The role played by miRNAs as a non-coding RNA in tumors has become a hot research topic in recent years [
13]. Starting with the first discovered miRNAs-lin-4 in 1993, many researchers subsequently started to explore the generation, structure, and function of miRNAs. Studies on a variety of tumors such as chronic lymphocytic leukemia, breast cancer, colorectal cancer, and lung cancer revealed that miRNA expression is abnormal in most tumor cells [
14]. It is believed that miRNAs can play an important role in the diagnosis of many tumors [
15]. It has been found that miRNAs are closely related to the occurrence and progression of prostate cancer, and it is believed that miRNAs are important in both the early diagnosis and malignant progression of prostate cancer [
16]. Therefore, in this study, we screened miRNAs with differential expression in prostate cancer by miR microarray, and verify the potential of the miRNAs in prostate cancer diagnosis and explore the role of the validated miRNAs in the malignant progression of prostate cancer to open a new path for miRNA-based treatment of prostate cancer.
Prostate cancer risk factors and biomarkers
The gold standard for the diagnosis of prostate cancer is puncture biopsy of prostate tissue, but puncture biopsy is a costly and invasive test, which has an impact on patients’ lives [
17]. In fact, in case of transrectal biopsy, the risk of complications requiring hospital admission ranges from 0.1 to 2.5% [
18] being in most of the cases secondary to urinary tract infection (UTI), fever or sepsis. Loeb et al. [
19] reported a cumulative increase in the risk of having a complication where each additional biopsy was associated with a 1.7-fold increase in overall hospitalizations and a 1.7-fold increase in serious infectious complications. Clinical complications and hospital admissions following transrectal prostate biopsy have increased during the last years primarily due to an increasing rate of infections [
20]. Carignan et al. [
21] in 5.798 submitted to transrectal prostate biopsy demonstrated an increased incidence of infections from 0.52% in 2002–2009 to 2.15% in 2010–2011. Pietro Pepe et al. [
22] retrospectively evaluated the clinical complications of 8500 patients who underwent prostate biopsy in more than 20 years of clinical practice and found that clinical complications followed by prostate biopsy biologically involved 35.9% of the patients. Urinary tract infection with over was the most frequent cause of hospital recovery (33.4% of the cases). Loeb et al. [
23] in a random sample of Medicare participants in Surveillance, Epidemiology, and End Results (SEER) regions from 1991 to 2007 found that prostate biopsy was associated with a 2.65-fold increased risk of hospitalization secondary to infections within 30 days compared to the control population. Therefore, there is a need to accurately include patients for puncture biopsy. Currently, the indications for prostate puncture biopsy are mainly PSA, rectal examinations, and imaging examinations, among which PSA, as a clinically common prostate cancer-specific marker, is widely used in the fields of early tumor diagnosis, prediction of recurrence, and clinical monitoring [
24]. However, the indications for prostate puncture biopsy in clinical practice are still controversial. Since 1997, Catalona et al. [
25] advocated prostate biopsy for men with a PSA value in the 2.5- to 4.0-ng/ml range, believing that the use of this parameter increases the detection rate of curable forms of PCa. More recently [
26], they reported on 6691 men who underwent PSA-based screening for PCa, using a mathematical model to adjust for verification bias, they estimated that 82% of the cancers in younger men and 65% of those in older men would be missed with a PSA cutoff value of 4.1 ng/ml. The considerable number of clinically significant cases with PSA ≥ 4 ng/ml has led to suggest that the PSA cut-off value should be lowered to 2.5 ng/ml in order to increase its sensitivity [
27], although this increases the risk of overdiagnosis [
28]. F. Aragona et al. [
29] found that when PSA ≥ 10 is used as the diagnostic standard for prostate cancer, it can only detect 48.2% of prostate cancer patients. If PSA ≥ 4 is used as the diagnostic standard for prostate cancer, it can detect 91.2% of prostate cancer patients, but its specificity for diagnosing prostate cancer is particularly low. Some studies [
30] have shown that elevated peripheral circulating PSA concentrations are not only seen in prostate cancer, but they may also be caused by the effects of indwelling catheterization, urethral manipulation, prostatitis, and urinary tract infection. Therefore, the specificity and sensitivity of PSA in the peripheral circulation for the diagnosis of prostate cancer is poor, and it is prone to misdiagnosis and underdiagnosis, which affects the early diagnosis and treatment of prostate cancer. Digital rectal examination is also an important method in the examination of prostate cancer, especially for asymptomatic prostate cancer patients, which is of great significance in the diagnosis and staging of prostate cancer. However, a digital rectal examination is difficult to reach tumors in the central and transitional regions, especially smaller tumor lesions, and has strong subjectivity. Due to the fact that rectal digital examination can only detect changes in prostate volume and larger palpable tumor masses, 80–90% of prostate cancer detected are advanced prostate cancer in T3 and T4 stages, and the accuracy of reported results is inconsistent. Aragona et al. [
29] found that out of 36 patients with abnormal rectal digital examination, only 8 (22.8%) were diagnosed with prostate cancer, out of 779 patients with abnormal rectal digital examination and PSA, 589 (75.6%) were diagnosed with prostate cancer. Some studies suggest that for early prostate cancer with a PSA of 2.5–10 ng/ml, rectal digital examination is not correlated with prostate biopsy and pathological staging. Multi-parameter magnetic resonance imaging technology and other imaging examinations have the ability to perform multi-parameter, multi-sequence, and non-invasive imaging and have been widely applied in the detection and evaluation of prostate cancer. A study found that the detection rate for prostate cancer [
31] increased with the use of mpMRI reducing the risk of overdiagnosis in comparison with systematic prostate biopsy (17 vs. 28%). The use of mpMRI in clinical practice allowed to reduce the number of needle biopsy cores performed during prostate biopsy, the reduction of needle cores could reduce prostate biopsy complications. A study [
22] showed that complications following transrectal prostate biopsy were directly correlated with the number of needle cores resulting equal to 17.4% (235 cases), 38.7% (1.751 cases), and 55.3% (1.455 cases) in patients who underwent 12 vs. 18 vs. > 24 cores (
p = 0.001), respectively; however, Hajdinjak [
32] and Borofsky et al. [
33] found that mpMRI can also miss clinically significant prostate cancer and may underestimate the volume of prostate cancer tissue. Underestimating the size of the lesion is also a serious issue, especially when doctors rely solely on targeted puncture results for nerve preservation surgery, which may result in residual tumors or positive margins. In addition, mpMRI single fixation of the lesion cannot guarantee that the rest of the prostate is tumor free. Therefore, in addition to PSA, rectal digital examination, and imaging examination, it is necessary to discover new indications for prostate puncture biopsy, to screen patients suitable for prostate puncture biopsy, and to reduce the harm caused by overtreatment.
The results of this study showed that f/t ratio ≤ 0.18, miRNA-222 ≤ 0.69, and miRNA-1248 ≤ 0.94 are independent risk factors for prostate cancer, and it is considered that f/t ratio ≤ 0.18, miRNA-222 ≤ 0.69, and miRNA-1248 ≤ 0.94 can be used as one of the reference indicators for patients before puncture biopsy, when patients are stratified into low-risk and high-risk by these factors. High-risk patients can be considered for puncture biopsy, and low-risk patients can avoid unnecessary invasive tests such as puncture biopsy to reduce the impact of puncture biopsy on patients’ lives to some extent.
The f/t ratio refers to the ratio of free prostate-specific antigen to total prostate-specific antigen. Previous studies have shown [
34] that the risk of prostate cancer is negatively correlated with the f/t ratio and increases with a decrease in the f/t ratio. A study [
35] showed that patients with f/tPSA < 0.2 were 3.84 (1.28–11.56) times more likely to develop prostate cancer than those with f/tPSA > 0.2. It has been shown [
36] that f/tPSA < 0.16 is an independent influence on prostate cancer, when 0.16 was used as a diagnostic criterion for prostate cancer, the sensitivity and specificity of f/t ratio were 71.64% and 67.57%. The results of this study are consistent with them, and patients with f/t ratio < 0.18 are more likely to develop prostate cancer than those with f/t ratio > 0.18.
miRNA-222 is a member of the miRNA family, which plays different roles in different cellular microenvironments and tumors [
37‐
40]. Most studies suggest that miRNA-222 is a “cancer-promoting factor” that plays a pro-cancer role in different tumors. miRNA-222 expression levels are significantly elevated in colon, kidney, gastric, breast, pancreatic, bladder, liver, and multiple myeloma tumors [
41‐
46]. Although miRNA-222 plays a pro-cancer role in most tumors, it has also been reported that miRNA-222 expression levels are significantly reduced in certain tumor tissues and play a cancer-suppressive role. Liu et al. [
47] found that overexpression of miRNA-222 inhibited metastasis and invasion of tongue squamous carcinoma cells and played a cancer-suppressive role. O’Hara et al. [
48] found that miRNA-222 expression levels were downregulated in Kaposi’s sarcoma and exudative lymphoma. The results of the present study are consistent with them, and patients with low miRNA-222 expression levels were more likely to develop prostate cancer than those with high miRNA-222 expression levels. Zohreh Heydari et al. [
49] found in their latest study that miRNA-222 was significantly upregulated in the circulating plasma of prostate cancer patients, but not significantly upregulated in prostate hyperplasia patients. The results of our study are the opposite, possibly because prostate biopsy may affect the expression of miRNA in the peripheral blood circulation. A study comparing the expression profiles of miRNAs in the circulation before and after tumor resection in lung squamous cell carcinoma found that the expression levels of miRNA-205 in plasma significantly decreased 7–10 days after tumor resection [
50]. Analysis shows that among 46 miRNAs with different expressions in peripheral blood monocyte of non-small cell lung cancer patients, 42 miRNAs are down regulated after tumor resection of lung cancer [
51]. It is controversial that some scholars have reported only slight differences in the preoperative and postoperative levels of miRNA-34a in non-small cell lung cancer, with let-7c expression levels increasing after surgery, while miRNA-202 and miRNA-769p showed no significant changes before and after surgery. Follow-up observation of miRNA expression in the plasma of lung cancer patients was conducted from preoperative to postoperative 18 months, it was found that the miRNA expression profile showed specific fluctuations, and the level of miRNA expression was correlated with postoperative time [
52]. Therefore, these studies suggest that miRNAs in the circulation may originate from the release of tumor cells [
53]. However, the source of miRNA in peripheral blood is not yet fully understood. In our study, the subjects were all divided into groups after the results of the prostate biopsy were obtained, and then blood was drawn to test the expression level of miRNA-222. Other studies may include patients who underwent prostate biopsy before taking blood to test the expression of miRNA-222, some patients who met the inclusion criteria first take blood samples and undergo prostate biopsy before grouping, without considering the changes in miRNA-222 expression before and after biopsy. Other reasons for the differences in the results of these studies may be that some patients with different disease progression may be included as the research subjects, and the miRNA expression levels of patients with different disease progression may also be different. Michele Salemi et al. [
54] found an increased expression of miRNA-132 and miRNA-212 in the index case of prostatic adenocarcinoma compared to normal prostate tissue and a lower expression of miR-132 and miR-212 in metastatic lymph nodes compared to primitive PCa and normal prostate tissue. Furthermore, some limitations in our work might influence the results, which may reduce the credibility of our findings, including the low sample size, and one center sampling. Further studies are required to investigate the diagnostic value of plasma miRNA-222-3p in prostatic hyperplasia and prostatic cancer patients at a larger scale, combined with PSA as a known marker for prostatic cancer. There are also reports that miRNA-222-3p is downregulated in metastatic prostate cancer tissue compared to local prostate cancer tissue [
55]. However, another study showed that miRNA-222-3p was upregulated in metastatic prostate cancer tissue [
56]. Therefore, the research results of miRNA-222 in prostate cancer are still contradictory. In addition, the release control of miRNA-222-3p in the body fluids (serum, plasma, and urine) of prostate cancer patients is still unclear, and there are still some unresolved issues.
miRNA-1248 is also a member of the miRNA family, and it plays different roles in different tumors [
57]. Yuhao et al. [
58] found that miRNA-1248 was significantly different in intrahepatic cholangiocarcinoma tissues and paraneoplastic tissues by miRNA microarray, and qRT-PCR was used to detect the expression of miRNA-1248 in 139 cancer and normal patient tissues, verifying that miRNA-1248 expression was upregulated in intrahepatic cholangiocarcinoma tissues. Tanic M et al. [
59] found that miR-1248 can be used as a genetic test standard in hereditary breast cancer. A study [
60] showed that miR-1248 could inhibit the proliferation of gastric cancer cells and induce apoptosis of gastric cancer cells for cancer suppression purposes. Xiaoyuan et al. [
61] found that the expression of miRNA-1248 in the plasma of liver cancer and non-hepatocellular carcinoma patients differed by high-throughput sequencing. qRT-PCR was used to detect the expression of miRNA-1248 in the plasma of 139 cancer and normal patients, and it was verified that miRNA-1248 expression was down-regulated in the plasma of liver cancer patients. The results of this study were consistent with it, and patients with low miRNA-1248 expression levels were more likely to develop prostate cancer than those with high miRNA-1248 expression levels.
Regulation of prostate cancer by miRNA-222
It has been found that certain miRNAs can promote or inhibit the metastatic and invasive ability of tumor cells. Also, some miRNAs affect the apoptosis of tumor cells by promoting or inhibiting the proliferation ability of cells. It plays a crucial role in the malignant progression of prostate cancer [
62]. It has been reported that antagonists and agonists of miRNAs have been synthesized to affect the biological function of prostate cancer cells by blocking or restoring the function of specific miRNAs [
63]. Alternatively, miRNA or anti-miRNA molecules can be delivered into the body through a vector vehicle, and these small molecules can induce changes in the biological functions of the targeted cells by systemic or local administration. If these antitumor molecules specifically alter the biological specificity of tumor cells, then these molecules may be harmless to normal cells. The application of these antitumor molecules in vivo opens new paths for miRNA-based therapy for prostate cancer.
In this study, miRNA microarray technology was used to identify 13 miRNAs that differ in prostate cancer tissues from non-prostate cancer tissues. By reviewing the literature, we selected four known miRNAs that have rarely been studied in prostate cancer (hsa-miRNA-1248, hsa-miRNA-144-3p, hsa-miRNA-3651, hsa-miRNA-222), the present study found that the expression of hsa-miRNA-222 among the above four miRNAs was consistent in microarray results, tissue samples, and blood samples. In addition, although miRNA-222 has been shown to be one of the cancer suppressor or cancer-promoting miRNAs in malignant tumors such as colon cancer, kidney cancer, gastric cancer, breast cancer, pancreatic cancer, bladder cancer, liver cancer, multiple myeloma, tongue squamous carcinoma, Kaposi's sarcoma and exudative lymphoma, its role in prostate cancer is unclear. Bin Gui et al. [
64] found in their latest study that miRNA-222, as a carcinogenic gene for prostate cancer, promotes the proliferation of prostate cancer cells and the development of castration-resistant prostate cancer (CRPC) in the early stages, indicating that miRNA-222 has a cancer-promoting effect. Other studies have shown that miRNA-222 expression is downregulated in metastatic prostate cancer and CRPC specimens, indicating that miRNA-222 has tumor-inhibitory effects [
65‐
67]. The expression and biological function of miRNA-222 in prostate cancer are still controversial. Therefore, in this study, miRNA-222 was used as a research target to explore the regulation of miRNA-222 in prostate cancer and to provide a new basis for miRNA-222-based treatment of prostate cancer.
Previous studies have indicated that miRNA-222 has a complex function in different tumors and has important applications in the early diagnosis, treatment, and prognosis prediction of malignant tumors. Overexpressed miRNA-222 is associated with invasive ability, apoptosis, and cell proliferation in a variety of tumor cells [
43,
44]. It was found that upregulation of miRNA-222 expression can cause increased invasion, growth, and metastatic ability of tumor cells and even lead to conditions such as drug resistance [
68,
69]. Zhang et al. [
70] found that overexpressed miRNA-222 in gastric cancer increased the invasion and proliferation ability of gastric cancer cells by regulating the expression of PTEN. It was also found that miRNA-222 overexpressed in gastric cancer cells increased the proliferative ability of gastric cancer cells by regulating the expression of RECK [
71]. The above studies suggest that miRNA-222 expression in gastric cancer may become a biomarker and target for targeted therapy in gastric cancer. Liu et al. [
47] found that miRNA-222 overexpressed in tongue squamous cell carcinoma exerted cancer-suppressive effects by reducing the expression of peroxisome 2 and matrix metalloproteinase 1 and inhibiting the metastatic and invasive ability of cells. The results of the present study are consistent with this finding. The invasive, proliferative, and migratory abilities of miRNA-222 overexpressed PC-3 cells were significantly inhibited, and the apoptosis rate was significantly increased. It can be concluded that miRNA-222 plays an important role in inhibiting the malignant progression of prostate cancer, which provides a new direction and basis for the study of new therapeutic targets and new biomarkers for prostate cancer.