogene TCAM1P in cervical cancer via integrated bioinformatics analysis.

BACKGROUND: HPV as the main cause of cervical cancer has long been revealed, but the detailed mechanism has not yet been elucidated. The role of testis/cancer antigen in cervical cancer has been revealed. However, there are no reports about the statement of testis/cancer-specific non-coding RNA. In this study, we first proposed TCAM1P as a testis/cancer-specific pseudogene, and used a series of experimental data to verify its relationship with HPV, and analyzed its diagnosis value of high-grade cervical lesions and the mechanism of their high expression in cervical cancer. This provides a new direction for the prevention and treatment of cervical cancer. METHODS: The specific expression of pseudogenes in each tissue was calculated by "TAU" formula. ROC curve was used to judge the diagnosed value of TCAM1P for high-grade lesions. The proliferation ability of cells was measured by CCK8. The expression of TCAM1P, HPV E6/E7 were detected by qRT-PCR. The binding for RBPs on TCAM1P was predicted by starbase v2.0 database, then RIP assay was used to verify. Besides, Gene Ontology (GO) and KEGG enrichment analysis were performed with "clusterprofiler" R package. RESULTS: TCAM1P was specifically high-expressed in normal testicular tissue and cervical cancer. Interesting, with the severity of cervical lesions increased, the expression of TCAM1P increased, and TCAM1P could effectively diagnose high-grade cervical lesions. Besides, the expression of TCAM1P was HPV dependent, with highest expression in HPV-positive cervical cancer tissues. Furthermore, RIP assay showed that EIF4A3 regulated the expression of TCAM1P through binding with it. CCK8 assay showed that TCAM1P promoted the proliferation and the Gene ontology (GO) and KEGG Pathway enrichment analysis same suggested that TCAM1P is involved in multiple ways in cell proliferation including Cell cycle, DNA replication and etc. CONCLUSIONS: In this study, we firstly proposed that TCAM1P is cancer/testis pseudogene and is regulated by HPV E6/E7 and EIF4A3. TCAM1P promotes the proliferation of cervical cancer cells and acts as promoter in cervical cancer. Otherwise, TCAM1P promote proliferation through regulating cell cycle and DNA replication, but more evidence needs to be provided to reveal the mechanism by which TCAM1P plays a role in cervical cancer.

Ammonia Emissions from Croplands Decrease with Farm Size in China.

Farm size affects nitrogen fertilizer input and agricultural practices, which are key determinants of ammonia (NH3) emissions from croplands. However, the degree to which NH3 emissions are associated with changes in farm size is not well understood yet despite its crucial role in achieving agricultural sustainability in China, where agricultural production is still dominated by smallholder farms. Here we provide a first analysis of the relationship between farm size and NH3 emissions based on 863 000 surveys conducted in 2017 across China. Results show that NH3 emissions (kg ha-1) on average decrease by 0.07% for each 1% increase in average farm size. This change occurs mainly due to a reduction in nitrogen fertilizer use and the introduction of more efficient fertilization practices. The largest reduction in NH3 emissions is found in maize, with less pronounced changes in rice cultivation, and none for wheat production. Overall lower NH3 emissions factors can be observed in the north of China with increasing farm size, especially in the northeast, the opposite pattern was found in the south. National total NH3 emissions could be approximately halved (1.5 Tg) in a scenario favoring a conversion to large-scale farming systems. This substantial reduction potential highlights the potential of such a transition to reduce NH3 emissions, including benefits from a socioeconomic point of view as well as for improving air quality.

Genetic diagnosis of common fetal renal abnormalities detected on prenatal ultrasound.

OBJECTIVES: This retrospective study aimed to investigate the correlations between phenotypes of fetal renal abnormalities on prenatal ultrasound and genetic aetiologies detected using chromosomal microarray analysis (CMA) and whole-exome sequencing (WES). METHODS: Fetuses with renal abnormalities were subjected to CMA and were further analysed by WES when CMA-negative. The detection rates for chromosomal abnormalities and monogenic variants among different types of isolated renal abnormalities and those with extrarenal abnormalities (non-isolated cases) were determined and compared. RESULTS: CMA detected chromosomal abnormalities in 78 of 577 fetuses (13.52%). WES detected monogenic variants in 31 of 160 fetuses (19.38%) that had non-diagnostic CMA results. In cases of isolated hyperechogenic kidney, polycystic kidney disease, and multicystic dysplastic kidney, the detection rates of copy number variants (CNVs) by CMA and monogenic variants by WES were not significantly different (p > 0.05). However, monogenic variants were more frequently detected than CNVs when kidney abnormalities were accompanied by reduced amniotic fluid (p < 0.05). Other renal abnormalities identified on prenatal ultrasound had different detection rates. CONCLUSIONS: Our findings contribute to the overall knowledge of genetic variants associated with prenatally identified renal anomalies and may aid in decision making regarding prenatal genetic testing options for affected pregnancies.

RPL34-AS1-induced RPL34 inhibits cervical cancer cell tumorigenesis via the MDM2-P53 pathway.

Ribosomal proteins (RPs) are important components of ribosomes and related to the occurrence and development of tumors. However, little is known about the effects of the RP network on cervical cancer (CC). In this study, we screened differentially expressed RPL34 in CC by high-throughput quantitative proteome assay. We found that RPL34 acted as a tumor suppressor and was downregulated in CC and inhibited the proliferation, migration, and invasion abilities of CC cells. Next, we verified that RPL34 regulated the CC through the MDM2-P53 pathway by using Act D medicine, MDM2 inhibitor, and a series of western blotting（WB）assays. Moreover, an antisense lncRNA, RPL34-AS1, regulated the expression of RPL34 and participated in the tumorigenesis of CC. RPL34 can reverse the effect of RPL34-AS1 in CC cells. Finally, by RNA-binding protein immunoprecipitation (RIP) assay we found that eukaryotic initiation factor 4A3 (EIF4A3), which binds to RPL34-AS1, regulated RPL34-AS1 expression in CC. Therefore, our findings indicate that RPL34-AS1-induced RPL34 inhibits CC cell proliferation, invasion, and metastasis through modulation of the MDM2-P53 signaling pathway, which provides a meaningful target for the early diagnosis and treatment of CC.

Profiling and analysis of multiple constituents in Crataegi Fructus before and after processing by ultrahigh-performance liquid chromatography quadrupole time-of-flight mass spectrometry.

RATIONALE: Crataegi Fructus (CF) is one of the most commonly used herbal medicines with a long history of clinical applications. CF is often processed to minimize gastric membrane irritation, although differently processed products can have different biological effects. The purpose of this study was to comprehensively identify the chemical composition of CF, determine the changes caused by processing, and elucidate the active constituents causing the clinical effects. This study aimed to define a theoretical basis for intensive mechanistic studies of CF processing and its reasonable clinical applications. METHODS: An optimized ultrahigh-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC/QqTOFMS) method in positive and negative ion modes, coupled with multivariate statistical analyses, was developed for the identification and analysis of chemical components in raw and processed products of CF. RESULTS: A total of 87 compounds were identified, including 61 marker compounds that were found to be primary contributors to the significant differences (p < 0.01) between raw and processed products using principal component analysis, t-test, and Venn analysis. The conversion mechanism for a subset of the changed compounds was inferred by analyzing 25 unique differential components between the raw and processed CF. CONCLUSIONS: A rapid and efficient analytical method for identifying the chemical components in CF before and after processing was successfully established. We show how the changes in the chemical constituents in processed CF could be investigated using multivariate statistical analysis methods, and thus facilitate understanding of the processing mechanism of CF.

Analysis of the chemical composition changes of Gardeniae Fructus before and after processing based on ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry.

Gardeniae Fructus, the dry fruit of Gardenia jasminoides Ellis, has been widely used for the treatment of different diseases. Although four types of processed Gardeniae Fructus products, characterized by differing effects, are available for clinical use, little is known regarding the respective processing mechanisms. In this study, ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry combined with multivariate statistical analysis was applied to characterize the chemical profiles of the differently processed Gardeniae Fructus products and to determine differences in their chemical compositions, thereby enabling us to identify those active compounds associated with the observed clinical effects. A total of 125 compounds were accordingly identified, among which, 56 were established as primary contributors to the significant differences (P < 0.01) between crude and processed Gardeniae Fructus, based on t-test analysis. Furthermore, the potential mechanisms underlying the chemical transformations that occurred during processing were discussed. The findings of this study may not only contribute to the more effective quality control of Gardeniae Fructus but also provide basic information for elucidating the mechanisms underlying the changes in chemical constituents in response to processing, and provide a basis for further investigations of Gardeniae Fructus processing mechanisms.

A new method for detecting Na+ , K+ -ATPase activity by ICP-MS: Quantitative analysis on the inhibitory effect of rhein on Na+ , K+ -ATPase activity by ICP-MS in HCT116 cells.

Rhein is an active component from Chinese herbal medicine. It can cause diarrhea by inhibiting Na+ , K+ -ATPase activity on intestinal epithelial cells, thus decreasing the re-absorption of Na+ from intestinal tract to blood. However, when this Na+ , K+ -ATPase inhibition was quantitated by a colorimetric method that measures ATPase-catalyzed release of inorganic phosphorus, the data obtained were inconsistent and showed great variation. We developed a novel method using inductively coupled plasma mass spectrometry (ICP-MS) to quantitate the amount of intracellular Rb+ . This method largely mimics the 86 RbCl tracer flux assay, but it uses non-radioactive RbCl as a flux substrate. The results demonstrated that this method has better precision and accuracy than the conventional colorimetric method. More importantly, this method is free from radioactive substances, which is expected to make it safer and more convenient than the radioactive 86 RbCl tracer flux method. In conclusion, the ICP-MS method for Na+ , K+ -ATPase activity determination is novel and accurate. It can also provide a reference for studying the transport of other metal ions across membranes under biological conditions.

Williams syndrome transcription factor promotes proliferation and invasion of cervical cancer cells by regulating PI3K/Akt signaling pathway.

OBJECTIVE: This study aimed to investigate the expression of Williams Syndrome transcription factor (WSTF) in cervical cancer (CC) tissues and cells, the effect on the proliferation, migration, invasion, and the molecular mechanism of WSTF in CC cells to find a new biomarker. MATERIALS AND METHODS: The expression of WSTF in tissues was detected by real-time quantitative polymerase chain reaction (RT-qPCR) and/or immunohistochemistry. Human CC cell lines and human normal cervical epithelial cell lines were detected by RT-qPCR. Lentivirus-mediated gene transfected in Siha/CaSki cells. The transfection efficiency of lentivirus was observed by a fluorescence microscope, RT-qPCR, and western blot. After transfection, the proliferation of Siha/CaSki cells was detected by CCK-8 assay and colony formation assay. The migration and invasion of Siha/CaSki cells were detected by transwell assay and wound healing assay. Western blot assay were used to detect the expression of WSTF and PI3K/Akt-related proteins in Siha/CaSki cells. RESULTS: The expression of WSTF in CC tissues was higher than that in adjacent tissues (p < 0.05). The expression of WSTF in CC cells was higher than that in normal cervical epithelial cells (p < 0.01). Downregulation of WSTF expression could inhibit the proliferation, migration, and invasion of CC cells (p < 0.01). WSTF overexpression activates PI3K/Akt signaling pathway (p < 0.01). CONCLUSION: WSTF is highly expressed in CC tissues and cells, and downregulation of WSTF can inhibit the proliferation, invasion, and migration of CC cells by activating the PI3K/Akt signaling pathway. WSTF is a very promising new biomarker for CC.

Fertilizer overuse in Chinese smallholders due to lack of fixed inputs.

Fertilizer overuse by smallholder farmers is widespread in China, leading to significant financial losses and threatening the environment. Understanding what mechanism behind this is critical for agricultural and environmental sustainability. By using a fixed effect panel model of over 20,000 rural households in China from 1995 to 2016, we found that the low ratio of fixed inputs such as machinery and knowledge to total inputs is the key factor leading to over-fertilization in smallholder farms. Low fixed input can result in or interact with nutrient-unbalanced fertilization, low agricultural income ratio and more cash crops that further aggravate fertilizer overuse. Smallholders lack fixed inputs, then compensate by over-applying fertilizer to attempt to achieve their yield goals. Thus, improving fixed input via increasing the average farm size to 3.8 ha or advanced service rental could save not only 45% fertilizers but also increase 16% agricultural net profit, benefiting agricultural and environmental sustainability.

A Slug-dependent mechanism is responsible for tumor suppression of p53-stabilizing compound CP-31398 in p53-mutated endometrial carcinoma.

Mutation in the tumor suppressor gene p53 is the most frequent molecular defect in endometrial carcinoma (EC). Recently, CP-31398, a p53-stabilizing compound, has been indicated to possess the ability to alter the expression of non-p53 target genes in addition to p53 downstream genes in tumor cells. Herein, we explore the alternative mechanisms underlying the restoration of EC tumor suppressor function in mutant p53 by CP-31398. A p53-mutated EC cell was constructed in AN3CA cells with restored or partial loss of Slug using lentiviral vectors, followed by treatment with 25 µM CP-31398. A p53-independent mechanism of CP-31398 was confirmed by the interaction between mouse double minute 2 homolog (MDM2) and Slug AN3CA cells treated with IWR-1 (inhibitor of Wnt response 1). Furthermore, the AN3CA cells were treated with short hairpin RNA against Slug, Wnt-specific activators (LiCl) or inhibitors (XAV-939) followed by CP-31398 treatment. Moreover, AN3CA cell proliferation and apoptosis were examined. A tumorigenicity assay was conducted in nude mice. CP-31398 could promote the apoptosis of p53-mutated EC cells, while Slug reversed this effect. Slug ubiquitination was found to occur via binding of Slug to MDM2 in AN3CA cells. We found that CP-31398 increased the GSK-3ß, p-Slug, Puma, Wtp53, and Bax expressions whereas Wnt, Mtp-53, Slug, Bcl-2, and Ki-67 expressions were decreased. However, these findings were reversed following the activation of the Wnt pathway and overexpression of Slug. Finally, the in vivo experimental evidence confirmed that CP-31398 with depleted Slug suppressed tumor growth by downregulating the Slug. Collectively, CP-31398-regulated Slug downregulation represses the p53-mutated EC via the p53/Wnt/Puma pathway.

Role of CXCL12-CXCR4 axis in ovarian cancer metastasis and CXCL12-CXCR4 blockade with AMD3100 suppresses tumor cell migration and invasion in vitro.

Ovarian cancer (OC) is a lethal gynecologic tumor, which brings its mortality to the head. CXCL12 and its receptor chemokine receptor 4 ( CXCR4) have been found to be highly expressed in OC and contribute to the disease progression by affecting tumor cell proliferation and invasion. Here, in this study, we aim to explore whether the blockade of CXCL12-CXCR4 axis with AMD3100 (a selective CXCR4 antagonist) has effects on the progression of OC. On the basis of the gene expression omnibus database of OC gene expression chips, the OC differentially expressed genes were screened by microarray analysis. OC (nonmetastatic and metastatic) and normal ovarian tissues were collected to determine the expressions of CXCL12 and CXCR4. A series of AMD3100, shRNA against CXCR4, and pCNS-CXCR4 were introduced to treat CAOV3 cells with the highest CXCR4 was assessed. Cell viability, apoptosis, migration, and invasion were all evaluated. The microarray analysis screened out the differential expression of CXCL12-CXCR4 in OC. CXCL12 and CXCR4 expressions were increased in OC tissues, particularly in the metastatic OC tissues. Downregulation of CXCR4 by AMD3100 or shRNA was observed to have a critical role in inhibiting cell proliferation, migration, and invasion of the CAOV3 OC cell line while promoting cell apoptosis. Overexpressed CXCR4 brought significantly promoting effects on the proliferation and invasiveness of OC cells. These results reinforce that the blockade of CXCL12-CXCR4 axis with AMD3100 inhibits the growth of OC cells. The antitumor role of the inhibition of CXCL12-CXCR4 axis offers a preclinical validation of CXCL12-CXCR4 axis as a therapeutic target in OC.

Effects of shRNA-mediated silencing of PSMA7 on cell proliferation and vascular endothelial growth factor expression via the ubiquitin-proteasome pathway in cervical cancer.

This study aims to evaluate the effects of PSMA7 silencing on cervical cancer (CC) cell proliferation and vascular endothelial growth factor (VEGF) expression through the ubiquitin-proteasome pathway. CC tissues (n = 43) and normal tissues (n = 27) were first collected from patients. Human CC cell line (SiHa) and human normal cervical epithelial cells (H8) were obtained and classified into the normal, blank, negative control (NC), PSMA7-shRNA1, and PSMA7-shRNA2 groups, respectively. In situ hybridization was used to detect the expressions of wild-type and mutant p53 proteins. Immunofluorescence assay was carried out to test the activity of 20S proteasomes. Reverse transcription quantitative polymerase chain reaction and Western blot analysis were both performed to determine the expressions of PSMA7, ubiquitin, P27, P53, and VEGF in sample tissues and cells. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was used to analyze cell proliferation rates, and flow cytometry was used to analyze the cell cycle and the apoptotic rate. Compared with normal tissues, CC tissues showed increased expression levels of PSMA7, ubiquitin, p53, VEGF as well as increased activity of 20S proteasomes but exhibited a decrease in p27 expression. Compared with the blank and NC groups, the PSMA7-shRNA1 and PSMA7-shRNA2 groups all had decreased expression levels of PSMA7, ubiquitin, p53, and VEGF as well as decreased cell proliferation, 20S proteasomes activity, and cell number in the S phase, increased p27 expression, cell apoptosis and cell number in the G0/G1 phase. Our study demonstrated that PSMA7 silencing can suppress CC cell proliferation and VEGF expression in addition to promoting cell apoptosis through inhibiting the UPP signaling pathway.

CP-31398 inhibits the progression of cervical cancer through reversing the epithelial mesenchymal transition via the downregulation of PAX2s.

CP-31398, a styrylquinazoline, emerges from a screen for therapeutic agents that restore the wild-type DNA-binding conformation of mutant p53 to suppress tumors in vivo, but its effects on cervical cancer (CC) remain unknown. Hence, this study aimed to explore the effects CP-31398 has on the CC cells and to investigate whether it is associated with paired box 2 (PAX2) expression. CC cells were treated with different concentrations of CP-31398 (1, 2, 4, 6, 8, and 10 µg/ml) to determine the optimum concentration using fluorometric microculture cytotoxicity assay. After constructing the sh-PAX2 vector, CC cells were transfected with sh-PAX2 or treated with CP-31398. The effects of CP-31398 or PAX2 silencing on CC cell proliferation, apoptosis, invasion, and migration were evaluated. Epithelial mesenchymal transition (EMT)-related genes such as E-cadherin, vimentin, N-cadherin, snail, and twist in CC cells were detected. Tumor formation experiment in nude mice was performed to observe tumor growth. The optimum concentration of CP-31398 was 2 µg/ml. PAX2 was overexpressed in CC cells. CC cells treated with CP-31398 or treated with sh-PAX2 inhibited proliferation, invasion, and migration but promoted apoptosis with decreased PAX2 expression. The EMT process in CC cells was also reversed after treatment with CP-31398 or sh-PAX2. Moreover, the tumor formation experiment in nude mice revealed the inhibitory activity of CP-31398 in CC tumor in nude mice by suppressing PAX2. Our results provide evidence that CP-31398 could inhibit EMT and promote apoptosis of CC cells to curb CC tumor growth by downregulating PAX2.

Performance of p16/Ki67 immunostaining, HPV E6/E7 mRNA testing, and HPV DNA assay to detect high-grade cervical dysplasia in women with ASCUS.

BACKGROUND: Atypical squamous cell of undetermined significance (ASCUS) is a common cervical cytological diagnosis. At present, HPV DNA assay is used to triage these patients, but its lower specificity brings a series of problems. The purpose of this study was to evaluated the value of p16/Ki67 immunostaining, HPV E6/E7 mRNA testing in triaging women with ASCUS by comparing HPV DNA assay. METHODS: Liquid based cytology specimens were collected from 300 patients. P16/Ki67 immunocytochemistry using the CINtec® Plus Kit and HPV E6/E7 mRNA testing by QuantiVirus®HPV E6/E7 mRNA assay used the same cytology sample. Detection rates of each test were evaluated against histopathology. RESULTS: All assays yielded a high sensitivity for the detection of CIN3+ (100% (86.7-100) for HPV DNA assay, 88.0% (70.0-95.8) for HPV E6/E7 mRNA testing and 100% (86.7-100) for p16/Ki67 immunocytochemistry) and CIN2+ (98.2% (90.2-99.7) for HPV DNA assay, 87.0% (75.6-93.6) for HPV E6/E7 mRNA testing, 98.2% (90.2-99.7) for p16/Ki67 immunocytochemistry). The specificity to detect high grade dysplasia was highest for p16/Ki67 immunocytochemistry (74.2% (68.7-79.0) in CIN3+ and 82.5% (77.3-86.8) in CIN2+), followed by HPV E6/E7 mRNA testing (39.6% (34.0-45.5) in CIN3+ and 42.7% (36.7-48.9) in CIN2+) and HPV DNA assay (16.0% (12.1-20.8) in CIN3+ and 17.5% (13.2-22.7) in CIN2+). CONCLUSIONS: p16/Ki67 immunostaining and HPV E6/E7 mRNA testing, especially the former, may be promising tools in triage of ASCUS.

Prognostic and diagnostic validity of p16/Ki-67, HPV E6/E7 mRNA, and HPV DNA in women with ASCUS: a follow-up study.

BACKGROUND: We evaluated the prognostic and diagnostic ability of p16/Ki-67 immunocytochemistry, HPV E6/E7 mRNA testing and HPV DNA assay in triaging ASCUS to find a way to manage cervical lesions more effectively. METHODS: We conducted a prospective study through follow-up. The detection methods of the three factors: p16/Ki-67 immunocytochemistry conducted by using the CINtec® Plus Kit, E6/E7 mRNA testing by QuantiVirus®HPV E6/E7 mRNA assay and DNA by Hybrid Capture 2 assay. RESULTS: One hundred three women with ASCUS satisfied requirements and completed the entire follow-up process. All CIN2+ occurred in women who were mRNA positive at baseline, none in mRNA negative. 100% (6/6) patients with CIN2+ were HPV DNA assay positive, 100% (6/6) were HPV E6/E7 mRNA testing positive and 50.0% (3/6) were p16/Ki-67 immunocytochemistry positive. The risk ratio of E6/E7 mRNA test was 57.306 (95% CI 0.077-42,400.545). For endpoint of CIN2+, the sensitivity between HPV DNA assay and HPV E6/E7 mRNA testing is no statistical difference, but statistical difference exists between HPV E6/E7 mRNA testing vs. p16/Ki-67 immunocytochemistry (χ2 = 5.718, P = 0.023) and HPV DNA assay vs. p16/Ki-67 immunocytochemistry (χ2 = 5.718, P = 0.023). The specificity of E6/E7 mRNA testing, p16/Ki-67 and DNA assay in triaging ASCUS was 44.33, 75.26 and 11.34% respectively and is all statistical difference (χ2 = 26.277, P < 0.001(HPV DNA assay vs. HPV E6/E7 mRNA testing), χ2 = 19.297, P < 0.001(HPV E6/E7 mRNA testing vs. p16/Ki-67 immunocytochemistry), χ2 = 80.707, P < 0.001(HPV DNA assay vs. p16/Ki-67 immunocytochemistry). The expression level of 2097.09 copies/ml was the optimal cut-off value for HPV E6/E7 mRNA testing to diagnose CIN2+, the sensitivity and specificity was 61.1 and 68.2%. CONCLUSIONS: High expression of HPV E6/E7 mRNA could be a good candidate as a diagnostic biomarker to triage ASCUS superseding HPV DNA. p16/Ki-67 immunocytochemistry is suggested to be a good tool to triage ASCUS, but it reduced the sensitivity of diagnosis when improves the diagnostic specificity.

SHH gene silencing suppresses epithelial-mesenchymal transition, proliferation, invasion, and migration of cervical cancer cells by repressing the hedgehog signaling pathway.

The study aimed to investigate the mechanism by which the sonic Hedgehog (SHH) gene silencing acts upon epithelial-mesenchymal transition (EMT), proliferation, invasion, and migration of cervical cancer (CC) cells via the Hedgehog signaling pathway. RT-qPCR and Western blotting were all employed to detect the SHH mRNA and protein expressions. HeLa and CasKi cells were cultured and subsequently divided into the blank, negative control (NC), and SHH-RNAi groups. A cell counting kit-8 (CCK-8) assay was utilized for cell proliferation. Cell migration and invasion ability were evaluated through scratching test and Transwell assay. The mRNA and protein expressions of the Hedgehog signaling pathway-related factors were detected using RT-qPCR and Western blotting, respectively. After tumor xenograft in nude mice, tumor growth was subsequently observed. SHH mRNA and protein expressions were greater in the SHH-RNAi group than in the blank and NC groups. Compared with the blank group and NC groups, the SHH-RNAi group displayed inhibited levels of proliferation, migration, invasion abilities, as well as a decreased in the Hh signaling pathway-related factors, as well as a reduction in the mRNA and protein expressions of N-cadherin and Vimentin, however, on the contrary increased expressions of E-cadherin were observed. Following tumor xenograft in nude mice, tumor growth was exhibited vast levels of inhibition, particularly in the SHH-RNAi group in comparison to the blank and the NC groups. During the study it was well established that SHH gene silencing suppresses EMT, proliferation, invasion, and migration of CC cells through the repression of the Hedgehog signaling pathway.